Comparative proteomic analysis of neutrophils from patients with microscopic polyangiitis and granulomatosis with polyangiitis.

Both microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) belong to ANCA-associated vasculitis (AAV), in which neutrophils play a key role in their pathology. In this study, in order to discriminate between MPA and GPA, protein profiles of peripheral blood polymorphonuclear cells (PMNs) of 11 MPA patients and 9 GPA patients and 10 healthy controls (HC) were analyzed by 2D-DIGE. In all the 864 spots detected, intensity of 55 spots was significantly different (p<0.05) among the three groups by ANOVA. 31 out of the 55 spots were identified by mass spectrometry. Orthogonal partial-least-squares-discriminate analysis revealed that the abundance profile of the protein spots discriminated the AAV group from the HC group, and the MPA group from the GPA group completely. 13 protein spots were considered as biomarker candidates to distinguish between MPA and GPA. In those, spots whose intensity was higher in MPA than in GPA included actin with various pI values, while a considerable part of spots whose intensity was higher in GPA were proteins related with the activity of neutrophils. Among the candidate proteins, ROC analysis showed that a combination of neutrophil gelatinase-associated lipocalin and a-kinase anchor protein 7 isoforms beta had a high diagnostic potential. BIOLOGICAL SIGNIFICANCE: In this study, protein profiles of polymorphonuclear cells (PMNs) of microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) patients and healthy controls (HC) were investigated by 2D-DIGE, and MS analysis. As a result, we found that the protein profiles of PMNs were useful for distinguishing between patients (MPA and GPA) and HC, and between patients with MPA and patients with GPA. Especially, we found that the 13 protein spots that consisted of 10 proteins considerably contributed to the discrimination between MPA and GPA. This is the first to demonstrate that protein profiles of PMNs are different among MPA, GPA and healthy control. The 10 proteins we identified in this study would be new biomarkers for the diagnosis of the diseases, and may be reflect the pathology difference between MPA and GPA.

Altered posttranslational modification on U1 small nuclear ribonucleoprotein 68k in systemic autoimmune diseases detected by 2D Western blot.

Anti-ribonucleoprotein (anti-RNP) antibodies are one of the representative autoantibodies detectable in patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). Generally, posttranslational modifications (PTMs) on autoantigens are proposed to be involved in the production of autoantibodies. In this study, we tried to detect the alteration in PTMs on a U1 small nuclear RNP 68k subunit (U1-68k), a major antigen of anti-RNP antibodies. Peripheral blood mononuclear cells (PBMCs) were obtained from patients with MCTD, SLE, and rheumatoid arthritis (RA), and from healthy donors. U1-68ks in the PBMCs were detected by 2D Western blot (WB), where extracted nuclear proteins were separated by 2DE, followed by the detection of U1-68k using WB. More than 20 PTM isoforms were detected with different molecular weights of 65.0 , 66.5, and 68.0kDa, and different pIs between 6.0 and 8.5. Importantly, the relative intensity of the spot with 66.5 kDa and pI 7.5 was significantly increased in the MCTD and SLE groups compared to the RA and healthy groups. Further, this U1-68k isoform, in particular, in its RS domain, was found to have significantly decreased phosphorylation compared to the other isoforms. The PTM alternation may be one of the steps to generate the anti-RNP antibodies.

Proteomic analysis of bone marrow-adherent cells in rheumatoid arthritis and osteoarthritis.

AIM: To elucidate the pathophysiology of rheumatoid arthritis (RA) as well as osteoarthritis (OA), we analyzed protein profiles of bone marrow-derived adherent cells (BMACs) from patients with these diseases. METHODS: Proteins, extracted from BMACs from three RA and three OA patients, were comprehensively analyzed by 2-dimensional differential image gel electrophoresis (2D-DIGE). Then a part of the detected proteins, differently expressed between the two diseases, were identified by mass spectrometric analysis. RESULTS: 2D-DIGE analysis detected more than 1600 protein spots in both RA and OA BMACs. Out of these, expression of 340 spots was significantly altered between the diseases (more than 1.5-fold: RA > OA, 26 spots; OA > RA, 314 spots; P < 0.05). Eleven protein spots the intensity of which were significantly altered by more than 2.0-fold were identified, which included vimentin and annexin A5 as increased proteins in RA rather than in OA. As increased proteins in OA compared to RA, alpha chain of collagen VI, a membrane anchor for acetylcholine esterase, heat shock protein 27, caldesmon and cytoskeletal proteins, such as beta actin and alpha tubulin, were identified. CONCLUSIONS: We here report different protein profiles of BMACs between RA and OA for the first time. BMACs possessing differently expressed proteins may be involved in the pathophysiology of the two diseases.

[A case of primary Sjögren's syndrome complicated by chronic progressive myositis].

The patient was a 64-year-old woman with a nearly 20-year history of sicca symptoms, having been given a diagnosis of primary Sjögren's syndrome. Three years previously, she experienced difficulty in walking up a slope and had leg malaise, which insidiously progressed to an inability to go up and down stairs. This disability brought her to our hospital, where her muscle strength was examined by manual muscle testing, and she was found to have reduced muscle strength in proximal muscles like the thigh muscles and the neck flexor muscles. Blood studies revealed elevated ESR, increased serum IgG, mildly increased myogenic enzymes, and positive results for anti-SS-A and -SS-B antibodies. MRI scans disclosed extensive muscle atrophy as well as fatty degeneration in the thigh. A biopsy of the quadriceps femoris muscle provided a diagnosis of myositis based on the finding of muscle fibers of unequal size, nuclear centralization, and inflammatory cell infiltration into muscle fibers. CD4-positive lymphocytes were the predominant inflammatory cells. We diagnosed the case as myositis in primary Sjögren's syndrome based on the clinical course and laboratory findings. She recovered well with steroid medication. It is noteworthy that myositis associated with primary Sjögren's syndrome presents with mild symptoms and unremarkable laboratory data but may run a chronic progressive course.