Heterogeneous Nuclear Ribonucleoprotein A2, ¢ñ (hnrnpa2, ¢ñ,) And Chemokine Receptor 3 (of Cxcr3) In Rheumatic Diseases In Clinical Research

Clinical research of Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 in RA and comparison with import Anti-RA33 antibody reagent boxBackgroudHeterogeneous nuclear ribonucleoprotein is the important nucleic acid-binding protein in RNA processing. In addition, increasing evidence shows that hnRNP is an important target of the autoimmune response in rheumatic diseases and anti-hnRNP autoantibodies has specificity in rheumatic diseases. In fact, anti-hnRNP autoantibody in RA mainly directs to the hnRNP A2 protein, which is also termed as anti-RA33 antibody. Anti-RA33/hnRNP A2 antibody is not only valuable diagnostic marker but may also allow additional insights into the pathogenesis of rheumatic diseases.Objective and MethodsIn this study, the RA33 antigen was partially purified from Ehrlich ascites carcinoma cell nuclear extracts by affinity chromatography on heparin sepharose CL-6B. The sera from different patients including 137 patients with Reumatoid Arthritis(RA), 48 patients with Systemic LupusErythematosus(SLE), 39 patients with Sjogren's Syndrome(pSS), 24 patients with Mixed Connective Tissue Disease (MCTD) , 25 patients with Systemic Sclerosis(SSc), 28 patients with seronegative spondyloarthropathies(SPA), 42 other diffused rheumatic diseases, and 40 healthy controls were detected by ELISA with the purified recombinant hnRNP A2 protein. To verify the reliability of self-made reagent box by comparing with the result detected by the imported Anti-RA33 antibody reagent box. Clinical characters and laboratory tests are evaluated between anti-hnRNP A2/RA33 antibody positive and negative RA patients to study the significance of the antibody in RA.ResultsIt shows that the sensitivity and specificity of anti-hnRNP A2/RA33 in RA is 37.23% and 85.44%. The positivity of anti-hnRNP A2/RA33 in SLE, SS, MCTD, SSc, SPA, other CTD, is 20.83%,20.51%,25%,8%,3.57%,7.14% respectively. The positivity of anti-hnRNP A2/RA33 in RA is obviously higher than that of other diseases(p＜0.05).The positivity of anti-hnRNP A2/RA33 antibody is 43.75% in early RA patients. The positivity of anti-hnRNP A2/RA33 antibody in RA patients detected by self-made hnRNP A2 protein has no significant difference in comparison with that detected by imported anti-RA33 antibody reagent box, and the total coincident rates of two testing methods is 86.88% in patients of rheumatic diseases. In addition, the studies show that age, disease duration, morning stiffness, erythrocyte sedimentation rate, C-reactive protein, joint function, X-ray classification, RF titer, APF, AKA, and anti-CCP have no significant difference between positive and negative anti-hnRNP A2/RA33 antibody RA patients group(p＞0.05).ConclusionIn a word, we extracted and partial-purified RA33 antigen and hnRNP A2 had been cloned, expressed and purified. HnRNP A2 protein detected by ELISA is a reliable method for early diagnosis of RA. Compared with imported anti-RA33 antibody reagent box, the two detecting results have no significant difference. It shows that self-made anti-RA33 antibody reagent box can be reliably used in clinical test. Clinical research of Heterogeneous nuclear ribonucleoprotein (hnRNP)Ⅰin SScBackgroudHeterogeneous nuclear ribonucleoprotein (hnRNP) is one of the most important nucleic acid binding proteins in RNA processing and it also acts as important targets of the autoimmune response in rheumatic diseases. It has been reported in 1990s that hnRNPⅠprotein, as one member of hnRNPs, played an important role in the muture and splice process of mRNA. The first evidence of human autoantibodies directed to the hnRNP I has been proved in 1996 and it was suggested anti-hnRNPⅠautoantibodies were associated with the clinical features of systemic sclerosis(SSc). SSc is an autoimmune disease characterized by a high frequency of circulating autoantibodies directed to a variety of cellular components. Main autoantibodies in SSc sera include antibodies against centromere and DNA topoisomerase I. Anti-topo I antibody is mainly detected in diffused SSc and has a low positivity in it , whereas anticentromere antibody(ACA) not only identifies a subset of limited SSc(CREST syndrome), but also has positive immunoreactivity in other inflammatory diseases. It seems necessary to find out more sensitive and specific autoantibody present in SSc. ObjectiveTo assess the presence of autoantibodies directed against the epitopes of hnRNPⅠin SSc as well as other CTDs and analyze the clinical significance of them.MethodsTwo polypeptides synthesized by the biologic technical software based on the animo acids sequence of hnRNPⅠ,and these were named as I-1 and I-2 respectively. The sera from different patients including 113 SSc, 45 SLE, 37 pSS, 20 MCTD, 50 RA, 28 SPA, 30 other diffused rheumatic diseases, and 54 controls were detected by ELISA with the synthetic polypeptides.ResultsThe positivity of anti-I1 and anti-I2 were higher in SSc than in other groups(p＜0.05).The sensitivity and specificity of anti-I1 in SSc were 43.4% and 88.6%, and the sensitivity and specificity of anti-I2 in SSc were 47.8% and 87.6%. There were no significant difference between the two peptides(p＞0.05). Clinical characters and laboratory indexes were also compared to assess the clinical significance of the antibodies in SSc. It showed that age, erythrocyte sedimentation rate, digestive tract involvement, pulmonary involvement had no significant difference between positive and negative anti-I antibody groups in SSc patients (P＞0.05). But the positive groups had obviously shorter disease duration compared with negative groups (p＜0.05) . There was no statistically significant association between anti-I antibody and ANA, ACA, anti-Scl-70 in SSc patients(p＞0.05).ConclusionIt was showed by ELISA that the biologic technical software was helpful to design the epitopes of different protein at a certain extent. Polypeptides of I-1_264-292 and I-2(441-461) had clinical significance in SSc, particularly in the early stage of the disease duration, suggesting an early apprarance of these disease antibodies. Clinical research of chemokine receptor 3 in Rheumatoid ArthritisBackgroundRheumatoid arthritis (RA) is a systemic autoimmune disease. It is characterized by joint inflammation and synoviocyte proliferation, which always lead to joint destruction eventually. Chemokines are a series of inducible secretary cytokines which play an important role in local infiltration of inflammatory cells in RA and are closely associated with synovitis, tissue damage, neovascularization. All of these biological effects are performed by binding to receptors. Elsewhere, chemokine receptor 3 (CXCR3) is a marked chemokine receptor in RA.ObjectiveTo detect the expression of CXCR3 mRNA in peripheral blood mononuclear cells (PBMC) in the patients with active RA patients, relieved RA patients, other diffused rheumatic diseases patients and healthy control cases, respectively. Their clinical data were compared to explore the role of chemokine in the pathogenesis and progression of RA, and to investigate the relation between serum level of chemokine and common clinical indices of RA, to evaluate the possibility of these chemokine used clinically as a referrible index to assess the activity degrees and prognosis in patient with RA. MethodsWe selected continuously 51 patients of rheumatoid arthritis during November 2006 to March 2007. According to respective inclusive and exclusive standards, they were divided into four groups:28 patients in active rheumatoid arthritis group, 23 healthy controls in relieved RA group, 29 patients in other CTD group, and 32 patients in Healthy control group. The primers were designed which were specific for the mRNA of CXCR3. Then the mRNA expressions of CXCR3 mRNA in PBMC of 113 patients were detected respectively by using real- time fluorescence quantitative polymerase chain reaction (RFQ- PCR).ResultsThere was significant difference in expression levels of CXCR3 mRNA among three groups(P＜0.05).Comparison between each two groups displayed that expression levels of CXCR3 mRNA, in clinic active RA group, were higher than those of relieved RA patients, other diffused rheumatic diseases patients and healthy control cases (P＜0.05). Expression levels of CXCR3 mRNA had no significant difference among relieved RA patients, other diffused rheumatic diseases patients andhealthy control cases (P＞0.05). Expression levels of CXCR3 mRNA have high positive correlation with serum levels of ESR and CRP in clinic active RA group (P＜0.05). In addition, RF titer, APF, AKA, and anti-CCP have no significant correlation with expression levels of CXCR3 mRNA in RA patients (p＞0.05).ConclusionsThis assay is sensitive, reproducible and suitable for clinical practice. The mRNA expressions of CXCR3 were significantly elevated in RA patients, which suggests that CXCR3 might be involved in the pathogenesis of RA. The mRNA expressions of CXCR3 in active RA patients were higher than those of relieved RA patients. It infers that CXCR3 plays an important role during pathogenesis and progression of RA, and CXCR3 maybe considered as an index of the activity, therapeutic effects and prognosis of RA.