PHYSICAL PROPERTIES OF THE RED CELL AGGLUTININS IN ACQUIRED HEMOLYTIC ANEMIA

The sera of 8 patients with acquired hemolytic anemia associated with elevated levels of cold agglutinins were studied by various procedures of zone electrophoresis. The agglutinating activity was found associated with proteins of variable mobility in the different cases. The majority represented "fast" γ-globulins. The 4 sera with the highest titers of cold agglutinins showed distinguishable abnormal electrophoretic components. The titers correlated with the height of the abnormal components. Ultracentrifugal analysis of the electrophoretic fractions indicated that the cold agglutinins were associated with proteins having a sedimentation coefficient of approximately 19 S. The abnormal component from the serum with the highest biological activity showed almost no contamination with lower molecular weight proteins. The amount of 19 S material found correlated with the titer of agglutinating activity. The high molecular weight character of the cold agglutinins was confirmed by procedures of density gradient zone centrifugation. The biological activity sedimented with proteins of the 19 S class in all the sera including those of relatively low titer with which no abnormal electrophoretic components were observed. Dissociation of the abnormal high molecular weight components was possible by means of certain sulfhydryl compounds. This resulted in disappearance of cold agglutinin activity. Some of the cases could be classified as macroglobulinemias because of the very large content of high molecular weight components. However, the same disease picture occurred without recognizable elevation of these components. The sera of 3 patients with severe acquired hemolytic anemia of the warm type associated with warm incomplete antibodies failed to show similar abnormal electrophoretic components and the antibody activity sedimented with proteins of the 7 S class

THE PRECIPITIN REACTION BETWEEN DNA AND A SERUM FACTOR IN SYSTEMIC LUPUS ERYTHEMATOSUS

The sera of certain patients with systemic lupus erythematosus contain an antibody-like substance capable of reacting with highly purified DNA preparations from widely divergent sources. Precipitin reactions have been demonstrated by double diffusion in agar and quantitative precipitin curves have been obtained. Complement was observed to be fixed in the reaction. Evidence was obtained that the serum factor possessed antigenic properties similar to those of γ-globulins and migrated with this fraction on zone electrophoresis. The interaction of this factor with DNA exhibited certain specific characteristics which differ considerably from non-specific reactions between DNA and proteins in general. The DNA-precipitating factor appeared to be one of a number of related factors reacting with nuclear constituents of many different cells. It differed in certain respects from the "LE factor" which is responsible for the formation of "LE cells." The accumulated evidence, although not yet conclusive, favors the concept that the precipitating factor represents an antibody to DNA, and that it is one of a number of autoantibodies elicited in this disease

INTERACTION OF AGGREGATED γ-GLOBULIN WITH B LYMPHOCYTES

Specific binding of aggregated γ-globulin to a subpopulation of lymphocytes was demonstrated. This subpopulation was identified as the Ig-staining or B lymphocytes. The binding was irreversible and independent of complement, pH, temperature, protein content of the medium, and divalent cations. Aggregates of large size were needed for optimal visualization. Evidence was obtained that the site on the lymphocyte membrane responsible for binding aggregates was distinct from surface Ig

MEMBRANE IMMUNOGLOBULINS OF B LYMPHOCYTES : Inability to Detect Certain Characteristic IgM and IgD Antigens

Hemagglutination and fluorescent antibody studies have provided strong evidence for the unavailability or absence of specific antigenic sites on membrane-bound IgM which are present in serum and intracellular IgM. Antisera specific for different parts of the molecule indicated that a portion but not all of the Fc was involved. Absorption experiments with normal and leukemic viable B lymphocytes failed to remove a population of Fc antibodies found in IgM-specific antisera. Similar findings were made for IgD, the other major membrane immunoglobulin of human peripheral blood B cells. Various interpretations of these observations are discussed. The most likely possibility appears that the C-terminal portion of the heavy chains of the immunoglobulin molecule is buried in the membrane

THE STABILIZATION OF SERUM LIPID EMULSIONS BY SERUM PHOSPHOLIPIDS

Clarity of high lipid sera is closely correlated with elevated proportions of serum phospholipids, and lipemia (milkiness) with low proportions of phospholipids. Clear high lipid sera occur uniquely in obstructive jaundice, both intra- and extrahepatic in origin. Destruction of the polar nature of serum "lecithin" by enzymatic hydrolysis, using Cl. welchii lecithinase, results in a degree of lipemia which is linearly proportional to total lipid content in clear or lipemic high lipid or normal lipid sera. Even in grossly lipemic sera, a significant proportion of the serum lipids is masked in particles of invisible size. Enzymatic removal of the stabilizing properties of serum "lecithin" unmasks this hitherto invisible fraction. The concentration of serum phospholipids available for complex formation with serum proteins appears to be an important factor in determining particle size of serum lipids and hence of their occurrence in serum as masked or as visible particles. The implications of these findings for studies of the genesis of atheromatosis are discussed

IMMUNOLOGICAL STUDIES CONCERNING THE NEPHRITIS OF SYSTEMIC LUPUS ERYTHEMATOSUS

Antibodies were eluted from the isolated glomeruli prepared from the kidneys of 10 patients with the nephritis of systemic lupus erythematosus. Antibodies reacting primarily with buffer extracts of nuclei were eluted by acid treatment, and antibodies reacting mainly with DNA and nucleoprotein were eluted with deoxyribonuclease. Quantitative immunochemical studies revealed a high concentration of antinuclear antibody per milligram of γ-globulin in glomerular eluates compared with that in the corresponding serums. The γ-globulin of two eluates was found to consist predominantly of antinucleoprotein antibody. The selective elution of antinuclear antibodies was also indicated by the absence of other serum antibodies in the eluates. DNA antigen was demonstrated in the glomeruli of two kidneys with nephritis by means of isolated anti-DNA antibody labeled with fluorescein. In one of these cases, anti-DNA antibodies were also found concentrated in the glomeruli and, in the second, circulating anti-DNA antibodies were demonstrated in the patient's serum. The immunochemical evidence for the high specific activity of antinuclear antibodies and the association of DNA antigen with DNA antibody in glomeruli add further support for the antigen-antibody complex hypothesis for renal injury in systemic lupus erythematosus

THE CARBOHYDRATE OF γ-GLOBULIN AND MYELOMA PROTEINS

Various preparations of γ-globulin homogeneous in the ultracentrifuge showed a similar content of hexose, hexosamine, fucose, and sialic acid. Subfractionation of Fr. II γ-globulin by zone electrophoresis revealed multiple components of different mean mobilities but containing similar amounts of carbohydrate. Gamma globulin isolated directly from normal serum by zone electrophoresis showed a heavy component in addition to the usual 7 S material. The heavy component (s20, w = 19 S) concentrated by preparative ultracentrifugation was found to be considerably richer in carbohydrate than the rest of the γ-globulin and accounted for small differences in carbohydrate content between different preparations of γ-globulin. Pathological sera with marked elevation in γ-globulin showed a carbohydrate-protein ratio for the γ-globulin similar to that found for the corresponding 7 S fraction in normal serum. This was only partially true of the myeloma proteins with a mobility in the γ-globulin region. Certain of these proteins showed slight but significant differences. The myeloma proteins of faster mobility (β-myelomas) contained considerably more carbohydrate. The possible role of these carbohydrates in accounting for some of the mobility and immunological differences in the myeloma proteins is discussed. The pathological proteins found in two cases of macroglobulinemia showed a high carbohydrate content similar to but slightly lower than the normal 19 S component of γ-globulin

IDIOTYPIC DETERMINANTS OF IMMUNOGLOBULIN M DETECTED ON THE SURFACE OF HUMAN LYMPHOCYTES BY CYTOTOXICITY ASSAYS

The complement-mediated cytotoxicity assay has been used to demonstrate surface immunoglobulin determinants on human peripheral blood lymphocytes. In two individuals with monoclonal serum IgM components, idiotypic antisera demonstrated similar components on a significant proportion of the peripheral blood lymphocytes

IMMUNOLOGICAL RELATIONSHIPS AMONG THE MYELOMA PROTEINS

An immunological study of 21 myeloma sera was carried out to determine their relationship to components of normal γ-globulin and to each other. Ten proteins were separated for detailed characterization. Every one of the myeloma proteins studied was immunologically different, indicating individual specificity. The γ-type myeloma proteins were all related to normal γ-globulin or a fraction thereof, and although each was distinct from the others, all were related. The β-type myeloma proteins likewise were related to one another, though individually specific. Evidence was found favoring the view that some of the γ-type myeloma proteins are related to the β-type myeloma proteins although most of them were completely unrelated. Likewise the results indicated a definite but distant relationship between the β-myeloma proteins and normal γ-globulin. Certain of the β-myeloma proteins were more closely related to a fraction of normal serum of similar electrophoretic mobility. It is suggested that most multiple myeloma proteins fall into two groups or families of cross-reacting abnormal proteins, one closely related to normal γ-globulin, the other distantly related to γ-globulin with marked individual specificities

AN UNUSUAL PROTEIN COMPONENT OF HIGH MOLECULAR WEIGHT IN THE SERUM OF CERTAIN PATIENTS WITH RHEUMATOID ARTHRITIS

In the sera of a number of patients with rheumatoid arthritis an unusual, high molecular weight protein component could be detected by direct ultracentrifuge analysis of whole serum. This material sedimented more rapidly than the normal 19 S component and reached concentrations up to 340 mg. per cent. Similar components were not observed in a limited control series. The high molecular weight material was present in the γ-globulin fraction of serum and joint fluid. It had an S20,w of approximately 22 S and could be dissociated into 2 fractions, one of which had a sedimentation coefficient of approximately 19 S. Evidence for a direct relationship between the 22 S component and the γ-globulin precipitation test was obtained. The latter reaction was found to occur in the presence of altered, aggregated γ globulin. Absorption of serum with altered γ globulin removed the 22 S component. There also appeared to be a connection with the sheep cell agglutination reaction and the latex fixation test. The 22 S fraction was always observed in sera giving the most positive tests. Procedures of density gradient and repeated preparative ultracentrifugation demonstrated that each of these reactions was caused by a high molecular weight fraction. The relationship between the unusual protein complex and various 19 S γ-globulins and 19 S antibodies is discussed