Biochemical and histological characterization of antigens preferentially expressed on the surface and cytoplasm of breast carcinoma cells identified by monoclonal antibodies against the human milk fat globule.

The preparation of monoclonal antibodies (MAbs) against the human milk fat globule membrane with preferential binding to breast carcinoma cells is described. Using BALB/c mouse myeloma cells; inter-specific, intra-strain, and inter-strain hybridomas were isolated that identified three different components of the human milk fat globule of approximately 46,000, and 70,000 daltons and a mucin-like glycoprotein complex (NPGP) ranging from 400,000 to over a million daltons, respectively. Three MAbs (BrE1, BrE2, BrE3) identified the latter component which consists of at least three different size molecules for which the aforementioned MAb's have different binding specificities. MAbs, BrE2 and BrE3, bound to normal breast epithelial cells but to a lesser extent than to tumors and only at the apical surface facing the lumen, while they bound breast carcinomas strongly, and often in the cytoplasm as well as on the surface. Higher concentrations of BrE3 were required to stain normal breast compared to breast tumors. BrE1 also stained breast carcinomas both on the surface and cytoplasmically but did not stain normal breast tissue. The MAb, Mc13, as well as the previously reported MAb McR2, both against the 70,000 dalton component, did not significantly stain either normal or cancerous breast tissue in histological sections but did bind significantly to cultured breast epithelial cells and to the milk fat globule membrane. The MAbs, Mc8 and Mc3, reported previously to be against the 46,000 dalton component, stained histologically only malignant breast tissue but only weakly; however, they bound strongly to intact breast carcinoma cells and breast cell membrane preparations with a radioimmunobinding assay. These MAbs should be useful in characterizing the surface of breast epithelial cells, studying surface alterations in malignancy, and possibly in breast cancer diagnosis and therapy.

Human milk-fat globule membrane derived mucin is a disulfide-linked heteromer.

The human milk-fat globule membrane (HMFG) contains a number of antigens also expressed on breast tumors. The dominant antigens are a mucin of MW greater than 400,000 and a group of antigens of MW 67,000-70,000. The mucin was separated with monoclonal antibodies against HMFG and a MW 70,000 doublet was found to be associated with the mucin under non-reducing conditions, the latter identifiable by another set of monoclonal antibodies. Immunoprecipitation of the mucin and analysis of the precipitated material using Western blots and identification of transferred material with monoclonal antibodies demonstrated that the MW 70,000 protein and the mucin were co-precipitated and linked by reducible disulfide bonds. Treatment with detergents, protein-dissociating agents and glycosidases did not release the component from the mucin. Mucins in HMFG membrane and breast tumor cells may be associated therefore to disulfide-linked linker proteins similar to those described for intestinal and gastric mucins, that would co-purify with the mucin under non-reducing conditions.

In vitro cytotoxicity and antibiotic activity of polymyxin B nonapeptide.

Polymyxin B nonapeptide, prepared by enzymic removal of the fatty acyl diaminobutyric acid side chain from polymyxin B, was about 100-fold less toxic to K562 cells than polymyxin B. MICs of polymyxin B nonapeptide against a test panel of bacteria were 2- to 64-fold lower than those of polymyxin B.

Natural killer cell-mediated lysis involves an hydroxyl radical-dependent step.

The role of oxygen radicals in lysis of K562 target cells by human natural killer (NK) cells was determined by addition of scavengers of these free radicals. Lysis was greatly reduced under hypoxic conditions. Superoxide dismutase and cytochrome c, scavengers of superoxide anions, and catalase and scavengers of hypochlorite had no effect on lysis. Of 15 hydroxyl radical scavengers tested, 13 inhibited lysis. These were not toxic, because cell morphology and spontaneous chromium release were not affected and preculture with scavengers was not inhibitory. These scavengers differed widely in structure, but degree of inhibition of lysis correlated with their rate constants (k) for reaction with hydroxyl radical (k vs log inhibitor concentration required to decrease lysis by 50%: r = -0.9202, p less than 0.001), showing that inhibition was due to inactivation of the hydroxyl radical. Target cell binding was not reduced at concentrations that inhibited lysis. Inhibitors of the lipoxygenase pathway also decreased lysis, suggesting this pathway to be the source of hydroxyl radicals. In view of the reported requirements for hydroxyl radical-mediated lipid peroxidation for optimal secretory activity in a number of cell types, it appears that the generation of hydroxyl radicals by NK cells is required for delivery of cytotoxic factors.

Depressed natural killer and lectin-induced cell-mediated cytotoxicity in cholesterol-fed guinea pigs.

Dunkin-Hartley and Hartley guinea pigs were fed a diet containing 1% cholesterol (C+) or a control diet (C-). The C+-fed guinea pigs showed a decrease in antitumor effector cell levels as measured by an in vitro 18-hour 51Cr release assay. Natural killer (NK) activity fell rapidly after initiation of cholesterol feeding, decreasing to 25.6% of control levels by 2 weeks. While the interferon inducer polyinosinic-polycytidylic acid increased NK activity as much as 3.6-fold in controls, the NK levels in C+-fed animals were not increased. NK activity was lower in both spleen and peripheral blood of C+-fed animals and against K562, MOLT-3, HL-60, and Raji target cells. Lectin-dependent cell-mediated cytotoxicity was increased in the C+-fed group over, the first 1-2 weeks on the diet, but it dropped to low levels by 6 weeks. Lipoprotein preparations from plasmas of both C+- and C--fed animals inhibited NK cell activity, but suppression was not due to lipoprotein cholesterol content. On the basis of lipoprotein protein, lipoproteins from C--fed animals were more suppressive. The results suggest that the decrease in cytotoxicity induced by dietary cholesterol is due to more than the high levels of plasma and lipoprotein cholesterol.

Involvement of hydroxyl free radical, but not superoxide, in the cytolytic pathway of natural killer cells. Revision of an earlier hypothesis.

Our earlier hypothesis suggested that NK effectors, in response to tumor cells, generate superoxide anion (O2.-) which was necessary for NK cytolysis to proceed. This event could be detected by chemiluminescence. More recent data suggests that O2.- is not necessary for NK cytolysis and that chemiluminescence is the result of an NK-tumor cell-monocyte interaction. Hydroxyl radicals however do contribute to the NK-mediated cytolytic process. The source of OH. is unknown but does not appear to be generated by the NADPH oxidase system. We suggest that hydroxyl radical generation is a necessary event in NK cytolysis but we do not yet know if it acts at the level of NK activation, delivery of the lethal hit or in autolysis by the tumor cells.

Effects of dietary cholesterol on antibody-dependent phagocytosis and cell-mediated lysis in guinea pigs.

The effect of dietary cholesterol on antibody-dependent phagocytosis and cell-mediated cytotoxicity (ADCC) by peritoneal cells and on the susceptibility to lysis of erythrocytes was studied in the guinea pig. We found that peritoneal cells from cholesterol-fed animals (CHOL PEC) demonstrated a decreased ability to both phagocytose and lyse antibody-coated (Ab) guinea pig erythrocytes than did those from control guinea pigs (CONT PEC). This decrease was equal in groups fed cholesterol for 5 1/2-13 weeks, preanemic or anemic, and with normal or enlarged spleens. Dose response curves varying Ab concentration showed that CHOL PEC required higher concentrations of Ab to effect phagocytosis and lysis than did CONT PEC. Dietary cholesterol, while rapidly inducing morphological changes such as spurring in guinea pig erythrocytes, was found not to affect the susceptibility of the cells to lysis or phagocytosis in this assay system. These findings suggest that the increased incidence of infection in cholesterol-fed guinea pigs may be due to impaired phagocytic function and that the anemia observed in guinea pigs after 8-10 weeks of feeding cholesterol is not due to increased antibody-dependent removal of spurred erythrocytes by the phagocytic system.

Effects of the composition of peritoneal dialysis fluid on chemiluminescence, phagocytosis, and bactericidal activity in vitro.

Commercial fluid used for peritoneal lavage in peritonitis and in peritoneal dialysis suppressed the activity of peripheral blood leukocytes as measured by chemiluminescence, phagocytosis, and bacterial killing. Suppression was found to be due to the low pH and high osmolality of the fluid. The pH was adjusted to noninhibitory levels in vivo within 30 min, whereas osmolality changes were less rapid and remained at inhibitory levels for fluids of high dextrose concentration (4.25%). Chemiluminescence was the most sensitive assay for inhibitory effects of pH and osmolality, as well as for urea and heparin. The metabolic waste product urea at levels normally found in dialysate and heparin at concentrations routinely added to fluid inhibited only chemiluminescence, whereas creatinine and added insulin were not inhibitory. High fluid volume also resulted in a decrease in efficiency of bacterial killing. These results suggest some changes to be made in the treatment of peritonitis and in peritoneal dialysis.

Immunological senescence. II. Normal in vitro colony formation by B cells from old mice.

The in vitro frequency and proliferative capacity of B cells and B-cell precursors in old mice was assessed. The mitogenic response to the B-cell mitogens lipopolysaccharide (LPS) and dextran sulphate decline later and at a slower rate than the T-cell response to phytohaemagglutinin and concanavalin A. The response to precursor B-cell mitogen dextran sulphate was not depressed in the bone marrow of aging mice. In addition, the dose response and kinetics of LPS and mercaptoethanol stimulated B-cell colony formation in agar was identical in spleen cells from young and old mice. These results indicate that the intrinsic proliferative capacity of B cells from old mice is normal.

Immunological senescence. I. The role of suppressor cells.

The in vitro anti-SRBC response of several murine strains declined markedly with age in parallel with an increase in the activity of suppressor cells in the spleen and bone marrow which prevented early events during the induction of the immune response. These suppressor cells released soluble mediators and lacked the characteristics of mature T cells or macrophages. In addition the suppressor cell in the bone marrow could be removed on anti-Ig columns and fractions of old splenic suppressor cells sedimenting at 0.32 cm/h were greatly enriched in surface Ig bearing cells. Old immunodepressed mice did not lack potentially immunocompetent cells since the antibody response of old spleen cells could be restored by specifically activated T cells or lipopolysaccharide which act on B cells. These results suggest that a rise in the activity of non-T suppressor cells in the spleen and bone marrow may account, in part, for the depression in humoral immunity observed in aging mice.