Biomedical subjects
Publications and source records attributed to D Roelcke.
Cold agglutinins of anti-Pr specificity were detected in two newborn infants suffering from serologically ascertained rubella embryopathy, an IgM kappa anti-Pr(a), titer 64, and an IgM lambda anti-Pr1, titer 16. The cases are rare examples of cold agglutinin production in newborns; a possible relationship between anti-Pr specificity and rubella infection is discussed.
Purified monoclonal human IgM cold agglutinins (CA) of different specificities (anti-I, anti-i, anti-Pr) were investigated for their complement-activating capacity in a homologous system. Incubation of human RBC with excess of IgM CA in the cold, and subsequently with human serum at 37 degrees C, resulted in striking differences in hemolysis. Hemolysis did not correlate to the amount of antibodies bound to RBC at 4 or 20 degrees C. Despite the hemolytic inefficiency of anti-i and anti-Pr CA tested, C1 fixation and subsequent activation of the classical pathway of complement could be assessed in all cases. Absolute numbers of C3 molecules bound to RBC, exceeding the critical level to initiate the terminal sequence of the complement cascade, could not fully explain the differences in the hemolytic activity of the CA. Since C8 binding protein (C8bp) carries I determinants it is hypothesized that anti-I-induced complement-mediated hemolysis might also be favored by the binding of the autoantibody to and probably steric hindrance of this major regulatory protein of the terminal complement sequence. The prominent role of homologous restriction of complement-mediated lysis as a protective mechanism can also be deduced from the fact that rabbit as well as rat serum as a source of heterologous complement lysed cold agglutinin-sensitized red blood cells more efficiently than human serum.
Serologic evidence of anti-I and anti-Fl cold agglutinins occurring in mycoplasma infections led to the isolation of I/Fl glycoprotein from human erythrocyte membranes. Mycoplasma pneumoniae bound to purified I/Fl glycoprotein in a dose-dependent fashion depending on sialylated carbohydrate determinants. This was shown by the decreased binding of mycoplasmas to either sialidase-treated I/Fl glycoprotein (dot blot analysis) or sialidase-treated erythrocytes (hemagglutination test). Structural properties of the receptor for optimal binding could be explored by hemagglutination inhibition assays. Glycophorins were excluded as receptors. These results indicate that Fl (and I) antigens are receptors for M. pneumoniae.
To investigate the molecular basis of the autoimmune response to the related i and I carbohydrate antigens, we studied cold agglutinins (CA) from B-cell clones and from the peripheral circulation of patients with lymphoproliferative syndromes. Sequence analyses of expressed variable region genes indicate that both anti-i and anti-I specificities from B-cell clones from two patients are encoded by the VH4.21 or a very closely related VH4 heavy chain gene, whereas the expressed light chain genes differed. The anti-i-secreting B-cells express unmutated germline-encoded VH4.21 and VKI gene sequences. The VH region gene encoding anti-I has the closest homology (97%) to the VH4.21 germline gene and differs at the protein level by only three amino acids. In contrast, while the VL region gene encoding anti-I is most homologous (96%) to the VKIII, kv328 germline gene, there are seven amino acid differences due to nonrandom replacement mutations, which suggests a role for antigen-mediated selection in the anti-I response of this individual. These studies were extended by a structural survey of 20 additional serum CA using antipeptide antibodies specific for determinants in VH and VL regions. All anti-I and anti-i CA were shown to express VH4 heavy chains, and 14 of 17 CA expressed a previously described VH4 second hypervariable region determinant, termed VH4-HV2a. We also found that 13 of 14 anti-I CA used VKIII light chains, while the anti-i CA used light chains from at least three VL families. Taken together, the data show that anti-i and anti-I CA probably both derive from the VH4.21 gene (or a closely related gene). Furthermore, the restricted VH and different VL gene use in anti-i and anti-I CA may reflect the close structural relationship of the i and I antigens.
Postinfection cold agglutinins (CAs) with anti-Sia-b1 (i.e., anti-Sialo-branched) specificity frequently occurring together with anti-I CAs recognize antigenic determinants that are present on Oh red cells and are partially destroyed by endo-beta-galactosidase on the red cell surface. They differ markedly from the monoclonal anti-Sia-b1 FI CA, which recognizes an epitope not expressed on Oh cells, and resistant to the enzyme. On the other side, Sia-b1 determinants reacting with postinfection CAs share the characteristics of I determinants, with the exception that Sia-b1 determinants require sialyl groups as an immunodominant component. Five sera containing coexisting anti-Sia-b1 and anti-I CAs were tested against Oh and enzyme-treated group O red cells. The results are consistent with a postinfection autoimmune response against a sialo-type 2 structure common for Sia-b1 and I determinants, which could serve as a receptor for Mycoplasma pneumoniae.
Since 1986 only anti-HBc negative blood donors had been admitted for blood donations at the Heidelberg University Blood Bank. The maximum ALT-level for blood donors was set at less than or equal to 30 U/l. Four of 1514 blood donors (0.26%) were found to be anti-HCV positive. The routine screening of blood donors for anti-HBc in combination with an ALT-level less than or equal to 30 U/l leads to the observed lower prevalence compared to the rate found in German blood donors (0.26%/0.42% [1]).
The prevalence of anti-HCV in patients after kidney transplantation was tested by HCV-Antibody-ELISA (Ortho Diagnostics). In addition, reactive samples were tested by HCV-EIA (Abbott Laboratories), neutralization, anti-HBc (Corzyme, Abbott) and by HBs-Ag (Auszyme, Abbott). 27 of 271 patients (10%) were anti-HCV positive. Receiving more than one kidney graft (TPL) or the transfusion of more than four blood units (BU) increases the risk of HCV infection four times (OR: 4.1; p less than 0.01) or 2.5 times (OR: 2.5; p less than 0.05), respectively, compared with one TPL or less than 4 BU. Receiving more than one kidney graft and transfusion of more than four BU raises the risk of HCV infection 6.8 times. 52% of anti-HCV positive patients were anti-HBc positive, 48% were anti-HBc negative as well as HBs-Ag negative.
The antigens Fl and Vo, recognized by cold agglutinins, are protease-resistant differentiation antigens like I and i antigens. In contrast to Ii, Fl and Vo are sialidase-susceptible antigens like the protease-resistant Gd antigens. Because I/i antigens are branched/linear type-2 (poly-N-acetyllactosamine or neolacto series) chains which are cleaved from red cells by endo-beta-galactosidase, the effect of the enzyme on Fl, Vo and Gd antigens was studied. Fl and Gd antigens, previously shown to be sialylated branched (Fl), and linear and branched (Gd) type-2 sequences, were not inactivated on red cells by the enzyme, whereas Vo antigenicity was completely abolished. The data suggest that Vo antigen is expressed on sialylated linear poly-N-acetyllactosamine sequence.
Human red cells (RBC) were treated with endo-beta-galactosidase from Bacteroides fragilis removing type 2 polylactosamine chains [Galssl-4GlcNAc]n from the RBC surface by cleaving internal Galssl-4Glc(NAc) bonds of linear lactosamine sequences. I and i built up by unsubstituted branched and linear chains were inactivated. The sialo-antigen Vo resembling i antigen expression on adult, newborn and i adult RBC was also inactivated, demonstrating that the Vo determinant is created by sialylation of linear chains. Fl and Gd antigens known to be sialylated polylactosamine chains were not inactivated. Because Gd antigenicity is increased by increasing the length of sugar sequences and Vo determinants are obviously short chain sequences, the action of endo-beta-galactosidase could depend on the length of sialylated sequences on the RBC membrane.
Incomplete warm hemolysins (IWHs) form an independent class of red blood cell (RBC) autoantibodies. We studied eight sera in which autoantibodies with the characteristic features of IWHs were demonstrated. The case reports of those patients revealed that IWHs were predominantly associated with a serious course of autoimmune hemolytic disease. In four sera we found a combination of IWHs and cold agglutinins with the specificity anti-I. The cold agglutinins could be separated from the IWHs by affinity chromatography with immobilized I-active RBC material. The binding of IWHs to RBCs was demonstrated on the RBC surface with a modified enzyme-linked immunoassay (APAAP-EIA: monoclonal anti-immunoglobulin antibodies + bridging antibody + alkaline phosphatase/anti-alkaline phosphatase complexes). With the APAAP-EIA technique and different primary anti-immunoglobulin antibodies we found that seven sera contained IgM-IWHs and one contained IgG-IWHs. In three sera, IgM-IWHs with monotypical kappa light chains were detected.
The pathogenesis of autoimmune hemolytic anemias induced by incomplete warm hemolysins (IWHs) is only poorly understood. Sera from eight patients with mostly severe IWH-related autoimmune anemias allowed us to address some key elements of the pathogenetic process. With the use of a modified enzyme-linked immunoassay we could show that (i) IWHs bound to native RBCs and (ii) IWHs initiated the activation of the complement cascade on native RBCs up to the level of C3. The interaction of IWHs with their corresponding antigens on red blood cell membranes, which were pretreated with different enzymes, showed that despite serological uniformity there are at least two different types of IWHs: (i) IWHs reacting with phospholipase susceptible antigens (six of the studied sera) and (ii) IWHs reacting with antigens which are hardly, if at all, phospholipase susceptible (two of the studied sera). Aside from Rh antibodies, most IWHs apparently belong to a second category of RBC antibodies whose corresponding antigens can be inactivated by phospholipase treatment of the cell membranes. IWHs showed no specificity for Rh antigens or the antigens of the I/i-system.
Autoantibodies against red cells optimally reacting at 0 degree C, ie, CA, are normally found with low titers in the serum of human adults. High-titer CA may be induced by certain infectious agents, including M pneumoniae, EBV, CMV, and rubella virus, or may develop on the basis of chronic (malignant) B cell lymphoproliferation. The main clinical manifestation of cold agglutination is AIHA. Antigens and antibodies of cold agglutination are the best characterized reaction partners of a human autoimmune process. CA may recognize I and i antigens, which are lipid- and protein-linked branched and linear N-acetyl-lactosamine chains, respectively. They are precursors of the ABH blood group antigens and are converted into H by fucosylation. An alternative substitution by sialylation creates Gd, Fl, and probably Vo/Li antigens. CA with anti-Pr and anti-Sa specificities recognize 0-glycans with immunodominant sialyl groups on glycophorins. Several Pr subspecificities can be identified by chemically modified sialyl groups on glycophorins. Because CA in chronic lymphoproliferation are monoclonal antibodies, structure-specificity-interrelations of the antibodies could be identified by primary structure analyses of the N-terminal variable regions of H and L chains and by studies on CA idiotypes. Interrelations between distinct CA specificities and particular infectious agents could explain cold agglutination as a response to receptors for the agents or to the binding sites of antibodies against the agents. Interrelations also existing between certain CA isotypes (Ig classes and L chain types) and CA specificities could be a basis for the elucidation of the enigmatic etiology of chronic (malignant) monoclonal cold agglutination.
A new low frequency red cell antigen JFV has been found in two families, one of German and one of Dutch origin. JFV is inherited as an autosomal dominant character. Family studies showed that JFV is not controlled by the Rh, MNS, Lu, Fy, or Jk loci and that it is not X- or Y-borne.
192 sera containing cold agglutinins of apparent anti-I specificity were reinvestigated for concomitant cold agglutinins (CA) against sialic acid-dependent antigens. 35 cases of additional anti-F1 and 3 cases of additional anti-Gd were detected. 53% of cases with coexisting anti-I and anti-F1/Gd CA had a clinical diagnosis of pneumonia, in 39% IgM antibodies against Mycoplasma pneumoniae could be demonstrated. Since F1 and Gd antigens are identical with the structures identified as receptors for M. pneumoniae, the findings support the hypothesis that postinfectious CA are directed against the receptor of the infectious agent.
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The complement-mediated lysis is inefficient when complement and target cells are homologous with regard to the species. In erythrocytes from patients suffering from paroxysmal nocturnal hemoglobinuria (PNH), the species restriction is lost: PNH-erythrocytes (PNH-E) are susceptible to lysis by human complement. In human erythrocytes (huE) the species restriction is ascribed to an integral membrane protein, designated C8-binding protein (C8bp). In the present study, we tested membranes of PNH-E type III for the presence of C8bp. A protein with C8-binding capacity could not be detected. C8bp, which was isolated from the membrane of huE, inhibited the lysis of PNH-E by C5b-9 as well as the C9 polymerization. Thus, addition of C8bp restored the species restriction in PNH-E. In conclusion, we propose that lack of C8bp might represent the defect in PNH-E type III membranes, which is responsible for their enhanced lytic susceptibility towards lysis by the late complement components.
The first cold agglutinin occurring in an adult patient with fresh varicella infection is described. The antibody caused a hemolytic crisis. It had anti-Pr 1h specificity. It belonged to the rare cold agglutinins of the IgG class and was a kappa-monotypic (monoclonal) antibody.
An intrinsic membrane protein with a m.w. of 65,000 that can bind human C8 has been identified after separation of human erythrocyte membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransfer to nitrocellulose sheets. The protein, tentatively designated as the C8-binding protein (C8bp) could be isolated from papain-treated erythrocyte (E) membranes by phenol-water extraction and isoelectric focusing. In a functional assay, with chicken (ch) E as target cells, C8bp inhibited the lysis of ch E C5b67 intermediates by human C8 and C9, whereas the lysis by rabbit C8 and C9 was not affected. Because the decay accelerating factor (DAF) from human erythrocyte membranes also inhibits the activity of C3/C5 convertases in an homologous system, we tested whether or not a DAF activity was present in C8bp. C8bp, however, did not accelerate the decay of the classic C3 convertases. Thus, it appears that C8bp and DAF are two different factors of E membranes with a similar molecular size inhibiting different sites of the activation cascade of complement while they can function synergistically to minimize the self-inflicted damage by complement.