Discovery of Two Novel Immunoepitopes and Development of Peptide-based Sarcoidosis Immunoassay.

RATIONALE: Sarcoidosis is a systemic granulomatous disorder associated with hypergammaglobulinemia and the presence of autoantibodies. The specific antigens initiating granulomatous inflammation in sarcoidosis are unknown and there is no specific test available to diagnose sarcoidosis. To discover novel sarcoidosis antigens, we developed a high-throughput T7 phage display library derived from the sarcoidosis cDNA and identified numerous clones differentiating sarcoidosis from other respiratory diseases. After clone sequencing and homology search, we identified two epitopes (Cofilinµ and Chain A) that specifically bind to serum IgGs of sarcoidosis patients. OBJECTIVES: To develop and validate an epitope-specific IgG-based immunoassay specific for sarcoidosis. METHODS: We chemically synthesized both immunoepitopes (Cofilinµ and Chain A), and generated rabbit polyclonal antibodies against both neoantigens. After extensive standardization, we developed a direct peptide ELISA and measured epitope-specific IgG in sera of 386 subjects including, healthy controls (n=100), three sarcoidosis cohorts (n=186), pulmonary tuberculosis (n=70) and lung cancer (n=30). MEASUREMENTS AND MAIN RESULTS: To develop a model to classify sarcoidosis from other groups, data were analyzed using five-fold cross-validation when adjusting for confounders. The Cofilinµ IgGs model yielded a mean sensitivity, specificity, and positive and negative predictive value (PPV, NPV) of 0.97, 0.9, 0.9 and 0.96, respectively. Those same measures for Chain A IgG antibody were 0.9, 0.83, 0.84 and 0.9 respectively. Combining both biomarkers improved AUC, sensitivity, specificity, PPV and NPV. CONCLUSIONS: These results provide a novel immunoassay for sarcoidosis. The discovery of two neoantigens facilitates the development of biospecific drug discovery and the sarcoidosis-specific model.

Modulatory role of macrophage migration inhibitory factor on cytokines and clinical features of sarcoidosis.

Sarcoidosis is a systemic granulomatous disease of unknown etiology with significant heterogeneity in organ manifestations and clinical course. Subjects with sarcoidosis share several features such as, non-necrotizing granuloma, hypergammaglobulinemia, increased local and circulating inflammatory cytokines. Macrophage migration inhibitory factor (MIF) is a pluripotent chemokine modulating cellular function. Study included healthy controls (n = 28) and sarcoidosis patients (n = 65). Sera and BAL of sarcoidosis patients were collected and patients were followed longitudinally for 3 years, and demographics, stages, pulmonary function tests, and organ involvements were recorded. We evaluated MIF in the serum and bronchoalveolar lavage (BAL) fluid of sarcoidosis patients in association with clinical features and cytokines, IL-18, IL-10, IL-6, IFN-γ. We found serum MIF had a positive correlation with IL-10 and IFN-γ and % predicted total lung capacity (%TLC). Serum IL-18 had a significant positive correlation with serum lysozyme, but a negative correlation with %TLC and %DLCO. We identified two groups of sarcoidosis subjects with distinct clinical and cytokine features. A group with prominent extrapulmonary involvement, and low serum MIF, IL-10 and IFN-γ and a group with elevated serum MIF, IL-10 and IFN-γ levels. Our work provides understanding of phenotypic diversity in association with heterogeneity in cytokine landscape in sarcoidosis.