Long-term control and reversal of inhibitor formation in BALB/c mice with hemophilia A. (A) Feeding (HC and C2 material) and FVIII administration schedule for prevention of inhibitor formation. Numbers in circles indicate time-points for blood collection. Inhibitor titers in BU per milliliter (B) and IgG1 titers against FVIII (C) at weeks 8, 12, and 21 of the experiment for FVIII-fed mice (n = 5, back square symbols) are compared with control mice (which were fed with WT plant material; n = 7; gray diamonds). Statistically significant differences between these groups for specific time points are indicated (*P < .05; **P < .01; ***P < .001, as calculated by unpaired 2-tailed Student t-test; data are averages ± SEM). A third group of mice (n = 7) was also fed with FVIII material, and FVIII was administered IV once/week starting 1 month after initiation of the oral tolerance regimen. However, FVIII feeding and treatment were continued for the remaining duration of the experiment (ie, 20 weeks of FVIII feeding; these mice are labeled as “FVIII continuously fed” and graphed with black triangle symbols and dotted line in B and C; data are averages ± SEM). (D) FVIII administration and feeding schedule for reversal of inhibitor formation. Inhibitor formation was induced by repeated weekly IV injections of FVIII as indicated. Mice were divided into 2 groups with similar average inhibitor titers. Control mice (n = 5) did not receive any further treatment. The second group (“FVIII fed”; n = 4) was fed with FVIII plant material twice per week for the following 3 months. Inhibitor titers in BU per milliliter (E) and IgG1 titers against FVIII (F) are graphed for weeks 5, 9, 13, and 17 of the experiment, as explained earlier.