Epinostics

Autoimmune liver disease-related autoantibody profiles (AIH, PBC, PSC) have been compared to profiles obtained from (i) “liver-associated autoimmune diseases (Omenn syndrome, ALPS)”, as well as from (ii) “non-liver autoimmune diseases (SLE, scleroderma) and (iii) from non-autoimmune liver diseases (e.g. alcohol induced chirrosis). The general objective was to determine epitopes that react with disease-related autoantibodies. Hereto, epitopes present in linear peptide sequences have been taken in order to assess their diagnostic values and therapeutic potential. The latter was analyzed by studying anti-anti-MPO anti-idiotypic Abs in the context of vasculitidis including Churg-Strauss syndrome, microscopic polyangiitis and idiopathic necrotizing and crescentic glomerulonephritis. Patient sera of autoimmune liver diseases studied have been subjected to 48729 different linear peptides most of them representing peptide sequences derived from known autoantigens.

The main goal of the Epinostics project was to determine immune responses against various autoantigens related to autoimmune liver diseases (primary biliary cirrhosis (PBC), autoimmune hepatitis (AIH)). A full epitope map of these epitopes has been stored in the EpiMap-database at the Rostock Epitope Screening Center (ESC). Serum samples were tested with molecular based assays for various autoantibodies, clinical data concerning biomaterial were collected and analyzed, and B cell epitope mapping was performed using overlapping peptides and in house ELISA. Dominant anti-myeloperoxidase (MPO) epitopes from sera of patients with vasculitis were analyzed by affinity purification of anti-MPO from patients with vasculitis and from IVIG preparation. Several synthetic peptides derived from MPO sequences encompass immunodominant epitopes in small vessle vasculitis paving the way to therapeutic vaccination strategies. C57BL/6 mice were immunized with recombinanthuman-MPO in order to induce mouse anti-MPO and pathology resembling the human pANCA associated vasculitis. IgM, IgG and IgA BCR repertoires in primary biliary cirrhosis (PBC) patients were analyzed by means of quantitative RT-PCR and CDR3-spectratyping. PBMC from 35 PBC patients and 18 normal controls were analyzed. Quantitative B cell repertoire analysis of IgM from healthy donors showed the preferential usage of VH3a, VH3b and VH4 families. Very similar VH family usage was observed in IgM B cells from PBC patients. In Stiff-Person-Syndrome (SPS) long-lived plasma cells and rituximab-resistant memory B cells seem to be the main source of potentially pathogenic GAD65-specific autoantibodies. In autoimmune lymphoproliferative syndrome (ALPS) patients, serum concentration of FasL and IL10 have been found to be increased in patients carrying Fas mutations. Using mass spectrometric analysis, specifically enriched mono-specific antibody pools bound to selected peptides were characterized as monoclonal, oligoclonal, and polyclonal, respectively. Pools of peptides were identified to be preferentially bound by antibodies present in sera of AIH and PBC patients. The diagnostic value of distinct peptides are currently evaluated in ongoing ELISA testings. Peptides representing proteins such as Ro-52 and PDC-E2 have been scanned to determine linear epitopes using sera from PBC and AIH patients. Linear peptides derived from more than 59 different proteins in case of PBC and 49 different proteins in case of AIH have been subjected to ongoing validation processes to determine their diagnostic value. In summary, each human serum encompasses individual-specific antibodies that have to be discriminated from diseaserelated antibodies. Furthermore, peptides of diagnostic relelvance as well have to be specific in comparison to antibodiy signatures of related diseases. Besides these criteria, one has to take into account that each autoimmune disease is a polygenic disease most likely representing different disease-subtypes together with different epitope profiles as well.