这项工作得到意大利囊性纤维化基金会项目FFC＃18/2013，FFC＃29/2015和意大利囊性纤维变性联盟的支持，该联盟通过威尼托分公​​司 - 协奏曲Veneta Lotta控制Fibrosi Cistica Onlus。

描述的所有动物实验由维罗纳大学实验研究服务部门间动物实验室内动物福利委员会批准，并遵守欧洲指令2010/63 UE，意大利D.Lgs 26/2014和修订版"实验动物护理和使用指南"，华盛顿特区:国家科学院出版社，1996年。该方案和实验由美国国家卫生研究院（n 273/15）批准。动物可以自由进入标准的啮齿动物食物和软化的自来水，并且适应环境至少5天到当地的生殖条件（室温:20-24℃;相对湿度:40-70％;光暗周期:12小时）在任何治疗之前。 体内基因递送<ol><li>使用层流罩来制备体内递送试剂/核酸复合物。 </li><li>定义实验方案and参数遵循制造商的体内指示。 <ol><li>每只小鼠使用200μL复合物的总注射体积。 </li><li>从悬浮在无内毒素水中的40μgDNA开始;优化范围可达60μg。 注意:根据制造商的说明，注射体积中核酸的最终浓度不得超过0.5μg/μL。 </li><li>使用N / P比为6-8（每克核酸为0.12-0.16μL的递送试剂）。计算相应的输送试剂体积。 注意:复合物应该是阳离子的，用于有效的细胞进入。 N / P比定义为每个磷酸核酸（P）的体内递送试剂上的氮残基数（N），并表示复合物内离子平衡的量度。 </li></ol></li><li>在5％葡萄糖（最终浓度）中稀释计算量（参见步骤1.2.1）核酸使用10％葡萄糖储备溶液（提供）和无菌水。确保稀释体积是最终进样体积的一半。轻轻旋转或通过上下移动混合。 </li><li>使用10％葡萄糖储备溶液（提供）和无菌水将计算量（见步骤1.2.3）稀释为5％葡萄糖（终浓度）的一半注射体积。轻轻涡旋，旋转13,000 xg 15秒。 </li><li>将上述稀释的递送试剂一次性加入到稀释的核酸中。通过温和涡旋混合，并以13,000 xg的速度旋转15秒。 </li><li>在室温下将混合物从步骤1.5中孵育15分钟。 注意:从这一点开始，复合物在室温下稳定4小时，在4℃下储存多达7天。 </li><li>使用在室温下平衡的复合物进行尾静脉注射。将小鼠尾巴放入温水（50-53℃）30秒以允许静脉扩张。 <ol><li>将鼠标放在约束装置内。在20-30°角的尾静脉插入一个27至30号的针头，缓慢注射200μL。完成后，取下针头并向注射部位施加压力。 注意:注射期间尾部轻微的凸起表示定位不正确。如果发生这种情况，请取出针头并重复上一个位置附近的过程。 </li></ol></li><li>在静脉内注射后24和48 h，通过体内 BLI（见步骤2）显示基因表达。 </li></ol> 2. 体内 BLI 注意事项:事先准备，在DPBS中制备15 mg / mL D-荧光素的新鲜溶液，使用0.22μm过滤装置对其进行过滤灭菌，并将其储存在-20°C。 <ol><li>将小鼠放入透明有机玻璃麻醉室。确保异氟醚室已满。准备麻醉动物时，确保泵（left）和室（右）开关打开。将异氟烷表盘转换为2.5％的感应和2％的维护。 IVIS室内的动物在图像采集期间保持在2.5％异氟烷麻醉下。 </li><li>在小鼠完全麻醉后，在成像前15分钟通过腹膜内途径注射10mL / kg体重的D-萤光素溶液。 注意:应该对D荧光素进行动力学研究以确定给予D-荧光素后信号峰的时间。 </li><li>打开体内成像系统，并通过用一块黑色卡片衬垫来准备成像室（完成时丢弃）。根据需要放置鼻锥，以进行正确的麻醉（每只小鼠使用一个锥体）。 注意:将麻醉用于仪器的管是分开的，使得相同浓度的麻醉被引导到位于成像系统内部的麻醉歧管。 </li><li>从b转移小鼠（最多5个）牛到连接到成像系统中的歧管的鼻锥并且关闭门。图像采集持续5分钟。 </li><li>使用制造商的软件获取BLI，如下所示。 <ol><li>初始化软件在体内成像系统采集控制面板中，在Luminescent旁边加上一个复选标记。确认激励滤波器设置为阻塞，发射滤波器设置为打开。 </li><li>点击箭头:对于发光成像模式，选择5分钟的曝光时间，Binning 8和F / Stop 1;对于照相成像模式，选择Binning 4和F / stop 8。 </li><li>从"视野"下拉列表中，选择D，19厘米，主题高度为1.5厘米。准备好获取图像时单击"获取"。 </li></ol></li><li>图像采集完成后，将鼠标放回笼子中。 </li><li>使用制造商的软件量化从特定地区发射的光子。 <ol><li>点击<strong&gt;工具调色板中的ROI工具。在ROI工具中 ，从类型下拉列表中选择测量ROI 。 </li><li>点击Square图标，绘制一个平方的ROI，其尺寸适当，以覆盖一只动物的胸部。复制并粘贴每只动物的ROI，以获得具有相同尺寸的ROI。在"ROI工具"面板中，单击" 测量ROI"以获取ROI中总强度的度量。 </li><li>观察在会话期间在图像或序列中创建的所有ROI的ROI测量数据（每行一个ROI）。单击导出并选择文件将被保存的文件夹。 </li></ol></li></ol> 3.炎症刺激的老鼠挑战注意:在用促炎症刺激进行小鼠攻击之前，通过体内 BLI检查基线激活（参见步骤2）。至少7天必须在体内基因传递和摩尔之间通过挑战来让轻度和短暂的炎症消失。 <ol><li>准备用于气管内滴注的设备（参见图1和图2 ）。 <ol><li>将5 ml一次性注射器与弹簧（C，E），100μL注射器（B）和一次性量规（D）连接到三通旋塞阀（A）。将系统放在支架（H）上。将系统放在支架（H）上。 </li><li>将PE190微型医疗管（F）连接到一次性量规（D）和笔世纪（G）。 </li><li>向5 mL注射器中注入800μL空气，然后转动3路旋塞。 注意:通过在100μl注射器中吸出50μl向铜管填充铜绿假单胞菌培养物上清液。 </li></ol></li></ol><img alt="图1" class="xfigimg" src="/files/ftp_upload/55499/55499fig1.jpg" /> 图1:示意图代码气管内装置的单一组成部分。 （A） 3路旋塞阀， （B） 100μL汉密尔顿注射器， （C）一次性5 mL注射器， （D）一次性计量器， （E）一次性5 mL带弹簧的注射器， （F） PE190微型医用管， （G）笔世纪和（H）支持。 <a href="http://ecsource.jove.com/files/ftp_upload/55499/55499fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55499/55499fig2.jpg" /> 图2:组装的气管内装置的表示。 标识与图1相同。 <a href="http://ecsource.jove.com/files/ftp_upload/55499/55499fig2large.jpg" target="_blank">请点击</a>这里可以查看这个数字的较大版本。 <ol start="2"><li>使用设在2.5％异氟烷与氧气混合的异氟烷蒸发器室麻醉小鼠。 </li><li>监测动物，评估3-5分钟麻醉后的效果。 注意:要确认鼠标完全麻醉，请仔细监测以下标志:呼吸速度减慢，颈部捡起时手臂伸展不足，后肢刺激时缺乏反应。再等几分钟，再检查一下，如果不符合这些条件，则继续下一步。 </li><li>将麻醉的小鼠放在有机玻璃插管平台上，将其悬挂在门牙上，将其放置在电线上。 </li><li>用左手打开喉镜（右手研究人员），并抓住一双平头镊子。使用喉镜尖和镊子轻轻撬开口。 </li><li>拉舌头和h使用镊子将其老化到侧面。将喉镜刀片引导到嘴巴的后面。保持喉镜以90°的角度非常缓慢的按压直到气管的开口可见。握住喉镜就位。 </li><li>另一方面，将输送管连接到PE管的末端并将其插入气管。旋转三通阀以递送接种物。尽快将气管拔出气管。将鼠标悬挂几秒钟，让接种物吸入肺部。 注意:如果气管阻塞太久，小鼠会窒息死亡。 </li><li>从平台上移除鼠标。恢复时间可能会因应变而有所不同;仔细监测小鼠，确保动物在手术后30分钟内完全清醒。 </li><li>腹膜内注射150 mg / kg D-荧光素，并在气管内4,24和48 h后使用体内成像系统对肺进行成像滴注刺激。使用制造商的软件量化从特定地区发射的光子。 </li></ol>

<ol>
	<li>Barnes, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+approaches+to+asthma-chronic+obstructive+pulmonary+disease+overlap+syndromes.">Therapeutic approaches to asthma-chronic obstructive pulmonary disease overlap syndromes.</a> J Allergy Clin Immunol. 136 (3), 531-545 (2015).</li><li>Cohen-Cymberknoh, M., Kerem, E., Ferkol, T., Elizur, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+inflammation+in+cystic+fibrosis:+molecular+mechanisms+and+clinical+implications.">Airway inflammation in cystic fibrosis: molecular mechanisms and clinical implications.</a> Thorax. 68 (12), 1157-1162 (2013).</li><li>Dhooghe, B., Noel, S., Huaux, F., Leal, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+inflammation+in+cystic+fibrosis:+pathogenesis+and+novel+therapies.">Lung inflammation in cystic fibrosis: pathogenesis and novel therapies.</a> Clin Biochem. 47 (7-8), 539-546 (2014).</li><li>Durham, A. L., Caramori, G., Chung, K. F., Adcock, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+anti-inflammatory+therapeutics+in+asthma+and+chronic+obstructive+lung+disease.">Targeted anti-inflammatory therapeutics in asthma and chronic obstructive lung disease.</a> Transl Res. 167 (1), 192-203 (2015).</li><li>Sagel, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+biomarkers+of+airway+inflammation+in+cystic+fibrosis.">Noninvasive biomarkers of airway inflammation in cystic fibrosis.</a> Curr Opin Pulm Med. 9 (6), 516-521 (2003).</li><li>Starkey, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+models+of+infectious+exacerbations+of+airway+inflammation.">Murine models of infectious exacerbations of airway inflammation.</a> Curr Opin Pharmacol. 13 (3), 337-344 (2013).</li><li>Cacalano, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+and+B+cell+expansion+in+mice+that+lack+the+murine+IL-8+receptor+homolog.">Neutrophil and B cell expansion in mice that lack the murine IL-8 receptor homolog.</a> Science. 265 (5172), 682-684 (1994).</li><li>Simonet, W. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+impaired+neutrophil+migration+in+mice+overexpressing+human+interleukin-8.">Long-term impaired neutrophil migration in mice overexpressing human interleukin-8.</a> J Clin Invest. 94 (3), 1310-1319 (1994).</li><li>Stellari, F. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+transiently+transgenized+mice+with+a+bovine+interleukin+8+(CXCL8)+promoter/luciferase+reporter+construct.">In vivo imaging of transiently transgenized mice with a bovine interleukin 8 (CXCL8) promoter/luciferase reporter construct.</a> PLoS One. 7 (6), e39716 (2012).</li><li>Stellari, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+the+lung+inflammatory+response+to+Pseudomonas+aeruginosa+and+its+modulation+by+azithromycin.">In vivo imaging of the lung inflammatory response to Pseudomonas aeruginosa and its modulation by azithromycin.</a> J Transl Med. 13, 251 (2015).</li><li>Stellari, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+monitoring+of+lung+inflammation+in+CFTR-deficient+mice.">In vivo monitoring of lung inflammation in CFTR-deficient mice.</a> J Transl Med. 14 (1), 226 (2016).</li><li>De Simone, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+genetic+background+influences+the+response+to+the+opportunistic+Pseudomonas+aeruginosa+infection+altering+cell-mediated+immunity+and+bacterial+replication.">Host genetic background influences the response to the opportunistic Pseudomonas aeruginosa infection altering cell-mediated immunity and bacterial replication.</a> PLoS One. 9 (9), e106873 (2014).</li></ol>

作者没有什么可以披露的。

在以前的工作11中 ，显示了bIL-8-Luc依赖性BLI和BAL标记之间的对比。它依赖于小鼠品系12的敏感程度差异。因此，bIL-8-Luc模型首次应用于不同的小鼠品系需要对BLI和更标准化的炎症标记物进行初步的炎症反应研究。 小鼠转染引起轻度肺部炎症和bIL-8-Luc的活化，这可以通过BLI在DNA注射后3〜4天可以检测到（ 图4 ）。 1周后完全消失<su...

慢性肺部疾病，如哮喘，慢性阻塞性肺疾病（COPD），囊性纤维化（CF）和支气管扩张，其特征在于气道炎症。气道炎症的特征在于水肿，细胞浸润，T淋巴细胞和肥大细胞活化，气道分泌增加和胶原沉积过多。 CF是多系统疾病，其主要死因和发病原因是肺细菌感染与肺部恶化加重。肺功能的下降预测显着较差的结果1，2，3，4 。 呼吸道的炎症状态通常通过评估在炎症过程中引发的免疫标记来观察，所述免疫标记来源于下呼吸道和上呼吸道，例如痰液，其提供可变的resULTS。支气管镜也进行5 。鼠模型是研究以气道炎症为特征的疾病的发病机理和进展的有价值的工具，并且尚未确定有效的治疗或治疗方法。肺部感染和炎症的动物模型已被用于研究哮喘和宿主 - 病原体相互作用，包括模拟人类病症的化学物质的作用（ 例如，香烟烟雾暴露，LPS，弹性蛋白酶，卵白蛋白，poly I:C 等 ，作为以及上述的组合） 6 。炎症相关参数的测量需要牺牲动物，因为需要侵入性方法来测量诸如细菌负荷，肺中的细胞因子和收集的支气管肺泡灌洗液（BAL）液体等因素。此外，通常需要进行组织学检查。获得关于炎症反应动力学的信息的可能性需要使用numerous小鼠因此，在技术，道德，经济和运营基础上，无需牺牲动物就可以获得这种信息的技术是有价值的。 IL-8是炎症过程中的重要参与者，将白细胞募集到发炎组织。它代表了炎症途径激活研究的分子读数。 MIP-2和KC可以是小鼠中人IL-8的功能同源物。小鼠仅表达一种潜在的IL-8受体，即人CXCR2,7,8的同系物，但它们能够调节驱动报告基因的异源IL-8基因启动子。在观察到牛IL-8启动子/荧光素酶报告基因构建体可以在小鼠中反式激活之后，最近开发了肺炎症鼠模型。该功能允许利用生物发光成像（BLI）监测生活中的炎症反应小数9 。 该模型已经适应于研究由细菌外源产物（ 例如 LPS或细菌株释放的产物）或TNFα10,11引起的炎症。药物发现过程的重点是开发和优化可以治疗肺部疾病如CF，哮喘和COPD的新旧抗炎分子。这些新的化学实体必须在可以与特定临床表型相关联的动物模型中快速方便地进行测试，以便于智能临床试验的设计。

<table border="0" cellpadding="0" cellspacing="0" width="1536">
	<tbody>
		<tr height="30">
			<td height="30" style="height:30px;width:347px;">FMT 2500 Fluorescence Tomography System</td>
			<td style="width:457px;">Perkin Elmer Inc.</td>
			<td style="width:147px;">Experimental Builder</td>
			<td style="width:587px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">IVIS Lumina serie II Pre-clinical In Vivo Imaging System</td>
			<td style="width:457px;">Perkin Elmer Inc.</td>
			<td>Experimental Builder</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">MMPsense 750 FAST</td>
			<td style="width:457px;">Perkin Elmer Inc.</td>
			<td style="width:147px;">NEV10001EX</td>
			<td>Protect from light, store the probe at 4 &#176;C</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Female inbred BalbC</td>
			<td style="width:457px;">Harlan Laboratories Italy</td>
			<td style="width:147px;"></td>
			<td>Prior to use, animals were acclimatized for at least 5 days to the local vivarium conditions</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">bIL-8-Luc plasmid</td>
			<td style="width:457px;">Department of Medical Veterinary Science, University of Parma, Italy</td>
			<td style="width:147px;"></td>
			<td>Store the plasmid at -20 &#176;C</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">pGL3basic vector</td>
			<td style="width:457px;">Promega</td>
			<td style="width:147px;">E1751</td>
			<td>Store the vector at -20 &#176;C</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">JetPEI DNA transfection reagent</td>
			<td style="width:457px;">Polyplus transfection</td>
			<td style="width:147px;">201B-001G</td>
			<td>The DNA and JetPEI mix was formulated with a final N/P ratio of 7</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">D-luciferin potassium salt 1g</td>
			<td style="width:457px;">Perkin Elmer Inc.</td>
			<td align="right" style="width:147px;">122796</td>
			<td>Protect from light, store at -20 &#176;C</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Living Image software</td>
			<td style="width:457px;">Caliper Life Sciences,</td>
			<td>Experimental Builder</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Isoflurane</td>
			<td style="width:457px;">ESTEVE spa</td>
			<td style="width:147px;">571329.8</td>
			<td>Do not inhale</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Bio-Plex Cytokine Assay Kit</td>
			<td style="width:457px;">Bio-Rad Laboratories</td>
			<td style="width:147px;">M60-009RDPD</td>
			<td>Store the unopened kit at 4 &#176;C</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Automated cell counter</td>
			<td style="width:457px;">Dasit XT 1800J</td>
			<td>Experimental Builder</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Penn-century model DP-4M Dry power insufflator</td>
			<td style="width:457px;">Penn-century</td>
			<td>DPM-EXT</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Gas anesthesia system XGI-8</td>
			<td style="width:457px;">Perkin Elmer Inc.</td>
			<td>Experimental Builder</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">PE190 micro medical tubing</td>
			<td style="width:457px;">2biological instruments snc</td>
			<td style="width:147px;">BB31695-PE/8</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Syringe without needle 5ml</td>
			<td style="width:457px;">Terumo</td>
			<td style="width:147px;">SS*05SE1</td>
			<td>Cut the boards of the piston by a scissors</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Hamilton 0,10 ml (model 1710)</td>
			<td style="width:457px;">Gastight</td>
			<td align="right">81022</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Discofix 3-way Stopcock</td>
			<td style="width:457px;">Braun</td>
			<td align="right">4095111</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Syringe with needle 1ml</td>
			<td style="width:457px;">Pic solution</td>
			<td align="right">3,071,260,300,320</td>
			<td>Use without needle</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:347px;">Plastic feeding tubes 18ga x 50mm</td>
			<td style="width:457px;">2biological instruments snc</td>
			<td>FTP-18-50</td>
			<td>Cut obliquely the tip&#160;</td>
		</tr>
	</tbody>
</table>

an il-8 transiently transgenized mouse model for the in vivo long-term monitoring of inflammatory responses

气道炎症通常与细菌感染相关，代表肺部疾病的主要决定因素。各种因素的促炎能力的体内测定是具有挑战性的，并且需要终端程序，例如支气管肺泡灌洗和用于原位分析的肺的去除，排除了在同一只小鼠中的纵向可视化。在这里，通过在异源IL-8牛启动子的控制下，表达荧光素酶报告基因的瞬时转基因小鼠气管内滴注铜绿假单胞菌培养上清（SN）诱导肺部炎症。通过在滴注后2.5至48小时的时间范围内的体内生物发光图像（BLI）分析来监测肺中的荧光素酶表达。该过程可在2-3个月内重复多次，从而允许评估同一小鼠的炎症反应需要终止动物。这种方法允许实时监测在肺中起作用的促炎症因子和抗炎因子，并且似乎适合于功能和药理学研究。

将bIL-8-Luc瞬时转基因小鼠模型用于体内监测用含有分泌的毒力因子的浓缩细菌上清液（30x）攻击的小鼠的肺部炎症。诱导的炎症反应可通过体内成像检测，因为BLI信号的增加。尽管BLI信号在5和24小时之间达到最高峰，并且在48小时仍然是可检测的，但在滴注后2.5小时可清楚地检测出促炎活性。 <img alt="图3" class="xfig...

Watch this Scientific Journal Video about An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses at JoVE.com

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

这里描述的方法允许通过非侵入性生物发光成像（BLI）在小鼠肺中IL-8启动子依赖性炎症激活的可视化。荧光素酶报告基因构建体的交付时间可以将相同的动物多次进行BLI多至两个月。

IL-8瞬时转化的小鼠模型<em&gt;体内</em&gt;长期监测炎症反应

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

an-il-8-transiently-transgenized-mouse-model-for-vivo-long-term

Research

JoVE Journal

Immunology and Infection

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

7.3K 次观看.  University of Verona. 这里描述的方法允许通过非侵入性生物发光成像（BLI）在小鼠肺中IL-8启动子依赖性炎症激活的可视化。荧光素酶报告基因构建体的交付时间可以将相同的动物多次进行BLI多至两个月。 

视频: An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

浆液性卵巢癌原位模型在免疫小鼠，为在体内肿瘤显像和肿瘤免疫反应的监测

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

科研

免疫与感染

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

使用的EpiAirway模型，用于描述长期宿主 - 病原体相互作用

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

区分功能基因表达的作用，通过骨髓移植免疫和非免疫细胞在小鼠结肠炎

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

荧光目标阵列的T细胞反应评价中的应用<em&gt;在体内</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

长期培养的干扰素γ酶联免疫检测方法的评估效应和记忆T细胞应答的牛应用

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

一个<em&gt;体外</em&gt;模型在HIV合并感染的背景下测量免疫应答疟疾

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release cytokines and other mediators of inflammation. For a long time, humoral and cellular mechanisms have been studied for their role in regulating the immune response, but recent advances in the field of immunology and neuroscience have also unraveled specific neural mechanisms with interesting therapeutic potential. The so-called cholinergic anti-inflammatory pathway (CAP) has been described to control innate immune responses and inflammation in a very potent manner. In the early 2000s, Tracey and collaborators developed a technique that stimulates the vagus nerve and mimics the effect of the pathway. The methodology is based on the electrical stimulation of the vagus nerve at low voltage and frequency, in order to avoid any side effects of overstimulation, such as deregulation of heart rate variability. Electrical devices for stimulation are now available, making it easy to set up the methodology in the laboratory. The goal of this research was to investigate the potential involvement of prostaglandins in the CAP. Unfortunately, based on earlier attempts, we failed to use the original protocol, as the induced inflammatory response either was too high or was not suitable for enzymatic metabolism properties. The different settings of the original surgery protocol remained mostly unchanged, but the conditions regarding inflammatory induction and the time point before sacrifice were improved to fit our purposes (i.e., to investigate the involvement of the CAP in more limited inflammatory responses). 
The modified version of the original protocol, presented here, includes a longer time range between vagus nerve stimulation and analysis, which is associated with a lower induction of inflammatory responses. Additionally, while decreasing the level of lipopolysaccharides (LPS) to inject, we also came across new observations regarding mechanistic properties in the spleen.

Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

增加了恢复时间，并减少LPS管理研究迷走神经刺激机制的有限炎症反应

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.