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.

Synergy between subpopulations of mouse spleen cells in the in vitro generation of cell-mediated cytotoxicity: evidence for the involvement of a non-T cell.

Two subpopulations separated from normal spleen have been shown to synergize as responding cells in the in vitro induction of specific cell-mediated cytotoxicity during the mixed lymphocyte culture (MLC). The synergizing populations are a nylon wool column-adherent and a nylon wool column-nonadherent fraction, enriched for B lymphocytes and T lymphocytes, respectively. When a mixture of these fractions is used as the responding cell population in MLC, greater cytotoxicity is generated than would be expected from the sum of activities generated in the two subpopulations sensitized separately. The synergy appears to occur at the sensitization rather than the effector phase. The synergizing cell which is contained in the nylon-adherent subpopulation is distinct from the cytotoxic effector T lymphocyte, is resistant to lysis by rabbit antimouse brain serum, and is unresponsive to phytohemagglutinin; its synergizing function could not be replaced by either plastic-adherent spleen cells or peritoneal exudate cells. These results suggest a role of a non-T-cell nonmacrophage population in the generation of cytotoxic activity.

Antigenic heterogeneity of human immunoglobulin A proteins.

Two types of human IgA myeloma proteins were distinguished by immunochemical tests. Seven of 51 IgA-myeloma proteins contained an an tigenic determinant that was not de tected in the other 44 proteins. The distinctive antigenic site was not dem onstrated on either the heavy or light polypeptide chains.

Electrophoretic heterogeneity of the polypeptide chains of human G-myeloma proteins.

The light and heavy polypeptide chains derived from human Gmyeloma proteins are electrophoretically heterogeneous as judged by disc electrophoresis of the polypeptide chains in urea-acrylamide gels. Individual myeloma proteins contained as many as eight light-chain and nine heavy-chain components.

B-cell alloantigens determined by the H-2 linked Ir region are associated with mixed lymphocyte culture stimulation.

Cell surface antigens, controlled by genes located in the Ir region of the murine major histocompatibility complex, are shown serologically to be expressed preferentially on bone marrow-derived lymphocytes. These antigens may play a major role as stimulators in mixed lymphocyte cultures.

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.

Metabolic properties of IgG subclasses in man.

Metabolic properties of the four subclasses of human IgG were investigated by performing 47 turnover studies in individuals with normal IgG serum concentrations, as well as in patients with an increased level of one of the subclasses. Studies in 12 subjects with normal IgG serum concentration showed that the average biologic half-life of G(1), G(2), and G(4) was 21 days, while that of G(3) was only 7.1 days. Fractional catabolic rates of G(1), G(2), and G(4) were 6.9 to 8% of the intravascular pool per day. G(3), however, had a higher fractional catabolic rate, amounting to 16.8% of the intravascular pool per day. Distribution of the subclasses was such that the intravascular compartment contained 51-54% of the total body pools of G(1), G(2), and G(4), but 64% of the total body pool of G(3).The short survival and high fractional catabolic rate of G(3) is an inherent property of these molecules, and is not due to denaturation during isolation and radiolabeling. This was demonstrated by studies of a patient with a serum G(3)-myeloma protein. The survival of her own protein, separately labeled either in vivo with guanidoarginine-(14)C or in vitro with (125)I, was determined in the patient. Survivals of the in vivo and in vitro labeled proteins were identical.G(1) and G(3) serum concentrations and synthetic rates were determined. The mean serum concentration of G(1) was 6.8 mg/ml and that of G(3) was 0.7 mg/ml, while their synthetic rates were 25.4 and 3.4 mg/kg per day respectively. The low serum concentration of IgG(2) thus results from a combination of high catabolic and low synthetic rates. Studies in 10 patients with multiple myeloma showed that an elevated serum concentration of any IgG subclass was associated with shortened biologic half-life and increased fractional catabolic rate of all subclasses. The implications of this concentration-catabolism relationship are discussed. The serum concentration of nonmyeloma IgG was usually low in myeloma patients and the synthesis of nonmyeloma IgG was somewhat decreased, suggesting that low serum concentrations of nonmyeloma IgG result from decreased synthesis, as well as from an increased fractional catabolic rate.

Lymphocytes in patients with variable immunodeficiency and panhypogammaglobulinemia. Evaluation of B and T cell surface markers and a proposed classification.

Peripheral blood lymphocytes from 15 patients with variable immunodeficiency and severe panhypogammaglobulinemia were evaluated for B and T cell surface markers. B cells were enumerated by immunofluorescent detection of both surface immunoglobulin (Ig) and the ability to bind aggregated Ig complexes. T cells were identified by their ability to form nonimmune rosettes with sheep red blood cells. Four distinct patterns were observed which were designated types I-IV. Type I: six patients had normal percentages (8.5-19.0%) of Ig-bearing B lymphocytes. Type II: four patients were observed to have B lymphocytes (4.5-15.0%) which lacked fluorescence-detectable surface Ig. Type III: the peripheral blood of these four patients contained a subpopulation (11.3-20.0%) of lymphocytes which apparently lacked both B and T cell markers ("null" cells). Type IV: one patient's blood was characterized by a subpopulation (18.0-22.0%) of lymphocytes which bore both B and T cell markers. Patients of each type had some clinical features in common. It is concluded that evaluation of lymphocyte surface markers provides a means of separating patients with variable immunodeficiency and panhypogammaglobulinemia into distinct groups which appear to differ in the nature of their fundamental defect.

Human monoclonal gamma G-cryoglobulins with anti-gamma-globulin activity.

Seven human gammaG-myeloma proteins which were also cryoglobulins were studied with respect to their reactivity with gammaG-globulins as well as with regard to their antigenic classification within the gammaG-heavy chain subclasses. Five of the seven cryoglobulins studied were positive in at least two of the three tests used to assay for anti-gamma-globulin activity. One protein was only weakly positive in one test system and another was negative in all test systems. The structures which were recognized by the cryoglobulins were localized to the Fc-fragment. Only primate gammaG-globulins contained these antigenic determinants and in some cases the cryoglobulin appeared to show specificity for one human heavy chain subclass over the others. Antigenic analysis revealed that four of the five cryoglobulins with definite antibody activity belonged to the gammaG3-subclass, the fifth belonged to the gammaG1-subclass. The two cryoglobulins which reacted only weakly or failed to combine with gammaG-globulins were both of the gammaG1-subclass. These findings taken together with the localization of the combining site to the Fab-fragment suggests that many of these cryoglobulins may represent antibodies to gammaG-globulin, and that the cryoprecipitate in these cases represents antigen-antibody complexes of such a nature that they precipitate only in the cold.

Structural identity of Bence Jones and amyloid fibril proteins in a patient with plasma cell dyscrasia and amyloidosis.

The partial amino acid sequence of the amyloid fibril protein isolated from the small intestine of a patient with plasma cell dyscrasia and associated amyloidosis has been determined and compared with the sequence of the kappa-type Bence Jones protein isolated from the urine of the same patient. Identical sequences were observed for the 27 amino-terminal residues that could be compared. The C-terminal tryptic peptide of the amyloid protein was identical with that of the Bence Jones protein. Apparent molecular weights and amino acid compositions of the Bence Jones and amyloid proteins were similar. It appears, therefore, that the predominant protein present in the amyloid deposits in this patient was an intact kappa-type light polypeptide chain that was identical with the urinary Bence Jones protein.

Immunoglobulin E in immunologic deficiency diseases. I. Relation of IgE and IgA to respiratory tract disease in isolated IgE deficiency, IgA deficiency, and ataxia telangiectasia.

Serum immunoglobulin E concentration was studied in normal children and adults, in 25 patients with isolated IgA deficiency, and in 44 patients with ataxia telangiectasia using a double antibody radioimmunoassay. The geometric mean IgE level of the normal adult population studied was 105 ng/ml, with a broad 95% interval (5-2045 ng/ml). Individuals with concentrations less than 15 ng/ml were considered to be IgE deficient. IgE deficiency, defined in this way, was observed in 7 of 73 normal adults and was not found to be associated with respiratory tract disease.80% (35 of 44) of patients with ataxia telangiectasia (AT) were IgE deficient, 66% were IgA deficient, and 57% had combined IgE and IgA deficiencies. Although 45% of the patients with AT had respiratory tract disease, there was no correlation found between IgE deficiency or combined IgE and IgA deficiency and respiratory tract disease in these patients.11 of 25 individuals with isolated IgA deficiency were also IgE deficient. All 11 patients with both IgA and IgE deficiency were uniformly asymptomatic. However, there was an extremely high incidence (71%) of respiratory tract disease in IgA-deficient individuals who were not IgE deficient. These data fail to support the concept of a protective role for IgE in respiratory tract immunity. The possible role of IgE in the pathogenesis of respiratory tract disease in IgA-deficient patients is discussed.

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.

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.