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1.  Unique Correlation Between Mutated Citrullinated Vimentine IgG Autoantibodies and Markers of Systemic Inflammation in Rheumatoid Arthritis Patients 
Rheumatoid arthritis (RA) is the most common inflammatory systemic autoimmune disease, primarily affecting the peripheral joints. Anti-mutated citrullinated vimentin autoantibodies (anti-MCV) of IgG isotype were shown to be a useful diagnostic marker of RA especially in RA patients who were anti-cyclic citrullinated protein autoantibodies (anti-CCP) negative. Nevertheless, published data correlates rheumatoid factor (RF), anti-CCP or anti-MCV antibodies with either erythrocyte sedimentation rate (ESR) or serum C-reactive protein (CRP) as markers of disease activity, not investigated the possible correlations of RA autoantibodies towards ESR and CRP in comparison. Herein, we aim to evaluate the usefulness of anti-MCV as a dependable marker in established RA compared with anti-CCP and RF antibodies and to examine correlations between RF, anti-CCP and anti-MCV antibodies towards ESR and serum CRP. Serum RF-IgA, RF-IgM, anti-CCP and anti-MCV levels were measured in 30 patients with RA and 40 patients with other autoimmune diseases (non-RA) compared with 20 normal subjects. Specificity, sensitivity and AUC for RF antibodies, anti-CCP and anti-MCV were calculated towards RA diagnosis. Our results showed that ESR and CRP had significantly higher values in both RA and non-RA patients compared with our healthy controls with observed significant increment in RA patients compared with non-RA patients. An important finding from our study is that 33.3 % of RA patients were anti-CCP negative but being positive towards anti-MCV. Also, in-between 36.7 up to 40 % of RA patients were RF-IgA and RF-IgM negative while being anti-MCV positive. Anti-MCV antibodies showed the highest specificity and sensitivity (97.5 and 86.6 %, respectively) towards RA diagnosis with the highest AUC value (0.920) compared with anti-CCP and RF antibodies. Correlation analyses revealed that there was no significant correlation between ESR along with CRP towards RF-IgA, RF-IgM and anti-CCP while profound highly significant correlation exhibited between ESR and CRP towards anti-MCV data (r = 0.879 and 0.994, respectively). Thus, our data suggest that the assessment of serum anti-MCV autoantibodies along with ESR and CRP considered as a simple laboratory regime for monitoring RA patients to assess and follow-up disease activity. The addition of anti-MCV autoantibodies to serologic markers in the ACR/EULAR classification criteria for RA will add points for patients with negative anti-CCP and RF antibodies.
PMCID: PMC3689334  PMID: 24426223
Rheumatoid arthritis; Anti-mutated citrullinated vimentine antibodies; Specificity; Sensitivity; C-reactive protein; Erythrocyte sedimentation rate
2.  Antibodies to mutated citrullinated vimentin for diagnosing rheumatoid arthritis in anti-CCP-negative patients and for monitoring infliximab therapy 
Arthritis Research & Therapy  2008;10(6):R142.
Antibodies against cyclic citrullinated peptides (CCPs) are useful for diagnosing rheumatoid arthritis (RA). Antibodies to mutated citrullinated vimentin (MCV) were described recently in RA. The aims of this study were to evaluate the usefulness of anti-MCV for diagnosing RA in anti-CCP-negative patients and to monitor anti-MCV titres during infliximab therapy for RA.
We studied two groups of RA patients, one with (n = 80) and one without (n = 76) anti-CCP antibodies. The specificity of anti-MCV was evaluated by investigating 50 healthy controls and 158 patients with other rheumatic diseases (51 psoriatic rheumatism, 58 primary Sjögren syndrome, and 49 ankylosis spondylitis). Serum anti-MCV and anti-CCP titres were measured in 23 patients after 6, 12, 18, and 24 months of infliximab treatment. Anti-CCP2 and anti-MCV levels were assayed using a commercial enzyme-linked immunosorbent assay. IgM rheumatoid factor was determined by nephelometry.
In accordance with the cutoff values recommended by the manufacturer, the specificity of anti-MCV antibodies was 90.9%. We adjusted the cutoff values to obtain the same specificity as that of anti-CCP antibodies (94.2%). With this optimal cutoff, anti-MCV antibodies were found in 11.8% (9/76) of RA patients without anti-CCP, and similarly, anti-CCP antibodies were found in 11.2% (9/80) of RA patients without anti-MCV. Anti-MCV antibodies were positive in 6 patients who tested negative for both anti-CCP and rheumatoid factor. Anti-MCV titres were significantly decreased after 18 and 24 months of infliximab therapy compared with baseline (P < 0.01) as a significant decrease of anti-CCP levels occurred only at 24 months (P < 0.04). Moreover, an anti-MCV decrease was significantly associated with DAS28 (disease activity score using 28 joint counts) improvements 12 months into therapy.
Our results suggest that anti-MCV antibodies may be valuable for diagnosing RA in anti-CCP-negative patients without replacing them as an equivalent number of anti-CCP-positive RA patients test negative for anti-MCV. Moreover, anti-MCV antibodies could be useful for monitoring the effects of infliximab therapy.
PMCID: PMC2656247  PMID: 19077182
3.  Diagnostic value of antibodies against a modified citrullinated vimentin in rheumatoid arthritis 
Antibodies directed against citrullinated vimentin are members of the family of autoantibodies reactive with citrullinated proteins and are among the most specific serological markers for the diagnosis of rheumatoid arthritis (RA). This study was performed to test the diagnostic value of a newly developed enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against a genetically modified citrullinated vimentin (anti-MCV) in comparison with a second-generation anti-cyclic citrullinated peptides (anti-CCP2) ELISA test system. Blinded sera from 631 patients (409 consecutive out-patients and 222 randomly selected stored sera) with RA (n = 164) and non-RA (osteoarthritis [n = 120], polymyalgia rheumatica/giant cell arteritis [n = 80], spondyloarthritis [n = 36], and other inflammatory rheumatic or non-inflammatory disease [n = 67]) were tested for the presence of anti-MCV and anti-CCP2 antibodies according to the manufacturers' instructions. The diagnostic performance of the anti-MCV was comparable with the anti-CCP2 assay for the diagnosis of RA according to the calculated area under the curve (0.824; 95% confidence interval (CI) 0.778–0.870 versus 0.818; 95% CI 0.767–0.869) as analysed by receiving operating characteristic curve. When categorised with a cutoff value of 20.0 U/ml (as recommended by the manufacturer), sensitivity and specificity of the anti-MCV ELISA were 69.5% (95% CI 61.9%–76.5%) and 90.8% (86.9%–93.8%), respectively, compared with 70.1% (62.5%–77.0%) and 98.7% (96.7%–99.6%) of the anti-CCP2 assay. Using the cutoff values of 19.0 U/ml and 81.5 U/ml for the anti-MCV test to obtain a sensitivity and specificity identical to the anti-CCP2 assay, showed a reduced specificity (89.8%; 85.8%–92.9%) and sensitivity (53.7%; 45.7%–61.5%), respectively, of the anti-MCV ELISA compared with the anti-CCP2 test. In conclusion, the serum ELISA testing for anti-MCV antibodies as well as the anti-CCP-2 assay perform comparably well in the diagnosis of RA. In the high-specificity range, however, the anti-CCP2 assay appears to be superior to the anti-MCV test.
PMCID: PMC1779400  PMID: 16859519
4.  Evaluation of Anti-Mutated Citrullinated Vimentin Antibodies, Anti-Cyclic Citrullinated Peptide Antibodies and Rheumatoid Factor in Omani Patients with Rheumatoid Arthritis 
Rheumatoid factor (RF) is currently used in the diagnosis of rheumatoid arthritis (RA). The discovery of anticitrullinated protein autoantibodies has led to the development of various new tests, such as anti-cyclic citrullinated peptide (anti-CCP) antibodies, and anti-mutated citrullinated vimentin (anti-MCV) antibodies, to diagnose RA. The aims of this study were to determine the sensitivity and specificity of anti-MCV antibodies in comparison with anti-CCP antibodies and RF in Omani Arab patients with RA and compare our findings with published values from different ethnic groups. The sensitivity of anti-MCV antibodies was 72% with 87% specificity. For anti-CCP antibodies the sensitivity was 52% and the specificity was 97%. The sensitivity of RF was 57% with 94% specificity. Anti-CCP antibodies have higher diagnostic specificity and positive predictive value than RF and anti-MCV antibodies. Anti-MCV antibodies have the highest sensitivity when compared to anti-CCP antibodies and RF. Anti-MCV antibodies do not appear to be very useful in the diagnosis of RA. However, long-term study is required to find out whether anti-MCV antibodies can be used as predictive test for incidence of RA.
PMCID: PMC3426236  PMID: 22934118
5.  Antibodies to Mutated Citrullinated Vimentin in Rheumatoid Arthritis: Diagnostic Value, Association with Radiological Damage and Axial Skeleton Affection 
Early definitive diagnosis and effective treatment are mandatory in rheumatoid arthritis (RA) as it can halt the disease progression and subsequent joints destruction.
To investigate the diagnostic and prognostic value of anti-mutated citrullinated vimentin (anti-MCV) and its correlation with disease activity, peripheral and axial skeleton affection in RA patients.
Patients and methods:
A total of 123 patients with different rheumatic diseases were enrolled in a prospective-two year study at Ain Shams University hospital: 64 patients with RA and 59 patients with other rheumatic diseases as controls. RA patients were fulfilling the traditional and the new ACR/EULAR diagnostic criteria for RA. They have been followed up for two years. At baseline, all RA patients were subjected to: Clinical assessment of disease activity by taking full histories, general and local examination, measurement of 28 joint count of tender and swollen joints with calculation of disease activity score (DAS-28) for each patient. Complete blood count, erythrocytes sedimentation rate, C-reactive protein and rheumatoid factor titers were performed. Anti-MCV IgG immunoglobulins’ assay was performed at the study endpoint by ELISA. RA patients were then classified into; anti-MCV positive and anti-MCV negative groups for statistical comparison. Plain X-ray was performed on the peripheral joints and scored by the Simple Erosion Narrowing score (SEN-score). Magnetic Resonance Imaging (MRI) scans were carried out to 22 RA patients on cervical and lumbosacral regions.
Anti-MCV antibodies were found to be of high sensitivity (79.6%) and specificity (96.6%) in diagnosing RA. The area under the curve was 0.893 at 95% confidence interval (CI), confers an odds ratio of 23.5. Anti-MCV positive RA patients had significantly higher DAS-28 and SEN-scores than anti-MCV negative patients; who were found to have more benign disease with lower incidence of erosions (P < 0.05). MRI scans revealed that; 17/22 (77%) had cervical joints involvement while, 8 (36%) had lumbo-sacral joint lesions (P < 0.05), both were correlated significantly with aggressive peripheral joint disease.
Anti-MCV antibodies are promising diagnostic and prognostic marker in RA, with high sensitivity and specificity. They may identify a subset of RA patients with aggressive early erosive disease. The axial skeleton—especially the cervical spine—could be affected in RA and this was correlated with aggressive peripheral joints’ disease. MRI scanning is a sensitive method for detecting axial skeleton involvement in RA, in attempt for better disease control and outcomes.
PMCID: PMC2989636  PMID: 21124694
rheumatoid arthritis; RA; anti-mutated citrullinated vimentin; anti-MCV
6.  Greater prevalence of seropositivity for anti-cyclic citrullinated peptide antibody in unaffected first-degree relatives in multicase rheumatoid arthritis-affected families 
This study determined the prevalence and determinants of seropositivity for rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP) antibody, and anti-mutated citrullinated vimentin (anti-MCV) antibody in unaffected first-degree relatives (FDRs) of rheumatoid arthritis (RA) patients.
A total of 337 subjects (135 with RA and 202 FDRs) were enrolled in this case-control study. Serum RF, anti-CCP antibody, and anti-MCV antibody were assayed. Subjects in multicase families (≥ 2 affected FDRs within the same family) were identified. Multivariate logistic regression analysis was used to identify risk factors associated with RA-related autoantibodies.
Seropositivity for RF, anti-CCP antibody, or anti-MCV antibody was detected in 14.4%, 5.0%, or 13.4% of unaffected FDRs, respectively. Anti-CCP antibody seropositivity was more prevalent in FDRs in multicase families (17.8%) than in those not in multicase families (1.3%, p < 0.0001). Significant correlations between RA-associated autoantibodies were detected in the FDR group (between RF and anti-CCP antibody: r = 0.366, p < 0.0001; between RF and anti-MCV antibody: r = 0.343, p < 0.0001; and between anti-CCP antibody and anti-MCV antibody: r = 0.849, p < 0.0001). After adjustment for age and sex, anti-CCP antibody seropositivity in FDRs was significantly associated with being in a multicase family (odds ratio, 49.8; 95% confidence interval, 5.6 to 441.6).
The association between anti-CCP antibody seropositivity in unaffected FDRs and being in a multicase family suggests that genetic and/or environmental factors may increase the risk for RA development in unaffected FDRs.
PMCID: PMC3543960  PMID: 23345996
Rheumatoid arthritis; First-degree relative; Rheumatoid factor; Citrullinated antigen
7.  Comparison of Two Assays to Determine Anti-Citrullinated Peptide Antibodies in Rheumatoid Arthritis in relation to Other Chronic Inflammatory Rheumatic Diseases: Assaying Anti-Modified Citrullinated Vimentin Antibodies Adds Value to Second-Generation Anti-Citrullinated Cyclic Peptides Testing 
BioMed Research International  2014;2014:198198.
Determination of anti-citrullinated peptide antibodies (ACPA) plays a relevant role in the diagnosis of rheumatoid arthritis (RA). To date, it is still unclear if the use of several tests for these autoantibodies in the same patient offers additional value as compared to performing only one test. Therefore, we evaluated the performance of using two assays for ACPA: second-generation anti-citrullinated cyclic peptides antibodies (anti-CCP2) and anti-mutated citrullinated vimentin (anti-MCV) antibodies for the diagnosis of RA. We compared three groups: RA (n = 142), chronic inflammatory disease (CIRD, n = 86), and clinically healthy subjects (CHS, n = 56) to evaluate sensitivity, specificity, predictive values, and likelihood ratios (LR) of these two assays for the presence of RA. A lower frequency of positivity for anti-CCP2 was found in RA (66.2%) as compared with anti-MCV (81.0%). When comparing RA versus other CIRD, sensitivity increased when both assays were performed. This strategy of testing both assays had high specificity and LR+. We conclude that adding the assay of anti-MCV antibodies to the determination of anti-CCP2 increases the sensitivity for detecting seropositive RA. Therefore, we propose the use of both assays in the initial screening of RA in longitudinal studies, including early onset of undifferentiated arthritis.
PMCID: PMC4082878  PMID: 25025037
8.  Human Merkel cell polyomavirus infection II. MCV is a common human infection that can be detected by conformational capsid epitope immunoassays 
Merkel cell polyomavirus (MCV) is a newly-discovered human tumor virus found in ∼80% of Merkel cell carcinoma (MCC). The rate of MCV infection among persons without MCC is unknown. We developed a MCV virus-like particle (VLP) enzyme-linked immunoassay (EIA) that does not cross-react with human BK or murine polyomaviruses. Peptide mapping of the MCV VP1 gene and immunoblotting with denatured MCV VLP are less sensitive than the MCV EIA in detecting MCV antibodies suggesting antibody reactivity in this assay primarily targets conformational but not linear epitopes. Among MCC patients, all 21 (100%) patients tested with MCV-positive tumors had high serum MCV IgG but not high MCV IgM levels. Only 3 of 6 (50%) MCC patients with MCV-negative tumors were positive for MCV antibodies. Sera from most adults, including 107 of 166 (64%) blood donors, 63 of 100 (63%) commercial donors and 37 of 50 (74%) systemic lupus erythematosus patients, show evidence for prior MCV exposure. Age-specific MCV prevalence was determined by examining a cross-sectional distribution of 150 Langerhans cell histiocytosis (an unrelated neoplasm) patient sera. MCV prevalence increases from 50% among children age 15 years or younger to 80% among persons older than 50 years. We did not find evidence for vertical transmission among infants. Although past exposure to MCV is common among all adult groups, MCC patients have a markedly elevated MCV IgG response compared with control patients. Our study demonstrates that MCV is a widespread but previously unrecognized human infection.
PMCID: PMC2747737  PMID: 19499548
Merkel cell polyomavirus; Merkel cell carcinoma; virus-like particles; enzyme-linked immunosorbent assay; serologic assay
9.  Higher Levels of Autoantibodies Targeting Mutated Citrullinated Vimentin in Patients with Psoriatic Arthritis Than in Patients with Psoriasis Vulgaris 
Antibodies against citrullinated proteins/peptides (ACPAs), and especially antibodies targeting mutated citrullinated vimentin (anti-MCVs), are novel biomarkers of rheumatoid arthritis (RA). Whereas ACPAs are specific and sensitive markers for RA, there have hardly been any reports relating to ACPAs in psoriatic arthritis (PsA) or in psoriasis without joint symptoms (PsO). The aim of the present study was to investigate the prevalence of anti-MCVs in PsA and PsO. Serum anti-MCV titers were measured in 46 PsA and 42 PsO patients and in 40 healthy controls by means of a commercial enzyme-linked immunosorbent assay. The potential correlations of the serum autoantibody levels with several clinical and laboratory parameters were examined. The anti-MCV levels in the PsA patients were significantly higher than those in the PsO group. Among the clinical variables, the presence of tender knee joints and nail psoriasis was significantly associated with anti-MCV positivity in the PsA patients. Higher anti-MCV titers in the PsO patients were associated with a more severe disease course and with the early onset of psoriatic skin symptoms. Our results suggest that anti-MCVs can be used as novel markers in the diagnosis of PsA and in a subset of PsO patients.
PMCID: PMC3614022  PMID: 23573111
10.  Antibodies to mutated citrullinated vimentin and disease activity score in early arthritis: a cohort study 
The aim of our study was to investigate the association between arthritic disease activity and antibodies to mutated citrullinated vimentin (anti-MCV), because such a relation has been suggested.
Anti-MCV levels were measured in 162 patients with early arthritis (123 with rheumatoid arthritis and 39 with undifferentiated arthritis) at baseline and at 1 and 2 years of follow up. Disease activity was measured using the disease activity score (Disease Activity Score based on 28 joints [DAS28]) and serum C-reactive protein. General estimation equation analysis was used to assess the relation between anti-MCV levels and DAS28 over time.
Both, anti-MCV levels and DAS28 exhibited a significant decrease during the first and second year. However, the association between anti-MCV levels and DAS28, adjusted for dependency on sequential measurements within one individual, was very low (β = 0.00075). In a population of patients with rheumatoid arthritis or undifferentiated arthritis, anti-MCV had a specificity of 92.3% and a sensitivity of 59.3% when using the recommended cut-off of 20 U/ml. Specificity and sensitivity of antibodies against second-generation cyclic citrullinated peptide, using the recommended cut-off value of 25 U/ml, were 92.1% and 55.3%, respectively. Anti-MCV-positive early arthritis patients had significantly higher Sharp-van der Heijde score, erythrocyte sedimentation rate and C-reactive protein levels than did anti-MCV-negative patients at all time points (P < 0.005), but DAS28 was higher in anti-MCV-positive patients at 2 years of follow up only (P < 0.05).
Because the correlation between anti-MCV levels and parameters of disease activity was very low, we conclude that it is not useful to monitor disease activity with anti-MCV levels.
PMCID: PMC2374444  PMID: 18226202
11.  Anti-Cyclic Citrullinated Peptide (Anti-CCP) and Anti-Mutated Citrullinated Vimentin (Anti-MCV) Relation with Extra-Articular Manifestations in Rheumatoid Arthritis 
Journal of Immunology Research  2014;2014:536050.
We evaluated the association between anti-cyclic citrullinated peptide antibodies (anti-CCP) and anti-mutated citrullinated vimentin antibodies (anti-MCV) with the presence of extra-articular (ExRA) manifestations in 225 patients with rheumatoid arthritis (RA). Ninety-five patients had ExRA and 130 had no ExRA. There was no association of anti-CCP and anti-MCV levels with the presence of ExRA as total group (P = 0.40 and P = 0.91, resp.). Making an analysis of individual manifestations, rheumatoid nodules were associated with positivity for rheumatoid factor (RF); (P = 0.01), anti-CCP (P = 0.048), and anti-MCV (P = 0.02). Instead, RF, anti-CCP, or anti-MCV were not associated with SS, chronic anemia, or peripheral neuropathy. Levels of anti-CCP correlated with the score of the Health Assessment Questionnaire-Disability Index (HAQ-Di) (r = 0.154, P = 0.03), erythrocyte sedimentation rate (ESR); (r = 0.155, P = 0.03), and RF (P = 0.254, P < 0.001), whereas anti-MCV titres only correlated with RF (r = 0.169, P = 0.02). On adjusted analysis, ExRA was associated with longer age (P = 0.015), longer disease duration (P = 0.007), higher DAS-28 score (P = 0.002), and higher HAQ-DI score (P = 0.007), but serum levels of anti-CCP and anti-MCV were not associated. These findings show the need to strengthen the evaluation of the pathogenic mechanisms implied in each specific ExRA manifestation.
PMCID: PMC3997885  PMID: 24804270
12.  Separate and overlapping specificities in rheumatoid arthritis antibodies binding to citrulline- and homocitrulline-containing peptides related to type I and II collagen telopeptides 
Our objective was to find out if there are antibodies binding to homocitrulline-containing type I and II collagen carboxyterminal telopeptides in sera of patients with rheumatoid arthritis (RA), and if these antibodies cross-react with citrulline and homocitrulline in the same peptide sequence.
A total of 72 RA and 72 control sera were analyzed for binding using enzyme-linked immunosorbent assay to citrulline- or homocitrulline-containing type I and II collagen carboxyterminal telopeptides, as well as to cyclic citrullinated peptide (CCP) and to mutated citrullinated vimentin (MCV). Specificities of the antibodies were tested using inhibition-ELISA.
Of the RA sera, 39 (54%) and 41 (57%) were positive for binding to CCP and MCV, respectively. Further, 34 (47%) and 30 (42%) of the patients had specific antibodies binding to and being inhibited by citrulline-containing type I collagen telopeptides and by citrulline-containing type II collagen carboxyterminal telopeptides, respectively. The corresponding figures regarding homocitrulline-containing type I and homocitrulline-containing type II collagen telopeptides were 16 (22%) and 14 (19%). Most of the patients, who were seropositive for citrullinated peptides, showed binding in multiple assays. A total of 10 (14%) RA patients were positive for all the tested peptide pairs, while 28 (39%) of them had antibodies that contained overlapping specifities between citrulline and homocitrulline in the same peptide sequence.
Antibodies to both citrulline and homocitrulline containing type I and II collagen telopeptides can be found in sera of RA patients. These antibodies are not constant from one RA patient to another, but contain separate or overlapping specificities within the same peptide sequence varying between individuals. Our results suggest some relationship between citrulline and homocitrulline-recognizing antibodies, since homocitrulline antibodies exist mainly in individuals seropositive to anti-CCP and anti-MCV.
Electronic supplementary material
The online version of this article (doi:10.1186/s13075-014-0515-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4320812  PMID: 25573503
13.  Ectopic Lymphoid Structures Support Ongoing Production of Class-Switched Autoantibodies in Rheumatoid Synovium 
PLoS Medicine  2009;6(1):e1.
Follicular structures resembling germinal centres (GCs) that are characterized by follicular dendritic cell (FDC) networks have long been recognized in chronically inflamed tissues in autoimmune diseases, including the synovium of rheumatoid arthritis (RA). However, it is debated whether these ectopic structures promote autoimmunity and chronic inflammation driving the production of pathogenic autoantibodies. Anti-citrullinated protein/peptide antibodies (ACPA) are highly specific markers of RA, predict a poor prognosis, and have been suggested to be pathogenic. Therefore, the main study objectives were to determine whether ectopic lymphoid structures in RA synovium: (i) express activation-induced cytidine deaminase (AID), the enzyme required for somatic hypermutation and class-switch recombination (CSR) of Ig genes; (ii) support ongoing CSR and ACPA production; and (iii) remain functional in a RA/severe combined immunodeficiency (SCID) chimera model devoid of new immune cell influx into the synovium.
Methods and Findings
Using immunohistochemistry (IHC) and quantitative Taqman real-time PCR (QT-PCR) in synovial tissue from 55 patients with RA, we demonstrated that FDC+ structures invariably expressed AID with a distribution resembling secondary lymphoid organs. Further, AID+/CD21+ follicular structures were surrounded by ACPA+/CD138+ plasma cells, as demonstrated by immune reactivity to citrullinated fibrinogen. Moreover, we identified a novel subset of synovial AID+/CD20+ B cells outside GCs resembling interfollicular large B cells. In order to gain direct functional evidence that AID+ structures support CSR and in situ manufacturing of class-switched ACPA, 34 SCID mice were transplanted with RA synovium and humanely killed at 4 wk for harvesting of transplants and sera. Persistent expression of AID and Iγ-Cμ circular transcripts (identifying ongoing IgM-IgG class-switching) was observed in synovial grafts expressing FDCs/CD21L. Furthermore, synovial mRNA levels of AID were closely associated with circulating human IgG ACPA in mouse sera. Finally, the survival and proliferation of functional B cell niches was associated with persistent overexpression of genes regulating ectopic lymphoneogenesis.
Our demonstration that FDC+ follicular units invariably express AID and are surrounded by ACPA-producing plasma cells provides strong evidence that ectopic lymphoid structures in the RA synovium are functional and support autoantibody production. This concept is further confirmed by evidence of sustained AID expression, B cell proliferation, ongoing CSR, and production of human IgG ACPA from GC+ synovial tissue transplanted into SCID mice, independently of new B cell influx from the systemic circulation. These data identify AID as a potential therapeutic target in RA and suggest that survival of functional synovial B cell niches may profoundly influence chronic inflammation, autoimmunity, and response to B cell–depleting therapies.
Costantino Pitzalis and colleagues show that lymphoid structures in synovial tissue of patients with rheumatoid arthritis support production of anti-citrullinated peptide antibodies, which continues following transplantation into SCID mice.
Editors' Summary
More than 1 million people in the United States have rheumatoid arthritis, an “autoimmune” condition that affects the joints. Normally, the immune system provides protection against infection by responding to foreign antigens (molecules that are unique to invading organisms) while ignoring self-antigens present in the body's own tissues. In autoimmune diseases, this ability to discriminate between self and non-self fails for unknown reasons and the immune system begins to attack human tissues. In rheumatoid arthritis, the lining of the joints (the synovium) is attacked, it becomes inflamed and thickened, and chemicals are released that damage all the tissues in the joint. Eventually, the joint may become so scarred that movement is no longer possible. Rheumatoid arthritis usually starts in the small joints in the hands and feet, but larger joints and other tissues (including the heart and blood vessels) can be affected. Its symptoms, which tend to fluctuate, include early morning joint pain, swelling, and stiffness, and feeling generally unwell. Although the disease is not always easy to diagnose, the immune systems of many people with rheumatoid arthritis make “anti-citrullinated protein/peptide antibodies” (ACPA). These “autoantibodies” (which some experts believe can contribute to the joint damage in rheumatoid arthritis) recognize self-proteins that contain the unusual amino acid citrulline, and their detection on blood tests can help make the diagnosis. Although there is no cure for rheumatoid arthritis, the recently developed biologic drugs, often used together with the more traditional disease-modifying therapies, are able to halt its progression by specifically blocking the chemicals that cause joint damage. Painkillers and nonsteroidal anti-inflammatory drugs can reduce its symptoms, and badly damaged joints can sometimes be surgically replaced.
Why Was This Study Done?
Before scientists can develop a cure for rheumatoid arthritis, they need to know how and why autoantibodies are made that attack the joints in this common and disabling disease. B cells, the immune system cells that make antibodies, mature in structures known as “germinal centers” in the spleen and lymph nodes. In the germinal centers, immature B cells are exposed to antigens and undergo two genetic processes called “somatic hypermutation” and “class-switch recombination” that ensure that each B cell makes an antibody that sticks as tightly as possible to just one antigen. The B cells then multiply and enter the bloodstream where they help to deal with infections. Interestingly, the inflamed synovium of many patients with rheumatoid arthritis contains structures that resemble germinal centers. Could these ectopic (misplaced) lymphoid structures, which are characterized by networks of immune system cells called follicular dendritic cells (FDCs), promote autoimmunity and long-term inflammation by driving the production of autoantibodies within the joint itself? In this study, the researchers investigate this possibility.
What Did the Researchers Do and Find?
The researchers collected synovial tissue from 55 patients with rheumatoid arthritis and used two approaches, called immunohistochemistry and real-time PCR, to investigate whether FDC-containing structures in synovium expressed an enzyme called activation-induced cytidine deaminase (AID), which is needed for both somatic hypermutation and class-switch recombination. All the FDC-containing structures that the researchers found in their samples expressed AID. Furthermore, these AID-containing structures were surrounded by mature B cells making ACPAs. To test whether these B cells were derived from AID-expressing cells resident in the synovium rather than ACPA-expressing immune system cells coming into the synovium from elsewhere in the body, the researchers transplanted synovium from patients with rheumatoid arthritis under the skin of a special sort of mouse that largely lacks its own immune system. Four weeks later, the researchers found that the transplanted human lymphoid tissue was still making AID, that the level of AID expression correlated with the amount of human ACPA in the blood of the mice, and that the B cells in the transplant were proliferating.
What Do These Findings Mean?
These findings show that the ectopic lymphoid structures present in the synovium of some patients with rheumatoid arthritis are functional and are able to make ACPA. Because ACPA may be responsible for joint damage, the survival of these structures could, therefore, be involved in the development and progression of rheumatoid arthritis. More experiments are needed to confirm this idea, but these findings may explain why drugs that effectively clear B cells from the bloodstream do not always produce a marked clinical improvement in rheumatoid arthritis. Finally, they suggest that AID might provide a new target for the development of drugs to treat rheumatoid arthritis.
Additional Information.
Please access these Web sites via the online version of this summary at
This study is further discussed in a PLoS Medicine Perspective by Rene Toes and Tom Huizinga
The MedlinePlus Encyclopedia has a page on rheumatoid arthritis (in English and Spanish). MedlinePlus provides links to other information on rheumatoid arthritis (in English and Spanish)
The UK National Health Service Choices information service has detailed information on rheumatoid arthritis
The US National Institute of Arthritis and Musculoskeletal and Skin Diseases provides Fast Facts, an easy to read publication for the public, and a more detailed Handbook on rheumatoid arthritis
The US Centers for Disease Control and Prevention has an overview on rheumatoid arthritis that includes statistics about this disease and its impact on daily life
PMCID: PMC2621263  PMID: 19143467
14.  Quantitation of Human Seroresponsiveness to Merkel Cell Polyomavirus 
PLoS Pathogens  2009;5(9):e1000578.
Merkel cell carcinoma (MCC) is a relatively uncommon but highly lethal form of skin cancer. A majority of MCC tumors carry DNA sequences derived from a newly identified virus called Merkel cell polyomavirus (MCV or MCPyV), a candidate etiologic agent underlying the development of MCC. To further investigate the role of MCV infection in the development of MCC, we developed a reporter vector-based neutralization assay to quantitate MCV-specific serum antibody responses in human subjects. Our results showed that 21 MCC patients whose tumors harbored MCV DNA all displayed vigorous MCV-specific antibody responses. Although 88% (42/48) of adult subjects without MCC were MCV seropositive, the geometric mean titer of the control group was 59-fold lower than the MCC patient group (p<0.0001). Only 4% (2/48) of control subjects displayed neutralizing titers greater than the mean titer of the MCV-positive MCC patient population. MCC tumors were found not to express detectable amounts of MCV VP1 capsid protein, suggesting that the strong humoral responses observed in MCC patients were primed by an unusually immunogenic MCV infection, and not by viral antigen expressed by the MCC tumor itself. The occurrence of highly immunogenic MCV infection in MCC patients is unlikely to reflect a failure to control polyomavirus infections in general, as seroreactivity to BK polyomavirus was similar among MCC patients and control subjects. The results support the concept that MCV infection is a causative factor in the development of most cases of MCC. Although MCC tumorigenesis can evidently proceed in the face of effective MCV-specific antibody responses, a small pilot animal immunization study revealed that a candidate vaccine based on MCV virus-like particles (VLPs) elicits antibody responses that robustly neutralize MCV reporter vectors in vitro. This suggests that a VLP-based vaccine could be effective for preventing the initial establishment of MCV infection.
Author Summary
For more than 50 years it has been known that some polyomavirus types can induce cancer in experimental animals. However, associations between the various polyomaviruses known to chronically infect most humans and the development of cancer have been difficult to uncover. Last year, DNA from a new human polyomavirus, called Merkel cell polyomavirus (MCV), was found embedded in an uncommon form of skin cancer called Merkel cell carcinoma. Emerging evidence indicates that most adults display detectable immune responses to MCV, suggesting that most individuals eventually become infected with the virus. In this study, we investigate antibodies that directly bind the protein coat of MCV, thereby obstructing its ability to penetrate cultured cells. We found that the magnitude of antibody responses against MCV varies dramatically among normal adults. Interestingly, patients suffering from MCV-associated Merkel cell carcinoma display uniformly strong antibody responses against the virus. This suggests that the development of Merkel cell carcinoma is preceded by an unusually robust MCV infection. It is currently unclear whether MCV infection may also be associated with additional diseases aside from Merkel cell carcinoma. Quantitation of immune responsiveness to the virus, using techniques reported here, could help identify such links.
PMCID: PMC2734180  PMID: 19750217
15.  Comparison of sensitivity and specificity of anti-CCP and anti-MCV antibodies in an Iranian cohort of patients with rheumatoid arthritis 
Background: Anti-CCP is a test commonly used for the diagnosis of rheumatoid arthritis. The aim of this study was to determine the diagnostic values of ACCP compared to anti-MCV in rheumatoid arthritis patients in north of Iran.
Methods: The serum samples of 150 RA patients and 75 controls, with the mean age of 49.6+11.8 and 48.8+12 years respectively, were tested using the commercially available ELISA kits for ACCP and anti-MCV. Sensitivity, Specificity were determined and Roc curve were used for comparison between these two groups.
Results: The sensitivity of ACCP versus anti-MCV was 85% and 81%, respectively. Specificity was 96% and 95%, respectively. In the RA patients, ACCP was positive in 127 (84.7%) and anti MCV in 121 (80.7%) cases. In the control group, these parameters were positive in 3 (4%) and 4 (5.3%) (p<0.0001 and p<0.0001, respectively). The correlation coefficient for ACCP and anti-MCV was calculated at 0.63 (p<0.001). The area under the curve for ACCP was 0.941±0.015 (p<0.001), anti-MCV was 0.902±0.02 (p<0.001). The measure of agreement (Kappa) for these variables was 0.81. In these patients, there was no correlation between DAS28 and the positivity of these tests.
Conclusion: It was concluded, compared to ACCP, anti-MCV has approximately the same accuracy for the diagnosis of rheumatoid arthritis and it does not have additional value.
PMCID: PMC3755833  PMID: 24009964
Rheumatoid arthritis; ACCP; Anti-MCV
16.  Rheumatoid arthritis associated autoantibodies in patients with synovitis of recent onset 
Arthritis Research  2000;2(3):236-243.
An inception cohort of 238 patients having peripheral joint synovitis of less than 12 months duration was evaluated clinically and followed prospectively for 1 year to determine the clinical significance of a number of rheumatoid arthritis (RA) associated autoantibodies. Serum samples collected at the time of the initial evaluation were tested for rheumatoid factor (RF) and antibodies to Sa (anti-Sa), RA-33, (pro)filaggrin [antifilaggrin antibody (AFA)], cyclic citrullinated peptide (anti-CCP), calpastatin, and keratin [antikeratin antibody (AKA)]. RF had a sensitivity of 66% and a specificity of 87% for RA. Anti-Sa, AFA, and anti-CCP all had a specificity of more than 90%, but a sensitivity of less than 50% for this diagnosis. Overall, there was a high degree of correlation between AFA, AKA, anti-Sa or anti-CCP, this being highest between anti-Sa and anti-CCP (odds ratio, 13.3; P < 0.001). Of the 101 patients who were positive for at least one of these four autoantibodies, 57% were positive for only one. Finally, anti-SA identified a subset of predominantly male RA patients with severe, erosive disease. Anti-SA, AFA and anti-CCP are all specific for early RA but, overall, have little additional diagnostic value over RF alone. Although these antibodies may preferentially recognize citrullinated antigens, the modest degree of concordance between them in individual patient sera suggests that it is unlikely a single antigen is involved in generating these responses.
A spectrum of autoantibodies is now known to be specifically associated with RA. There continues to be uncertainty as to what stage of the disease each of these autoantibodies develop, and whether they are associated with unique clinical features.
To help address these questions, a spectrum of autoantibodies known to be associated with RA in a cohort of patients with early synovitis was evaluated.
An inception cohort of 238 patients having peripheral joint synovitis of less than 12 months duration was evaluated clinicially then followed prospectively for 1 year. Patients were classified as having RA on the basis of fulfilling the 1987 criteria. Serum samples collected at the time of the initial evaluation were tested for anti-Sa and anti-RA-33 using immunoblotting, and to (pro)filaggrin (AFA), anti-CCP, and calpastatin (anti-RA-1) using enzyme-linked immunosorbent assay techniques. AKA were detected using immunoflurescence on human epidermal tissue. RF was tested by nephelometry. HLA-DRB1 alleles were determined using sequence specific primers. Initial and 1 year radiographs were evaluated for the presence of erosions.
Of the 238 patients with synovitis of recent onset in the cohort, 106 (45%) met RA criteria, 102 (96%) of whom met the criteria on their initial visit. Diagnoses in the remaining patients included 22 (9%) with reactive arthritis, 14 (6%) with psoriatic arthritis or another form of spondylarthropathy, 11 (5%) with another well-defined rheumatic diagnosis, and 85 (36%) with undifferentiated arthritis. The RA patients were significantly older than the nonRA patients (46 ± 13 versus 39 ± 13; P < 0.001), had higher mean swollen joint count (13.8 ± 9.7 versus 2.3 ± 2.3; P < 0.001), and higher C-reactive protein (CRP) level (1.9 ± 1.9 versus 1.6 ± 2.4; P < 0.01). Table 1 summarizes the prevalence of the various RA associated antibodies in patients diagnosed as having RF-positive (RF+) RA, RF-negative (RF-) RA, and nonRA. Regarding the characteristics of these tests, RF had the highest sensitivity at 66%, and all the other antibodies individually were less than 50% sensitive. AFA, anti-Sa, anti-CCP were greater than 90% specific for RA, while RF and AKA were 80-90% specific, and anti-RA-33 and anti-RA-1 was not specific for this diagnosis. The data further indicate that adding any one of AFA, AKA, anti-Sa, or anti-CCP to RF increases the specificity for RA from 80 to 90%. In the absence of RF, the presence of one or more of these antibodies carried a sensitivity of only 31% for RF- RA, with anti-Sa being the most specific at 98%. Overall, there was a high degree of correlation between AFA, AKA, anti-Sa or anti-CCP, this being highest between anti-Sa and anti-CCP (odds ratio, 13.3; P < 0.001). Despite this high level of correlation, of the 101 patients who were positive for at least one of these four autoantibodies, 57% were positive for only one, suggesting considerable variability in individual reactivity patterns.
RA has been shown in multiple populations to be associated with HLA-DRB1 alleles encoding for the shared epitope (SE). In this study, as illustrated in Table 2, the presence of each of these autoantibodies was significantly associated with having two shared epitope alleles, even when only the RA patients were considered.
Patients with anti-Sa antibodies were predominantly male (61% versus 28%; P<0.01), had significantly higher swollen joint counts (18 ± 12 versus 13 ± 9; P=0.02), and higher CRP levels (2.6 ± 3 mg/dl versus 1.6 ± 1.4 mg/dl; P=0.03) at the initial visit. Despite subsequently begin treated with significantly higher doses of prednisone (4.8 ± 6.0 mg/day versus 1.8 ± 3.3 mg/day; P<0.01), and more disease modifying antirheumatic drug therapy (1.4 ± 0.8 versus 0.9 ± 0.7 disease modifying antirheumatic drugs; P<0.01), the anti-Sa-positive RA patients had a higher frequency of erosions than the rest of the RA patients (60% versus 33%; P=0.03). Neither RF nor SE were associated with the disease severity measures, and analyses evaluating all the other autoantibodies failed to reveal a similar trend.
Despite a well-documented lack of specificity, RF continues to be a central part of the definition of RA, primarily because of its favourable sensitivity profile. In our cohort, RF had a sensitivity of 66%, a specificity of 87%, and an overall accuracy of 78% for the diagnosis of RA. AFA, anti-Sa, anti-CCP were all highly specific for this diagnosis, and when any of them were present in conjunction with RF, the specificity for RA approached 100%. Potentially of more importance to the clinician is the diagnostic value of these antibodies when RF is not detectable. Our data indicate that only 31% of RF- RA patients had any of AKA, AFA, anti-Sa or anti-CCP, and that anti-Sa was the most specific for this diagnosis. This modest level of sensitivity suggests that testing for this spectrum of autoantibodies carries little advantage over RF alone in diagnosing early RA.
AFA, AKA, and antiperinuclear factor (APF) have all been proposed to identify a common antigen present in the skin protein (pro)filaggrin. It has continued to be puzzling why a skin antigen would be targeted relatively specifically in a disorder that is primarily articular. A potential explanation for this may relate to the demonstration that citrulline appears to be an essential constituent of the antigenic determinants recognized by AKA, APF, and AFA. The citrulline rich (pro)filaggrin molecule makes an ideal substrate for detecting this reactivity. Moreover, the SA antigen, which, unlike (pro)filaggrin, is detectable in rheumatoid synovium, has recently been shown to also be citrullinated. It is thus possible that AKA, AFA, APE, and anti-Sa all recognize one or more citrullinated antigens. Despite this possibility, the modest degree of concordance between them in individual patient sera suggests that it is unlikely that a single antigen is involved in generating these responses.
This study provides evidence suggesting that anti-Sa antibodies appear to be a marker for a subset of early RA patients whose disease may be more severe and erosive. Moreover, it was determined that anti-Sa, AFA, and anti-CCP were all highly associated with SE, particularly two copies. We examined a spectrum of potential RA severity indicators including the number of swollen joints, CRP level, and presence of early radiographic erosions. Our data indicate that anti-Sa was more highly associated with these measures of RA severity than any other parameter, including the most accepted prognostic indicators, RF and SE.
In conclusion, it is demonstrated that antibodies directed against putatively citrullinated antigens including SA, filaggrin, keratin, and CCP are the most specific for RA, and are detectable early in the disease course. It will be of interest to find out whether the cumulative prevalence of specific autoantibody subsets tends to increase over time, as this would suggest that the mechanisms underlying the development of these reactivities continue to evolve over the course of the arthropathy.
PMCID: PMC17811  PMID: 11056669
autoantibodies; early synovitis; human leukocyte antigen; rheumatoid arthritis; spondylarthropathy
17.  Diagnostic value of anti-human citrullinated fibrinogen ELISA and comparison with four other anti-citrullinated protein assays 
We studied the diagnostic performance of the anti-human citrullinated fibrinogen antibody (AhFibA) ELISA for rheumatoid arthritis (RA) in a consecutive cohort (population 1) and evaluated the agreement between the AhFibA ELISA and four other assays for anti-citrullinated protein/peptide antibodies (ACPAs) as well as rheumatoid factor in patients with longstanding RA (population 2). Population 1 consisted of 1024 patients with rheumatic symptoms; serum samples from these patients were sent to our laboratory for ACPA testing within the context of a diagnostic investigation for RA. Ninety-two of these patients were classified as having RA according to the American College of Rheumatology criteria and 463 were classified as non-RA patients. Population 2 consisted of 180 patients with longstanding RA and was used to assess agreement and correlations between five ACPA assays: anti-cyclic citrullinated peptide (CCP)1 and anti-CCP2 antibodies were detected using a commercially available ELISA, AhFibA using ELISA, and anti-PepA and anti-PepB antibodies using line immunoassay. Applying previously proposed cut-offs for AhFibA, we obtained a sensitivity of 60.9% and a specificity of 98.7% in population 1. Receiver operating characteristic curve analysis could not detect a significant difference in diagnostic performance between the AhFibA ELISA and anti-CCP2 assay. Performing a hierarchical nearest neighborhood cluster analysis of the five different ACPA assays in population 2, we identified two clusters: a cluster of anti-pepA, anti-pepB and anti-CCP1, and a cluster of AhFibA and anti-CCP2. In conclusion, we found that AhFibA and anti-CCP2 antibodies had similar diagnostic performance. However, disagreement between ACPA tests may occur.
PMCID: PMC1779401  PMID: 16859515
18.  Fingertip rapid point-of-care test in adult case-finding in coeliac disease 
BMC Gastroenterology  2013;13:115.
Coeliac disease (CD), due to its protean clinical manifestation, is still very under diagnosed in adults and delays in diagnosis may take years and even decades. Simple tools to find cases in primary care may help to identify patients for further diagnostic tests. We have evaluated the usefulness of an on site rapid fingertip whole blood point-of-care test (POCT) for such a purpose.
As CD is known to run within families, we tested 148 healthy relatives of 70 Romanian index cases with biopsy-proven CD (87% of all first-degree family members, median age 36 years) for the presence of circulating autoantibodies. In addition to performing the POCT (which measures blood erythrocyte self-TG2-autoantibody complexes) on site, blood was drawn for later evaluations of serum IgA-class endomysial antibodies (EMA). EMA-positive sera were further tested for transglutaminase 2 antibodies (TG2-IgA). All serological parameters were analyzed blindly in a centralized laboratory that had no knowledge of the on site POCT result. Endoscopic small intestinal biopsies was recommended for all POCT- or EMA-test positive subjects.
In on site testing the POCT was positive in 12/148 first-degree relatives (8%) and all these subjects were also serum EMA-positive. A positive EMA test was found only in one other subject. All remaining 135 healthy first-degree relatives were negative for both POCT and EMA. Four subjects positive for both POCT and EMA were negative for TG2-IgA. Ten out of thirteen of the antibody-positive subjects agreed to undergo endoscopy. The POCT was found to be positive in 8/9 first-degree relatives having coeliac-type mucosal lesions of grade Marsh 2 (n = 3) or Marsh 3 (n = 6). The three POCT-positive subjects not agreeing to undergo endoscopy were also both EMA- and TG2-IgA-positive.
The fingertip whole blood rapid POCT might fulfill the unmet need for a simple and cheap case-finding biomarker for early detection and presumptive diagnosis of CD. Confirmatory studies are warranted in adult case-finding in specialized outpatient clinics and in primary care.
PMCID: PMC3717015  PMID: 23849178
Coeliac disease; Healthy relatives; Case-finding; Point-of-care test
19.  Role of anti-citrullinated protein antibodies in diagnosis and prognosis of rheumatoid arthritis 
Antibodies to citrullinated proteins/peptides (ACPAs) are the second serological marker to have recently been included in the 2010 ACR/EULAR Rheumatoid Arthritis (RA) Classification Criteria, which are focused on early diagnosis and therapy. This review discusses their history and some clinical aspects of ACPAs, focusing on the diagnostic utility of anti-cyclic citrullinated peptide (anti-CCP) antibodies as a marker of RA as compared to the widely used rheumatoid factor (RF). Simultaneously, this review aims to raise physician awareness and interest in anti-citrullinated vimentin antibody (anti-Sa/anti-MCV), another member of the ACPA family, which appears to have a better predictive value as a marker of RA than anti-CCP or RF and correlates closely with disease activity and therapeutic response among patients with RA.
PMCID: PMC3258718  PMID: 22291756
antibodies to citrullinated protein/peptide; anti-cyclic citrullinated peptide; anti-mutated citrullinated vimentin; anti-Sa; rheumatoid arthritis; American College of Rheumatology
20.  Circulating immune complexes contain citrullinated fibrinogen in rheumatoid arthritis 
There is increasing evidence that autoantibodies and immune complexes (ICs) contribute to synovitis in rheumatoid arthritis (RA), yet the autoantigens incorporated in ICs in RA remain incompletely characterised.
We used the C1q protein to capture ICs from plasma derived from human RA and control patients. Antibodies specific for immunoglobulin were used to detect ICs, and fibrinogen antibodies were used to detect fibrinogen-containing ICs. RA and control plasma were separated by liquid chromatography, and fractions then characterised by ELISA, immunoblotting and mass spectrometry. Immunohistochemical staining was performed on rheumatoid synovial tissue.
C1q-immunoassays demonstrated increased levels of IgG (p = 0.01) and IgM (p = 0.0002) ICs in plasma derived from RA patients possessing anti-cyclic citrullinated peptide (CCP+) autoantibodies as compared with healthy controls. About one-half of the anti-CCP+ RA possessed circulating ICs containing fibrinogen (p = 0.0004). Fractionation of whole RA plasma revealed citrullinated fibrinogen in the high molecular weight fractions that contained ICs. Positive correlations were observed between fibrinogen-containing ICs and anti-citrullinated fibrinogen autoantibodies, anti-CCP antibody, rheumatoid factor and certain clinical characteristics. Immunohistochemical staining demonstrated co-localisation of fibrinogen, immunoglobulin and complement component C3 in RA pannus tissue. Mass spectrometry analysis of immune complexes immunoprecipitated from RA pannus tissue lysates demonstrated the presence of citrullinated fibrinogen.
Circulating ICs containing citrullinated fibrinogen are present in one-half of anti-CCP+ RA patients, and these ICs co-localise with C3 in the rheumatoid synovium suggesting that they contribute to synovitis in a subset of RA patients.
PMCID: PMC2575608  PMID: 18710572
21.  The use of citrullinated peptides and proteins for the diagnosis of rheumatoid arthritis 
The presence or absence of antibodies to citrullinated peptides/proteins (ACPA) is an important parameter that helps a clinician set a diagnosis of early rheumatoid arthritis and, hence, initiate treatment. There are several commercial tests available to measure ACPA levels, although it can be difficult to decide what the best test for a given clinical question is. We analyzed literature data in which the diagnostic and other properties of various ACPA tests are compared. The results show that for diagnostic purposes the CCP2 test has the highest specificity, the highest sensitivity in stratified studies and the highest positive predictive value. For the prediction of future joint destruction the CCP2, MCV, and CCP3 tests may be used. The ability to predict the likelihood of not achieving sustained disease-modifying antirheumatic drug-free remission was highest for the CCP2 test. Finally, the levels of anti-CCP2 and anti-CCP3 (and possibly anti-mutated citrullinated vimentin) in rheumatoid arthritis patients are not significantly influenced by TNFα blocking agents.
PMCID: PMC2875630  PMID: 20236483
22.  Evidence of fibrinogen as a target of citrullination in IgM rheumatoid factor-positive polyarticular juvenile idiopathic arthritis 
Several studies have noted the significance of measuring anti-cyclic citrullinated peptide (CCP) antibodies in juvenile idiopathic arthritis (JIA) as an important indicator for destructive disease, as is the case in rheumatoid arthritis (RA). While the role of anti-CCP antibodies in RA and JIA has become better understood, the identity of the target proteins of this modification has remained elusive. In this study, we evaluated serum from patients with various subtypes of JIA to investigate the presence of anti-deiminated (citrullinated) fibrinogen and anti-citrullinated α-enolase antibodies, and their association with RF and anti-CCP antibody isotypes.
Sera were obtained from 96 JIA patients, 19 systemic lupus erythematosus (SLE) patients, and 10 healthy children. All sera were measured for antibodies against citrullinated and native fibrinogen and α-enolase by an enzyme linked immunosorbent assay (ELISA). In addition, all sera were assayed for anti-CCP antibody isotypes and rheumatoid factor (RF) isotypes by ELISA. The relationship between anti-citrullinated fibrinogen and anti-α-enolase antibodies and disease activity and joint damage were also investigated. All results were correlated with clinical and laboratory parameters using Spearman's rho correlation coefficient. Multiple logistic regression analysis was utilized to identify which variables were associated with joint erosions and diagnosis of JIA.
Thirty-one JIA patients (32%) demonstrated reactivity to citrullinated fibrinogen and 9 (9%) to citrullinated α-enolase. Reactivity to citrullinated fibrinogen and α-enolase was predominantly found in IgM RF-positive polyarthritis patients. Fourteen JIA patients reacted with native α-enolase and a higher percentage of SLE patients reacted with citrullinated α-enolase when compared to JIA patients. Anti-citrullinated fibrinogen antibodies correlated with the presence of IgG anti-CCP antibodies and IgA and IgM RF. The presence of anti-citrullinated α-enolase antibodies correlated with IgA anti-CCP antibodies. IgG anti-CCP antibodies were significantly associated with joint damage and anti-citrullinated fibrinogen antibodies were strongly associated with JIA when compared to control groups. Anti-citrullinated fibrinogen antibodies demonstrated high sensitivity (81%) for IgM RF-positive polyarticular JIA. IgG anti-CCP antibodies had the highest specificity (95%) for JIA, with anti-citrullinated fibrinogen antibodies, IgA anti-CCP antibodies and IgA RF all following at 84%.
JIA patient sera exhibited strong reactivity to anti-citrullinated fibrinogen antibodies and demonstrated high sensitivity and specificity for JIA, primarily in IgM RF-positive polyarthritis patients. Fibrinogen is one of several protein targets for citrullination in JIA.
PMCID: PMC3071779  PMID: 21439056
23.  Profiling anti-cyclic citrullinated peptide antibodies in patients with juvenile idiopathic arthritis 
Anti-citrullinated protein/peptide antibodies (ACPA), have high specificity for rheumatoid arthritis (RA). Some children with juvenile idiopathic arthritis (JIA), phenotypically resemble RA and test positive for rheumatoid factor (RF) a characteristic biomarker of RA. We investigated the prevalence of ACPA and its relationship to other serologic markers associated with RA in a well-characterized JIA cohort.
Cases were 334 children with JIA, 30 of whom had RF + polyarticular JIA. Sera from all cases and 50 healthy pediatric controls were investigated by ELISA at a single time point for anti-cyclic citrullinated peptide (anti-CCP) IgG, RF IgM, IgA and IgG, anti-RA33 IgG, and antinuclear antibodies (ANA). Comparisons between cases and controls were made using Chi-square or Fisher exact tests and T-tests.
The prevalence of RF was 8% among controls, and 12% among cases (ns). The prevalence of ACPA was 2% in controls and 14.3% in cases (OR 8.2, p <0.01). Children who were ACPA-positive and RF-negative (n = 23) had a significantly earlier onset-age (4.6 years vs. 12.1 years, p <0.00001) and had fewer HLA-DRB1 shared epitope alleles than those positive for both RF and ACPA (n = 25). Prevalence of anti-RA33 was not different between cases and controls.
ACPAs are detectable in 14% of children with JIA. Children with positive ACPA but negative RF are frequent, and may define a distinct subset of children with JIA. ACPA testing should be included in the classification of JIA.
PMCID: PMC3490766  PMID: 22931121
Juvenile idiopathic arthritis; Serologic markers; Cyclic citrullinated peptide; Rheumatoid arthritis
24.  The Relationship of Antibodies to Modified Citrullinated Vimentin and Markers of Bone and Cartilage Destruction in Rheumatoid Arthritis 
Objective. To make individualised decisions regarding treatment is one of the most important challenges in clinical practise, and identification of sensitive and specific markers of prognosis is an important research question. The main objective of this study was to evaluate relationships between the level of autoantibodies, radiographic changes and laboratory markers of bone, and cartilage destruction. Methods. A total of 114 RA patients were examined. The serum concentration of IgM RF, antibodies to cyclic citrullinated peptide (anti-CCP), modified citrullinated vimentin (anti-MCV), matrix metalloproteinase 3 (MMP-3), and cartilage oligomeric matrix protein (COMP, ng/mL) were measured. The van der Heijde-modified Sharp Score was used to quantify the radiologic changes. Results. Among the patients who were high-positive for anti-MCV, the value of total modified Sharp score (mTSS) (96.5; 66–120) was higher as well as the joint space narrowing (82; 60.5–105.5), and a higher level of MMP-3 was recorded more frequently (56%) in comparison with negative/low-positive patients (57; 31–88, 50; 29–82, 31% resp., P < 0.05). The level of COMP was also higher among patients high-positive for anti-MCV (9.7; 8.1–13.1 and 6.8; 5.4–10.7, resp., P = 0.02). Conclusion. A high positive level of anti-MCV as contrasted with anti-CCP and IgM RF is associated with more pronounced destructive changes in the joints.
PMCID: PMC4009265  PMID: 24839444
25.  Presence and utility of IgA-class antibodies to cyclic citrullinated peptides in early rheumatoid arthritis: the Swedish TIRA project 
The present study was carried out to assess whether IgA-class antibodies against cyclic citrullinated peptides (IgA anti-CCP) in recent-onset rheumatoid arthritis add diagnostic and/or prognostic information to IgG anti-CCP analysis.
Serum samples were obtained from 228 patients with recent-onset (<12 months) rheumatoid arthritis at the time of inclusion in the Swedish TIRA cohort (Swedish Early Intervention in Rheumatoid Arthritis). Sera from 72 of these patients were also available at the 3-year follow-up. Disease activity and functional ability measures (erythrocyte sedimentation rate, serum C-reactive protein, 28-joint count Disease Activity Score, physician's assessment of disease activity, and the Swedish version of the Health Assessment Questionnaire) were registered at inclusion and at regular follow-ups during 3 years. An IgA anti-CCP assay was developed based on the commercially available IgG-specific enzyme immunoassay from EuroDiagnostica (Arnhem, the Netherlands), replacing the detection antibody by an anti-human-IgA antibody. A positive IgA anti-CCP test was defined by the 99th percentile among healthy blood donors.
At baseline, a positive IgA anti-CCP test was observed in 29% of the patient sera, all of which also tested positive for IgG anti-CCP at a higher average level than sera containing IgG anti-CCP alone. The IgA anti-CCP-positive patients had significantly higher disease activity over time compared with the IgA anti-CCP-negative patients. After considering the IgG anti-CCP level, the disease activity also tended to be higher in the IgA anti-CCP-positive cases – although this difference did not reach statistical significance. The proportion of IgA anti-CCP-positive patients was significantly larger among smokers than among nonsmokers.
Anti-CCP antibodies of the IgA class were found in about one-third of patients with recent-onset rheumatoid arthritis, all of whom also had IgG anti-CCP. The occurrence of IgA-class antibodies was associated with smoking, and IgA anti-CCP-positive patients had a more severe disease course over 3 years compared with IgA anti-CCP-negative cases. Although IgA anti-CCP analysis does not seem to offer any diagnostic information in addition to IgG anti-CCP analysis, further efforts are justified to investigate the prognostic implications.
PMCID: PMC2575621  PMID: 18601717

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