Differential abnormality in cell-cycle stage of peripheral B cells from patients with systemic lupus erythematosus

Summary In order to clarify the stage of abnormal activation of systemic lupus erythematosus (SLE) B cells, we investigated the cell-cycle phase of SLE B cells by flow-cytometric analysis. This study uses the simultaneous flow-cytometric analysis of cellular DNA content and incorporated bromodeoxyuridine, an analogue of thymidine, as indicators for DNA synthesis to detect activated B cells in peripheral blood of patients with SLE. In active SLE, the percentage of B cells increased in the S and G₂M phase and decreased in the G₀G₁ phase as compared with normal control subjects. In inactive SLE, the percentage of B cells in the S phase also increased. Active SLE B cells did not progress through the cell cycle with the stimulation of anti-IgM plus PMA or SAC plus BSF, although normal B cells transit into S and G₂M phase with such stimulation. These data suggest that SLE B cells are already activated and shifted to a matured state and that for this reason the B cells were poorly responsive to mitogen.     

Estrogen receptor expression by peripheral blood mononuclear cells of patients with systemic lupus erythematosus

To investigate the influence of sex hormones on the development of systemic lupus erythematosus (SLE), we examined the estrogen receptor (ER) expression by peripheral blood mononuclear cells (PBMC) in patients with SLE using the real-time quantitative polymerase chain reaction (TaqMan) method. The expression of messenger RNA (mRNA) for ER alpha (ERa) was increased and expression of ER beta (ERb) mRNA was decreased in PBMC from SLE patients compared with PBMC from normal controls. These findings may be useful for elucidation of the pathophysiology of SLE.     

系统性红斑狼疮患者外周血单个核细胞T细胞受体重组删除环水平

目的 通过检测系统性红斑狼疮(SLE)患者外周血单个核细胞(PBMC)T细胞受体重组删除环(TREC)水平,探讨其与临床表现间的关系及SLE的发病机制.方法 收集21例SLE组患者和22例年龄、性别相匹配的健康对照组一般临床资料;采集2组受试者外周静脉血,分离PBMC,实时荧光定量PCR检测TREC水平,结果以"TREC拷贝数/1000 PBMC"表示.结果 SLE组患者外周血PBMC中TREC水平为(9.6±7.5)拷贝/1000 PBMC,明显低于健康对照组[(16.1±11.1)拷贝/1000 PBMC,P=0.033].健康对照组PBMC中TREC水平与年龄呈负相关(r=-0.614,P=0.002),而SLE组无这种相关性.SLE组患者PBMC中TREC水平与SLEDAI评分呈负相关(r=-0.656,P=0.001),与皮肤黏膜受累旱负相关(r=-0.620,P=0.003),与其他临床表现及实验室指标无明显关联.结论 SLE患者外周血TREC水平降低提示SLE患者胸腺近期输出产物比例减少,这可能与该疾病造成胸腺近期输出功能减弱和(或)T细胞外周增殖增加有关.胸腺近期输出产物在外周T细胞池巾的含量减少,可能埘SLE患者免疫功能异常产生影响.     

PDCD5在系统性红斑狼疮患者外周血单个核细胞中表达的初步研究

目的研究程序性死亡因子5（PDCD5）在系统性红斑狼疮（SLE）患者外周血单个核细胞（PBMC）中的表达情况,探讨其可能作用机制。方法应用免疫细胞化学和蛋白免疫印迹法检测45例SLE活动期患者、22例稳定期患者和50例健康对照者PBMC中PDCD5蛋白的细胞定位和表达情况。结果SLE活动期患者PDCD5蛋白的阳性表达率高于正常对照组（P〈0.01）;活动期患者PDCD5蛋白的表达水平高于稳定期患者和正常对照,稳定期患者高于正常对照（P〈0.01）。SLE活动期患者PBMC中PDCD5的表达与SLE疾病活动指标SLEDAI、补体C3、抗ds-DNA抗体滴度具有相关性。结论PDCD5可能作为淋巴细胞凋亡的正性调控基因,在SLE发生和发展中具有重要意义。     

系统性红斑狼疮患者外周血单个核细胞B细胞刺激因子的异常表达及其临床意义

目的研究系统性红斑狼疮(SLE)患者外周血单个核细胞中B淋巴细胞刺激因子(Blys)的表达情况,并探讨Blys的转录水平与狼疮活动性和狼疮肾炎(LN)的关系,分析Blys在狼疮发病中的作用。方法应用半定量反转录-聚合酶链反应(RT-PCR)法检测活动期组(26例)和缓解期组(22例)SLE患者外周血单个核细胞(PBMCs)BlysmRNA的表达水平,以健康志愿者(20例)作为对照;同时将BlysmRNA的表达水平与患者临床检验指标进行相关分析;采用流式细胞仪技术检测20例SLE患者和16例健康对照膜结合型Blys蛋白水平。结果SLE患者PBMCsB1ysmRNA的表达明显高于正常人(P<0.01),活动期患者的表达高于缓解期(P<0.05);BlysmRNA的表达与狼疮活动指数(SLEDAl)和蛋白尿呈正相关,与补体C3、C4水平呈负相关;抗dsDNA抗体阳性和尿蛋白阳性的SLE患者BlysmRNA的表达高于阴性患者(P<0.05);而且SLE患者PBMCs中Blys的平均荧光强度也显著增加(P<0.05)。结论SLE患者PBMC的膜Blys和BlysmRNA表达均增强,且与狼疮活动性及尿蛋白呈正相关,提示Blys可能参与SLE及狼疮肾炎的发病过程,且有望成为临床观察和疗效评价的新指标。     

人重组B细胞刺激因子对系统性红斑狼疮外周血B细胞功能的影响

目的 探讨人重组B细胞刺激因子(rhBlys)对系统性红斑狼疮(SLE)患者外周血B细胞增殖和分泌功能的影响. 方法 分离22例SLE患者和20名健康志愿者的外周血细胞,分为3组:rhBlys 加抗IgM抗体组、抗IgM抗体组和培养基组.培养72 h收集B细胞及培养上清.MTT法检测B细胞增殖,ELISA法检测培养上清IgG、IgM和抗ds-DNA抗体浓度. 结果 ①SLE患者外周血B细胞在rhBlys与抗IgM抗体的协同作用下,增殖显著增强(0.36±0.15对比0.21±0.05,P＜0.01),强于单用抗IgM抗体组(0.36±0.15对比0.24±0.08,P＜0.05);②SLE患者外周血B细胞在rhBlys与抗IgM抗体的协同作用下,IgG、IgM和抗ds-DNA抗体分泌显著增强,强于培养基组(1 207.04±200.84对比801.16±116.22,P＜0.01;375.11±78.20对比208.15±47.96,P＜0.01;159.27±42.65对比102.21±32.54,P＜0.01);也强于单用抗IgM抗体组(1 207.04±200.84对比926.54±134.64,P＜0.05;375.11±78.20对比236.50±53.72,P＜0.05;159.27±42.65对比114.16±33.25,P＜0.05);③SLE患者B细胞的增殖和培养上清IgG、IgM和抗ds-DNA抗体的分泌均高于健康对照者. 结论 本实验结果进一步证实Blys是B细胞强共刺激剂,Blys与抗IgM抗体对B细胞的增殖和分泌功能有协同增强的作用.SLE患者与健康对照相比可能存在B细胞数量上的异常或本身免疫耐受的缺陷.这为干预Blys在SLE患者B细胞增殖和分泌功能中的作用,寻找新的治疗方法提供了依据.     

免疫清除性化疗结合自体外周血造血干细胞移植治疗重症系统性红斑狼疮

目的 观察免疫清除性化疗结合自体外周血造血干细胞移植（移植）治疗重症系统性红斑狼疮（SLE）的疗效及安全性。方法 重症SLE4例，分别合并狼疮性肾炎，狼疮脑，狼疮心或股骨头坏死，干细胞的动员采用环磷酰胺加重组人粒细胞集落因子（G－CSF）；预处理为回输前3天每天应用环磷酰胺50mg/kg 回输后3天每天应用抗胸腺细胞球蛋白（ATG）5mg/kg。观察移植前后临床症状，体征，狼疮相关抗体等指标的改变，并动态观察移植后免疫功能的重建。结果 移植后患者的临床症状完全缓解，狼疮相关抗体全部转阴，移植后患者的免疫功能均明显降低，约半年后恢复正常，但不伴有临床症状的复发。结论 免疫清除性化疗结合自体外周血造血干细胞移对难治性SLE有明显的疗效，尤其适用对各种药物治疗无效者，治疗是安全的，远期疗效还需长期随访。     

系统性红斑狼疮患者体液免疫和ENA酶谱变化及意义

目的　探讨体液免疫和可提取性核抗原 (ENA)酶谱在系统性红斑狼疮 (SL E)稳定期和活动期中的变化及意义。方法　应用散射速率比浊法检测 5 2例 SL E患者 (下称实验组 )以及 2 4例正常对照者 (对照组 )的Ig G、Ig A、Ig M、C3、C4 等水平 ,应用免疫印记技术检测 ENA酶谱。结果　实验组血清 Ig G、Ig A水平明显高于对照组 ,其中活动期 (实验 B组 )血清 Ig G、Ig A、Ig M高于稳定期患者 (实验 A组 )。实验组血清 C3、C4 水平明显低于对照组 ,且实验 B组 C3水平明显低于实验 A组 ,差异有显著性 (P<0 .0 1)。 4 5 .4 % SL E患者抗 SSA、抗 SSB同时阳性 ,抗 ds DNA阳性率为 32 .7%。抗 r RNP和抗 Sm抗体在实验 A、B组的阳性率有显著差异 (P<0 .0 1,<0 .0 5 ) ,其他抗体均无明显差异。结论　 SL E患者行体液免疫指标及 ENA酶谱测定可提示其疾病进展情况。     

系统性红斑狼疮患者血管内皮细胞损伤标志物检测及其临床意义

目的探讨血管内皮细胞损伤与系统性红斑狼疮(SLE)疾病活动、肾脏损伤的关系。方法应用酶联免疫吸附试验(ELISA)检测31例SLE患者和10例健康对照者血浆可溶性血栓调节蛋白(sTM)、血管假性血友病因子(vWF)水平。结果SLE组血浆sTM、vWF水平明显高于对照组;SLE组血浆sTM、vWF水平均与疾病活动指数(SLEDAI)呈显著正相关,与24小时尿蛋白定量呈正相关。结论血管内皮损伤可能在SLE发病机制中起重要作用,血浆sTM、vWF水平可作为判断SLE疾病活动性及肾脏损伤程度的指标。     

Activated peripheral blood mononuclear cells detected in lupus patients using cDNA coding for proliferating cell nuclear antigen

 The expression of proliferating cell nuclear antigen (PCNA) mRNA in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) was measured by dot blot hybridization using a PCNA cDNA, and correlated with the percentage of PCNA-positive cells detected immunohistochemically using a monoclonal anti-PCNA antibody. PCNA-positive PBMCs were detected in 72.2% of SLE patients (n = 36), which is significantly more than among healthy controls. In addition, among those in whom PCNA expression was detected, the percentage of PBMCs expressing PCNA was significantly higher in SLE patients (mean 2.5% vs. 0.15%). The level of PCNA mRNA was increased in PBMCs from 83.3% of SLE patients, and was significantly correlated with the percentage of PCNA-positive cells (r = 0.54, P < 0.01) and with the disease activity score (r = 0.56, P < 0.01). A longitudinal study of two SLE patients confirmed that PCNA mRNA expression and the percentages of PCNA-positive cells varied in parallel with disease activity. Thus, an analysis of activated PBMCs from SLE patients using PCNA cDNA may be a useful method by which to estimate SLE disease activity. Received: January 25, 2002 / Accepted: March 30, 2002 Acknowledgments This work was supported in part by a 1999 research grant from the Mixed Connective Tissue Disease Research Committee of Japan, Ministry of Health and Welfare, and a 1999 Grant-in-Aid for Scientific research (C) (2), Ministry of Education. Correspondence to: Y. Takasaki     

SLE患者血清及外周血单个核细胞中IL-12、IL-18的检测及临床意义

目的探讨系统性红斑狼疮（SLE）患者IL-12、IL-18的表达及与疾病活动的关系。方法应用ELISA法以及半定量RT-PCR法分别测定SLE活动组、缓解组和对照组血清IL-12、IL-18水平以及外周血单个核细胞中的IL-12 mRNA和IL-18 mRNA的表达。结果SLE活动组IL-18 mRNA、IL-18的表达较SLE缓解组和正常对照组明显升高（P均〈0.05）。血清IL-18水平与IL-12水平呈负相关（r=-0.406,P=0.019）。SLE患者SLE疾病活动指数与IL-12、IL-12 mRNA呈负相关（r=-0.617,P〈0.05;r=-0.512,P〈0.05）,与IL-18、IL-18 mRNA呈正相关性（r=0.817,P〈0.05;r=0.806,P〈0.01）。结论SLE活动组患者中存在IL-18异常高表达和IL-12异常低表达,二者之间呈负相关,在SLE发病和发展中可能起不同作用。     

IgG and IgM anti-endothelial cell antibodies in patients with collagen-vascular disorders

Summary A cellular enzyme-linked immunosorbent assay (ELISA) was used to detect circulating anti-endothelial cell antibodies (AECA) in sera of patients suffering from rheumatoid arthritis (RA), Felty's syndrome (FS), systemic lupus erythemtosus (SLE), progressive systemic sclerosis (PSS) and those with a lupus anticoagulant (LA). IgG AECA were detected in RA, FS, SLE and LA sera, while IgM AECA were only detected in RA and FS. AECA were not specific for endothelial cells. However, IgG binding to endothelial cells and dermal fibroblasts was F(ab) mediated while in all other cell types tested, nonspecific binding most likely via the Fc region occurred. In RA and FS rheumatoid factor was shown to augment immunoglobulin binding to endothelial cells. Additional studies revealed that some of these pathological sera also contained cytotoxic IgG autoantibodies which fixed complement and damaged human umbilical vein endothelial cells in vitro. These studies suggest that a group of autoantibodies, present in a variety of collagen vascular disorders, react with endothelial cells and thus may be important aetiopathogenic factors in the vasculopathies associated with these disorders.     

Anti-Sm and anti-DNA antibodies in paired serum and synovial fluid samples from patients with SLE

Summary Antibodies to nuclear antigens (ANAs) are frequently found in the serum of patients with connective tissue diseases (CTDs). Particularly systemic lupus erythematosus (SLE), and have been implicated in the immune-complex mediated pathogenesis of these diseases. In this study we have compared the occurrence of precipitating ANAs in paired samples of serum and synovial fluid from patients with different CTDs. Of the 30 patients examined 3 had precipitating ANAs in their serum only, 1 in the synovial fluid only, and 3 had antibodies in both serum and synovial fluid. Precipitating ANAs in synovial fluid were found in 3/6 patients with SLE, 1 patient with RA/Sjogren's syndrome overlap, and one patient with RA/SLE overlap. Of the other 15 patients with RA, 2 had precipitating antibodies only in their serum. Two of the SLE patients had anti-Sm antibody, one in serum only and the other in both serum and synovial fluid. Detection by ELISA of class specific anti-Sm antibodies in serum or synovial fluid paralleled the occurrence of antidenatured DNA antibodies when both specificities occurred together. One SLE patient did show evidence in synovial fluids of elevated concentrations of specific antibody classes to individual antigens; however, elevated levels were more frequently found in serum. Local production of ANAs does not, therefore, appear to be a feature of synovial fluids from SLE patients.     

New onset of lupus nephritis in two patients with SLE shortly after initiation of treatment with belimumab

PurposeBelimumab is currently approved for the treatment of patients with active SLE despite standard treatment. However, it has not been formally tested for patients with lupus nephritis because such patients had been excluded from the clinical trials. In this report, we present two patients with SLE who developed lupus nephritis de novo shortly after belimumab treatment initiation; both patients improved rapidly upon belimumab discontinuation.ResultsThe first patient (a 30-year-old female, with a 15-year disease duration, receiving prednisolone, hydroxychloroquine, and azathioprine, with no previous history of nephritis that was repeatedly anti-dsDNA negative) had exacerbation of a facial butterfly-like rash developed after 3 months of belimumab treatment initiation. Concomitantly, her urinalysis became abnormal for the first time during her long follow-up (15–20 red blood cells per hpf, and a 24-h urine protein of 1600 mg), and a renal biopsy documented the diagnosis of a Class III (WHO classification). Her anti-dsDNA titers became highly positive for the first time. Belimumab was discontinued and her proteinuria and abnormal urinalysis reverted to normal rapidly, and before MMF administration was approved by local regulatory authorities. Our second patient (a 38-year-old female with a 19-year disease duration) was being treated with prednisone and azathioprine. Two months following belimumab treatment initiation, she became edematous and had an active urine sediment (50–60 rbc per hpf, dysmorphic, and a 24-h urine protein levelabove 6000 mg) for the first time during her disease course. Her renal biopsy was compatible with a Class V membranous nephritis. Belimumab was discontinued and MMF (2 g/d) was substituted for azathioprine with her urinary protein declining to 2.7 g/d just 10 days afterwards.ConclusionsIn this report, apart from our two patients, we discuss the relevant literature consisting of a handful of studies and case reports. The studies analyze patients with renal involvement treated with belimumab and are inconclusive. There are only a few case reports in which belimumab along with other agents had a potential benefit, although not straightforward. There is only one case report with striking similarities to the two patients with SLE we report herein. It could be claimed that belimumab was unable to prevent the appearance of lupus nephritis during a potentially serious disease exacerbation. Certainly, a causative association between belimumab treatment and the de novo appearance of lupus nephritis cannot be claimed because of our report. However, a potential association between belimumab treatment and the development of such a serious manifestation cannot be entirely excluded. In support of the latter hypothesis is the quick resolution/significant reduction of proteinuria shortly after belimumab discontinuation and before other treatment measures had any reasonable effect. Studies evaluating the potential usefulness of belimumab in patients with lupus nephritis are currently ongoing; until then, one should keep in mind unanswered questions as far as renal safety is concerned.     

自体外周血干细胞移植治疗难治性系统性红斑狼疮的临床研究

目的探讨自体外周血干细胞移植(APBSCT)治疗难治性系统性红斑狼疮(SLE)的临床疗效。方法对难治性SLE患者进行APBSCT治疗。采用环磷酰胺(CTX) 粒细胞集落刺激因子(G-CSF)方案动员,CS-3000血细胞分离机采集外周血干细胞并保存于-80℃冰箱;用CTX50mg/(kg.d)×3～4d方案预处理后,解冻后回输冻存的干细胞的治疗方法。观察APBSCT前后临床表现及免疫学指标的变化。结果动员后获得单个核细胞数(MNC)(3.383～3.704)×108/kg;CD34 细胞数(4.4～11.12)×106/kg。3例均获得造血重建,中性粒细胞>0.5×109/L、血小板>20×109/L的中位数时间分别是8.3d、10d。移植后患者临床症状消失,1例并肾功能不全、难治性高血压和重度贫血的患者移植后恢复正常。3例患者自身抗体转阴或滴度减低,尿蛋白定性消失,SLE疾病活动指数(SLE-DAI)由移植前的平均15分下降为移植后6个月的平均4分。结论APBSCT治疗难治性SLE安全有效,近期疗效好,远期疗效有待进一步观察。     

乙型肝炎患者外周血单个核细胞端粒酶活性的研究

目的 了解乙型肝炎患者外周血单个核细胞（PBMC）端粒酶活性的表达情况。方法 通过扩增端粒重复序列（TRAP）及光度酶联免疫法，分别检测健康人及各类乙型肝炎患者PBMC的端粒酶水平。结果 各组患者PBMC在植物血凝素（PHA）刺激前均有端粒酶活性的表达，以急性型肝炎组最高，重型肝炎组最低，二者差别具有显著性（P〈0．001）。PHA刺激后与刺激前比较各组端粒酶活性均有显著性升高（P〈0．001），刺激后的端粒酶水平以重型肝炎组为最低，与其他三组比较差别具有显著性（P〈0．05）。慢性重型乙型肝炎经胸腺五肽（TP5）治疗后端粒酶活性显著增高（P〈0．05）。结论 HBV急性感染期PBMC的端粒酶水平升高；慢性感染期PBMC的端粒酶水平在体内被抑制。TP5具有免疫调节作用，能使过低的端粒酶水平趋向于正常。     

Systolic and diastolic heart function in SLE patients

Cardiovascular pathology is frequent in systemic lupus erythematosus (SLE). Left ventricular (LV) diastolic dysfunction is its common findings. The aim of the study was to assess the systolic and diastolic function of the left ventricle (LV) in SLE patients without clinically evident cardiovascular disease, using pulsed Doppler echocardiography. Another purpose was to estimate whether there is a correlation between the duration and severity of SLE and the degree of LV diastolic dysfunction. A comparison of the average values of echocardiographic measurements was made between the SLE group and control group, which constituted healthy volunteers. No statistically significant differences in systolic heart function between groups were observed, except for lower values of the fractional shortening (SF 35.9 ± 1.2 and 37.1 ± 0.9, P = 0.01) in SLE patients, particularly in long (more than 10 years) disease duration (34.9 ± 0.6 vs. 37.0 ± 0.8, P < 0.005) and the value of SLE Disease Activity Index (SLEDAI) higher than six points (35 ± 0.9 vs. 37.1 ± 0.5, P < 0.01) Left atrial end-systolic diameter (LA) was greater (3.69 ± 0.37 vs. 3.5 ± 0.28, P < 0.05) and the ejection fraction (EF) was lower (64.6 ± 1.5 vs. 66.3 ± 1.3, P < 0.05) in SLE subjects of long disease duration than in the controls. SLE patients demonstrated significantly higher late diastolic velocity (A’) and lower E’/A’ ratio than the control group. No differences were observed in A’ values between SLE subset of short disease duration and the controls. Isovolumetric relaxation time in turn was significantly longer and E/A ratio as well as E’/A’ ratio lower in SLE of long disease duration versus the short one. In older patients, peak velocity at the time of atrial contraction (A) and A’ values were higher and peak early velocity wave (E), early diastolic velocity (E’), E/A ratio and E’/A’ ratio lower than in the younger subset. Increased the value of SLEDAI correlated with increased A’ and decreased E, E/A ratio and E’/A’ ratio in SLE subjects. Further analysis concerning the strong connection of these parameters with patients’ age, however, revealed no statistically significant correlation between SLEDAI values and LV diastolic function parameters. In long (>10 years) disease duration LV diastolic properties were worse.     

Nature of IgG anti-lymphocyte autoantibody-reactive molecules shed from activated T cells in systemic lupus erythematosus

Summary Shedding of cell-surface antigens that react with anti-lymphocyte autoantibodies in systemic lupus erythematosus (SLE) is well-recognized, but the nature of such molecules is unknown. The present investigation demonstrates the rapid shedding of three IgG antibody target molecules of M_r 55 000, 37 000, and 32 000 from the surface of mitogen-activated peripheral T cells during brief incubation at 37°C. Sera lacking IgG anti-lymphocyte antibodies stained none of the three antigens. Absorption of antibody-positive sera with viable HSB-2 cells, a primitive T-cell line lacking HLA antigens and many CD antigens characteristic of mature peripheral T cells, eliminated staining of the shed molecules. These data delineate the number and estimated molecular mass of anti-lymphocyte autoantibody target molecules that are shed from the surface of T cells, and provide further insight into potential mechanisms by which anti-lymphocyte antibodies contribute to the pathogenesis of SLE and related disorders.