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Background: Recombinant human (rh) interferon alpha2a (IFN-α2a) therapy is successfully used for the treatment of Behçet's disease (BD) uveitis refractory to conventional immunosuppressive treatment. Purpose: Our aim in this study was to investigate the frequency and clinical significance of anti-IFN-α antibodies and autoantibodies during recombinant human rhIFN-α2a therapy in patients with BD uveitis. Methods: This comparative, cross-sectional, serological screening study included 30 BD patients treated with rhIFN-α2a (Group 1), 29 BD patients treated with conventional immunosuppressive agents (Group 2), 29 BD patients who received only colchicine (Group 3), and 30 healthy subjects (Group 4). Anti-IFN-α-binding antibodies and autoantibodies, including anti-nuclear antibody, anti-thyroid peroxidase antibody, and anti-cardiolipin antibody, were measured in serum samples. Antibody seropositivity was compared between study groups. Retrospective clinical data were compared between antibody-positive and antibody-negative patients. Results: A significantly higher proportion of patients in Group 1 had anti-interferon-α (26.6 %) and autoantibody (30 %) seropositivity compared to the other groups. No correlation was found between seropositivity for anti-interferon-α and other autoantibodies. No significant difference was found in cumulative dose of IFN-α, duration of IFN-α therapy, time to first uveitis attack, or attack rate between anti-interferon-α antibody-positive and antibody-negative patients in Group 1. Uveitis attacks were observed in 22 % of autoantibody-positive and 71 % of autoantibody-negative patients in Group 1 ( p = 0.018). Conclusions: Patients with BD uveitis develop anti-IFN-α-binding antibodies and autoantibodies during treatment with rhIFN-α2a. While the clinical relevance of anti-IFN-α-binding antibodies remains unclear in this study, induction of autoimmunity was found to be associated with a tendency for better therapeutic response. [ABSTRACT FROM AUTHOR]