D. Roelcke

Hepatitis C-virus (HCV) antibodies in patients after kidney transplantation.

SummaryThe hepatitis C-virus (HCV) is the main etiologic agent of posttransfusion hepatitis (PTH). Most patients depending on hemodialysis need transfusion of blood before kidney transplantation. Of 272 patients after kidney transplantation, 27 (10%) were found to be anti-HCV-ELISA-positive (HCV-Antibody-ELISA, Ortho Diagnostics). The antibodies could be neutralized by HCV C-100-3 antigen. Eight of 22 patients (36%) who had more than one kidney transplantation were classified anti-HCV positive [30% (8/27) of all anti-HCV positive patients]. The number of transfused blood units ranged from 0 to 99 BU. Receiving more than one kidney graft or the transfusion of more than 5 units of blood increased the risk for HCV infection 3.5 or 4.1 times, respectively, compared with one transplantation or less than 5 units of blood. No significant interactions were seen between these two variables. Of the anti-HCV positive patients, 48% were anti-HBc negative as well as HBs-antigen negative, 52% were anti-HBc positive.

[The spontaneous in vitro increase in the antigenicity of the human beta 1C-A globulin. Normal course, induction and inhibition].

ZusammenfassungDie spontane in-vitro-Antigenitätssteigerung des humanen β1-C/A-Globulins wird auf die Wirkung eines thermolabilen Prinzips zurückgeführt, da Wärmebehandlung des Serums die Antigenitätssteigerung zu unterbinden vermag. Dasselbe Prinzip greift in den Transformationsablauf des Proteins ein. Proteasen heben die durch Erwärmung herbeigeführte Antigenitätspermanenz auf und bedingen ein dem spontanen Modifikationenablauf sehr ähnliches Verhalten des β1-C/A-Globulins. Ihre hier erstmals mitgeteilte antigenitätssteigernde Wirkung auf ein Serumprotein finder ihre Parallele in der Blutgruppenserologie, wo die Steigerung der Antigenität verschiedener Erythrozytenantigene sowie deren Erschließbarkeit durch Proteasen zur Reaktion mit inkompletten Antikörpern seit langem bekannt ist.SummaryThe spontaneous in-vitro-increase in antigenicity of the human β1-C/A-globulins is traced to the effect of a thermolabile principle, because the warm treatment of the sera permits the inhibition of the antigenicity increase. The same principle takes action into the course of the protein transformation. Proteases neutralize the antigenicity permanence by the previously undertaken warm treatment and cause a course of modification that is very similiar to the spontaneous activity of the β1-C/A-globulins. The antigenicity increase phenomen of a serum protein given for the first time here finds its parallel in the blood grouping serology, where the increase of antigenicity of various erythrocyte antigens, as well as their preparation by proteases to react with incomplete antibodies has been known for a long time.

Gc-Darstellung mittels Hydroxylapatit-Säulen-Chromatographie Immunelektrophoretische Untersuchungen unter besonderer Berücksichtigung des α2-Bereiches

ZusammenfassungEin einfaches Verfahren zur Gc-Globulindarstellung aus nativem menschlichen Serum wird beschrieben. Die Auftrennung des Serums erfolgt an Hydroxylapatit-Säulen unter Verwendung vonSörensen-Puffern. Die Gc-Fraktion enthält noch einen Teil des Albumins, der durch Rechromatographie bis auf sehr geringe Reste eliminiert wird. Die Gc-Proteinausbeute ist nahezu quantitativ.Weiterhin werden Beobachtungen über das prinzipielle Verhalten der Serum-Proteine, insbesondere der des α2-Bereiches, bei der Hydroxylapatit-Chromatographie mitgeteilt.SummaryA simple method to prepare Gc-globulin from native human serum is described. The serum is separated on hydroxylapatit columns usingSörensen buffers. The Gc fraction contains part of the albumin which can be eliminated by means of rechromatography, leaving traces of albumin only. The output of Gc protein is almost quantitative. Furthermore, observations on the fundamental behaviour of the serum proteins during the hydroxylapatit chromatography with special reference to the α2-globulins are reported.Ein einfaches Verfahren zur Gc-Globulindarstellung aus nativem menschlichen Serum wird beschrieben. Die Auftrennung des Serums erfolgt an Hydroxylapatit-Saulen unter Verwendung vonSorensen-Puffern. Die Gc-Fraktion enthalt noch einen Teil des Albumins, der durch Rechromatographie bis auf sehr geringe Reste eliminiert wird. Die Gc-Proteinausbeute ist nahezu quantitativ.

Autoimmune hemolytic anemia by coexisting anti-I and anti-Fl cold agglutinins.

SummaryIn association with atypical pneumonia, a patient developed acute severe autoimmune hemolytic anemia. Hemoglobin temporarily was only 7.0 g/100 ml, so that the patient needed red blood cell (RBC) transfusion. Hemolysis was found to be caused by high titer cold agglutinins (CA), which occurred transiently during the acute period of the disease. CA of two different specificities, anti-I and anti-Fl, were demonstrated in the patients serum. Antibodies of the two specificities were clearly separated by absorption/elution experiments using neuraminidase (RDE)-treated RBC. They were distinguished by serologic means: Both anti-I and anti-Fl react more strongly with adult RBC than with newborn and i adult RBC; in contrast to anti-I, anti-Fl does not agglutinate RDE-treated cells. Inhibition experiments showed that I-active substances prepared from papainized RBC exhibited both I and Fl antigenic activity. By RDE-treatment of I-active substances, Fl-activity was markedly reduced, while I-activity was increased.

Monoclonal anti-idiotype antibodies against lymphoma-associated cold agglutinins

SummaryHybridomas secreting monoclonal antibodies against purified cold agglutinins (CA) from lymphoma patients were screened by a cold hemagglutination inhibition test. Supernatants from positive clones were further tested against several purified CA and paraproteins of different immunoglobulin (Ig) classes. It was shown that most monoclonal antibodies raised by immunization with CA had reactivity against the constant region of IgM. However, clone H-1 produced an anti-idiotypic antibody that reacted exclusively with the CA used for immunization. Using this anti-idiotype antibody, the idiotype could be demonstrated on 25% of the patients peripheral mononuclear cells. So far, the idiotype could not be demonstrated on the patients T-cells. Monoclonal antibodies against lymphoma idiotypes are powerful tools for studying the immunobiology of these malignancies and may be useful as specific therapeutic reagents.

Die Unterteilbarkeit menschlicher Lipoproteine niederer Dichte (LDL) hinsichtlich ihrer Lp(a)-Eigenschaft durch Ionenaustauscher-Säulenchromatographie

ZusammenfassungEin Subfraktionierungsverfahren für menschliche Lipoproteine niederer Dichte (LDL) mittels AE-Zellulose-Ionenaustauscher-Säulenchromatographie wird angegeben, durch das Lp(a+)-LDL in jeweils 2 Lp(a-)- und 2 Lp(a+)-, also insgesamt 4 Subfraktionen, aufgeschlüsselt werden können. Mit dieser Methode lassen sich ca. 60% Lp(a)-inaktiver LDL von ca. 40% Lp(a)-aktiven Lipoproteinen niedriger Dichte bei Lp(a+)-LDL als Ausgangssubstanz abtrennen.Mit zunehmender LDL-Affinität zu AE-Zellulose wird zunehmende Lp(a)-Aktivität der Subfraktionen angetroffen. Die Lp(a)-aktiven Fraktionen weisen einen auf etwa 150% erhöhten relativen Peptidanteil im Vergleich zu den Lp(a)-inaktiven Subfraktionen auf. Die absolute Proteinmenge der Lp(a)-inaktiven Hauptfraktion ist jedoch mindestens doppelt so hoch wie die der Lp(a)-aktiven Hauptkomponente, so daß die Lp(a)-Nachweisbarkeit nicht von der Konzentration eines (immunologisch) homogenen Peptidanteils der einzelnen Subfraktionen abhängen kann.Immunisierungsversuche bestätigen die immunologische Heterogenität der 4 Subfraktionen, die neben der chromatographischen Heterogenität besteht. Beide Befunde weisen darauf hin, daß innerhalb individueller LDL nicht nur Lipid-, sondern auch Peptid-Heterogenität herrscht.Eine von dem Trennmodus an AE-Zellulose offenbar unabhängige LDL-Unterteilung kann mit DEAE-Zellulose-Säulenchromatographie erreicht werden. Damit ist eine weitgehende Differenzierung humaner LDL mittels Ionenaustausch möglich.SummaryA method is described to subfractionate human low-density lipoproteins (LDL) by means of AE-cellulose-ionexchange-column-chromatography, by which the Lp(a+)-LDL can be separated into on each side 2 Lp(a-)- and 2 Lp(a+)-, which means together 4 subfractions. By this method about 60% of Lp(a)-inactive LDL can be separated from about 40% Lp(a)-active low-density lipoproteins taking Lp(a+)-LDL as basic substance.With increasing LDL-affinity to AE-cellulose we meet with increasing Lp(a)-activity of the subfractions. The Lp(a)-active fractions show an about 50% higher relative peptid component in comparison with the Lp(a)-inactive subfractions. The absolute protein quantity of the Lp(a)-inactive main fraction however is at least twice as high as that of the Lp(a)-active main component, so that the detection of Lp(a) cannot depend from the concentration of an (immunologically) homogeneous peptid component of the single subfractions.Immunization tests prove the immunological heterogenity of the 4 subfractions existing beside the chromatographic one. These two findings suggest that there is within individual LDL not only a lipid- but also a peptid heterogeneity.A LDL-subfractionation obviously independend from the mode of separation with AE-cellulose was obtained by DEAE-cellulose columns. So a farreaching differentiation of human LDL by means of ion exchange is possible.

C8 binding protein bears I antigenic determinants

SummaryC8 binding protein (C8bp) is an integral membrane glycoprotein of peripheral blood cells, which inhibits the C5b-9-mediated lysis in a homologous system. In the present study, we analyzed the carbohydrate portion of the C8bp. We found that C8bp is associated with I antigenic determinant, a sugar sequence found on human erythrocytes of adults. To assess whether or not the sugar residues are essential for the C8bp function, I determinant was cleaved off from the isolated C8bp by endo-β-galactosidase (E.C. 3.2.2.103) that hydrolyses internal β-galactosidic-linked-N-acetyllactosamine residues. Enzyme treatment removed I-antigen, the inhibitory function of C8bp, however, was not affected. When intact erythrocytes were treated with endo-β-galactosidase, I-antigen was lost and the lytic insusceptibility of human erythrocytes to homologous C5b-9 could not be abolished. Thus, I-antigen is associated with the C8bp, but its presence is not required for the homologous species restriction.

A further subspecificity within human monoclonal anti-Pr cold agglutinins

SummaryA human monoclonal IgM-K cold agglutinin Ad, reacting with immunodominant neuraminyl groups on red cell membrane sialoglycoproteins, as typical for anti-Pr CA, was studied for its subspecificity. Inhibition experiments with chemically modified red cell sialoglycoproteins and agglutination studies with animal red cells showed that the CA Ad did not fit into the subspecificities anti-Pr1h,-Pr1d,-Pr2,-Pr3. Cold agglutinin Ad presents a new specificity within human monoclonal antibodies against immunodominant neuraminyl groups.

New antigenic determinant (Sa) on human lymphocytes and phagocytes

SummaryPresence of antigenic determinants reacting with homogeneous IgM/k cold agglutinin (CA) of a new specificity, tentatively called Sa, was investigated by bithermic cytotoxicity assay and by immunofluorescence. CA Sa killed on average 38% allogeneic peripheral blood lymphocytes (PBL) and up to 74% of autologous PBL. There was preferential kill of B-PBL compared to T-PBL. Some preference toward B cells was also noted using tonsillary B and T lymphocytes. Cytotoxic activity of CA Sa against chronic lymphocytic leukemia cells of B-type was almost equal to that of potent anti-I CA and much stronger than anti-i CA. Presence of additional B-cytotoxic factor in the serum was excluded by the use of red blood cell eluate composed solely of homogeneous CA. Thymocytes and helper-type T cells from a patient with T cell chronic lymphocytic leukemia were very susceptible to the cytotoxic action of Sa. CA Sa killed 39% of monocytes, but there was almost no kill of polymorphonuclear leukocytes. Lymphocytotoxicity of CA Sa was abolished by sialyllactose and was not influenced by I-active glycoproteins. Comparison of CA Sa to CA of other specifities showed marked differences, supporting the view that Sa has new, previously unrecognized specificity.

Human cold agglutinins against "cryptic" erythrocyte antigens.

ZusammenfassungZwei Beispiele humaner IgM-Kälteagglutinine werden beschrieben, die humane Erythrozyten in vitro erst nach Enzymbehandlung agglutinierten. Proteasen waren am wirksamsten, doch konnte der gleiche Effekt auch mit Neuraminidase erzielt werden. Die Kälteagglutinine banden sich nicht auf native Erythrozyten, sie sind gegen „kryptische“ Determinanten gerichtet. Sie gehören zum Komplex der Anti-I/-i Kälteagglutinine, zeigen jedoch einen „neuen“ Typ von I/i-Determinanten an. Diese Determinanten waren auf nativen Erythrozyten eines Patienten mit kongenitaler dyserythropoietischer Anämie unmittelbar für die Antikörper zugänglich. Enzymbehandlung von Testerythrozyten erwies sich damit nicht nur als geeignet zur Differenzierung von Kälteagglutinin-Spezifitäten, sie ist auch Voraussetzung für den Nachweis des „neuen“ Typs von Kälteagglutininen, die in vivo offenbar autoimmunhämolytische Anämien auslösen können.SummaryTwo examples of human IgM cold agglutinins agglutinated human RBC only after enzyme treatment in vitro. Proteases were optimally effective, neuraminidase was also effective. The cold agglutinins did not coat native RBC but were directed against “cryptic” RBC determinants. The cold agglutinins belonged to the anti -I/-i complex indicating a “new” type of I/i determinants. They were strongly accessible to cold agglutinin interaction on native RBC of a patient with congenital dyserythropoietic anaemia. Enzyme treatment of RBC was shown to be not only suited for defining cold agglutinin specificities but also essential for detecting the “new” type of cold agglutinins, obviously causing autoimmune haemolytic anaemia in vivo.