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This AMC antibody is the gold standard reagent for the detection of citrullinated peptides and is a polyclonal antibody that specifically recognizes chemically modified peptidyl-citrulline residue. After chemical treatment, an anti-chemically modified citrulline (anti-modified citrulline, AMC) rabbit polyclonal immunoglobulin (Ig) G antibody (ROI004, SHIMA Laboratories) was used to detect citrullinated peptides. The proteins in the samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by western blotting and membrane chemical treatment. This study was approved by the Medical Ethics Review Committee of Tokyo Medical University. Saliva was collected after rinsing the mouth with water and stored in a freezer at -20☌ until subsequent experimentation. Serum was collected by centrifugation of whole blood at 3000 rpm for 10 min and stored in a -20☌ freezer until subsequent experimentation. Written informed consent was obtained from all individuals for participation in the study. For saliva from healthy individuals, samples were used for experiments in the order of collection, and 10 subjects were used for experiments to detect citrullinated peptides in saliva by Western blot, and the other 10 subjects for semi-quantitative study of the citrullinated peptide identified in saliva by mass spectrometry.

, saliva samples were collected from participants with no subjective symptoms of periodontal disease (11 patients with RA and 20 healthy participants). Materials and methods SamplesĪfter screening based on a questionnaire developed by Sugihara et al. This study aimed to identify previously unidentified citrullinated peptides in human saliva through a comprehensive investigation of human saliva, and to discuss their clinical significance. Although there have been several reports on ACPAs in the saliva, there are limited reports on citrullinated peptides as autoantigens in saliva. Īnti-citrullinated peptide antibodies (ACPAs) have been detected in the sera of patients with RA and are widely used for RA diagnosis because of their excellent sensitivity and specificity. Although microorganisms do not normally express PAD, Porphyromonas gingivalis (Pg), a pathogen of periodontal disease, has been reported to produce Porphyromonas PAD (PPAD) and citrullinated peptides in periodontal tissues. PAD2 is ubiquitously expressed in several tissues, including skeletal muscles and the spleen, brain, and secretory glands, whereas PAD4 is primarily expressed in neutrophils and monocytes. PAD2 and PAD4 are primarily involved in the pathogenesis of RA and other immune diseases. Human citrullinated enzymes have five isoforms: PAD1, PAD2, PAD3, PAD4 (identical to PAD5), and PAD6, with each isoform exhibiting a different tissue localization and substrate profile. Ĭitrullinated peptides are produced by the post-translational modification of arginine residues in proteins to citrulline residues by the action of PAD in the presence of calcium ions. Filaggrin undergoes citrullination by peptidylarginine deiminase (PAD) during epithelial cell differentiation and plays an important role in the physiological function of epithelial cells. As a citrullinated peptide present in normal mucosal tissues, citrullinated filaggrin is reportedly expressed in the mucosal epithelial cells of the skin, oral cavity, esophagus, and nasal cavity. Recent studies have indicated that in the pre-onset period, immune responses to citrullinated peptides in mucosal membranes, such as those present in the lungs and intestinal tract, may play an important role in the transition from pre-onset RA to clinically evident RA. Fibrin (fibrinogen), vimentin, fibronectin, and α-enolase have been reported as citrullinated peptides present in the joint synovium and joint fluid of patients with RA. Citrullinated peptides are thought to be involved in the pathogenesis of RA. Rheumatoid arthritis (RA) is an autoimmune disease, in which the synovium is the primary site of the disease.

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