Advanced glycation end products contribute to the immunogenicity of IFN-β pharmaceuticals  Angelina Bozhinov, PhD, Yordan Handzhiyski, PhD, Krasimir Genov,

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Advanced glycation end products contribute to the immunogenicity of IFN-β pharmaceuticals  Angelina Bozhinov, PhD, Yordan Handzhiyski, PhD, Krasimir Genov, MD, PhD, Vera Daskalovska, MD, DSc, Toshimitsu Niwa, MD, PhD, Ivan Ivanov, DSc, Roumyana Mironova, PhD  Journal of Allergy and Clinical Immunology  Volume 129, Issue 3, Pages 855-858.e6 (March 2012) DOI: 10.1016/j.jaci.2011.10.035 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 IFN-β1a and IFN-β1b have undergone structural alterations. A, SE-HPLC chromatogram of Betaferon. B, Native PAGE (lane 1) and SDS-PAGE (lane 2) of Betaferon. C, Immunoblot of Betaferon SE-HPLC fraction no. 1 with an anti–IFN-β antibody. D, Silver-stained SDS polyacrylamide gel of Rebif. In Fig 1, C and D, degradation and covalent aggregation are indicated by solid arrows, and M stands for molecular mass standards in kilodaltons. Journal of Allergy and Clinical Immunology 2012 129, 855-858.e6DOI: (10.1016/j.jaci.2011.10.035) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Titers of anti–IFN-β (A, C, and E) and anti-AGE (B, D, and F) antibodies. Data are means ± SDs (n = 2) and are representative of 3 independent experiments. Sera from patients with multiple sclerosis with average titers greater than the mean + 3 SDs of the control group (dashed line) were considered positive (black and gray bars). G, Inhibition of the reactivity of sera R6 (solid circles) and B7 (solid triangles) to IFN-β by AGE-BSA. H, Anti–IFN-β antibody–positive sera in which the reactivity to IFN-β was significantly (P < .05) inhibited by AGE-BSA. In Fig 2, G and H, inhibition is presented as the percentage difference in sera reactivity to IFN-β in the presence of either AGE-free (100%) or AGE-modified BSA. Data are means from 8 replicates, and the difference was tested for statistical significance by using the paired 2-tailed Student t test. ∗P < .05, ∗∗P < .001, and ∗∗∗P < .0001. Journal of Allergy and Clinical Immunology 2012 129, 855-858.e6DOI: (10.1016/j.jaci.2011.10.035) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Correlation of the levels of anti–IFN-β antibodies with the levels of anti-AGE antibodies in the sera of patients with multiple sclerosis treated with Avonex (A), Rebif (B), and Betaferon (C). Correlation coefficients were calculated with the CORREL function of Microsoft Office Excel 2003 (Microsoft, Redmond, Wash), and their statistical significance was evaluated (http://faculty.vassar.edu/lowry/rsig.html). Journal of Allergy and Clinical Immunology 2012 129, 855-858.e6DOI: (10.1016/j.jaci.2011.10.035) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions