| Mouse Model of Dengue Virus Infection with Serotypes 1 and 2 Clinical Isolates
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Author:
Date:
2016-12-05
[Abstract] Dengue is a global public health threat caused by infection with any of the 4 related dengue virus serotypes (DENV1-4). Clinical manifestations range from self-limiting febrile illness, known as dengue fever (DF), to life-threatening severe diseases, such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Most cases of DHF/DSS are associated with secondary heterotypic infections through a phenomenon that is described as antibody-dependent enhancement of infection (ADE). There are an estimated 400 million human infections and several hundred thousand cases of severe dengue occurring yearly. At present, however, there are no approved antiviral drugs against DENV infection. The lack of a suitable animal model has hampered the evaluation of novel antiviral candidates for DENV ...
[摘要] 登革热是由4种相关登革热病毒血清型（DENV1-4）的任一种感染引起的全球公共卫生威胁。临床表现的范围从自限性发热疾病，称为登革热（DF），到危及生命的严重疾病，如登革热出血热（DHF）或登革热休克综合征（DSS）。大多数DHF/DSS病例通过描述为抗体依赖性增强感染（ADE）的现象与继发性异型感染相关。估计每年有4亿人感染和数十万例严重登革热病例。然而，目前，还没有批准的抗DENV感染的抗病毒药物。缺乏合适的动物模型阻碍了DENV感染的新抗病毒候选物的评价。由于DENV在免疫活性小鼠中不良地建立感染，已将AG129小鼠（缺乏I型和II型IFN [干扰素]受体）和小鼠适应的DENV2株应用于能够繁殖人类感染的几种主要病理的登革动物模型。最近，我们开发了具有临床分离株DENV1和DENV2的新的小鼠模型，其将用于药物测试和登革热发病机理研究（Watanabe等人，2016）。在这里我们描述建立临床分离株的登革热小鼠模型的细节;从体外材料制备到体内病毒感染。值得注意的是，由于DENV在小鼠中的感染性在病毒株之间不同，不是所有临床分离株都可以诱导严重的登革热。
关键字：登革热病毒，致命的小鼠模型，临床病毒，病毒感染的抗体依赖性增强，药物测试
[背景] 为了克服DENV在啮齿动物细胞中不能很好复制的缺点，多年来已经进行了许多努力来开发模拟人类登革热感染的小动物模型。近交小鼠模型系统允许实验可变性最小化，并且遗传工程小鼠模型能够再现动物中登革热临床症状的一些方面。过去的研究显示，用DENV2临床分离物感染的AG129小鼠（缺乏I型和II型IFN受体）感染瘫痪的迹象，这是中枢神经系统受累的病症，在人类病例中是罕见的（Shresta等人。，2004）。或者，产生可在AG129小鼠中诱导人类DHF/DSS样疾病的小鼠适应的DENV2毒株，并已用于登革热研究（Shresta等人，2006; ...
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| Visualization of Intracellular Tyrosinase Activity in vitro
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Author:
Date:
2016-04-20
[Abstract] Melanocytes produce the melanin pigments in melanosomes and these organelles protect the skin against harmful ultraviolet rays. Tyrosinase is the key cuproenzyme which initiates the pigment synthesis using its substrate amino acid tyrosine or L-DOPA (L-3, 4-dihydroxyphenylalanine). Moreover, the activity of tyrosinase directly correlates to the cellular pigmentation. Defects in tyrosinase transport to melanosomes or mutations in the enzyme or reduced intracellular copper levels result in loss of tyrosinase activity in melanosomes, commonly observed in albinism. Here, we describe a method to detect the intracellular activity of tyrosinase in mouse melanocytes. This protocol will visualize the active tyrosinase present in the intracellular vesicles or organelles including melanosomes.
[摘要] 黑素细胞在黑素体中产生黑色素，这些细胞器保护皮肤免受有害的紫外线。 酪氨酸酶是使用其底物氨基酸酪氨酸或L-DOPA（L-3,4-二羟基苯丙氨酸）引发颜料合成的关键铜酶蛋白酶。 此外，酪氨酸酶的活性与细胞色素沉着直接相关。 酪氨酸酶转运到黑素体中的缺陷或酶中的突变或降低的细胞内铜水平导致黑素体中酪氨酸酶活性的丧失，通常在白化病中观察到。 在这里，我们描述了一种方法来检测小鼠黑素细胞中酪氨酸酶的细胞内活性。 该协议将使存在于细胞内囊泡或细胞器（包括黑素体）中的活性酪氨酸酶可视化。
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| In vitro Assessment of Immunological Synapse Formation by Flow Cytometry
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Author:
Date:
2016-03-20
[Abstract] In adaptive immune system, formation of immunological synapse between T cells and antigen presenting cells (dendritic cells, B cells, and macrophages) or target cells (tumor cells and viral-infected cells) is critical for the execution of T cell immune responses via cytokine secretion or direct killing activity. Here, we describe the practical methods that directly measure the number of conjugates as a result of immunological synapse formation between T cells and superantigen-loaded B cells or between cytotoxic T cells and antigen-loaded target cells by dual-color flow cytometry.
[摘要] 在适应性免疫系统中，T细胞和抗原呈递细胞（树突细胞，B细胞和巨噬细胞）或靶细胞（肿瘤细胞和病毒感染细胞）之间的免疫突触的形成对通过细胞因子执行T细胞免疫应答至关重要 分泌或直接杀死活动。 在这里，我们描述了通过双色流式细胞术直接测量T细胞和加载超抗原的B细胞之间或细胞毒性T细胞和抗原负载的靶细胞之间的免疫突触形成的结合物数量的实际方法。
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