我们感谢彼得Staeheli提供B6.A2G-MX1小鼠和Semra Ozcelik酒店的技术咨询。 这项工作是由德国研究基金会通过SFB829和KN536 / 16，和科隆财富计划/科隆大学医学院，支持。

伦理声明。 表皮片从处死的动物的制备是严格按照Landesamt献给NATUR，UMWELT和Verbraucherschutz，北莱茵 - 威斯特法伦（德国）的指南的建议。该研究是通过LANUV NRW（编号8.84-02.05.20.13.018）。 1.准备仪器仪表及文化传媒<ol><li>培养表皮片在Dulbecco改良的Eagle培养基（DMEM）/高葡萄糖含谷氨酰胺二肽，10％FCS，100IU / ml青霉素，和100μg/ ml链霉素（以下简称“DMEM”）。 </li><li>对表皮片的制备中，仔细地溶解分散酶II粉末（5毫克/毫升）中加热的PBS（高达37℃）。过滤通过0.22微米的过滤器将溶液直接将其用于治疗。 </li><li>对于表皮整体编制机构 13，准备拦截的buff尔用0.5％奶粉，从冷水鱼皮和0.5％的Triton X-100的TBS 0.25％明胶。允许物质通过至少2小时温育该混合物在RT下在旋转混合器溶解。还在PBS制备0.2％吐温20洗涤缓冲液。为固定，制备3.4％和在PBS中的4％甲醛。 </li><li>用于冷冻切片的免疫染色，制备封闭缓冲液，用5％正常山羊血清和0.2％吐温20在PBS中。对于固定，准备0.5％甲醛的PBS。 </li></ol>从鼠皮2制备表皮片的<ol><li>从新生儿背部皮肤感染的研究表皮的制备<ol><li>放置一个断头小鼠（出生后1至3天）成夹层菜和切断四肢和尾靠近躯干，以允许从完全躯干有效地除去皮肤的。在去除四肢，尽量保持切口尽可能小的直径。 </li><li>修正了曲细镊子一躯干ND轻轻移动钝头剪刀皮肤下面从颅沿后尾鳍到皮肤从躯干分离。狭缝沿背部皮肤。 </li><li>对于FACS分析，角质形成细胞或准备RNA的分离，用钳子固定的组织和钝头剪刀推下躯干的躯干剥离完整的皮肤。对于冰冻切片或整个坐骑的准备只拿个背部皮肤。 </li><li>印章的背部皮肤，用手术刀为1-2枚约10×10毫米。把碎片在一个6孔板以〜1毫升无菌PBS中的一个孔中。确保真皮侧面的底部。 </li><li>更换PBS 1.5毫升分散酶II（5毫克/毫升的PBS）和孵育在37℃或者30分钟（对于整个坐骑）或O / N在4℃。用PBS洗3次。轻轻地取出表皮为使用一个镊子来解决下面的真皮和其他钳解除表皮完整片。用双眼做这一步EAS年。转移表皮片成DMEM符合其对底部的基底的一面。立即感染实验使用的床单。 注：使用从新生儿皮肤的表皮准备主要为FACS分析，角质形成细胞或准备RNA的隔离。由于其脆弱性，编制感染薄板的冰冻切片或整个坐骑不太适合。 </li></ol></li><li>成人尾部皮肤感染研究表皮的制备<ol><li>安乐死的小鼠在1至3个月的颈椎脱臼的年龄。取尾巴，而不是背部皮肤，因为在背面的长嵌入毛囊阻碍完好表皮片的分离。切断从处死小鼠的尾部，用手术刀切开纵向它。 </li><li>剥去骨头的皮肤，用手术刀把它切成约5×5mm的碎片。竖起来4件于一体的，并在2.1.5所述继续进行孵化分散酶II 注：使用成人泰l湿准备冰冻切片或整个坐骑。可替代地，以立即使用，尾部可以存储或在4℃下输送大约24小时，而不会失去显著感染效率。如果尾巴跟上至48小时，表皮片可以几乎不感染HSV-1。 </li></ol></li><li>表皮用于FACS分析的解离 注：检测通过FACS分析表面蛋白质的表达需要适当的细胞解离技术不损害细胞表面和所关心的蛋白。 <ol><li>孵育1.5毫升/孔的重组胰蛋白酶的基于细胞解离15分钟，室温溶液和15分钟，在37℃的新生表皮片在一个6孔板与基底侧。 </li><li>通过加入3ml DMEM停止孵育。通过使用弯曲的细镊子轻轻移动表皮在6孔板使得角质得到溶解解离的角质形成细胞。收集细胞悬液，重复此步骤3次总是用新鲜的DMEM。 注：此分离程序是合适的检测的Nectin-1角质形成细胞的表面上。 </li><li>可替代地，孵育上无酶细胞解离溶液的表皮片（CDS）15分钟，在室温和15分钟，在37℃。孵育另外15分钟，在RT下轻轻冲洗掉角质吹打CDS上下。 </li><li>收集细胞悬浮液，并使用新鲜的DMEM重复冲洗步骤3次。 注：此分解适用于检测HVEM的表面表达。在CDS溶液的缺点是，更少的细胞解离比用重组胰蛋白酶的基于细胞解离溶液。除了感染表皮片用重组胰蛋白酶的基于细胞解离溶液的离解后表面表达，核或细胞质表达如ICP0表达可以通过FACS进行分析。 </li></ol></li><li>表皮解离隔离角质形成细胞或前道RNA <ol><li>放新生儿表皮片在一个6孔板与朝其含有1.5毫升/孔的重组胰蛋白酶的基于细胞解离溶液的培养皿底部的基底侧。孵育在室温30分钟。添加3 ml的DMEM中，并通过使用弯曲的细镊子轻轻移动的菜表皮角化细胞解离。 </li><li>收集细胞悬浮液，并使用新鲜的DMEM重复此步骤3次。使用该细胞悬浮液中提取RNA或文化原代鼠角质形成细胞。 </li></ol></li></ol> 3.感染表皮片与HSV-1 <ol><li>制备的HSV-1重量17株11的溶液在不大于500微升DMEM中以下，由病毒溶液在其上表皮片是浮更换介质。这个时间点被定义为时间0 12。在一个24孔板的一个孔，在37℃下进行表皮片的感染。病毒滴度的计算是基于所估计的数细胞在每表皮片的基底层的（大约每5×5毫米片2×10 5个细胞）。 </li><li>感染了HSV-1在约100噬斑形成单位（PFU）/细胞并更换含病毒介质由DMEM，在1小时圆周率 注：如果从新生儿皮肤表皮片的，记住，张开始孵化过程中解离。 </li></ol> 4.可视化感染的细胞<ol><li>表皮整体机构 13 <ol><li>修复表皮片用3.4％的甲醛无论是2小时在室温或O / N在4℃。洗两次，用PBS和0.5％奶粉，从冷水鱼皮和0.5％的Triton X-100的TBS 1小时在室温14 0.25％明胶阻塞。 </li><li>孵育板O / N小鼠抗ICP0（单克隆抗体11060）15稀释1:60在封闭缓冲液在室温下。以可视化的中间纤维网络中同时孵育兔多克隆抗鼠标角蛋白14（100纳克/毫升）。 </li><li>期间4小时，在室温洗涤4〜5次，用PBS-0.2％吐温20。孵育O / N与AF488标记的抗小鼠IgG（1微克/毫升），AF555缀合的抗兔IgG（1微克/毫升），和DAPI（4&#39;，6-二脒基-2-苯基）（100纳克/毫升）在封闭缓冲液在室温下。 </li><li>用PBS-0.2％Tween 20的4小时在室温洗。山表皮片与他们的标本幻灯片顶底侧，嵌入荧光封固剂，盖上盖玻片。 </li></ol></li><li>表皮冷冻切片<ol><li>成人表皮片材在室温下固定在PBS中的4％甲醛20分钟，洗涤2次，用PBS。在组织中嵌入固定片冷冻介质和冷冻在液氮中。切8-10微米的部分用冷冻切片机。 </li><li>固定再次用在PBS中的0.5％甲醛在室温10分钟的部分中，洗涤2次，用PBS，用30分钟，在室温在PBS 5％正常山羊血清和0.2％吐温20块，然后染色60分钟以米乌斯抗ICP0（单克隆抗体11060）15稀释1:60在封闭缓冲液，随后3次用封闭缓冲液洗涤。 </li><li>然后用AF488标记的抗小鼠IgG（1微克/毫升）和DAPI（100毫微克/毫升）培养在封闭缓冲液在室温45分钟，洗3次。荧光封固剂嵌入部分，盖上盖玻片。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

当从C57BL / 6成人皮肤的表皮片材被感染HSV-1在约100 PFU /细胞，我们观察到感染几乎所有细胞中的基底层中的滤泡表皮而较低的病毒剂量关联与少感染的细胞和较慢的感染的进展。在一般情况下，毛囊显示可变数目的感染的细胞;而大多数的相当小的角化细胞衬显影毛囊感染，成人毛囊仅实施发胚芽被完全感染。取决于如何平缓的表皮由分散酶II处理前进的分离，毛囊的部件会丢失。在大多数情况下?...

单纯疱疹病毒（HSV）可引起一系列疾病从轻度无并发症的皮肤粘膜损伤到危及生命的感染人类。单纯疱疹病毒1型（HSV-1）是显性与口面部感染和脑炎相关，而HSV 2型（HSV-2）更可能导致生殖器感染1。虽然在了解HSV是如何进入细胞的培养，引发感染并产生病毒的后代显著的进步，我们知道一点关于病毒的入侵途径（S）进入组织细胞水平2。单纯疱疹病毒的皮肤或粘膜感染，小鼠的研究，兔和豚鼠已用作动物模型。皮肤感染成立通过皮内注射或通过刮擦中病毒的存在的皮肤，和疾病发展率与病毒的产生。这些方法有助于理解疾病的发病机制的各个方面，并用于评估抗病毒药。研究HSV感染的Tissu酒店E级，人体器官皮肤模型已经应用。作为感染的速率被限制在这些筏培养，研究调查感染，病毒传播和抗病毒成分的影响仅有限数量的已出版3-6。 为了表征细胞的决定因素，在HSV-1感染的完整上皮细胞发挥 ​​作用，我们建立了一个协议，用于小鼠表皮7的体外感染的研究。皮肤从新生儿或从成年小鼠的尾部制备。由于HSV-1能不感染完整的皮肤样本，它被淹没在含病毒的培养基中，我们通过分散酶II处理分离的真皮表皮。含病毒介质上浮动的表皮片材后，感染的细胞可以在不同的时间感染后（PI）的7被可视化的表皮基底层。现有可视感染个体细胞的启动病毒复制一第二病毒产生，我们用针对所述感染的细胞蛋白质0（ICP0），这是在HSV-1感染所表示的第一蛋白之一的抗体。 ICP0的细胞定位在早期感染穿过不同的阶段。而ICP0存在于核灶期间病毒基因表达的早期阶段，ICP0至细胞质的重新定位指示随后感染8的相位。 我们使用表皮片的体外感染测定从不同小鼠模型感染期间测试各种细胞因子的潜在作用。为了解决Rac1的作为肌动蛋白动力学的关键调节的影响，我们感染小鼠的表皮具有 ​​角化细胞特异性缺失RAC1基因9。此模型使我们能够研究对HSV-1感染在表皮角化层中的效率缺陷的Rac1的后果。以对照同窝感染表皮重新比较vealed无显著差异，这表明不存在的Rac1的有感染在表皮7的基底层开始没有影响。使用进一步小鼠模型使我们能够解决该细胞受体介导进入表皮。从任一的Nectin-1-或HVEM缺陷小鼠用HSV-1感染表皮片显露最初的病毒进入组织强烈依赖的Nectin-1 10的存在。此外，我们的结果表明，HVEM也可以有效地少充当受体在鼠表皮，虽然比的Nectin-1 10。 为了解决感染的细胞中的表皮层的空间分布，我们想象在组织切片和表皮全样载（图1）ICP0表达。在完整皮肤的冷冻切片，没有ICP0表达细胞中检测到（ 图1）。与此相反，表皮片的冷冻切片表明细胞质ICP0表达在基底层已经3小时圆周率（ 图1）。在稍后时间，病毒扩散到基底层可以可视化。感染细胞在基底层的空间分布可以很容易地跟随在表皮全样载（ 图1）。在感染了HSV-1在100 PFU /细胞，在滤泡表皮的基底角质形成细胞的大约50％显示在1.5小时丕ICP0表达在这个时间点最感染的细胞表达核ICP0。 ICP0的再定位至细胞质指示早期基因表达的稍后阶段存在于几乎所有细胞在3小时圆周率（ 图1）。在体外 HSV-1感染的可视化感染细胞的这些模式提供了有力的实验来研究对病毒的进入和扩散在组织中删除/突变的细胞成分的抑制剂或效果。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM/high glucose/GlutaMAX</td>
			<td>Life Technologies</td>
			<td align="right">31966047</td>
			<td>needed for cultivation of epidermal sheets</td>
		</tr>
		<tr>
			<td>dispase II powder</td>
			<td>Roche</td>
			<td align="right">4942078001</td>
			<td>has to be solved in heated PBS</td>
		</tr>
		<tr>
			<td>enzyme-free cell dissociation solution</td>
			<td>Sigma</td>
			<td>C5914</td>
			<td>needed for very gentle dissociation of epidermal sheets</td>
		</tr>
		<tr>
			<td>TrypLE select cell dissociation solution</td>
			<td>Life Technologies</td>
			<td>12563-029</td>
			<td>needed for dissociation of epidermal sheets</td>
		</tr>
		<tr>
			<td>chelex 100 resin</td>
			<td>Bio-Rad</td>
			<td>142-2832</td>
			<td>needed for chelation of polyvalent metal ions from the fetal calf serum</td>
		</tr>
		<tr>
			<td>gelatin from cold water fish skin&#160;</td>
			<td>Sigma</td>
			<td>G7765</td>
			<td>needed for minimization of non-specific antibody binding</td>
		</tr>
		<tr>
			<td>Keratin 14 Polyclonal Antibody (AF64) (conc.: 1 mg/ml)</td>
			<td>Covance</td>
			<td>PRB-155P</td>
			<td>used to visualize the intermediate filament keratin 14 which is a marker of the basal layer of the epidermis</td>
		</tr>
		<tr>
			<td>Mouse IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 488 conjugate (conc.: 2 mg/ml)</td>
			<td>Life Technologies</td>
			<td>A-11029</td>
			<td>used as secondary antibodies</td>
		</tr>
		<tr>
			<td>Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 555 conjugate (conc.: 2 mg/ml)</td>
			<td>Life Technologies</td>
			<td>A-21429</td>
			<td>used as secondary antibodies</td>
		</tr>
		<tr>
			<td>4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI dihydrochloride)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (conc.: 0.1 mg/ml)</td>
			<td>Sigma</td>
			<td align="right">36670</td>
			<td>used to counterstain the nucleus</td>
		</tr>
	</tbody>
</table>

ex vivo infection of murine epidermis with herpes simplex virus type 1

进入其人类宿主，单纯疱疹病毒1型（HSV-1），必须克服的粘膜表面，皮肤或角膜的屏障。初始进入期间HSV-1的目标的角质，并建立在该上皮，之后是神经元的潜伏感染的主要感染。激活后，病毒可出现皮肤水泡或粘膜溃疡粘膜部位变得明显。如何HSV-1侵入皮肤或黏膜和达到其受体知之甚少。调查入侵路线的HSV-1的成表皮组织在细胞水平上，我们建立鼠表皮的离体感染模型，它代表的原发性和复发性感染中的皮肤的部位。该测定法包括鼠皮肤的制备。表皮从真皮层经分散酶II处理分离。浮动的表皮片含病毒的培养基上后，将组织是固定的，感染后可在不同的时间感染后由可视化用针对HSV-1的立即早期蛋白ICP0的抗体染色感染细胞。 ICP0表达细胞，可以在基底角质形成细胞层在1.5小时感染后观察到了。较长感染次，感染的细胞在基底层层检测，表明感染并不限定于基底角质形成细胞，但病毒扩散到其他的层中的组织。使用各种小鼠模型的表皮片，感染协议允许确定向HSV-1侵入到组织的细胞成分的参与。另外，该测定是合适的，以测试在组织抑制剂，与初始条目的步骤干扰，细胞至细胞的传播和病毒生产。在这里，我们详细描述的体外感染协议和使用的Nectin-1-或HVEM缺陷型小鼠呈现我们的结果。

该方法的挑战是，制备表皮片到其中HSV-1可以从基底层穿透。的关键步骤是从真皮的表皮由分散酶II处理，这取决于该小鼠品系，需要调整的分离。分散酶II的浓度的范围可以从2.5至5毫克/毫升，并孵育20至45分钟的时间。的中间丝蛋白的角蛋白14的染色很容易地允许预测所述基底表皮层是否可以感染或是否会显示出感染的细胞（图2）只有非常有限。理想的是，角蛋白14染色的表皮整体坐...

Watch this Scientific Journal Video about Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1 at JoVE.com

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

鼠表皮离体感染单纯疱疹病毒1型

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

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Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

11.0K 次观看.  University of Cologne. The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

视频: Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

体外广角T细胞在小鼠淋巴结切片和共聚焦显微镜成像

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

科研

免疫与感染

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

神经元的初步培养体系的研究疱疹病毒潜伏期和活化

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

跨体内迟发型变态反应检测方法的抗原特异性调节

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

体内人淋巴组织和女性生殖器粘膜与人体免疫机能丧失病毒1和 Histoculture 的感染

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

原发性鸡初囊-细胞培养模型研究传染性囊内病病毒发病机制

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

免疫荧光共聚焦显微镜对单纯疱疹病毒1蛋白核细胞质转移的时间分析

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is ...

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

非酒精性脂肪性肝炎合并2型糖尿病的一种先进的小鼠模型

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

体外ELISA测试评估狂犬病疫苗效力

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...