Yuji Hataya

Drug-induced neutropenia associated with anti-neutrophil cytoplasmic antibodies (ANCA):possible involvement of complement in granulocyte cytotoxicity

Although antineutrophil antibodies are thought to be involved in drug‐induced neutropenia, neither the precise mechanisms nor the particular antigens on the neutrophil surface have yet been clarified. Recently, we examined a patient with Graves’ disease who developed antineutrophil cytoplasmic antibodies (ANCA) after propylthiouracil treatment and exhibited neutropenia. Because several target antigens of ANCA are expressed on the surface of neutrophils, it was suggested that ANCA might contribute to neutropenia. The patient’s serum bound specifically to neutrophils and HL‐60 cells differentiated into granulocytes, and lysed the HL‐60 cells via a complement‐mediated mechanism. Furthermore, two representative ANCA antigens, proteinase 3 and myeloperoxidase, significantly inhibited both the binding and cytotoxicity of the serum. Finally, tumour necrosis factor‐α, which is known to up‐regulate cell surface expression of several ANCA antigens, enhanced both the binding and cytotoxicity of the serum. These findings suggest that ANCA induced by propylthiouracil contributed to leucopenia through a complement‐mediated mechanism.

Recombinant monoclonal thyrotropin-stimulation blocking antibody (TSBAb) established from peripheral lymphocytes of a hypothyroid patient with primary myxedema

Anti-TSH receptor antibodies (TRAbs) have been known to be involved in Graves’ disease and primary hypothyroidism. We previously isolated and reconstituted immunoglobulin (Ig) genes of Epstein-Barr virus-transformed B cell clones producing monoclonal TRAbs obtained from Graves’ patients. In the present study, we performed a similar experiment using a B cell clone, 32A-5, derived from a patient with primary hypothyroidism. The variable region genes of Ig heavy (H) and light (L) chains were isolated and sequenced from the 32A-5 clone. A significant number of somatic mutations were found in variable regions of H and L chain gene segments. Each pair of H and L chain cDNAs was ligated into an expression vector for IgG1 production and stably introduced into myeloma cells. The transfectants were injected ip into BALB/c mice to yield ample volume of the antibody for following applications. Interactions of recombinant 32A-5 with Graves’ sera with varying thyroid-stimulating antibody (TSAb) activities were studied. The recombinant antibody tended to suppress TSAb activities in 10 of 15 Graves’ sera, in which four were significantly inhibited. In summary, this is the first study to analyze human monoclonal TSH-stimulation blocking antibodies (TSBAb) at the molecular level. Use of human recombinant monoclonal TSBAb may be an analytical tool for molecular-basis etiology and an alternative therapeutic path for Graves’ disease.