Name: an immunohistopathologic study to profile the folate receptor beta macrophage and vascular immune microenvironment in giant cell arteritis
Uploaded: 2019-02-08T12:30:00
Description: Watch this Scientific Journal Video about An Immunohistopathologic Study to Profile the Folate Receptor Beta Macrophage and Vascular Immune Microenvironment in Giant Cell Arteritis at JoVE.com

这项工作是在道格拉斯·斯泰尔博士、玛丽安·克林格、安·本科、风湿病系/医学司以及宾州立大学医学院分子和组织病理学核心实验室的支持下得以完成的。

所述所有方法都得到了宾州机构审查委员会的批准。
1. 获得时间动脉活检后的组织病理学准备及处理
请注意:颞动脉活检 (tab) 是在局部麻醉和无菌条件下由认证的外科医生进行的。手术后, 获得3厘米动脉部分在受影响较大的一侧, 标本被固定在福尔马林立即 24小时, 分为3至4毫米的部分, 并嵌入石蜡, 注意适当的对齐组织在块然后在室温下储存。...

<ol>
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gca 是成人中最常见的血管炎, 其病理特征体现了 t 辅助淋巴细胞和活化巨噬细胞的有力组合, 也可以在其他肉芽肿性疾病或血管病, 如结节病和肉芽肿性多血管炎2,3。在 gca 中, 颞动脉提供了一个中到大大动脉的良好表示, ta 活检提供了一个相当大的样本, 可以存储在石蜡块多年, 从而创造了机会, 研究新兴诊断和像 frb 这样的治疗靶点在血管微环境中。我?...

巨细胞动脉炎 (gca) 是一种针对主动脉及其分支并影响老年人的中大动脉炎症性疾病。它表现为轻度至重度缺血性并发症, 如头痛、下巴疼痛、视力下降、中风和组织坏疽。诊断是由高炎症标志物, 如红细胞沉降率 (esr) 和一个独特的组织病理学模式的颞动脉活检 (tab)1证实。gca 是最常见的成人血管炎, 获得诊断的颞动脉的可及性比其他血管病变具有优势, 从而使人们能够更容易地研究其发病机制。tab 上的典型发现包括巨噬细胞 (巨噬细胞) 和 t 淋巴细胞浸润, 这些血管层在内膜、培养基和肌细胞的所有维度层中都有浸润, 同时破坏了通常将这些血管层分开的弹性层隔间2。目前的证据表明, gca 涉及一种未知的抗原, 它激活血管外膜中的树突状细胞, 然后招募辅助 t (th) 细胞, 特别是分泌白细胞介素-17 和干扰素伽玛 (ifg)。ifg 然后招募和激活巨噬细胞产生细胞因子和蛋白酶。这些包括促炎细胞因子;包括 il-12, 它相互激活 th1 细胞, 从而提供一个正反馈回路;肿瘤坏死因子和白细胞介素-6, 引起关节炎和发烧和金属蛋白酶, 损害弹性层。糖皮质激素 (gc) 构成标准的慢性治疗, 通过衰减 th17 il-17 而不是免疫反应的 thiifg 臂 3, 4,部分控制细胞因子通路.不幸的是, 停止 gc 导致疾病复发。作为一种替代方法, 甲氨蝶呤已被重新用于 gca 治疗, 但尽管更敏感的生物标志物尚未用于治疗监测5,6, 但所需的临床终点并没有一致实现。.2017年, 白细胞介素6阻滞剂 tocilizumab 被批准用于治疗 gca, 因为它有效地显示了疾病控制和类固醇的保护效果7,8。
到目前为止, gca 还没有很好的生物标志物。因此, 很难监测疾病活动和对现有药物的滴定剂量, 以避免不利影响, 减轻社会成本负担。因此, 必须寻找与疾病活动相关的候选生物标志物, 提供新的关于发病机制的见解, 并在治疗决策中提供帮助。
巨噬细胞活化失调是 gca 发病机制的关键因素。因此, 治疗 gca 的有效方法可能是选择性靶向活化巨噬细胞。叶酸受体β (frb) 是一种糖基磷脂酰肌醇糖蛋白, 锚定在正常粒细胞细胞、髓样白血病细胞和活化巨噬细胞中表达的细胞膜上。它的配体, 叶酸, 是一种必需的维生素, 在其减少的形式, 这使得细胞 dna 合成, 甲基化和修复9。frb 在自身免疫性疾病和癌变过程中被诱导表达。其表达仅限于类风湿关节炎单核细胞或促炎性 m1 巨噬细胞的表面, 或在固体器官和骨髓恶性肿瘤中的抗炎 m2 巨噬细胞 10, 11,12,13. 除了其作为活化巨噬细胞的生物标志物的潜在作用外, frb 还可以通过一个多步骤的过程, 从细胞内的受体与叶酸、抗叶酸或叶酸结合药物开始, 从而实现选择性药物运输表面, 然后将所需药物内化、释放并最终输送到内部细胞机械 12、14、15、16.与 frb 在癌细胞和活化巨噬细胞中表达相比, 在正常造血细胞中表达的 frb 无法结合叶酸, 从而确保 frb/folt 介导的药物传递仅限于 frb 阳性炎症或癌细胞。
在我们之前的研究中, 我们首次证明了 frb 在 gca 巨噬细胞中的表达, 其表达与 cd688+ 和内皮素受体β巨噬细胞相关, 这对 gca 的发病机制17有一定的贡献。在本报告中, 我们描述了用于评估 frb 巨噬细胞的组织病理学和免疫组织化学方法, 并分析了总淋巴细胞和巨噬细胞计数, 以深入了解 frb 在 gca 血管景观中的位置。

<table>
	<tbody>
		<tr>
			<td>Microtome</td>
			<td>Reichert-Jung</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>plain and frosted microscope slides and cover slips</td>
			<td>Fisher Scientific&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical rack</td>
			<td>Fisher Scientific&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Olympus BX60&#160; microscope</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone in distilled water</td>
			<td></td>
			<td></td>
			<td>concentrations 100%, 90%, 70%</td>
		</tr>
		<tr>
			<td>Tris-buffered saline solution (TBS)&#160;</td>
			<td>Dako Wash BufferS3006</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3% hydrogen peroxide in TBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Diaminobenzidine (DAB)</td>
			<td>Sigma Fast Tablet set</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemical Permount&#160; Mounting Medium</td>
			<td>Fisher Scientific&#160;</td>
			<td>SP-15-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Harleco Gill&#39;s III Hematoxylin</td>
			<td>Fiisher Scientific 23-750-019</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Harleco Eosin Y 1% Alcoholic Stock Solution</td>
			<td>Fisher Scientific&#160;</td>
			<td>23-749-977</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM citric acid monohydrate (pH 6.0)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal rabbit anti-folate receptor beta&#160;</td>
			<td>Manohar Ratnam</td>
			<td></td>
			<td>optimized at 1:800</td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-human CD68</td>
			<td>Dako&#160;</td>
			<td>M071801</td>
			<td>optimized at 1:200</td>
		</tr>
		<tr>
			<td>Polyclonal rabbit anti-CD3</td>
			<td>Agilent Dako</td>
			<td>A0452</td>
			<td>optimized at 1:100</td>
		</tr>
		<tr>
			<td>Polymer goat anti- rabbit/mouse secondary antibody</td>
			<td>Dako</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Placental tissue</td>
			<td></td>
			<td></td>
			<td>&#160;for FRB positive control</td>
		</tr>
	</tbody>
</table>

an immunohistopathologic study to profile the folate receptor beta macrophage and vascular immune microenvironment in giant cell arteritis

巨细胞动脉炎 (gca) 是一种影响老年人的中大型动脉慢性免疫介导的疾病。gca 表现为关节炎和头痛、中风或视力下降的闭塞症状。巨噬细胞和 t 辅助淋巴细胞浸润血管壁, 产生促炎症反应, 导致血管损伤和缺血。到目前为止, 还没有 gca 生物标志物可以监测疾病活动和指导治疗反应。
叶酸受体β (frb) 是一种糖基磷脂甾醇蛋白, 锚定在细胞膜上, 通常表达在骨髓细胞系和大多数骨髓性白血病细胞, 以及在肿瘤和类风湿滑膜巨噬细胞,它的表达与疾病的严重程度有关。frb 结合叶酸化合物、叶酸结合剂和抗叶酸药物的能力使其成为癌症和炎症性疾病研究中的一个可吸毒靶点。本报告描述了组织病理学和免疫组化方法, 用于评估 frb 的表达和分布与 gca麻醉正理学。
用血红素和 eosin 对 gca 和正常对照组的福尔马林固定和石蜡包埋的颞动脉活检进行染色, 以回顾组织组织学特征并确定其病理学特征。免疫组织化学用于检测 frb、cd68 和 cd3 的表达。进行了微观分析, 以量化10个选定的高功率场部分的阳性染色细胞的数量及其各自在动脉壁的位置。
淋巴缺血细胞 (lh) 炎症伴内膜增生和弹性片受干扰在 gca 中被发现, 对照组没有发现。lh 浸润由大约60% 的淋巴细胞和40% 的巨噬细胞组成。frb 表达仅限于巨噬细胞, 占 cd688+ 巨噬细胞总数的 31%, 并局部到媒体和外膜。在控件中没有看到 frb。
该方案证明了 frb 巨噬细胞相对于 gca 血管免疫微环境的独特的数值和空间模式。

病理组织学发现
正常标本中的 h & e 染色显示正常动脉解剖, 内膜内皮细胞, tunica 培养基中的平滑肌细胞, 以及包括成纤维细胞和供体血管的异质胶原蛋白基质, 称为 vasa vasorum在 tunica 外膜。没有发现炎症浸润。gca 正颞动脉表现为中度至重度炎症, 具有巨噬细胞、淋巴细胞、偶尔的浆细胞和多核巨细胞聚集。注意到?...

Watch this Scientific Journal Video about An Immunohistopathologic Study to Profile the Folate Receptor Beta Macrophage and Vascular Immune Microenvironment in Giant Cell Arteritis at JoVE.com

An Immunohistopathologic Study to Profile the Folate Receptor Beta Macrophage and Vascular Immune Microenvironment in Giant Cell Arteritis

该协议说明了使用组织病理学检查和免疫组织化学来描述叶酸受体β巨噬细胞及其与全免疫细胞浸润的关系, 在颞动脉活检在巨细胞动脉炎。

巨细胞动脉炎叶酸受体贝塔巨噬细胞和血管免疫微环境的免疫组织病理学分析

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis ...

这种免疫组织化学和组织病理学协议可用于检查叶酸受体β，叶酸受体β是一种候选蛋白质，对巨型细胞动脉炎的血管微环境具有潜在的预测和治疗价值。该协议经过时间考验，被广泛使用，可用于探索叶酸受体β属性在时间动脉组织中，即使在长期存储后。为了获得组织部分，使用微原子切开四到五微米厚的石蜡嵌入时间动脉，在获得时将每个部分漂浮在40摄氏度的水浴中，以消除皱纹。使用玻璃显微镜幻灯片每张幻灯片可捕获三个部分，并在加热板上加热幻灯片，在 60 摄氏度的烤箱中加热 60 分钟。当部分粘附在幻灯片上时，将幻灯片放入垂直机架中，在 37 摄氏度下干燥 24 小时。第二天，将幻灯片转移到幻灯片容器中，在 4 摄氏度下存储至少 12 小时。接下来，负载滑入滑架，用两个5分钟，100%二甲苯浸入和下降，5分钟每个浓度乙醇浸入系列，每30秒搅拌10秒水合组织。70%乙醇浸入后，在双蒸馏水中冲洗两次幻灯片5分钟，每30秒搅拌10秒。对于抗原回收，将机架转移到一个玻璃容器中，其玻璃容器内有 200 微升 95 摄氏度，每升柠檬酸缓冲液为 10 毫摩尔，使用 30 分钟。在孵化结束时，让滑梯冷却到室温20分钟，然后用自来水冲洗5分钟。为了去除内源性过氧化物酶活性，首先使用疏水标记在每个组织样本周围画一个圆圈，然后将样品孵育为200微升的3%过氧化氢10分钟，然后每次洗涤的300微糖中洗3次三分缓冲盐水溶液（TBSS）。最后一次洗涤后，轻轻涂抹每张幻灯片，去除多余的缓冲液，并在每张幻灯片上加入200微升的原抗体鸡尾酒，并在室温下在潮湿室中孵育一小时。在孵育结束时，丢弃盖滑，用三个两分钟的洗涤液在新鲜的TBSS中冲洗幻灯片。去除多余的缓冲液后，将200微升适当的生物素化二次抗体溶液和盖滑到每张幻灯片上，在室温下在潮湿室中孵育45分钟。丢弃盖滑后，使用 TBSS 冲洗幻灯片，如演示。删除任何多余的缓冲区。并在室温下用200微升DAB基板缓冲液标记各部分10分钟。在孵育结束时，用 TBSS 和自来水清洗幻灯片 10 分钟，然后用赤氧树脂冲刷 3 分钟。然后，将 70 微升适当的安装介质添加到每个幻灯片上，然后缓慢地将玻璃盖条翻到安装介质上，以避免在盖滑降低到位时产生气泡。对于组织病理学分析，将幻灯片放在光学显微镜的舞台上，并评估第一组织部分的血管结构。然后，量化巨噬细胞相对于每节淋巴细胞数的数量。对于免疫组织化学分析，检查每个组织部分的叶酸受体β染色模式，并量化叶酸受体β阳性、CD68阳性和CD3阳性细胞的数量，每节随机选择10个高功率场。血液氧林和雌激素染色在正常标本揭示了一个正常的动脉解剖，内皮细胞在图尼卡内皮瘤，平滑肌肉细胞在图尼卡介质，和异质胶原蛋白基质，其中包括成纤维细胞和喂食血管称为血管血管在图尼卡出现。巨型细胞动脉过敏性时态动脉表现出中度至重度炎症，具有聚集物和巨噬细胞，或组织细胞、淋巴细胞、偶尔的血浆细胞和多核巨细胞。还注意到发光变窄，并伴有刺激增厚和 90% 的内部弹性层的破坏。CD3阳性淋巴细胞和CD68阳性巨噬细胞也存在于所有巨型细胞动脉炎阳性标本中，在所有活检中观察到类似的染色模式。叶酸受体β的染色仅限于巨噬细胞和多核巨型细胞，这些细胞优先地局部化到出现体和介质。值得注意的是，叶酸受体β约占巨噬细胞总数的30%。时间动脉活检处理、切割和脱瓦需要小心灵巧，以便执行后续步骤。幻灯片解释需要经验丰富的心血管病理学家的专业知识。在处理标本和试剂时，提醒观看者使用接触和呼吸预防措施，如手套和通风罩。

Research

JoVE Journal

Immunology and Infection

Seven Steps to Stellate Cells

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

科研

免疫与感染

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

寄生蜂简介<em&gt;果蝇</em&gt;和Antiparasite的免疫反应的

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

一个<em在体外</em&gt;模型研究人类巨噬细胞分化和极化的异质性

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

淋巴结脂肪垫对嵌合体的淋巴结基质细胞起源研究的一代

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

在体外和体内模型来研究细菌粘附于血管壁下流动条件

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

开发和鉴定人的中性粒细胞源性巨噬细胞新型亚群<em&gt;体外</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

制备光学质量玻璃表面研究巨噬细胞融合

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

果蝇、果蝇细胞免疫应答的评估

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...