Celiac disease is associated with endomysial antibodies (EmA), which have recently been reported to be directed to tissue transglutaminase (tTG). To demonstrate binding of antibodies to recombinant tTG, human tTG was cloned, expressed by in vitro transcription/translation and used to develop novel radioligand assays for combined and single detection of immunoglobulin A (IgA) and G (IgG)-specific antibodies. IgA and IgG-tTGA were found in 43 (95.6%) of 45 patients with newly-diagnosed celiac disease verified by biopsy. In addition, all 30 sera from patients with gastrointestinal symptoms and positive EmA were positive for IgA-tTGA, and all but one serum (96.7%) had antibodies of the IgG class. Receiver-operating characteristic analysis including 574 sera from healthy controls revealed a specificity of 99.5%. By means of these new assays, we identified all patients with endomysial antibodies and achieved, at equal specificity, an even improved sensitivity (95.6%) as compared to EmA (91.1%) detected by the standard immunofluorescence test. Here, we have provided direct evidence that recombinant tTG is a major target of antibodies in celiac disease. Our data suggest that tTGA measured by radioligand assay have the power to overcome the limitations of the EmA-test. This new strategy may considerably facilitate large-scale screening for silent and latent celiac disease.

SUMMARYThe enzyme tissue transglutaminase (tTG) has been recently identified to represent a highly sensitive and specific target of autoantibodies in coeliac disease. To characterize autoantigenic epitopes, we generated novel tTG deletion mutants by polymerase chain reaction, produced radiolabelled fragments by in vitro transcription/translation, immunoprecipitated the mutants using sera from patients with coeliac disease, and related the binding data with putative structural and functional domains of human tTG. We show that tTG antibody positive sera display a heterogeneous autoantibody response covering distinct regions of the molecule. The N-terminal and C-terminal third of tTG, comprising amino acid (aa) 1±281 and aa 473±687, harbour the dominant epitopes (67´4% and 69´4% positive), whereas the catalytic region is of minor antigenicity (22´5% positive). Autoantibodies directed to one, two and three domains were observed in 36´7%, 28´6% and 22´4% of patients, respectively. Comparative analysis revealed the presence of strictly conformational epitopes which were dependent on the N-terminus (aa 1±12) or the intact b-barrel domains in the C-terminus (aa 473±497, aa 649±687). In conclusion, we here demonstrate for the first time that the humoral autoimmunity is directed against distinct functional tTG domains. The spectrum of autoantibodies indicates that the native folded protein may be the target of autoantibodies.

Aims/hypothesis. Dendritic cells (DCs) are professional antigen presenting cells involved in the initiation of primary immune responses and the preservation of peripheral tolerance. The aim of this study was to develop a DC vaccine for autoantigen-specific prevention of autoimmune diabetes. Methods. Splenocytes from diabetes-prone NOD mice were cultured in conditioned media to obtain a homogeneous DC sub-population for vaccination experiments. These cells were used to modulate autoimmune responses in NOD mice after synchronization of diabetes with cyclophosphamide. After immunisation with insulin-pulsed DCs the incidence of diabetes, the insulitis grade and the cytokine production was examined. Results. The long-term culture of splenocytes resulted in the generation of a cell line, termed NOD-DC1, which have a phenotype of myeloid DCs (CD11c, CD11b, DEC-205), express MHC class II and costimulatory molecules (CD40, CD80, CD86). The NOD-DC1 cells have preserved functional activity shown by the detection of a high antigen uptake capacity, the induction of a mixed lymphocyte reaction and stimuli-dependent IL-6 and TNF-α secretion. Vaccination with insulin-pulsed NOD-DC1 cells results in an antigen-specific prevention of diabetes. This was mediated by a reduction of the severity of insulitis and a decrease of T helper 1 effector cells. Conclusion/interpretation. We describe the generation of a DC line which confers protection from diabetes in an antigen-specific way. Our data shows that autoantigen-loaded DCs can induce strong immunoregulatory effects supporting the hypothesis that DCs are promising candidates to develop novel vaccines for the prevention of autoimmune diabetes. [Diabetologia (2003[Diabetologia ( ) 46:1357[Diabetologia ( -1365