这项工作得到了葡萄牙天主教大学医学院的内部资金和科学与技术基金会 （FCT） 的外部资金支持，赠款为 UIDP/04279/2020、UIDB/04279/2020 和 EXPL/SAU-INF/0742/2021。

注意：此处描述的方案适用于BCG，但可以应用于任何分枝杆菌。当 BSL3 设施不可用时，BCG 可用作结核病实验的替代细菌22.使用卡介苗的以下程序应在生物安全 2 级 （BSL2） 实验室下进行，并遵循适当的生物安全指南和操作危险组 2 微生物的良好实验室规范。
1. 培养基制备
<ol><li>根据供应商的说明，准备补充有 10% （v/v） 油酸、白蛋白、葡萄糖和过氧化氢酶 （OADC） 富集的 Middlebrook 7H9 肉汤。用 0.05% （v/v） 的泰洛沙泊补充肉汤。 注意：Tyloxapol 是一种非离子液体聚合物，已用作表面活性剂以防止细菌团块形成16.</li>
	<li>根据供应商的说明制备补充有10%（v / v）OADC富集的Middlebrook 7H10固体培养基。</li>
	<li>每个方形培养皿 （120 mm x 120 mm） 分配 40 mL 培养基。让平板干燥，以尽量减少琼脂表面的冷凝。 注意：这种特定尺寸的培养皿对于允许从 96 孔板直接转座至少 96 个液滴至关重要。板的有效干燥将促进细菌悬浮液小液滴的电镀，并防止液滴扩散。</li>
	<li>制备 Roswell Park Memorial Institute 培养基 （RPMI 1640） 或 Dulbecco 改良 Eagle 培养基 （DMEM） 以产生感染培养基。无论哪种情况，都应用 10% 胎牛血清、1% L-谷氨酰胺和 1 mM 丙酮酸钠补充培养基。不要向培养基中加入青霉素和链霉素。</li>
</ol>2. 样品制备
<ol><li>从各种来源获取样品。通常，为了量化 CFU 以评估结核病疫苗的功效，请从已接种疫苗和未接种疫苗的动物组织中获取样本。例如，用小鼠肺和脾脏11或猕猴肺、胸和外周淋巴结、脾脏、肝脏、皮肤、血液、骨髓和支气管肺泡灌洗液23。或者，从感染 BCG18、19、20、24、25、26 的巨噬细胞/树突状细胞/中性粒细胞的体外培养物中获取样本。</li>
</ol>3. BCG培养物的生产
注：对于结核病疫苗的体内研究，目的是提高卡介苗的疗效。因此，通常使用接种卡介苗的组作为对照。用于人类疫苗接种的卡介苗菌株非常适合在动物模型中进行测试。在这种情况下，必须根据供应商的指示27 重组 BCG 培养物。然而，用于体内研究的 BCG 培养也可以在内部生产11。在几项研究11、16、18、19、20、26、28、29 中，使用以下方案，也可用于动物激发研究，已非常成功地生产用于体外感染方案的单细胞、均匀和高质量的 BCG 培养物，也可用于动物激发研究。
<ol><li>在37°C下，在7H9肉汤中培养50mL BCG，以200rpm搅拌。根据实验的需要改变体积。</li>
	<li>每天，持续 8-10 天，收集 100 μL 培养物，并通过在 1 mL 比色皿中加入 900 μL PBS 来稀释。然后继续测量细菌的光密度（λ=600nm处的OD;OD600）在分光光度计中。从这些值绘制一条增长曲线。确定培养物的对数中期（当 OD 每单位时间持续加倍时）。</li>
	<li>准备后续培养物并孵育至达到中/晚期对数生长阶段，如步骤3.1和3.2所示。使用在上一步中获取的值作为指导。确保培养物不会达到固定生长阶段（当OD开始稳定时），以保持活菌的高质量培养。</li>
	<li>在对数生长的中/后期收集培养物。以3000× g 离心10分钟。除去上清液。</li>
	<li>加入 10 mL PBS 清洗细菌。以3000× g 离心10分钟。除去上清液。</li>
	<li>用 5 mL 感染培养基重悬细菌。将试管置于超声浴中 15 分钟，全功率为 80 Hz。</li>
	<li>以1000× g 离心10分钟。收集上清液，避开沉淀，因为它富含细菌团块，在高质量的BCG培养物中应避免，并将其丢弃。</li>
	<li>测量上清液的OD。在这里，处于指数生长期的培养物，OD600 为 0.1，相当于 1 x 107 CFU/mL。 注意：每个实验室在开始实验之前应生成自己的 BCG 生长曲线，以使用分光光度计在 OD600 和 CFU 之间建立线性回归。请注意，分光光度计具有不同的光程距离，这可能会改变同一样品获得的读数。</li>
	<li>进行简单的计算，以确定要添加到每个宿主细胞培养物中的细菌数量。每个宿主细胞的细菌数量是感染的多重性 （MOI）。每个宿主细胞使用 10 个细菌的 MOI，这是用于 BCG 感染实验的最常见 MOI。</li>
</ol>4. 微菌落形成单元测定
注： 体内 或 体外 感染实验完成后，可以通过mCFU进行细菌计数。对于 体内 研究，必须首先在微珠打浆器或其他组织匀浆器中对样品进行均质化。对于感染卡介苗的巨噬细胞/树突状细胞/中性粒细胞的 体外 培养，必须使用非离子去污剂（例如，0.05% 非离子、非变性去污剂溶液）裂解样品。
<ol><li>使用 96 孔板进行连续稀释：根据 图 1A 中的方案，在无菌 96 孔板中对裂解物进行连续 10 倍稀释。将裂解物分布在 A 行和 E 行上。对于每个板，最大样品和/或重复次数为 24。</li>
	<li>向剩余的孔中加入 180 μLdH 2O，以进行连续稀释。</li>
	<li>使用 12 通道移液管，重悬 A 行中的裂解物，并将 20 μL 转移到 B 行（20 μL 裂解物 + 180 μL dH2O）。均匀化良好。依次对 B 行和 C 行重复此步骤，直到达到 D 行的最后一个稀释度。 注意：我们通常进行三次稀释（100、101、102、103），因此每组 12 个样品和/或重复使用 4 行板（AD 或 E-H）。</li>
	<li>微液滴铺板：根据 图1B，使用0.5-10 μL（首选细吸头）多通道移液器将5 μL从每排96孔板转移到固体介质方板上。</li>
	<li>在缓慢移液 5 μL 液滴的同时，让它们轻轻接触琼脂。这将有助于去除从尖端向琼脂方向的液滴，并减少液体滞留在尖端内的可能性。</li>
	<li>让液滴干燥，关闭琼脂平板，并在37°C下孵育，同时监测细菌生长。或者，将琼脂平板在密封的塑料袋中孵育，以防止平板干燥。</li>
	<li>微菌落计数：孵育约6-10天后，检查肉眼可见的单个菌落（图2）。</li>
	<li>使用倒置光学显微镜或放大镜的最低放大倍率物镜（4倍或更低）计数菌落。应在菌落数低于 300 且高于 30 的稀释液中进行计数。或者，使用相机拍摄液滴的照片以在计算机上手动计数菌落，或使用 ImageJ 等软件自动计数菌落计数。</li>
	<li>要以 CFU/mL 表示细胞数，请使用以下公式： <img alt="Equation 1" src="/files/ftp_upload/65447/65447eq01.jpg" style="margin: auto;"> 其中 C = 计数的菌落数，V = 以 μL 为单位的铺板体积，Dil = 计数菌落的稀释度 （100， 101， 102， 103）。例如，如果在稀释 102 的 5 μL 液滴中计数 30 个菌落，则： <img alt="Equation 2" src="/files/ftp_upload/65447/65447eq02.jpg" style="margin: auto;"></li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65447/65447fig01.jpg"> 图 1.mCFU协议的示意图。 （A） 在 96 孔板中对含 BCG 的裂解物进行连续 10 倍稀释。（B） 含有固体培养基的方形培养皿，并由 96 个液滴覆盖，每个液滴 5 μL。使用多通道移液器直接从96孔板中移液液滴。用 BioRender.com 创建。 <a href="https://www.jove.com/files/ftp_upload/65447/65447fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65447/65447fig02.jpg"> 图2.孵育 10 天后 BCG 的微菌落形成单位。在左边，一个方形培养皿的照片，上面覆盖着96个5 μL的液滴，如图1B所示。 在右侧，3 个液滴的单个照片对应于原始裂解物 （100） 和两个稀释液 （101， 102）。照片使用配备 18-55 毫米变焦镜头（板）或 105 毫米微距镜头（液滴）的 DSRL 相机拍摄。<a href="https://www.jove.com/files/ftp_upload/65447/65447fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
5. 斐济的微菌落形成单位计数 （ImageJ）
注：mCFU 方法允许对大量样品进行 CFU 定量。可以记录液滴的图片以进行后验分析，以促进菌落计数。为此，几种摄影设备可以产生足够质量的图像。这些包括数码相机、网络摄像头、相机连接显微镜和放大镜以及手机。ImageJ 等免费图像分析软件提供了在这些图像中手动或自动进行菌落计数的可能性。为了演示这两种方法，将使用 Fiji，它是 ImageJ 的发行版，它打包了几种用于科学图像分析的工具 30。斐济可以从 https://fiji.sc/ 下载。
<ol><li>手动计数方式<ol><li>在斐济打开包含 mCFU 的图像。选择 插件>分析>细胞计数器。</li>
		<li>在“细胞计数器”菜单上，选择 “初始化 ”，然后选择一个计数器（例如，键入 1）。</li>
		<li>通过单击每个菌落继续。每次单击都会显示在图片上，并将更新计数器（图 3）。若要撤消意外点击，请选择 “删除”。</li>
		<li>注册计数器上显示的值。单击 “重置 ”按钮重置计数并打开新图像以计数其他样本。 注意：有关此插件的更多说明，请参见 https://imagej.net/plugins/cell-counter。</li>
	</ol></li>
	<li>自动计数方式<ol><li>在斐济打开包含 mCFU 的图像。选择 “映像>键入 > 8 位”。这会将图像转换为 8 位灰度图像。</li>
		<li>在工具栏中选择 “椭圆 ”工具，并在具有菌落的区域周围绘制一个椭圆（图4A）。椭圆可以在绘制后进行调整。</li>
		<li>选择 “编辑”>“清除外部”，以消除外部区域的任何干扰（图4B）。选择 图像>调整>阈值。</li>
		<li>移动阈值菜单中的滑块，直到菌落显示为红色并且背景噪音最小化（图4C）。</li>
		<li>选择 “应用 ”并退出阈值窗口。生成黑白图像（图4D）。</li>
		<li>选择 “分析”（Analyze） >“分析粒子”（Analyze Particles）。在分析粒子窗口中，指定菌落面积（介于 1 和无穷大之间，以平方像素为单位）和圆度（介于 0 和 1 之间，其中 1 为完美圆; 图4E）。</li>
		<li>在显示弹出菜单中选择 轮廓 。在结果窗口中查看显示 结果 ，了解每个菌落的详细测量值。选中 “清除结果 ”以擦除任何先前的测量值。选中 “汇总 ”框以显示测量的汇总结果（图4E）。</li>
		<li>通过选择“ 确定”启动分析器。将出现一个新窗口，显示所有已检测和计数的轮廓菌落。结果窗口显示每个菌落的详细信息，汇总结果窗口显示计数的总菌落（图4F）。 注意：大小和圆度的设置将随图像的分辨率和放大倍率以及菌落的大小和形状而变化。重复该过程几次，直到找到检测所有菌落的最佳设置。有关分析粒子插件的更多说明，请参见 https://imagej.nih.gov/ij/docs/menus/analyze.html#ap。</li>
	</ol></li>
</ol><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65447/65447fig03.jpg"> 图3.使用斐济软件上的细胞计数仪插件对 mCFU 进行计数的手动方法。 蓝点表示用户已点击的菌落。右侧的菜单显示到目前为止计数的菌落数（计数为 41）。 <a href="https://www.jove.com/files/ftp_upload/65447/65447fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65447/65447fig04.jpg"> 图4.一种使用 Fiji 软件计算 mCFU 的自动化方法。 （甲、乙）使用椭圆形选择工具选择具有菌落的感兴趣区域，并使用清除外部命令删除外部区域。（C、D）使用阈值工具生成菌落的黑白图像。（E、F）使用分析颗粒工具对菌落数量进行量化。 <a href="https://www.jove.com/files/ftp_upload/65447/65447fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

使用 Micro-CFU 方法加速结核病测定

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DP 和 PJGB 声明，该研究是在没有任何可被解释为潜在利益冲突的商业或财务关系的情况下进行的。

结核病是一个重要的公共卫生问题，其重要性日益增加，特别是在低收入和中等收入国家。在COVID-19大流行期间，诊断和治疗结核病的医疗机构中断，对新发病例的发病率产生了负面影响1。此外，必须紧急解决多药和广泛耐药的结核分枝杆菌菌株以及结核分枝杆菌和艾滋病毒的合并感染，以控制这一流行病1,34。替代或改?...

结核病（TB）是全球单一传染性病原体结 核分枝杆菌 （M.tb）死亡的主要原因，其死亡人数超过任何其他病原体。2021年，结核病导致160万人死亡，在2019-2021年大流行期间被COVID-19超过1。此外，根据世界卫生组织的 2022 年全球结核病报告，COVID-19 大流行是导致新发结核病病例增加的原因。世卫组织还报告说，在此期间，被诊断患有结核病的人数大幅下降，这可能会进一步增加结核病病例数1。
卡介苗 （BCG） 是致病性牛分枝杆菌的减毒活菌株，100 多年前首次用作疫苗。这是唯一的结核病疫苗，也是世界上仍在使用的最古老的许可疫苗2,3。目前，有 12 种疫苗处于临床试验的不同阶段4，数十种疫苗正在临床前开发中 5,6。结核病疫苗的临床前评估包括安全性和免疫原性评估7，这可以在斑马鱼、小鼠、豚鼠、兔子、牛和非人灵长类动物等各种动物模型中获得 8,9,10。此外，评估疫苗诱导对结核分枝杆菌感染和/或传播的保护能力，即疫苗效力，需要在体内进行结核分枝杆菌攻击5,11。有趣的是，卡介苗疫苗接种通过训练有素的免疫机制 12,13 诱导影响其他细菌和病毒病原体存活的非特异性效应14。为了量化受感染动物的活细菌负荷，选择的方法是通过菌落形成单位 （CFU） 测定法 5,15 对细菌菌落进行计数。CFU是一个单位，用于估计在特定生长条件下形成菌落的微生物（细菌或真菌）的数量。CFU起源于可存活和可复制的微生物，每个菌落中活微生物的绝对数量难以估计。尚不确定菌落是否起源于一种或多种微生物。CFU单位反映了这种不确定性，因此在同一样品的重复中可以观察到很大的变异性。这种耗时的检测需要经过培训的专业技术人员在生物安全 3 级 （BSL3） 设施、大量实验室和培养箱空间中工作，需要 4 到 6 周才能完成，并且容易受到污染。
在这项研究中，我们描述了一种优化的菌落计数方法，即 micro-CFU （mCFU），并提供了一种简单快速的解决方案来分析结果 15,16,17,18,19,20。mCFU 检测所需的试剂数量减少了 10 倍，潜伏期缩短了 3 倍，需要 1 到 2 周才能完成，减少了实验室空间和试剂成本，并将与大量 M.tb 相关的健康和安全风险降至最低。我们建议将这种方法普遍用于疫苗功效测定的临床前研究，最终缩短结核病疫苗的开发时间。最后，这种优化的CFU计数方法不仅用于定量分枝杆菌，还用于定量其他细菌，如大肠杆菌和Ralstonia solanacearum21。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well plates</td>
			<td>VWR</td>
			<td>734-2781</td>
			<td></td>
		</tr>
		<tr>
			<td>DSLR 15-55 mm lens</td>
			<td>Nikon</td>
			<td>AF-P DX NIKKOR 18-55mm f/3.5-5.6G VR</td>
			<td></td>
		</tr>
		<tr>
			<td>DSLR camera</td>
			<td>Nikon</td>
			<td>D3400</td>
			<td></td>
		</tr>
		<tr>
			<td>DSLR macro lens</td>
			<td>Sigma</td>
			<td>MACRO 105mm F2.8 EX DG OS HSM</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji Software</td>
			<td></td>
			<td>https://fiji.sc/</td>
			<td>Fiji is an open-source software supported by several laboratories, institutions, and individuals. All the required plugins are included.</td>
		</tr>
		<tr>
			<td>Igepal CA-630</td>
			<td>Sigma-Aldrich</td>
			<td>18896</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook 7H10</td>
			<td>BD</td>
			<td>262710</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook 7H9</td>
			<td>BD</td>
			<td>271310</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (0.5 - 10 &#181;l)</td>
			<td>Gilson</td>
			<td>FA10013</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (20 - 200 &#181;l)</td>
			<td>Gilson</td>
			<td>FA10011</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycobacterium bovis BCG&#160;</td>
			<td>American Type Culture Collection</td>
			<td>ATCC35734</td>
			<td>strain TMC 1011 [BCG Pasteur]</td>
		</tr>
		<tr>
			<td>OADC enrichment</td>
			<td>BD</td>
			<td>211886</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>NZYTech</td>
			<td>MB25201</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Gibco</td>
			<td>21875091</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer UV-6300PC</td>
			<td>VWR</td>
			<td>634-6041</td>
			<td></td>
		</tr>
		<tr>
			<td>Square Petri dish 120 x 120 mm</td>
			<td>Corning</td>
			<td>BP124-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyloxapol</td>
			<td>Sigma-Aldrich</td>
			<td>T8761</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound bath Elma P 30 H</td>
			<td>VWR</td>
			<td>142-0051</td>
			<td></td>
		</tr>
	</tbody>
</table>

micro-colony forming unit assay for efficacy evaluation of vaccines against tuberculosis

结核病 （TB） 是全球传染性病原体导致死亡的主要原因，2022 年造成 160 万人死亡，仅在 2019-2021 年大流行期间被 COVID-19 超越。该病由 结核分枝杆菌 （M.tb）引起。 牛分枝杆菌 卡介苗（BCG）是唯一的结核病疫苗，是世界上获得许可的最古老的疫苗，仍在使用。目前，有12种疫苗处于临床试验阶段，数十种疫苗处于临床前开发阶段。在临床前研究中，用于评估结核病疫苗疗效的首选方法是通过菌落形成单位 （CFU） 测定法对细菌菌落进行计数。这种耗时的检测需要 4 到 6 周才能完成，需要大量的实验室和培养箱空间，试剂成本高，并且容易受到污染。在这里，我们描述了一种优化的菌落计数方法，即微 CFU （mCFU），它为分析 结核分枝杆 菌疫苗的功效结果提供了一种简单而快速的解决方案。mCFU 检测所需的试剂数量减少了 10 倍，潜伏期缩短了 3 倍，需要 1 到 2 周才能完成，减少了实验室空间和试剂成本，并将与大量 M.tb 相关的健康和安全风险降至最低。此外，为了评估结核病疫苗的功效，可以从各种来源获得样本，包括感染分枝杆菌的接种疫苗动物的组织。我们还描述了一种优化的方法，用于感染研究生产单细胞、均匀和高质量的分枝杆菌培养物。最后，我们建议将这些方法普遍用于疫苗功效测定的临床前研究，最终缩短结核病疫苗开发的时间。

这里描述的mCFU测定将可以从单个培养皿中检索到的信息量增加到至少96倍。图 5 描述了两种药物递送方法的比较，用于重新利用沙奎那韦 （SQV）31,32 作为宿主定向药物治疗结核病。在该测定中，使用四种不同的结核分枝杆菌菌株感染原代人巨噬细胞。结核分枝杆菌葡萄牙国立卫生研究院的Ricardo Jorge博士（INSA）从活?...

Watch this Scientific Journal Video about Optimizing CFU Determination for Efficient Assessment of TB Vaccine Efficacy and Antigen Presentation Analysis at JoVE.com

作者聚焦：优化 CFU 测定以有效评估结核病疫苗功效和抗原呈报分析 

菌落形成单位 （CFU） 的测定是定量细菌的金标准技术，包括 结核分枝杆菌，结核分枝杆 菌可能需要数周时间才能形成可见菌落。在这里，我们描述了一种用于 CFU 测定的微型 CFU，它提高了时间效率，减少了实验室空间和试剂成本，并可扩展到中高通量实验。

用于结核病疫苗功效评估的微集落形成单位测定

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during ...

CFU测定对于衡量免疫反应对细菌的功效是必要的。然而，它的通量非常低，当应用于结核分枝杆菌等致病菌时，它变得繁琐且昂贵。当我们需要测试大型治疗面板时，这是一个很大的限制。该研究的重点是结核病和结核病疫苗的评估。它探讨了针对结核分枝杆菌的疫苗的安全性、免疫原性和有效性。该研究提出了一种优化的 micro-CFU 方法，用于计数细菌菌落以评估临床前研究中的疫苗功效。该协议旨在通过从类似于常规培养皿的小液滴中产生结果来优化CFU测定。从如此小的样品中量化细菌活力，使我们能够使用传统方法所需的一小部分材料和时间来分析数千个数据点。我们的研究旨在剖析抗原呈递机制，并确定最相关的抗原以优化抗原呈递。这将为我们设计和开发更好的疫苗提供信息，以改进或取代目前的结核病疫苗，即卡介苗。

micro-colony-forming-unit-assay-for-efficacy-evaluation-vaccines

Research

JoVE Journal

Medicine

Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis

2.0K 次观看.  Universidade Católica Portuguesa. CFU测定对于衡量免疫反应对细菌的功效是必要的。然而，它的通量非常低，当应用于结核分枝杆菌等致病菌时，它变得繁琐且昂贵。当我们需要测试大型治疗面板时，这是一个很大的限制。该研究的重点是结核病和结核病疫苗的评估。它探讨了针对结核分枝杆菌的疫苗的安全性、免疫原性和有效性。该研究提出了一种优化的 micro-CFU 方法，用于计数细菌菌落以评估临床?...

视频: 作者聚焦：优化 CFU 测定以有效评估结核病疫苗功效和抗原呈报分析 

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

快速点护理依诺肝素的抗凝血剂功效的测定全血中

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

科研

医学

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

光动力疗法治疗骨肉瘤细胞的细胞毒功效<em&gt;体外</em

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

评价疗效<em&gt;小时。幽门螺杆菌</em&gt;蛋白HP-NAP为膀胱癌的治疗在原位小鼠模型的治疗工具

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

超超磁氧化铁纳米探针在肺外结核检测的合成、表征及应用

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

利用斑马鱼胚胎对化合物毒性的快速评价

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

Biotribological Testing and Analysis of Articular Cartilage Sliding against Metal for Implants

Osteochondral defects in middle-aged patients might be treated with focal metallic implants. First developed for defects in the knee joint, implants are now available for the shoulder, hip, ankle and the first metatarsalphalangeal joint. While providing pain reduction and clinical improvement, progressive degenerative changes of the opposing cartilage are observed in many patients. The mechanisms leading to this damage are not fully understood. This protocol describes a tribological experiment to simulate a metal-on-cartilage pairing and comprehensive analysis of the articular cartilage. Metal implant material is tested against bovine osteochondral cylinders as a model for human articular cartilage. By applying different loads and sliding speeds, physiological loading conditions can be imitated. To provide a comprehensive analysis of the effects on the articular cartilage, histology, metabolic activity and gene expression analysis are described in this protocol. The main advantage of tribological testing is that loading parameters can be adjusted freely to simulate in vivo conditions. Furthermore, different testing solutions might be used to investigate the influence of lubrication or pro-inflammatory agents. By using gene expression analysis for cartilage-specific genes and catabolic genes, early changes in the metabolism of articular chondrocytes in response to mechanical loading might be detected.

This protocol describes the preparation, biotribological testing, and analysis of osteochondral cylinders sliding against metal implant material. Outcome measures included in this protocol are metabolic activity, gene expression and histology.

植入物关节软骨滑向金属的生物摩擦学测试与分析

Osteochondral defects in middle-aged patients might be treated with focal metallic implants. First developed for defects in the knee joint, implants are ...

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

The present protocol describes a simple and efficient method for collecting blood from the subclavian vein in rats. It enables rapid, timely, and easily identifiable sampling without anesthesia and obtains high-quality blood through repetitive sample collection.

意识大鼠锁骨下静脉的重复血液采样

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to ...

使用 M-ROSE 加强肺部感染治疗

Microbiological Rapid On-Site Evaluation for Pulmonary Infectious Diseases

The prompt initiation of empirical anti-infective therapy is crucial in patients presenting with unexplained pulmonary infection. Although imaging acquisition is relatively straightforward in clinical practice, its lack of specificity often necessitates additional time-consuming tests such as sputum culture, bronchoalveolar-lavage fluid culture, or genetic sequencing to identify the underlying etiology of the disease accurately. Moreover, the limited efficacy of empirical anti-infective treatment may contribute to antibiotic misuse. Recent advancements in interpreting microbial background on rapid on-site evaluation (ROSE) slides have enabled clinicians to promptly obtain samples through bronchoscopy (e.g., alveolar lavage, mucosal brushing, tissue clamp), facilitating bedside staining and interpretation that provides essential microbial background information. Consequently, this establishes a foundation for developing targeted anti-infection treatment and individualized drug therapy plans. With a better understanding of which pathogens are causing infections in real-time, physicians can avoid unnecessary broad-spectrum antibiotics contributing to antibiotic resistance. Establishing a rapid and standardized M-ROSE workflow within respiratory medicine departments or intensive care units will greatly assist physicians in formulating accurate treatment strategies for patients, which holds significant clinical implications.

作者聚焦：使用 M-ROSE 实时推进病原体检测和疾病评估

The protocol here demonstrates a fast and standardized microbiological rapid on-site evaluation (M-ROSE) workflow, including three steps: slide making, staining, and interpretation. This protocol will help physicians make rapid clinical decisions.