Myositis-Associated Autoantibodies in Patients With Juvenile Myositis Are Associated With Refractory Disease and Mortality.

OBJECTIVE: Myositis-associated autoantibodies (MAAs) have been associated with overlap myositis, certain disease manifestations such as interstitial lung disease (ILD), and worse prognosis in the idiopathic inflammatory myopathies. MAAs overall remain largely uncharacterized in patients with juvenile-onset myositis. Moreover, it is unknown whether the number of MAAs is associated with disease severity. METHODS: Patients with juvenile myositis in cross-sectional natural history studies who underwent testing for myositis autoantibodies were included. Demographics, myositis autoantibodies, clinical characteristics, medications received, and outcomes of those with and without MAAs were compared. Multivariable logistic regression was performed to determine whether the number of MAAs detected was associated with severe disease features. RESULTS: Among 551 patients, 36% had an MAA and 13% had more than one MAA. Among those who were MAA positive, there was a higher frequency of overlap myositis (18% vs 5.9%, P < 0.001). MAA positivity was associated with certain clinical features, including Raynaud phenomenon (odds ratio [OR] 2.44, 95% confidence interval [CI] 1.41-4.28) and ILD (OR 3.43, 95% CI 1.75-6.96), as well as a chronic disease course (OR 1.72, 95% CI 1.10-2.72) and mortality (OR 3.76, 95% CI 1.72-8.43). The number of MAAs was also associated with mortality (OR 1.83, 95% CI 1.16-2.86). CONCLUSION: MAAs were prevalent in a large cohort of patients with juvenile myositis. ILD, refractory disease, and mortality were associated with MAA positivity. Prospective studies are needed to determine whether early detection of MAAs may lead to improved outcomes for patients with juvenile myositis.

Immunoprecipitation-western blot for proteins of low abundance.

Combining the procedures of immunoprecipitation and immunoblotting can help to overcome some of the limitations of each separate procedure. Immunoblotting can identify immunoprecipitated proteins more specifically and with higher sensitivity than nonspecific protein stains or autoradiography. Immunoprecipitation can enrich proteins of interest to improve sensitivity for detection when compared with immunoblotting of whole cell extracts. Recently, immunoprecipitation-blotting helped to characterize a new autoantibody, anti-p155, and to test for the presence of the autoantibody in patient sera to study its clinical associations. The procedure for immunoprecipitation-blotting, with specific reference to this autoantibody test ("reverse" immunoprecpitation-blotting), is reported here in detail.

SDS-PAGE for 35S Immunoprecipitation and Immunoprecipitation Western Blotting.

This report discusses recent methods of sample preparation and gel electrophoresis for 35S immunoprecipitation (IP) and IP western blotting. In both methods, IP is used to obtain purified proteins, and sodium dodecyl sulfate polyacrylamide gel electrophoresis is used to separate the proteins on a gel. In 35S IP, the proteins are radiolabeled and visualized on film by fluorography; in IP blotting, proteins are transferred onto nitrocellulose paper, and antibodies are used to detect specific proteins. A similar IP and SDS-PAGE method can be used for both procedures, but IP blotting has the potential advantages of improvement in sensitivity for low-abundance proteins and enhanced specificity for identification of proteins from a mixture. Some of the technical adaptations discussed here to facilitate IP blotting and avoid loss of beads or purified proteins may also be useful for 35S IP.

Immunoprecipitation: Western blot for proteins of low abundance.

Combining the procedures of immunoprecipitation and immunoblotting can help overcome some of the limitations of each separate procedure. Immunoblotting can identify immunoprecipitated proteins more specifically and with higher sensitivity than nonspecific protein stains or autoradiography. Immunoprecipitation can enrich proteins of interest to improve sensitivity for detection when compared with immunoblotting of whole cell extracts. Recently, immunoprecipitation-blotting helped us characterize a new autoantibody, anti-p155, and to test for the presence of the autoantibody in patient sera to study its clinical associations. The procedure for immunoprecipitation-blotting, with specific reference to this autoantibody test ("reverse" immunoprecipitation-blotting), is reported here in detail.

SDS-PAGE for ³5S immunoprecipitation and immunoprecipitation western blotting.

This report discusses recent methods of sample preparation and gel electrophoresis for (35)S immunoprecipitation (IP) and IP western blotting. In both methods, IP is used to obtain purified proteins, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is used to separate the proteins on a gel. In (35)S IP, the proteins are radiolabeled and visualized on film by fluorography; in IP blotting, proteins are transferred onto nitrocellulose paper, and antibodies are used to detect specific proteins. A similar IP and SDS-PAGE method can be used for both procedures, but IP blotting has the potential advantages of improvement in sensitivity for low-abundance proteins and enhanced specificity for identification of proteins from a mixture. Some of the technical adaptations discussed here to facilitate IP blotting and avoid loss of beads or purified proteins may also be useful for (35)S IP.

A novel autoantibody to a 155-kd protein is associated with dermatomyositis.

OBJECTIVE: In polymyositis and dermatomyositis (DM), identified autoantibodies occur in <50% of adult patients and in a smaller proportion of children. This study was undertaken as part of a larger effort to define novel autoantibodies that assist in the clinical evaluation of myositis. METHODS: Sera from children and adults satisfying criteria for idiopathic inflammatory myopathies and from patients with other connective tissue diseases (CTDs), patients with noninflammatory myopathies, and healthy individuals were tested for autoantibodies by immunoprecipitation (IP). A previously unrecognized autoantibody that immunoprecipitated a 155-kd protein along with a weaker 140-kd protein was seen. When the presence of this anti-p155 autoantibody in test sera was suggested based on IP results, it was confirmed by immunoblotting of immunoprecipitates. RESULTS: Sera from 51 of 244 myositis patients (21%), including 30 with juvenile DM (29%), 5 with juvenile CTD-associated myositis (33%), 8 with adult DM (21%), 6 with cancer-associated DM (75%), and 2 with adult CTD-associated myositis (15%), were found to have anti-p155 autoantibody. One of 49 patients with lupus, and none of 89 others without myositis, had anti-p155. Caucasian patients with anti-p155 had a unique HLA risk factor, DQA1*0301 (odds ratio 5.4, corrected P = 0.004). In adults with anti-p155, of several clinical features assessed only the frequency of V-sign rash was increased, but patients with this antibody were clinically distinct from those with autoantibodies to aminoacyl-transfer RNA synthetases. CONCLUSION: A newly recognized autoantibody, anti-p155, is associated with DM and cancer-associated DM, and is one of the most common autoantibodies in this condition, occurring as frequently in children as in adults. The clinical features and immunogenetics associated with anti-p155 differ from those associated with antisynthetases.