WP 6. Application to other rheumatic diseases : Myositis.

This area of work will be lead by Partner 13 (Prague) with input from Partner 1 (KI), Partner 2, Partner 3 (Berlin-Charité), Partner 4 (Vienna), Partner 6 (Amsterdam), Partner 7 (London), Partner 8 (Nijmegen), Partner 9 (Montpelier), Partner 10 (Zurich), Partner 11 (Lund) , Partner 12 (Umeå), Partner 14 (Manchester), Partner 19 (Warsaw), partner 20 (Heraklion) and Partner 25 (BMD)

•  Patients with definite and probable poly- and dermatomyositis according to the classification criteria of Bohan and Peter will be included in the AUTOCURE project. Clinical data, autoantibody profiles and genetic data of clinically well characterised patients with poly- and dermatomyositis will be collected by different partners (P1, P2, P3, P4, P6, P7, P8, P9, P10, P11, P13 and P14) and assembled in a common database. DNA samples have already been collected to some extent and additional DNA samples will be collected. We aim to collect DNA samples, clinical information and autoantibody profiles from 600 patients. From each partner DNA samples from age, sex and ethnicity matched non-related healthy controls will be collected.

•  Presence of certain candidate gene polymorphisms of interest for disease susceptibility will be performed by PCR methods by partners P1, P13 and P14 and association studies will be carried out.

•  A European multi-centre controlled trial with methotrexate versus placebo in patients with polymyositis or dermatomyositis according to Bohan and Peter criteria with partners 13 and P1 as principal investigators has just been launched. We plan to enrol 100 patients with new onset disease over 2-3 years. It is a one year trial. The primary endpoint is the accumulated corticosteroid dose. Secondary endpoints include detection of prognostic markers. In this study we will collect clinical outcome measures and peripheral blood and DNA will be collected for genetic profiling. In at least two centres (P1 and P13) muscle biopsies will be taken before and after treatment.

•  Search for potential key molecular pathways will be performed by cDNA microarrays to analyse differential gene expression in peripheral blood and muscle biopsy from patients with myositis in different phases of disease in comparison with healthy controls. Muscle biopsies are to some extent already available at Partners 1 and 13. The next approach will be through analysis of muscle biopsy samples collected in the European multi-centre trial on the treatment of myositis with methotrexate and corticosteroids. A unique strength in our study will be the analyses for gene expression profiling on paired muscle samples, before and after treatment from the same individual on at least 10 patients. Gene expression analyses will be combined with proteomics and immunohistochemistry to identify protein expression in muscle tissue. Molecular data will be correlated with clinical data on muscle function. The cDNA will be generated from total RNA isolated from peripheral blood or muscle tissues, labelled with two-colour fluorescence and hybridised onto Affymetrix gene chips and spotted cDNA microarrays containing probes of the explored genes (cytokines/chemokines and their receptors, growth and transcriptions factors, members of signal pathways, hormones, pro- and anti-apoptotic molecules). This will be performed in collaboration with P 12 and 13 and information that has been developed from studies of animal models and from studies of synovial tissue from patients with rheumatoid arthritis will be used. The control tissue samples will be generated by mixing tissues from a small number of control individuals.

•  The potential role of IL-1 in causing muscle weakness will be tested in an in vitro system in which we will test the effect of recombinant IL-1 on muscle strength and endurance on both whole normal mouse muscles and on single muscle fibres in a validated system (partner 1). Patients with treatment resistant poly- and dermatomyositis will be treated with an IL-1 blockade which is licensed for treating patients with rheumatoid arthritis in a pilot study. The effect will be evaluated both on clinical symptoms and on molecular expression in muscle tissue by performing repeated muscle biopsies. If the result is positive we will continue with a placebo controlled trial with IL-1 blockade.

•  We will start to test the hypothesis that auto-antigens are modified in muscle tissue in patients with myositis with investigations of the ubiquitously expressed histidyl-tRNA synthetase, Jo-1. During the first 18 months of the project a) Picorna viral affected cells will be analysed for modification of the Jo-1 antigen by proteomics technique including mass spectrometry at the proteomics facility by partner 8. The picorna modified autoantigens will be tested against sera from myositis patients. A large number of myositis sera from several of the partners have already been collected by Partner 8. b) Muscle tissue from myositis patients will be tested for expression of modified anti-Jo-1 antigen by 2D gel electrophoresis and immunohistochemistry. We already have 5 different anti-Jo-1 monoclonal antibodies available by partner 8. These need to be further characterised. Muscle biopsies from patients with or without anti-Jo1 antibodies as well as from healthy individuals will be tested for posttranslational changes of the Jo-1 antigen.