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Lew PH, Rahman MT, Safii SH, Baharuddin NA, Bartold PM, Sockalingam S, Kassim NLA, Vaithilingam RD. Antibodies against citrullinated proteins in relation to periodontitis with or without rheumatoid arthritis: a cross-sectional study. BMC Oral Health 2021; 21:360. [PMID: 34284769 PMCID: PMC8293567 DOI: 10.1186/s12903-021-01712-y] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2021] [Accepted: 07/07/2021] [Indexed: 11/10/2022] Open
Abstract
Background Previous studies have reported conflicting findings between serum anti-citrullinated protein antibodies (ACPA) levels in rheumatoid arthritis (RA) participants with and without periodontitis (Pd). This study aimed to analyse possible correlations between serum ACPA levels and clinical parameters in Pd and RA participants. Methods Full mouth periodontal examination (probing pocket depth, clinical attachment levels, gingival bleeding index, visual plaque index) was conducted and serum samples obtained from 80 participants comprising RA, Pd, both RA and Pd (RAPd) and healthy individuals (HC). Erythrocyte sedimentation rates (ESR) and periodontal inflamed surface area (PISA) were obtained. Serum samples were analysed for ACPA quantification using enzyme-linked immunosorbent assay (ELISA). Results Median levels (IU/mL) of ACPA (interquartile range, IQR) in RAPd, RA, Pd and HC groups were 118.58(274.51), 102.02(252.89), 78.48(132.6) and 51.67(91.31) respectively. ACPA levels were significantly higher in RAPd and RA as compared to HC group (p < 0.05). However, ACPA levels of any of the groups were not correlated with any clinical periodontal and RA parameters within the respective groups. Conclusions At individual level, the amount of serum ACPA seem to have an increasing trend with the diseased condition in the order of RAPd > RA > Pd > HC. However, lack of any significant correlation between the serum ACPA levels with the clinical Pd and RA parameters warrants further studies to investigate the causal link between RA and Pd for such a trend. Further studies involving more inflammatory biomarkers might be useful to establish the causal link between Pd in the development and progression of RA or vice versa. Supplementary Information The online version contains supplementary material available at 10.1186/s12903-021-01712-y.
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Affiliation(s)
- Pit Hui Lew
- Department of Restorative Dentistry, Faculty of Dentistry, University of Malaya, 50603, Kuala Lumpur, Malaysia
| | | | - Syarida Hasnur Safii
- Department of Restorative Dentistry, Faculty of Dentistry, University of Malaya, 50603, Kuala Lumpur, Malaysia
| | - Nor Adinar Baharuddin
- Department of Restorative Dentistry, Faculty of Dentistry, University of Malaya, 50603, Kuala Lumpur, Malaysia
| | | | - Sargunan Sockalingam
- Department of Rheumatology, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia
| | - Noor Lide Abu Kassim
- Kulliyyah of Education, International Islamic University Malaysia, 53100, Kuala Lumpur, Malaysia
| | - Rathna Devi Vaithilingam
- Department of Restorative Dentistry, Faculty of Dentistry, University of Malaya, 50603, Kuala Lumpur, Malaysia.
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Scherer HU, Huizinga TWJ, Krönke G, Schett G, Toes REM. The B cell response to citrullinated antigens in the development of rheumatoid arthritis. Nat Rev Rheumatol 2018; 14:157-169. [DOI: 10.1038/nrrheum.2018.10] [Citation(s) in RCA: 89] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
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3
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Fouda AI, Rageh I, Hashaad NI, Hamza S. Synovial fluid anti-citrulline-containing peptide antibody and its role in the diagnosis of rheumatoid arthritis. EGYPTIAN RHEUMATOLOGY AND REHABILITATION 2017. [DOI: 10.4103/err.err_5_17] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022] Open
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4
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Gudowska M, Gindzienska-Sieskiewicz E, Gruszewska E, Cylwik B, Sierakowski S, Szmitkowski M, Chrostek L. Independence of carbohydrate-deficient isoforms of transferrin and cyclic citrullinated peptides in rheumatoid arthritis. REVISTA BRASILEIRA DE REUMATOLOGIA 2017; 57:185-189. [PMID: 28535888 DOI: 10.1016/j.rbre.2016.01.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2015] [Accepted: 01/05/2016] [Indexed: 06/07/2023] Open
Abstract
OBJECTIVE The aim of this study was to assess the relationship between the two types of posttranslational modifications of proteins in RA: glycosylation on the example of carbohydrate-deficient transferrin and citrullination by means of autoantibodies to cyclic citrullinated peptides. METHODS The study was carried out in 50 RA patients. CDT was measured using N Latex CDT immunonephelometric test, the results were presented in absolute and relative units. Anti-CCP were measured using the chemiluminescent method and rheumatoid factor by immunoturbidimetric method. RESULTS 80% of RA patients were positive for anti-CCP, 70% for RF and 62% for both, anti-CCP and RF. The level of %CDT was significantly elevated, but absolute CDT level was not changed. The mean absolute CDT concentration was higher in anti-CCP positive patients than that in anti-CCP negative. CDT (absolute and relative concentration) did not correlate with anti-CCP and RF. However, serum RF significantly correlated with anti-CCP. %CDT did not correlate with anti-CCP, but absolute level correlated with anti-CCP only in anti-CCP negative and RF negative patients. CDT did not correlate with RF, but solely with anti-CCP in anti-CCP negative patients. Anti-CCP correlated with DAS 28 only in anti-CCP negative RA, but CDT (absolute and relative units) correlated with DAS 28 in all patients and in anti-CCP positive RA. CONCLUSIONS These results suggest that the changes in CDT and anti-CCP concentrations are not associated with oneself and indicate on the independence of these posttranslational modifications in rheumatoid arthritis. Only the alterations in transferrin glycosylation reflected the activity of RA.
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Affiliation(s)
- Monika Gudowska
- Medical University of Białystok, Department of Biochemical Diagnostics, Białystok, Poland
| | | | - Ewa Gruszewska
- Medical University of Białystok, Department of Biochemical Diagnostics, Białystok, Poland
| | - Bogdan Cylwik
- Medical University of Białystok, Department of Pediatric Laboratory Diagnostics, Białystok, Poland
| | - Stanislaw Sierakowski
- Medical University of Białystok, Department of Rheumatology and Internal Diseases, Białystok, Poland
| | - Maciej Szmitkowski
- Medical University of Białystok, Department of Biochemical Diagnostics, Białystok, Poland
| | - Lech Chrostek
- Medical University of Białystok, Department of Biochemical Diagnostics, Białystok, Poland.
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Independência de isoformas de transferrina deficiente em carboidrato e peptídeos citrulinados cíclicos na artrite reumatoide. REVISTA BRASILEIRA DE REUMATOLOGIA 2017. [DOI: 10.1016/j.rbr.2016.01.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022] Open
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6
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Umeda N, Matsumoto I, Tanaka Y, Kawaguchi H, Ebe H, Kagami Y, Ishigami A, Sumida T. Anti-cyclic citrullinated glucose-6-phosphate isomerase peptide-7 (CCG-7) antibodies were suppressed by biologics treatment and deposited to citrullinated proteins in CD68-positive cells in the RA synovium. Mod Rheumatol 2017; 27:914-916. [DOI: 10.1080/14397595.2016.1270388] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Affiliation(s)
- Naoto Umeda
- Department of Internal Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan
- Department of Rheumatology, Tsuchiura Kyodo General Hospital, Tsuchiura, Japan
| | - Isao Matsumoto
- Department of Internal Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan
| | - Yuki Tanaka
- Department of Internal Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan
| | - Hoshimi Kawaguchi
- Department of Internal Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan
| | - Hiroshi Ebe
- Department of Internal Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan
| | - Yayoi Kagami
- Molecular Regulation of Aging, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan
| | - Akihito Ishigami
- Molecular Regulation of Aging, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan
| | - Takayuki Sumida
- Department of Internal Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan
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Coeliac disease and rheumatoid arthritis: similar mechanisms, different antigens. Nat Rev Rheumatol 2015; 11:450-61. [PMID: 25986717 DOI: 10.1038/nrrheum.2015.59] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Rheumatoid arthritis (RA) and coeliac disease are inflammatory diseases that both have a strong association with class II HLAs: individuals carrying HLA-DQ2.5 and/or HLA-DQ8 alleles have an increased risk of developing coeliac disease, whereas those carrying HLA-DR shared epitope alleles exhibit an increased risk of developing RA. Although the molecular basis of the association with specific HLA molecules in RA remains poorly defined, an immune response against post-translationally modified protein antigens is a hallmark of each disease. In RA, understanding of the pathogenetic role of B-cell responses to citrullinated antigens, including vimentin, fibrinogen and α-enolase, is rapidly growing. Moreover, insight into the role of HLAs in the pathogenesis of coeliac disease has been considerably advanced by the identification of T-cell responses to deamidated gluten antigens presented in conjunction with predisposing HLA-DQ2.5 molecules. This article briefly reviews these advances and draws parallels between the immune mechanisms leading to RA and coeliac disease, which point to a crucial role for T-cell-B-cell cooperation in the development of full-blown disease. Finally, the ways in which these novel insights are being exploited therapeutically to re-establish tolerance in patients with RA and coeliac disease are described.
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Humphreys JH, van Nies JAB, Chipping J, Marshall T, van der Helm-van Mil AHM, Symmons DPM, Verstappen SMM. Rheumatoid factor and anti-citrullinated protein antibody positivity, but not level, are associated with increased mortality in patients with rheumatoid arthritis: results from two large independent cohorts. Arthritis Res Ther 2014; 16:483. [PMID: 25471696 PMCID: PMC4272533 DOI: 10.1186/s13075-014-0483-3] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2014] [Accepted: 11/05/2014] [Indexed: 11/16/2022] Open
Abstract
Introduction This study aimed to investigate rheumatoid factor (RF) and anti-citrullinated protein antibody (ACPA) status and levels as predictors of mortality in two large cohorts of patients with early inflammatory arthritis (EIA). Methods Data from the Norfolk Arthritis Register (NOAR) and Leiden Early Arthritis Clinic (EAC) cohorts were used. At baseline, patients had demographic data and smoking status recorded; RF, ACPA and inflammatory markers were measured in the local laboratories. Patients were flagged with national death registers until death or censor date. Antibody status was stratified as negative, low or high positive by RF and ACPA levels individually. In addition, patients were grouped as seronegative, RF positive, ACPA positive or double antibody (RF and ACPA) positive. Cox regression models explored associations between antibody status and mortality adjusting for age, sex, smoking status, inflammatory markers and year of enrolment. Results A total of 4962 patients were included, 64% were female. Median age at onset was 56 (NOAR) and 54 (EAC) years. In NOAR and EAC respectively, 35% and 42% of patients were ACPA/RF positive. When antibody status was stratified as negative, low or high positive, there were no consistent findings between the two cohorts. Double antibody positivity was associated with excess mortality in both cohorts compared to seronegative patients: NOAR and EAC respective adjusted HR (95% confidence interval) 1.35 (1.09 to 1.68) and 1.58 (1.16 to 2.15). Conclusions Patients with EIA who are seropositive for both RF and ACPA have increased mortality compared to those who are single positive or seronegative. Antibody level in seropositive patients was not consistently associated with excess mortality. Electronic supplementary material The online version of this article (doi:10.1186/s13075-014-0483-3) contains supplementary material, which is available to authorized users.
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van der Woude D, Toes REM, Scherer HU. How undifferentiated arthritis evolves into chronic arthritis. Best Pract Res Clin Rheumatol 2014; 28:551-64. [PMID: 25481549 DOI: 10.1016/j.berh.2014.10.013] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
Undifferentiated arthritis (UA) is a frequently occurring clinical presentation with a variable outcome. While some forms of UA will spontaneously remit, other forms will progress to chronic arthritis; an outcome that would preferably be prevented. Which immunological factors are normally at the basis of resolution of inflammation, and what, on the other hand, causes inflammation to persist? This review provides an overview of the immunological mechanisms involved in these two scenarios, including specific examples of how these mechanisms apply, or can be influenced in rheumatic diseases. Furthermore, what do we know about risk factors for chronic arthritis, such as the development of autoantibodies? The recent years have provided many insights concerning risk factors for autoantibody-positive versus autoantibody-negative rheumatoid arthritis, which are discussed along with a possible pathophysiological model incorporating autoantibodies into the larger process of disease development. Finally, the evolution of the autoantibody response over time is described.
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Affiliation(s)
- D van der Woude
- Department of Rheumatology, Leiden University Medical Center, The Netherlands.
| | - R E M Toes
- Department of Rheumatology, Leiden University Medical Center, The Netherlands.
| | - H U Scherer
- Department of Rheumatology, Leiden University Medical Center, The Netherlands.
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Maher J. Role of the clinical immunology laboratory in disease monitoring. World J Immunol 2013; 3:18-30. [DOI: 10.5411/wji.v3.i2.18] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/22/2013] [Accepted: 05/17/2013] [Indexed: 02/05/2023] Open
Abstract
Immunological investigations provide useful information to guide diagnosis of several disorders. Many such tests are also commonly repeated at intervals, in an effort to facilitate disease monitoring. In general however, immunology test results are often slow to alter. Furthermore, audit activity has indicated that repeated testing accounts for a substantial workload in many immunology services, which may waste resources and compromise the efficient completion of necessary tests. Consequently, the need and appropriate minimum interval between repeated testing requires critical evaluation. In this review, the clinical utility of repeated performance of several common immunology investigations has been evaluated, based upon published evidence. In some cases (e.g., paraprotein quantification, or measurement of anti-glomerular basement membrane antibodies), repeated testing provides vital clinical information and can be justified on a frequent and individualized basis. However, many other investigations provided by immunology services provide less valuable information when used to aid disease monitoring rather than diagnosis. It is hoped that the data summarized here will facilitate a more evidence-based approach to repeated testing. Such information may also assist with the local implementation of demand management strategies based upon setting of minimum retesting intervals for these investigations.
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Umeda N, Matsumoto I, Ito I, Kawasaki A, Tanaka Y, Inoue A, Tsuboi H, Suzuki T, Hayashi T, Ito S, Tsuchiya N, Sumida T. Anti-citrullinated glucose-6-phosphate isomerase peptide antibodies in patients with rheumatoid arthritis are associated with HLA-DRB1 shared epitope alleles and disease activity. Clin Exp Immunol 2013; 172:44-53. [PMID: 23480184 DOI: 10.1111/cei.12033] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/08/2012] [Indexed: 02/06/2023] Open
Abstract
To identify and characterize anti-citrullinated glucose-6-phosphate isomerase (GPI) peptide antibodies in patients with rheumatoid arthritis (RA). Nine GPI arginine-bearing peptides in human GPI protein were selected and cyclic citrullinated GPI peptides (CCG-1-9) were constructed. Samples were obtained from RA (n = 208), systemic lupus erythematosus (SLE) (n = 101), Sjögren's syndrome (SS; n = 101) and healthy controls (n = 174). Antibodies against CCG-1-9 were measured, and anti-citrullinated α-enolase-1 (CEP-1), -cyclic citrullinated peptides (CCP) and -GPI proteins antibodies were also examined. Patients with RA were genotyped for HLA-DRB1. The numbers of shared epitope (SE) alleles were counted and compared with those of the autoantibodies. Rabbit GPI was citrullinated with rabbit peptidylarginine deiminase and immunoblot analysis of RA sera performed. The levels of autoantibodies were compared before and after treatment with TNF antagonists in 58 RA patients. Anti-CCG-2, -4 and -7 antibodies were detected in 25·5, 33·2 and 37·0% patients with RA, respectively, and these antibodies were very specific for RA (specificity, 98·1-99·7%). Altogether, 44·2, 86·1 and 13·9% of RA sera were positive for anti-CEP-1, -CCP and -GPI protein antibodies, respectively. Anti-CCG-2, -4 and -7 antibodies were correlated with anti-CCP and anti-CEP-1 antibodies and with the presence of HLA-DRB1 SE alleles. Citrullinated GPI protein was detected using RA sera. Treatment with tumour necrosis factor antagonists reduced significantly the levels of anti-CCG-2 and -7 but not of anti-CEP-1 antibodies. This is the first report documenting the presence of anti-CCG antibodies in RA. Anti-CCG-2 and -7 antibodies could be considered as markers for the diagnosis of RA and its disease activity.
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Affiliation(s)
- N Umeda
- Department of Internal Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan
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Esalatmanesh K, Jamali R, Jamali A, Jamali B, Nikbakht M. Serum anti-cyclic citrullinated peptide antibodies may predict disease activity in rheumatoid arthritis. Rheumatol Int 2012; 32:3799-3805. [PMID: 22187060 DOI: 10.1007/s00296-011-2282-3] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2011] [Accepted: 12/10/2011] [Indexed: 10/14/2022]
Abstract
To define the relationship between serum anti-cyclic citrullinated peptide antibodies (anti-CCP) and disease activity, and to construct a new disease activity index by using anti-CCP in rheumatoid arthritis (RA). One hundred and five RA patients were included. Disease activity based on DAS28-ESR and serum anti-CCP was measured. There was correlation between serum anti-CCP and DAS28-ESR. (R (2) = 0.71, P value < 0.01). New disease activity index was developed by replacing anti-CCP with ESR in DAS28-ESR. There was correlation between new model and DAS28-ESR. (R (2) = 0.91, P value < 0.01) The new composite index best cut-off values corresponding to DAS28-ESR values of 2.6, 3.2, and 5.1 were 3.21, 3.38, and 4.74, respectively. There was agreement between new model and DAS28-ESR for determination of patients in different disease activity categories. (Kappa = 0.71, P value < 0.01). The new disease activity index that applies serum anti-CCP may predict disease activity in RA.
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Affiliation(s)
- Kamal Esalatmanesh
- Internal Medicine Ward, Division of Rheumatology, Shahid Beheshti Hospital, Kashan University of Medical Sciences, Kashan, Iran
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Lipinska J, Brózik H, Stanczyk J, Smolewska E. Anticitrullinated protein antibodies and radiological progression in juvenile idiopathic arthritis. J Rheumatol 2012; 39:1078-87. [PMID: 22382337 DOI: 10.3899/jrheum.110879] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/28/2022]
Abstract
OBJECTIVE The aim of the study was to investigate whether determination of anticitrullinated protein antibodies (ACPA) provides predictive information on severity of disease course and joint destruction in children with juvenile idiopathic arthritis (JIA). METHODS Sera from 74 children with JIA were examined for ACPA using the ELISA test. To assess joint destruction, plain radiographs of both hands were scored twice according to the Steinbrocker scale: at the beginning of observation and after 8.9 to 15.2 months (median 11.5 months) of the followup. Correlations between ACPA serum levels and the disease characteristics (type of JIA onset, disease activity, disease duration, radiological status) were investigated. RESULTS Twenty-six out of 74 examined children with JIA (35.0%) were ACPA-positive [> 5 relative units (RU)/ml]. ACPA were present in all types of JIA onset, including 36.6% of children with early stage JIA (disease duration < 6 months). All of the IgM-rheumatoid factor (RF)-positive children with polyarticular type of JIA onset were simultaneously positive for ACPA. ACPA levels correlated positively with disease activity at the beginning of the study (rho = 0.7196; p < 0.0001) and after followup (rho = 0.2485; p = 0.0486). Disease duration did not significantly affect ACPA serum levels. ACPA levels correlated positively with radiological joint destruction in children with JIA, both at the beginning of the study (rho = 0.4599; p = 0.0004) and after the followup period (rho = 0.5523; p < 0.0001). CONCLUSION ACPA were superior to IgM-RF in diagnosing JIA and provided predictive information on severity of disease course and radiological outcome.
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Affiliation(s)
- Joanna Lipinska
- Department of Pediatric Cardiology and Rheumatology, Medical University of Lodz, Lodz, Poland.
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Kuna AT. Mutated citrullinated vimentin antibodies in rheumatoid arthritis. Clin Chim Acta 2011; 413:66-73. [PMID: 22037509 DOI: 10.1016/j.cca.2011.10.020] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2011] [Revised: 10/11/2011] [Accepted: 10/13/2011] [Indexed: 12/17/2022]
Abstract
Rheumatoid arthritis (RA) is the most common inflammatory systemic autoimmune disease, primarily affecting the peripheral joints. The past decade has been marked with revolutionary changes both in the therapeutic and diagnostic perspectives of RA. The discovery of an RA-specific citrullination-driven immune reaction gave a substantial contribution in the diagnostic approach to RA. Efforts directed towards the identification of the antigenic target specifically recognized by these autoantibodies resulted in the identification of vimentin in citrullinated form as the potential native antigen, among other proteins. Furthermore, it was found that the mutation of vimentin represents an independent trigger of antigenic properties, in addition to citrullination. As a result of this discovery, a commercial ELISA using mutated citrullinated vimentin (MCV) was developed. Increasingly, data now support the use of anti-MCV in RA diagnosis and prognosis for errosion. This review summarizes the research to date on the use of anti-MCV in RA diagnosis and prognosis and its potential use as a therapeutic marker. The pathologic role of these antibodies in RA disease is also discussed.
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Affiliation(s)
- Andrea Tesija Kuna
- Clinical Institute of Chemistry, University Hospital Sestre Milosrdnice, Zagreb, Croatia.
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