EZH2 deficiency attenuates Treg differentiation in rheumatoid arthritis.

The chromatin modifier enhancer of zeste homolog 2 (EZH2) methylates lysine 27 of histone H3 (H3K27) and regulates T cell differentiation. However, the potential role of EZH2 in the pathogenesis of rheumatoid arthritis (RA) remains elusive. We analyzed EZH2 expression in PBMC, CD4+ T cells, CD19+ B cell, and CD14+ monocytes from active treatment-naïve RA patients and healthy controls (HC). We also suppressed EZH2 expression using EZH2 inhibitor GSK126 and measured CD4+ T cell differentiation, proliferation and apoptosis. We further examined TGFß-SMAD and RUNX1 signaling pathways in EZH2-suppressed CD4+ T cells. Finally, we explored the regulation mechanism of EZH2 by RA synovial fluid and fibroblast-like synoviocyte (FLS) by neutralizing key proinflammatory cytokines. EZH2 expression is lower in PBMC and CD4+ T cells from RA patients than those from HC. EZH2 inhibition suppressed regulatory T cells (Tregs) differentiation and FOXP3 transcription, and downregulated RUNX1 and upregulated SMAD7 expression in CD4+ T cells. RA synovial fluid and fibroblast-like synoviocytes suppressed EZH2 expression in CD4+ T cells, which was partially neutralized by anti-IL17 antibody. Taken together, EZH2 in CD4+ T cells from RA patients was attenuated, which suppressed FOXP3 transcription through downregulating RUNX1 and upregulating SMAD7 in CD4+ T cells, and ultimately suppressed Tregs differentiation. IL17 in RA synovial fluid might promote downregulation of EZH2 in CD4+ T cells. Defective EZH2 in CD4+ T cells might contribute to Treg deficiency in RA.

Good outcome of severe lupus patients with high-dose immunosuppressive therapy and autologous peripheral blood stem cell transplantation: a 10-year follow-up study.

OBJECTIVES: This study aimed to examine the long-term efficacy, remission and survival of patients with severe systemic lupus erythematosus (SLE) after the combination treatment with high-dose immunosuppressive therapy (HDIT) and autologous peripheral blood stem cell transplantation (APBSCT). METHODS: Chinese patients with severe SLE receiving combination therapy with HDIT and APBSCT in Peking Union Medical College Hospital were enrolled from July 1999 to October 2005. Disease activity, treatment, and adverse effects of these patients were evaluated. The 10-year overall survival and 10-year remission survival were also analysed. RESULTS: Among the 27 patients, one patient failed to collect enough CD34+ cells and data was missing for two patients. In the end, 24 patients were included in the final analysis. After APBSCT, one patient died, two patients achieved partial remission and 21 (87.5%) achieved remission at 6 months. The median follow-up duration of the 23 patients was 120 months. Fourteen patients had completed a ten-year follow-up. The median proteinuria level of the 14 patients with LN with ten years of follow-up significantly decreased from 4.00 g/24 hours at pre-treatment to 0.00g/24 hours at year 5 and 0.00 g/24 hours at year 10 (both p=0.001). The 10-year overall survival rate and 10-year remission survival rate were both 86.0% (95% CI: 71.1-100.9%). After a median follow-up for 120 months, 16 patients (66.7%) remained in remission, 4 patients were lost to follow-up, 2 patients died and 1 patient remained active. CONCLUSIONS: The combination of HDIT and APBSCT may be an option to improve the survival of severe lupus patients.

Comparison of Tripterygium wilfordii Hook F with methotrexate in the treatment of active rheumatoid arthritis (TRIFRA): a randomised, controlled clinical trial.

OBJECTIVES: To compare the efficacy and safety of Tripterygium wilfordii Hook F (TwHF) with methotrexate (MTX) in the treatment of active rheumatoid arthritis (RA). METHODS: Design: a multicentre, open-label, randomised controlled trial. All patients were assessed by trained investigators who were unaware of the therapeutic regimen. INTERVENTION: 207 patients with active RA were randomly allocated (1:1:1) to treatment with MTX 12.5 mg once a week, or TwHF 20 mg three times a day, or the two in combination. At week 12, if reduction of the 28-joint count Disease Activity Score (DAS28) was <30% in the monotherapy groups, the patient was switched to MTX+TwHF. The primary efficacy point was the proportion of patients achieving an American College of Rheumatology (ACR) 50 response at week 24. RESULTS: 174/207 (84.1%) patients completed 24 weeks of the trial. In an intention-to-treat analysis, the proportion of patients reaching the ACR50 response criteria was 46.4% (32/69), 55.1% (38/69) and 76.8% (53/69), respectively, in the MTX, TwHF and MTX+TwHF groups (TwHF vs MTX monotherapy, p=0.014; MTX+TwHF vs MTX monotherapy, p<0.001). Similar statistically significant patterns at week 24 were found for ACR20, ACR70, clinical Disease Activity Index good responses, EULAR good response, remission rate and low disease activity rate. Significant improvement in the Health Assessment Questionnaire and 36-item Short-Form Health Survey questionnaire scores from baseline to week 24 was seen in each treatment arm (p<0.05), though no significant difference was found among the treatment arms (p>0.05). The result of per-protocol analysis agreed with that seen in the intention-to-treat analysis. Seven, three and five women in the TwHF, MTX and combination groups, respectively, developed irregular menstruation (TwHF vs MTX monotherapy, p=0.216). CONCLUSIONS: TwHF monotherapy was not inferior to, and MTX+TwHF was better than, MTX monotherapy in controlling disease activity in patients with active RA. TRIAL REGISTRATION NUMBER: NCT01613079.

Induction of endothelial cell apoptosis by anti-alpha-enolase antibody.

OBJECTIVE: To assess the prevalence of anti-alpha-enolase antibody in systemic autoimmune diseases in Chinese patients and its role in endothelial cell apoptosis. METHODS: The reactivity of anti-alpha-enolase antibody in a variety of autoimmune disorders in Chinese patients was evaluated by dot blot assay. Endothelial cell apoptosis was investigated by in vitro incubation of endothelial cells with IgG purified from anti-alpha-enolase antibody-positive sera, with or without pre-incubation with recombinant alpha-enolase. RESULTS: Anti-alpha-enolase antibody was prevalent in different systemic autoimmune diseases with relatively high reactivity in Chinese patients. In vitro incubation of endothelial cells with IgG containing anti-alpha-enolase antibody induced apoptosis in a time- and dose-dependent manner. Apoptosis was partly inhibited by pre-incubation of the endothelial cells with recombinant alpha-enolase. CONCLUSIONS: Our data suggest that alpha-enolase is a common auto-antigen recognized by anti-endothelial cell antibodies in connective tissue disease. Interaction between alpha-enolase and its autoantibody plays a role in endothelial cell apoptosis. Changes other than cell killing may contribute to the pathogenesis of endothelial damage and microvascular lesions.

The safety and effectiveness of a chloroform/methanol extract of Tripterygium wilfordii Hook F (T2) plus methotrexate in treating rheumatoid arthritis.

OBJECTIVES: The objective of the study was to assess the safety and effectiveness of the chloroform/methanol extract of Tripterygium wilfordii Hook F (T2) plus methotrexate (MTX) in treating patients with rheumatoid arthritis (RA). METHODS: One hundred sixty-six patients with RA, who started the combination therapy of T2 (20 mg b.i.d. or t.i.d.) and MTX (10-12.5 mg/wk), were enrolled, and these patients were followed up for at least 1 year. Demographics, disease severity, markers of disease activity before and after the combination therapy, and incidence of adverse events were evaluated. RESULTS: The patients were predominantly female (n = 134, 81%) with a mean age of 58.0 (SD, 7.9) years (range, 39-79 years) and a mean disease duration of 55.0 (SD, 72.2) months (range, 0-456 months). A total of 161, 161, 146, and 85 patients had received at least 1, 3, 12, and 24 months of the combination of T2 and MTX, with a total of 4162 patient-months' exposure to the combination therapy. The combination therapy reduced tender and swollen joint counts, morning stiffness, inflammatory indices such as ESR and CRP, and improved disease activity as measured by the DAS28 significantly by 3 months as well as 12 months (P < 0.05). Most of the adverse events noted during this study were mild. Menstrual irregularity occurred in 72.7% (16/22) of premenopausal female. Only 10 (6.0%) and 8 (4.8%) subjects withdrew because of adverse events or lack of efficacy, respectively. Severe infections were very rare. CONCLUSION: T2 plus MTX is an effective and relatively safe treatment for RA patients.

[A study of T cell recombination excision circles levels in peripheral blood mononuclear cells of systemic lupus erythematosus patients].

OBJECTIVE: To compare the T cell receptor recombination excision cycle (TREC) levels in peripheral blood mononuclear cells (PBMC) of systemic lupus erythematosus (SLE) patients with normal age- and gender- matched controls. To investigate the correlations between TREC levels of SLE patients and their clinical features. METHODS: We studied TREC levels in peripheral blood mononuclear cells (PBMC) of 21 SLE patients and 22 normal age- and sex-matched controls. TREC concentration was determined by real-time quantitative polymerase chain reaction (real-time qPCR) as the number of TREC copies/1000 PBMCs. The clinical features of the SLE patients such as systemic lupus erythematosus disease activity index (SLEDAI), ESR, C reaction protein (CRP), ANA, anti-dsDNA and complement levels and organ involvement were recorded and assessed. RESULTS: SLE patients had lower TREC levels [(9.6±7.5) copies/1000 PBMC] than controls [(16.1±11.1) copies/1000 PBMC, P=0.033]. There was an inverse correlation between age and TREC levels in controls (r=-0.614, P=0.002) but not in SLE patients. There was an inverse correlation between SLEDAI and TREC levels in SLE patients (r=-0.656, P=0.001) and TREC levels seemed to have relations to skin lesions (r=-0.620, P=0.003). No other clinical association was observed between TREC levels and clinical and laboratory SLE manifestations. CONCLUSION: SLE patients had lower TREC levels than normal controls and there is a tendency that TREC level is reversely correlated with disease activity. The decrease PBMC TREC level is indicative of a low proportion of recent thymic emigrant (RTE) in SLE and could be caused by decreased RTE output and/or by increased peripheral T cell proliferation in this disease. The under-representation of RTE in the peripheral T cell pool may play a role in the immune tolerance abnormalities observed in SLE.

[The soluble expression and identification of single-chain fragment V antibodies against SSA antigen epitopes from the pHEN2 phagemid library].

OBJECTIVE: To obtain the soluble single-chain fragment V (ScFv) monoclonal antibodies (McAbs) against the SSA antigen epitopes. METHODS: Three octapeptides (60000 SSA antigen residues 482-493 termed as P1 epitope, residues 310-323 termed as P2 epitope and residues 230-241 termed as P3 epitope) were synthesized on the lysine frame. The McAbs were panned by coating the corresponding as targets. The specificity, affinity and gene sequences of the positive clones were assessed. Soluble single-chain fragment V antibodies special for SSA antigen epitopes were expressed and then identified. RESULTS: After 5 rounds of panning, reactive scFv clones contained full-length scFv antibodies coding regions were obtained, with sufficient affinity and specificity for respective antigen peptides. The absorbance values at 410 nm of the fusion protein of anti-P1-P3 activity with the corresponding peptides were 1.43±0.23, 0.82±0.31 and 0.80±0.25, and there was also statistically significant difference in the cross reactions (P<0.01). Three clones were successfully expressed and then purified by His-bind resin. The activity in vivo of soluble ScFv antibodies was identified to be positive by the indirect immune-fluorescence assay on Hep-2 cells. CONCLUSION: Soluble ScFv McAbs against corresponding SSA antigen peptides with high affinity, specificity and activity in vivo were obtained, which are to be competent enough for epitopes expression on the target organs.

[Expression of SSA antigen epitopes in minor salivary glands from patients with primary Sjögren's syndrome].

OBJECTIVE: To investigate the correlation between the differential expression of 60 000 SSA epitopes in minor salivary glands (MSG) from patients with primary Sjögren's syndrome (pSS) and glandular inflammation. METHODS: The screening and soluble expression of single-chain fragment V monoclonal antibodies (Scfv McAb) against SSA antigen epitopes (P1: 480 - 494, P2: 310 - 323 and P3: 230 - 241) were performed by phagemid antibody expression system. The expression of epitopes was detected by immunohistochemical assay (IH) in minor salivary glands from these patients. The correlation between epitopes expression and glandular inflammation was analyzed quantitatively. RESULTS: Immunohistochemical assay of MSG with McAb against P1-P3 epitopes showed that the epithelial cells of glandular tubes and striated duct were stained in membrane and cytoplasm. The expression of P1 epitope was higher than the other two in grading score (chi(2) = 12.94, P < 0.01). And a positive correlation was found between the extent of glandular infiltration and the grade of P1 epitope expression (t = 2.27, P < 0.05) but not with P2 or P3 epitope. CONCLUSION: Aberrant redistribution of intracellular SSA antigen epitopes onto the cell membrane of involved cells may break the immune tolerance and thus induce the production of pathogenic autoantibodies involved in triggering and maintaining the tissue-specific autoimmune response in pSS MSG. A significantly high membranous expression of P1 and a positive correlation between P1 and the inflammation of MSG suggest that P1 may be the dominant determinant of the antigen-driven immune response in pSS.

[Analysis of risk factors for reversible posterior leukoencephalopathy syndrome in lupus].

OBJECTIVE: To analyze the different risk factors for reversible posterior leukoencephalopathy syndrome (RPLS) in systemic lupus erythematosus (SLE). METHODS: The clinical and imaging characteristics of 2 cases were described in details. The literature reports for 33 cases of SLE and RPLS were reviewed systematically. The etiologies of SLE and RPLS were analyzed for all 35 cases. RESULTS: Thirty-five cases of SLE patients were complicated with RPLS, 32 cases suffered hypertension, 3 cases had no hypertension, 1 case was explicitly related with rituximab and 1 case was related with cyclosporine therapy. CONCLUSION: SLE complicated by RPLS has different causes, such as hypertension, SLE involving central nervous system and drug neurotoxicity.

[Characteristics of patients with primary Sjögren's syndrome and non-Hodgkin's lymphoma: analysis of 9 cases].

OBJECTIVE: To characterize the clinical patterns of expression, laboratory serologic parameters and lymphomatous histological characteristics in patients with primary Sjögren's syndrome (pSS) who subsequently developed non-Hodgkin's lymphoma (NHL). METHODS: The authors analyzed 9 pSS patients (8 females, 1 male) who developed NHL. Five patients had received glucocorticoids, four of whom had received at least one immunosuppressive drugs (methotrexate, glucosidorum tripterygll totorum, cyclophosphamide and imuran). A protocol form was used to record the main characteristics of pSS and NHL. RESULTS: Eight patients fulfilled the American-European Consensus Criteria (AECC). The main SS manifestations were painless parotid enlargement (n = 7), six of whom were unilateral; the main immunologic features were positive rheumatoid factor (RF) in all examined patients and hyperimmunoglobulinemia (n = 7). The main manifestations of NHL were splenomegaly (n = 7) and lymphadenopathy (n = 5). The main histological subtypes were mucosa-associated lymphoid tissue (MALT) lymphoma (n = 4) and diffuse large B cell lymphoma (n = 2). None of the patients with MALT lymphoma had a nodal primary location. Eight patients had an extranodal primary location, most frequently in salivary gland (n = 4) and lung (n = 4). CONCLUSION: Patients with pSS and NHL are clinically characterized by a high frequency of painless unilateral parotid enlargement, splenomegaly, lymphadenopathy, an immunologic pattern dominated by the presence of high-titer RF and hyperimmunoglobulinemia, a predominance of MALT lymphomas and an elevated frequency of primary extranodal involvement.

Comparison of the impact of Tripterygium wilfordii Hook F and Methotrexate treatment on radiological progression in active rheumatoid arthritis: 2-year follow up of a randomized, non-blinded, controlled study.

BACKGROUND: Tripterygium wilfordii Hook F (TwHF) alone or in combination with methotrexate (MTX) has been shown to be more effective than MTX monotherapy in controlling the manifestations in subjects with disease-modifying antirheumatic drug (DMARD)-naïve active rheumatoid arthritis (RA) over a 6-month period. The long-term impact of these therapies on disease activity and radiographic progression in RA has not been examined. METHODS: Patients with DMARD-naïve RA enrolled in the "Comparison of Tripterygium wilfordii Hook F with methotrexate in the Treatment of Active Rheumatoid Arthritis" (TRIFRA) study were randomly allocated into three arms with TwHF or MTX or the two in combination. Clinical indexes and radiographic data at baseline and year 2 was collected and compared using an intent-to-treat (ITT) and a per-protocol (PP) analysis. Two radiologists blinded to the treatment scored the images independently. RESULTS: Of 207 subjects 109 completed the 2-year follow up. The number of subjects withdrawing from the study and the number adhering to the initial regimens were similar among the three groups (p > = 0.05). In the ITT analysis, proportions of patients reaching American College of Rheumatology 50% (ACR50) response criteria were 46.4%, 58.0% and 50.7% in the MTX, TwHF and MTX + TwHF groups (TwHF vs MTX monotherapy, p = 0.004). Similar patterns were found in ACR20, ACR70, Clinical Disease Activity Index good responses, European League Against Rheumatism good response, remission rate and low disease activity rate at year 2. The results of the PP analysis agreed with those in the ITT analysis. The changes in total Sharp scores and joint erosion and joint space narrowing during the 2 years were associated with changes in disease activity measured by the 28-joint count Disease Activity Score and were comparable among the three groups (p > 0.05). Adverse events were similar in the three treatment groups. CONCLUSIONS: During the 2-year therapy period, TwHF monotherapy was not inferior to MTX monotherapy in controlling disease activity and retarding radiological progression in patients with active RA. TRIAL REGISTRATION: This is a follow-up study. Original trial registration: ClinicalTrials.gov , NCT01613079 . Registered on 4 June 2012.

Peripheral blood CD34+ cell mobilization in 42 patients with severe autoimmune disease.

OBJECTIVE: To evaluate the feasibility and safety of peripheral CD34+ cell mobilization in patients with severe autoimmune disease. METHODS: Forty-two patients underwent a total of 46 mobilizations by the regimen of cyclophosphamide 2-3 g/m2+ recombinant human granulocyte colony stimulating factor (rhG-CSF) 5 microg x kg(-1) x d(-1). The positive selection of CD34+ cell was performed through the CliniMACS. RESULTS: In 8.1 +/- 2. 3 days after administration of cyclophosphamide, the peripheral white blood cell and mononuclear cell (MNC) decreased to the lowest level. In 3.7 +/- 1.6 days after injection of rhG-CSF, the peripheral absolute MNC and CD34+ cell counts were 0.95 x 10(9)/L and 0.035 x 10(9)/L, respectively. After 2.4 +/- 0.6 times of leukapheresis, there gained 4.46 x 10(8)/kg of MNC and 5.26 x 10(6)/kg of CD34+, respectively. After mobilization, the underlying diseases were ameliorated more or less. In systemic lupus erythematosus (SLE) patients, SLE Disease Activity Index (SLEDAI) decreased from a median of 17 to 3 (P < 0.01). In rheumatic arthritis patients, an American College of Rheumatology criteria for 20% (ACR20) response was achieved in all five patients. Totally, 17.4% of patients whose absolute neutrophil count < 0.5 x 10(9)/L suffered infection, and 31.0% of patients had bone pain after the injection of rhG-CSF. Two patients suffered severe complications, one with acute renal failure and recovered by hemodialysis, the other died of thrombotic thrombocytopenic purpura. Failed mobilization occurred in three patients. CONCLUSIONS: Sufficient CD34+ cells can be mobilized by low dose of cyclophosphamide and rhG-CSF. CD34+ cell mobilization for treatment of severe autoimmune disease not only is appropriate in both effectiveness and safety but ameliorates disease also.

[Clinical analysis of 29 patients with clinically amyopathic dermatomyositis].

OBJECTIVE: To explore the clinical features and prognosis of clinically amyopathic dermatomyositis (C-ADM). METHODS: The clinical data, including skin lesion, muscle involvement, and lung disease, of 29 patients with C-ADM, 6 males and 23 females with a male/female ratio of 1:3.83, aged 44 +/- 8, were analyzed. Skin biopsy was taken in 5 patients and lung HRCT was done in all the patients. RESULTS: The mean age at onset was (44 +/- 8) years. All patients presented with characteristic skin lesions such as Gottron's papulae (91.3%); heliotropic periorbital erythremia (75.9%); V-sign rash (37.9%); shawl-sign rash (24.1%); and periungual erythema and telangiectasias (20.7%). Gottron's papule was most commonly seen on the dorsal aspect of elbow (86.2%), proximal interphalangeal joints (58.6%), metacarpophalangeal joints (48.3%), patellae (23.8%), hip (20.7%), shoulder (13.8%), and ankle (9.5%). The cutaneous histopathologic pictures of these patients were all compatible with the skin lesions of dermatomyositis. Interstitial lung disease (ILD) was found by lung HRCT in 19 patients (65.5%). All patients received steroids combined with immunosuppressants, but rapidly progressive ILD happened to some of the patients and finally led to death due to respiratory failure in 5 of them. CONCLUSION: C-ADM is most commonly seen in the middle-aged women. ILD is the commonest respiratory problem arising in C-ADM patients and can be fatal, therefore should be properly treated.

[A prospective and randomized study of two conditioning regimens in treatment of severe systemic autoimmune diseases with autologous peripheral blood stem cell transplantation].

OBJECTIVE: To investigate the differences in immune reconstitution, hematopoietic reconstitution, efficacy, and complication between the two conditioning regimens with or without total body irradiation (TBI) in patients with refractory and severe autoimmune diseases (AID) who receiving autologous peripheral blood stem cell transplantation (APBSCT). METHODS: Thirty-two AID patients, 5 males and 27 females, aged 29 (15 - 49), underwent APBSCT. The CD34(+) cells were mobilized with cytoxan (CTX) + granulocyte-colony stimulating factor (G-CSF) and selected by clinical magnetic activated cell sorting (CliniMACS). The conditioning regimen included CTX + antithymocyte globulin (ATG) in 11 patients and CTX + TBI in 21 patients. All the patients were followed up for more than 12 months. RESULTS: The median time of granulocyte recovery were 11 and 9 days in the CTX + TBI and CTX + ATG groups respectively (P = 0.003), the median time of platelet recovery were 13 and 8 days respectively (P = 0.001). In both groups, the lymphocyte subsets were recovered with the inverted CD4/CD8 ratio 12 months after transplantation. Relapse was seen in 3 cases of the CTX + TBI group (14.3%), and 2 cases of the CTX + ATG group (18.2%), and the rest of patients remained free of AID. During transplantation incidence of bacteria infection occurred in 5 of the 21 cases in the CTX + TBI group (23.8%) and in 2 of the 11 cases of the CTX + ATG group (18.2%) respectively; viral infection occurred in 1 of the 21 cases of the CTX + TBI group (4.8%) and in 2 of the 11 cases of the CTX + ATG group (18.2%) respectively. The number of radiated parotitis was 4 among the 21 patients of the CTX + TBI group (19%) and was 3 among the 12 patients of the CTX + ATG group (25%). Serum sickness reaction occurred in 3 of the 12 patients of the CTX + ATG group (25%). Bacterial and viral infections were cured soon after antibacterial or antiviral therapy, no fatal bleeding occurred due to thrombocytopenia in both groups. CONCLUSION: The conditioning regimen of TBI + CTX delays the hematopoietic reconstitution compared with the ATG + CTX regimen in treating AID. The regimen of CTX + TBI can be better tolerated, but there are no significant differences in efficacy and immune reconstitution among these two regimens.

[Changes of lymphocyte subsets in autologous hemopoietic stem cell transplantation for severe/refractory autoimmune disease].

OBJECTIVE: To investigate the dynamic changes of lymphocyte subsets before and after autologous hemopoietic stem cell transplantation (HSCT) in severe/refractory autoimmune disease (AID) and study the post-transplantation immunological reconstitution in AID. METHODS: Thirteen patients with severe/refractory AID who registered for HSCT from April 2003 to April 2005 in Peking Union Medical College Hospital, including 8 patients with systemic lupus erythematosus, 4 patients with rheumatoid arthritis, and 1 patient with primary Sjögren's syndrome (pSS) were enrolled in this study. Blood samples were collected before/after mobilization, before conditioning, and 2 weeks, 1 month, 3 months, 6 months, 12 months, and 18 months post-transplantation. Lymphocyte subsets were tested by flow cytometry as follows: T cell (CD3 +), B cell (CD19 +), natural killer (CD3-CD16 + CD56 +), Th (CD3 + CD4 +), Tc (CD3 + CD8 +), naïve T (CD4 + CD45RA), memory T (CD4 + CD45RO), and CD4/CD8 ratio. RESULTS: Lymphocyte subsets for SLE patients were severely abnormal compared to normal or RA patients (both P < 0.01). B cell reconstituted to normal level within 18 months, meanwhile NK and T cell remained low. The repopulations of Th and naive T cell were delayed, which caused the up-side-down of CD4/CD8 ratio and low level of naYve T cell percentage for a relatively long time. CONCLUSIONS: Lymphocyte subsets abnormality in SLE patients are more severe than in RA patients. Although most autoimmune T/B cell in the grafts and patients can be effectively removed after transplantation, nonmyeloablative conditioning may be a risk for the relapse of AID. The long-term inhibition of CD4 + T cell may be related with the relief of AID after transplantation.

Contribution and underlying mechanisms of CXCR4 overexpression in patients with systemic lupus erythematosus.

Aberrant expression of CXCR4 has been indicated to play a role in the pathogenesis of systemic lupus erythematosus (SLE), but the mechanism of CXCR4 dysregulation in SLE is unclear. This study is aimed to explore the clinical significance and possible mechanisms of abnormal CXCR4 expression on B cells from patients with untreated SLE. Expression of CXCR4 on peripheral B cells was determined by flow cytometry and western blotting. Freshly isolated B cells were cultured with exogenous interleukin 21(IL-21) in the presence or absence of CD40 ligand (CD40L) plus anti-IgM antibody (aIgM), and changes in CXCR4 expression were detected. Involvement of phosphatidylinositol 3 kinase (PI3K)/Akt and Janus kinase/Signal transducer and activator of transcription (JAK/STAT) signaling pathways was assessed by adding blocking agents Ly294002 and AG490. Since CD63 is reported to mediate endosomal recruitment of CXCR4 and BCL6 is capable of silencing CD63 gene transcription, we also measured BCL6 and CD63 gene transcription with real-time PCR. It was shown that CXCR4 expression on B cells was significantly upregulated in SLE patients, especially in those with lupus nephritis, and was positively correlated with SLE Disease Activity Index scores and negatively with the serum complement 3 levels (P<0.05). Downregulation of CXCR4 by IL-21 was intact. In contrast, a similar effect of aIgM plus CD40L in downregulating CXCR4 expression was defective in SLE patients but was restored by co-stimulation with IL-21 in vitro. Both Ly294002 and AG490 promoted downregulation of surface CXCR4 expression on B cells from SLE patients (P=0.078 and P=0.064). Furthermore, B cells from SLE patients exhibited diminished CD63 mRNA and enhanced BCL6 mRNA expression (both P<0.05). To sum up, CXCR4 was overexpressed on SLE B cells, positively correlating with disease activity and kidney involvement. Overactivation of the PI3K/Akt and JAK/STAT pathways as well as defective CD63 synthesis may contribute to CXCR4 dysregulation in SLE.

Evaluation of efficacy and safety of diacerein in knee osteoarthritis in Chinese patients.

OBJECTIVE: To evaluate the efficacy and safety of diacerein in patients with knee osteoarthritis (OA). METHODS: A total of 223 patients satisfying the American College of Rheumatology criteria for knee OA were chosen for this 17-week, randomized, double-dummy, diclofenac sodium-controlled trial, with diacerein dosage of 100 mg/d and diclofenac sodium of 75mg/d. Efficacy and safety of both drugs were evaluated. RESULTS: Totally 106 patients in the diacerein group and 107 patients in the diclofenac group were considered qualified for the evaluation. After 12 weeks of treatment, the total effective rates of patients/physicians' overall assessment in diacerein and diclofenac groups were 65.4%/61.6% and 61.2%/61.2%, respectively (P > 0.05). The primary efficacy parameter [visual analog scale (VAS) assessment of pain on 20 metres walking] and the secondary efficacy parameters [tenderness on palpation, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and 36-item Short-Form (SF-36) Health Survey] significantly improved compared with baseline in both groups (P < 0.05). In the follow-up period, there were no obvious changes in above parameters in diacerein group. However, in diclofenac group, pain on 20 metres walking, tenderness on palpation, and WOMAC became aggravated after withdrawing the drug for 4 weeks (P < 0.05). Moreover, the consumption of paracetamol was significantly lower in diacerein group than in diclofenac group during follow-up (P < 0. 001). The incidences of related adverse events were 35.7% in diacerein and 45.1% in diclofenac group, respectively. Mild-to-moderate gastrointestinal disorders were the most frequent adverse events. CONCLUSIONS: Diacerein is as effective as diclofenac sodium in treating patients with knee OA. Furthermore, it has better extended effect and a good safety profile. It is generally well tolerated and has no severe adverse effect.

[Detection of anti-Scl-70 antibody by recombinant fusion peptide enzyme-linked immunosorbent assay: a preliminary study].

OBJECTIVE: To detect anti-Scl-70 antibody with enzyme-linked immunosorbent assay (ELISA) using a recombinant fusion peptide which comprises 207 - 765 amino acid fragment of Scl-70 as antigen. METHODS: cDNA encoding Scl-70 antigen fragment from aa207 to aa765 from human placenta cDNA first strand was amplified with PCR. The obtained cDNA was inserted into expression vector Pet-42b and transferred into E.coli BL21 (DE3) for expression of his-tagged fusion peptide. After Ni(2+)-NTA affinity purification, the antigenicity was identified with Western blotting. Serum samples from 36 systemic sclerosis (SSc) patients, 20 patients with other connective tissue disorder (CTD) and 30 normal controls were retrospectively tested with ELISA. RESULTS: A soluble fusion peptide was expressed and purified. The antigenicity was confirmed with Western blotting using standard positive anti-Scl-70 antibody serum. Of the 36 serum samples from SSc patients, 5 of 6 samples showing positive reaction with natural Scl-70 antigen in double immunodiffusion (DID) assay also recognized the recombinant fusion peptide with ELISA, only one serum sample which showed positive anti-Scl-70 in DID displayed a negative result in ELISA assay. On the contrary, 3 patients with negative anti-Scl-70 in DID showed positive results in ELISA assay using this recombinant peptide. All the serum samples from patients with other CTD showed negative results in ELISA assay. CONCLUSION: The recombinant antigen fragment contains major epitope regions in natural Scl-70 antigen. Detection of anti-Scl-70 antibody with ELISA using the recombinant peptide can improve the sensitivity and has a potential role in determining its clinical association.

[Study of the proteins associated with Sa antigen].

OBJECTIVE: To study the proteins associated with Sa antigen, a target of the anti-Sa antibodies specific for rheumatoid arthritis, and to elucidate the nature of these proteins. METHODS: Sa antigen was extracted from fresh human placental tissue by anion exchange chromatography and subjected to SDS-PAGE electrophoresis. Serum samples were collected from 155 patients with connective tissue diseases, including rheumatoid arthritis (71 cases), ankylosing spondylitis (11 cases), psoriatic arthritis (7 cases), reactive arthritis (7 cases), juvenile idiopathic arthritis (4 cases), osteoarthritis (5 cases), polymyalgia rheumatica (6 cases), gout (6 cases), systemic lupus erythematous (7 cases), Sjogren's syndrome (10 cases), adult onset Still's disease (3 cases) and Sa antibodies were detected by immunoblotting. The gel bands corresponding to the stained bands were excised, trypsin-digested in gel, and analyzed by LC-ESI-MS/MS. Once identified, the protein was recombinate and expressed in Escherichia coli, and the antibodies were detected by immunoblotting. Then the protein was citrullinated to detect the antibodies again. RESULTS: Immunoblotting showed anti-Sa antibodies, band (s) with apparent molecular weight of 50 000 (and) 55 000, in 34 of the 71 patients of rheumatoid arthritis and 4 of the 84 patients of other rheumatic diseases, with a sensitivity rate of 47.9% and a specificity rate of 95.2%. The target protein was identified as vimentin. The positive rate of anti-vimentin antibody was statistically different between the RA patients and the patients with other rheumatic diseases (P = 0.005), with a sensitivity rate of 36.6% and a specificity rate of 83.3%, respectively. But there was no obvious correlation between anti-vimentin antibody and anti-Sa antibodies (Kappa = 0.316). The positive rate of anti-citrullinated vimentin antibody was significantly higher in the RA patients than in the patients with other rheumatic diseases (P < 0.01), with a sensitivity rate of 49.3%. There was a high correlation between anti-citrullinated vimentin antibody and anti-Sa antibodies (Kappa = 0.746), albeit a low specificity rate (86.9%). CONCLUSION: Citrullinated vimentin is closely correlated with Sa antigen.

[Characteristics of airway involvement in relapsing polychondritis].

OBJECTIVE: To investigate the characteristics of airway involvement in relapsing polychondritis (RP). METHODS: The clinical data, including clinical manifestations, respiratory function test, computerized tomography (CT), and bronchoscopy of 38 out of the 56 RP patients who had airway involvement, 20 males and 18 females, with the mean onset age of 45 +/- 11 (27 - 71), and 3 RP patients without airway involvement were retrospectively analyzed. Three patients out of the 16 RP patients who did not have respiratory involvement but underwent respiratory function test, CT, and bronchoscopy were used as controls. RESULTS: The symptoms of airway involvement included cough, expectoration, hoarseness, feeling of suffocation, asthma, and dyspnea. Obstructive disturbance of ventilation was found in 10 and mixed disturbance of ventilation was seen in 2 of the 18 RP patients with airway involvement. The forced expiratory volume in one second, ratio of forced expiratory volume in one second to forced vital capacity, peak expiratory flow, FEF50/FIF50, and maximal mild-expiratory flow of the patients with airway involvement were 1.4 L +/- 0.23 L, 46.0 +/- 4.86, 3.3 L/s +/- 0.67 L/s, 0.3 +/- 0.08, and 0.4 L/s +/- 0.18 L/s respectively, all significantly lower than those of the RP patients without airway involvement (2.5 L +/- 0.09 L, 83.7 +/- 2.24, 6.9 L/s +/- 0.52 L/s, 1.3 +/- 0.51, and 2.8 L/s +/- 0.73 L/s, all P = 0.01). Flow volume loop showed remarkable decrease of PEF and formation of a plateau in the expiratory phase in the RP patients with airway involvement. CT performed in 27 RP patients with airway involvement showed trachea stenosis in 11, and thickened airway wall in 8 of them. Bronchoscopy performed in 23 patients with airway involvement showed inflammation in 16, destruction of tracheobronchial cartilage in 6, collapsed tracheobronchial wall in 7, tracheal stenosis in 15, left major bronchial stenosis in 13, and right major bronchial stenosis in 12 of them. CONCLUSION: Respiratory function test is sensitive in early detection of airway involvement in RP. Bronchoscopy and CT are useful in evaluation of the severity of airway involvement in patients with RP.