HIV/AIDS患者外周血B细胞数量以及TLR9 mRNA表达水平的研究

目的 探讨HIV-1感染后外周血B细胞数量的变化,以及B细胞TLR9 mRNA表达水平与HIV-1感染疾病进展的关系.方法 采集HIV/AIDS患者EDTA抗凝静脉血,荧光抗体染色后用流式细胞仪检测HIV/AIDS患者B细胞数量.密度梯度离心法分离外周血单个核细胞,应用MACS磁珠分选系统分选CD19+B细胞,并采用荧光定量实时PCR技术检测B细胞TLR9 mRNA水平.结果 HIV/AIDS患者B细胞数量显著低于健康对照组(P＜0.01),与CD4+T细胞数量呈正相关(r=0.534,P=0.006).HIV/AIDS患者外周血B细胞TIR9 mRNA的表达明显低于健康对照组(P=0.023),与CD4+T细胞数量呈正相关(r=0.390,P=0.040).结论 HIV感染可降低HIV/AIDS患者外周血B细胞数量以及B细胞TLR9 mRNA的表达量,B细胞数量和B细胞TLR9 mRNA的表达量均可能与疾病进展相关.     

TLR与足细胞损伤

TLRs是一类重要的模式识别受体家族,主要调节天然免疫反应.研究发现在肾脏固有细胞及间质细胞都有TLR表达,其介导的炎症反应参与了许多肾脏疾病的发生.多种足细胞标志蛋白的发现加快了足细胞表面分子的研究进程.目前已发现足细胞表面有TLR的表达,且TLR的表达与足细胞的损伤有关.文章综述了已经发现的TLR成员及其与足细胞损...     

慢性乙型肝炎病毒感染树鼩Kupffer细胞TLR2和TLR4的表达及其意义

目的探索慢性乙型肝炎病毒(HBV)感染树鼩Kupffer细胞Toll样受体(TLR)家族中的TLR2和TLR4在mRNA水平的表达情况及其对Kupffer细胞功能的影响。方法树鼩分为确定慢性感染HBV的树鼩、疑似慢性感染HBV的树鼩和未接种HBV的正常对照树鼩。全部动物定期抽血和进行肝活检手术,采用实时荧光定量PCR(qRT-PCR)分析血清和肝组织的HBV DNA水平;对手术切取的树鼩肝组织进行Kupffer细胞的分离、纯化和原代培养,采用qRT-PCR检测TLR2、TLR4以及TNF-α的mRNA表达水平;采用迁移实验及溶酶体荧光探针等方法分析TLR2和TLR4对Kupffer细胞迁移能力及溶酶体数量的影响。结果确定慢性感染HBV的树鼩TLR2 mRNA和TLR4 mRNA表达水平均低于疑似慢性感染HBV的树鼩和未接种HBV的正常对照树鼩(P0.05),表达水平均与动物肝组织的HBV DNA拷贝数呈负相关(P0.05),与Kupffer细胞的细胞迁移数、溶酶体密度及TNF-αmRNA表达水平呈正相关(P0.05)。结论 Kupffer细胞中的TLR2和TLR4可能通过影响Kupffer细胞功能而参与树鼩HBV感染后肝脏病变的慢性化发展过程。     

B族链球菌不同菌株对血小板的活化作用

 目的: 探索B族链球菌(GBS)不同菌株对血小板活化的作用及可能的机制。方法: 来自脓毒症伴血小板减少患者分离鉴定的6株GBS作为诱导剂,分别采用血小板聚集仪、流式细胞术、扫描电镜和Western blotting方法检测它们对血小板聚集率以及血小板膜蛋白CD62P、TLR2和TLR4表达的影响;进一步用TLR2和TLR4单克隆抗体封闭血小板表面TLR2和TLR4以检测它们与GBS诱导的血小板活化的关系。结果: 6株GBS中有3株可诱导血小板聚集,且能明显上调血小板TLR2和CD62P的表达(P < 0.05),但对血小板TLR4的表达没有影响;封闭血小板TLR2后,GBS诱导的血小板聚集率明显下降。结论: 部分GBS菌株可诱导血小板活化,血小板TLR2可能在这个过程中起重要作用。     

急性白血病患者浆细胞样树突状细胞TLR9的表达及功能分析

目的 检测Toll样受体9(TLR9) mRNA在急性白血病(AL)患者外周血浆细胞样树突状细胞(pDCs)内的表达水平及pDCs的功能.方法 免疫磁珠分选13例初诊未治急性髓细胞白血病(AML)、11例急性淋巴细胞白血病(ALL)患者和15例健康对照外周血pDCs,real time-PCR检测pDCs内TLR9mRNA表达水平;用CpG ODN2216与pDCs共培养24h,ELISA检测上清液中IFN-α、IL-6、TNF-α水平.结果 AML-pDC和ALL-pDCs产生的IFN-α、IL-6、TNF-α水平分别为[(378.24±89.96)pg/mL,(57.98±29.68)pg/mL,(60.24±26.75) pg/mL]、[(352.56±67.34) pg/mL,(68.78±31.45) pg/mL,(53.67±25.98) pg/mL]明显低于正常对照组[(685.86±102.16) pg/mL,(91.25±32.12) pg/mL,(86.65±28.69)pg/mL] (P＜0.05);AML-pDC、ALL-pDC中TLR9 mRNA的表达水平分别为0.34±0.25、0.41±0.23,均明显低于正常对照组(P＜0.05).结论 急性白血病患者pDCs内TLR9mRNA水平明显降低,可能与患者pDCs功能缺陷有关.     

Toll样受体信号介导细胞凋亡的研究进展

细胞凋亡在病毒、细菌等感染的过程中和肿瘤等疾病的发生发展中起至关重要的作用。Toll样受体(TLR)存在于巨噬细胞、肿瘤细胞等细胞表面,能直接识别并结合病原微生物和宿主细胞表面的病原相关分子模式,然后通过髓样分化因子88/Fas相关死亡结构域/caspase-8、TIR结构域接头蛋白/蛋白激酶/干扰素调节因子和核因子κB路径等信号途径对巨噬细胞、肿瘤细胞等细胞的凋亡起调节作用。随着对TLR介导的细胞凋亡中Fas相关死亡结构域、TIR结构域接头蛋白等多种信号分子的深入研究,有助于了解它们在细胞凋亡中的作用,并为感染和肿瘤等疾病的分子靶向治疗提供新的目标。     

腺样体扁桃体肥大相关B细胞免疫活化调控机制的研究进展

B细胞通过其表面分布的 B细胞受体 （BCR） 识别外界抗原,是机体产生保护性抗体与免疫记忆的关键步骤。 B细胞免疫活化调控与诸多上呼吸道疾病是密切相关的。儿童常见疾病腺样体扁桃体肥大 （ATH）,其特征性病理本质为淋巴滤泡的增生,但其确切机制尚未明确。本文综述静息态下维持B细胞存活的BCR滋养信号研究,阐述了B细胞免疫活化及产生快速高效免疫记忆的调控机制,尤其是活化早期分子事件,强调mIgG-tail对记忆性抗体应答的相关作用。B细胞活化调控过程出现异常可破坏免疫稳态平衡,导致疾病的发生。本文总结了B细胞免疫活化调控与ATH的机制关联,尝试探讨信号转导通路失调或突变对ATH的可能影响。旨在深入理解ATH的致病机理,以期挖掘潜在研究突破口,寻找ATH新的诊疗靶点。     

Ⅰ型干扰素参与调节SLE外周T细胞活化和TLR表达谱的研究

血清高水平Ⅰ型干扰素(typeⅠinterferon)是系统性红斑狼疮(SLE)患者重要的病理特征之一,外周高水平IFN-α对T细胞的免疫调节作用值得深入探讨。本研究首先比较分析了SLE患者外周血T细胞活化和TLR分子表达格局,结果显示SLE患者外周血CD4~+或CD8~+T细胞和正常人相比呈现出更加活化的表型变化,其表面活化标志CD69和HLA-DR表达阳性率均高于正常人;分析T细胞表达TLR分子格局发现,SLE患者较正常人T细胞中TLR分子的表达有明显升高,其中CD4~+T细胞中TLR8和TLR9的升高明显,而在CD8~+T细胞中明显升高的TLR分子有TLR3和TLR8;结合SLE病理状态下外周高水平IFN-α的持续存在,我们进一步分析了IFN-α对正常T细胞活化和TLR分子表达谱的影响,结果显示IFN-α协同TCR信号可以促进T细胞的活化,并上调T细胞中部分TLR分子的表达,其中CD4~+和CD8~+T细胞中TLR8的表达均明显上升。综合分析SLE患者和经IFN-α活化的正常T细胞的活化和TLR分子表达谱的变化格局,提示SLE病理状态下高水平的Ⅰ型干扰素可以与T细胞的持续活化有关,其对T细胞表达TLR分子格局的影响,特别是与核酸类分子配体相关的TLR分子的表达增高为内源性核酸类配体参与T细胞的活化提供了分子基础。     

B细胞激活因子研究进展

B细胞激活因子BAFF是新近发现的TNF超家族成员,因其在B细胞发育及自身免疫病中起关键作用而日益受人关注。BAFF对外周B细胞发育及功能十分重要。BAFF信号的缺失导致外周B细胞大量减少,B细胞发育在T1B向T2B转换阶段被阻断。BAFF介导TD及TI抗原诱导的抗体应答。BAFF转基因小鼠发生系统性红斑狼疮样表型。在天然狼疮小鼠及部分红斑狼疮患者血清中可溶性BAFF蛋白水平增高。老年的BAFF转基因小鼠还出现干燥综合征样表型。这些研究结果提示,BAFF是参与某些自身免疫病发病的重要致病因子。     

Toll-like receptor driven B cell activation in the induction of systemic autoimmunity

Studies over the past decade have demonstrated a key role for pattern recognition receptors in the activation of autoreactive B cells. Self reactive B cells that manage to escape negative selection often express relatively low affinity receptors for self antigens (ignorant B cells), and can only be activated by integrating a relatively weak BCR signal with signals from additional receptors. Members of the toll-like receptor (TLR) gene family, and especially the nucleic acid binding receptors TLR 7, 8 and 9, appear to play a key role in this regard and promote the production of autoantibodies reactive with DNA- or RNA-associated autoantigens. These autoantibodies are able to form immune complexes with soluble or cell-bound ligands, and these immune complexes can in turn activate a second round of proinflammatory cells that further contribute to the autoimmune disease process. Recent data have emerged showing a pathogenic role for TLR7, with an opposing, protective role for TLR9. Targeting these disregulated pathways offers a therapeutic opportunity to treat autoimmune diseases without crippling the entire immune system. Further understanding of the role of specific receptors, cell subsets, and inhibitory signals that govern these TLR-associated pathways will enable future therapeutics to be tailored to specific categories of autoimmune disease.     

人TLR4的B细胞优势表位设计、合成及其免疫原性检测

目的　鉴定TLR4的优势抗原表位 ,为抗人TLR4单克隆抗体制备奠定基础。方法　分别应用Hoop&Woods亲水性参数、抗原性参数、可及性参数和抗原表位的软件分析对TLR4B细胞表位进行预测 ,最后用抗原性指数的方法进行综合评述。合成针对该表位的多肽 ,以此多肽为免疫原免疫小鼠 ,对其免疫原性进行检测。结果　预测TLR4的B细胞优势表位为 189～ 2 0 2氨基酸序列 :NH2 HKLTLRNNFDSLNV COOH ,此多肽能诱导机体产生较高的抗体滴度 ,多抗具有较高的特异性。结论　预测的TLR4短肽是B细胞的优势表位 ,以此制作抗人TLR4单克隆抗体是可行的。     

Expression of bcl-x during mouse B cell differentiation and following activation by various stimuli

We have studied the expression of the novel anti-apoptotic protein bcl-x during mouse B cell differentiation and activation. We find that bcl-x is expressed throughout all stages of B cell differentiation in the bone marrow, and is only down-regulated in mature (sIgD⁺) B cells. Immature peripheral B cells express low levels of bcl-x even in adult animals, whereas mature resting B cells do not. Mature B cells re-express the protein following activation, achieving maximal levels after 36–48 h. The highest levels of bcl-x are observed with potent comitogenic stimuli (such as anti-CD40 + anti-Ig): B cells first express bcl-x in the G1 phase of the cell cycle and contain maximal levels in S phase. In addition, B cells from CBA/N mice, which do not proliferate when stimulated with anti-Ig, anti-CD40, or both, exhibited only low levels of the protein following culture with these stimuli. To investigate the functional significance of bcl-x in activated B cells, we tested their sensitivity to apoptosis induced by the Ca²⁺ ATPase inhibitor thapsigargin: B cell blasts activated with anti-CD40 and anti-Ig were resistant to this agent. The available data therefore suggest that bcl-x fulfils two roles in B cells: it promotes survival of immature B cells (which lack bcl-2) and secondly, it apparently plays an additional role in protecting activated mature B cells (perhaps those in germinal centers) from apoptotic stimuli.     

B淋巴细胞刺激因子的研究进展

肿瘤坏死因子超家族成员B淋巴细胞刺激因子(BAFF)通过和隶属与肿瘤坏死因子受体超家族成员的BCMA、TACI和BAFF-R的结合,在调节外周成熟B淋巴细胞的发育过程中起着重要的作用。由于在多种自身免疫性疾病中的异常表达,BAFF和其受体也被认为是治疗自身免疫性疾病的重要治疗靶点。     

TLR9 responses of B cells are repressed by intravenous immunoglobulin through the recruitment of phosphatase

One way for intravenous Ig (IVIg) to affect responses of the B cells might be to operate through their TLR7 and TLR9. We confirm the ability of TLR agonists to induce CD25 expression in B cells. For this to occur, sialylated Fc-gamma of IgG included in the IVIg preparation are required. As a result, IVIg suppresses TLR-induced production of the proinflammatory IL-6, but not that of the anti-inflammatory IL-10. That is, IVIg mimics the effects of the MyD88 inhibitor. Finally, as we previously showed that IVIg induces CD22 to recruit the inhibitory SHP-1, we established that this enzyme was also involved in IVIg-induced inhibition of TLR9 signaling. This is the first report to demonstrate such a mechanism underlying the negative impact of IVIg on B lymphocytes.     

结核分枝杆菌脂糖ManLAM对CE蛋白抗原诱导的B细胞活化的影响

目的甘露糖修饰的脂阿拉伯甘露聚糖(mannose-capped lipoarabinomannan,ManLAM)是结核分枝杆菌细胞壁的主要成分之一,本研究拟探究ManLAM对CE蛋白诱导B细胞抗结核免疫应答的作用及潜在机制。方法磁珠分选活动性肺结核患者B细胞,ManLAM协同CE蛋白离体刺激,CFSE检测增殖,流式检测B细胞凋亡及活化,ELISA检测细胞因子及抗体亚型分泌水平,酶联斑点试验检测B细胞分化抗体分泌细胞的水平。结果ManLAM抑制CE蛋白诱导的B细胞增殖、活化、促炎因子释放和分化为CE蛋白特异性IgG分泌细胞,但对凋亡和分化为IgM分泌细胞无显著影响。ManLAM可通过TLR2抑制CE蛋白特异性IgG1和IgG3的分泌,诱导IgG4的分泌。结论本研究证实ManLAM可抑制B细胞抗结核免疫应答,为结核分枝杆菌免疫逃逸提供了新的理论依据。     

调节性B细胞与自身免疫性疾病

以往认为,B细胞通过产生特异性抗体提呈抗原产生共刺激分子活化T细胞、并通过细胞因子发挥免疫作用.但是近年来研究发现,B细胞对免疫应答和炎性反应有调节作用,这种B细胞被命名为调节性B细胞(Br).Br通过分泌调节性的细胞因子IL-10和TGF-β影响B细胞和致病性T细胞之间的相互作用而抑制病理性免疫反应.因而了解Br的生物学功能、活化机制以及在自身免疫性疾病中的作用具有重要意义.     

Negative regulation of signaling by a soluble form of toll-like receptor 9

Nucleic acid structures are highly conserved through evolution and when self nucleic acids are aberrantly detected by toll‐like receptors (TLRs) they contribute to autoimmune disease. For this reason, multiple regulatory mechanisms exist to prevent immune responses to self nucleic acids. TLR9 is a nucleic acid‐sensing TLR that is regulated at multiple levels including association with accessory proteins, intracellular localization and proteolytic processing. In the endolysosomal compartment TLR9 is proteolytically processed to an 80 kDa form (p80) and this processing is a prerequisite for activation. Here, we identified a soluble form of TLR9 (sTLR9) generated by a novel proteolytic event that cleaved TLR9 between amino acids 724–735. Similar to p80, sTLR9 was generated in endosomes. However, generation of sTLR9 was independent of the cysteine protease cathepsin B, active at acidic pH, but partially dependent on cathepsin S, a protease active at neutral pH. Most importantly, sTLR9 inhibited TLR9‐dependent signaling. Altogether, these data support a model where an intrinsic proteolytic processing mechanism negatively regulates TLR9 signaling. A proper balance between the independent proteolytic events probabably contributes to regulation of TLR9‐mediated innate immunity and prevention of autoimmune disease.     

FCRL3 promotes TLR9‐induced B‐cell activation and suppresses plasma cell differentiation

Fc receptor‐like (FCRL) molecules are preferentially expressed by B lymphocytes and possess tyrosine‐based immunoregulatory function. Although they generally inhibit B‐cell receptor signaling, their influence on other activation pathways remains largely unexplored. In humans, FCRL3 encodes a type I transmembrane protein harboring both cytoplasmic ITAM and ITIM elements that can repress B‐cell receptor activation. Despite this inhibitory property, mounting associations for FCRL3 with autoimmune and lympho‐proliferative disorders imply a role for it in promoting B‐cell pathogenesis. Here, we explore the influence of FCRL3 on B‐cell responses to innate TLR9 stimulation. A detailed survey of blood B‐cell populations found that FCRL3 expression increased as a function of differentiation and was higher among memory subsets with innate‐like features. FCRL3 ligation augmented CpG oligodeoxynucleotide TLR9‐mediated B‐cell proliferation, activation, and survival, but surprisingly, abrogated plasma cell differentiation and antibody production. Although FCRL3 amplified the NF‐κB and mitogen‐activated protein kinase signaling cascades, it halted CpG triggered BLIMP1 induction in an ERK‐dependent fashion. These findings indicate that FCRL3 differentially modulates innate signaling in B cells and provide new insight into the potential of this disease‐associated receptor to counter‐regulate adaptive and innate immunity.     

B cell subsets in postmenopausal women and the effect of hormone replacement therapy

Objectives: In elderly subjects the capacity for antibody production is depressed. This immunosenescence state of humoral immunity is associated with the occurrence of autoimmune disorders involving CD5⁺ B (B-1) cells. Since estrogen is capable of stimulating the production of autoantibodies, this sex steroid hormone may be a contributing cause of the higher incidence of autoimmune diseases in women. In the present study, B cell subsets in women during the postmenopausal period was determined. The effect of hormone replacement therapy (HRT) on B cell subsets was examined to establish whether the administration of gonadal hormones influence humoral immunity in postmenopausal women. Methods: Forty six untreated pre- and postmenopausal women and 39 women on HRT were studied. The proportion of B-1 (CD5⁺) and conventional CD5⁻ B (B-2) lymphocytes was determined by two-color flow cytometry. Serum autoantibodies to a nuclear antigen and to interleukin (IL)-1 were measured by immunofluorescence and by radioimmunoassay, respectively. Thirteen women were examined prospectively before and during HRT. Results: In late postmenopausal women (≥30 years postmenopausal period), the proportion of B-2 cells was significantly reduced (p<0.01) compared to those of premenopausal and perimenopausal women. HRT induced a significant (p<0.01) increase in the percentage of B-2 cells, while that of B-1 cells remained unchanged. HRT did not affect autoantibody production. Conclusion: HRT may retard the progress of immunosenescence by increasing the production of B-2 cells. Moreover, HRT appears not to increase the risk of autoimmune diseases developing in postmenopausal women.