Familial hypercatabolic hypoproteinemia. A disorder of endogenous catabolism of albumin and immunoglobulin.

The metabolism of albumin and IgG was investigated in two siblings, products of a first-cousin marriage, a female aged 34 yr and a male aged 17, who had a marked reduction in their respective serum concentrations of IgG (1.3 and 3.1 mg/ml) and albumin (19 and 21 mg/ml). The metabolism of radioiodinated IgG and albumin was studied in the two patients. The total circulating and body pools of IgG were less than 28% of normal. The IgG synthetic rates were within the normal range. However, the IgG survival was short, with their respective fractional catabolic rates increased fivefold to 31% and 36% of the intravenous pool per day (normal, 6.7 +/- 2%/d). Furthermore, the patients had reduced total body pools, normal synthetic rates, and increased fractional catabolic rates for albumin. There was no proteinuria or abnormality of renal or liver function. In addition, the patients did not have circulating antibodies directed toward IgG, IgA, or albumin. Furthermore, both patients had normal fecal 51Cr-labeled albumin tests, thus excluding excessive gastrointestinal protein loss. We propose that these siblings have a previously unrecognized familial disorder characterized by reduced serum concentrations of IgG and albumin caused by a defect in endogenous catabolism, leading to a short survival of these proteins that is associated in this family with chemical diabetes and a skeletal deformity.

Specific interaction of myeloma tumor cells with hapten-bearing liposomes containing methotrexate and carboxyfluorescein.

Hapten-bearing liposomes containing methotrexate and the fluorescent solute carboxyfluorescein were incubated with murine myeloma tumor cells expressing surface immunoglobulin with affinity for the hapten. Liposomes bearing the dinitrophenyl hapten became bound to MOPC 315 myeloma tumor cells, and liposomes bearing the phosphorylcholine hapten became bound in much larger amounts to TEPC 15 cells. In each case, fluorescence microscopy showed a patchy surface pattern, indicating intact liposomes at the cell surface. Few liposomes were bound to cells in the presence of excess soluble hapten or to cells lacking the relevant surface immunoglobulin. Cell-associated liposomes were quantitated by use of the fluorescence-activated cell sorter, and the pharmacological effect of the methotrexate was assessed from measurement of incorporation of radiolabeled deoxyuridine into the cells. Little inhibition of deoxyuridine incorporation was observed, because contents of the bound liposomes did not enter the cytoplasmic compartments of the cells.

Receptor-mediated endocytosis of antibody-opsonized liposomes by tumor cells.

Specific receptor-mediated delivery of the contents of small, sonicated liposomes was studied with three murine tumor cell types: an IgG Fc receptor-negative nonphagocytic line (EL4); an Fc receptor-positive phagocytic line (P388D1); and an Fc receptor-positive nonphagocytic line (P388). The liposomes (formed from phosphatidylcholines, cholesterol, and dinitrophenyl-substituted phosphatidylethanolamine) contained carboxyfluorescein as a fluorescent marker and methotrexate as a pharmacologic agent. Binding and internalization of the liposomes were observed by fluorescence microscopy and measured by flow microfluorometry. The hapten-derivatized lipid was used as a binding point on the liposome for the antibody-combining site of the immunoglobulin. In the presence of IgG anti-dinitrophenyl, but not F(ab')2 or IgA anti-dinitrophenyl, liposomes bound to the Fc receptor-bearing cells. The liposomes underwent endocytosis by the P388D1 cells and, to a lesser extent, by the P388 cells. As measured by depression of [3H]deoxyuridine incorporation, methotrexate in IgG-opsonized liposomes had a much greater pharmacologic effect on the P388D1 cells than did the same amount in unopsonized liposomes or in free solution. This observation indicates that an appropriately chosen drug, incorporated in liposomes, can exert its effect on a cytoplasmic target after endocytosis. P388 cells showed a moderate effect of the drug in liposomes. Neither P388 nor P388D1 cells bound or ingested unopsonized liposomes, and the Fc receptor-negative EL4 line neither bound nor ingested opsonized liposomes. The data demonstrate specific interaction of opsonized liposomes with the cells' IgG Fc receptor.

Studies on in vivo priming of the TNP-reactive cytotoxic effector cell system. I. Comparison of the effects of intravenous inoculation with TNP-conjugated cells on the development of contact sensitivity and cell-mediated lympholysis.

A comparison was made of the effects of i.v. inoculation of trinitrophenyl-(TNP) conjugated syngeneic cells on the subsequent in vitro generation of TNP-reactive effector cell activity and on the in vivo development of TNP-contact sensitivity. The administration of syngeneic TNP-conjugated spleen cells before 2,4,6-trinitro-1-chlorobenzene (TNCB) painting abolished the capability of animals to develop TNP-contact sensitivity. In contrast, the same treatment resulted in an appreciable augmentation in the generation of TNP-reactive cytotoxic effector cell activity as measured by subsequent in vitro sensitization with TNP-conjugated cells. The possible mechanisms by which enhanced TNP-reactive cytotoxic effector cell activity was elicited under conditions identical to those that induced unresponsiveness for TNP-contact sensitivity are discussed.

Binding of antigen-bearing fluorescent liposomes to the murine myeloma tumor MOPC 315.

Small unilamellar lipid vesicles bearing the DNP-hapten on their surfaces and containing the water-soluble fluorescent dye carboxyfluorescein were formed by sonication. These vesicles were incubated with cells from the murine myeloma tumor MOPC 315, which secrete and also bear on the cell surface an immunoglobulin with affinity for the nitrophenyl hapten. At 0 degrees C the cells bound an average of several thousand vesicles at saturation. This binding was specific for the nitrophenyl hapten on the vesicle since it was abolished by an excess of soluble nitrophenyl derivative, by omission of the hapten from the vesicle, or by substitution for MOPC 315 of a tumor lacking receptors for the nitrophenyl hapten. Specific binding of vesicles was greater when cells were incubated at 37 degrees C. The study suggests that ligand-bearing vesicles can be a useful marker for cell surface immunoglobulin. However, in spite of the ability to "target" vesicles to cell surface determinants, binding did not result in increased delivery of vesicle contents to the cytoplasm.

Characterization of the nonphagocytic adherent cell from the peritoneal cavity of normal and BCG-treated mice.

A distinctive subpopulation of nonphagocytic, tightly adherent cells (NPAC) comprised approximately 6% of the adherent peritoneal cells from untreated mice, and about 18% of those from mice previously given BCG i.p. A separation procedure based on adherence and lack of phagocytosis was devised. Isolated NPAC were morphologically intermediate between small lymphocytes and macrophages. They were positive for nonspecific esterase, negative for peroxidase, positive for surface IgM, and negative for surface IgG1, IgG2 and IgA. When capped, their surface IgM regenerated in vitro. NPAC had demonstrable Fc receptors but not EAC receptors. They resisted killing by an anti-macrophage serum, were negative by immunofluorescence with an anti-T cell reagent, and incorporated increased amounts of thymidine in response to LPS but not to PHA. They were more readily killed with anti-Ia serum and complement than macrophages, but less readily than splenic B cells. NPAC appeared to represent a subpopulation of B lymphocytes which contaminates some preparations previously regarded as "macrophages" and which may be ressponsible for some of the activities previously ascribed to "macrophages".

Modifications and evaluation of double antibody radioimmunoassay of human carcinoembryonic antigen.

Double antibody radioimmunoassay of carcinoembryonic antigen (CEA), a cancer-associated antigen of the human digestive system, was subjected to certain modifications and critically evaluated. Modifications pertained to: (a) the production of a high titer goat anti-CEA antiserum that was rendered highly specific by solid phase immunoabsorption with cyanogen bromide-activated Sepharose conjugates of normal plasma liver, and colon perchloric acid-soluble glycoprotein antigens: (b) the introduction of suitable alterations in the experimental conditions of radioiodination procedure to minimize and to prevent breakdown of the antigen, thus prolonging the storage of the labeled antigen; (c) the extended incubation period of CEA-anti-CEA immune reaction; and (d) the use of sodium acetate buffer, pH 6.1. Furthermore, the use of an automatic pipetting station for accurate and rapid reagent dispensation and statistical analysis of the radioimmunoassay data on a modern computer to ensure strict quality control of the assay provided some definite improvement over the existing assay.

Differential stimulation of murine lymphoma growth in vitro by normal and BCG-activated macrophages.

Peritoneal macrophages from mice infected with Bacille Calmette-Guérin (BCG) and from normal mice were examined for their effects in vitro on thymidine uptake by 10 murine lymphomas, a murine fibroblast line, and a guinea pig hepatoma. Only the murine fibroblast line showed growth inhibition in the presence of BCG macrophages. For the majority of tumors, normal macrophages were profoundly stimulatory to tumor cell DNA synthesis, while BCG macrophages were much less stimulatory, without being frankly inhibitory. The effect of 2-mercaptoethanol on tumor cell growth was also studied. All lymphomas stimulated to grow more rapidly in vitro by normal macrophages were stimulated to a similar degree by 2-mercaptoethanol.

The relationship of IgA and IgE deficiency.

Studies of IgE deficiency and IgE levels in selective IgA deficiency and ataxia-telangiectasia are reviewed. Isolated IgE deficiency and combined IgE-IgA deficiency do not predispose individuals to recurrent respiratory tract disease. In contrast, IgA-deficient patients with normal or elevated levels of IgE frequently have recurrent respiratory infections. Although IgE deficiency and combined IgE-IgA deficiency occur frequently in ataxia-telangiectasia, these deficiences do not appear to play a primary role in the pathogenesis of sinopulmonary disease in these patients. These observations are discussed in relation to recent evidence concerning the regulation of the IgE-antibody response.