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  • Title: Detection of anti-type 3 muscarinic acetylcholine receptor autoantibodies in the sera of Sjögren's syndrome patients by use of a transfected cell line assay.
    Author: Gao J, Cha S, Jonsson R, Opalko J, Peck AB.
    Journal: Arthritis Rheum; 2004 Aug; 50(8):2615-21. PubMed ID: 15334476.
    Abstract:
    OBJECTIVE: Sjögren's syndrome (SS) is an autoimmune disease affecting primarily the salivary and lacrimal glands, leading to dry mouth and dry eyes. Recent studies have suggested that autoantibodies reactive with the type 3 muscarinic acetylcholine receptors (M3Rs) expressed on salivary and lacrimal gland cells may be highly specific for SS. To test this hypothesis, we constructed a cell line expressing the human M3R gene in order to screen for anti-M3R autoantibodies in sera from SS patients. METHODS: Complementary DNA encoding the open-reading frame (ORF) of the human M3R gene was amplified, ligated into the pcDNA5/FRT/V5-His-TOPO TA vector, and then used to transform Escherichia coli bacteria. Plasmid DNA containing the M3R ORF with the correct orientation was transfected into Flp-In Chinese hamster ovary (CHO) cells using Flp recombinase-mediated site-specific recombination. An M3R-transfected CHO cell line, selected and propagated in hygromycin, was used to detect anti-M3R autoantibodies in SS patient and healthy control sera by flow cytometry. RESULTS: Testing of sera for the presence of anti-M3R autoantibodies bound to CHO-transfected cells revealed the presence of anti-M3R autoantibodies in SS patients (9 of 11) but not in healthy controls (0 of 11). Although the anti-M3R autoantibodies detected in patient sera were of multiple isotypes, the most consistently detected were IgG1, IgG3, and IgA. CONCLUSION: Using a newly constructed cell line expressing human M3R, anti-M3R autoantibodies were easily detected in sera from SS patients. These autoantibodies were skewed toward the IgG1, IgG3, and IgA isotypes, probably recognizing a tertiary epitope created by extracellular domains of the receptor protein. Anti-M3R autoantibodies represent a highly promising clinical marker for the identification of SS.
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