Molecular biological studies of the mechanism of the development of vasculitis in Kawasaki disease.

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The etiology and pathogenesis of Kawasaki disease (KD) remain unknown. Previously we reported that auto-antibodies against a 70 kDa protein from smooth muscle cells derived from human coronary artery (SMC) were detected in the sera of patients with KD. In this study, we tried to identify the specific antigen(s) that react with circulating auto-antibodies against SMC by immunoscreening of the cDNA expression library.1)Immunoscreening of a cDNA expression library : Approximately 3×10^5 plaques from the cDNA library derived from SMC (Uni-ZAP cDNA library, Stratagene) were used to screen for clones showing immunoreactivity with sera from four patients with KD, which yielded auto-antibodies that were strongly positive in preliminary Western immunoblots. Bound auto-antibodies were detected using a second antibody separately with horse-radish peroxide-conjugated rabbit anti-human IgA and IgM (Dako). The screening of the cDNA library were performed three times, and positive clones were isolate … More d. 2)DNA sequence analysis : Cloned phage DNAs were converted into plasmid DNAs using the methods of the in vivo excision. PCR reactions were performed using these plasmid DNAs as templates and both T3 and T7 as specific primers, and then the nucleotide sequences of each cloned cDNA insertion were analyzed with an automated sequencer ABI Prism 310 Genetic Analyzer. Corresponding proteins were searched by Blast analysis using the GenBank sequence database. Results : Eight positive clones were identified by the screenings. Seven clones and two clones were isolated using IgA and IgM as the second antibody, respectively. One clone was isolated by both IgA and IgM. Although the sizes of the identified proteins varied widely, three proteins were around 70kDa. In addition, two proteins including one around 70kDa in size were independently isolated from the sera of different patients with KD. Conclusions : Our results suggest that some of these proteins may be a specific antigen(s) against the auto-antibodies in the sera of KD patients and it may be useful for understanding the mechanisms by which auto-antibodies may cause the systemic vasculitis in KD. Further studies will be required to definite the specific antigen recognized by the circulating auto-antibodies in KD. Less