Associazione 意大利每 la Ricerca Cancro (2016 号 18842), Cariplo 基金会 (赠款 2014-0655), 乔瓦尼博物馆每 la Ricerca 牛津 (FRRB) 支持。 伊兹获得 NIH 赠款 1R01AI121066-01A1, 1R01DK115217, HDDC P30 DK034854 补助金, 哈佛医学院弥尔顿发现, CCFA 高级研究奖 (412708), 埃莉诺和迈尔斯海岸第五十周年奖学金计划, Cariplo 基金会 (2014-0859)。

所有程序均经机构动物护理和使用委员会 (IACUC) 批准, 并在波士顿儿童医院儿童医院动物资源部的监督下运作, 或经意大利卫生部并在米兰大学 Bicocca 的机构动物护理委员会下进行。
1.白念珠菌制剂
<ol><li>文化C. 白念珠菌, 应变 CAF3-113, 在含有丰富培养基的管 (酵母萃取蛋白胨葡萄糖 (YEPD), 1% (w/v) 酵母提取物, 2% (w/v) Bacto 蛋白胨, 2% (瓦特/v) 葡萄糖) 补充苷 (50 毫克/升) 在25摄氏度。在这些条件下,白色念珠菌生长为酵母。 注: 其他c. 白念珠菌可使用, 如临床分离c. 白色念珠菌 SC531413。因为我们最近证明了溃疡过程是由先天免疫系统的活化引起的14, 理论上每一个C. 白念珠菌保持其细胞壁成分和结构, 以及有能力发起菌丝预测, 可以使用。</li>
	<li>使用细胞计数器分析仪对细胞浓度进行计数, 以此来监测文化。</li>
	<li>当它达到大约 8 x 106细胞/毫升的浓度时, 收获它的文化。或者, 使用分光光度计测量 OD600。 注: 通常, OD600 = 1 的值对应于 3 x 107细胞/毫升的酵母浓度, 但建议滴定。</li>
	<li>并用重悬 YEPD-苷培养基中的培养, 以 HEPES (50 毫米, pH 7.5) 为缓冲剂, 孵育37°c 的培养, 诱导菌丝形成。用100X 放大显微镜检查菌丝的形成。在37摄氏度培养后, 菌丝形成可见5小时;然而, 当菌丝百分比达到总培养的近95% 时, 在孵化后的37摄氏度使用细胞16小时。</li>
	<li>为了进一步丰富制剂的菌丝浓度, 离心机整除数的培养在 3300 x g 5 分钟. 弃上清, 并并用重悬在无菌 PBS 中的颗粒浓度 1 x 108菌丝/毫升。</li>
</ol>2. 深部真皮注射
<ol><li>24 h 在感染程序之前, 麻醉小鼠腹腔注射氯胺酮 (80-100 毫克/千克) 和甲苯噻嗪 (10 毫克/千克), 然后用电动剪刀剃掉老鼠的侧面。 注: 在任何麻醉过程中, 将老鼠放在加热灯下, 直到有效地恢复。</li>
	<li>休息的小鼠24小时, 以避免任何炎症由于剃须过程。</li>
	<li>24小时后剃须程序, 麻醉小鼠与腹腔注射氯胺酮 (80-100 毫克/千克) 和甲苯噻嗪 (10 毫克/千克)。</li>
	<li>检查爪子反射, 以确保小鼠被深深麻醉。通过牢牢捏爪来评估爪子反射。如果麻醉达到了, 动物不会感到疼痛, 也不会移动。</li>
	<li>用0.3 毫升胰岛素注射器在深真皮中注射白色念珠菌菌丝, 其最终容积为50µL/注射液, 对应 5 x 106菌丝/注射液。<ol>
<li>用两个手指拉紧剃的侧面的皮肤, 以便直接在深真皮中注入体积。用针朝上的斜角插入 10-半角的针。</li>
		<li>以这个角度, 病原体将被注入大约 300-500 µm 的深度。为了确保注射良好, 检查注射的体积是否形成一个肿块, 在注射后吸收几分钟。 注: 注射后形成的肿块约为1厘米2。这是将被删除的区域, 在指定的时间点, 无论是分子或组织学分析。</li>
		<li>为少于 24 h 的时间点 (推荐) 标记传染区域与标记笔, 因为与射入形成的凹凸持续几分钟, 在之后它被吸收。对于24小时或以上的时间点, 这一步骤是不需要的, 因为溃疡或囊肿将明显可见。</li>
	</ol>
</li>
</ol>3. 组织学染色标本的收集和准备
<ol><li>根据制度程序弄死老鼠。</li>
	<li>使用手术钳, 拉在注射部位的边界, 以前标记与标记笔的皮肤。使用手术剪刀, 通过切割的皮肤后, 标记线的感染部位。 注意: 小心用圆尖剪刀将皮肤与腹膜分开。</li>
	<li>将皮肤浸泡在一次性的基模中, 填充最佳切削温度 (OCT) 化合物, 与皮肤的内部面朝上。在液氮中冷冻样品, 直到化合物变成白色。不要让液氮覆盖样品之前, 它完全冻结, 因为这将损害样品。 注意: 小心!在步骤3.3 中, 戴上防护罩, 防止液态氮。</li>
	<li>如果样品不立即用于幻灯片准备, 迅速储存在-80 摄氏度。 注: 样品可以无限期储存在-80 摄氏度。</li>
</ol>4. 幻灯片的制作
<ol><li>将样品切成两半, 从样品的中心开始为组织学准备切片。</li>
	<li>用恒温器切开5µm 厚片。 注: 对于皮肤样品, 恒温器的温度不应高于摄氏-25 摄氏度。较高的温度会导致 H OCT 部分熔化, 因此样品在切割过程中不会保持在正确的位置。</li>
	<li>使用带正电荷的显微镜幻灯片收集切片。</li>
	<li>如果在准备后不久不使用收集的切片, 请将其存储在摄氏-20 摄氏度。此时, 切片可以无限期地存储在-20 摄氏度。</li>
</ol>5. 苏木精 & 伊红染色
<ol><li>浸泡在鳃的苏木素4分钟, 使幻灯片着色. 在自来水中冲洗幻灯片5分钟。</li>
	<li>浸泡在1分钟内, 用浸没在红 Y 溶液中的幻灯片来着色. 用蒸馏水冲洗幻灯片, 然后继续执行该协议。</li>
	<li>浸泡在连续增加的乙醇溶液浓度 (50%, 70%, 80%, 95%, 100%) 脱水。在每个乙醇溶液中浸泡十五年代的幻灯片。</li>
	<li>通过浸泡在组织学清除剂中, 清除幻灯片至少2分钟。</li>
	<li>使用安装介质和盖子滑块装入幻灯片。</li>
	<li>以40X 倍的比例获取带有显微镜幻灯片扫描仪的幻灯片图像。</li>
</ol>6. 周期性酸希夫 (PAS) 染色
<ol><li>用≥99.5% 丙酮固定在室温下的幻灯片1分钟. 在自来水中冲洗幻灯片1分钟。</li>
	<li>浸泡在周期性酸溶液中 (1 克/dL), 以5分钟的方式对幻灯片进行着色. 在3的蒸馏水中冲洗幻灯片。</li>
	<li>浸泡在席夫的试剂15分钟, 使幻灯片着色. 在自来水中冲洗幻灯片5分钟。</li>
	<li>浸泡在鳃的苏木素5分钟, 使幻灯片着色. 在三十年代的自来水中冲洗滑梯。</li>
	<li>浸泡3的100% 乙醇的变化, 脱水的幻灯片。每一次将幻灯片浸入十五年代。</li>
	<li>通过浸泡在组织学清除剂中, 清除幻灯片至少2分钟。</li>
	<li>使用安装介质和盖子滑块装入幻灯片。</li>
	<li>使用显微镜幻灯片扫描仪获取幻灯片的图像。</li>
</ol>7. RNA 提取的样品收集和准备
<ol><li>根据制度程序弄死老鼠。删除步骤3.2 中的外观示例。</li>
	<li>将样品浸入2毫升的盖管中, 用1毫升的试剂胍硫氰酸盐-苯酚-氯仿萃取液。 注意: 协议可以在这里暂停, 样品可以存储在-80 摄氏度。</li>
	<li>用手术钳将样品切成大约0.2 厘米2块。 注意: 小心!大多数 RNA 分离试剂是有毒和诱变的。步骤7.3 和 7.4必须在通风罩下完成。</li>
	<li>以20振荡/秒的速度在20分钟内粉碎样品与珠磨房. 或者, 可以使用机械波特。</li>
	<li>检查样品的有效均匀性, 寻找是否有完整的皮肤片。如果样品不是完全均匀的, 重复此步骤。</li>
	<li>离心样品在 1.6万 x g 1 分钟保持上清液 RNA 提取和丢弃颗粒。执行此步骤可消除可能阻止抽取列的毛发和碎片。</li>
	<li>使用 rna 纯化柱14进行 rna 提取。</li>
	<li>使用分光光度计评估 RNA 的纯化和浓度。如果 RNA 提取物不立即用于 cDNA 准备, 保存样品在-80 °c。</li>
</ol>

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作者没有什么可透露的。

在这里, 我们描述了一种C. 白念珠菌感染的方法, 以研究在深层真皮真菌入口引发的炎症过程。
虽然皮肤脓肿的形成是一个相对罕见的事件后, C. 白色念珠菌感染15, 直接在深真皮中注射真菌不仅可以研究真菌驱动脓肿的形成, 而且还分析特异免疫参与控制真菌扩散的细胞。在这里描述的方法, 可以研究免疫细胞之间的相互作用, 以及免疫细胞的招?...

人体上覆盖着极高数量的微生物。皮肤表面是大约100万细菌的栖所每平方厘米1。然而, 这个数字并没有反映出殖民皮肤的各种微生物。除了细菌外, 人体还被许多真菌物种所殖民, 包括白色念珠菌, 它能够在粘膜和皮肤2级存活。
近年来, 被诊断为真菌感染的人的百分比大大增加。这主要是由于免疫机能丧失者的数量增加,即HIV 阳性患者和3移植后经过化疗或免疫抑制剂的患者。在美国进行的一项监测研究中, Wisplinghoff等人发现, 9.5% 的医院血流感染是由念珠菌4种引起的。由于真菌感染的增加, 特别是由于血液感染中发现的念珠菌的比例升高, 了解这种病原体如何逃脱免疫系统的控制是非常重要。
白色念珠菌是一种二形真菌, 生长在不同形态形态, 如酵母, blastospores, 假菌丝和菌丝, 取决于环境条件5。在其菌丝的形式,白色念珠菌显示其最高的侵袭能力, 并有能力穿透上皮6。
用几种实验方法对白念珠菌感染进行了研究。最常见的感染模型是静脉注射白色念珠菌酵母7。然而, 这个模型没有考虑到所有的过程中发生的真菌传播到血液中。另一种模型利用C. 白念珠菌侵入上皮细胞的能力。这种方法, 也称为砂纸模型8, 是由 Gaspari等在1998年开发的9, 并包括使用沙纸擦伤皮肤, 从而消除角质层之前, 应用C. 白念珠菌。这个过程允许真菌穿透上皮, 从而能够分析这种病原体的侵袭能力。最后, 其他模型的胃肠道10和呼吸道感染11已被用于不同的研究。
伤口的形成 (如在沙纸模型中) 导致了几个途径的活化, 包括免疫细胞的招募和活化, 以促进愈合过程12。这可以改变或掩盖对病原体特别诱发的免疫反应, 从而导致混淆结果。
在这里, 我们描述一种皮肤感染的方法, 避免最初的伤口形成和诱导的基底炎症环境。为了保持完整的上皮结构, 我们直接在深真皮中注射菌丝形式的白色念珠菌。尽管单个注射可以引起轻度炎症, 但与在沙纸模型中的开放性伤口的形成相比, 炎症的数量受到限制和限制。我们在这里描述的方法允许研究真菌感染和传播的免疫反应, 同时避免机械损伤造成的过度和预先存在的炎症环境。

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Euroclone</td>
			<td>ECB4053L</td>
			<td>warm in 37 &#176;C bath before use</td>
		</tr>
		<tr>
			<td>H-OCT compound</td>
			<td>histo-line laboratories</td>
			<td>R0030</td>
			<td></td>
		</tr>
		<tr>
			<td>Gill&#39;s Hematoxilyn</td>
			<td>histo-line laboratories</td>
			<td>09-178-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y solution, alcoholic</td>
			<td>histo-line laboratories</td>
			<td>09-209-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>scharlau</td>
			<td>ET00232500</td>
			<td></td>
		</tr>
		<tr>
			<td>Citro-HISTOCLEAR</td>
			<td>histo-line laboratories</td>
			<td>R0050</td>
			<td></td>
		</tr>
		<tr>
			<td>Eukitt, mounting medium</td>
			<td>bio-optica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>sigma-aldrich</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr>
			<td>PAS staining system</td>
			<td>sigma-aldrich</td>
			<td>395B-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Peptone</td>
			<td>BD</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Yeast Extract</td>
			<td>BD</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-Glucose anhydrous for molecular biology</td>
			<td>Applichem PanReac</td>
			<td>50-99-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Merck Millipore</td>
			<td>8451</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Applichem PanReac</td>
			<td>A1070,0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-Lock tubes 2 mL</td>
			<td>eppendorf</td>
			<td>30121597</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Life Technologies</td>
			<td>15596018</td>
			<td>Toxic, corrosive and mutagen. Use all precaution needed</td>
		</tr>
		<tr>
			<td>Rneasy Mini Kit</td>
			<td>QIAGEN</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>liquid nitrogen</td>
			<td></td>
			<td></td>
			<td>Wear eye protection</td>
		</tr>
		<tr>
			<td>Instrument</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coulter Counter-Particle Count</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5415 R</td>
			<td>eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MC 3000 Microtome Cryostat</td>
			<td>histo-line laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TissueLiser</td>
			<td>QIAGEN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.3 ml Insulin Syringe with a 30G x 8mm needle&#160;</td>
			<td>BD</td>
			<td>324826</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Base mould disposable</td>
			<td>histo-line laboratories</td>
			<td>2781</td>
			<td></td>
		</tr>
		<tr>
			<td>Positively charged bio microscope slides</td>
			<td>bio-optica</td>
			<td>09-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slips 24 x 50 mm</td>
			<td>thermo scientific</td>
			<td>11911998</td>
			<td></td>
		</tr>
	</tbody>
</table>

deep dermal injection as a model of candida albicans skin infection for histological analyses

皮肤是身体的一个非常扩展的器官, 并且, 由于这个大表面, 它持续暴露于微生物。皮肤损伤很容易导致真皮的感染, 进而导致病原体向血液中传播。了解免疫系统在早期如何对抗感染, 以及宿主如何消除病原体是确定今后治疗干预的基础的重要步骤。在这里, 我们描述了一种白色念珠菌感染模型, 可以可视化的过程中发生的早期感染, 包括当病原体已经通过了上皮屏障, 以及由C. 白念珠菌诱发的免疫反应入侵。我们利用这种感染模型进行组织学分析, 显示了渗透皮肤的免疫细胞以及病原体的存在和定位。在感染后收集的样本可以处理 RNA 提取。

通过直接在深部真皮中注射病原体, 组织结构形态保持不变 (图 1A)。
皮肤结构完整性的维护允许检测免疫细胞及其在感染部位的定位。图 1B显示的放大倍数显示, 脓肿主要由中性粒细胞组成, 其中含有病原体的传播, 其受感染部位的限制。如中性粒细胞的招聘, 可以形成脓肿, 从而避?...

Watch this Scientific Journal Video about Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses at JoVE.com

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

深部真皮注射液作为白色念珠菌皮肤感染模型的组织学分析

在这里, 我们描述了一个协议, 允许皮肤标本的组织学和分子分析后,白色念珠菌皮注射液。该协议维护皮肤的结构完整性, 并允许组织居民或新招募的免疫细胞的定位以及病原体的分布。

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

deep-dermal-injection-as-model-candida-albicans-skin-infection-for

Research

JoVE Journal

Immunology and Infection

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

10.6K 次观看.  University of Milano-Bicocca. 在这里, 我们描述了一个协议, 允许皮肤标本的组织学和分子分析后,白色念珠菌皮注射液。该协议维护皮肤的结构完整性, 并允许组织居民或新招募的免疫细胞的定位以及病原体的分布。

视频: Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

一个96孔微孔板为基础的方法监测的形成和抗真菌的药敏试验白色念珠菌生物膜

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

科研

免疫与感染

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

使用<em&gt;大蜡螟</em&gt;作为模式生物来研究<em&gt;嗜肺军团菌</em&gt;感染

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

研究微生物群落<em&gt;在体内</em&gt;:主机之间介互动的典范<em&gt;白色念珠菌</em&gt;和<em&gt;铜绿假单胞菌</em&gt;在航空

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

下腔静脉狭窄: 深静脉血栓形成的小鼠模型

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

全自动微流控装置在白色念珠菌生物膜形成中的可视化

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Using Robotic Systems to Process and Embed Colonic Murine Samples for Histological Analyses

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of experimental models needs to be as rigorous as that applied for human samples. Indeed, drawing reliable and accurate conclusions is critically influenced by the quality of tissue section preparation. Here, we describe a protocol for histopathological analysis of murine tissues that implements several automated steps during the procedure, from the initial preparation to the paraffin embedding of the murine samples. The reduction of methodological variables through rigorous protocol standardization from automated procedures contributes to increased overall reliability of murine pathological analysis. Specifically, this protocol describes the utilization of automated processing and embedding robotic systems, routinely used for the tissue processing and paraffin embedding of human samples, to process murine specimens of intestinal inflammation. We conclude that the reliability of histopathological examination of murine tissues is significantly increased upon introduction of standardized and automated techniques.

Lack of standardization for murine tissue processing reduces the quality of murine histopathological analysis as compared to human specimens. Here, we present a protocol to perform histopathological examination of murine inflamed and uninflamed colonic tissues to show the feasibility of robotic systems routinely used for processing and embedding&#160;human samples.

利用机器人系统对结肠小鼠样本进行处理和嵌入进行组织学分析

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of ...

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J&#183;cm-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcome of red light application on the growth of Candida albicans biofilm. A non-coherent red light device with the wavelength of 635 nm and energy density of 87.6 J&#183;cm-2 was applied throughout the growth of Candida albicans biofilms for 48 h.

红光光疗法调节白色念珠菌生物膜生长

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

使用荧光原位杂交对毛尿胃肠道中的念珠菌的可视化

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

澄清和成像念珠菌生物膜

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...