Mason, Lesley Jane; (2004) An investigation of factors affecting the binding and pathogenicity of human anti-DNA antibodies. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

High affinity IgG anti-DNA antibodies are believed to be important in the pathogenesis of lupus nephritis. However, some anti-dsDNA antibodies appear relatively harmless. This thesis examines factors which determine the pathogenicity of anti-DNA antibodies and their ability to damage the kidney. Chapter Three compares the pathological effect of implanting anti-DNA antibody RH- 14 in 2 month old SCID mice, 'leaky' 8 month old SCID mice and 'non-leaky' Rag-1-/- mice. RH-14 deposition resulted in hyaline thrombi associated with fibrin in 8 month old SCID mice but not in 2 month old mice. However, these thrombi were not associated with greater pathology than had been observed in 2-month old mice implanted with RH-14. Foot process effacement was not observed in the 8 month old mice. Chapter Four describes how the CDRs were interchanged between the lambda chains of two human hybridoma derived anti-DNA antibodies (B3 and 33.HI 1) and a human antiphospholipid antibody (UK4). The chimeric light chains were paired with the heavy chain of B3 and whole IgG molecules were produced using a transient expression system. As predicted by computer modelling, arginine residues as positions 27a (B3 VλCDR1) and 92 (33.H11 VλCDR3) enhanced antibody binding to DNA, whilst an arginine at position 94 (UK4 VλCDR3) blocked binding. The requirement of a cofactor, present in cell supernatant, was shown for the binding of affinity purified and DNase I treated recombinant anti-DNA antibodies to dsDNA. Chapter Five describes stable CHO cell lines producing recombinant B3 and its mutants which were implanted into SCID mice to assess the pathogenicity of the different IgG molecules. This system had too much inherent background pathology to assess the pathogenicity of the anti-DNA IgG. Chapter Six of this thesis demonstrates that two pathogenic human anti-dsDNA monoclonal antibodies bind to α-actinin, but not a non-pathogenic anti-dsDNA antibody. Patients with SLE had significantly higher binding to α-actinin than healthy controls. A greater proportion (6/10) of anti-DNA antibodies purified from the sera of lupus patients with renal disease bound to α-actinin than those purified from patients without renal disease (2/8).

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