抗类风湿性关节炎54kD和36kD自身抗体在类风湿性关节炎诊断 …

目的：研究类风湿性关节炎（ＲＡ）中抗类风湿性关节炎５４ｋＤ和３６ｋＤ自身抗体（抗ＲＡ５４和ＲＡ３６）的情况。方法：应用免疫印迹技术（ＩＢＴ），在过筛和检测盐水可提取核抗原（ＥＮＡ）的自身抗体谱过程中，发现不仅可检测到抗ＲＡ５４，还可检测到抗ＲＡ３６自身抗体，并对１６９例各种不同风湿性疾病患者测定ＥＮＡ。结果：抗ＲＡ５４和抗ＲＡ３６自身抗体均仅见于类风湿性关节炎（ＲＡ）组（ｎ＝８０），阳性率分别为１     

抗RA54自身抗体的识别及其临床意义

本文应用免疫印迹技术（ＩＢＴ），以兔胸腺的盐水提取物（ＲＴＥ）为抗原，在类风湿关节炎（ＲＡ）患者血清中检测到与ＲＴＥ中５４ｋＤ蛋白多肽反应的一种新自身抗体，暂命名为抗ＲＡ５４。通过对３６５３例各种不同风湿性疾病患者的检测，观察到抗ＲＡ５４主要见于ＲＡ组，阳性率为１４．５％，对ＲＡ的诊断有高度特异性（Ｐ〈０．００１）。     

红细胞抗原免疫印迹法在抗SSA抗体分型中的应用

建立了以人红细胞为ＳＳＡ抗原来源的免疫印迹法（ＲＢＣ－ＩＢＴ）检测ＳＳＡ抗体。此法的敏感性略低于常规的对流免疫电泳法（ＣＩＥ），但可检出部分ＣＩＥ中阴性的ＳＳＡ抗体阳性血清，而且尚可鉴定出ＳＳＡ抗体的两种亚型（５２ＫＤ和６０ＫＤ）。检测了干燥综合症（ＳＳ）、系统性红斑狼疮（ＳＬＥ）及类风湿性关节炎（ＲＡ）等三组病人血清，其中ＳＳ和ＳＬＥ两组在ＳＳＡ抗体亚型的分布上差异显著。抗５２ＫＤＳＳＡ抗体主要分布于ＳＳ组（Ｐ＜０．０５），而抗６０ＫＤＳＳＡ抗体主要见于ＳＬＥ组（Ｐ＜０．０５），ＲＢＣ－ＩＢＴ中，非特异或不明显色带极少见，而ＳＳＡ抗体带清晰、易辨。     

免疫印迹技术检测抗中性粒细胞胞浆抗体的应用研究

用免疫印迹技术（ＩＢＴ）检测了１０１例不同原因的血管炎、ＳＬＥ和类风湿性关节炎（ＲＡ）患者血清中的抗中性粒细胞胞浆抗体（ＡＮＣＡ），ＡＮＣＡ与凝胶电泳时２９ＫＤａ处的中性粒细胞胞浆抗原形成一条明显的沉淀带。韦格纳氏／多血管炎组患者的阳性率为５４．５％（６／１１例），显著高于其他血管炎、ＳＬＥ和ＲＡ患者的１４．２％（３／２１例），４．２％（２／４８例）和０％（０／２１例）。３５份正常人血清全呈阴性。用ＡＮＣＡ阳性血清作用于中性粒细胞，在间接免疫荧光检测时均呈胞浆型（Ｃ－ＡＮ－ＣＡ）。此法特异、简便、易于推广应用，有助于血管炎的诊断。     

广东汉族类风湿关节炎某些易感基因研究

为了探讨类风湿关节炎（ＲＡ）的遗传易感基因，用多聚酶链反应－聚丙烯酰胺凝胶电泳（ＰＣＲ－ＰＡＧＥ）和银染色作ＨＬＡ－ＤＱＡ１基因分型，对１０６例健康人和５０例ＲＡ患者进行检测。结果显示：广东汉族共检出６种ＤＱＡ１等位基因，ＲＡ患者组ＤＱＡ１＊０１０１等位基因显著增高（ＲＲ＝２．３３４、Ｐ＜０．００５、ＥＦ＝０．１５４）；ＤＱＡ１＊０１０２明显减少（ＲＲ＝０．０６８、Ｐ＜０．０１、ＰＦ＝０．５７７）；对ＲＡ组中的３１例ＤＲ４阳性患者的ＤＱＡ１基因分析显示，ＤＲ４与ＤＱＡ１＊０３０１连锁的频率显著高于健康人组（Ｐ＜０．００５）。提示ＤＱＡ１＊０１０１对ＲＡ有易感作用，而ＤＱＡ１＊０１０２有遗传抵抗作用；ＤＱＡ１和ＤＲ４基因型检测可能为预测ＲＡ易感者和估计预后提供理论依据。     

类风湿关节炎患者RA33／36抗体,角质蛋白抗体及抗核 …

目的 对类风湿关节炎（ＲＡ）患者血清中的ＲＡ３３／３６抗体，角质蛋白抗体（ＡＫＡ）及抗核周因子（ＡＰＦ）进行检测，并对这些抗体间的相关性进行探讨。方法 采用免疫印迹法检测血清ＲＡ３３／３６抗体；采用ＩＩＦ法检测ＡＫＡ及ＡＰＦ抗体；采用卡方检验及Ｆｉｓｈｅｒ＇ｓ检验对结果进行统计学比较。结果 ＲＡ患者血清中ＲＡ３３／３６抗体阳性率为３５．６％（１６／４５），ＡＫＡ（１：８０）阳性率为３１．１％（１４     

中国汉人HLA—DQA1基因对类风湿关节炎的遗传易感性研究

用ＰＣＲ－ＰＡＧＥ方法，结合高灵敏的银染色作ＨＬＡ－ＤＱＡ１等位基因分型，研究ＤＱＡ１基因对类风湿关节炎（ＲＡ）的遗传易感性。选择无亲缘关系的广东籍汉族健康者１０６例和５０例ＲＡ患者。发现该方法测的６种ＨＬＡ－ＤＱＡ１等位基因中，ＲＡ组ＤＱＡ１＊０１０１（２７％，ＲＲ＝２．３３４，Ｐ＜０．００５，ＥＦ＝０．１５４）等位基因明显增高；而ＤＱＡ１＊０１０２（１％，ＲＲ＝０．０６８，Ｐ＜０．０１，ＰＦ＝０．５７７）明显下降；ＤＱＡ１的２种纯合子基因型（０１０１／０１０１和０３０１／０３０１）在ＲＡ组明显增高（Ｐ值分别小于０．０２５和０．００５）。上述结果显示：ＨＬＡ－ＤＱＡ１＊０１０１对ＲＡ有遗传易感作用，ＤＱＡ１＊０１０２等位基因有遗传抵抗作用；ＤＱＡ１基因型的检测对预测ＲＡ易感者和判断预后及疗效可能提供理论依据。     

钙调素特异性免疫复合物的测定与意义

在研制出钙调素（ＣａＭ）和抗钙调素（抗ＣａＭ）血清的基础上，建立了检测ＣａＭ特异性免疫复合物（ＣａＭ－ＩＣｓ）的ＥＬＩＳＡ法．经检测２６名ＳＬＥ、２８名类风湿性关节炎（ＲＡ）、１５名甲亢、２６名慢性活动性肝炎（ＣＡＨ）患者及１００名正常人血清中ＣａＭ－ＩＣｓ阳性率分别为６１．５％、６７．９％、４６．７％、６９．２％和０％．患者ＣａＭ－ＩＣｓ的出现常与抗ＣａＭ自身抗体的出现同步（ｘ２＝６０．１２，Ｐ＜０．００１）。     

类风湿关节炎患者RA33/36抗体，角质蛋白抗体及抗核周因子的相关性初步研究

目的对类风湿关节炎（ＲＡ）患者血清中的ＲＡ３３／３６抗体，角质蛋白抗体（ＡＫＡ）及抗核周因子（ＡＰＦ）进行检测，并对这些抗体间的相关性进行探讨。方法采用免疫印迹法检测血清ＲＡ３３／３６抗体；采用ＩＦ法检测ＡＫＡ及ＡＰＦ抗体；采用卡方检验及Ｆｉｓｈｅｒｓ检验对结果进行统计学比较。结果ＲＡ患者血清中ＲＡ３３／３６抗体阳性率为３５．６％（１６／４５），ＡＫＡ（１８０）阳性率为３１．１％（１４／４５），ＡＰＦ的阳性率为５３．３％（２４／４５）。结论虽然ＲＡ患者血清中可出现上述三种抗体，但三者间无相关性。     

广东汉族人类风湿关节炎易感性与HLA-DRB1基因相关性研究

目的探讨ＨＬＡ－ＤＲＢ１基因与类风湿关节炎（ＲＡ）相关性。方法采用ＰＣＲ－ＳＳＰ方法对４７例广东汉族人ＲＡ患者进行ＨＬＡ－ＤＲＢ１基因分型，并与相应人群健康者１０２例结果比较。结果ＨＬＡ－ＤＲ４基因在ＲＡ组显著增高（３５．１％，ＲＲ＝３．５５，Ｐ＜０．００５，ＥＦ＝０．２５２），ＤＲ１６在ＲＡ组也高于正常（ＲＲ＝２．５７，Ｐ＜０．０５）；而ＤＲ９基因在ＲＡ组显著减少（Ｐ＜０．００５）。３１例ＤＲ４＋患者患病年龄较早，病情较重（类风湿因子阳性率和Ⅱ期ＲＡ骨关节Ｘ线改变者显著高于ＤＲ４－患者，Ｐ值分别＜０．０５和０．０２５）。结论广东汉族人ＲＡ易感性与宿主ＤＲ４基因密切相关，ＨＬＡ－ＤＲ４可能是一个对判断病情和预后有价值的实验指标。     

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.     

Increased Expression of Activation-Induced Cytidine Deaminase is Associated with Anti-CCP and Rheumatoid Factor in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is associated with higher levels of autoantibodies and IL-17. Here, we investigated if ectopic lymphoid follicles and peripheral blood mononuclear cells (PBMCs) from RA patients exhibit increased activation-induced cytidine deaminase (AID), and if increased AID is correlated with serum levels of autoantibodies and IL-17. The results of immunohistochemical staining showed that organized AID⁺ germinal centres were observed in six of the 12 RA synovial samples, and AID⁺ cells were found almost exclusively in the B-cell areas of these follicles. Aggregated but not organized lymphoid follicles were found in only one OA synovial sample without AID⁺ cells. Significantly higher levels of AID mRNA ( Aicda ) detected by RT-PCR were found in the PBMCs from RA patients than PBMCs from normal controls ( P  < 0.01). In the PBMCs from RA patients, AID was expressed predominately by the CD10⁺IgM⁺CD20⁺ B-cell population and the percentage of these cells that expressed AID was significantly higher than in normal controls ( P  < 0.01). AID expression in the PBMCs correlated significantly and positively with the serum levels of rheumatoid factor (RF) ( P  ≤ 0.0001) and anti-cyclic citrullinated peptide (CCP) ( P  = 0.0005). Serum levels of IFN-γ ( P  = 0.0005) and IL-17 ( P  = 0.007), but not IL-4, also exhibited positive correlation with the expression of AID. These results suggest that the higher levels of AID expression in B cells of RA patients correlate with, and may be associated with the higher levels of T helper cell cytokines IFN-γ and IL-17, leading to the development of anti-CCP and RF.     

Anti-MBL autoantibodies in patients with rheumatoid arthritis: prevalence and clinical significance

Occurrence of autoantibodies in patients' sera is the characteristic feature of autoimmune disorders. We assessed the presence of anti-mannose binding lectin (MBL) autoantibodies in the sera of 107 rheumatoid arthritis (RA) patients and 121 control subjects by enzyme immunoassay. Elevated levels of anti-MBL autoantibodies in the sera of RA patients (P<0.0001) was detected for the first time. The ratios of anti-MBL positive in RA patients and controls were respectively 60.7% and 1.65%. Experiments were then designed to understand the functional relevance of these autoantibodies. An inverse correlation of anti-MBL autoantibodies with serum MBL levels (P=0.001) and MBL complex activity (P=0.02) was observed without genetic association between MBL polymorphisms and anti-MBL autoantibody secretion. A significant increase (P=0.038) in the level of anti-MBL autoantibodies was observed in 23 synovial fluid samples in comparison to the serum samples. Moreover, the anti-MBL autoantibodies were found to be more often present in the sera of RA patients (60.75% sensitivity, 98.35% specificity and 0.913 area under the ROC curve) in comparison to the IgM and IgG isotypes of rheumatoid factors (RF). Anti-MBL autoantibodies were still positive in 25.23% RA patients when both the RF isotypes were negative. Also, in RA patients, at all stages of disease activity and joint deformity, anti-MBL autoantibodies were more often present than both the RF isotypes. Therefore, the significant presence of anti-MBL autoantibodies enunciates that anti-MBL autoantibodies might have a diagnostic value; however, more studies are needed to confirm the role of anti-MBL autoantibodies in the diagnosis of rheumatoid arthritis.     

Anti‐citrullinated glucose‐6‐phosphate isomerase peptide antibodies in patients with rheumatoid arthritis are associated with HLA‐DRB1 shared epitope alleles and disease activity

To identify and characterize anti‐citrullinated glucose‐6‐phosphate isomerase (GPI) peptide antibodies in patients with rheumatoid arthritis (RA). Nine GPI arginine‐bearing peptides in human GPI protein were selected and cyclic citrullinated GPI peptides (CCG‐1–9) were constructed. Samples were obtained from RA (n = 208), systemic lupus erythematosus (SLE) (n = 101), Sjögren's syndrome (SS; n = 101) and healthy controls (n = 174). Antibodies against CCG‐1–9 were measured, and anti‐citrullinated α‐enolase‐1 (CEP‐1), ‐cyclic citrullinated peptides (CCP) and ‐GPI proteins antibodies were also examined. Patients with RA were genotyped for HLA‐DRB1. The numbers of shared epitope (SE) alleles were counted and compared with those of the autoantibodies. Rabbit GPI was citrullinated with rabbit peptidylarginine deiminase and immunoblot analysis of RA sera performed. The levels of autoantibodies were compared before and after treatment with TNF antagonists in 58 RA patients. Anti‐CCG‐2, ‐4 and ‐7 antibodies were detected in 25·5, 33·2 and 37·0% patients with RA, respectively, and these antibodies were very specific for RA (specificity, 98·1–99·7%). Altogether, 44·2, 86·1 and 13·9% of RA sera were positive for anti‐CEP‐1, ‐CCP and ‐GPI protein antibodies, respectively. Anti‐CCG‐2, ‐4 and ‐7 antibodies were correlated with anti‐CCP and anti‐CEP‐1 antibodies and with the presence of HLA‐DRB1 SE alleles. Citrullinated GPI protein was detected using RA sera. Treatment with tumour necrosis factor antagonists reduced significantly the levels of anti‐CCG‐2 and ‐7 but not of anti‐CEP‐1 antibodies. This is the first report documenting the presence of anti‐CCG antibodies in RA. Anti‐CCG‐2 and ‐7 antibodies could be considered as markers for the diagnosis of RA and its disease activity.     

Prevalence of systemic autoimmune rheumatic diseases and clinical significance of ANA profile: data from a tertiary hospital in Shanghai,China

It is necessary and useful to explore prevalence of various systemic autoimmune rheumatic diseases (SARDs) in patients with suspicion of having SARDs and to characterize antinuclear antibodies (ANA) profile for identifying different populations (SARDs and non‐SARDs). A total of 5024 consecutive patients with available medical records were investigated, whose sera had been tested for ANA profile, including ANA, anti‐dsDNA and anti‐extractable nuclear antigen (ENA) antibodies, between 31 January 2012 and 26 March 2014. Only 594 (11.8%) patients were diagnosed with SARDs of those suspected with SARDs. The prevalence of systemic lupus erythematosus (SLE) was highest (3.2%), followed by rheumatoid arthritis (RA) (2.5%), primary Sjögren's syndrome (pSS) (1.7%), ankylosing spondylitis (AS) (1.5%), etc. Of females, SLE also showed the highest prevalence (6%), while of males, AS showed the highest prevalence (1.9%). The prevalence of most SARDs was closely associated with age, except mixed connective tissue disease (MCTD), and the variation characteristics among different age groups were different among various SARDs. The prevalence of ANA was significantly increased in most SARD patients [especially in SLE, systemic sclerosis (SSc) and MCTD]. For anti‐ENA antibodies, in contrast to some autoantibodies associated with multiple SARDs (e.g. anti‐SSA, SSB, nRNP), others were relatively specific for certain diseases, such as anti‐dsDNA, Sm, histone, nucleosome and Rib‐P for SLE, anti‐SCL‐70 for SSc and anti‐Jo‐1 for polymyositis/dermatomyositis (PM/DM). Of note, ANA profile appeared to be of little significance for AS, ANCA‐associated vasculitis (AAV), polymyalgia rheumatic (PMR), adult‐onset Still's disease (ASD) and Behcet's disease (BD). The younger were more likely to have the presence of anti‐dsDNA, Sm, histone or Rib‐P for SLE, and anti‐SSA for RA or MCTD. No significant differences for frequencies of ANA and anti‐ENA autoantibodies were found between sexes in most SARDs, with the exception of RA and AS. The present study suggests that, of patients with SARDs‐like clinical manifestations, the proportion of those with true SARDS is small, for most of whom tests for autoantibodies are necessary and useful to help make a prompt and precise diagnosis.     

Comparative analysis of autoantibodies targeting peptidylarginine deiminase type 4, mutated citrullinated vimentin and cyclic citrullinated peptides in rheumatoid arthritis: associations with cytokine profiles,clinical and genetic features

Antibodies against cyclic citrullinated peptides (anti‐CCP) are widely used for diagnosis of rheumatoid arthritis (RA). We performed a comparative analysis of antibodies targeting the citrullinating enzyme peptidylarginine deiminase type 4 (anti‐PAD4) and mutated citrullinated vimentin (anti‐MCV) with anti‐CCP autoantibodies in RA patients and examined their relationships with clinical parameters, cytokine profiles and the PADI4 gene. Autoantibodies were examined by enzyme‐linked immunosorbent assay (ELISA) in sera of 170 RA patients and 103 controls. Cytokine profiles were measured using a multiplex system. PADI4 polymorphisms (89G > A, 90T > C and 92G > C) were genotyped by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). Anti‐PAD4, anti‐MCV and anti‐CCP autoantibodies were detected in 24, 61 and 74% of RA patients, respectively. Positive correlations were observed between anti‐PAD4 and disease duration; anti‐CCP and erythrocyte sedimentation rate (ESR); anti‐MCV and ESR and C‐reactive protein. Anti‐MCV antibodies were associated with high disease activity score 28 (DAS‐28) in early RA. Concentrations of T helper type 1 (Th1) [tumour necrosis factor (TNF)‐α, interleukin (IL)‐12, IL‐2, IL‐1β], Th2 (IL‐4, IL‐6, IL‐10, IL‐13) and Th17 (IL‐17) cytokines were higher in RA than in controls. Th2 and, to a lesser extent, Th1‐related cytokines, showed positive correlations with anti‐MCV and anti‐CCP. The GTG haplotype in PADI4 was associated with anti‐CCP and anti‐MCV, but not anti‐PAD4 antibodies. In conclusion, anti‐PAD4 antibodies are detected mainly in established RA, which is in contrast to the early detection of antibodies against citrullinated peptide/proteins (ACPAs). Among autoantibodies, anti‐MCV appear to perform better as markers of disease activity. Furthermore, anti‐CCP and anti‐MCV are associated genetically with the citrullinating enzyme PAD4 and are related strongly to Th1 and Th2 cytokines, suggesting a feed‐forward loop between cytokines and ACPA production.     

核不均一性胞核核糖核蛋白Ⅰ(hnRNPI)抗原表位在系统性硬化症中临床意义的研究

目的探讨核不均一性胞核核糖核蛋白I(hnRNPI)抗原表位多肽在系统性硬化症(systemic sclerosis,SSc)中的临床意义,初步建立简便快捷的ELISA检测方法,为系统性硬化症的早期诊断寻找新的临床指标。方法根据已知的hnRNPI蛋白的氨基酸序列,应用不同的蛋白质抗原表位图谱分析软件对其进行表位分析,经比对筛选后,化学合成hnRNPI短肽序列2个,分别命名为I-1_(264-292)及I-2_(441-461),作为抗原对临床上包括硬皮病在内的多种结缔组织病患者血清相应抗体进行ELISA检测,包括SSc 42例、系统性红斑狼疮(SLE)102例、干燥综合征(SS)26例、混合型结缔组织病(MCTD)16例、未分化结缔组织病(UCTD)13例,其他结缔组织病(CTD)30例、类风湿关节炎(RA)26例及正常对照54例。结果抗hnRNPI-1及抗hnRNPI-2多肽抗体在SSc组中阳性率均明显高于其他疾病组(P<0.05),在SSc中敏感性分别为47.62%及38.1%,特异性分别为93.43%及91.08%,二者之间差异无统计学意义(P>0.05)。另外,除了与SSc患者病程相关外,该二抗体均与发病年龄、临床症状、器官受累、ESR、抗Scl-70抗体、抗着丝点抗体及抗核仁抗体之间未发现有统计学意义的相关性(P>0.05)。结论I-1及I-2分别是位于hnRNPI蛋白表面的抗原表位之一,具有抗原性,其抗体对SSc的临床诊断具有较高的敏感性及特异性,且在病程早期具有更高的阳性率,有助于SSc的早期诊断。