This work was supported in part by the Center for Integration of Medicine and Innovative Technology (CIMIT) under the U.S. Army Medical Research Acquisition Activity Cooperative Agreement (CIMIT No. 14-1894 to TD) and the National Institutes of Health (1R21AI109172 to TD). YW was supported by an ASLMS Student Research Grant (BS.S02.15). We are grateful to Tayyaba Hasan, PhD at the Wellman Center for her co-mentorship for YW.

1.细菌培养的制备<ol><li>添加7.5毫升脑心浸液（BHI）培养基中至50mL离心管中。种子鲍曼不动杆菌细胞在BHI培养基，然后孵育鲍曼不动杆菌培养在轨道培养箱（37℃）18小时。 </li><li>离心细胞的培养物在3,500×g下5分钟，除去上清液，并在磷酸盐缓冲盐水（PBS）洗涤小球。 </li><li>重悬在新鲜的PBS中的细菌颗粒和彻底吸管的悬浮液。 </li><li>收集100μL的细菌悬浮液，并使用新鲜的PBS作1:10稀释。 </li><li>转移稀释到1.5mL半微量比色杯，并测量在600nm（OD 600 nm）的波长的光密度（OD）。 </li><li>根据稀释的测量OD 600nm的值与稀释倍数（10）计算原始（未稀释的）悬浮液的OD 600纳米在PBS中。 </li><li>调整日在PBSë原始悬浮液至OD 600纳米 = 0.6（对应于10 8 CFU / mL的细胞密度）。 </li></ol>通过生物发光甲导致了烧伤感染的2小鼠模型。 鲍曼不动杆菌 <ol><li>年龄7-8周，体重17〜19克使用成年雌性BALB / c小鼠。允许小鼠实验开始前适应环境实验室条件至少3天。保持在12小时光照/黑暗周期的小鼠的21℃室温下，给他们的食物和水随意 。 </li><li>麻醉小鼠用氯胺酮 - 赛拉嗪混合物的腹膜内注射（100mg / kg的 - 20毫克/千克）。轻轻触摸每只小鼠用棉签的睑;不存在所述眼睑反射的暗示适当的麻醉深度。盖鼠标眼睛兽医药膏，以防止干燥眼睛在麻醉过程中。 </li><li>剃须背面的小鼠用5揭露尽可能多的皮肤尽可能0刀理发器。 </li><li>放置在35毫米培养皿中的小鼠的腹部下方的盖，以保持他们的背部在相对水平的位置。 </li><li>使用10&quot; ×10&quot; ，220 VAC热板在250毫升烧杯（80％满）烧开水。直到达到与水热平衡浸入黄铜块（1 1cm×1cm的横截面）放入烧杯中。热平衡通常需要&lt;5分钟，并通过在烧杯中的水的再沸腾指示。 </li><li>在此之前创建烧伤，辖先发制人镇痛药（为0.1mg / kg的丁丙诺啡的皮下注射），用于缓解疼痛。 </li><li>先发制人镇痛药10分钟后，轻轻按下加热黄铜块对小鼠为7秒背面的剃毛区以诱导烧伤创面。 注:为避免热损伤到工作人员，在执行烧录程序时戴上热防护手套。 </li><li>管理通过subcutaneou 0.5毫升的无菌盐水的注射液，以防止脱水。 </li><li>五分钟后的热损伤的诱导，接种含有5×10 6 CFU的PBS细菌悬浮液的50微升到使用移液管的小鼠烫伤。通过在皮肤上的锯齿形运动移动无菌棉签涂抹在灼伤等分在过火面积尽可能均匀地分布在细菌细胞。 </li><li>细菌接种后，立即进行生物发光成像的感染的烧伤，如第4说明。 </li><li>放置小鼠上的水加热外科床（37℃，回收区），直到小鼠已经完全从麻醉中恢复。房子与在生物安全等级-2动物室的每笼5只的最大数量的小鼠。 </li><li>施用镇痛药（0.1mg / kg的丁丙诺啡的皮下注射），每天两次对烧伤后的前三天。 </li></ol> 3.抗菌蓝色光疗法对于A＆＃160; 杆菌感染小鼠<ol><li>在细菌接种后24个小时开始ABL治疗。 </li><li>使用的发光二极管（LED），其具有用于照射ABL 415nm处的峰值发射。安装在一个散热片上的LED，以防止对小鼠18所照射的区域的热效应。固定LED与夹子连接器的光学支承杆，以允许LED以向上和向下移动。 </li><li>打开电源/能量计和按波长按钮选择415纳米。重置电源/能量计减去背景（环境光）。 </li><li>将电源/电能表右侧的LED下。穿蓝色光护目镜。开启LED光并调整LED孔（即来自LED会聚的光的透镜）和功率/能量计的光传感器（2厘米直径）之间的距离，使得光斑覆盖的整个区域光传感器。 </li><li>仔细调节LED驱动器和记录鲍威的阅读R /电能表。根据读取计算的辐照度:辐照度=读数（W）/面积（cm 2）。通过调整LED驱动器调节LED 100毫瓦/厘米2的辐照度。 </li><li>关闭LED。测量LED孔和功率/能量计的光传感器之间的距离。 </li><li>麻醉小鼠用氯胺酮 - 赛拉嗪混合物的腹膜内注射（100mg / kg的 - 20毫克/千克）。不存在所述眼睑反射的暗示适当的麻醉深度。 </li><li>随机划分小鼠成ABL治疗组（n = 10）和未处理的对照组（n = 10）。 </li><li>对于ABL处理组，覆盖小鼠的眼睛用铝箔以避免过度暴露于光。将鼠标烧伤直属LED，用鼠标腹部下方的35毫米陪替氏培养皿的盖子，以保持他们的背部在水平位置。 </li><li>在一个正方形皮氏二取代，在步骤3.5，用鼠标提到的功率/能量计SH。调整鼠标回的位置处，如步骤所讨论的LED孔和鼠标烧伤的表面之间的距离是等于LED孔和电源/能量计的光传感器（水平之间的高度3.5）。 </li><li>以100mW / cm 2的辐照度照射感染的烧伤。递送ABL在剂量72焦耳/厘米2，直到360达到焦耳/ cm 2的总剂量（ 例如 ，0，72，144，216，288和360焦耳/厘米2）。各光剂量后，执行生物发光成像的小鼠，如第4所讨论的。 </li><li>对于未处理的对照组，执行鼠标烧伤生物发光成像，如第4所讨论的，使用如用于ABL处理组相同的时间间隔。 </li><li>小鼠的恢复之前，放置在水加热手术床上的小鼠以防止热量散失和低温。观察活动和老鼠，直到复苏的眼睑反射。后日小鼠电子回收，安置他们2-3个笼子。 </li><li> ABL治疗后，执行生物发光成像，如第4对第3天所讨论的，每日然后隔天以监测感染的小鼠的时间生物负担。 </li></ol> 4.感染的生物发光成像小鼠<ol><li>图像使用低光成像系统，其包括增强型电荷耦合器件照相机，摄像机控制器，一个样品室，以及图像处理器19中的小鼠。 </li><li>麻醉小鼠用氯胺酮 - 赛拉嗪混合物的腹膜内注射（100mg / kg的 - 20毫克/千克）。轻触用棉签小鼠的眼睑;不存在所述眼睑反射的暗示适当的麻醉深度。 </li><li>启动实时成像软件。在显示的控制面板，单击初始化。等到温度框的颜色变为绿色，表明阶段的温度在试样腔室已经达到了37℃。 </li><li>放置在成像系统的样本腔室中的阶段（37℃）的小鼠中，与直接在相机下的感染的烧伤。 注:当烧伤变得干燥细菌的生物发光可以降低。因此，建议在成像之前润泽用PBS小鼠烧伤。 </li><li>在控制面板中，把一个对号旁边的"发光"。选择"自动曝光"，使成像的曝光时间将通过基于生物发光强度的活体成像软件进行优化。 </li><li>从"查看现场的"下拉列表中选择"C"。选择"扫描中间范围"选项，让软件判断焦距。将一个复选标记旁边为叠加。 </li><li>点击"获取"来捕捉图像。在"编辑图像标签"框中，单击"确定"; "图像窗口"和"收费调色板"将出现。 </li><li>设置自动选择自动ROI参数。 </li><li>量化生物发光强度为相对发光单位（的RLU）和伪彩色尺度从粉红（最强烈）到蓝色（至少激烈）19，20显示生物发光。 </li><li>计算在不同的基于生物发光强度分析的时间点在小鼠烧伤细菌的存活分数。在给定时间点的细菌的存活分数=在该时间点测量到的生物发光强度/生物发光强度ABL曝光17前右测量。 </li></ol> 5.小鼠安乐死<ol><li>是该实验的终点为如下:（a）如由生物发光损失作为通过成像确定的感染的分辨率; （B）系统的感染的发生通过生物发光的传播烧成以外如所指示一个;在比较的≥15％体重（c）中损失到正常年龄匹配的小鼠中，或从疼痛和痛苦由缺乏响应于手动刺激，不动，一个不能吃或喝所指示的痛苦。在研究过程中，如果我们遇到这样的情况，我们不确定鼠标是否已经完全达到了奄奄一息的状态，根据我们的定义所概述，和/或我们是不确定的，如果我们需要安乐死它的情况，我们会联系CCM兽医人员行动计划; （d）中，否则将小鼠研究开始后30天被安乐死。 </li><li> 重新定位到小鼠关闭笼子专门为安乐死和通过递送二氧化碳安乐死的小鼠（CO 2）的压缩气体进入笼中。 <ol><li>打开CO 2罐或稳压阀以启动气体的流动。验证读取调节器根据刊登的单元的指令正确的PSI（磅每平方英寸），并调整重新根据需要gulator到正确的PSI，通常不高于5磅。 </li><li>慢慢填写。流速应置换每分钟腔室/笼体积的不超过30％（对于典型的鼠笼，〜2L /分钟;对于鼠笼，〜7.5升/分钟）。 </li><li>等待约3-5分钟为动物停止移动或呼吸;眼睛应该是固定和扩张。关闭CO 2罐或调节阀，停止CO 2的流动。 </li><li>确保心脏没有摸着拇指和食指之间的胸口跳动。确保有通过触摸眼球没有眨眼反射。 <ol><li>如果有一个心跳或眨眼反射，重复安乐死过程或使用剪刀来打开胸腔以创建气胸（动物必须是非响应于脚趾捏之前执行此过程）。 </li></ol></li></ol></li></ol>

<ol>
	<li>Gibran, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Summary+of+the+2012+ABA+Burn+Quality+Consensus+conference.">Summary of the 2012 ABA Burn Quality Consensus conference.</a> J Burn Care Res. 34 (4), 361-385 (2013).</li><li>Sommer, R., Joachim, I., Wagner, S., Titz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+to+control+infections:+anti-biofilm+strategies+against+gram-negative+bacteria.">New approaches to control infections: anti-biofilm strategies against gram-negative bacteria.</a> Chimia (Aarau). 67 (4), 286-290 (2013).</li><li>Peleg, A. Y., Seifert, H., Paterson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acinetobacter+baumannii:+emergence+of+a+successful+pathogen.">Acinetobacter baumannii: emergence of a successful pathogen.</a> Clin Microbiol Rev. 21 (3), 538-582 (2008).</li><li>Uppu, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amide+side+chain+amphiphilic+polymers+disrupt+surface+established+bacterial+bio-films+and+protect+mice+from+chronic+Acinetobacter+baumannii+infection.">Amide side chain amphiphilic polymers disrupt surface established bacterial bio-films and protect mice from chronic Acinetobacter baumannii infection.</a> Biomaterials. 74, 131-143 (2016).</li><li>Schaber, 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=Pseudomonas+aeruginosa+forms+biofilms+in+acute+infection+independent+of+cell-to-cell+signaling.">Pseudomonas aeruginosa forms biofilms in acute infection independent of cell-to-cell signaling.</a> Infect Immun. 75 (8), 3715-3721 (2007).</li><li>Hoiby, N., Bjarnsholt, T., Givskov, M., Molin, S., Ciofu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+of+bacterial+biofilms.">Antibiotic resistance of bacterial biofilms.</a> Int J Antimicrob Agents. 35 (4), 322-332 (2010).</li><li>Lebeaux, D., Ghigo, J. M., Beloin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm-related+infections:+bridging+the+gap+between+clinical+management+and+fundamental+aspects+of+recalcitrance+toward+antibiotics.">Biofilm-related infections: bridging the gap between clinical management and fundamental aspects of recalcitrance toward antibiotics.</a> Microbiol Mol Biol Rev. 78 (3), 510-543 (2014).</li><li>Akers, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms+and+persistent+wound+infections+in+United+States+military+trauma+patients:+a+case-control+analysis.">Biofilms and persistent wound infections in United States military trauma patients: a case-control analysis.</a> BMC Infect Dis. 14, 190 (2014).</li><li>Burmolle, 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=Biofilms+in+chronic+infections+-+a+matter+of+opportunity+-+monospecies+biofilms+in+multispecies+infections.">Biofilms in chronic infections - a matter of opportunity - monospecies biofilms in multispecies infections.</a> FEMS Immunol Med Microbiol. 59 (3), 324-336 (2010).</li><li>. National strategy on combating antibiotic-resistant bacteria Available from: <a target="_blank" href="https://www.whitehouse.gov/sites/default/files/docs/carb_national_strategy.pdf">https://www.whitehouse.gov/sites/default/files/docs/carb_national_strategy.pdf</a> (2014)</li><li>Dai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+for+Acinetobacter+baumannii+burn+infections+in+mice.">Photodynamic therapy for Acinetobacter baumannii burn infections in mice.</a> Antimicrob Agents Chemother. 53 (9), 3929-3934 (2009).</li><li>Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+blue+light+therapy+for+multidrug-resistant+Acinetobacter+baumannii+infection+in+a+mouse+burn+model:+implications+for+prophylaxis+and+treatment+of+combat-related+wound+infections.">Antimicrobial blue light therapy for multidrug-resistant Acinetobacter baumannii infection in a mouse burn model: implications for prophylaxis and treatment of combat-related wound infections.</a> J Infect Dis. 209 (12), 1963-1971 (2014).</li><li>Dai, T., 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=Ultraviolet+C+light+for+Acinetobacter+baumannii+wound+infections+in+mice:+potential+use+for+battlefield+wound+decontamination?.">Ultraviolet C light for Acinetobacter baumannii wound infections in mice: potential use for battlefield wound decontamination?.</a> J Trauma Acute Care Surg. 73 (3), 661-667 (2012).</li><li>Dai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+light+for+infectious+diseases:+Propionibacterium+acnes,+Helicobacter+pylori,+and+beyond?.">Blue light for infectious diseases: Propionibacterium acnes, Helicobacter pylori, and beyond?.</a> Drug Resist Updat. 15 (4), 223-236 (2012).</li><li>Yin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+based+anti-infectives:+ultraviolet+C+irradiation,+photodynamic+therapy,+blue+light,+and+beyond.">Light based anti-infectives: ultraviolet C irradiation, photodynamic therapy, blue light, and beyond.</a> Curr Opin Pharmacol. 13 (5), 731-762 (2013).</li><li>Haisma, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+and+antimicrobial+responses+to+methicillin-resistant+Staphylococcus+aureus+in+an+in+vitro+wound+infection+model.">Inflammatory and antimicrobial responses to methicillin-resistant Staphylococcus aureus in an in vitro wound infection model.</a> PLoS One. 8 (12), e82800 (2013).</li><li>Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+Blue+Light+Inactivation+of+Gram-Negative+Pathogens+in+Biofilms:+In+Vitro+and+In+Vivo+Studies.">Antimicrobial Blue Light Inactivation of Gram-Negative Pathogens in Biofilms: In Vitro and In Vivo Studies.</a> J Infect Dis. 213 (9), 1380-1387 (2016).</li><li>Chen, D., Shen, Y., Huang, Z., Li, B., Xie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-Emitting+Diode-Based+Illumination+System+for+In+Vitro+Photodynamic+Therapy.">Light-Emitting Diode-Based Illumination System for In Vitro Photodynamic Therapy.</a> Int J Photoenergy. 2012 (2), (2012).</li><li>Demidova, T. N., Gad, F., Zahra, T., Francis, K. P., Hamblin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+photodynamic+therapy+of+localized+infections+by+bioluminescence+imaging+of+genetically+engineered+bacteria.">Monitoring photodynamic therapy of localized infections by bioluminescence imaging of genetically engineered bacteria.</a> J Photochem Photobiol B. 81 (1), 15-25 (2005).</li><li>Hamblin, M. R., Zahra, T., Contag, C. H., McManus, A. T., Hasan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+monitoring+and+treatment+of+potentially+lethal+wound+infections+in+vivo.">Optical monitoring and treatment of potentially lethal wound infections in vivo.</a> J Infect Dis. 187 (11), 1717-1725 (2003).</li><li>Rowan, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burn+wound+healing+and+treatment:+review+and+advancements.">Burn wound healing and treatment: review and advancements.</a> Critical Care. 19, 243 (2015).</li><li>Marx, D. E., Barillo, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+in+medicine:+The+basic+science.">Silver in medicine: The basic science.</a> Burns. 40 (Supplement 1), S9-S18 (2014).</li><li>Heyneman, A., Hoeksema, H., Vandekerckhove, D., Pirayesh, A., Monstrey, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+silver+sulphadiazine+in+the+conservative+treatment+of+partial+thickness+burn+wounds:+A+systematic+review.">The role of silver sulphadiazine in the conservative treatment of partial thickness burn wounds: A systematic review.</a> Burns. 42 (7), 1377-1386 (2016).</li><li>Roberts, 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=Individualised+antibiotic+dosing+for+patients+who+are+critically+ill:+challenges+and+potential+solutions.">Individualised antibiotic dosing for patients who are critically ill: challenges and potential solutions.</a> Lancet Infect Dis. 14 (6), 498-509 (2014).</li><li>Dai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+light+eliminates+community-acquired+methicillin-resistant+Staphylococcus+aureus+in+infected+mouse+skin+abrasions.">Blue light eliminates community-acquired methicillin-resistant Staphylococcus aureus in infected mouse skin abrasions.</a> Photomed Laser Surg. 31 (11), 531-538 (2013).</li><li>Uppu, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amide+side+chain+amphiphilic+polymers+disrupt+surface+established+bacterial+bio-films+and+protect+mice+from+chronic+Acinetobacter+baumannii+infection.">Amide side chain amphiphilic polymers disrupt surface established bacterial bio-films and protect mice from chronic Acinetobacter baumannii infection.</a> Biomaterials. 74, 131-143 (2016).</li><li>Donlan, R. M., Costerton, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms:+survival+mechanisms+of+clinically+relevant+microorganisms.">Biofilms: survival mechanisms of clinically relevant microorganisms.</a> Clin Microbiol Rev. 15 (2), 167-193 (2002).</li><li>Olsen, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm-specific+antibiotic+tolerance+and+resistance.">Biofilm-specific antibiotic tolerance and resistance.</a> Eur J Clin Microbiol Infect Dis. , (2015).</li><li>Song, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototoxic+effect+of+blue+light+on+the+planktonic+and+biofilm+state+of+anaerobic+periodontal+pathogens.">Phototoxic effect of blue light on the planktonic and biofilm state of anaerobic periodontal pathogens.</a> J Periodontal Implant Sci. 43 (2), 72-78 (2013).</li><li>Rosa, L. P., da Silva, F. C., Viana, M. S., Meira, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+effectiveness+of+455-nm+blue+LED+to+reduce+the+load+of+Staphylococcus+aureus+and+Candida+albicans+biofilms+in+compact+bone+tissue.">In vitro effectiveness of 455-nm blue LED to reduce the load of Staphylococcus aureus and Candida albicans biofilms in compact bone tissue.</a> Lasers Med Sci. 31 (1), 27-32 (2015).</li><li>Guffey, J. S., Wilborn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+bactericidal+effects+of+405-nm+and+470-nm+blue+light.">In vitro bactericidal effects of 405-nm and 470-nm blue light.</a> Photomed Laser Surg. 24 (6), 684-688 (2006).</li><li>Enwemeka, C. S., Williams, D., Enwemeka, S. K., Hollosi, S., Yens, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+470-nm+light+kills+methicillin-resistant+Staphylococcus+aureus+(MRSA)+in+vitro.">Blue 470-nm light kills methicillin-resistant Staphylococcus aureus (MRSA) in vitro.</a> Photomed Laser Surg. 27 (2), 221-226 (2009).</li><li>Bumah, V. V., Masson-Meyers, D. S., Cashin, S. E., Enwemeka, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wavelength+and+bacterial+density+influence+the+bactericidal+effect+of+blue+light+on+methicillin-resistant+Staphylococcus+aureus+(MRSA).">Wavelength and bacterial density influence the bactericidal effect of blue light on methicillin-resistant Staphylococcus aureus (MRSA).</a> Photomed Laser Surg. 31 (11), 547-553 (2013).</li><li>Maclean, M., MacGregor, S. J., Anderson, J. G., Woolsey, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+bacterial+pathogens+following+exposure+to+light+from+a+405-nanometer+light-emitting+diode+array.">Inactivation of bacterial pathogens following exposure to light from a 405-nanometer light-emitting diode array.</a> Appl Environ Microbiol. 75 (7), 1932-1937 (2009).</li><li>Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+lens-microneedle+array+for+percutaneous+light+delivery.">Optical lens-microneedle array for percutaneous light delivery.</a> Biomedical Optics Express. 7 (1o), 4220-4227 (2016).</li></ol>

The authors declare that they have no competing financial interests.

ABL是用于治疗感染的新方法。由于其作用机制不同于化疗的完全不同，它更是一个理疗。介导抗微生物效果的药剂是蓝色光照射（400-470纳米）。随着蓝光LED的发展，我们获得了进入的MDR感染的有效和简单的基于光抗菌方法。 在这个协议中，我们描述了造成MDR， 鲍曼不动杆菌的生物发光应变烧伤感染的小鼠模型的发展。与使用生物发光的细菌，感染的程度可以是非侵?...

烧伤感染，这是因为皮肤的热损伤的频传，继续成为发病率和死亡率1的一个重要原因。烧伤感染的管理已经通过多药耐药性（MDR）的细菌菌株2的增加出现了进一步的损害，由于大量使用抗生素。一个重要的MDR革兰阴性菌是鲍曼不动杆菌 ，这是已知的与最近的战斗创伤相关联且几乎所有可用的抗生素耐药3。生物膜的损伤灶的存在已经报告了4，5，被认为是加剧耐受抗生素和宿主防御6，7，引起持续感染的8，9。因此，有一种压力机，加工摹需要替代治疗的发展。在最近公布的国家战略打击抗生素耐药细菌 ，替代疗法，抗生素的发展已注意到由美国10政府的作用。 光为基础的抗菌剂的方法，由名称所指示的，需要与或不与其它药剂的光照射。这些方法包括抗微生物光动力学疗法（APDT），紫外线C（UVC）照射，和抗微生物蓝光（ABL）。在以前的研究中，他们已经杀死MDR菌株11，12，13显示出可喜的成效。在这三种光为基础的方法，ABL已吸引近几年越来越多的关注，由于无需使用光敏剂14的其固有的抗菌性能。在COMPARISON到APDT，ABL仅涉及使用光的，而需要APDT光和光敏剂的组合。因此，ABL是简单且廉价的14。相比于UVC，ABL被认为是细胞毒性和遗传毒性对宿主细胞15要少得多。 此协议的目的是研究ABL对小鼠模型引起MDR 鲍曼不动杆菌烧伤感染的治疗功效。我们用生物发光致病细菌培养烧伤感染，允许的实时细菌负荷的无创监测的新的小鼠模型。相比，体液/组织提取和随后的镀敷和菌落计数16传统的方法，这种技术提供了精确的结果。组织取样的过程中可能引入的实验误差的另一个来源。由于细菌的发光强度成线性比例的受文者积水细菌CFU 17，我们可以直接测量光照射一定剂量后的细菌的存活。通过监测在活的动物中实时接收光处理中的细菌负荷，的细菌杀死动力学可使用小鼠的显著减少数量来表征。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>IVIS&#160;</td>
			<td>PerkinElmer Inc, Waltham, MA</td>
			<td>IVIS Lumina Series III</td>
			<td>Pre-clinical in vivo imaging</td>
		</tr>
		<tr>
			<td>Light-emitting diode LED</td>
			<td>VieLight Inc, Toronto, Canada&#160;</td>
			<td>415 nm</td>
			<td>Light source for illumination</td>
		</tr>
		<tr>
			<td>Power/energy meter</td>
			<td>Thorlabs, Inc., Newton, NJ</td>
			<td>PM100D</td>
			<td>Light irradiance detector</td>
		</tr>
		<tr>
			<td>Mouse&#160;</td>
			<td>Charles River Laboratories, Wilmington, MA</td>
			<td>BALB/c</td>
			<td>7-8 weeks age, 17-19 g weight</td>
		</tr>
		<tr>
			<td>Acinetobacter baumannii&#160;</td>
			<td>Brooke Army Medical Center, Fort Sam Houston, TX</td>
			<td>Clinical isolate</td>
			<td>Engineered luminescent strain</td>
		</tr>
		<tr>
			<td>Insulin Syringes</td>
			<td>Fisher Scientific</td>
			<td>14-826-79</td>
			<td>BD Lo-Dose U-100 Insulin Syringes for injection</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>721016</td>
			<td>0.9% Sodium Chloride</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline, 1X Solution</td>
			<td>Fisher Scientific</td>
			<td>BP24384&#160;</td>
			<td>A standard phosphate buffer used in many biomolecular procedures</td>
		</tr>
		<tr>
			<td>Brain Heart Infusion</td>
			<td>Fisher Scientific</td>
			<td>B11059</td>
			<td>Bacterial culture medium</td>
		</tr>
		<tr>
			<td>Falcon 15mL Conical Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-70C</td>
			<td>For bacterial suspension centrifuge</td>
		</tr>
		<tr>
			<td>Benchtop Incubated Orbital Shakers</td>
			<td>Laboratory Supply Network, Inc, Atkinson, NH&#160;</td>
			<td>Incu-Shaker Mini</td>
			<td>For culturing of bacteria</td>
		</tr>
		<tr>
			<td>Inoculating Loops</td>
			<td>Fisher Scientific</td>
			<td>22-363-605&#160;&#160;</td>
			<td>For smearing bacterial inoclum on burn surface of mice</td>
		</tr>
		<tr>
			<td>Fisher Scientific Redi-Tip Pipet Tips, 1-200&#181;L</td>
			<td>Fisher Scientific</td>
			<td>02-707-502</td>
			<td>Pipet Tips</td>
		</tr>
		<tr>
			<td>Thermo Scientific&#160;Sorvall Legend X1 Centrifuge</td>
			<td>Fisher Scientific</td>
			<td>75-004-220</td>
			<td>For bacterial suspension seperation</td>
		</tr>
		<tr>
			<td>Brass Block</td>
			<td>Small Parts, Inc., Miami, FL</td>
			<td>10 mm by 10 mm&#160;</td>
			<td>For creation of burns in mice</td>
		</tr>
		<tr>
			<td>Extreme Dragon PBI/Kevlar High-Heat Gloves</td>
			<td>Superior Glove Works Ltd, Cheektowaga, NY</td>
			<td>PBI83514&#160;</td>
			<td>Heat Resistant Gloves</td>
		</tr>
		<tr>
			<td>Greiner dishes</td>
			<td>Sigma-Aldrich Co. LLC</td>
			<td>P5112-740EA</td>
			<td>35 mm &#215;10 mm</td>
		</tr>
		<tr>
			<td>Corning Digital Hot Plate</td>
			<td>Cole-Parmer Instrument Company, LLC</td>
			<td>UX-84301-65</td>
			<td>10&#34; x 10&#34;, 220 VAC, for boiling water&#160;</td>
		</tr>
		<tr>
			<td>Mouse/Rat Thin Line Water Heated Surgical Bed</td>
			<td>E-Z Systems</td>
			<td>EZ-211</td>
			<td>Prevents heat loss and hypothermia during surgery</td>
		</tr>
	</tbody>
</table>

in vivo investigation of antimicrobial blue light therapy for multidrug-resistant acinetobacter baumannii burn infections using bioluminescence imaging

烧伤感染仍是发病率和死亡率的重要原因。多药耐药（MDR）细菌的日益兴起，导致了传统抗生素治疗的频繁故障。替代治疗迫切需要解决MDR细菌。 一个创新的非抗生素的办法，抗菌蓝光（ABL），已经显示出对MDR感染的有前途的有效性。 ABL的作用机制尚不清楚。它通常假设在细菌中天然存在的内源性光敏发色团（ 例如 ，无铁卟啉，黄素， 等 ）由ABL，这反过来又产生细胞毒性活性氧类（ROS）通过光化学方法激发。 不同于另一基于光的抗菌方法中，抗微生物光动力学疗法（APDT），ABL疗法不需要外源photosensitiz的参与呃。所有它需要生效是蓝色光的照射;因此，它是简单和廉价的。 ABL的受体是在细菌中内源性细胞的光敏剂，而不是DNA。因此，ABL被认为是少得多基因毒性对宿主细胞不是紫外线C（UVC）照射，这直接导致在宿主细胞中的DNA损伤。 在本文中，我们提出了一个协议，以评估ABL治疗的烧伤损伤的小鼠模型MDR 鲍曼不动杆菌感染的效果。通过使用生物发光的工程菌株，我们能够无创监测感染的实时程度在活的动物。这种技术也用于监测动物感染的空间分布的有效工具。

我们使用的是鲍曼不动杆菌菌株是MDR临床分离株，如先前12,17报道。该菌株是由luxCDABE歌剧11的转染由生物发光。 图1A示出了从代表性小鼠连续细菌发光图像烧伤感染5×10 6 鲍曼不动杆菌 ，并在细菌接种后24个小时暴露于单个ABL曝光。有代表性的小鼠皮肤的组织切片的革?...

Watch this Scientific Journal Video about In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging at JoV

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

在抗菌蓝光治疗的体内研究的多药耐药鲍曼不动杆菌感染烧伤利用生物发光成像

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

in-vivo-investigation-antimicrobial-blue-light-therapy-for-multidrug

Research

JoVE Journal

Immunology and Infection

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

9.2K 次观看.  Massachusetts General Hospital, Harvard Medical School. Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

视频: In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Multivirus特异性T细胞的生成，预防/治疗异基因造血干细胞移植后的病毒感染

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

科研

免疫与感染

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like allergic reactions 1. In clinical practice, diagnosis of drug hypersensitivity is mainly performed by patient history, as skin testing is not reliable and oral provocation testing bears life-threatening risks for the patient 2. Hence, evidence for an underlying IgE-mediated pathomechanism is hard to obtain. 
Here, we present an in vitro method based on the use of human basophils derived from drug-hypersensitive patients that mimics the allergic effector reaction in vivo. As basophils of drug-allergic patients carry IgE molecules specific for the culprit drug, they become activated upon IgE receptor crosslinking and release allergic effector molecules. The activation of basophils can be monitored by the determination of the upregulation of CD63 surface expression using flow cytometry 3. 
In the case of low molecular weight drugs, conjugates are designed to enable IgE receptor crosslinking on basophils. As depicted in Figure 1, two representatives of NSAIDs, PP and DF, are covalently bound to human serum albumin (HSA) via a carboxyl group reacting with the primary amino group of lysine residues. DF carries an intrinsic carboxyl group and, thus, can be used directly 4, whereas a carboxyl group-containing derivative of PP had to be organochemically synthesized prior to the study 1.
The coupling degree of the low molecular weight compounds on the protein carrier molecule and their spatial distribution is important to guarantee crosslinking of two IgE receptor molecules. The here described protocol applies high performance-size exclusion chromatography (HPSEC) equipped with a sequential refractive index (RI) and ultra violet (UV) detection system for determination of the coupling degree.
As the described methodology may be applied for other drugs, the basophil activation test (BAT) bears the potential to be used for the determination of IgE-mediated mechanisms in drug hypersensitivity. Here, we determine PP hypersensitivity as IgE-mediated and DF hypersensitivity as non-IgE-mediated by BAT.

Basophil activation test is a potent tool for the detection of IgE-dependent allergies in vitro. Here, an optimized protocol for basophil activation test is used to investigate drug hypersensitivity. A method for the efficient production of covalent drug-protein conjugates and their physicochemical characterization is described.

嗜碱粒细胞激活测试IgE介导的药物过敏机制的调查

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

生物发光成像的NADPH氧化酶活性在不同的动物模型

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

4D多模态成像<em&gt;柠檬酸rodentium</em在小鼠感染

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

无创<em&gt;体内</em炎性MMP活性的使用可激活荧光成像剂&gt;荧光光学成像

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

来到光明的一面: 体内糖尿病无毛小鼠慢性伤口生物膜感染的监测

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

微链 rna 的共聚焦成像和模式识别受体在负感 rna 病毒感染中的应用

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

利用流细胞测定人类白细胞测定阿斯珀吉鲁斯紫锥体烟曲的噬菌体

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to assess the purity of the bilayers. The OM has an increased overall density compared to the inner membrane, largely due to the presence of lipooligosaccharides (LOS) and lipopolysaccharides (LPS) within the outer leaflet. LOS and LPS molecules are amphipathic glycolipids that have a similar structure, which consists of a lipid-A disaccharolipid and core-oligosaccharide substituent. However, only LPS molecules are decorated with a third subunit known as the O-polysaccharide, or O-antigen. The type and amount of glycolipids present will impact an organism&#8217;s OM density. Therefore, we tested whether the membranes of bacteria with varied glycolipid content could be similarly isolated using our technique. For the LPS-producing organisms, Salmonella enterica serovar Typhimurium and Escherichia coli, the membranes were easily isolated and the LPS O-antigen moiety did not impact bilayer partitioning. Acinetobacter baumannii produces LOS molecules, which have a similar mass to O-antigen deficient LPS molecules; however, the membranes of these microbes could not initially be separated. We reasoned that the OM of A. baumannii was less dense than that of Enterobacteriaceae, so the sucrose gradient was adjusted and the membranes were isolated. The technique can therefore be adapted and modified for use with other organisms.

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

Gram-negative bacteria produce two spatially segregated membranes. The outer membrane is partitioned from the inner membrane by a periplasm and a peptidoglycan layer. The ability to isolate the dual bilayers of these microbes has been critical for understanding their physiology and pathogenesis.

将葛兰阴性细菌的细胞包络分离到内膜和外膜分数，对阿辛托巴基的培养素进行技术调整

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to ...

Longitudinal Follow-Up of Urinary Tract Infections and Their Treatment in Mice using Bioluminescence Imaging

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, due to increasing microbial resistance, the efficacy of the most used antibiotics has declined. To find alternative treatment options, there is a great need for a better understanding of the UTI pathogenesis and the mechanisms that determine UTI susceptibility. In order to investigate this in an animal model, a reproducible, non-invasive assay to study the course of UTI is indispensable.
For years, the gold standard for the enumeration of bacterial load has been the determination of Colony Forming Units (CFU) for a particular sample volume. This technique requires post-mortem organ homogenates and serial dilutions, limiting data output and reproducibility. As an alternative, bioluminescence imaging (BLI) is gaining popularity to determine the bacterial load. Labeling pathogens with a lux operon allow for the sensitive detection and quantification in a non-invasive manner, thereby enabling longitudinal follow-up. So far, the adoption of BLI in UTI research remains limited.
This manuscript describes the practical implementation of BLI in a mouse urinary tract infection model. Here, a step-by-step guide for culturing bacteria, intravesical instillation and imaging is provided. The in vivo correlation with CFU is examined and a proof-of-concept is provided by comparing the bacterial load of untreated infected animals with antibiotic-treated animals. Furthermore, the advantages, limitations, and considerations specific to the implementation of BLI in an in vivo UTI model are discussed. The implementation of BLI in the UTI research field will greatly facilitate research on the pathogenesis of UTI and the discovery of new ways to prevent and treat UTI.

This manuscript describes the intravesical administration of uropathogenic bacteria with a lux operon to induce a urinary tract infection in mice and subsequent longitudinal in vivo analysis of the bacterial load using bioluminescence imaging.

使用生物发光成像对小鼠尿路感染及其治疗的纵向随访

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, ...