An in vitro Co-culture System for the Activation of CD40 by Membrane-presented CD40 Ligand versus Soluble Agonist
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Author:
Date:
2018-07-05
[Abstract] One fundamental property of the TNR receptor (TNFR) family relates to how ‘signal quality’ (the extent of receptor ligation or cross-linking) influences the outcome of receptor ligation, for instance the induction of death in tumour cells. It is unequivocal that membrane-presented ligand (delivered to target cells via cell-surface presentation by co-culture with ligand-expressing third-party cells) induces a greater extent of carcinoma cell death in vitro in comparison to non-cross-linked agonists (agonistic antibodies and/or recombinant ligands). The CD40 receptor epitomises this fundamental property of TNF receptor-ligand interactions, as the extent of CD40 cross-linking dictates cell fate. Membrane-presented CD40 ligand (mCD40L), but not soluble agonists (e.g., ...
[摘要] TNR受体(TNFR)家族的一个基本特性涉及“信号质量”(受体连接或交联的程度)如何影响受体连接的结果,例如肿瘤细胞中的死亡诱导。毫无疑问,膜呈递配体(通过与表达配体的第三方细胞共培养通过细胞表面呈递递送至靶细胞)在体外诱导更大程度的癌细胞死亡非交联激动剂(激动性抗体和/或重组配体)。 CD40受体集中体现了TNF受体 - 配体相互作用的这种基本特性,因为CD40交联的程度决定了细胞命运。膜呈递CD40配体(mCD40L),但不是可溶性激动剂(例如,激动性抗CD40抗体),诱导高水平的促炎细胞因子分泌并导致恶性肿瘤细胞广泛死亡(细胞凋亡)但不是正常的)上皮细胞。在本文中,我们描述了通过mCD40L激活CD40并随后检测细胞凋亡的各种特征(包括细胞膜透化,DNA片段化,半胱天冬酶活化)以及细胞内细胞死亡介质检测的共培养系统(包括衔接蛋白,促凋亡激酶和活性氧,ROS)。
【背景】TNFR及其配体在调节淋巴组织以及上皮(尤其是癌)细胞中的细胞增殖或死亡中的作用已经在广泛研究中,因为它们诱导细胞死亡(主要通过细胞凋亡)的能力代表了有希望的目标。用于癌症治疗。然而,重要的是,当以可溶性对膜结合形式存在时,TNFR激动剂引发细胞死亡的能力存在明显差异。当作为单独治疗施用时,可溶性激动剂通常表现出相对低的细胞毒性效力,而膜呈递的配体似乎是优越的(Albarbar ...
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An Optimized Method for the Production Using PEI, Titration and Neutralization of SARS-CoV Spike Luciferase Pseudotypes
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Author:
Date:
2017-08-20
[Abstract] The protocol outlined represents a cost-effective, rapid and reliable method for the generation of high-titre viral pseudotype particles with the wild-type SARS-CoV spike protein on a lentiviral vector core using the widely available transfection reagent PEI. This protocol is optimized for transfection in 6-well plates; however it can be readily scaled to different production volumes according to application. This protocol has multiple benefits including the use of readily available reagents, consistent, high pseudotype virus production Relative Luminescence Units (RLU) titres and rapid generation of novel coronavirus pseudotypes for research into strain variation, tropism and immunogenicity/sero-prevalence.
[摘要] 概述的方案代表了使用广泛可用的转染试剂PEI在慢病毒载体核心上产生具有野生型SARS-CoV刺突蛋白的高滴度病毒假型颗粒的成本效益,快速和可靠的方法。 该协议针对6孔板中的转染进行了优化; 然而,根据应用,它可以容易地扩展到不同的生产量。 该方案具有多种益处,包括使用容易获得的试剂,一致的,高假型病毒生产相对发光单位(RLU)滴度,并快速产生用于研究菌株变异,趋向性和免疫原性/血清流行性的新型冠状病毒假型。 【背景】假型病毒颗粒(PV)的生产和使用对于许多病毒是广泛建立的,并且在血清学,监测和疫苗开发领域的应用是多方面的(Temperton等人,2015; Carnell等人,2015)。 PV已经被证明是研究病毒包膜糖蛋白突变对血清学结果,病毒向性和免疫原性研究的影响的有力工具,特别是当与表位信息结合时。 PV是嵌合病毒构建体,其中一个病毒的外(表面)糖蛋白与另一病毒的复制缺陷病毒核心组合。 ...
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Reporter Assay for Semen-mediated Enhancement of HIV-1 Infection
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Author:
Date:
2016-07-20
[Abstract] Semen contains amyloid fibrils that enhance HIV-1 infection (Münch et al., 2007; Kim et al., 2010; Roan et al., 2011; Arnold et al., 2012; Usmani et al., 2014; Roan et al., 2014). Positively charged semen amyloids capture negatively charged viral particles and increase their attachment rates to the cell surface resulting in enhanced fusion and infection (Roan et al., 2009). Since semen is highly cytotoxic, we developed an assay that allows quantification of the infection enhancing activity of semen while minimizing its cell damaging activity. Here, we describe two protocols that allow the quantification of the infectivity enhancing activity of semen using a reporter cell line (TZM-bl cells) or peripheral blood mononuclear ...
[摘要] 精液含有增强HIV-1感染的淀粉样蛋白原纤维(Münch等,2007; Kim等,2010; Roan等,2011; Arnold等,2012; Usmani等,2014; Roan et al。 ,2014)。 带正电的精液淀粉样蛋白捕获带负电的病毒颗粒并增加其对细胞表面的附着率,导致增强的融合和感染(Roan等人,2009)。 由于精液具有高度的细胞毒性,我们开发了一种能够定量精液感染增强活性同时最大限度降低其细胞损伤活性的测定方法。 在这里,我们描述允许使用报道细胞系(TZM-bl细胞)或外周血单核细胞(PBMC)定量精液的感染性增强活性的两种方案。
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