共信号分子CD40/CD40L质粒标准品的构建

背景：目前,对CD40/D40L的研究主要集中在可溶性和蛋白方面,对其在转录水平上的研究文献不多,可能与其无商品化质粒标准品有关。 目的：构建检测CD40/CD40L mRNA表达水平差异的标准品。 设计、时间及地点：单一样本实验,于2009-04在苏州大学附属第一医院检验科实验室完成。 材料：取手术切除的子宫肌瘤组织,经患者知情同意。 方法：以手术切除的子宫肌瘤组织细胞的总RNA为模板、Random 6mers为引物反转录合成cDNA,用该cDNA为模板PCR扩增人CD40/CD40L基因相应的cDNA片断,构建PMD-18-CD40/CD40L重组质粒,鉴定测序后,用荧光定量PCR制作标准曲线。 主要观察指标：①RNA质量检测结果。②扩增的目的片段电泳结果。③CD40/CD40L基因测序结果。④标准品的扩增情况。 结果：组织提取的总RNA完整性良好,构建的CD40/CD40L质粒经PCR扩增后分别得到136 bp,200 bp的清晰条带,测序结果与目的片段完全一致,且质粒的原始浓度为2.66×1013、2.45×1013 copies/mL,倍比稀释至2.0×106 copies/mL均能得到良好的标准曲线(R2=1)。 结论：实验成功构建了CD40/CD40L基因荧光定量PCR标准质粒。     

The role of CD40 in CD40L- and antibody-mediated platelet activation

Our initial finding that CD40- and CD40 ligand (CD40L)-deficient mice displayed prolonged tail bleeding and platelet function analyzer (PFA-100) closure times prompted us to further investigate the role of the CD40-CD40L dyad in primary hemostasis and platelet function. Recombinant human soluble CD40L (rhsCD40L), chemical cross-linking of which suggested a trimeric structure of the protein in solution, activated platelets in a CD40-dependent manner as evidenced by increased CD62P expression. CD40 monoclonal antibody (mAb) M3, which completely blocked rhsCD40L-induced platelet activation, also prolonged PFA-100 closure times of normal human blood. In contrast, CD40 mAb G28-5 showed less potential in blocking rhsCD40L-induced CD62P expression and did not affect PFA-100 closure times. However, when added to the platelets after rhsCD40L, G28-5 significantly enhanced the platelet response by causing clustering of, and signaling through, FcgammaRII. Similarly, higher order multimeric immune complexes formed at a 1/3 molar ratio of M90, a CD40L mAb, to rhsCD40L induced strong Fcgamma RII-mediated platelet activation when translocated to the platelet surface in a CD40-dependent manner, including the induction of morphological shape changes, fibrinogen binding, platelet aggregation, dense granule release, microparticle generation and monocyte-platelet-conjugate formation. The results suggest that CD40 may play a role in primary hemostasis and platelet biology by two independent mechanisms: First, by functioning as a primary signaling receptor for CD40L and, second, by serving as a docking molecule for CD40L immune complexes. The latter would also provide a potential mechanistic explanation for the unexpected high incidence of CD40L mAb-associated thrombotic events in recent human and animal studies.     

Systemic upregulation of CD40 and CD40 ligand mRNA expression in multiple sclerosis

It is increasingly clear that the CD40 and CD40 ligand (CD40L) receptor-ligand pair mediates a crucial activation signal in both cell-mediated and humoral immune responses. Here, we detected mRNA levels of CD40 and CD40L in non-stimulated peripheral blood mononuclear cells in 46 patients with multiple sclerosis (MS) and 46 healthy controls by a competitive RT - PCR procedure allowing quantification without previous culture or antigenic stimulation. The levels of CD40 and CD40L mRNA were markedly increased in MS patients (P<0.0001) compared with healthy controls. There was no difference between clinical MS subgroups or stage of disease. Our findings indicate that, although MS is an organ specific disorder, an increased signaling via the CD40 and CD40L pathway may be present at the systemic level. The nature of this upregulation, whether primary or secondary to the organ-specific autoimmune response, is yet to be determined. Since interference with CD40/CD40L is an effective way to interfere with autoimmune model diseases such as experimental autoimmune encephalomyelitis, it may be relevant to investigate further the role of these molecules in the pathogenesis of MS.     

Neutrophil CD40 enhances platelet-mediated inflammation

INTRODUCTION: CD40 is a transmembrane protein expressed on monocytes, macrophages, endothelial cells, and platelets. Platelets are the richest source of soluble CD40 ligand (sCD40L) and interact with monocytes and endothelial cells via CD40. While CD40 was recently reported to be present on neutrophils, the detailed mechanism of its interaction with platelets via CD40-CD40L has not been examined. MATERIALS AND METHODS: The existence of neutrophil CD40 was verified by real-time PCR and western blot. Platelet sCD40L release was measured by ELISA. Neutrophil superoxide generation was measured by chemiluminescence and confocal microscopy. The neutrophil-platelet conjugates were measured by flow cytometry. RESULTS AND CONCLUSION: The presence of neutrophils enhances stimulation-induced platelet release of sCD40L. The addition of platelets leads to an enhancement of neutrophil superoxide and reactive oxygen species (ROS) generation. The specificity of the CD40-CD40L pathway in the neutrophil-platelet interaction was confirmed by using recombinant soluble CD40L (rsCD40L) and an anti-CD40L antibody. The involvement of the PI3 kinase/Akt pathway in neutrophil superoxide production was revealed by using LY294002 in isolated neutrophils/platelets experiments, as well as during whole blood aggregation-mediated neutrophil-platelet conjugation. N-acetylcysteine, a scavenger of ROS, eliminates both neutrophil superoxide generation and sCD40L release from activated platelets. These data suggest that activated neutrophils release ROS in a PI3 kinase-dependent manner, contributing to platelet activation and further sCD40L release in a redox-controlled positive feed-back loop. In conclusion, our results define a new pathway by which platelets and neutrophils interact and modulate each other's function, and may be relevant in understanding acute thrombo-inflammatory processes.     

Membrane-associated CD40L and sCD40L in atherothrombotic disease

Recent data suggests that CD40L is involved in the pathogenesis of atherothrombotic disease. This review will focus on the history of CD40L, its role in platelet-mediated pathogenesis of atherothrombotic disease, its association to clinical syndromes and procedures, and its role in determining plaque rupture.     

Induction of beta-chemokine secretion by human brain microvessel endothelial cells via CD40/CD40L interactions

beta-chemokines play an important role during the course of central nervous system (CNS) inflammation. Using primary cultures of human cerebral microvascular endothelial cells, we detected increased monocyte chemoattractant protein-1 (MCP-1) and regulated upon activation normal T cell expressed and secreted (RANTES) production following incubation with soluble CD40L. These results suggest a potential mechanism by which activated CD40L positive T cells may enhance beta-chemokine expression and thus influence the recruitment of mononuclear cells across the human blood-brain barrier.     

Expression of CD40 induces neural apoptosis

The tumor necrosis factor receptor superfamily includes 12 members, some of which (e.g., tumor necrosis factor receptor I and FAS) induce cell death triggered by ligand binding. Another member of the superfamily, the neurotrophin receptor p75^NTR, induces neural apoptosis, with apoptosis being inhibited by binding of ligand to the receptor. As such, it is a candidate for the mediation of neurotrophin dependence. Here, we show that CD40, a superfamily member that is closely related to p75^NTR, also induces neural apoptosis, but apoptosis is inhibited by binding of the G28-5 monoclonal antibody to CD40. These results provide further support for a model in which some members of the tumor necrosis factor receptor superfamily induce apoptosis triggered by ligand binding, whereas other members may, at least under certain conditions, induce apoptosis in the absence of ligand binding, with apoptosis being inhibited by binding of ligand or monoclonal antibody. J. Neurosci. Res. 50:383–390, 1997. © 1997 Wiley-Liss, Inc.     

Death receptor-mediated apoptosis in human malignant glioma cells: modulation by the CD40/CD40L system

CD40, a TNF-R-related cell surface receptor, is shown here to be expressed by glioma cells in vitro and in vivo. Glioma cell lines expressing low levels of CD40 at the cell surface resist cytotoxic effects of CD40L. CD40 gene transfer sensitizes glioma cells to CD40L. Inhibition of protein synthesis potentiates cell death which involves CD40 clustering and caspases 8 and 3 processing. CD40-transfected LN-18 cells acquire resistance to CD95L. In contrast, subtoxic concentrations of CD40L strongly sensitize these cells for TNF-alpha-induced apoptosis. Bispecific CD40xCD95 antibodies specifically kill glioma cells, disclosing the property of endogenous CD40 to facilitate death signalling.     

外周血淋巴细胞CD40／CD40L表达与多发性肌炎／皮肌炎发病关系

目的:初步探讨协同刺激分子CD40/CD40L在多发性肌炎(PM)/皮肌炎(DM)发病机制中的作用。方法:采用流式细胞学检测PM/DM患者外周血淋巴细胞CD40、CD40L的表达,并与正常健康人对照。结果:CD40、CD40L在正常健康人外周血淋巴细胞几乎无表达,但在PM/DM患者表达增高,PM患者CD40平均荧光强度为10.24±4.15,CD40L平均荧光强度为1.35±0.77;DM患者CD40平均荧光强度为13.67±5.83,CD40L平均荧光强度为1.73±0.98。PM/DM患者比较差异无显著性(P>0.05)。结论:CD40/CD40L的表达可能参与了PM/DM的发病。     

CD40L/CD40 bidirectional signaling is a major regulator of neuronal morphology in the developing nervous system

正Appropriate nervous system function depends on a precise but plastic neural architecture.Neuronal morphology determines how neurons interact with each other and with other cells.Every kind of neuron has its own morphological characteristics,which are determined by both intrinsic and extrinsic factors.In addition to intrinsic genetic programmes and patterns of neural activity,a variety of extrinsic factors regulate the growth and branching of neural processes and synaptogenesis.     

中国东北地区CD40及CD40L基因多态性与散发帕金森病的相关性分析

目的 探究CD40基因rs1883832位点及CD40L基因rs1126535位点单核苷酸多态性与帕金森病(Parkinson disease,PD)的相关性。方法 本研究纳入285名中国东北地区汉族健康人和396例PD患者。根据其发病年龄将PD组患者再分为早发PD组(发病年龄≤50岁)和晚发PD组(发病年龄 50岁),收集一般临床资料,提取外周血基因组DNA,利用MALDI-TOF-PEX技术检测rs1883832位点及rs1126535位点多态性分布情况,分析其与帕金森病的相关性。结果 EOPD组与对照组rs1883832位点基因型分布有统计学差异(P 0. 05),等位基因频率在两组间没有统计学差异。PD组和LOPD组与对照组在rs1883832位点及rs1126535位点上,基因型及等位基因型频率无统计学差异。结论 CD40基因rs1883832位点单核苷酸多态性与中国东北地区汉族早发帕金森病相关,T等位基因可能是EOPD的危险因素。     

Thrombin regulates CD40 expression in microglial cells

Microglial cells are the innate immune cells of the central nervous system and quickly respond to injury by proliferation, cytokine release, and increased cell surface antigen expression. Thrombin is a multifunctional serine proteinase, which has the capability to activate microglial cells. Here, we report that pharmaceutical-grade thrombin dose-dependently increases the expression of CD40 in N9 microglial cells. This effect is blocked by a thrombin inhibitor, mimicked by thrombin receptor-activating peptide and modified by mitogen-activated protein kinase pathway inhibitors. Thrombin-induced CD40 regulation might play a role in diseases with breakdown of the blood-brain barrier such as multiple sclerosis or stroke.     

Behavioral effects of CD40-CD40L pathway disruption in aged PSAPP mice

We have shown that, when an amyloid-beta peptide (Abeta) overproducing transgenic mouse model (PSAPP) of Alzheimer's disease (AD) is treated with a depleting antibody against CD40L, it causes marked attenuation of Abeta pathology associated with decreased amyloidogenic processing of amyloid precursor protein (APP) and increased cerebral clearance of Abeta. Here, we report that, when PSAPP mice receive a regimen of anti-CD40L antibody commencing at an age associated with initial Abeta deposition, they demonstrate superior spatial memory on the standard water maze and radial arm water maze tasks, as well as exhibiting superior non-spatial memory in the object recognition test, as compared to control PSAPP mice. Furthermore, PSAPP mice treated with an anti-CD40L antibody regimen commencing at an age associated with extensive Abeta deposition demonstrate superior spatial memory on the standard water maze task, as compared to control PSAPP mice. Disruption of CD40L activity has beneficial effects on pathology and cognitive behavior in the PSAPP mouse model, providing support for the therapeutic potential of interrupting the CD40-CD40L interaction in AD.     

Diurnal variation of soluble CD40 ligand in patients with acute coronary syndrome. Soluble CD40 ligand and diurnal variation

Introduction

We sought to investigate whether day-night variations occur in the concentration of circulating soluble CD40 ligand in patients with acute coronary syndrome, as this may have practical implications.

Materials and Methods

We assessed 70 consecutive ST-segment elevation myocardial infarction patients admitted into the Coronary Care Unit and 50 control subjects. Each subject was studied under strictly controlled light/dark conditions. Blood samples were drawn at 09:00 h (light phase) and 02:00 h (dark phase). Nocturnal blood samples were drawn by a trained nurse, with the help of a minute torch with a dim red light in order to avoid any direct lighting on the patient during sleep. The soluble CD40 ligand was measured using a commercially available ELISA.

Results

Soluble CD40 ligand levels showed no diurnal variations in control subjects. In the ST-segment elevation myocardial infarction group, however, soluble CD40 ligand concentration (pg/mL) in the light phase was significantly higher than that in the dark phase (167.3 ± 63.2 vs 118.9 ± 48.3 pg/mL, p < 0.001).

Conclusions

The study shows for the first time the existence of diurnal variations in soluble CD40 ligand levels in ST-segment elevation myocardial infarction patients, which indicates the need for standardizing the time of blood sampling for the assessment of this molecule, at least in studies involving ST-segment elevation myocardial infarction patients.     

Differences of soluble CD40L in sera and plasma: implications on CD40L assay as a marker of thrombotic risk

INTRODUCTION: Soluble CD40L (sCD40L) ELISA has emerged as a promising predictor of poor outcomes in acute coronary syndrome. Yet many blood processing techniques have been used with little consideration of their effect on the results. METHODS: We measured sCD40L by ELISA in 10 patients with thrombocytopenia and 12 with normal or high platelet counts and 8 healthy controls using three sampling techniques: serum clotted on ice (serum-I) or at room temperature (serum-RT) and platelet poor plasma (PPP). RESULTS: Serum-RT samples, compared to serum-I, gave significantly higher CD40L values (p=0.003), demonstrating that ex vivo sCD40L release by activated platelets is inhibited by cold temperature. Although serum-I and PPP were comparable in patients with normal platelet counts, serum-I gave significantly higher values than PPP in the thrombocytosis group (p=0.01), suggesting that cold inhibition is insufficient in the latter group. To estimate the fraction of sCD40L that was microparticle-bound CD40L (mp-CD40L), 16 samples underwent 0.1-microm filtration. 50.6% of sCD40L was mp-CD40L in serum-RT, whereas 21.3% and 29.9% were observed in serum-I and PPP, respectively. Lastly, plasma sCD40L was assayed in 46 patients with and 35 without thrombosis. Plasma sCD40L did not correlate with platelet count in non-thrombotic, non-inflammatory patients but did (p<0.01) in those with thrombosis. CONCLUSIONS: Sample processing and temperature profoundly affect sCD40L assay. Serum-I and PPP minimize the release of sCD40L ex vivo and better represent sCD40L in vivo. However, PPP may be preferable particularly in patients with thrombocytosis. The existence of mp-CD40L highlights the importance of centrifuge conditions.     

The role of platelets CD40 ligand (CD154) in acute coronary syndromes

Background

Despite of the proof of the biological function of CD154 on platelets, there has been little information about its role either in patients with stable angina or in those with acute coronary syndrome (ACS).

Objective

This study aimed to investigate the expression of CD154 on platelets and its role in ACS.

Methods

The study included 50 patients with ACS (24 patients with acute myocardial infarction (AMI) and 26 patients with unstable angina (UA)), 20 patients with stable angina (SA) and 18 healthy volunteers. CD154 and CD62 expression on platelets were analyzed by flow cytometry. Their relations to the clinical and laboratory data were assessed in the studied group.

Results

Patients with AMI and UA had higher levels of platelets CD154 and CD62 as compared to those with SA and among patients with AMI, UA and SA versus healthy volunteers. Platelets CD154 showed significant positive correlations with the studied pro-inflammatory markers (Ox-LDL, CRP and fibrinogen), segmental wall motion score and the studied risk factors. There were significant negative correlations between platelet CD154 and serum nitric oxide among patients.

Conclusions

CD154 may be used as a marker of thrombo-embolic events. Nitric oxide may have an anti-atherogenic effect. There is an association between platelet activation and severity of coronary artery disease among patients with ACS.     

Increased T cell expression of CD154 (CD40-ligand) in multiple sclerosis

CD154 (CD40-ligand, gp39), expressed on activated T cells, is crucial in T cell-dependent immune responses and may be involved in the pathogenesis of multiple sclerosis (MS). We studied cerebro-spinal fluid and peripheral blood T cell expression of CD154 in MS by flow cytometry. Patients with secondary progressive MS (SPMS) had constitutive CD154 expression on CD4 and CD8 T cells in blood. Constitutive CD154 expression was not observed in patients with relapsing-remitting MS (RRMS) or clinically isolated syndromes (CIS) suggestive of demyelinating disease. After ex vivo activation CD154 was, however, expressed on a higher percentage of T cells from patients with CIS or RRMS than from healthy control subjects. These results suggest involvement of CD154 in the pathogenesis of MS, and the shift from a relapsing-remitting to a secondary progressive disease course may be associated with constitutive, systemic CD154 expression.