Idiotypic network: possible explanation of seronegativity in a patient with rheumatoid arthritis

In order to explain long term seronegativity in certain patients with rheumatoid arthritis (RA), we looked for the presence of anti-idiotypic antibodies against rheumatoid factors (RFs). From a patient's serum with classical but seronegative RA, we isolated a low quantity of the IgG fraction which partially recognized polyclonal RF idiotypes. The purified Fab'2 anti-idiotypic antibodies were able to inhibit up to 48% of in vitro RF production by pokeweed mitogen stimulated lymphocytes from a patient's peripheral blood with seropositive RA. The two main conclusions of this study are: (1) this single patient with seronegative RA has serum antibodies directed against RF idiotypes and (2) these anti-idiotypic antibodies could be implicated in the generation of seronegativity.     

Novel autoantibody markers for early and seronegative rheumatoid arthritis

Approximately one-third of rheumatoid arthritis (RA) patients are seronegative for the 2 serological RA markers, rheumatoid factor (RF) and antibodies against cyclic citrullinated peptides (ACCP). Moreover, the sensitivities of both markers are lower in the diagnostically important early disease phase. The aim of this study was to identify additional autoantibody markers for early RA and for RF-negative, ACCP-negative (seronegative) RA.We screened an RA synovium cDNA phage display library with autoantibodies in plasma from 10 early (symptoms of maximum 1 year) and 10 seronegative (RF-negative, ACCP-negative) RA patients with validation in 72 additional RA patients and 121 controls (38 healthy controls, 43 patients with other inflammatory rheumatic diseases, 20 osteoarthritis patients and 20 subjects with mechanical joint complaints). Fourteen novel autoantibodies were identified that showed a 54% sensitivity and 90% specificity for RA. For 11 of these autoantibodies, an exclusive presence was demonstrated in RA patients (100% specificity, 37% sensitivity) as compared to controls. All early RA patients were positive for at least one of the identified autoantibodies and antibody-positivity was associated with a shorter disease duration (P = 0.0087). 52% of RA patients who initially tested negative for RF and ACCP, tested positive for at least one of the 14 novel autoantibodies, resulting in a 19% increase in sensitivity compared to current serological testing. Moreover, 5 identified autoantibodies were detected more frequently in seronegative RA patients, indicating that these autoantibodies constitute novel candidate markers for this RA subtype. We demonstrated that the targets of 3 of these 5 autoantibodies had an increased expression in RA synovial tissue compared to control synovial tissue, pointing towards a biological rationale for these auto antibody targets in RA.In conclusion, we identified novel candidate autoantibody markers for RA that can be detected in early and seronegative RA patients indicating the potential added value for RA diagnostics.     

A basic study of determination matrix metalloproteinase-3 and carbohydrate in rheumatoid factor in serum for diagnosis of rheumatoid arthritis

The examination of rheumatoid factor (RF), one of the diagnostic marker of rheumatoid arthritis (RA), showed negative about 25% of patients with RA. We analyzed a matrix metalloproteinase-3 (MMP-3) and a carbohydrate in rheumatoid factor (CA.RF) for diagnosis of RA: the former is used the kit "Panaclear MMP-3[Plate]" and the latter is used the kit "Picolumi CA.RF". The basic study of these reagents showed satisfactory results. In 73.3% of seronegative RA showed positive on both MMP-3 and CA.RF levels in serum, respectively. We found that these examinations might be useful for diagnosis of RA, especially during seronegative RA.     

Immune complexes in rheumatoid synovitis: a mixed staining immunofluorescence study

Complexes of IgG—rheumatoid factor (RF) and IgG—β_1C were demonstrated in the synovial membrane obtained from patients with rheumatoid arthritis (RA) by a mixed immunofluorescence method that shows simultaneously the two components of the complex.

IgG—RF was found only in the synovia of cases with circulating RF, while IgG—β_1C deposits could be detected in both seropositive and seronegative cases.

These findings are discussed in connection with the pathogenesis of rheumatoid synovitis in the light of the immune complex pathogenetic mechanism.

     

Antibody to streptococcal cell wall peptidoglycan-polysaccharide polymers in seropositive and seronegative rheumatic disease

An ELISA has been developed for serum antibodies to streptococcal cell wall peptidoglycan-polysaccharide polymers (PG-GSP). A significantly increased prevalence of serum anti-PG-GSP antibody was found in juvenile chronic arthritis and both seropositive and seronegative rheumatoid arthritis (RA), compared with ankylosing spondylitis, systemic lupus erythematosus, myeloma and healthy controls. Anti-PG-GSP antibody was always of the IgG class and there was no correlation of anti-PG-GSP levels with C reactive protein, rheumatoid factor (RF) or anti-streptolysin O titres. There was no direct cross-reaction of RF with PG-GSP, nor did the presence of IgM-RF significantly interfere with the assay. Examination of paired serum and synovial fluid samples offered no evidence for local production of anti-PG-GSP antibody in synovial tissue. These data are compatible with an increased systemic immunization by bacterial fragments in RA.     

HLA class II genes in Latvian patients with juvenile rheumatoid arthritis

PCR-based HLA genotyping was used to analyze the association of HLA-DR and -DQ genes in 127 juvenile rheumatoid arthritis patients and 111 population-based controls from Latvia. The results show DQA1*03 to be positively associated in overall patients and DRB1*01-DQA1*0101-DQB1*0501 to be negatively associated with JRA in overall patients and in polyarthritis patients compared to controls. These data indicate the immunogenetic heterogeneity in the JRA patients, in the disease subgroups and in different ethnic groups. Rheumatoid factor (RF) was assayed in patients ( n = 119) and controls ( n =98). RF was present in patients (7/119, 6%) compared to controls (5/98, 5%). None of the DQA1, DQB1 alleles, DQ and DR-DQ haplotypes was associated in seropositive patients compared to seropositive controls. DR1-DQ5 (DQA1*0101-B*0501) was decreased in seronegative patients (11/111, 10%) compared to seronegative controls (24/105, 23%), but the difference was not significant after correction of the p value.     

Rheumatoid factor avidity in patients with rheumatoid arthritis: Identification of pathogenic RFs which correlate with disease parameters and with the gal(O) glycoform of IgG

The standard ELISA for measuring rheumatoid factor (RF) binding was modified by treatment after the RF-Fc interaction with 2 M guanidine, which allowed a measurement of the avidity of the interaction. Incubation with 4 M guanidine eliminated RF binding. There was a direct correlation (r= 0.99) between the avidity as measured by the modified guanidine ELISA, and the dissociation constant for monoclonal RFs, as measured by competitive ELISA. Of the seropositive rheumatoid arthritis (RA) patients tested, 47% had high-avidity RFs (8% RF binding remaining after guanidine treatment). Tender joint count scores were significantly higher in the high avidity group (p=0.05), whereas there was no significant difference in the ages, disease duration, sedimentation rate, RF titer or serum Ig levels compared to those with low-avidity RFs. Additionally 58% of those with high-avidity RFs had subcutaneous nodules, compared to 40% of the low-avidity group. A significantly higher number of nodules was present in the high-avidity RF group compared to those with low-avidity RFs (p=0.03). Interestingly, the RF avidity was significantly higher in isolated immune complexes (IC), compared to that in circulating IgM RFs (p=0.01). The RF avidity correlated with the presence of the glycoform of IgG lacking galactose in both circulating and IC-derived IgG (p=0.003 and 0.009 respectively). Information about the strength of binding to Fc identifies a subgroup of IgM RFs that are likely pathological in patients with RA, as well as a specific glycoform of the target antigen.     

The association between HLA genes and radiological erosions in Malaysian patients with rheumatoid arthritis

OBJECTIVE: To assess the relationship between the HLA-DRB1 genes with disease severity as assessed by radiological erosions in Malaysian patients with rheumatoid arthritis (RA). METHODS: In this cross-sectional study, we studied 61 RA patients who fulfilled the ACR criteria for the diagnosis of RA. HLA-DRB1 genotyping was performed by sequence specific primer (SSP) - PCR. Radiological grading and erosive score of the hands and wrists was calculated according to the Larsen-Dale method. Demographic data and treatment given to the patients were obtained from their case records. RESULTS: Fifty-six females and five males were studied from three ethnic groups. In 57 patients with erosions, rheumatoid factor was detected in 80%, HLA-DR4 in 40%, HLA-DRB1*0405 in 24% and shared epitope (SE) in 31%. The median delay in starting DMARDs was 24 months. The presence of rheumatoid factor, HLA-DR4 and HLA-DRB1*0405 were not significantly associated with a worse erosive score. Patients who possessed the SE had a higher erosive scores, compared to those who did not (p = 0.05). Concurrently, a delay in starting DMARD was associated with a high erosive score (p = 0.023, r = 0.348). However, after adjustment for the delay in starting DMARD, SE was no longer significantly associated with the erosive score. CONCLUSIONS: In these patients, the delay in starting DMARDs had a greater influence on the erosive score than SE alone. Whilst we cannot discount the contribution of the SE presence, we would advocate early usage of DMARDs in every RA patient to reduce joint erosions and future disability.     

IgG rheumatoid factors and anti-nuclear antibodies in rheumatoid vasculitis.

We studied the distribution and characteristics of circulating rheumatoid factors (RF) and anti-nuclear antibodies (ANA) in 30 rheumatoid arthritis (RA) patients who had polyarthritis alone (group I), 28 RA patients with polyarthritis and extra-articular disease (group II), 28 RA patients with systemic vasculitis (group III) and 60 healthy matched controls. IgG RF occurred more frequently and in higher serum titres in group III (100%) than RA patients in group I (40%), or in group II (18%) or in normal controls (5.8%). The serum titre of IgM RF was higher in vasculitis patients than in other RA patients. ANA were found in 74% of all RA patients and although the frequency did not differ in the three patient groups, the serum titre was significantly higher in the vasculitis group. Antibodies to extractable nuclear antigen were found only in group III (18.7%). Antibodies to histones were also more prevalent in group III than in the other RA groups. The serological abnormalities in rheumatoid vasculitis differed quantitatively as well as qualitatively from other RA patients.     

类风湿关节炎四种自身抗体的检测及意义

为评估类风湿因子(rheumatoid factor,RF)、抗环瓜氨酸肽(cyclic citrullinated pepdide,CCP)抗体、抗Sa抗体和抗角蛋白抗体(anti-keratin antibody,AKA)自身抗体对类风湿关节炎(rheumatoid arthritis,RA)诊断的意义,采用速率散...     

Glycosylation of random IgG distinguishes seropositive and seronegative rheumatoid arthritis

The N-glycosylation of human immunoglobulins, especially IgGs, plays a critical role in determining affinity of IgGs towards their effector (pro- and anti-inflammatory) receptors. However, it is still not clear whether altered glycosylation is involved in only antibody-dependent disorders like seropositive rheumatoid arthritis (RA) or also in pathologies with similar clinical manifestations, but no specific autoantibodies like seronegative RA. The clarification of that uncertainty was the aim of the current study. Another study aim was the detection of specific glycan forms responsible for altered exposure of native glycoepitopes. We studied sera from seropositive RA (n?=?15) and seronegative RA (n?=?12) patients for exposure of glycans in native IgG molecules, followed by determination of specific glycans by capillary electrophoresis with laser-induced fluorescent detection (CE-LIF). Aged-matched groups of normal healthy donors (NHD) and samples of intravenous immunoglobulin IgG preparations (IVIG) served as controls. There was significantly stronger binding of Lens culinaris agglutinin (LCA) and Aleuria aurantia lectin (AAL) lectins towards IgG from seropositive RA compared to seronegative RA or NHD. CE-LIF analysis revealed statistically significant increases in bisecting glycans FA2BG2 (p?=?.006) and FABG2S1 (p?=?.005) seropositive RA, accompanied by decrease of bisecting monogalactosylated glycan FA2(6)G1 (p?=?.074) and non-bisecting monosialylated glycan FA2(3)G1S1 (p?=?.055). The results suggest that seropositive RA is distinct from seronegative RA in terms of IgG glycan moieties, attributable to specific immunoglobulin molecules present in seropositive disease. These glycans were determined to be bisecting GlcNAc-bearing forms FA2BG2 and FABG2S1, and their appearance increased the availability of LCA and AAL lectin-binding sites in native IgG glycoepitopes.     

Increased Number of IgG Fc Receptors on Monocyte-Enriched Peripheral Blood Leucocytes from Patients with Rheumatoid Arthritis

The number of free Fc receptors (FcR) per cell and the association constant (K_ass) for the binding of monomeric IgG were determined for monocyte-enriched peripheral blood mononuclear cells, isolated from 16 patients with active classical rheumatoid arthritis (RA) and from 15 normal healthy donors. The assay system was based on binding under equili brium conditions of ¹²⁵I-labelled monomeric rabbit IgG to monocytes purified from peripheral blood on a continuous gradient of Petcoll. Monocytes from 14 untreated RA patients (6 seropositive, 8 seronegative) expressed on the average 4.8±1.3 × 10⁴ FcR/cell. This number was significantly higher (P<0.01) than that found in the control group (3.46±0.7 × 10⁴ FcR/cell). There was also a significant difference between the mean K _ass of the RA group and the control group-2.1±0.7 × 10³1/mol and 2.6±1.0 × 10³ 1/mol, respectively (0.05 >P> 0.01). Two seropositive RA patients receiving systemic treatment with penicillamine expressed the same number of FcR/cell as the mean of the control group (3.6 ± 10⁴). Levels of circulating immune complexes (CIC) and of the complement-factor C3 split product C3d were also measured. No correlation was found between the number of FcR/cell and the concentration of C3d, but there was a weak correlation between the number of FcR/ccll and the level of CIC.     

The Distribution of Class-Specific Rheumatoid Factors Is Similar in Rheumatoid and Pre-Illness Sera

The occurrence of IgM, IgG, and IgA class rheumatoid factors (RF) was compared by means of ELISA in 21 sera from patients with established rheumatoid arthritis (RA) and in 22 pre-illness sera from subjects who 4 months to 5 years later developed seropositive rheumatoid arthritis. The positive cases were virtually confined to three groups of about equal size: positive for all three RF classes, positive for IgM-RF and IgG-RF, and positive for IgM-RF only. Thus, the distribution of the RF isotypes was similar in rheumatoid and pre-rheumatoid sera.     

The association between HLA genes and radiological erosions in Malaysian patients with rheumatoid arthritis

Objective: To assess the relationship between the HLA-DRB1 genes with disease severity as assessed by radiological erosions in Malaysian patients with rheumatoid arthritis (RA).

Methods: In this cross-sectional study, we studied 61 RA patients who fulfilled the ACR criteria for the diagnosis of RA. HLA-DRB1 genotyping was performed by sequence specific primer (SSP)—PCR. Radiological grading and erosive score of the hands and wrists was calculated according to the Larsen–Dale method. Demographic data and treatment given to the patients were obtained from their case records.

Results: Fifty-six females and five males were studied from three ethnic groups. In 57 patients with erosions, rheumatoid factor was detected in 80%, HLA-DR4 in 40%, HLA-DRB1*0405 in 24% and shared epitope (SE) in 31%. The median delay in starting DMARDs was 24 months. The presence of rheumatoid factor, HLA-DR4 and HLA-DRB1*0405 were not significantly associated with a worse erosive score. Patients who possessed the SE had a higher erosive scores, compared to those who did not (p = 0.05). Concurrently, a delay in starting DMARD was associated with a high erosive score (p = 0.023, r = 0.348). However, after adjustment for the delay in starting DMARD, SE was no longer significantly associated with the erosive score.

Conclusions: In these patients, the delay in starting DMARDs had a greater influence on the erosive score than SE alone. Whilst we cannot discount the contribution of the SE presence, we would advocate early usage of DMARDs in every RA patient to reduce joint erosions and future disability.     

Raising rheumatoid factor cutoff helps distinguish rheumatoid arthritis

The presence of rheumatoid factor (RF) is one of the clinical criteria for the diagnosis of rheumatoid arthritis (RA). The cutoff point of RF assays is usually based on a reference level obtained from normal subjects in the same population as the patients. We evaluated 63 rheumatoid arthritis (RA), 25 other arthritis patients and 110 blood donors. Their rheumatoid factors (RF) ranged from < 9.9 to 2,264, < 9.9 to 262, and < 9.9 to 66 mIU/ml, respectively. The sensitivity at different cutoff points of 15, 20, and 25 mIU/ml was 92.1%, 90.5%, and 88.9%, respectively. The specificity at the same cutoff points was 81.5%, 84.4%, and 85.2%, respectively. Having minimally sacrificed the sensitivity, we recommend using a higher RF cutoff to increase specificity.     

Usefulness of anti-cyclic citrullinated peptide antibodies (anti-CCP) for the diagnosis of rheumatoid arthritis

Rheumatoid factor (RF) has been commonly used as a marker of rheumatoid arthritis (RA). RF can be detected in 60-80% of RA patients, but the specificity is low against other rheumatic diseases patients. We evaluated the diagnostic accuracy of anti-cyclic citrullinated peptide antibody (anti-CCP), a new diagnostic test for RA. Anti-CCP demonstrated higher sensitivity (81.0%) and specificity (92.4%). By the receiver operating characteristic (ROC) curve analysis, anti-CCP was superior to other markers (ie. RF, CARF, IgG-RF, and MMP-3). In early RA patients (RA patients who had had disease symptoms for < 2 years), sensitivity was 68.8%. Positivities of anti-CCP in RA patients became higher as the advance of stage defined by the Steinbrocker classification. We concluded that anti-CCP is a very valuable tool for the diagnosis of RA. Moreover, anti-CCP is a useful for finding RA of recent onset.     

Rheumatoid factor plaque-forming cells in rheumatoid synovial tissue.

Cells producing rheumatoid factor (RF) were readily detected in vitro by means of a haemolytic plaque assay system employing sheep erythrocytes (SRBC) sensitized with reduced and alkylated rabbit IgG anti-SRBC antibody as target cells. Rheumatoid factor-producing, plaque-forming cells (RF-PFC) were observed in all of the synovial tissue cell preparations from seropositive rheumatoid arthritis patients studied. The numbers of RF-PFC varied considerably without any direct correlation with serum titres of RF antibody activity. However, high numbers of RF-PFC were never found in patients with low rheumatoid factor titres whereas, high and low numbers of RF-PFC were found among the patients with high RF titres. Synovial tissue cell preparations from a group of seronegative patients, with only one exception, failed to exhibit RF-PFC.     

抗环瓜氨酸肽抗体、葡萄糖6-磷酸异构酶及类风湿因子的检测及对类风湿关节炎的诊断价值

目的为寻找敏感性高、特异性强的诊断类风湿关节炎(RA)的实验指标,选择抗环瓜氨酸肽(CCP)抗体、葡萄糖6-磷酸异构酶(GPI)和类风湿因子(RF)并探讨对RA的诊断价值。方法抗CCP抗体和GPI试验采用酶联免疫吸附(ELISA)法,RF用胶乳凝集法,检测了125例RA患者(RA组)、56例其他风湿性疾病患者(对照组)和36例健康体检者(正常组)血清中上述3者的浓度。结果抗CCP抗体、GPI及RF,在RA组分别是:(83.6±45.9)RU/ml、(2.8±2.3)μg/ml和(320.4±208.6)IU/ml;在对照组分别是:(36.2±15.3)RU/ml、(0.19±0.06)μg/ml和(36.3±12.5)IU/ml;在正常组分别是:(19.2±8.6)RU/ml、(0.16±0.08)μg/ml和(19.2±6.5)IU/ml。在RA组中,3者的敏感度分别是64.8%、73.6%和69.6%,特异性分别是92.0%、72.8%和74.4%。结论 RA患者血清抗CCP抗体、GPI和RF显著高于其他疾病患者(P<0.01)。抗CCP抗体和GPI有可能成为诊断RA的新指标、RA的血清标志物,对提高诊断RA的敏感性和特异性,具有良好的临床研究和推广价值。     

表达Foxp3的调节性T细胞在类风湿关节炎发病中的意义

目的：研究表达Foxp3的调节性T细胞在类风湿关节炎（RA）发病中的意义及其与类风湿关节炎临床特征的相关性。方法：提取外周血总RNA，并逆转录为cDNA。应用实时荧光定量PCR法对RA治疗组（n=25）、RA未治疗组（n=25）及健康对照组（n=30）外周血Foxp3 mRNA含量进行检测，并研究其与RA患者病情活动程度、抗环瓜氨酸（CCP）抗体、C反应蛋白（CRP）、血沉（ESR）及类风湿因子（RF）的关系。结果：RA患者的Foxp3 mRNA含量明显低于正常对照组（P〈0．01）；RA未治疗组Foxp3表达水平明显低于RA治疗组（P〈0．01）。RA患者Foxp3表达水平与DAS28评分、抗CCP抗体及ESR水平呈明显负相关（P〈0．05），而与CRP及RF无明显相关性。结论：RA患者存在表达Foxp3的调节性T细胞数量减少和／或功能降低，这种调节性T细胞亚群的异常可能参与了RA的发病和病变进展。