Name: anti-nuclear antibody screening using hep-2 cells
Uploaded: 2014-06-23T18:15:00.000+00:00
Duration: 13 min 1 s
Description: Watch this Scientific Journal Video about Anti-Nuclear Antibody Screening Using HEp-2 Cells at JoVE.com

我们感谢卡桑德拉科比用于执行IIF实验和卡罗尔布氏她的专家技术审查。

1，样本选择和准备基板<ol><li>从包装中取出试剂，让每一个项目，以达到室温。制备的试剂和根据方向插入稀释病人血清。注：NOVA精简版幻灯片有条形码，并可以很容易地集成到自动化系统与实验室信息系统LIS结合。此过程说明了手动滑动处理;高通量实验室，然而，可以选择自动滑动处理设备。 </li></ol> 2，添加控件和样品<ol><li>分配1滴阳性对照和1滴的阴性对照，以适当的滑动孔中。吸取20-25微升稀释的患者血清其余井。处理一个幻灯片的时间。 </li><li>将滑动（收费）用湿纸巾在底部染色容器。覆盖容器，并培育在滑动（S）处理30分钟。注意：在潮湿条件下将防止基板干燥。孔中的干燥会导致伪迹染色。在这段温育期，在患者的血清中的抗核抗体将结合到被固定在每个孔中的细胞的抗原。 </li><li>培养结束后，用洗涤缓冲液缓流冲洗掉的血清。为了避免损坏基板，并防止样品井之间交叉，角度滑动略，以避免直接引导流上的孔中。点击关闭多余的洗涤缓冲液，然后将滑入含有洗涤缓冲液约5分钟染色壶。 </li></ol>荧光共轭3。加<ol><li>一个接一个，从洗涤缓冲液去除幻灯片和轻轻拍打，以去掉多余的洗涤缓冲液。申请1滴荧光结合物（FITC标记的抗人IgG）的到各孔中。孵育的幻灯片在加湿容器30分钟，并且一定要更换染色容器盖。注意：对于ANA检测，使用与IgG Fc特异性结合的建议。所述缀合物是光敏感的，并且盖子将保护载玻片暴露于光中除了保持湿润的环境。在这段温育期，该偶联物将结合到病人的已结合到细胞抗原抗核抗体。这个偶合物结合导致荧光的孔中的存在。 </li><li>冲洗，洗如上所述的幻灯片。 </li><li>放置盖玻片在纸巾上，并应用安装介质中的连续线到盖玻片的底部边缘。 </li><li>从洗涤缓冲液去除每张幻灯片，然后点击幻灯片轻轻去除多余的洗涤缓冲液。触摸滑动的下边缘到盖玻片的边缘。 </li><li>轻轻降低滑动到盖玻片在盖玻片上的安装介质流向没有气泡形成在滑动的顶部边缘的方式。注：合作verslipping，包括使用安装介质的最佳量，是一种技术，需要实践来完善。 </li></ol>正面和负面的结果4。鉴定手动解读<ol><li>查看幻灯片用荧光显微镜，坐落在一个黑暗的房间。用20X或25X的目标进行扫描整个井，以评估细胞分布和荧光的均匀性</li><li>切换到40X的目标做出关​​于积极和模式的最终解释权。 </li><li>看看阳性和阴性对照。使用反应分级标准从1 +到4 +级阳性。注：阴性对照可能不会出现完全黑暗的，但往往会显示低水平的非特异性荧光。阳性对照将显示明亮的苹果绿色荧光在细胞核中（ 图1）。 </li></ol>自动化的解读注意：在additi上人工判读，负载和扫描幻灯片由NOVA查看自动荧光显微镜或其他自动数字成像仪;无需暗室是必要的。通过选择合适的滑动式创建项目后，仪器采集和存储单元的高分辨率数字图像在每个孔中。此外，NOVA查看测量荧光强度，并进行分类的结果为正或负，并且提供了模式识别为阳性样品。图像是由一个高分辨率的电脑显示器上的操作者观看，允许最终解释权归NOVA查看结果，修订和确认。报告可以在确定的业绩产生。数码显微镜应与有经验的实验室技术人员一起使用，因为它的目的是在结果的解释提供帮助。

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作者加布里埃拉洛科什，卡桑德拉科比，卡罗尔布氏，和安娜伊斯拉米是INOVA诊断的雇员。

Screening by HEp-2 cells is a critical first step in the diagnosis of patients with SARD. However, IIF methods lack harmonization. Sources of variability include preparation of slides, conjugate specificity, and efficiency of the fluorescent microscope and experience of the reader. Despite these concerns, IIF remains the &#8220;gold standard&#8221; for ANA testing. HEp-2 cells contain a large variety of autoantigens, and provide the ideal substrate for the detection of these autoantibodies. In some laboratories, IIF has been replaced with solid phase screening methods such as multiplex assay or ELISA. The shortcoming of such tests is that they do not display the full range of antigens to be sufficiently sensitive. As a consequence, true positive patients may be missed which can have deleterious consequences In addition to the issues of treatment delay and wrong diagnosis, additional healthcare costs may occur due to the repetition of confirmatory tests or unnecessary diagnostic investigations. Given the inherent challenges to IIF, it is paramount to perform this technique properly to avoid subjectivity in results.
To ensure accurate interpretation and reporting of results for ANA screening, it is vital to use the highest quality substrate. When selecting a HEp-2 substrate, it is critical that the cells be optimized to express clinically relevant epitopes in their native protein state. Transfected or otherwise modified HEp-2 cell lines may not allow proper antibody-antigen binding. In addition, when several different autoantibodies are present simultaneously, recombinant cells may mask one or more patterns. High number of mitotic cells should also be present in the substrate. Adequate number of mitotic cells allows quick and accurate identification of patterns. 
In addition to the attributes of the substrate, the objective of this video protocol is to describe the IIF technique by showing critical steps such as the addition of sample, slide washing, addition of conjugate, cover slipping, and determination of positive and negative results. Results can be compromised if proper technique is not used for each of these steps. Proper washing is important to remove all unbound antibody. Some patients display very high amounts of autoantibodies and in these cases it is important to wash the serum from the wells such that it does not contaminate other wells. 
Although IIF has traditionally been a very labor intensive and subjective laboratory method, new technologies such as slide processors, barcoded slides and automated digital microscopy can greatly simplify the workflow, increase the consistency and reduce the sources of variability of interpretation.

术语抗核抗体（ANA）描述了多种与细胞的细胞核的成分包括DNA，蛋白质和核糖1,2反应的自身抗体。的Hep-2细胞，天然蛋白质阵列与数百抗原，提供了理想的底物为ANA 1的检测。全日空的人血清中检测是一个重要的筛选工具结缔组织疾病，IIF是全日空测试1的参考方法。最近，IIF在HEp-2细胞已被替换与抗原特异性免疫测定法和复用方法的一些实验室。由于担忧假阴性结果和缺乏透明度，临床医生，美国风湿病学院成立了一个特别工作组的结论是，IIF使用HEp-2细胞应该是“金标准”ANA筛查1。 由于主观ANA筛查的性质，HEp-2细胞的质量是在tegral到结果的准确和自信的报告。的大量有丝分裂的细胞的存在，最优细胞形态，足够汇合，以及相关抗原的表达是特别重要的。 IIF模式识别用作在患者以帮助诊断的重要工具。了解各种图案的意义使临床医生和实验室工作人员进行适当的后续测试，以确认诊断。例如，均相ANA模式可以发生在抗dsDNA或染色质的抗体的存在，并可能与全身性红斑狼疮（SLE）3相关联。从另一个方面，它最近已描述了所谓的致密的细斑点图案（DFS），其常见于常规样品的高达12％，主要是被用抗DFS70抗体有关。这些自身抗体，当隔离（无其他疾病特异性ANA）发现未与全身性自身免疫性风湿性疾病相关的&lt;燮&gt; 4-9。从而确证试验抗DFS70抗体可以帮助减少不必要的反射测试，提供了相当大的成本节约，并减轻病人的焦虑。 鉴于IIF是第一行筛选方法来检测自身抗体，它是最重要的，该用户能够理解如何试剂和组织基质的选择可影响结果。由于IIF技术本质上是主观的，重要的是，所用的试剂提供最高质量的结果。 该视频协议的这个目标是使用户熟悉执行IIF法，与ANA相关的常见模式所需的正确步骤，并引进新的进步，可以简化实验室工作流程和规范的结果。

<table><tbody><tr><td>NOVA Lite HEp-2 IgG (DAPI conjugate)</td><td>INOVA Diagnostics</td><td>708102</td><td></td></tr><tr><td>NOVA View Instrument</td><td>INOVA Diagnostics</td><td>NV2000</td><td></td></tr><tr><td>QUANTA Link Workstation</td><td>INOVA Diagnostics</td><td>LINK010</td><td></td></tr><tr><td>QUANTA Link Workstation License</td><td>INOVA Diagnostics</td><td>LINK001</td><td></td></tr><tr><td>Barcode Scanner</td><td>INOVA Diagnostics</td><td>LINK019</td><td></td></tr></tbody></table>

anti-nuclear antibody screening using hep-2 cells

对ANA检测风湿病立场声明的美国大学规定使用IIF作为全日空筛选1的金标准方法。虽然IIF是在专家手中一个很好的筛选试验，处理和阅读IIF的技术难点幻灯片 - 如劳动密集型幻灯片处理，人工读数，需要有经验的，训练有素的技术人员和使用黑暗的房间 - 使IIF法难以适应现代化，自动化实验室的工作流。 对高品质的ANA筛查的关键的第一步是仔细滑动处理。这个过程是劳动密集的，并且需要充分理解的过程中，以及关注细节和经验。 幻灯片阅读是通过荧光显微镜在黑暗的房间进行，由受过训练的技术人员谁是熟悉的各种图案，在细胞周期的背景下完成的和间期和分裂细胞的形态。但IIF是第一线筛检工具，可持续农业和农村，了解步骤正确执行此技术是至关重要的。 近年来，数字成像系统已经开发的IIF幻灯片自动读数。这些系统，如NOVA查看自动荧光显微镜，旨在简化日常IIF的工作流程。 NOVA查看获取并存储井的高分辨率数字图像，从而从解释分离图像采集;图像被视为一种解释型的高分辨率电脑显示器。它存储的图像以供将来参考，并通过提供对图像的荧光光强数据支持运营商的解释。它也预先进行分类的结果为正或负，并且提供了模式识别为阳性样品。总之，它省去了暗室，以及自动化和简化的IIF读写，程序纳克/口译工作流程。最重要的是，它增加了阅读器和读数之间的一致性。此外，与使用条形码的幻灯片，抄写错误是通过提供样品的可追溯性和积极的患者识别淘汰。这会导致增加病人的数据完整性和安全性。 此视频的总体目标是展示IIF程序，包括幻灯处理，识别常见的IIF模式，并引进了新的进步，以简化和协调这项技术。

当评估细胞染色结果喉癌Hep-2，五大核模式是最常见的：均匀，斑点，着丝粒，核仁和核点。这些模式的自身抗体结合至细胞核的特定成分的结果。虽然ANA检测是特异性的核结构，具有对细胞质结构自身抗体相关的细胞质图案也可以被观察到。在某些情况下，一些自身抗体可能存在于样品中，并且图像可能会显示为混合模式。 还有其它的，不经常观察到的，并且可以由IIF方法检测常细胞周期的特定模式;最近，所谓的密细斑点花纹具有特殊临床意义已经在某些ANA阳性血清描述。 均质模式要确定一个均匀的图案，扫描以及和识别有丝分裂或分割细胞。有丝分裂细胞的染色质凝集（ 图2，橙色箭头）显示出固体的均匀荧光这往往比在静息细胞nuclei.In均质模式，休眠细胞表现出均匀，整个核的弥漫性荧光（ 图更加明显2，白箭头）。这个特征模式往往是抗dsDNA抗体10的结果。 斑点型在粗斑点图案，有丝分裂的细胞显示没有染色简明的染色体区域（ 图3，橙色箭头）。在静息细胞中表现出粒状荧光在整个核。核中的斑点可以被定义为粗或细。粗斑点图案往往是抗Sm和抗RNP 11的结果。 在细斑点花纹，静息细胞显示细腻或弥漫性斑点整个细胞核，通常在统一ðistribution（ 图4中 ，白色箭头）。微细斑点图案往往是抗SSAand抗SSB 12的结果。 密集的细斑点型它现在认识到，致密细斑点图案（DFS）是常见的常规试验，并且多达12％的样品是阳性抗DFS70自身抗体。然而，这些自身抗体，当孤立地发现，不与全身性自身免疫性风湿性疾病4 -6关联。这些抗体多见于健康人，病人承载无关的可持续农业和农村6种疾病。验证性测试抗DFS70抗体可以帮助减少不必要的反射测试，提供可观的成本节约，并减轻病人的焦虑。 由于DFS模式是难以识别和ANA阳性会导致焦虑，以患者和医生，这是强烈建议鉴定​​后，进行确证试验<html> fying模式暗示抗DFS70抗体的澄清全日空的临床意义positivty 5,6。 在这个模式中，有丝分裂细胞（ 图5，橙色箭头）显示中期染色体阳性染色斑点，而静息细胞的细胞核（ 图5，白箭头）展览整个细胞核均匀分布的细小斑点。 着丝粒模式要识别着丝粒的模式，扫描井，并确定有丝分裂或分裂的细胞。在有丝分裂细胞（ 图6，橙色箭头），这些离散斑点成为什么经常被描述为一个“中期栏”密切相关。在着丝粒的模式，休眠细胞（ 图6，白箭头）显示整个细胞核分布约40-60离散斑点。着丝粒模式是抗着丝粒蛋白A中的结果，B，C抗体13。 步“&gt;核仁模式一些核仁图案被漫细胞质染色中的有丝分裂细胞中（ 图7，橙色箭头）和负极染色体区域相关联，而其他nucleolear图案显示染色体区域的阳性染色。核仁模式与核仁的同质或斑点染色（ 图7，白色箭头）相关联，随着弱斑点或核质的均匀染色。这些模式都与抗RNA聚合酶III相关联，抗核仁纤维，anti-Th/To和anti-PM/Scl抗体14,15。 核圆点图案核点图案与负中期有丝分裂的细胞（ 图8，橙色箭头），并与在静止细胞的细胞核几个离散斑点（ 图8中白色箭头）相关联。这个特征模式往往是SP-100，PML，或P80科林自身抗体的结果。这些抗体可以作为<html> sociated原发性胆汁性肝硬化和自身免疫性肝炎16。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig1highres.jpg" width="500" /> 图1：阴性对照（左）：电池显示低级别非特异性荧光，但没有具体的核染色阳性对照（右）：细胞显示苹果绿核荧光。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig2highres.jpg" width="500" /> 图2。同质格局。有丝分裂细胞（orangearrow）表明固体荧光。静息细胞显示均匀，弥漫性染色（whitearrow）。 “宽度=”500“/&gt; 图3。粗斑点型。有丝分裂细胞（橙色箭头）显示阴性染色。静息细胞表现出明显的斑点染色（白色箭头）。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig4highres.jpg" width="500" /> 图4。细斑点图案。有丝分裂细胞（橙色箭头）显示阴性染色。静息细胞表现出明显的细斑点染色（白色箭头）。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig5highres.jpg" width="500" /> 图5。密细斑点花纹。有丝分裂细胞（橙色箭头）显示出细粒状固体染色。静息细胞表现出非常精细的，弥漫性斑点状染色（whitearrow）。 图6。着丝粒的模式。有丝分裂细胞（orangearrow）显示均匀，离散斑点。静息细胞（whitearrow）显示每个细胞核40-60离散斑点。 <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig7highres.jpg" width="500" /> 图7。核仁格局。有丝分裂细胞（orangearrow）出现大簇颗粒染色。静息细胞nucleishows在核仁荧光（whitearrow）。 <img alt="图8" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig8highres.jpg" width="500" /> 图8。核圆点图案。有丝分裂细胞（orangearrow）出现负值。静息细胞显示了几个DISCR在细胞核中（whitearrow）ETE斑点。此外，nucelar点图案可以与自身抗体线粒体抗原（红色箭头）相关的细胞质染色共存。

Watch this Scientific Journal Video about Anti-Nuclear Antibody Screening Using HEp-2 Cells at JoVE.com

Anti-Nuclear Antibody Screening Using HEp-2 Cells

间接免疫荧光（IIF）的测定法在传统上被用于抗核抗体（ANA）的人血清中的检测。这些抗体的存在可以在全身性自身免疫性风湿病（SARD）帮助诊断。该协议演示了如何有效地进行IIF技术，准确检测这些自身抗体。

抗核抗体筛选使用Hep-2细胞

The American College of Rheumatology position statement on ANA testing stipulates the use of IIF as the gold standard method for ANA screening1. Although ...

anti-nuclear-antibody-screening-using-hep-2-cells

Research

JoVE Journal

Biology

134.3K 次观看.  INOVA Diagnostics, Inc.. 间接免疫荧光（IIF）的测定法在传统上被用于抗核抗体（ANA）的人血清中的检测。这些抗体的存在可以在全身性自身免疫性风湿病（SARD）帮助诊断。该协议演示了如何有效地进行IIF技术，准确检测这些自身抗体。 

视频: Anti-Nuclear Antibody Screening Using HEp-2 Cells

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

用于鉴定抗体依赖性细胞毒性修饰化合物的高内涵筛选检测

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells ...

科研

癌症研究

A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load but not result in complete remission. An efficient hepatocyte model for HBV infection would offer a true-to-life viral life cycle that would be crucial for the screening of therapeutic agents. Most available anti-HBV agents target lifecycle stages post viral entry but not before viral entry. This protocol details the generation of a competent hepatocyte model capable of screening for therapeutic agents targeting pre-viral entry and post viral entry lifecycle stages. This includes the targeting of sodium taurocholate cotransporting polypeptide (NTCP) binding, cccDNA formation, transcription, and viral assembly based on imHC or HepaRG as host cells. Here, the HBV entry inhibition assay used curcumin to inhibit HBV binding and transporting functions via NTCP. The inhibitors were evaluated for binding affinity (KD) with NTCP using isothermal titration calorimetry (ITC)-a universal tool for HBV drug screening based on thermodynamic parameters.

We present a protocol to screen anti-hepatitis B virus (HBV) compounds targeting pre- and post-viral entry lifecycle stages, using isothermal titration calorimetry to measure binding affinity (KD) with host sodium taurocholate cotransporting polypeptide. Antiviral efficacy was determined through the suppression of viral lifecycle markers (cccDNA formation, transcription, and viral assembly).

一种通过牛磺胆酸钠共转运多肽作为治疗靶点检查乙型肝炎病毒进入的感受态肝细胞模型

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load ...

生物化学

Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies available for ANA screening, indirect immunofluorescence (IIF) using Human epithelial cells-2 (HEp-2) substrate remains the primary and recommended method because of its superior sensitivity. HEp-2 substrates can detect a multitude of patterns resulting from autoantibody binding to various protein and nucleic acid autoantigens distributed throughout the nucleus and cytoplasm of the cells. The great diversity of monospecific and mixed patterns resulting from positive reactions on HEp-2 substrate also complicate the interpretation and accuracy of reporting. One specific example which received utmost attention recently is the dense fine speckled 70 (DFS70) pattern resulting from autoantibodies that specifically bind to a protein called lens epithelium derived growth factor (LEDGF). Lack of clear association with a specific systemic autoimmune disease and high prevalence in healthy populations have made accurate interpretation of DFS70 pattern important. Accurate distinction of DFS70 pattern from disease-associated patterns using conventional HEp-2 substrate is challenging. Moreover, frequent co-occurrence of DFS70 pattern along with disease-associated patterns such as homogeneous, speckled, and mixed homogeneous-speckled patterns complicate the IIF interpretation. The goal of this paper is to demonstrate the utility of a novel engineered HEp-2 IIF substrate that retains all advantages of conventional HEp-2 substrate while simultaneously providing the ability to distinguish DFS70 pattern with high confidence in both monospecific and mixed ANA positive examples. The new substrate is further able to unmask disease-associated ANA patterns previously concealed by DFS70 pattern.

DFS70 autoantibodies mimic common disease-associated antinuclear antibody patterns making accurate interpretation challenging when using conventional HEp-2 substrates. The protocol describes advantages of novel engineered HEp-2 substrate over conventional HEp-2 in ANA screening and distinguishing DFS70 patterns with high confidence in both monospecific and mixed ANA positive cases.

Monospecific 和混合 DFS70 模式在 HEp-2 ELITE/DFS70 击倒底物筛选中的同时鉴别

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies ...

免疫与感染

Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The characterization of these anti-drug antibodies (ADA), especially those that may neutralize the biological activity of the drug, termed neutralizing antibodies (NAbs), is crucial in understanding the effects of these antibodies on the drug&#39;s pharmacological profile. This protocol describes a cell-based flow cytometry method to detect factors that neutralize the cellular uptake of a representative lysosomal ERT in human matrix. The protocol consists of three procedures: screening, a confirmatory step, and titer assays to detect, identify, and establish the relative level of neutralizing antibody titer in subject samples.
In this method, samples are first mixed with the fluorophore-conjugated ERT product, then incubated with cells [e.g., human T lymphocytes (Jurkat cells)] that express a cell-surface cation-independent mannose 6-phosphate receptor (CI-M6PR), and finally, analyzed with a flow cytometer. A sample without NAbs will result in the uptake of the fluorophore-conjugated ERT product via CI-M6PR, whereas, the presence of NAbs will bind to the drug and interfere with the CI-M6PR binding and uptake. The amount of the fluorophore-conjugated ERT internalized by the Jurkat cells is measured by flow cytometry and evaluated as the percentage (%) signal inhibition compared to the response obtained in the presence of a representative drug-na&#239;ve matrix. In the confirmatory step, the samples are pre-incubated with ERT-conjugated magnetic beads to deplete drug-specific factors that bind to the drug (such as NAbs) prior to an incubation with cells. Samples that screen and confirm positive for drug-specific NAbs in the assay are then serially diluted to generate an antibody titer. Semi-quantitative antibody titers may be correlated with measurements of drug safety and efficacy.

Here we present a cell-based flow cytometry method to detect neutralizing antibodies or other factors that interfere with the cellular uptake of enzyme replacement therapies in a human matrix, such as cerebral spinal fluid (CSF) or human serum.

细胞法检测酶替代疗法中细胞吸收的抗体

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The ...

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

乙型肝炎病毒感染原代肝细胞和库珀细胞的 "肝片上" 培养

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

诱导BALB/c小鼠药物诱导的自身免疫性肝炎研究其致病机制

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293T&#160;overexpressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines.&#160;HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.&#160;&#160;

This manuscript describes a detailed protocol for Hepatitis B virus (HBV) infection in novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and traditional hepatic cells (HepG2-NE, expressing human NTCP).

非肝细胞293T-NE-3NRS细胞中乙型肝炎病毒感染的建模

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV ...

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.

We present a robust, cost-effective, and flexible method for measuring changes in hepatocyte number and nuclear ploidy within fixed/cryopreserved tissue samples that does not require flow cytometry. Our approach provides a powerful sample-wide signature of liver cytology ideal for tracking the progression of liver injury and disease.

小鼠肝细胞核球状估计的高通量原位方法

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as ...

医学

Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently inhibit viral replication, they have no direct effect on the expression of viral proteins transcribed from a viral covalently closed circular DNA (cccDNA). As high viral antigen load may play a role in this chronic and HBV-related carcinogenesis, the goal of HBV treatment is to eradicate viral proteins. HBV regulatory protein X (HBx) binds to the host DNA damage-binding protein 1 (DDB1) protein to degrade structural maintenance of chromosomes 5/6 (Smc5/6), resulting in activation of viral transcription from cccDNA. Here, using a split luciferase complementation assay system, we present a comprehensive compound screening system to identify inhibitors of the HBx-DDB1 interaction. Our protocol enables easy detection of interaction dynamics in real time within living cells. This technique may become a key assay to discover novel therapeutic agents for treatment of HBV infection.

Here, we present a method for screening anti-hepatitis B viral agents that inhibit the HBx-DDB1 interaction using a split luciferase assay system. This system allows easy detection of protein-protein interactions and is suitable for identifying inhibitors of such interactions.

使用分体路西酶测定系统识别 HBx-DDB1 相互作用的抑制剂

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently ...

通过线粒体-ALCAM 分选纯化人 iPSC 来源的肝细胞

Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells

Human embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications in cell-based regenerative medicine for treating severely diseased organs due to their unlimited proliferation and pluripotent properties. However, differentiating human ES/iPS cells into 100% pure target cell types is challenging due to their high sensitivity to the environment. Tumorigenesis after transplantation is caused by contaminated, proliferating, and undifferentiated cells, making high-purification technology essential for the safe realization of regenerative medicine. To mitigate the risk of tumorigenesis, a high-purification technology has been developed for human iPS cell-derived hepatocytes. The method employs FACS (fluorescence-activated cell sorting) using a combination of high mitochondrial content and the cell-surface marker ALCAM (activated leukocyte cell adhesion molecule) without genetic modification. 97% &plusmn; 0.38% (n = 5) of the purified hepatocytes using this method exhibited albumin protein expression. This article aims to provide detailed procedures for this method, as applied to the most current two-dimensional differentiation method for human iPS cells into hepatocytes.

作者聚焦:推进从人诱导多能干细胞中纯化肝细胞用于再生医学

Hepatocytes derived from pluripotent stem cells can be purified through cell sorting, using a combination of mitochondrial and activated leukocyte cell adhesion molecule (ALCAM, also known as CD166) staining.

活线粒体染色在细胞分选中纯化人诱导多能干细胞肝细胞的应用

Human embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications in cell-based regenerative medicine for treating severely ...