Anti-cyclic citrullinated peptide-2 (CCP2) autoantibodies and extra-articular manifestations in Greek patients with rheumatoid arthritis

The objective of our study was to establish whether there is an association between rheumatoid arthritis with extra-articular manifestations (exRA) and anti-cyclic citrullinated peptide 2 (anti-CCP2) antibodies in Greeks. A retrospective study of 220 Greek patients with RA, 95 with exRA and 125 without extra-articular manifestations (cRA). Serum anti-CCP2 antibodies and IgM rheumatoid factor (RF) were measured. CCP2(+) were 65.3% of exRA and 58.4% of cRA patients. RF(+) were 69.5% of exRA and 60.0% of cRA patients. Among exRA patients, 37.9% had high serum anti-CCP2 antibody levels (>100 IU/ml) compared to 21.6% cRA patients (p = 0.008). Serositis and pulmonary fibrosis were found to be associated with high levels of anti-CCP2 antibodies (52.9 vs 26.6%, p = 0.02 and 63.6 vs 26.8%, p = 0.008, respectively). Serum RF levels were 265.0 ± 52.0 IU/ml (mean ± SEM) in exRA and 205.1 ± 40.6 (mean ± SEM) in cRA (NS). High serum RF levels (>268 IU/ml) were more likely to have sicca syndrome. In Greek patients with rheumatoid arthritis (RA), high serum anti-CCP2 antibodies are associated with serositis and pulmonary fibrosis. Therefore, anti-CCP2 antibodies have prognostic significance in patients with RA.     

Prevalence and clinical significance of antibodies to citrullinated fibrinogen (ACF) in Chinese patients with rheumatoid arthritis

It has been reported that citrullinated fibrin(ogen) deposits in the inflamed joints played an important role in the pathogenesis of rheumatoid arthritis (RA). Although antibodies to citrullinated fibrinogen (ACF) have been detected in the sera of RA patients, the associations between ACF and RA remain unclear. In this study, human fibrinogen was citrullinated by peptidylarginine deiminase in vitro, and the ACF were detected by an enzyme-linked immunosorbent assay in rheumatic patients, including 183 RA, 121 systemic lupus erythematosus, 48 osteoarthritis, and 108 healthy controls. The prevalence of ACF was determined, and the associations between ACF and RA were evaluated. It was shown that the sensitivity and specificity of ACF in RA were 67.21 and 84.84%, respectively. There were significant correlations between ACF and erythrocyte sedimentation rate, anti-cyclic citrullinated peptide antibody, and anti-keratin antibodies (AKA). In radiographic progression, the RA patients with ACF had higher scores than those without ACF according to the Sharp–van der Heijde method. In addition, ACF was often positive in the RA patients who were IgM rheumatoid factor negative or AKA negative or anti-perinuclear factor negative. The results indicate that ACF assay is helpful for the diagnosis of RA.     

Influence of HLA-DR genes on the production of rheumatoid arthritis-specific autoantibodies to citrullinated fibrinogen

OBJECTIVE: Antibodies directed against citrullinated fibrinogen are highly specific for rheumatoid arthritis (RA). This study was undertaken to test whether RA-associated HLA-DR alleles are associated with anti-citrullinated fibrinogen in RA patient sera and whether replacement of arginyl by citrullyl residues on fibrinogen peptides modifies their binding to HLA-DR molecules and their recognition by T cells. METHODS: Antikeratin, antifilaggrin, and anti-citrullinated fibrinogen antibodies were assayed in RA patients who had undergone HLA-DR typing. Direct assays were performed to investigate binding of citrullinated or native fibrinogen peptides (encompassing the entire alpha- and beta-chains of fibrinogen) to purified HLA-DR molecules. T cell proliferative responses to citrullinated or native fibrinogen peptides were measured in RA patients and controls. RESULTS: HLA-DRB1*0404 was associated with anti-citrullinated fibrinogen in RA sera (P = 0.002). For the RA-associated alleles HLA-DRB1*0401 and HLA-DR1, there was a nonsignificant trend toward association (P = 0.07). Multiple peptides from the alpha- and beta-chains of fibrinogen bound many HLA-DR alleles; DRB1*0404 was the best fibrinogen peptide binder. Citrullination did not influence fibrinogen peptide binding to HLA-DR or fibrinogen peptide recognition by T cells. Peripheral blood T cells that recognized native or citrullinated fibrinogen peptides were common in RA patients but not in healthy controls. CONCLUSION: The RA-associated HLA-DRB1*0404 allele is also associated with production of antibodies to citrullinated fibrinogen. DRB1*0401 and DRB1*01 tend to be associated with anti-citrullinated fibrinogen, but this is not statistically significant. Citrullination of fibrinogen peptide does not influence peptide-DR-T cell interaction. Finally, T cell proliferation in response to citrullinated or uncitrullinated fibrinogen peptides is frequent in RA patients and very infrequent in controls.     

Citrullinated fibrinogen detected as a soluble citrullinated autoantigen in rheumatoid arthritis synovial fluids

BACKGROUND: Anti-citrullinated protein antibodies (ACPA) are specifically and frequently detected in sera of patients with rheumatoid arthritis (RA). Citrullinated fibrin or fibrinogen is a candidate autoantigen of such antibodies. OBJECTIVE: To investigate the presence of citrullinated fibrinogen (cFBG) in the plasma or synovial fluid of patients with RA and control patients, and to determine cFBG levels and their relationship with serum markers for RA if it is present. METHODS: A sandwich enzyme linked immunosorbent assay (ELISA) to measure cFBG was established using monoclonal antibodies cF16.1 and cF252.1, generated by immunising mice with R16Cit and R252Cit, the fibrinogen Aalpha chain derived sequences with citrulline at position 16 and 252, respectively, and the presence of cFBG was further investigated with immunoprecipitation-western blotting. RESULTS: Positive signals were detected in 11/15 RA synovial fluids (RASFs), but not in osteoarthritis synovial fluids or RA plasma with sandwich ELISA for cFBG using cF16.1 and an anti-modified citrulline (AMC) antibody. The presence of cFBG in RASFs was confirmed by immunoprecipitation-western blotting. Furthermore, most RA sera strongly reacted against R16Cit. No relationship was seen between RASF cFBG levels and C reactive protein or anti-cyclic citrullinated peptide antibody levels of the paired sera. CONCLUSION: cFBG is detected as a soluble citrullinated autoantigen in RASFs and may therefore be a genuine candidate antigen for ACPA in patients with RA.     

Antibodies to several citrullinated antigens are enriched in the joints of rheumatoid arthritis patients

Objective

High titers of specific anti–citrullinated protein antibodies (ACPAs) are frequently present in the serum of rheumatoid arthritis (RA) patients, but their presence in synovial fluid is less well characterized. The purpose of this study was to compare the levels of antibody to 4 well‐defined citrullinated candidate RA autoantigens in serum and synovial fluid and to determine whether antibodies to one citrullinated antigen are dominant over another. Furthermore, we studied their relationships with mutated citrullinated vimentin (MCV), a newly identified RA‐specific serum assay, and the classic cyclic citrullinated peptide (CCP) in the synovial fluid of well‐defined HLA–DR groups.

Methods

Paired serum and synovial fluid samples from 290 RA patients and serum samples from 100 age‐ and sex‐matched healthy controls were analyzed for the presence of anti‐MCV and anti‐CCP antibodies and for reactivity to citrullinated fibrinogen, α‐enolase, type II collagen, and vimentin. A total of 219 of the 290 patients were genotyped for the HLA–DR shared epitope alleles.

Results

Significantly higher proportions of antibodies against all RA‐associated citrullinated antigens were found in synovial fluid as compared with serum. This was also true for the MCV and CCP responses but not for non–RA‐associated anti–tetanus toxoid antibodies. As expected, we found a high correlation between citrullinated vimentin and MCV responses. All synovial fluid ACPAs were predominantly associated with HLA–DRB1*04 alleles and were confined to the CCP+/MCV+ subset of patients.

Conclusion

MCV and CCP positivity represent a similar subset of RA patients, whereas ACPAs with different fine specificities fall into subgroups of anti‐CCP+/anti‐MCV+ patients. The levels of all specific ACPAs were elevated in synovial fluid, suggesting that there is local antibody production and/or retention of ACPAs at the site of inflammation governed by RA‐predisposing genes.

     

T cell responses to citrullinated self-peptides in patients with rheumatoid arthritis

Antibodies to citrullinated peptides(ACPA) have high specificity for diagnosis and prognosis in rheumatoid arthritis (RA). ACPA are of IgG isotype and have an association with shared epitope-bearing HLA DR allele, suggesting that T cell help is needed for their generation. In mice models, T cell reactive to citrullinated self-peptide have been reported however, the human data is limited. Patients with RA satisfying ACR criteria were included and peripheral blood obtained for lymphoproliferative assay, antibody level and HLA typing. Citrullinated (Cit) and native peptides of Vimentin and Aggrecan were used for stimulating peripheral blood mononuclear cells in 5-day cultures. A SI value above >2.0 was taken as significant. HLA typing was done by SSCP and ACPA were tested by ELISA. A total of 50 patients (45 females; mean age 42 years; mean duration of disease 7 years) with RA were included in the study. A total of 90 % were RF positive and 78 % were ACPA positive. A total of 28 patients showed response to Agg peptide with 21 of them showing higher response to CitAgg as compared to native Agg peptide as well as the median SI was higher with CitAgg (6.07 Vs. 5.09; p = 0.009). A total of 31 patients showed response to Vim peptide with response to native peptide being higher than CitVim peptide in 22 of the patients. There was no association of T cell response with presence of shared epitope. Nearly half the patients with RA show T cell response to aggrecan and vimentin peptides; however, citrullination is not crucial for T cell response.     

Serum cytokines in patients with rheumatoid arthritis

Serum cytokines such as interleukin 1 (IL-1), interferon (IFN-), and tumor necrosis factor (TNF) were measured in 40 patients with rheumatoid arthritis (RA). In the 40 patients studied, serum IL-1 was detected in 5 patients, IFN- in 10 patients, and TNF in 20 patients. The IL-1-positive group showed increased values of activity indices compared to the IL-1-negative group. Values of serum IFN- correlated well with the number of peripheral blood lymphocytes and CD3⁺ cells and with the percentage of CD3⁺ CD26⁺ cells. Values of serum TNF correlated positively with the number of peripheral blood monocytes and the percentage of CD3⁺ HLA-DR⁺ and CD3⁺ CD25⁺ cells. These results indicated that serum IL-1 in RA patients reflects the activity of RA, while the serum IFN- and TNF in RA patients may be related to circulating activated lymphocytes and monocytes, respectively.     

Serum anti-cyclic citrullinated peptide antibodies may predict disease activity in rheumatoid arthritis

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.     

环瓜氨酸多肽对类风湿关节炎T细胞激活作用的研究

目的观察环瓜氨酸多肽(CCP)对类风湿关节炎(RA)患者T细胞激活的影响,探讨其在RA发病中的作用。方法用四甲基偶氮唑蓝法检测CCP对29例RA患者、8例原发性干燥综合征(pSS)患者及16例骨关节炎(OA)患者外周血单个核细胞(PBMC)激活增殖反应的影响,并分析CCP特异性T细胞增殖反应与患者临床特点之间的相关性。结果RA患者T细胞增殖反应的阳性率和程度均显著高于pSS和OA患者,并与病程呈正相关,差异有统计学意义(P<0.05)。CCP特异性T细胞增殖反应与病程及类风湿因子(RF)、抗CCP抗体阳性率呈正相关,但与肿痛关节数、关节骨质破坏、血沉(ESR)、C反应蛋白(CRP)和IgG、IgA、IgM等指标无显著相关。结论CCP可刺激多数RA患者T细胞增殖,在病程早期即可出现,提示瓜氨酸化抗原可能是诱导RA发病并参与RA致病过程的抗原成分。     

Diagnostic value of anti-cyclic citrullinated Peptide antibody in patients with rheumatoid arthritis

OBJECTIVE: To explore the diagnostic value of anti-cyclic citrullinated peptide antibody (anti-CCP) detected by ELISA in patients with rheumatoid arthritis (RA). METHODS: The synthesized cyclic citrullinated peptide was used as substrate for ELISA. Anti-CCP antibody was detected by ELISA in 191 patients with RA, 132 with rheumatic diseases other than RA, and 98 with nonrheumatic diseases. The antiperinuclear factor (APF), anti-keratin antibody (AKA), rheumatoid factor (RF), and HLA-DR4 gene complex were also tested in each RA patient. The results of these tests were compared with anti-CCP antibody to examine the correlation between them. RESULTS: Ninety (47.1%) patients with RA, 4 (3.0%) with other rheumatic diseases, and 2 (2.0%) with nonrheumatic diseases were found to be anti-CCP antibody positive by ELISA. The sensitivity of anti-CCP antibody was 47.1%, with a high specificity (97.4%) in RA. Anti-CCP antibody correlated with APF, AKA, RF, and HLA-DR4 gene complex. CONCLUSION: A new modified anti-CCP antibody test had a moderate sensitivity (47.1%) but a high specificity (97.4%) in patients with RA and was found as a valuable supplement to diagnosis of RA. Anti-CCP correlated with APF, AKA, RF, and HLA-DR4 gene complex, but did not completely overlap with them. Anti-CCP antibody could be regarded as a new diagnostic marker for RA.     

Differential association of HLA-DRB1 alleles in Japanese patients with early rheumatoid arthritis in relationship to autoantibodies to cyclic citrullinated peptide

OBJECTIVE: To evaluate the role of HLA-DRB1 genotypes and antibodies to cyclic citrullinated peptides (anti-CCP antibodies) in the development and radiographic progression of Japanese patients with rheumatoid arthritis (RA). METHODS: One hundred and ten patients with early RA (88 female, 22 male) who visited our clinic within 1 year of symptom onset were examined for anti-CCP antibody levels and HLA-DRB1 genotypes. HLA-DRB1 genotypes were also determined in 265 healthy controls. Radiographic progression over a 2-year interval was evaluated using the Larsen's method in 66 patients. RESULTS: Among the 110 patients with early RA, 82 patients (74.5%) were anti-CCP positive. Carrier frequency of HLA-DRB1*0405 was significantly increased in RA patients with anti-CCP antibodies compared with controls and RA patients without anti-CCP antibodies (odds ratio [OR] 3.4, 95% confidence interval [95% CI] 2.0-5.7 and OR 3.3, 95% CI 1.3-8.6, respectively). Carriership of one or two SE alleles was significantly associated with production of anti-CCP antibodies (OR 2.7, 95% CI 1.1-6.7 and OR 9.3, 95% CI 1.1-78.2, respectively). On the other hand, allele frequency of HLA-DRB1*0901 was significantly increased in RA patients without anti-CCP antibodies compared with controls and RA patients with anti-CCP antibodies (OR 2.2, 95% CI 1.1-4.1 and OR 3.0, 95% CI 1.4-6.4, respectively). CONCLUSION: In Japanese patients with RA, HLA-DRB1 SE alleles are associated with production of anti-CCP antibodies and HLA-DRB1 alleles appear to be differently associated with early RA depending on anti-CCP positivity as in Caucasian patients with RA.     

Serum pyridoxal in patients with rheumatoid arthritis.

Abnormalities of tryptophan metabolism have been reported in patients with rheumatoid arthritis (RA) and it has been suggested that these abnormalities are the result of disordered vitamin B6 metabolism. Fasting serum pyridoxal, assayed by an automated microbiological system, was found to be below normal in 35 out of 42 patients with RA while a similar abnormality was found in 8 out of 35 patients with osteoarthrosis (OA). Within the RA group the abnormality could not be related to the age, sex, or drug therapy of individuals but of the 8 patients with OA and a low serum pyridoxal, 7 were receiving indomethacin either alone or in conjunction with aspirin.     

Serum aluminium concentrations in patients with rheumatoid arthritis

Aluminium (AlS) and zinc (ZnS) concentrations in serum were measured in patients with rheumatoid arthritis (RA) with and without Al-containing drugs and compared with levels in healthy controls. In RA patients without Al-containing antacids, the AlS levels did not differ from those of the healthy controls (mean 9.9, SEM 0.9 microgram/l and mean 8.6, SEM 0.5 microgram/l). However, the AlS levels in RA patients treated with Al-containing drugs were significantly (p less than 0.05) higher than in the other two groups (mean 14.1, SEM 1.1 microgram/l). Serum zinc (ZnS) levels in both RA groups were significantly (0.01 less than p less than 0.025) lower than in the healthy controls (mean 10.5, SEM 0.4 mumol/l and mean 10.2, SEM 0.4 mumol/l for the RA groups with and without Al-containing drugs, and in healthy volunteers mean 14.4, SEM 0.6 mumol/l). The difference in AlS levels might be due not only to the intestinal absorption of Al from Al-containing drugs, but also to a slightly impaired renal function, serum creatinine being slightly but significantly higher (p less than 0.05) in the Al-treated RA group than in non-Al-treated one. This difference in serum creatinine between the two RA groups might be explained by the more frequent use of cimetidine in the Al-treated RA group.     

Serum transferrin receptor levels in patients with rheumatoid arthritis are correlated with indicators for anaemia

To measure serum soluble transferrin receptor (s-TfR) levels in patients with rheumatoid arthritis (RA), sera were obtained from 50 Japanese RA patients and 20 healthy subjects. Both s-TfR and serum erythropoietin (EPO) levels were measured by enzyme-linked immunosorbent assay (ELISA). Routine laboratory tests were also performed, including peripheral blood analysis and determination of erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF), serum iron levels, total iron-binding capacity (TIBC) and serum ferritin levels. The s-TfR levels in the 50 RA patients (mean ± SD, 1801 ± 512 ng/ml) were significantly higher than those in the 20 control subjects (1316 ± 345 ng/ml). There were no differences in the values of s-TfR between men and women in either group, or between RA patients over and under 50 years old. Serum EPO levels in 47 RA patients were as low as 14.0 ± 10.1 mIU/ml (mean ± SD), ranging from 3.9 to 58.7 mIU/ml (normal range 2.8–17.2 mIU/ml), unrelated to low haemoglobin concentration. The s-TfR levels in RA patients showed negative correlations with red blood cell count, serum iron level and haemoglobin concentration, and positive correlations with ESR and serum EPO levels. However, there were no correlations between s-TfR level and markers of inflammation such as CRP, platelet count or RF titre. In conclusion, s-TfR level in RA patients could be a marker of erythropoiesis rather than of joint inflammation. Received: 14 September 2000 / Accepted: 15 March 2001