Such administration was chosen to avoid the difficulty of using adjuvants, and multiple injections were chosen to enhance disease prevention. when administered subcutaneously in the absence of adjuvant, can induce a dramatic humoral response leading to fatal anaphylaxis in CZC-25146 NOD mice. Introduction Type 1A diabetes (the immune-mediated form of diabetes mellitus) occurs in many species. The NOD mouse is a particularly well-studied animal model. T cells of NOD mice target multiple islet molecules, and a recent international workshop found consistent evidence for specific anti-insulin autoantibodies (1). There is evidence in both humans and NOD mice for intra- and intermolecular epitope spreading with progression to diabetes (2C5), and T cell clones reacting with multiple antigens in mice rapidly transfer diabetes (6). Our group has been particularly interested in autoimmunity directed at insulin following the discovery by Daniel, Wegmann, and colleagues that the majority of islet CD4+ infiltrating T cells recognize insulin and that, of such T cells, more than 90% react with the amino acids 9C23 of the insulin B chain (B:9-23) (6, 7). The NOD CD4+ T cell clones recognizing B:9-23 usually express the V13.3 chain variable segment with J45 or J34 but have no apparent restriction of the nDn junction (8). Two different epitopes, B:9-16 and B:13-23, which share only four amino acids, B:13-16 (EALY), within B:9-23, are recognized by the AV13S3 T cell clones (9). In the NOD mouse, CD4+ antiCB:9-23 T cells were present in islets at the earliest time studied (4 weeks of age); a NOD CD8 pathogenic T cell clone reacting with peptide B:15-23 has also been described (10). Even in BALB/c mice, administration of the B:9-23 peptide induces high levels of antibodies that react with intact insulin and are not absorbed by the peptide (11). This B:9-23 insulin sequence is identical in multiple species, including humans and mice. Human T cells reacting with B:9-23 were detected in new-onset and at-risk patients using an ELISPOT assay (12). Recently the crystal structure of the B:9-23 peptide bound to DQ8 was described (13). An altered-peptide ligand of the insulin peptide B:9-23 is able to suppress diabetes in NOD mice and is CZC-25146 in phase I clinical CZC-25146 trials in humans (12). Self-peptides have been considered poor immunogens, but a recent phase II clinical trial in patients with multiple sclerosis using an altered-peptide ligand of a myelin basic protein peptide resulted in immediate hypersensitivity reactions in a subset of patients (14). Similarly, mice immunized with the self-antigen myelin proteolipid CZC-25146 protein experienced anaphylactic shock (15). Following prevention studies of the B:9-23 peptide in adjuvant, we began studies of the subcutaneous (SQ) administration in saline of B:9-23 peptide. Such administration was chosen to avoid the difficulty of using adjuvants, and multiple injections were chosen to enhance disease prevention. In the first experiment, with a 100-g dose of peptide B:9-23, eight of ten mice died of apparent acute anaphylaxis minutes after injection during the seventh week of treatment. This led to the current study of insulin peptide induction of autoantibodies and anaphylaxis. Methods Mice and induction of anaphylaxis. Female NOD mice were obtained from Taconic (Germantown, New York, USA) and housed in the pathogen-free animal colony at the Barbara Davis Center for Childhood Diabetes. The mice were injected with HPLC-purified insulin peptide B:9-23 (Research Genetics, Huntsville, Alabama, USA). Briefly, the peptide was dissolved in sterile saline and adjusted to a neutral pH, at a concentration of 100 g in 100 l. A total of 100 g of B:9-23 CZC-25146 was injected subcutaneously CANPml in the scruff of the neck, starting at 4C6 weeks of age or at 11 weeks of age, and given on days 1C5 and 8 and then weekly thereafter. Alternatively, B:9-23, B:13-23, or tetanus toxin peptide (TT:830-843) was administered at a dose of 10 g in 100 l of saline, starting at 4 weeks of age or 10C11 weeks of age. Mice were monitored for clinical signs of anaphylaxis (see below). Consistently, onset of anaphylaxis occurred 6 weeks from the start of B:9-23 peptide immunization (100 g/dose) in the mice immunized subcutaneously at 4C6 weeks of age but was not observed when subcutaneous immunization started at 11 weeks of age. Five weeks from the start of B:13-23 peptide immunizations (10 g/dose), anaphylaxis occurred in the mice, regardless of age at immunization. There were no clinical signs of anaphylaxis in the mice immunized with B:9-23 or TT:830-843 at 10 g/dose regardless of age at.