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Detection of neutrophil extracellular traps in patients with systemic lupus erythematosus

Qiumei JIANG ; Bo YAN ; Jing GOU ; Yan XING

Chinese Journal of Rheumatology.2017;21(8):542-546,后插1. doi:10.3760/cma.j.issn.1007-7480.2017.08.008

Objective To assay the level of neutrophil extracellular traps (NETs) in systemic lupus erythematosus (SLE) patients and healthy controls and compare the difference between SLE group and control group, and to analyze between the level of NETs and related laboratory parameters. Methods Forty-four females patients with SLE were recruited as research subjects, with 24 cases in the active stage and 20 in stable stage. Forty-two healthy female volunteers matched in age were enrolled as control subjects. The fluorescence intensity of NETs in neutrophils was detected by fluorospectrophotometry. The concentration of neutrophil elastase in plasma was quantitatively detected. Statistical analysis of the difference of level of NETs between the SLE group and control group was conducted. Then the correlation between the fluorescence intensity of NETs and erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), and anti-double-stranded DNA (dsDNA), and anti-histone, and anti-nucleosome, and systemic lupus erythematosus disease activity index (SLEDAI) was analyzed respectively. The same tests were conducted for the level of NE. The results of the two groups were compared using analysis of variance and the relevance was analyzed using Spearman correlation analysis. NETs morphosis was observated by immunoflurescence method, and the difference of NETs between SLE group and control group was analyzed. Results ① The fluorescence intensity of NETs was significantly increased in SLE group(241±139) than that in the control group (173±135), (t=2.31, P<0.05).②The concentration of NE in the SLE group (102±47) was significantly higher than that in the control group (62±22), t= 4.38, P<0.01. No difference of NETs [(274±168) vs (211±102), t=1.52, P>0.05] and NE concentration [(104±43) vs (96±48), t=0.50, P>0.05] between SLE active stage and stable stage was detected. ③ The fluorescence intensity of NETs in SLE patients was positively correlated with SLEDAI, but had no obvious correlation with anti-dsDNA, anti-histone, anti-nucleosome, CRP or ESR, respectively. ④Images under confocal microscope showed that more NETs generated by PMN from SLE patients than that from controls. Conclusion The generation of NETs is enhanced in female patients with SLE. And NETs may relate to disease activity. However, NETs may not induce the production of autoantibodies.

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The efficacy of t acrolimus on the treatment of Takayasu arteritis

Hu CHEN ; Zhe CHEN ; Jing LI ; Yunjiao YANG ; Jiuliang ZHAO ; Min LIU ; Xinping TIAN

Chinese Journal of Rheumatology.2017;21(8):536-541. doi:10.3760/cma.j.issn.1007-7480.2017.08.007

Objective To explore the efficacy and safety of tacrolimus among Chinese Takayasu arteritis (TAK) patients. Methods This was a single center, prospective study of active TAK patients treated with tacrolimus. Clinical manifestations, white blood cell count, hemoglobin level, erythrocyte sedimentation rate (ESR), hypersensitivity C reactive protein (hsCRP), alanine and aspartate aminotransferase and serum creatinine were recorded before and during tacrolimus treatment. Vascular changes were repeated every 6 months during tacrolimus treatment. All data were analyzed by statistical product and service solutions (SPSS) 20.0 statistical software, unpaired t test and Fisher exact probability and Kruskal-Wallis H test were used for statistical analysis. Results A total of 19 consecutive patients with an average age of (26 ±6) years were analyzed in this study. Sixteen of them were women. Pulselessness, fatigue, asymmetric blood pressure and fever were the most common clinical findings. Cervical and subclavian artery were more vulnerable. The most common artery involvement pattern was Numano type Ⅰ, followed by type Ⅱa and type Ⅴ. The median tacrolimus dosage was 2(2, 3) mg. Tacrolimus was effective in 9 out of the 19 patients. Patients who responded to tacrolimus tended to have lower mean ESR [(33±29) mm/1 h vs (42±20) mm/1 h, t=-0.776, P=0.448] and hsCRP [(20 ±31) mg/L vs (54 ±45) mg/L, t=-1.758, P=0.099] levels. However, no statistical significance was observed. During tacrolimus treatment, no drug related side effect was observed. Conclusion Tacrolimus is an alternative and effective therapy for some of the TAK patients.

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An investigation of work disability and related factors in ankylosing spondylitis patients

Jun DU ; Qiongfang WEN ; Zhuo SUN ; Xiaojian JI ; Jinshui YANG ; Fei SUN ; Jianglin ZHANG ; Feng HUANG ; Jian ZHU

Chinese Journal of Rheumatology.2017;21(8):529-535. doi:10.3760/cma.j.issn.1007-7480.2017.08.006

Objective To study the characteristics of work disability and its influencing factors in patients with ankylosing spondylitis (AS). Methods The demographic data, work conditons and disease related characteristics of 277 patients with AS were recorded, and randomly selected from the Department of Rheumatology, Chinese PLA General Hospital from November 2014 to January 2016. Work and productivity activity impair-ment questionnaire (WPAI) was used to survey the work disability and productivity loss, then explore its in-fluencing factors and the relationships between patient-reported outcomes and WPAI scores. Logistic regression was used to analyze the associated factors of work disability. Multivariate linear regression was used to analyze the predictive factors of lose of work productivity. Results The prevalence of work disability was 30.3%. Twenty patients were unemployed because of working disability. Two hudreds patients were employed, with average 36.5 (24.0, 50.0) hours workingtime in the past week. Average AS related absenteeism was 4.4 (0, 10) hour. Average workproductivity loss was 26.4%(2.5%, 40.0%). Logistic multiple regression analysis showed that Bath AS disease activity index (BASDAI), SF-36 physical component summary (PCS) scores might be the important influencing factors among those clinical measures ( OR=1.270, 0.959). Presenteeism and overall work impairment were moderately correlated with patients' global assessment of disease activity (VAS), BASDAI, bath AS functional index (BASFI), SF-36 physical Functioning (PF), SF-36 body pain (BP) and SF-36 Physical Component Summary (PCS) (|r|=0.539-0.648). Linear multivariate analyses indicated that work presente-eismand absenteeismwere significantly associated with BASDAI (P<0.01). Conclusion High prevalence of work disability in patients with AS is noted, which is closely related with disease activity and body function;High attention should be paid to AS patients with work disability.

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Effects of SMYD3 and MLL5 on histone methylation of Foxp3 gene in children with Kawasaki disease

Jiehua MEI ; Qin WANG ; Guobing WANG ; Pengqiang WEN ; Mingguo XU ; Gen TANG ; Dong CUI ; Cong LIU ; Dongli MA ; Chengrong LI

Chinese Journal of Rheumatology.2017;21(8):518-523. doi:10.3760/cma.j.issn.1007-7480.2017.08.004

Objective To investigate the effects of SMYD3 and MLL5 on histone methylation of Transcription factor forkhead box protein 3 (Foxp3) gene and its roles in the immunological pathogenesis of Kawasaki disease (KD). Methods Forty-two children with KD and 26 age-matched healthy children were consented to participate in this study. Co-Immunoprec-ipitation and real-time polymerase chain reaction (PCR) was performed to determine Foxp3-associated histone methylation levels of H3K4me3 and H3K27me3, and binding levels of SMYD3 and MLL5 with Foxp3 gene in CD4+T cells. The proportion of CD4+CD25high Foxp3+cells (Treg) and protein levels of Foxp3, cytotoxic T lymphocyte associated antigen-4 (CTLA4), TGF-βRⅡand pSmad3 were analyzed by flow cytometry. Quantitative real-time PCR was used to evaluate levels of Foxp3, interleukin (IL)-10, GITR, TGF-βRⅠand RARαmRNA in CD4+T cells. Plasma concentrations of TGF-βand retinol acid (RA) were measured by enzyme-linked Immunosorbent assay. Independent-samples t-test was used as the statistical method in this study. Results ① The proportion of Treg, expression levels of Foxp3 and molecules associated with suppressive function of Treg cells(IL-10, GITR and CTLA4), and histone methylation levels of H3K4me3 associating with promoter, conserved non-coding DNA sequence (CNS) 1 and CNS2 of Foxp3 gene decreased remarkably during acute KD [Promoter:(5.4±1.8)%vs (9.1±2.2)%;CNS1:(2.6±0.9)% vs (3.8±1.1)%; CNS2: (2.4±0.8)% vs (4.2±1.0)%; t=5.50, 6.02, 9.56, 7.92, 7.97, 4.76, 7.73, 5.01, 8.66; P<0.05], and restored after intravenous immunoglobulins (IVIG) therapy [Promoter: (7.2 ±2.1)% vs (5.4 ±1.8)%; CNS1:(3.6±1.4)% vs (2.6±0.9)%; CNS2: (3.6±1.4)% vs (2.4±0.8)%; t=5.56, 4.59, 7.01, 6.04, 5.89, 4.83, 4.45, 4.00, 5.12; P<0.05]. Meanwhile, the nine former items in KD patients with coronary artery lesions (KD-CAL+) were lower than those without coronary artery lesions (KD-CAL-) [Promoter: (4.11±1.45)% vs (6.16±1.93)%; CNS1:(1.99±0.87)%vs (2.96±1.10)%;CNS2: (1.75±0.63)%vs (2.72±1.16)%;t=6.28, 3.24, 4.56, 3.69, 3.38, 4.40, 3.65, 3.00, 3.51; P<0.05]. No significant difference of H3K4me3 associated with CNS3 and H3K27me3 were found among the groups (t=1.03, 0.91, 1.48 and 0.79, 0.82, 1.53; P>0.05). ② Binding levels of SMYD3 and MLL5 with Foxp3 gene in CD4+T cells were down-regulated significantly during acute KD (t=6.63, 6.15; P<0.05), and restored to some extent after IVIG treatment (t=5.36, 4.56; P<0.05). Positive correlations between binding levels of SMYD3 and MLL5 and expression level of Foxp3 mRNA were detected in patients with acute KD (r=0.62、0.45, P<0.05). Furthermore, Binding levels of SMYD3 and MLL5 with Foxp3 gene in KD-CAL+group were lower than those in KD-CAL- group (t=4.11, 4.31; P<0.05). ③ Compared with healthy controls, plasma concentration of TGF-β and RA, and expressions of TGF-βRⅡ, TGF-βRⅠ, pSmad3 and RARα were down-regulated during acute KD (t=11.54, 12.81, 7.43, 16.10, 8.25, 12.06; P<0.05), and elevated remarkably after IVIG treatment (t=8.40, 6.24, 5.94, 11.78, 6.27, 8.30; P<0.05). Simultaneously, all the items aforementioned in KD-CAL+ group were found to be lower than those in KD-CAL-group (t=3.58, 3.30, 3.82, 5.27, 4.71, 3.78; P<0.05). Conclusion Hypomethylation of H3K4me3 associated with Foxp3 gene caused by insufficient binding levels of SMYD3/MLL5 may be involved with immune dysfunction in Kawasaki disease.

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Role of lupus murine B cells in abnormal development of T cell subsets

Jiyu JU ; Zhiwei XU

Chinese Journal of Rheumatology.2017;21(8):508-512. doi:10.3760/cma.j.issn.1007-7480.2017.08.002

Objective To study the effects of B cells from lupus prone Triple congenic (TC) mouse model on the differentiation and development of T cell subsets. Methods The spleen size and B cell numbers were measured, and surface CD40, CD86 and Ⅰ-Ab molecules on B cells as well as CD4+T cell subsets were detected using flow cytometry when the spontaneous systemic lupus erythematosus (SLE) model TC mice and control B6 mice were 6 months old. In addition, the chimera of TC B cells and B6 CD4+T cells or chimera of B6 B cells and B6 CD4+ T cells were transferred into B6.Rag-/- mice via intravenous injection. Then, T cell subsets in the spleen of recipient B6.Rag-/-mice were observed 7 days after cell transplantation. Results TC mice had significantly bigger spleen [(5337±934) mg] and more CD19+B cell number [(276.0±48.7)×107] than control B6 mice [spleen weight: (91±4) mg; B cell number: (6.4±0.3)×107](P<0.01). TC mice showed markedly increased CD40 [MFI (63.6±3.1)], CD86 [(MFI (18.96±0.44)] and Ⅰ-Ab [MFI (637±41) on spleen B cells compared with that of B6 mice [CD40 MFI: (36.6 ±2.0); CD86 MFI: (14.26 ±0.19); Ⅰ-Ab MFI: (307 ±23)] (P<0.01). In addition, TC mice revealed notably more Th1 subset [(36.54 ±4.22)%] in spleen than B6 mice [Th1 subset: (19.90±0.10)%] [P<0.01], but both strains had equivalent percentages of Th17 and IL-21+CD4+T cell populations (P>0.05). The recipient B6.Rag-/-mice transplanted with TC B cells had significantly more Th1 subset [(54.1±2.8)%] and IL-21+CD4+T cell population [(14.3±1.0)%], but less Th17 subset [(2.05±0.09)%] in spleen than the recipient B6.Rag-/-mice administered by B6 B cells [Th1 subset: (39.5±1.1)%; IL-21+CD4+T cell population:(7.5±1.2)%;Th17 subset:(6.45±1.10)%](P<0.01). Conclusion The B cells of lupus-prone TC mice exhibit a markedly hyper-activation in spleen, and promote CD4+T cells differentiation preferentially into Th1 subset and IL-21+CD4+T cell population, which may further contribute to SLE pathogenesis.

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Association between cytotoxic T lymphocyte associated antigen-4 gene polymorphism and ankylosing spondylitis susceptibility: a meta-analysis

Xiaomin LIU ; Yinhe CHEN

Chinese Journal of Rheumatology.2017;21(8):547-552. doi:10.3760/cma.j.issn.1007-7480.2017.08.009

Objective To explore the association between cytotoxic T lymphocyte associated antigen-4 (CTLA-4) gene polymorphism and susceptibility to ankylosing spondylitis (AS). Methods The case-control studies from Chinese Biomedical Database, Chinese National Knowledge Infrastructure, Wanfang, Weipu, PubMed, Cochrane Library, OvidSP, Wiley Online Library, Elsevier Science Direct, Springer Link databases for the association of CTLA-4 gene polymorphism with AS. The association strength was assessed with chi-squared test by Stata 12.0 software. Results Seven references of CTLA-4 gene +49A/G (rs231775) polymorphism were enrolled which included 1119 AS patients and 995 controls (healthy subjects or non-AS patients), which showed that there were no statistical difference between AS and control groups under recessive, dominant, co-dominant, additive and allele gene models. Five references of CTLA-4 gene-318C/T (rs5742909) polymorphism were enrolled which included 635 AS patients and 512 controls, which showed that there were no statistical difference between AS and control groups under recessive and additive gene models; however, there were statistical difference between AS and control groups under dominant model [ OR=1.651, 95%CI (1.052, 2.590), P=0.029], co-dominant model [OR=0.621, 95%CI (0.403, 0.957), P=0.031] and allele model [OR=1.587, 95%CI (1.068, 2.357), P=0.022]. Conclusion The meta analysis reveal that CTLA-4 gene rs231775 single nucleotide polymorphism is not associated with the susceptibility to AS; rs5742909 SNP is associated with the susceptibility to AS, which suggests that C→T mutation increases the risk of AS.

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Expression and clinical significance of long noncodi ng RNA AJ227913 in patients with gouty arthritis

Chengjiao YAO ; Xiaowu ZHONG ; Yufeng QING ; Yaxi YAO ; Yuanhong PENG ; Yangyang XU ; Hong YANG ; Wenguang XIE ; Jingguo ZHOU

Chinese Journal of Rheumatology.2017;21(8):524-528. doi:10.3760/cma.j.issn.1007-7480.2017.08.005

Objective To investigate the role of long noncoding RNA-AJ227913 in the pathogenesis of primary gout arthritis (GA). Methods The subjects were divided into three groups:30 acute gout patients (AGA), 30 non-acute gout patients (NAGA), 30 healthy controlsand 30 hyperuricemia patients (HUA). Real-time quantitative polymerase chain reaction (RT-qPCR) was employed to examine the expression of AJ227913 in peripheral blood mononuclear cells(PBMCs) from four groups. 100 μg/ml monosodium urate (MSU) was used to stimulate the peripheral blood of NAGA and healthy controls patients. Then the expression ofAJ227913 was detected by RT-qPCR. Kruskal-Wallis test, Mann-Whitney test, Spearman correlations were used for statistical analysis. Results The expression level of AJ227913 in the AGA group (0.0557 ±0.0156) was higher than that in the NAGA group (0.0223±0.018) and healthy controls group (0.0038±0.0013). There was significant difference between the NAGA group and healthy controls group (P>0.05). Compared with the control group, the expression of AJ227913 in NAGA group which were stimulated by MSU was significantly increased. The Spearman correlation analysis found that the AJ227913 expression levels in GA groups were correlated with UREA (r=0.608, P<0.01), CREA (r=0.337, P<0.05), CYSC (r=0.422, P<0.01). Conclusion Altered expression of AJ227913 may be involved in the inflammatory process of GA and the balance of uricacid.

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Changes and significance of CD5+B lymphocyte in patients with systemic lupus erythematosus

Junwei CHEN ; Xiaona JING ; Meihua HAO ; Zhaoyun LIANG ; Jinhua YANG ; Meng WU ; Shaoliang JIE ; Shengxiao ZHANG ; Yanfang CHU ; Chunmiao ZHAO ; Yue QIAO

Chinese Journal of Rheumatology.2017;21(8):513-517,后插1. doi:10.3760/cma.j.issn.1007-7480.2017.08.003

Objective To measure the number of lymphocytes, B lymphocytes, CD5+B lymphocytes and level of IL-10 in peripheral blood of patients with systemic lupus erythematosus (SLE), and analyze their effects in the disease. Methods In this study, 84 cases of patients with SLE were randomly selected and evaluated according to the activity index (SLEDAI). These cases were divided into low activity group (SLEDAI<9) and high activity group (SLEDAI≥9). Ten healthy individuals were selected as the control group at the same time. The number of peripheral blood lymphocytes, B lymphocytes, CD5 + B lymphocytes, erythrocyte sedimentation rate (ESR), C3, C4 and interleukin (IL)-10 levels in serum were measured respectively and the correlation between the above indexes and SLEDAI and complement levels were analyzed. Pair-wise comparison of means of groups was conducted with one-way ANOVA. Comparison between the two groups was conducted by LSD-t test. Correlations between variables were carried out using Spearman's rank correlation test. Results The total number of lymphocytes in SLE group was lower than that in normal control group ( F=7.216, P<0.001); The number of CD19+ B lymphocytes in SLE group was higher than that in normal control group (F=3.589, P=0.036). The number of CD5+B lymphocytes of peripheral blood [(2.5±0.6)%] in patients with systemic lupus erythematosus was significantly lower than that in the normal control group [(3.2 ±0.8)%], but the difference was not statistically significant (t=3.412, P=0.698). The number of CD5+B lymphocytes in the high activity group was significantly lower than that in the low activity group (t=7.365, P=0.027)and the normal control group (t=5.649, P=0.002). The number of CD5+ B lymphocytes was negatively correlated with SLEDAI score (r=-0.692, P=0.001) and positively associated with the level of complement 3 (r=0.305, P=0.038), but not with complement 4 and ESR (P>0.05). In addition, the level of serum IL-10 in whether the low activity group (t=1.935, P=0.031) or the high activity group (t=3.048, P=0.012) was all higher than the normal control group. The level of serum IL-10 in patients with systemic lupus erythematosus was positively associated with SLEDAI score (r=0.425, P=0.024) and ESR (r=0.479, P=0.008), but was negatively correlated with complement 4 (r=-0.359, P=0.031). Conclusion The total number of lymphocytes in patients with SLE decreases significantly, while B lymphocytes increases significantly. The number of CD5+ B lymphocytes and the serum IL-10 level are also changed. It maybe related to the patient's inflammatory environment, and the number of CD5+B lymphocytes and the serum IL-10 level may be associated with disease activity.

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The value of 18F-fluorodeoxyglucose posotron emission tomography/computed tomography for detecting the relapse of polychondritis in patients with fever of unknown origin

Liheng QIU ; Qian WANG

Chinese Journal of Rheumatology.2017;21(12):841-843. doi:10.3760/cma.j.issn.1007-7480.2017.12.011

Objective To analyze the value of 18F-fluorodeoxyglucose (FDG) posotron emission tomography/computed tomography (PET/CT) in the detection of relapsing polychondritis (RP) in fever of unknown origin (FUO) patients.Methods Retrospective analysis of the clinical data of 3 patients with FUO,who were final clinical diagnosed as RP,were conducted.Clinical symptoms,laboratory tests and 18F-FDG PET/CT image data were analyzed,and literature were reviewed.Results Three patients with FUO,2 had increased FDG uptake in PET/CT in multiple cartilages,including ear,nose,trachea/bronchial and rib cartilage (SUVmax 2.5-7.5),which were consistent with RP.One case with a history of RP,PET/CT detected pulmonary infla rrmatory lesions which might be the cause of fever.At the same time,PET/CT showed RP presentation in CT with nasal and airway structure changes,but there was no abnormal FDG uptake.Conclusion 18F-FDG PET/CT has an important clinical value in diagnosis and differential diagnosis of RP,and also in follow-up and clinical response observation.

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Clinical validation of the 2012 classification criteria for early rheumatoid arthritisin: a domestic multi-center cohort

Yucui LI ; 山西医学科学院山西大医院风湿免疫科 ; Wenqiang FAN ; Haiying CHEN ; Ping YU ; Huali MIAO ; Kunjie LI ; Jinrong XU ; Liufu CUI ; Gailian ZHANG

Chinese Journal of Rheumatology.2017;21(12):807-811. doi:10.3760/cma.j.issn.1007-7480.2017.12.004

Objective To evaluate the value of 2012 classification criteria for early rheumatoid arthritis (ERA),2010 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) classification criteria,and 1987 ACR classification criteria in the diagnosis of early rheumatoid arthritis (RA).Methods Patients who had at least one swollen and tender joint with disease duration no more than 2 years,and age more than 16 years were enrolled.The patients were diagnosed as RA or other non-RA by 2 experienced rheumatologists.The clinical and laboratory parameters were recorded.The sensitivity and specificity of three RA classification criteria were compared by McNemar test,The areas under the receiver operating characteristic curve (ROC) curve (AUC) of each RA classification criteria were analyzed using MedCalc software.Results Atotal of 310 patients were enrolled in this study,including 182ERA and 128 non-RA.The sensitivity(88.5％) of ERA criteria was much higher than that of the 1987 ACR criteria (45.6％,x2=75.013,P＜0.05),and not significantly different with the 2010 ACR/EULAR criteria (91.8％,X2=1.042,P＞0.05).The specificity of ERA criteria (91.4％) of 2010 ACR/EULAR criteria (87.5％,x2=1.8,P＞0.05) was similar to that of the 1987 ACR criteria (96.1％,x2=3.1,P＞0.05).The AUC of ERA criteria was 0.962 [95％CI(0.934,0.980)],which was slightly better than that of the 2010 ACR/EULAR criteria 0.959 [95％CI(0.931,0.978)],Z=0.380,P=0.7038,and much higher than that of the 1987 ACR criteria 0.885 [95％CI (0.845,0.919)],Z=4.517,P＜0.01.Conclusion Overall evaluation,the diagnostic value of ERA criteria is better than 1987 ACR and 2010 ACR/EULAR criteria in early rheumatoid arthritis.Compared to 2010 ACR/EULAR classification criteria,ERA criteria is more simple and practical.

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