TLR9、IFN-α和BAFF水平与系统性红斑狼疮的相关性研究

目的检测系统性红斑狼疮(SLE)患者外周血单个核细胞(PBMCs)Toll样受体(TLR)-9 mRNA及血清干扰素(IFN)-α、B细胞活化因子(BAFF)水平,探讨TLR9与SLE疾病活动的关系及与IFN-α、BAFF在SLE发病中可能的相互作用。方法应用实时荧光定量聚合酶链反应(real-time PCR)检测40例SLE患者及20例健康对照者PBMCs中TLR9 mRNA水平;采用酶联免疫吸附法(ELISA)检测60例SLE患者及20例健康对照者血清IFN-α、BAFF水平。结果 1SLE患者外周血单个核细胞TLR9 mRNA表达水平较健康对照组上调(P0.05);TLR9 mRNA表达水平与SLE疾病活动指数(SLEDAI)、血沉(ESR)呈正相关(P0.01,P0.05);与补体3(C3)呈负相关(P0.05)。2SLE患者血清中BAFF、IFN-α水平高于健康对照(P均0.05);BAFF、IFN-α水平均与SLEDAI呈正相关(P0.05)。3SLE患者中抗ds-DNA抗体阳性者TLR9 mRNA、BAFF与IFN-α水平高于阴性者(P均0.05)。4SLE患者外周血单个核细胞TLR9mRNA表达水平与血清中BAFF、IFN-α水平正相关(P0.01);血清中BAFF与IFN-α呈正相关(P0.05)。结论 SLE患者外周血单个核细胞TLR9 mRNA表达上调,血清中BAFF、IFN-α分泌增加,均与疾病活动相关。SLE患者外周血单个核细胞TLR9 mRNA表达与血清IFN-α、BAFF水平之间及BAFF和IFN-α之间均呈显著正相关,提示TLR9、BAFF及IFN-α参与了人类SLE的发病过程并相互调控,在SLE疾病活动维持中起重要作用。     

BR3 胞外区保守结构域附近刚性区域分析

目的:分析BR3胞外区保守结构域附近刚性区域在BR3结合BAFF过程中的作用。方法:将BR3胞外区分成包含Dx L结构域的BR3-1和其C端相邻的BR3-2,构建BR3-1-Fc和BR3-2-Fc重组质粒。将正确的BR3-1-Fc和BR3-2-Fc重组子转入大肠杆菌BL21中,诱导表达,并用蛋白A凝胶亲和层析柱纯化获得相应蛋白。ELISA检测其结合BAFF,BR3-1/2小肽对BR3-1-Fc与BAFF结合活性的影响。结果:表达纯化出纯度高达90%的BR3-1-Fc和BR3-2-Fc融合蛋白。融合蛋白可与BAFF结合,且具有剂量依赖性。BR3-2小肽在高浓度条件下对BR3-1-Fc与BAFF的结合起促进作用,300μg/ml时,促进作用达20%。结论:BR3受体胞外区保守Dx L结构域相邻的BR3-2刚性结构区域也可与BAFF结合,在BR3与BAFF的高亲和性能中起作用,为进一步设计高亲和性能BAFF拮抗肽奠定了理论基础。     

强直性脊柱炎患者外周血单个核细胞微小RNA的差异表达及其与免疫炎症反应的相关性

目的 检测强直性脊柱炎(AS)患者外周血单个核细胞(PBMC)中微小RNA(miRNA)的表达及其与免疫炎症反应的关系。方法 选取AS患者(AS组)及健康志愿者(对照组),每组15例。利用高通量测序技术筛选PBMC中miRNA表达,利用实时定量PCR检测6个差异表达miRNA水平,ELISA检测外周血细胞因子肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、 IL-17、 IL-23表达,Spearman法分析差异miRNA与疾病活动、免疫炎症指标的相关性。结果 与对照组相比,AS患者组共有44个miRNA差异表达,其中有22个上调,22个下调(差异倍数≥1);其中miR-1-3p、 miR-133a-5p表达明显上调,miR-127-5p、 miR-345-3p、 miR-136-3p表达明显下调;且AS患者TNF-α、 IL-1β、 IL-17、 IL-23表达明显升高,miR-1-3p与TNF-α、 C反应蛋白(CRP)、 Bath强直性脊柱炎疾病活动性指数(BASDAI)呈正相关,miR-133a-5p与TNF-α呈正相关,miR-127-5p与红细胞沉降率(ESR)...     

活动期强直性脊柱炎患者外周血效应性T细胞的比例增加且程序性死亡蛋白配体1(PD-L1)水平降低

目的分析强直性脊柱炎(AS)患者外周血CD4+CD25-效应性T细胞(Teff)和CD4+CD25+Foxp3+调节性T细胞(Treg)的比例以及两群细胞表面程序性死亡蛋白配体1(PD-L1)的表达情况。方法流式细胞术检测49例AS患者和31例健康对照者外周血中Teff和Treg的比例以及PD-L1在这两种细胞表面的表达水平。结果与健康对照组相比,活动期AS患者外周血中Teff的比例显著增加,Treg比例无明显变化;活动期AS患者外周血PD-L1+Teff的比例显著降低,且低于稳定期AS患者;活动期AS患者外周血Teff膜表面PD-L1的相对表达量也显著低于稳定期AS患者和健康对照组;AS患者外周血PD-L1+Treg的比例和Treg膜表面PD-L1的相对水平均显著低于健康对照组。结论活动期AS患者外周血中Teff比例增加,PD-L1水平降低。     

支气管哮喘患者粘附分子和细胞因子的变化及其意义

目的:探讨支气管哮喘患者外周血白细胞粘附分子β2整合素(β2-integrin熏CD18)、血清可溶性细胞间粘附分子-1(sICAM-1,CD54)、白细胞介素-6(IL-6)和白细胞介素-8(IL-8)的变化及其意义。方法:采用流式细胞仪技术检测外周血白细胞CD18和血清sICAM-1的表达;采用酶连接免疫吸附方法(ELISA)检测血清IL-6和IL-8水平。结果:(1)与正常对照组相比,支气管哮喘患者外周血白细胞CD18和血清sICAM-1表达显著增加穴P<0.05雪;(2)与正常对照组相比,支气管哮喘患者血清IL-6和IL-8水平显著增加穴P<0.05雪;(3)支气管哮喘患者外周血白细胞CD18和血清sICAM-1表达呈显著正相关(r=0.791熏P<0.05)。结论:支气管哮喘患者外周血白细胞CD18和血清sICAM-1表达以及IL-6和IL-8水平增加,可能是支气管哮喘重要的发病机制之一。     

流式细胞术检测HLA-B27及其临床意义

目的通过流式细胞术检测外周血白细胞HLA-B27的表达,并探讨其对强直性脊柱炎(AS)的诊断价值。方法利用流式细胞仪(FCM)检测41例AS患者、62例背痛及腰腿痛患者及112例健康体检者的外周血HLA-B27表达。结果 41例AS患者中,HLA阳性38例(92.6%);62例背痛腰腿痛患者中,HLA-B27阳性6例(9.67%);112例健康人群中,HLA-B27阳性2例(1.78%)。结论 AS患者HLA-B27阳性率明显增高,HLA-B27检测可视为临床支持AS诊断的重要依据。     

宁夏回族动脉粥样硬化患者外周血LCAT基因DNA甲基化与血脂水平的关系

目的探讨宁夏回族动脉粥样硬化(AS)患者外周血中卵磷脂胆固醇酰基转移酶(LCAT)基因启动子区甲基化水平及与血脂的关系。方法选择经颈部多普勒超声确诊的AS患者35例及正常者42例作为对照组,采用实时荧光定量PCR(qRT-PCR)检测外周血LCAT mRNA的表达,巢式降落式甲基化特异性PCR(nt-MSP)检测外周血LCAT基因DNA启动子区甲基化程度,全自动生化分析仪检测血脂水平。结果 AS组血清TC、TG和LDL水平显著高于对照组(P0.001,P0.001,P0.05);HDL水平低于对照组;与对照组相比,AS组外周血LCAT mRNA表达降低了61.2%(P0.01),同时LCAT基因甲基化程度升高了1.3倍(P0.05)。结论回族AS患者外周血中LCAT DNA高甲基化与AS的发生发展和血脂变化相关。     

特发性血小板减少性紫癜患者血清及树突状细胞分泌APRIL、BAFF研究

目的:通过探讨特发性血小板减少性紫癜(ITP)患者血清及外周血单个核细胞来源树突状细胞(MoDC)分泌的增殖诱导配体(APRIL)、B细胞活化因子(BAFF)特点,进一步明确ITP的发病机制。方法:研究16例初诊(初诊组)、14例治疗后获得有效(显效或良效,有效组)的ITP患者,14例健康对照组。ELISA法检测外周血清及MoDC经LPS活化后的上清APRIL、BAFF,荧光实时定量PCR测定活化后MoDC的BAFF、APRIL表达。结果:血清及DC培养上清APRIL、BAFF水平、MoDC的APRIL、BAFFmRNA表达量初诊组均明显高于对照组及有效组;除MoDC的BAFF mRNA与血小板计数之间,血清APRIL与MoDC的APRIL mRNA表达量之间无相关关系外,其余ITP患者血清、MoDC培养上清APRIL、BAFF、MoDC的APRIL、BAFF mRNA表达量之间分别有正相关关系,而与血小板计数之间均有负相关关系,对照组各参数之间除上清APRIL、BAFF分别与MoDC的APRIL、BAFF mRNA表达量之间有正相关关系外其余均无相关关系(均P0.05)。结论:DC分泌的APRIL、BAFF与ITP发病及病情进展有密切关系,DC可能通过分泌APRIL、BAFF起重要作用。     

增殖诱导配体在自身免疫病患者外周血中的表达

为检测增殖诱导配体(APRIL)在自身免疫病(SLE和RA)患者中的表达情况以及它与B细胞刺激因子(BAFF)表达、疾病预后、抗-dsDNA抗体之间的相关性。收集58名自身免疫病患者以及20名健康对照的外周血,用实时定量RT-PCR方法检测PBMC中APRIL和BAFF mRNA的表达,用ELISA方法检测血浆中APRIL和BAFF蛋白水平。同时,检测血浆中IgG、IgA、IgM、BF和抗-dsDNA抗体水平。结果同对照组相比,APRIL mRNA水平在自身免疫病患者中显著升高,与BAFF mRNA表达水平有相关性。与疾病初发组和治疗后缓解组相比,治疗后来缓解组患者APRIL mRNA表达显著升高。疾病组外周循环中BAFF蛋白显著升高,APRIL蛋白未见升高,APRIL和BAFF之间未见显著意义相关性,A-PRIL蛋白与抗-dsDNA和疾病活动度之间未见显著相关。检测APRIL mRNA含量是判断自身免疫病患者治疗情况和疾病预后的有用指标。循环中APRIL和BAFF蛋白的产生可能受到不同机制调节,在自身免疫病的发生、发展以及转归中所起的作用也不同,为研究APRIL的产生机制、表达水平和自身免疫病程度、早期诊断、预后等的关系打下基础。     

慢性乙型肝炎患者外周血单个核细胞Toll样受体3的表达降低

目的 探讨慢性乙型肝炎患者外周血单个核细胞Toll样受体3(TLR3)的表达及其临床意义.方法 分别采集慢性乙型肝炎患者和健康志愿者外周血,荧光定量PCR法检测血清HBV DNA复制水平;使用RT-PCR、流式细胞术以及免疫印迹技术分别检测外周血单个核细胞TLR3的mRNA、蛋白的表达;使用ELISA法检测血清中肿瘤坏死因子α(TNF-α)和干扰素β(IFN-p)水平.结果 慢性乙型肝炎患者外周血单个核细胞中的TLR3表达显著低于健康志愿者,且降低水平与血清HBV DNA复制水平相关;慢性乙型肝炎患者外周血TNF-α、IFN-β浓度显著低于健康志愿者,且降低的水平与血清HBV DNA复制水平相关.结论 慢性乙型肝炎患者外周血单个核细胞TLR3的表达与乙肝病毒的复制水平相关.     

Expression of BAFF and BR3 in patients with systemic lupus erythematosus

The objective of this study was to examine the relationship between the expression of B cell activating factor (BAFF) and BAFF receptor in patients with disease activity of systemic lupus erythematosus (SLE). Real-time RT-PCR was used to examine BAFF mRNA expression in peripheral blood monocytes of active and stable SLE patients and healthy controls. The percentage of BAFF receptor 3 (BR3) on B lymphocytes was measured by flow cytometry. Soluble BAFF levels in serum were assayed by ELISA. Microalbumin levels were assayed by an automatic immune analysis machine. BAFF mRNA and soluble BAFF levels were highest in the active SLE group, followed by the stable SLE group, and controls (P<0.01). The percentage of BR3 on B lymphocytes was downregulated in the active SLE group compared with the stable SLE group and controls (P<0.01). BAFF mRNA levels and soluble BAFF levels were higher in patients who were positive for proteinuria than in those who were negative (P<0.01). The percentage of BR3 on B lymphocytes was lower in patients who were positive for proteinuria than in those who were negative (P<0.01). The BAFF/BR3 axis may be over-activated in SLE patients. BAFF and BR3 levels may be useful parameters for evaluating treatment.     

Expression and localization of rabbit B-cell activating factor (BAFF) and its specific receptor BR3 in cells and tissues of the rabbit immune system

Rabbits are widely used for vaccine development, and investigations of human infectious and autoimmune diseases such as Systemic Lupus Erythematosus (SLE). For these applications, we cloned, sequenced and expressed rabbit B-cell Activating Factor (BAFF), and localized BAFF in cells and tissues of the rabbit immune system. The rabbit homolog of the human BAFF binding site (miniBR3 peptide) within the BAFF-specific receptor BR3 was synthesized. This 26-residue core domain binds to recombinant rabbit BAFF protein. Flow cytometric analyses using purified recombinant rabbit BAFF combined with real-time PCR findings revealed that BAFF detected on peripheral blood B-cells from normal rabbits is probably complexed to BAFF receptors rather than produced by the B-cells. BAFF was detected in developing appendix of young rabbits by immunohistochemical staining suggesting that BAFF plays a role during the period following birth when rabbit B-cell development and pre-immune antibody repertoire diversification and selection is occurring.     

A soluble BAFF antagonist, BR3-Fc, decreases peripheral blood B cells and lymphoid tissue marginal zone and follicular B cells in cynomolgus monkeys

BAFF (also known as BLyS), a member of the tumor necrosis factor superfamily, plays a critical role in the maturation and development of B cells. BAFF has three receptors on B cells, the most crucial of which is BR3. In this study, we demonstrate the biological outcome of BAFF blockade in cynomolgus monkeys using a soluble fusion protein consisting of human BR3 and human IgG1 Fc. In vitro, BR3-Fc blocked BAFF-mediated survival and proliferation of cynomolgus monkey B cells. Weekly treatment of cynomolgus monkeys with BR3-Fc for 13 to 18 weeks resulted in significant B-cell reduction in the peripheral blood and in lymphoid organs. CD21(high) B cells in lymphoid tissues, a subset analogous to human marginal zone B cells, expressed nearly twofold higher BR3 levels than did CD21(med) B cells. Lymphoid tissue flow cytometric analysis showed that BR3-Fc reduced this CD21(high) B-cell subset to a greater extent than it reduced CD21(med) B cells. Dual-label immunohistochemistry and morphometric image analysis supported these results by demonstrating that BR3-Fc reduced a significant proportion of the B cells within the splenic inner and outer marginal zones. These findings should prove very useful in guiding the desired therapeutic use of BR3-Fc for autoimmune diseases in the clinic.     

Specific forms of BAFF favor BAFF receptor-mediated epithelial cell survival

Although B cell activating factor (BAFF) and its receptor BR3 are produced and expressed by many cells, their role has been restricted to the lymphocyte lineage. Using various techniques (RT-PCR, indirect immunofluorescence, flow cytometry analysis), we observed the expression of BR3 and the production of BAFF by the human salivary gland cell line, by epithelial cells from biopsies of Sjögren's syndrome patients and their controls, but also by salivary gland epithelial cells in culture. To decipher the role of BAFF and BR3 on epithelial cells, BAFF and BR3 were neutralized by blocking antibodies or RNA specific inhibitor (siBR3) and epithelial cell survival was analyzed. Blocking BR3 promotes epithelial cell apoptosis in vitro. This apoptosis resulted in the nuclear translocation of PKCδ. BAFF neutralization by various anti-BAFF antibodies leads to different effects depending on the antibody used suggesting that only some forms of BAFF are required for epithelial cell survival. Our study demonstrates that BR3 is involved in the survival of cultured epithelial cells due to an autocrine effect of BAFF. It also suggests that epithelial cells produce different forms of BAFF and that only some of them are responsible for this effect.     

The Abnormal High Expression of B Cell Activating Factor Belonging to TNF Superfamily （BAFF） and Its Potential Role in Kidney Transplant Recipients

B cell activating factor belonging to TNF superfamily （BAFF） is a critical regulator of B cell maturation and survival In this present study, the expression characteristic of BAFF in kidney transplantation recipients was investigated, its potential significance was analyzed and peripheral blood of follow-up kidney transplant recipients was studied. Flow cytometric assay results showed that, cell-surface BAFF was significantly highly expressed on peripheral CD3＋ T lymphocytes in 〉 5 yrs group of kidney transplant recipients, compared with other groups （p 〈 0.05）. BAFF expression could be found on CD4＋ T cells and CD8＋ T cells. The BAFF mRNA levels in peripheral mononuclear cells were consistent with the protein levels. However, serum soluble BAFF levels were inter- individually different in each group. Stratified by renal function, it was found that cell-surface BAFF levels were significantly higher in those with abnormal renal function, compared with recipients with normal renal function （p 〈 0.05）. ELISA assay results showed that expression levels of cell surface BAFF were significantly correlated with anti-HLA I ＆ II antibodies. These results indicate that BAFF may be involved in the development of graft-loss and influences the long-time outcome of kidney allografl, likely mediated by interfering with immune response. Cellular ＆ Molecular Immunology. 2008;5（6）：465-470.     

实时荧光定量PCR方法检测非霍奇金淋巴瘤患者B淋巴细胞刺激因子表达水平

目的:建立实时荧光定量PCR方法(RFQ-PCR)检测非霍奇金淋巴瘤(NHL)患者B淋巴细胞刺激因子(BAFF)表达水平。方法:47例NHL患者及20例健康对照,采用实时荧光定量PCR方法检测其外周血单个核细胞中的B淋巴细胞刺激因子表达水平。结果:RFQ-PCR检测BAFF含量的示灵敏度为10 pg/ml,低浓度样本批内和批间变异系数(CV)分别为8.56%和11.32%,高浓度样本批内和批间CV分别为0.76%和4.58%。NHL患者和健康对照者BAFF mRNA表达量分别为(0.48±0.023,0.25±0.023),两者具有显著性差异(P=0.0001)。结论:RFQ-PCR检测BAFF mRNA含量的方法,具有较好的检测灵敏度和重复性。NHL患者BAFF mRNA高表达,提示BAFF可能在NHL发生发展中发挥重要作用。     

BAFF supports human B cell differentiation in the lymphoid follicles through distinct receptors

B cell-activating factor of the tumor necrosis factor family (BAFF/BLys) plays a critical role in B cell survival and immune responses through its three receptors: BAFF receptor (BAFF-R/BR3), transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA). Using specific antibodies, we have investigated the expression of BAFF-R on human tonsillar B cells and their functional roles in naive and germinal center (GC) B cell differentiation. Our studies show that BAFF-R is the dominant receptor on naive B cells. However, three receptors are differentially modulated during in vitro GC-B cell differentiation. BAFF-R expression increased initially and then decreased with a corresponding induction of TACI and BCMA expression during differentiation to plasma cells (PCs). Consistently, blocking of BAFF-R alone with specific mAb inhibited GC-B cell proliferation and PC generation in the early period of their differentiation, whereas depletion of BAFF with TACI-Ig exhibited consistent inhibition throughout the differentiation. Finally, histological and molecular analyses of human tonsil tissue revealed that follicular dendritic cells produce BAFF. In conclusion, BAFF in the GC plays an important role through more than one receptor, and the three known receptors are differentially modulated as GC-B cells differentiate to PCs.     

Characterisation and expression analysis of B-cell activating factor (BAFF) in spiny dogfish (Squalus acanthias): cartilaginous fish BAFF has a unique extra exon that may impact receptor binding

B-cell activating factor (BAFF), also known as tumour necrosis factor (TNF) ligand superfamily member 13B, is an important immune regulator with critical roles in B-cell survival, proliferation, differentiation and immunoglobulin secretion. A BAFF gene has been cloned from spiny dogfish (Squalus acanthias) and its expression studied. The dogfish BAFF encodes for an anchored type-II transmembrane protein of 288 aa with a putative furin protease cleavage site and TNF family signature as seen in BAFFs from other species. The identity of dogfish BAFF has also been confirmed by conserved cysteine residues, and phylogenetic tree analysis. The dogfish BAFF gene has an extra exon not seen in teleost fish, birds and mammals that encodes for 29 aa and may impact on receptor binding. The dogfish BAFF is highly expressed in immune tissues, such as spleen, and is up-regulated by PWM in peripheral blood leucocytes, suggesting a potentially important role in the immune system.     

Abnormal expression of BAFF and its receptors in peripheral blood and skin lesions from systemic lupus erythematosus patients

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disease that is characterized by abnormal T and B cells. B-cell activating factor (BAFF) has been suggested to play a crucial role in lupus by promoting the proliferation, differentiation, and survival of B cells. Increased serum levels of BAFF have been found in patients with lupus. However, the expression of BAFF and its receptors on immune cells and in skin has not been systematically reported before. Here, we report that SLE patients showed increased levels of BAFF on circulating CD3⁺ T cells and B-cell maturation antigen (BCMA) on CD14⁺ monocytes and dramatically increased expression of BAFF in lupus skin lesions compared with those of healthy controls. TACI was undetectable on circulating immune cells. An increased serum level of BAFF was also confirmed in lupus patients in this study. Our findings may provide a better understanding of the pathogenesis and predictors of BAFF antibody treatment response, as well as potential targets for skin therapies.     

B-cell activating factor of the tumour necrosis factor family expression in blood monocytes and T cells from patients with primary Sjögren's syndrome

We investigated B-cell activating factor of the tumour necrosis factor family (BAFF) level in peripheral blood mononuclear cells (PBMCs), monocytes and T cells from patients with primary Sjögren's syndrome (pSS) and controls both ex vivo and in vitro after cytokine stimulation. PBMCs, monocytes and T cells were isolated from 15 patients with pSS and 17 controls. Cells were cultured alone or with interferon (IFN)α, IFNγ and interleukin 10 (IL-10). T cells were stimulated with phytohaemagglutin and anti-CD3. BAFF protein was assessed by enzyme-linked immunosorbent assay. Ex vivo , no difference was observed in BAFF mRNA level in PBMCs and monocytes from patients and controls. Blood monocytes were the main cell type secreting BAFF both in patients and controls. In vitro , after IFNα stimulation, BAFF mRNA level was significantly higher in cells from patients than from controls (63.8 versus 20.7, P  = 0.03). T cells from patients secreted a higher level of BAFF protein than those from healthy donor cells (17.4 versus 2.9 pg/ml, respectively, P  = 0.04) but at a lower level than that from monocytes. Stimulation of T cells did not change BAFF secretion level. The induction of Th17 cells showed no increased BAFF expression. In conclusion, similar to epithelial cells, blood monocytes in patients with pSS show increased production of BAFF under IFNα, which confirms the involvement of IFNα in pSS. BAFF expression is also increased in blood T cells of such patients, independently of T-cell stimulation.