This study was supported by grants from The Committee for Research at the University of Antioquia (CODI), The Colombian Institute for Development of Science and Technology &#x2013; COLCIENCIAS and The Center for Development of Products against Tropical Diseases - CIDEPRO.

1。感染仓鼠 1。动物用于自交系男性和女性的黄金仓鼠（Mesocricetus鲫鱼 ），6-8周，体重140-160克。他们被安置在动物设施，在温度控制的住宿，饲喂标准啮齿类动物干制食品和水随意提供。所有的程序，涉及动物机构的实验动物使用的伦理委员会批准。之前与dermotropic 利什曼寄生虫的动物实验感染的性别鉴定，标志着加权按规范的程序。动物性别鉴定，检查，如乳腺线和ANO生殖器女性短距离的可视化，或睾丸和肛门和男性的包皮之间的距离更大的可视化鲜明的特点。然后，动物被标记穿耳洞或染色面积的皮肤用浸泡棉签苦味酸。耳穿孔，耳后，用70％酒精清洁利用老鼠的耳朵冲被刺穿。必须避免与血管的区域。建议镇静或麻醉与氯胺酮（50毫克/公斤）和Xilacine（20毫克/千克）腹腔容积260-300μL25-G的针混合9时01分。最后，动物被放置在一个精确的平衡条件的一个陷阱或方块，他们权衡。 2。寄生虫 promastigotes的dermotropic 利什曼物种，如属亚马孙 ，双相的诺维-MacNeal尼科尔（NNN）培养基中培养，在26°C。亚循环（固定）相promastigotes（5天）用于感染仓鼠。简言之，寄生虫收获，洗两次使用磷酸盐缓冲液（PBS），计数和接种男性或女性在0.1毫升PBS调整为1×10 7（男性）或1.5×10 7（女性）寄生虫，分别，然而，接种量可能会有所不同，根据利什曼物种（程序在视频显示）。 3。实验感染接种前一个面积的皮肤被剃。简言之，麻醉动物被放置在俯卧位，并用剪刀，把头发从两英寸的尾巴的基地。清理后用无菌生理盐水溶液剃光面积寄生虫接种皮内注射，直至形成丘疹。 4。临床随访动物进行监测，每7天到4 - 6周后接种病变的外观。不久，动物被固定在一个陷阱和接种面积的皮肤被触及。然后，形成溃疡的硬结区划定并用游标卡尺测量溃疡的宽度和长度。 2。仓鼠的治疗 1。 ADMInistration的化合物治疗计划开始时，动物们溃疡病变（接种后4-6周）。动物主要分布在5-6动物群体为每个要测试的化合物。化合物可以外用，口服，肌注或病灶路线管理。药物应用之前，仓鼠麻醉，用生理盐水或PBS清洗病灶​​区域。当化合物是通过局部或病灶，仓鼠是固定的陷阱，让暴露的面积治疗时，该化合物是由口服或肌肉注射航线管理的同时，仓鼠被牢固地固定颈部的基础。一）局部应用适用于：复合病变和动物，几分钟后返回笼子。 b）对于肌肉注射，注射的药物是（200μL最大）通过半腱半膜肌躯。 c）对于口腔ADMInistration，该药物的动物提供探头连接到1毫升注射器通过奥罗胃14G（200μL最大）。 d）对于病灶内注射毒品（100微升最大）正在逐步注入在使用26G计针的斜面放置下来溃疡基地。针旋转覆盖整​​个区域的病变。每天在10-20天的治疗管理，根据定义的治疗方案。 2。临床随访因为在这个实验模型的有效性取决于新antileishmanial化合物根据治疗后病灶的愈合和疤痕，每个动物进行临床监测每周在大院，长达三个月后申请结束。监测期间，病变的类型描述和硬结和溃疡面积测量与游标卡尺。到t的不同地区存在的病灶他接种部位以及病灶复发的外观也被描述和登记。每两个星期，动物加权和病变图。动物每天观察监测：一）外表（头发，协调，温度，眼睛，耳朵的位置，梳洗，排便，腹水存在，搅拌和脱水）和b）的行为（清醒，警惕，好奇，周到，保持同组，食品和饮料，硬期待赶上）。充血，炎症，咬和脱毛的存在，也观察到的复合应用程序的网站。 仓鼠也是在45天的治疗，以确定血液和血清学的价值可与相关化合物的毒性（见下文）结束后放血。 每次治疗的有效性进行评估比较前病变的大小和治疗后，使用下面的得分小号ystem： 治愈 （100％的面积和病变完全消失愈合）; 临床症状明显改善 （&gt; 50％的面积在减少病灶大小）; 临床失败 （增加病灶的大小）; 复发的病灶上固化后）。在研究结束时，吸入二氧化碳以前麻醉处死动物。死亡后，寄生虫学，组织学和血清学分析所需的样本采取。 3。寄生虫学考试 利什曼在从皮肤样本（病变或疤痕）的存在，肝，脾，肾的决定直接检查涂片染色吉姆萨1和这些组织病理检查（见下文）。 在病变部位的寄生虫负担极限稀释法估计，根据提图斯和同事（1985）2。简言之，一个组织的一小块是REMoved从每个动物，称重，匀浆冷PBS溶液用注射器柱塞。在4°C离心10分钟获得暂停在600克此后，被丢弃上清，并与抗生素1％和10％胎牛血清的RPMI 1640培养基中悬浮颗粒。转移到每个96口井的悬浮一百L的含50μL施耐德中NNN以中等覆盖微孔板保持在26°C。在这promastigotes可以通过考试检测倒置显微镜下一个月每星期的最高稀释可行的寄生虫在每个样品的数量确定。 4。皮损病理检查（或瘢痕）和其他组织一个从皮肤，肝脏和肾脏活检标本的第三部分是在10％福尔马林固定，石蜡包埋。从脾和心脏标本也可能是processeD。五微米的固定组织切片染色与haemotoxilin，曙红和使用200X，400X或1000X油浸研究微架构，细胞渗透的特点，和寄生虫的存在或没有光学显微镜下检查。显微照片和数字图像捕获。 5。治疗毒性评价有毒活性化合物的基础上跟进（见上文）在临床监测参数根据动物的身体和行为的条件。根据血液在血液样本，如上所述，在与haemotoxilin - 伊红染色的组织切片观察病理变化和代谢参数也决定毒性。 从心，最好的心室的血液样本。血液测试，血液转移对EDTA抗凝管和处理雅丁规范完整的血球计数的协议。血清学检查，血液转移到1.5离心瓶，离心获得血清肌酐，ALT和BUN水平的代谢分析测量使用柯达Ektachemdry化学3。 6。数据分析数据处理和未经处理的动物相比，使用方差的检验。意义设定为P &lt;0.05。 3。代表结果总结皮肤利什曼病的仓鼠模型进行了改进，利用该模型在药物疗效筛选目标。在仓鼠的皮肤皮肤原发感染后，病变的发展和寄生虫参数进行了研究。自交系的男性和女性的金黄地鼠（Mesocricetus鲫鱼 ），6-8周，皮内注射1×10 7（男性）或1.5×10 7（女）元L 的循环（固定相）promastigotes 亚马孙 。结节出现20 - 35天PI后4-6周丕成立感染后形成溃疡，疗效评价，两种剂量的五价锑（SBV）。仓鼠随机分为三组，每组5只动物中，葡甲胺锑酸盐在80或120 SBV毫克/千克每天在10天肌肉注射治疗。第三组用PBS肌肉注射安慰剂。 A组的三只动物是未经处理的，作为阴性对照。每次治疗的反应的治疗期结束后3个月随访。 1。临床课程后研究亚马孙感染金黄地鼠（Mesocricetus鲫鱼 ） 在背部皮肤感染的仓鼠不断发展，临床上明显的表皮溃疡，感染后4-6周。病变开始与小结节和溃疡结束根据感染后的时间和增加在大小达到被视为一种实验性药物的效果评价最优规模由第四至第六周后感染（18,99 54,7毫米2 4周35,55到92,71毫米，在6周2）（ 图1，图2）。病灶，随后保持长达20周后感染明显的溃疡，而在此时，实验终止（ 图1）。 感染由L.亚马孙并不影响仓鼠体重的变化从104,9 124,69克和129,71到134,02克，分别在未受感染和感染仓鼠（P&gt; 0.05，ANOVA）。在研究过程中获得的动物平均25,58±5,98％的体重。赛瑞肌酐和丙氨酸氨基转移酶（ALT）和血象参数值分别为未感染利什曼原虫感染仓鼠的参考值。 ONLy的BUN尿素氮（BUN）水平增加60％的受感染的动物。 病变的组织病理学分析与研究感染的仓鼠亚马孙显示，与那些未受感染的动物的差异。在一般情况下，从仓鼠感染的皮肤活组织切片检查显示没有炎症反应或任何病理改变（ 图3a），而那些感染与 L 亚马孙发展肉芽肿性皮炎和丰富的巨噬细胞浸润真皮（ 图3b）存在。 与 L感染仓鼠的组织病理学分析亚马孙和葡甲胺锑在80和120的SBV毫克/千克/天在10天的剂量治疗的感染过程相似，那些在对照组（感染和未经处理）的观测相关的更改。在一般情况下，仓鼠的皮肤样本呈轻微的浆水平（20％）和中性粒细胞（80％）中等陪同浸润淋巴细胞（80％），温和（20％）或严重（80％）（ 见表2）巨噬细胞的浸润。这些意见符合诊断的肉芽肿性皮炎，炎症性的事件，损害的真皮和下方的所在肌肉。这是主要的病变相关的感染，这是严重的表现与皮肤利什曼病在动物研究中的所有兼容。该协会统计相关时，卡方培，产生P &lt;0.05。在肾皮质和glomerules伴随着轻微的肾脏萎缩75％的动物，动物的60-100％的肾脏样本呈中度增生。这些意见与膜增生glomerulonefritis，先前已感染有关其他利什曼物种感染的诊断兼容。统计协会是大皮尔逊P &lt;0.05与卡方。 40-100％的肝脏各组imals表明在肝细胞轻微的空泡变化，可伴有生理过程，如肝细胞的糖原积累。其他如脂肪变性，纤维化和拥塞肝组织异常对应的感染过程。其他意见，如在肝细胞中的液泡改变，cardiomegalies，胞浆内eosinophiles夹杂物的存在，以及在肾管细胞空泡没有统计学相关（P&gt; 0.05）的测试化合物的毒性作用，并有可能由于在仓鼠的生理过程。 2。葡甲胺锑酸盐在仓鼠模型的疗效 表1总结了在不同的时间点，治疗后的临床表型感染仓鼠。 SBV 120毫克/公斤/天的剂量肌肉注射葡甲胺锑的重量在10天的治疗高效诱导完全回归的研究在所有动物的的亚马孙病变（ 图4）。 2和8周后治疗期间内治愈的比例是100％。然而，三个月后复发的病变观察20仓鼠％。当葡甲胺锑酸盐在80毫克SBV /公斤体重管理，彻底治愈观察，仅在3只动物。在其他两种动物的病变下降了33,9和69,0％，分别为（ 图5）。经过3个月内，L。亚马孙保持目前在葡甲胺锑酸盐治疗在SBV 80毫克/公斤/天的剂量，所有动物的皮肤，只有SBV 120毫克/公斤/天葡甲胺锑酸盐治疗的动物之一。治疗剂量的肌肉注射120 SBV毫克/公斤/天在10天的决定。 使用稀释法的限制，估计寄生虫数量呈显着减少为治疗组与MEGLUMINE锑比较负（未处理）的控制和车辆（安慰剂）治疗组（P &lt;0.001）。病变部位在未经处理的动物和用PBS治疗的，可行的寄生虫（每0.4至1.6毫克的皮肤组织的寄生虫）检测。只分离的动物，没有回应与葡甲胺锑酸盐治疗：一个120毫克/公斤/天（ 图5b）和80毫克/公斤/天（ 图5c）治疗仓鼠治疗的动物寄生虫。没有区别，观察葡甲胺锑比较后，未经处理或处理用PBS这些动物没有治愈的仓鼠寄生负载。 在皮肤活检病理组织学SBV 120毫克/千克/天肌肉注射葡甲胺锑酸盐治疗的仓鼠没有寄生虫观察，看到一些巨噬细胞和淋巴细胞浸润真皮（ 图6A，6B）。相反，当仓鼠我们重新处理与葡甲胺锑酸盐80 SBV毫克/公斤/天，广泛地被发现（ 图6C，6D），肉芽肿性皮炎真皮浸润的巨噬细胞的大量存在。 3。毒性使用相同的治疗方案疗效的研究，在80和120的SBV毫克/公斤体重，葡甲胺锑的毒性进行了评价。一般健康状况和体重监测的最后一次给药后3个月。治疗没有影响仓鼠体重变化从104,9 124,69和129,71到134,02 GR GR在未受感染和感染仓鼠，分别。动物获得的27,59±4,22％的体重平均在治疗期间及28,74±2,26％，在后续（P&gt; 0.05，ANOVA）。减肥没有观察到任何治疗组（数据未显示）。赛瑞肌酐和丙氨酸氨基转移酶（ALT）和血象参数值在未处理和葡甲胺锑酸盐治疗仓鼠的参考值是相似的。仅BUN尿素氮（BUN）水平增加20％，处理的动物。 与 L感染仓鼠的组织病理学分析亚马孙和与葡甲胺锑酸盐治疗剂量为80和120 SBV毫克/公斤/天10天期间发现没有相关的药物毒性的变化。 <img src="/files/ftp_upload/3533/3533table1.jpg" alt="表1" /> 磷酸盐缓冲液，B 处理后几天。 表1临床表型的研究 。 亚马孙实验仓鼠葡甲胺锑酸盐治疗后感染。 每次治疗的有效性进行了评估比较前和治疗后的病灶大小，使用以下的评分系统： 治愈 （100％的面积和病变完全消失愈合）临床症状明显改善 （&gt; 50％的面积在减少病灶大小）; 临床失败 （增加病灶的大小）;复发（病变治愈后激活） <img src="/files/ftp_upload/3533/3533table2.jpg" alt="表2" /> 答：不存在的迹象;适用：不适用; M：温和，M：中等，S：严重;中性粒细胞：中性粒细胞;国家安全法：无显着性病变，妊娠期糖尿病：肉芽肿皮肌炎; PGD的：Pyogranulomatous皮炎。 1。淋巴管; 2。矿产类材料。 表2。仓鼠的皮肤组织病理学实验感染与 L 亚马孙和葡甲胺锑酸盐治疗。 <img src="/files/ftp_upload/3533/3533fig1.jpg" alt="图1" /> 图1。皮肤病灶发展的历程后感染仓鼠皮下接种1-1.5×10 7 L。亚马孙亚循环（stationa<html> RY阶段）promastigotes在20周。 Y轴代表以毫米为单位的溃疡面积。 <img src="/files/ftp_upload/3533/3533fig2.jpg" alt="图2" /> 图2（b）和6个星期（三）发展中的金黄地鼠背皮肤溃烂病灶在2个星期（一），4周后的10×10 7皮内接种Photographyic历史。 <img src="/files/ftp_upload/3533/3533fig3.jpg" alt="图3" /> 图3（一）仓鼠未受感染和未经处理的：皮肤活检。观察无炎症反应，既没有显着的病理改变;（二）仓鼠感染和治疗肉芽肿性皮炎与多核巨细胞的存在（箭头加星号）和丰富的寄生虫（黑箭头）和巨噬细胞（白色箭头）浸润真皮。苏木素 - 伊红染色400X。 重新4“/&gt; 图4。摄影史上的临床反应的仓鼠感染与 L 亚马孙和SBV 120毫克/公斤/天在10天肌肉注射葡甲胺锑酸盐治疗。图片显示，在治疗（10天）（二）年底前病变的治疗（一）外观，并在后续：30天（C），60（d）和90（e）在治疗。 <img src="/files/ftp_upload/3533/3533fig5.jpg" alt="图5" /> 图5。葡甲胺锑酸盐在治疗皮肤利什曼病鼠的有效性。金黄地鼠感染与 L 亚马孙在背侧皮肤。感染6周后，他们是未经处理的（a）或在PBS单（二），葡甲胺锑120 SBV毫克/公斤/天（c）或80的SBV毫克/公斤/天（D）10天肌肉注射治疗。图表显示的比例减少病灶大小（0天）治疗结束，在15天，30，治疗结束后随访60 90。 P &lt;0.001为120或80 SBV毫克/公斤/日主场迎战车辆，而不是治疗。 <img src="/files/ftp_upload/3533/3533fig6.jpg" alt="图6" /> 图6。皮肤活检SBV 120毫克/公斤/天（A，B）和80毫克SBV /公斤/天（C，D）感染者和肌肉注射megumine antinmoniate治疗仓鼠。稀缺的巨噬细胞和淋巴细胞浸润真皮的存在。没有寄生虫遵守。苏木精 - 曙红染色200X（一），（三）具有丰富的巨噬细胞浸润，真皮广泛存在的1000倍（C）肉芽肿性皮炎。泡沫巨噬细胞与phagocyted寄生虫。苏木精 - 曙红染色200X（二），1000X（D）。

<ol>
	<li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+the+leishmaniasis.">Control of the leishmaniasis.</a> Report of a Meeting of the WHO Expert Committee on the Control of Leishmaniasis WHO. 949, (2010).</li><li>Titus, R. G., Marchand, M., Boon, T., Louis, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+limiting+dilution+assay+for+quantifying+Leishmania+major+in+tissues+of+infected+mice.">A limiting dilution assay for quantifying Leishmania major in tissues of infected mice.</a> Parasite Immunol. 7, 545-555 (1985).</li><li>Reynolds, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Kodak+Ektachem+dry+layer+technology+for+clinical+chemistry.">The Kodak Ektachem dry layer technology for clinical chemistry.</a> Upsala. J. Med. Sci. 91, 143-146 (1986).</li><li>Handman, E., Ceredig, R., Mitchell, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+cutaneous+leishmaniasis:+disease+patterns+in+intact+and+nude+mice+of+various+genotypes+and+examination+of+some+differences+between+normal+and+infected+macrophages.">Murine cutaneous leishmaniasis: disease patterns in intact and nude mice of various genotypes and examination of some differences between normal and infected macrophages.</a> Australian J. Exp. Biol. Med. Sci. 57, 9-29 (1979).</li><li>Hanson, W., Chapman, W., Waits, V., Lovelace, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Leishmania+(Viannia)+panamensis+lesions+and+relationship+of+numbers+of+amastigotes+to+lesion+area+antimony-treated+and+untreated+hamsters.">Development of Leishmania (Viannia) panamensis lesions and relationship of numbers of amastigotes to lesion area antimony-treated and untreated hamsters.</a> J. Parasitol. 77, 780-783 (1991).</li><li>Henao, H. H., Osorio, Y., Saravia, N. G., G&#243;mez, A., Travi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+toxicity+of+pentavalentantimonials+(Glucantime+and+Pentostam)+in+an+American+cutaneous+leishmaniasis+animal+model:+luminometry+application.">Efficacy and toxicity of pentavalentantimonials (Glucantime and Pentostam) in an American cutaneous leishmaniasis animal model: luminometry application.</a> Biom&#233;dica. 24, 393-402 (2004).</li><li>Travi, B. L., Martinez, J. E., Zea, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimonial+treatment+of+hamsters+infected+with+Leishmania+(Viannia)+panamensis:+assessment+of+parasitological+cure+with+different+therapeutic+schedules.">Antimonial treatment of hamsters infected with Leishmania (Viannia) panamensis: assessment of parasitological cure with different therapeutic schedules.</a> Trans. R. Soc. Trop. Med. Hyg. 87, 567-569 (1993).</li><li>Hommel, M., jaffe, C. L., Travi, B., Milon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+for+leishmaniasis+and+for+testing+anti-leishmanial+vaccines.">Experimental models for leishmaniasis and for testing anti-leishmanial vaccines.</a> Ann. Trop. Med. Parasitol. 89, 55-73 (1995).</li><li>Travi, B. L., Osorio, Y., Saravia, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammatory+response+promotes+cutaneous+metastasis+in+hamsters+infected+with+Leishmania+(Viannia)+panamensis.">The inflammatory response promotes cutaneous metastasis in hamsters infected with Leishmania (Viannia) panamensis.</a> J. Parasitol. 82, 454-457 (1996).</li><li>Travi, B. L., Osorio, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failure+of+Albendazole+as+an+alternative+treatment+of+cutaneous+Leishmaniasis+in+the+hamster+model.">Failure of Albendazole as an alternative treatment of cutaneous Leishmaniasis in the hamster model.</a> Memorias Instituto Oswaldo. 93, 515 (1998).</li><li>Travi, B. L., Osorio, Y., Melby, P. C., Chandrasekar, B., Arteaga, L., Saravia, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gender+is+a+major+determinant+of+the+clinical+evolution+and+immune+response+in+hamsters+infected+with+Leishmania+spp.">Gender is a major determinant of the clinical evolution and immune response in hamsters infected with Leishmania spp.</a> Infect Immun. 70, 2288-2296 (2002).</li><li>Osorio, Y., Melby, P. C., Pirmez, C., Chandrasekar, B., Guar&#237;n, N., Travi, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+site+of+cutaneous+infection+influences+the+immunological+response+and+clinical+outcome+of+hamsters+infected+with+Leishmania+panamensis.">The site of cutaneous infection influences the immunological response and clinical outcome of hamsters infected with Leishmania panamensis.</a> Parasite Immunol. 25, 139-148 (2003).</li><li>Osorio, Y., Bonilla, D. L., Peniche, A. G., Melby, P. C., Travi, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pregnancy+enhances+the+innate+immune+response+in+experimental+cutaneous+leishmaniasis+through+hormone-modulated+nitric+oxide+production.">Pregnancy enhances the innate immune response in experimental cutaneous leishmaniasis through hormone-modulated nitric oxide production.</a> J. Leukoc. Biol. 83, 1413-1422 (2008).</li><li>Espitia, C. M., Zhao, W., Saldarriaga, O., Osorio, Y., Harrison, L. M., Cappello, M., Travi, B. L., Melby, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duplex+real-time+reverse+transcriptase+PCR+to+determine+cytokine+mRNA+expression+in+a+hamster+model+of+New+World+cutaneous+leishmaniasis.">Duplex real-time reverse transcriptase PCR to determine cytokine mRNA expression in a hamster model of New World cutaneous leishmaniasis.</a> BMC Immunol. 22, 11-31 (2010).</li></ol>

没有利益冲突的声明。

Cutaneous leishmaniasis is endemic in the tropics and neotropics. It is often referred to as a group of diseases because of the varied spectrum of clinical manifestations, which range from small cutaneous nodules to gross mucosal tissue destruction. Most available drugs are costly, require long treatment regimens and are becoming increasingly ineffective, necessitating the discovery of new drugs.
The mouse model is widely used but has some disadvantages. Thus for example, on the basis of the l...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Type</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Ketamine </td>
			<td>Reagent</td>
			<td>Holliday &#x2013;Scott S.A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xilacine</td>
			<td>Reagent</td>
			<td>Synthesis</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Reagent</td>
			<td>GIBCO</td>
			<td>14190-136</td>
		</tr>
		<tr>
			<td>Digital caliper</td>
			<td>Equipment</td>
			<td>Fisher</td>
			<td>15-077-958</td>
		</tr>
		<tr>
			<td>Giemsa</td>
			<td>Reagent</td>
			<td>SIGMA</td>
			<td>GS1L-1L</td>
		</tr>
		<tr>
			<td>Formalin</td>
			<td>Reagent</td>
			<td>Nova lab</td>
			<td>11273</td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Reagent</td>
			<td>Pechiney Plastic Packaging</td>
			<td>PM-996</td>
		</tr>
		<tr>
			<td>Haemotoxilin</td>
			<td>Reagent</td>
			<td>Nova lab</td>
			<td>10870103</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>&nbsp;</td>
			<td>Nova lab</td>
			<td>10870203</td>
		</tr>
		<tr>
			<td>Kodak Ektachem dry chemistry</td>
			<td>Equipment</td>
			<td>Kodak Ektachem </td>
			<td>DT-60</td>
		</tr>
		<tr>
			<td>meglumine antimoniate</td>
			<td>Reagent</td>
			<td>Aventis</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscopy</td>
			<td>Equipment</td>
			<td>Nikon</td>
			<td>YS2-T</td>
		</tr>
	</tbody>
</table>

cutaneous leishmaniasis in the dorsal skin of hamsters: a useful model for the screening of antileishmanial drugs

传统上，仓鼠实验接种在鼻子或脚垫。然而，在这些网站溃疡并不总是发生，测量病灶大小，是一个很难的过程和动物显示困难，吃饭，呼吸，因为移动的病变。为了优化皮肤利什曼病，年轻的成年男性和女性的金黄地鼠，仓鼠模型（Mesocricetus鲫鱼 ）皮内注射在背侧皮肤与1至1.5×L0 7 promastigotes的利什曼物种及后续病变的进展进行评估，最多16周后感染。金黄地鼠被选中，因为它被认为是足够的生物模型，以评估对利什曼原虫的药物，因为它们很容易受到感染不同物种。皮肤感染仓鼠但控制慢性病灶，临床演变与人类观察到的迹象类似的结果。因此，建立Øf的感染程度测量根据病灶硬结和溃疡面积的大小是可行的。这种方法已被证明它的多功能性，易于管理，在接种跟进和表征不同处理后通过不同的方式，获得临床标本典型病变（溃疡），应用的治疗。动物的生命质量有关运动使用这种方法，还保存着寻找食物和水，游戏和社交活动。

Watch this Scientific Journal Video about Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs at JoVE.com

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

在仓鼠的背部皮肤：皮肤利什曼病Antileishmanial药物筛选的一个有用的模型

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

cutaneous-leishmaniasis-dorsal-skin-hamsters-useful-model-for

Research

JoVE Journal

Immunology and Infection

21.8K 次观看.  University of Antioquia. Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

视频: Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

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 ...

科研

免疫与感染

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Antileishmanial筛选对细胞内的寄生虫救援和转化实验<em&gt;杜氏利什曼原虫</em无鞭毛体在THP1人急性单核细胞白血病细胞株

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not associated with the protein in nature. Literature has provided numerous examples regarding the implementation of directed evolution to successfully alter molecular specificity and catalysis1. The primary advantage of utilizing directed evolution instead of more rational-based approaches for molecular engineering relates to the volume and diversity of variants that can be screened2. One possible application of directed evolution involves improving structural stability of bacteriolytic enzymes, such as endolysins. Bacteriophage encode and express endolysins to hydrolyze a critical covalent bond in the peptidoglycan (i.e. cell wall) of bacteria, resulting in host cell lysis and liberation of progeny virions. Notably, these enzymes possess the ability to extrinsically induce lysis to susceptible bacteria in the absence of phage and furthermore have been validated both in vitro and in vivo for their therapeutic potential3-5. The subject of our directed evolution study involves the PlyC endolysin, which is composed of PlyCA and PlyCB subunits6. When purified and added extrinsically, the PlyC holoenzyme lyses group A streptococci (GAS) as well as other streptococcal groups in a matter of seconds and furthermore has been validated in vivo against GAS7. Significantly, monitoring residual enzyme kinetics after elevated temperature incubation provides distinct evidence that PlyC loses lytic activity abruptly at 45 &deg;C, suggesting a short therapeutic shelf life, which may limit additional development of this enzyme. Further studies reveal the lack of thermal stability is only observed for the PlyCA subunit, whereas the PlyCB subunit is stable up to ~90 &deg;C (unpublished observation). In addition to PlyC, there are several examples in literature that describe the thermolabile nature of endolysins. For example, the Staphylococcus aureus endolysin LysK and Streptococcus pneumoniae endolysins Cpl-1 and Pal lose activity spontaneously at 42 &deg;C, 43.5 &deg;C and 50.2 &deg;C, respectively8-10. According to the Arrhenius equation, which relates the rate of a chemical reaction to the temperature present in the particular system, an increase in thermostability will correlate with an increase in shelf life expectancy11. Toward this end, directed evolution has been shown to be a useful tool for altering the thermal activity of various molecules in nature, but never has this particular technology been exploited successfully for the study of bacteriolytic enzymes. Likewise, successful accounts of progressing the structural stability of this particular class of antimicrobials altogether are nonexistent. In this video, we employ a novel methodology that uses an error-prone DNA polymerase followed by an optimized screening process using a 96 well microtiter plate format to identify mutations to the PlyCA subunit of the PlyC streptococcal endolysin that correlate to an increase in enzyme kinetic stability (Figure 1). Results after just one round of random mutagenesis suggest the methodology is generating PlyC variants that retain more than twice the residual activity when compared to wild-type (WT) PlyC after elevated temperature treatment.

A novel directed evolution method specific to the field of thermostability engineering was developed and consequently validated for bacteriolytic enzymes. After only one round of random mutagenesis, an evolved bacteriolytic enzyme, PlyC 29C3, displayed greater than twice the residual activity when compared to the wild-type protein after elevated temperature incubation.

耐高温溶菌酶的定向进化的一个新的筛选方法

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.

VIGS介导的正向遗传学筛选参与非寄主抗性基因鉴定


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

可视化痰生物膜发展影响使用腔室盖玻片型号

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides.
IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 &plusmn; 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 &#181;g/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 &#181;g/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

We describe the isolation of cardiac extracellular matrix from C57Bl/6J control mice, tight-skin mice, and tight-skin mice treated with the IRF5 inhibitory peptide. We also describe the vasodilation studies on the isolated vessels from C57Bl/6J, tight-skin mice and tight-skin mice treated with the IRF5 inhibitory peptide.

隔离容器的血管扩张和细胞外基质的紧皮肤小鼠隔离

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

虫媒病毒感染作为鉴定病毒免疫调节剂和宿主抗病毒因子的筛选工具

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

化学抑制剂库筛选 t 细胞受体信号的中介识别

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine models provide valuable mechanistic insight into these processes; however, no single model fully addresses all aspects of the wound healing response. Instead, it is ideal to use multiple models to address the different aspects of wound healing. Here, two different methods that address diverse aspects of the wound healing response are described. In the first model, polyvinyl alcohol sponges are subcutaneously implanted along the mouse dorsum. Following sponge retrieval, cells can be isolated by mechanical disruption, and fluids can be extracted by centrifugation, thus allowing for a detailed characterization of cellular and cytokine responses in the acute wound environment. A limitation of this model is the inability to assess the rate of wound closure. For this, a tail skin excision model is utilized. In this model, a 10 mm x 3 mm rectangular piece of tail skin is excised along the dorsal surface, near the base of the tail. This model can be easily photographed for planimetric analysis to determine healing rates and can be excised for histological analysis. Both described methods can be utilized in genetically altered mouse strains, or in conjunction with models of comorbid conditions, such as diabetes, aging, or secondary infection, in order to elucidate wound healing mechanisms.

Here, two murine wound healing models are described, one designed to assess cellular and cytokine wound healing responses and the other to quantify the rate of wound closure. These methods can be used with complex disease models such as diabetes to determine mechanisms of various aspects of poor wound healing.

使用多盐皮下聚乙烯醇海绵植入和外向尾皮肤伤口模型进行急性伤口愈合评估。

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine ...