title: Muscarinic type 3 receptor autoantibodies are associated with anti-SSA/Ro autoantibodies in Sjögren's syndrome.
abstract: Anti-muscarinic type 3 receptor autoantibodies (anti-M3R) are reported as potential inhibitors of saliva secretion in Sjögren's syndrome (SjS). However, despite extensive efforts to establish an anti-M3R detection method, there is no clinical test available for these autoantibodies. The purpose of this study was to propose inclusion of anti-M3R testing for SjS diagnosis through investigation of their prevalence using a modified In-Cell Western (ICW) assay. A stable cell line expressing human M3R tagged with GFP (M3R-GFP) was established to screen unadsorbed and adsorbed plasma from primary SjS (n=24), rheumatoid arthritis (RA, n=18), systemic lupus erythematosus (SLE, n=18), and healthy controls (HC, n=23). Anti-M3R abundance was determined by screening for the intensity of human IgG interacting with M3R-GFP cells by ICW assay, as detected by an anti-human IgG IRDye800-conjugated secondary antibody and normalized to GFP. Method comparisons and receiver-operating-characteristic (ROC)-curve analyses were performed to evaluate the diagnostic value of our current approaches. Furthermore, clinical parameters of SjS were also analyzed in association with anti-M3R. Anti-M3R was significantly elevated in SjS plasma in comparison with HC, SLE, or RA (P<0.01). SjS anti-M3R intensities were greater than two-standard deviations above the HC mean for both unadsorbed (16/24, 66.67%) and adsorbed (18/24, 75%) plasma samples. Furthermore, anti-M3R was associated with anti-SjS-related-antigen A/Ro positivity (P=0.0353). Linear associations for anti-M3R intensity indicated positive associations with focus score (R(2)=0.7186, P<0.01) and negative associations with saliva flow rate (R(2)=0.3052, P<0.05). Our study strongly supports our rationale to propose inclusion of anti-M3R for further testing as a non-invasive serological marker for SjS diagnosis.
authors.affiliation: Departments of Oral and Maxillofacial Diagnostic Sciences, University of Florida College of Dentistry, Gainesville, FL 32610, USA.
authors.forename: Jian
authors.initials: J
authors.lastname: Zuo
authors.affiliation: Departments of Oral and Maxillofacial Diagnostic Sciences, University of Florida College of Dentistry, Gainesville, FL 32610, USA.
authors.forename: Adrienne E G
authors.initials: AE
authors.lastname: Williams
authors.affiliation: Departments of Oral and Maxillofacial Diagnostic Sciences, University of Florida College of Dentistry, Gainesville, FL 32610, USA.
authors.forename: Yun-Jong
authors.initials: YJ
authors.lastname: Park
authors.affiliation: Departments of Oral and Maxillofacial Diagnostic Sciences, University of Florida College of Dentistry, Gainesville, FL 32610, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Choi
authors.affiliation: Department of Rheumatology and Clinical Immunology, University of Florida College of Medicine, Gainesville, FL 32610, USA.
authors.forename: Annie L
authors.initials: AL
authors.lastname: Chan
authors.affiliation: Department of Rheumatology and Clinical Immunology, University of Florida College of Medicine, Gainesville, FL 32610, USA.
authors.forename: Westley H
authors.initials: WH
authors.lastname: Reeves
authors.affiliation: Department of Rheumatology and Clinical Immunology, University of Florida College of Medicine, Gainesville, FL 32610, USA.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Bubb
authors.affiliation: Division of Rheumatology, Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Yun Jong
authors.initials: YJ
authors.lastname: Lee
authors.affiliation: Department of Physiology, School of Dentistry, Seoul National University School of Dentistry, Seoul, Republic of Korea.
authors.forename: Kyungpyo
authors.initials: K
authors.lastname: Park
authors.affiliation: Departments of Oral and Maxillofacial Diagnostic Sciences, University of Florida College of Dentistry, Gainesville, FL 32610, USA.
authors.forename: Carol M
authors.initials: CM
authors.lastname: Stewart
authors.affiliation: Departments of Oral and Maxillofacial Diagnostic Sciences, University of Florida College of Dentistry, Gainesville, FL 32610, USA. Electronic address: scha@dental.ufl.edu.
authors.forename: Seunghee
authors.initials: S
authors.lastname: Cha
mesh_terms: D000328:Adult; D000368:Aged; D001172:Arthritis, Rheumatoid / Q000175:diagnosis; D001323:Autoantibodies / Q000097:blood; D001324:Autoantigens / Q000276:immunology*; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000175:diagnosis; D008297:Male; D008875:Middle Aged; D011237:Predictive Value of Tests; D020733:RNA, Small Cytoplasmic / Q000276:immunology*; D012372:ROC Curve; D043587:Receptor, Muscarinic M3 / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012261:Ribonucleoproteins / Q000276:immunology*; D012698:Serologic Tests / Q000379:methods*; D012859:Sjogren's Syndrome / Q000175:diagnosis*; D000099207:SS-A Antigen
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D049452:Green Fluorescent Proteins; D020733:RNA, Small Cytoplasmic; D043587:Receptor, Muscarinic M3; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; C116006:RO60 protein, human; D000099207:SS-A Antigen
keywords: Autoantibodies; Muscarinic type 3 receptor; Sjögren's syndrome; Sjögren's syndrome antigen A