Preparation of Mosquito Salivary Gland Extract and Intradermal Inoculation of Mice
|
Author:
Date:
2017-07-20
[Abstract] Mosquito-transmitted pathogens are among the leading causes of severe disease and death in humans. Components within the saliva of mosquito vectors facilitate blood feeding, modulate host responses, and allow efficient transmission of pathogens, such as Dengue, Zika, yellow fever, West Nile, Japanese encephalitis, and chikungunya viruses, as well as Plasmodium parasites, among others. Here, we describe standardized methods to assess the impact of mosquito-derived factors on immune responses and pathogenesis in mouse models of infection. This protocol includes the generation of mosquito salivary gland extracts and intradermal inoculation of mouse ears. Ultimately, the information obtained from using these techniques can help reveal fundamental mechanisms of interaction between ...
[摘要] 蚊子传播的病原体是人类严重疾病和死亡的主要原因之一。蚊子唾液中的组分促进血液供体,调节宿主反应,并允许有效传播病原体,如登革热,紫草,黄热病,西尼罗河,日本脑炎和基孔肯雅病毒,以及疟原虫寄生虫等。在这里,我们描述了评估蚊子衍生因子对小鼠感染模型中免疫反应和发病机制的影响的标准化方法。该方案包括产生蚊子唾液腺提取物和皮内接种小鼠耳朵。最终,使用这些技术获得的信息可以帮助揭示病原体,蚊子和哺乳动物宿主之间的相互作用的基本机制。此外,该协议还可以帮助建立疫苗或治疗剂的临床前检测改进的感染挑战模型,以考虑通过蚊子传播的自然途径。 【背景】在探测血液的同时,蚊子接种有助于喂养的唾液,但如果蚊子曾经感染过感染个体,也可能含有病原体。蚊子唾液在建立感染,促进传播,调节免疫应答和加剧西尼罗河病毒发病过程中发挥重要作用(Schneider等人,2006; Styer等人)。 ,2011),登革热病毒(Cox等人,2012; Conway等人,2014; McCracken等人,2014; Schmid ,2016),基孔肯雅病毒(Agarwal等人,2016),Semliki Forest病毒(Pingen等人,2016) ,裂谷热病毒(Le Coupanec等人,2013)和疟原虫寄生虫(Schneider等人,2011)感染。许多重要问题仍然存在,需要改进动物模型。  而通过感染的蚊子接种最好地模仿自然传播,接种剂量的高度变异性和有限的昆虫设施的可用性导致这种程序的有限使用。此外,使用感染的蚊子时,不能控制唾液的含量和蚊子组分的存在或不存在。作为替代方案,未感染的雌性蚊子的“斑点喂养”,然后通过针对病原体进行真皮内接种,模拟了唾液在小鼠皮肤中的自然沉积并递送了一定剂量的病原体。 ...
|
|
VLA-4 Affinity Assay for Murine Bone Marrow-derived Hematopoietic Stem Cells
|
Author:
Date:
2017-02-20
[Abstract] Hematopoietic stem cells (HSCs) are defined by their functional ability to self-renew and to differentiate into all blood cell lineages. The majority of HSC reside in specific anatomical locations in the bone marrow (BM) microenvironment, in a quiescent non motile mode. Adhesion interactions between HSCs and their supporting BM microenvironment cells are critical for maintaining stem cell quiescence and protection from DNA damaging agents to prevent hematology failure and death. Multiple signaling proteins play a role in controlling retention and migration of bone marrow HSCs. Adhesion molecules are involved in both processes regulating hematopoiesis and stem- and progenitor-cell BM retention, migration and development. The mechanisms underlying the movement of stem cells from and to the ...
[摘要] 造血干细胞(HSC)由其自我更新的功能能力定义,并分化成所有血细胞谱系。大多数HSC位于骨髓(BM)微环境中的特定解剖位置,处于静止非运动模式。 HSCs与其支持的BM微环境细胞之间的粘附相互作用对于维持干细胞静止和保护免受DNA损伤因子阻止血液学失败和死亡至关重要。多重信号蛋白在控制骨髓HSCs的保留和迁移中起重要作用。粘附分子参与调节造血和干细胞和祖细胞BM保留,迁移和发育的两个过程。干细胞从骨髓移动到骨髓的机制尚未完全阐明,仍然是深入研究的对象。一个重要的方面是修饰干细胞保留,迁移和发育所需的干细胞和祖细胞的粘附分子的表达和亲和力。粘附力通过粘附分子的表达,亲和力和亲合力来调节。亲和力调节与分子结合识别和结合强度有关。在这里,我们描述了在我们的研究中使用的体外 FACS测定来研究整联蛋白α4亚类β1亚单位的表达,亲和力和功能也称为VLA-4)用于小鼠骨髓保留EPCR +长期复制HSC(LT-HSC)(Gur-Cohen等人,2015)。背景整合素是介导细胞和细胞 - ...
|
|
Adhesion Assay for Murine Bone Marrow Hematopoietic Stem Cells
|
Author:
Date:
2017-02-20
[Abstract] Hematopoietic stem cells (HSCs) are defined by their functional abilities to self-renew and to give rise to all mature blood and immune cell types throughout life. Most HSCs are retained in a non-motile quiescent state within a specialized protective microenvironment in the bone marrow (BM) termed the niche. HSCs are typically distinguished from other adult stem cells by their motility capacity. Movement of HSCs across the physical barrier of the marrow extracellular matrix and blood vessel endothelial cells is facilitated by suppression of adhesion interactions, which are essential to preserve the stem cells retained within their BM niches. Importantly, homing of HSCs to the BM following clinical transplantation is a crucial first step for the repopulation of ablated BM as in the case of ...
[摘要] 造血干细胞(HSC)由其自我更新的功能定义,并在整个生命中产生所有成熟的血液和免疫细胞类型。大多数HSC在被称为利基的骨髓(BM)的专门的保护性微环境内保持在非运动性静止状态。 HSC通常通过其运动能力与其他成体干细胞区分开来。通过抑制粘附相互作用促进骨髓细胞外基质和血管内皮细胞的物理屏障的移动,这是保留在其BM细胞壁内保留的干细胞所必需的。重要的是,在临床移植后将HSC归巢到BM是重建消融BM的关键的第一步,就像血液恶性肿瘤治疗策略一样。归位过程结束于HSC的选择性访问和锚定到其在BM内的专门的位置。粘附分子是在干细胞移植的情况下增强归巢或减少BM保留以从匹配供体的血液中收集动员的HSC的靶标。在HSC上功能表达并参与其归巢和保留的主要粘附蛋白是整合素α4β1(非常晚的抗原-4; VLA4)。在该方案中,我们引入了针对表达VLA4的鼠骨髓干细胞优化的粘附测定。该测定法在分离表达VLA4的贴壁细胞后,通过流式细胞术与HSC富集细胞表面标记物定量粘附的HSC。
背景 HSCs主要保留在BM中,并通过与其微环境(niche)的粘合相互作用来调节。以这种方式,HSC保持在非运动性静止状态,保护它们免受DNA损伤代理(Boulais和Frenette,2015; Mendelson和Frenette,2014; ...
|
|