据美国癌​​症协会（“督导小组”- 08 - 108 - 01），文森特J.丝高/美国脑瘤协会，和我野汗家庭基金会。

1。介质的制备<ol><li>媒体准备用10％胎牛血清（16140-071 - GIBCO公司）DMEM/F-12（1X），液体1:1（10565-042 - Invitrogen公司）0.6％的琼脂溶液纯净，颗粒（1.01614.1000 - EMD的）。无菌条件下均保持在整个实验过程。 </li><li>对于媒体胎牛血清5ML准备（16140-071 - GIBCO公司）加入到45mL的DMEM/F-12（1X），液体1:1（10565-042 - Invitrogen公司）50ml的最终体积。准备媒体在37 ° C的温度保持在水浴。 </li><li>琼脂溶液准备用热微波法。颗粒状的琼脂0.3克，溶解于50ml蒸馏水，利用微波水。琼脂溶液降温至37 ° C的温度保持水浴。 </li><li>我们用同等体积的1.2和1.3的步骤和准备原液媒体。的0.6％的琼脂（0.5ML）和10％FBS-D-MEM/F-12媒体（0.5ML）比例是1:1，在每孔6孔板的总体积为1 mL。 </li></ol> 2。组织处理<ol><li>胶质母细胞瘤（GBM）的组织，共收到后立即手术病理科。从病理学教研室组织被列为大紫荆勋章。 </li><li> patri板使用镊子小心被转移到组织和冰冷5ML 1XPBS洗3次。 </li><li>从解剖样品切取的标本连续切片，创建瘤块，用手术刀片（羽毛- 2976＃10）和forcep（Fisher Scientific这）（直径约10毫米）。 </li><li>块被转移到一个6孔板。每个井1ml含媒体提到在步骤1.4。该组织有足够的暴露在空气中不断组织可行的。 </li><li>瘤块，然后注射DMSO（5％）或TMZ网站（2.5纳米），16小时在37℃，含5％CO2的加湿空气。 0.1ml的二甲基亚砜（5％）或TMZ的瘤块注射3次，一致的方式处理不同的地方。这种药物被注射使用胰岛素注射器1ML 0.37X12.7mm 28G1 / 2（点安慰- 26027）。 </li><li> 16小时后洗净，用5 ml的3倍1XPBS。洗涤后的块固定与10毫升10％V / V 24小时，福尔马林50毫升管（Ricca化学公司）（BASIX - 5539802）和嵌入式4μm的部分石蜡处理。 </li><li>福尔马林固定部分是放置在烧杯中搅拌板与“无热”，并通过30分钟，在每一个解决方案下面的解决方案处理。 30％乙醇，50％的乙醇，75％的乙醇，80％乙醇，95％的乙醇，100％的乙醇，二甲苯等。 </li><li>组织抹杀纸巾和30分钟（58至60 ° C，费舍尔Paraplast石蜡）放置在熔化的石蜡。 </li><li>模具在使用Surgipath蓝丝带石蜡包埋，组织包埋组织被冷却到室温。 </li><li>石蜡包埋4μm的组织切片和费舍尔加幻灯片上。 </li></ol> 3。免疫组织化学免疫组织化学，如前面所述。 <ol><li> Deparaffinization和补液放置在机架的幻灯片，并执行以下步骤。二甲苯3X5分钟（5分钟的3倍），三五分钟的100％乙醇，95％为1X5分钟乙醇，70％乙醇为1X5分钟，运行3分钟自来水冲洗。 </li><li>抗原修复与热诱导的抗原表位的检索沉浸在1X在一个玻璃烧杯中的蒸馏水稀释的柠檬酸缓冲液（Thermo Scientific的AP9003 - 500）的幻灯片。煮沸15分钟上的一个热点板块（确定部分唐“T脱落幻灯片）。冷却30分钟，在室温下的解决方案。冲洗用1X PBS三五分钟的幻灯片。 </li><li>抑制过氧化内部沉浸在15分钟的玻璃烧杯中，用0.3％过氧化氢（费舍尔在蒸馏水科学BP2633 - 500稀释）在甲醇中的幻灯片（费舍尔科学- A - 452 - 4）。在1X PBS冲洗3X5分钟。 </li><li>封锁覆盖10％正常山羊血清组织。在潮湿中传输的幻灯片和1小时在室温下孵育。三五分钟，用1X PBS清洗的幻灯片。 </li><li>主要抗体孵育4 ° C的主要抗体在以下浓度：人类特定的Caspase - 3（1:1,000，研发系统，AF835），Ki67的（1:1，Dako公司，15626）与1X PBS稀释过夜。冲洗1X PBS于翌日3X10分钟的幻灯片。 </li><li>二次抗体反应封面用2-3滴HRP标记二抗（设想系统）的组织。在潮湿中的幻灯片和1小时在室温下孵育。用1X PBS清洗3X10分钟。 </li><li>检测发展为显色DAB试剂盒（载体实验室- SK - 4100）的初级抗体的检测，按照制造商的协议。 </li><li>反染色沉浸与苏木幻灯片（10-15小号<html> econds，确保不“T溢出民建联信号），运行3分钟自来水冲洗。 </li><li>脱水沉浸在下列顺序幻灯片，70％乙醇5分钟，95％乙醇5分钟，三五分钟100％的乙醇，二甲苯为3X5分钟。 </li><li>盖玻片permount安装试剂（费舍尔科学SP - 15 - 100）的幻灯片。 </li><li>使用奥林巴斯的荧光显微镜（DP - 72）.3.11）。在所有的实验，证实了特定的标签无一抗阴性对照。 </li></ol> 4。代表性的成果： 我们收集的多形性胶质母细胞瘤（GBM）的手术切除标本。患者接受第一次手术前化疗不包括替莫唑胺（TMZ）。 MRI的T1加权图- 1A钆增强的形象展示在正确的术前额叶增强肿瘤。术后的图像（图1B）确认次全切除手术后的病变。手术组织被送往病理部临床诊断的目的，并根据批准的机构审查委员会（IRB）在美国俄亥俄州立大学医学中心的协议处理，其余标本组织采购。苏木精和曙红染色（H＆E）表现出坏死的存在，pseudopallisading细胞及微血管增生（图1 - C）。因此，这些肿瘤的病理诊断为大紫荆勋章。值得注意的是，以下没有TMZ网站的潜伏期长达48小时的肿瘤外植体保持GBM的细胞结构。与此相反，潜伏期为72小时或更长的时间，我们注意到，在标本的凝结核数量增加，这表明一些肿瘤细胞开始发生凋亡。因此，潜伏期较长的肿瘤组织中，失去了肿瘤细胞，这是优先观察及其周围坏死区（数据未显示）的片状区域。 为了调查，如果这些肿瘤的外植体样品能够可靠地使用药物的疗效试验的目的，我们测试了TMZ的治疗效果。图2表示在这项研究中的实验流程。最近的一项研究表明，TMZ的瘤内注射对恶性胶质瘤的生长控制效果比全身给药对这种药物的 2更有效。经过16小时的潜伏期，这些外植体嵌入石蜡连续切片染色准备。 TMZ治疗GBM的组织免疫组织化学Ki - 67的阳性肿瘤细胞表现出与对照样品相比大幅减少。反过来，免疫组织化学凋亡标记，Caspase - 3的，没有取得任何TMZ治疗脑胶质瘤标本和对照样本（图3）之间的显着性差异。 与TMZ的治疗效果得到进一步的特点相结合，在体外培养或流式细胞仪之一植体检测。具体来说，我们试图确定对干细胞样肿瘤细胞（SCLTC）的影响。因此，下列与TMZ网站瘤的治疗，我们的表现领域形成实验和流式细胞仪检测这些肿瘤植。 TMZ治疗的标本分离成单个细胞，这些单细胞接种于低密度辅以碱性成纤维细胞生长因子和EGF的无血清培养基的96孔板（每微升1细胞或更低）。在对照组中，肿瘤从肿瘤植领域的形成7天的潜伏期后观察。鉴于这一领域的形成是一个SCLTC 3 4的财产，在药物治疗肿瘤领域的改变表示SCLTC效果。同样，流量仪，分离肿瘤与细胞表面标志，CD133植进行了验证SCLTC效果。如果SCLTC在GBM的不同种类的肿瘤细胞有选择性药物治疗根除，人们可能会预测，流式细胞仪检测CD133阳性的一部分降低治疗（数据未显示）。 <img src="/files/ftp_upload/2846/2846fig1.jpg" alt="图1"/> Figure.1磁共振图像（MRI）的病人的大紫荆勋章 。前置作业图- 1A和手术后的图- 1B的代表性人物。图- 1C是H＆E染色新鲜的大紫荆勋章的组织样本。原始的放大倍率，40X。 <img src="/files/ftp_upload/2846/2846fig2.jpg" alt="图2"/> 图。 2块培养实验的实验流图- 2A 。从病理学系的新鲜手术大紫荆勋章的图片。图2b显示了剥离瘤块，成与帮助手术刀片的10mm小块。图- 2C的药物（TMZ）治疗使用胰岛素注射器的瘤块。 E 3“/&gt; 图。 3免疫组织化学 ，免疫组织化学与DMSO或TMZ网站注入与激活Caspase - 3和Ki67的GBM的组织块。苏木被用于核染色。原始的放大倍率，40X。

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在目前的临床前实验模型的药物疗效和患者的疗效之间有一个差距。更具体地说，在脑肿瘤的研究领域，新的和可靠的方法，这将有助于填补之间存在的差距与当前方法的药物评价和患者的疗效。在这项研究中所描述的实验是一种额外的资产，如果没有一个解决方案，以方便灌装受影响的病人的实验数据和结果的差异。 优先扩大在体外细胞培养的培养条件下，无论某些肿瘤细胞类型，并表示只有一个亚群，整个肿瘤。此外，遗传和表型的人工细胞扩张发生转换，是与长期的细胞培养的必然。治疗恶性胶质瘤的主要障碍之一是肿瘤细胞的异质性，在一个单一的肿瘤和肿瘤样本，6，7间，。因此，选择性富集了肿瘤细胞的某些人口，无论一种或另一种，未必是一个合适的方法来确定不同种类的肿瘤细胞化疗药物的疗效。8月9日任何脑肿瘤的动物模型，从一个亚群的肿瘤细胞对药物的疗效棚灯上的一个亚群，而不是整个肿瘤。 一些局限性，另一方面，仍然在此瘤块法。一个这样的限制是相对较短的时间治疗。由于恶性肿瘤细胞被认为是获得耐大多数，如果不是全部的，随着时间的推移治疗，初步控制短期的肿瘤细胞的生长可能不能反映长期患者的预后，因为最近有报道用抗血管生成剂，贝伐单抗10。因此，本植体的检测，以维持一段较长时间的组织协议的改进是需要进一步调查。另一个问题是测试药物在肿瘤上SCLTC的一个特定的效果。鉴于，SCLTC相对抗药性恶性胶质瘤，新型抗癌药物SCLTC是必要的，potently消除的识别目前的治疗方法。在这方面，我们目前正在寻求结合起来， 在体外实验中 ，如神经球形成实验（数据未显示），随后这个植检测。我们希望了解更多的影响，如果有的话，通过这些组合分析SCLTC的候选药物。最后，用小块的肿瘤样本可能不能反映整个肿瘤细胞群体，是与传统的肿瘤细胞株或手术切除标本的原代培养的情况下。除了这些问题，保持基质肿瘤，包括血管利基，有助于在肿瘤细胞的特征的环境因素。因此，这个实验可能具有潜在的，以评估任何一个有关生理条件下的治疗策略。 在这里，我们建立了一个与GBM的手术切除标本植药物疗效测试的检测。事实上，随着测试的手术切除标本中，我们发现了TMZ的直喷降低在GBM的样品扩散。该组织块法理论上适用于其他固体肿瘤，并提供资产，以确定药物的疗效，病人的肿瘤上，希望这将有助于我们避免癌症在临床试验再次失败。

<table border="1"><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>胎牛血清，合格，热灭活</td><td> GIBCO公司</td><td> 16140-071 </td></tr><tr><td> D-MEM/F-12（1X），液体1:1 </td><td> Invitrogen公司</td><td> 10565-042 </td></tr><tr><td>琼脂纯净，颗粒</td><td>电解二氧化锰</td><td> 1.01614.1000 </td></tr><tr><td> DPBS染色</td><td> Invitrogen公司</td><td> 14190 </td></tr><tr><td> Hematoxyllin </td><td> Richalrd艾伦科学</td><td> 7211 </td></tr><tr><td> 10％W / V福尔马林的caspase - 3 </td><td> Ricca化学公司</td><td> 3810 </td></tr><tr><td> Caspase - 3的</td><td>研发体系</td><td> AF835 </td></tr><tr><td> Ki67的</td><td> Dako公司</td><td> 15626 </td></tr><tr><td>二甲基亚砜</td><td>西格玛</td><td> D2650 </td></tr><tr><td>设想系统抗鼠</td><td> Dako公司</td><td> 2012-07 </td></tr><tr><td>设想系统抗兔</td><td> Dako公司</td><td> 2011-12 </td></tr><tr><td> DAB - KIT </td><td>媒介实验室。 </td><td> SK - 4100 </td></tr></table> 表1材料 。 <table border="1"><tr><td> 设备名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> 6孔板</td><td>格雷纳的生物之一</td><td> 657160 </td></tr><tr><td>培养皿</td><td> VWR </td><td> 25384-302 </td></tr><tr><td>血清移液器</td><td>爱思进</td><td></td></tr><tr><td>过滤嘴</td><td>美国科学</td><td></td></tr><tr><td> 500微米聚酯网Netwe​​ll插入</td><td>配角</td><td> 3480 </td></tr><tr><td>基质胶基质</td><td> BD公司</td><td> 354230 </td></tr><tr><td>屋宇署10毫升注射器</td><td> BD公司</td><td> 309604 </td></tr><tr><td>注射针铝轮毂。 </td><td>泰科保健</td><td> 2000029 </td></tr><tr><td>组织培养瓶</td><td>康宁</td><td></td></tr><tr><td>离心管</td><td> BASIX </td><td> 5539800 </td></tr><tr><td>薄壁管</td><td>伊顿公学</td><td> 1107A </td></tr><tr><td>手术刀片不锈钢</td><td>羽毛</td><td> 2976＃10 </td></tr><tr><td>胰岛素注射器</td><td>舒适点</td><td> 26027 </td></tr><tr><td> 50毫升的平顶螺丝帽的管</td><td> BASIX </td><td> 5539802 </td></tr><tr><td>解剖镜下</td><td> Tritech </td><td></td></tr><tr><td>荧光显微镜</td><td>奥林巴斯</td><td> DP - 72 </td></tr><tr><td> Forcep </td><td>费舍尔科学</td><td></td></tr></table> 表2。设备。

method for novel anti-cancer drug development using tumor explants of surgical specimens

恶性胶质瘤目前的治疗方法只治标不治本的效果。治疗发展的一个障碍是由目前的药物的疗效试验和对患者的疗效确定的疗效差异。因此，评价药物疗效的新的和可靠的方法是必要的临床前研究阶段。，肿瘤组织，包括细胞株 ，在体外培养的癌细胞大量的表型，遗传和表观遗传的细胞培养人工环境所造成的改变，这可能没有反映在原地的原发肿瘤生物学。免疫缺陷小鼠移植瘤模型也有其局限性，即缺乏免疫系统和物种间的遗传和表观遗传在微环境的差异。在这里，我们展示了一种新的方法使用未解离肿瘤块恶性胶质瘤的手术标本，以评估治疗效果。为了验证这种方法，目前第一线化疗药物，替莫唑胺（TMZ），的数据进行了阐述。 我们用我们的实验恶性胶质瘤新鲜取出手术标本。 TMZ网站或其他候选药物孵化和手术切除标本的分析，我们进行瘤内注射。在这里，我们试图建立一个肿瘤组织块法作为一个平台，以确定新型抗癌疗法的疗效，使我们能够克服，至少当前的一些限制，并填补目前的实验存在的差距数据和实际的病人的肿瘤的疗效。这种方法可能有可能加速确定新的化疗药物的固体癌症治疗。

Watch this Scientific Journal Video about Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens at JoVE.com

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

在这里，我们的脑肿瘤的手术切除标本，被称为“肿瘤块法”建立了药物疗效检测的一种方法。使用这种方法，我们可以评估药物的疗效没有打破实体肿瘤的微环境。为了验证这种方法的可靠性，我们描述了具有代表性的数据，与我们当前的第一线化疗药物替莫唑胺治疗脑胶质瘤标本。

新型抗癌药物使用手术标本肿瘤植的发展方法


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

method-for-novel-anti-cancer-drug-development-using-tumor-explants

Research

JoVE Journal

Medicine

16.5K 次观看.  The Ohio State University Medical Center. 在这里，我们的脑肿瘤的手术切除标本，被称为“肿瘤块法”建立了药物疗效检测的一种方法。使用这种方法，我们可以评估药物的疗效没有打破实体肿瘤的微环境。为了验证这种方法的可靠性，我们描述了具有代表性的数据，与我们当前的第一线化疗药物替莫唑胺治疗脑胶质瘤标本。

视频: Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

从手术标本的人类胰岛细胞激光显微切割的改进方法

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

科研

医学

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Retinal detachment (RD) describes a separation of the neurosensory retina from the retinal pigmented epithelium (RPE). The RPE is essential for normal function of the light sensitive neurons, the photoreceptors. Detachment of the retina from the RPE creates a physical gap that is filled with extracellular fluid. RD initiates cellular and molecular adverse events that affect both the neurosensory retina and the RPE since the physiological exchange of ions and metabolites is severely perturbed. The consequence for vision is related to the duration of the detachment since a rapid reapposition of the two tissues results in the restoration of vision 1. The treatment of RD is exclusively surgical. Removal of vitreous gel (vitrectomy) is followed by the removal non essential part of the retina around the detached area to favor retinal detachment. The removed retinal specimens are res nullius (nothing) and consequently normally discarded. To recover RNA from these surgical specimens, we developed the procedure jouRNAl that allows RNA conservation during the transfer from the surgical block to the laboratory. We also standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for global gene expression analysis. The quality of the RNA was validated both by RT-PCR and microarray analysis. Analysis of the data shows a simultaneous involvement of inflammation and photoreceptor degeneration during RD.

We used retinal samples from retinectomy for a transcriptomic analysis of retinal detachment. We developed a procedure that allows RNA conservation between the surgical blocks and the laboratory. We standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for microarray analysis.

基因表达谱分析人视网膜手术标本杂志

Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load &#62;1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads &#60;1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.

Drug resistance testing for HIV-1 infected individuals failing antiretroviral therapy (ART) can guide future therapies and improve treatment outcomes. Optimizing individual and population health outcomes in high HIV prevalence but resource-limited settings will ultimately require affordable and accessible drug resistance genotyping and interpretation methods.

对于资源有限设置一个负担得起的HIV-1耐药性监测方法

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in ...

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.

A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

手术应激探索术后免疫抑制的机制和评估新围手术期免疫疗法的小鼠肿瘤模型

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

为获取原发性卵巢癌细胞固体样品的方法

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

在免疫抑制剂他克莫司对干血斑使用LC-MS / MS定量

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

动静脉内瘘失败的新型小鼠模型：在详细介绍手术过程

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

脑肿瘤/器官切片共培养系统研究肿瘤微环境和靶向药物治疗

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.