Analyse struktureller Eigenschaften humaner Antiphospholipidantikörper sowie Untersuchungen zu ihrer möglichen Beteiligung am Antiphospholipidsyndrom

Das Antiphospholipidsyndrom (APS) ist eine systemische Autoimmunerkrankung, die laborchemisch durch das Vorhandensein von Antiphospholipidantikörpern (APL), speziell Anti-Cardiolipin-Antikörpern (ACL), oder durch die Nachweisbarkeit des so genannten Lupus Antikoagulans (LA) und klinisch durch das Au...

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Bibliographic Details
Main Author: Buschmann, Catharina
Contributors: Lingelbach, Klaus (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2006
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Table of Contents: The antiphospholipid syndrome (APS) is an autoimmune disease characterised by antiphospholipid antibody (APL) titers, in particular by anti-cardiolipin (ACL) titers, or by the so-called lupus anticoagulant and by the clinical symptoms of venous or arterial thrombosis or in women by recurrent fetal loss. Although the underlying mechanisms causing the antiphospholipid syndrome are not completely understood, APL are believed to play a central role in the pathogenesis of the disease. APL are a heterogenous group of antibodies which can be detected by immunoassays or by functional coagulation assays. APL are able to bind negatively charged phospholipids, lipid-binding proteins or complexes of both. APL are found not only in the context of the autoimmune disease. They can be found transiently also after acute infections and are presumably part of the repertoire of poly-reactive natural autoantibodies for which a role in the innate immune defence is postulated. The difference between the so-called “pathogenic” APL, which can be observed in the context of the APS, and the non-pathogenic, natural antibodies or the infection-associated antibodies still remains to be eluciated. It is unclear, if the different types of APL are of the same origin and how their regulation is involved into the pathomechanism of the APS. The purpose of this thesis was to characterise APL of patients suffering from APS to gain insight into their possible role in the pathogenesis of the APS. As a central result three APL secreting monoclonal hybridomas could be established using B cells derived from APS patients: JGG9, HLC9 und HVA2. Their antibodies were characterised. Two of them, HLC9 and HVA2, show a binding profile against phospholipids which is characteristic for polyreactive natural autoantibodies. They are able to react with a wide range of different phospholipids without involvement of protein co-factors. In contrast, JGG9 binds a complex of cardiolipin and the protein co-factor ß2-glycoprotein I (ß2GPI). This binding profile is often defined as characteristic for pathogenic APL. Analyses of the coding DNA sequences and comparisons with underlying germline sequences made clear that JGG9 carries the sequence with the highest rate of mutations in the variable region. These mutations are unusual with respect to their location within the variable region and with respect to the type of mutation. A high insertion rate within the framework regions (FR) of the variable region results in a shift of the reading frame and therewith in a significant change of the amino acid sequence. Nevertheless structural sequence motifs defining certain areas of the variable region remain unchanged. This suggests that a functional antibody is expressed. The highly mutated sequence is not a result of somatic hypermutation of the antibody complementarity determining region (CDR), i.e. mutation do not seem to result from antigen driven processes. Pathogenity of the antibodies has not yet been demonstrated in animal models. In cellular assays with JGG9 and HL5B which is an additional monoclonal APL generated in our group no direct interaction of JGG9 or HL5B with monocytes could be detected although earlier work showed showed induction of tissue factor (TF) on monocytes by HL5B which is often described to be characteristic for APL. The results of the presented experiments contribute to further elucidation of structural characteristics of APL. Of particular interest is JGG9 with its unusual mutations. Even as the direct involvement of the described antibodies in pathogenic processes remains to be demonstrated the exceptional heavy symptoms of the patient JG from whom JGG9 was derived make the highly mutated JGG9 of interest for further analysis. Preliminary experiments and thoughts regarding the B-1 cells contribute to the recently started discussion on potential relationships between different subgroups of APL. A newly established method to differentiate between co-factor dependent and independent APL contribute significantly to the improvement of routine assays in the diagnosis of APS. The definition as well as the pathomechanisms leading to the APS are still under discussion. The results are discussed considering the different aspects of this discussion.