Name: a mouse model to assess innate immune response to staphylococcus aureus infection
Uploaded: 2019-02-28T14:00:00
Description: Watch this Scientific Journal Video about A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection at JoVE.com

这项工作得到了国家卫生研究院 r01 ai129302 (至 s. i. s.) 和药理学培训方案的支持: 从板凳到床头在加州大学戴维斯分校 (nih t32 gm099608 至 l. s. a.)。加州大学戴维斯分校的分子和基因组成像 (cmgi) 提供了出色的技术支持。

所有的小鼠研究都经过了加州大学戴维斯分校动物护理和使用机构委员会的审查和批准, 并根据《动物福利法》和《健康研究推广法》的准则进行。在与动物一起工作时, 一定要使用无菌手套。
1. 鼠标源和外壳
<ol><li>如前所述, 在 c57bl/6j 遗传背景下衍生 lysm-egfp 小鼠。在 c57bl/6j背景下, 通过将 lysm-egfp 小鼠与 myd88/小鼠杂交, 衍生 LysM---小鼠.</li...

<ol>
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利用荧光中性粒细胞记者鼠生物发光金黄色葡萄球菌感染的金黄色葡萄球菌感染模型, 结合整个动物体内光学成像的先进技术, 提高了我们对先天的认识对感染的免疫反应。此前使用 lysm-egfp 小鼠的研究表明, 在感染的前 24小时内, 多达 1 x 10 7只中性粒细胞被招募到金黄色葡萄球菌感染的伤口,而伤口招募的中性粒细胞将其半衰期从1.5天延长到5天对一个<e...

金黄色葡萄球菌(金黄色葡萄球菌) 占美国皮肤和软组织感染 (ssti) 的大多数。在过去20年中, 耐甲氧西林金黄色葡萄球菌(mrsa) 感染的发病率稳步上升 2, 这促使研究持久性的机制和发现新的治疗策略。mrsa 感染的护理标准是全身抗生素治疗, 但随着时间的推移, mrsa 对抗生素的耐药性越来越强 3, 这些药物会减少宿主的有益微生物群, 对健康造成负面影响, 尤其是在儿童4。临床前研究已经研究了治疗 mrsa 感染的替代策略5, 但将这些方法转化为临床已被证明具有挑战性, 因为出现的毒力因素, 阻止宿主免疫反应6。为了解剖驱动金黄色葡萄球菌 sst 的宿主病原体动力学, 我们结合无创和纵向读出的中性粒细胞的数量招募到伤口床与动力学措施的细菌丰度和伤口面积。
中性粒细胞是人类最丰富的循环白细胞, 也是细菌感染的第一反应者.中性粒细胞是宿主对金黄色葡萄球菌感染作出有效反应的必要组成部分, 因为它们具有杀菌机制, 包括产生活性氧、蛋白酶、抗菌肽和功能反应包括吞噬和中性粒细胞外陷阱产生8,9。人类患者的中性粒细胞功能的遗传缺陷, 如慢性肉芽肿病和 chediak-higashi 综合征, 显示出更容易感染金黄色葡萄球菌。此外, 遗传 (如先天性中性粒细胞减少症) 和后天 (如化疗患者中出现的中性粒细胞减少症) 缺陷的患者, 中性粒细胞数量的缺陷也极易感染金黄色葡萄球菌 10。鉴于中性粒细胞在清除金黄色葡萄球菌感染方面的重要性, 提高其免疫能力或调整其在金黄色葡萄球菌病变中的数量可能被证明是解决感染的有效策略。
在过去的十年里, 转基因小鼠与荧光中性粒细胞的记者已经开发, 以研究他们的贩运11,12.将中性粒细胞记者小鼠与整个动物成像技术相结合, 可以对组织和器官中的中性粒细胞进行时空分析。当与金黄色葡萄球菌的生物发光菌株结合使用时, 可以跟踪中性粒细胞的积累, 以应对金黄色葡萄球菌在细菌毒力因素的背景下的丰度和持久性, 而这些因素直接或间接地受影响组织中的中性粒细胞虫虫数13,14,15,16。
与人类相比, 小鼠对金黄色葡萄球菌毒力和免疫逃避机制的易感性较低。因此, 野生小鼠可能不是一个理想的动物模型, 以调查一个特定的治疗治疗治疗慢性金黄色葡萄球菌感染的疗效。缺乏 myd88 的小鼠 (即 myd88/-小鼠), 一种免疫功能低下的小鼠株, 缺乏功能白细胞介素-1 受体 (il-1r) 和 toll 样受体 (tlr) 信号, 显示出更容易感染金黄色葡萄球菌野生小鼠17和中性粒细胞贩运的损伤到皮肤中的金黄色葡萄球菌感染部位 18.myd88/小鼠中具有荧光中性粒细胞报告的小鼠株的研制为研究治疗金黄色葡萄球菌感染的疗效与目前中性粒细胞相比提供了一个替代模型记者鼠标。
在该方案中, 我们描述了免疫功能低下的 LysM-小鼠的金黄色葡萄球菌感染, 并与 lysm-egfp 小鼠比较了感染的时间过程和解决方法。lysm-EGFP×MyD88/小鼠出现不解决的慢性感染, 75% 的人在8天后死于感染。在最初的中性粒细胞招募中, 一个显著的缺陷发生在感染的炎症阶段的72小时以上, 在感染的后期招募的中性粒细胞减少了50%。lysm-EGFP×MyD88小鼠的易感性增加, 使这一特殊菌株成为一个严格的临床前模型, 以评估针对金黄色葡萄球菌感染的新治疗技术的有效性, 而目前的模型利用野生类型的小鼠, 特别是旨在提高先天免疫反应的技术, 以防止感染。

<table border="0" cellpadding="0" cellspacing="0" style="width:1101px;" width="1101">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">14 mL Polypropylene Round-Bottom Tube</td>
			<td style="width:301px;">Falcon</td>
			<td style="width:229px;">352059</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6mm Disposable Biopsy Punch</td>
			<td>Integra Miltex</td>
			<td>33-36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bioluminescent S. aureus</td>
			<td>Lloyd Miller, Johns Hopkins&#160;</td>
			<td>ALC 2906 SH1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Blood Agar, 5%, Hardy Diagnostics</td>
			<td>VWR</td>
			<td>10118-938</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buprenoprhine hydrochloride injectable</td>
			<td>Western Medical Supply</td>
			<td>7292</td>
			<td>0.3 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL/6J Mice</td>
			<td>Jackson Labratory</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloramphenicol (crystalline powder)</td>
			<td>Fisher BioReagents</td>
			<td>BP904-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS (1X)</td>
			<td>Gibco&#160;</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin Syringes</td>
			<td>Becton, Dickson and Company</td>
			<td>329461</td>
			<td>.35 mm (28 G) x 12.7 mm (1/2&#39;&#39;)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IVIS Spectrum In Vivo Imaging System</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Living Image Software &#8211; IVIS Spectrum Series</td>
			<td>Perkin Elmer</td>
			<td>128113</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LysM-eGFP Mice</td>
			<td>Thomas Graff Albert Einstein College of New York&#160;</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microvolume Spectrophotometer</td>
			<td>ThermoFisher Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MyD88 KO Mice</td>
			<td>Jackson Labratory</td>
			<td>009088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-woven sponges</td>
			<td>AMD- Ritmed Inc</td>
			<td>A2101-CH</td>
			<td>5 cm x 5 cm</td>
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			<td height="21" style="height:21px;">Povidone Iodine 10% Solution</td>
			<td>Aplicare</td>
			<td>697731</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Prism 7.0</td>
			<td>GraphPad Software</td>
			<td>License&#160;</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptic Soy Broth</td>
			<td>Becton, Dickson and Company</td>
			<td>211825</td>
			<td></td>
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a mouse model to assess innate immune response to staphylococcus aureus infection

金黄色葡萄球菌(金黄色葡萄球菌) 感染, 包括耐甲氧西林的污渍, 是一个巨大的负担, 医疗系统。随着金黄色葡萄球菌感染发病率每年攀升, 需要对其致病性进行更多的研究。传染病动物模型促进了我们对宿主病原体反应的认识, 并导致了有效疗法的发展。中性粒细胞在控制金黄色葡萄球菌感染的先天免疫反应中发挥着主要作用, 它形成了一个脓肿来围住感染, 并促进细菌的清除;浸润金黄色葡萄球菌皮肤感染的中性粒细胞的数量往往与疾病的结局有关。lysm-egfp 小鼠, 拥有增强的绿色荧光蛋白 (egfp) 插入溶菌酶 m (lysm) 启动子区域 (主要用中性粒细胞表示), 当与体内全动物荧光成像 (fli) 结合使用时, 提供了一种非侵入性和纵向向受伤皮肤的中性粒细胞移出的定量方法。当与生物发光金黄色葡萄球菌菌株和连续的体内全动物生物发光成像 (bi) 结合使用时, 可以在麻醉小鼠的感染, 从感染开始到解决或死亡。老鼠对金黄色葡萄球菌产生的一些毒力因素有更强的抵抗力, 这些因素有助于人类的有效定植和感染。免疫缺陷小鼠提供了一个更敏感的动物模型, 以检查持续的金黄色葡萄球菌感染和治疗能力, 以提高先天免疫反应。在此, 我们描述了 lysm-egfp 小鼠与野生类型 lysm-egfp 小鼠一起对 myd88 缺乏的小鼠 (lysm-EGFP×MyD88/小鼠) 的反应, 以调查金黄色葡萄球菌皮肤伤口感染。多光谱同时检测通过在体内使用 fli、体内 bli 使用的细菌负担以及随着时间的推移纵向和非侵入性伤口愈合, 能够研究中性粒细胞的招募动态。

LysM-/小鼠比 lysm-egfp 小鼠更容易感染金黄色葡萄球菌
本研究中使用的金黄色葡萄球菌菌株 alc2906 18 是用质粒构建的, 质粒包含了从活菌和活性代谢细菌产生生物发光信号的lux结构。当接种到小鼠体内时, 体内生物发光成像 (bl) 技术可用于纵向测量受感染伤口内的细菌负担。LysM--...

Watch this Scientific Journal Video about A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection at JoVE.com

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

介绍了一种实时检测小鼠皮肤损伤和金黄色葡萄球菌感染的先天免疫反应的方法。通过将 lysm-egfp 小鼠 (具有荧光中性粒细胞) 与 lysm-egfp 杂交免疫缺陷小鼠株进行比较, 提高了对感染的认识, 并提出了对抗感染的方法。

一种评价金黄色葡萄球菌感染先天免疫反应的小鼠模型

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

同时监测细菌负担和嗜中性粒细胞反应的技术可以提供对金黄色葡萄球菌持久性的机制和治疗策略的有效性的洞察。这种技术的主要优点是，在感染期间，可以在实时宿主中纵向测量多个参数。显示将伤口放在鼠标多苏姆的位置上、如何切除皮肤以及如何用金黄色葡萄球菌接种伤口至关重要。首先从冰面上80摄氏度的负温度储存中解冻生物发光葡萄球菌菌株。在将培养在 5% 牛血加盘上之前。在37摄氏度的夜间孵育。第二天早上，将三到四个单独的菌落从金黄色葡萄球菌盘子里转移到肉汤中，或TSB。辅以每毫升氯霉素10微克，在37摄氏度下过夜摇动孵育。第二天早上，在TSB的6毫升中稀释120微升的培养样品。每毫升氯霉素10微克，在37摄氏度下孵育两小时，每分钟200次旋转。当 600 纳米的光学密度达到 0.5 时，将三毫升细菌悬浮液转移到 10 毫升冰冷的 DPBS 中。小心地抽取上清液后，在颗粒中加入额外的冷冻 DPBS，彻底涡流重新悬浮的细菌。第二次离心后，将细菌颗粒重新悬浮在1.5毫升PBS冰上。为了验证细菌浓度，在新鲜 PBS 中，将细菌悬浮液的 100 微升连续稀释 1 x 10 到 4，1 x 10 到 5 倍。和板20微升每次稀释在个别阿加板。在37摄氏度过夜孵育后，通过总检查计算成菌组的数量，以计算前一天的细菌浓度。对于接受动物的切除皮肤损伤，在剃掉鼠标背面的一两英寸部分之前，请确认对手趾捏没有反应。轻轻擦拭以去除任何杂散的头发。并消毒暴露的皮肤，连续10%的波维酮碘，和70%乙醇浸泡纱布应用。从手术区中心向外移动，呈螺旋状模式。让皮肤干燥约一分钟后，在准备的手术区域中心用力按无菌六毫米冲孔活检。扭曲冲孔活检，在皮肤上创建圆形轮廓，完全切穿皮肤组织，以及至少一个部分的轮廓。注意不要切入底层筋膜或组织。然后使用无菌剪刀和钳子，按照冲孔活检印印的圆形图案切割表皮和真皮。对于金黄色葡萄球菌接种，用50微升的预制生物发光细菌注射剂填充28量胰岛素注射器。用手指把受伤动物的真皮拉到一边。将注射器与组织几乎平行，缓慢地将注射器插入组织，直到感觉到抵抗力突然降低。指示筋膜穿孔。将针头放在伤口中心，慢慢将注射剂的整个体积送来。确认消毒剂在伤口中心形成气泡，泄漏或分散最小。慢慢从动物中取出注射器。然后将动物返回到笼子的热量和监控，直到完全恢复。在受伤和感染后，每天在实验期间，将麻醉小鼠放在生物发光和荧光成像器中，伤口尽可能平坦。在成像器软件中，选择发光和照片作为成像模式。曝光时间应预设为一分钟的小型装箱，F/Stop 1 的发光，F/停止 8 的照片，并打开发射过滤器。然后单击"获取"以录制图像。对于体内荧光成像，请选择荧光和照片作为成像模式。曝光时间应预设为一秒的小型装箱，F/Stop 1 的荧光，F/Stop 8 的照片，以及 465 超过 30 纳米的激发和发射波长和 520 超过 20 纳米的发射波长，灯强度高。然后单击"获取"以录制图像。获得两组图像后，将动物返回到其笼子，进行监控，直到完全恢复。对于图像分析，在适当的图像分析软件程序中打开要量化的图像，并在整个伤口区域（包括周围皮肤）上放置默认的圆形区域。单击测量感兴趣区域，并记录伤口的均通量值，以允许绘制每个信号的均通量与时间。要测量伤口愈合，请将感兴趣的圆形区域设置在伤口边缘上，并测量伤口的面积（以厘米平方为单位）以图绘制基线与时间的分量变化。在这个代表性实验中，有条件EGFP表达骨髓分化原反应88，或 MyD88敲除小鼠，表现出比非有条件EGFP表达动物对金黄色葡萄球菌接种的易感性更大。在感染头八天观察到80%的致命性。两株小鼠在感染后立即减掉了约5%的体重。然而，条件EGFP表示小鼠恢复其原来的重量第二天。而 MyD88 小鼠没有。在金黄色葡萄球菌感染的情况下，伤口闭合在两个菌株之间没有不同。然而，与有条件的EGFP表达动物相比，无菌受伤的 MyD88 敲击小鼠在伤口愈合方面存在显著缺陷。整个动物成像显示更高的细菌负担，这从 MyD88 淘汰动物伤口的生物发光信号增加就证明，相比之下，有条件的EGFP表达动物。与有条件的EGFP表达动物相比，最初向 MyD88 敲击动物未感染伤口的中性粒细胞贩运减少了 40%。当1×10到第7个金黄色葡萄球菌形成单位在受伤后立即引入时，与有条件的EGFP表达动物相比，在淘汰动物中，嗜中性粒细胞的贩运明显减弱。在分配细菌之前，请确保注射器中没有气泡，并且针头的位置正确定位在伤口中心。动物组织可以在终点收获，以测量感兴趣的蛋白质的表达和细菌从伤口传播的细菌。金黄色葡萄球菌对人类具有传染性。因此，在处理细菌时应佩戴个人防护设备，在处理含有病原体的注射器时应谨慎行事。这项技术有助于探索使用生物疗法治疗金黄色葡萄球菌感染，提高宿主对感染的先天免疫反应。

Research

JoVE Journal

Immunology and Infection

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

皮下感染耐甲氧西林金黄色葡萄球菌（MRSA）的

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

科研

免疫与感染

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

耐甲氧西林实验性心内膜炎模型<em&gt;金黄色葡萄球菌</em&gt;（MRSA）的大鼠

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

金黄色葡萄球菌生长使用人血红蛋白作为铁源

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

呼吸爆发的响应为先天免疫健康的斑马鱼中的指标量化

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

一种研究食细胞诱导免疫应答的 mellonella 口服液模型

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

量化金黄色葡萄球菌对人多态核白细胞的细胞毒性

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

单核细胞分离研究人类粘膜淋巴组织中先天免疫反应

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...