Volume 131, Issue 1, Pages 85-96 (July 2006) Does Cross-Reactivity Between Mycobacterium avium paratuberculosis and Human Intestinal Antigens Characterize Crohn’s Disease?  Dimitrios Polymeros, Dimitrios P. Bogdanos, Richard Day, Dimitryi Arioli, Diego Vergani, Alastair Forbes  Gastroenterology  Volume 131, Issue 1, Pages 85-96 (July 2006) DOI: 10.1053/j.gastro.2006.04.021 Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 1 (A) Inhibition of antibody binding against MAP gsd230–244 by preincubation of serum with MAP gsd230–244 (◊), human GPg111–125 (□), and control peptide (○). (B) Inhibition of antibody binding against MAP ahpC20–34 by preincubation of serum with ahpC20–34 (♦), human TOG637–651 (■), and control peptide (○). On the vertical axis is the percentage of inhibition of antibody binding to the peptide in the presence of competitor peptide at different concentrations (0, 15, 30, 60, 120, 250, and 500 μg/mL). A decrease in binding of up to 85% is seen after preincubation with MAP and human intestinal mimics, but not with the control peptide in both sets of experiments. Gastroenterology 2006 131, 85-96DOI: (10.1053/j.gastro.2006.04.021) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 2 (A) Inhibition of MAP gsd230–244 by preincubation of individual serum samples (n = 5) with recombinant MAP gsd or human GPg or control antigen (cytochrome P450 IID6). (B) Inhibition of MAP ahpC20–34 by preincubation of individual serum samples (n = 5) with recombinant ahpC. Residual MAP peptide reactivity was detected by ELISA. Data are expressed as mean (±SD) absorbance values (n = 5) of triplicate experiments. Gastroenterology 2006 131, 85-96DOI: (10.1053/j.gastro.2006.04.021) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 Dot-blot analysis of antibody binding to recombinant MAP ahpC. Reactivity to MAP ahpC was found in all 5 MAP ahpC20–34/human TOG637–651 cross-reactive serum samples (lanes 2–6) but in none of the unreactive samples (lanes 7–11) from patients with CD. A MAP ahpC–specific dot revealed by monoclonal antipolyhistidine as primary antibody and horseradish-peroxidase–conjugated antimouse IgG as secondary antibody, followed by development with tetramethylbenzidine, was used as positive control (lane 1). Gastroenterology 2006 131, 85-96DOI: (10.1053/j.gastro.2006.04.021) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 4 Relative affinity indices (rai) of antipeptide antibody binding in MAP gsd230–244/human GPg111–125 reactive cases (n = 5) (left) and MAP ahpC20–34/human TOG637–651 reactive cases (n = 5) (right) expressed as mean (±SD). Experiments were performed in triplicate. * and **P < .05. Gastroenterology 2006 131, 85-96DOI: (10.1053/j.gastro.2006.04.021) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 5 Three-dimensional modeling structure of bovine glutathione peroxidase (grey color) with a (A) wire frame backbone colored by secondary structure and a (B) space fill backbone. The corresponding GPg111–125 mimic (A, white; B, black) is located within a helical domain in a solvent-accessible surface region of the protein compatible with antibody recognition on whole protein. The structure was analyzed with the Cn3D visualization tool. Gastroenterology 2006 131, 85-96DOI: (10.1053/j.gastro.2006.04.021) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions