感谢Therwa哈姆扎，约翰·E. TIDWELL，尼娜克洛维斯，和苏珊·史密斯的实验援助和苏珊·史密斯校对。作者还感谢提供临床分离的细菌和约翰·B·巴尼特博士对他的支持和使用的生物安全实验室在西弗吉尼亚大学微生物学，免疫学和细胞生物学系的约翰·托马斯博士。作者承认金融支持接骨和创伤护理基金会和美国国家科学基金会（＃1003907）。显微镜实验和图像分析技术在西弗吉尼亚大学的成像设备，这是支持部分的玛丽巴布伦道夫癌症中心和美国国立卫生研究院授予P20 RR016440。 动物使用的血液提请批准了西弗吉尼亚大学实验动物护理和使用委员会。所有的实验被处决符合所有相关guidelin的ES，法规和监管机构。

1。 PRP的制备和激活 1.1抽血<ol><li>麻醉家兔吸入异氟醚（2％O 2诱导和维护的1％）。 </li><li>绘制2毫升0.129中号柠檬酸三钠（一种抗凝血剂的溶液）放入20毫升注射器。的柠檬酸三钠溶液的制备通过1.897克柠檬酸三钠溶解在50毫升蒸馏H 2 O，并用0.22μm的无菌过滤器过滤。 </li><li>用70％乙醇消毒的兔耳。 </li><li>绘制血液（ 例如 5毫升）通过连接到注射器的蝴蝶针（25 G）从兔耳缘静脉。 </li><li>混血的柠檬酸三钠溶液中轻轻搅拌。血液和柠檬酸三钠溶液的体积比为9:1。 </li></ol> 1.2 PRP制备（ 图1） <ol><li>抗凝固处理的血液转移至50ml塑料离心管中。以10微升的等分试样血以确定基线血小板计数hemocytometry。 </li><li>离心血在300×g离心10分钟，在室温（RT）在离心分离机的摆​​动离开转子。设置加速和制动速度为低（ 图1）。 </li><li>离心分离后，血液被分离成三层。底层是主要的红血细胞，中间层（通常简称为“血沉棕黄层”）组成的浓缩血小板和白细胞，顶端层主要是等离子体，这是血液中的液体成分，和血小板（ 图1）。离心管小心地运送到细胞培养罩;不打扰层。塑料移液器使用1毫升到15毫升塑料管，将所有的血浆，棕黄色的大衣，和2-3毫米厚的红血细胞层。 </li><li>离心传输的示例的第二时间，以3,000 xg离心15分钟，在RT。顶层（上清液），贫血小板血浆（PPP）被视为d被转移到新的管中。 </li><li>获得PRP通过使用PPP，得到2.0×10 6血小板/μl（确定hemocytometry），在其它的血液试样中的血小板浓度调节。 </li></ol> 1.3 PRP激活<ol><li>准备PRP激活溶液用5毫升10％的氯化钙溶解5000 IU牛凝血酶的工作浓度1,000 IU / ml的。 </li><li>添加活化液PRP和PPP，并反复吹打形成PRP和PPP凝胶混合溶液。 PRP或PPP的激活溶液的体积比为1:4。 </li></ol> 2使用kill曲线法的PRP 的体外抗菌试验（ 图2） <ol><li>用无菌接种环，增加数个殖民地的S.黄色葡萄球菌从它的平板培养过夜到5毫升的Mueller Hinton肉汤（MHB）的塑料管中。涡简要然后在37℃下孵育2小时样品下，细菌的介质的光密度，使用分光光度计测定，并调整至光密度等于〜1×10 8 CFU / ml为根据预先确定的标准曲线。 </li><li> 100倍稀释液，使用PBS，得到1×10 6 CFU /毫升的接种物并放置在冰上。 </li><li>设置和无菌，一次性5 ml圆底聚苯乙烯管贴上标签，准备以下的样本组如表1中所示，在每个试管的最终体积为2ml。 </li><li>加入PRP和PPP，或PBS第一聚苯乙烯管中，然后由用于激活的凝血酶溶液（凝胶的形成）。接下来，添加MHB，然后S.黄色葡萄球菌接种物（1×10 6 CFU /毫升），得到的最终浓度为1×10 5 CFU /毫升。 </li><li>孵育在37℃下在150 rpm的搅拌与轨道管。 </li><li>在预先确定的时间点（ 例如，0，1，和2小时），每个试管中混合的解决方案通过重复吸移的（这步骤是非常重要的，因为细菌可能会被困在里面的PRP凝胶）。取10微升的样品，用无菌的0.9％的生理盐水稀释串联，和移液100微升等分的各稀释到胰蛋白酶大豆琼脂（TSA，用5％羊血）CFU计数板。 </li><li>文化的琼脂平板上，在37℃下过夜，然后计算和记录板菌落。标绘数据上logarithimic规模上的x轴和CFU /毫升在y-轴的时间（hr）。 </li></ol>

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作者宣称，他们没有竞争的金融利益。

富血小板血浆已被越来越多地用于临床的应用，由于其促进愈合的特性15-17。在本研究中中，PRP提出了一种新的方法预防感染。 PRP发现对MRSA，MSSA，A组链球菌 ， 淋病奈瑟菌有较强的抗菌性能。 PRP的主要优势在于，相对于传统的抗生素治疗，预防感染包括：（1）目前的抗生素治疗所面临的挑战，包括日 ​​益抗生素的耐药性14,18-20。 PRP可能是一个先进的替代品，因?...

种植体相关感染是一个显着的临床并发症。 金黄色葡萄球菌 （ 金黄色葡萄球菌 ）是孤立从植入物相关感染的最常见的微生物之一，。它是能够生产覆盖的植入物的表面，并可能会导致抗生素耐药性的感染1,2的生物膜。植入相关感染的治疗往往需要长期住院，反复清创术和静脉抗生素治疗时间延长。在抗生素有抗药性的情况下，去除植入物可能是必要的。上升抗生素对细菌的抵抗力也被称为中心疾病控制和预防中心（CDC）为“世界上最紧迫的健康问题之一。”随着时间的推移，如果没有新的有效的抗菌治疗的发展，它是多药耐药病原体与传统的抗生素是无法治愈的。植入相关的预防因此，感染是重要的和新的预防剂或方法是必要的，以防止这类感染。 富血小板血浆（PRP）是一个包含超过30的生长因子，可以帮助骨和骨移植愈合3-5的自体血浓度。已越来越多地在诊所报告，因为它的高浓度的各种生长因子，由血小板释放的PRP中的应用，以提高骨再生和软组织的成熟。 PRP几个特点表明，PRP可能还具有抗菌特性6-9。 PRP中包含了大量的血小板，白细胞高浓度的（其可以具有对细菌和真菌的宿主防御动作），和多个抗菌肽7,8,10。在最近的一大群心脏手术患者的研究中，据透露，术中使用PRP凝胶伤口愈合的显icantly的发病率下降的浅表和深部胸骨感染11。由于这些原因和观察，我们推测 ，PRP，除了其研究的愈合特性，具有抗菌性能。使用PRP，以防止感染的潜在优势，可包括：（ⅰ）PRP是不太可能诱导电阻相比，传统的抗生素治疗。 （ 二 ）PRP也有属性，促进愈合其中可能有一个协同效应对预防感染; PRP的愈合，促进属性可以提供密封，以防止细菌附着从而降低的几率为感染作为病原体和人类的细胞都竞相植入物表面12 ，13。 （ 三 ）PRP是固有的生物相容性，安全，无传染性疾病的风险。 我们的长期目标是使用PRP作为一种新的方法，以防止植入物相关infecti组件。本研究的目的是准备PRP使用两次离心的方法，检查PRP的体外抗菌性能，并描述协议，以评估这种抗菌性能。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Bovine thrombin</td>
 <td>King Pharmaceuticals, Inc </td>
 <td>60793-215-05</td>
 <td>Thrombin (bovine origin)</td>
 </tr>
 <tr>
 <td>Calcium chloride</td>
 <td>King Pharmaceuticals, Inc</td>
 <td>60793-215-05</td>
 <td>10% calcium chloride</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Sigma-Aldrich</td>
 <td>E7023</td>
 <td></td>
 </tr>
 <tr>
 <td>Isoflurane</td>
 <td>Baxter</td>
 <td>1001936060</td>
 <td></td>
 </tr>
 <tr>
 <td>Mueller Hinton broth</td>
 <td>Becton, Dickinson and Company</td>
 <td>275710</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate-buffered saline</td>
 <td>Sigma-Aldrich</td>
 <td>D8662</td>
 <td></td>
 </tr>
 <tr>
 <td>Tri-sodium citrate</td>
 <td>Sigma-Aldrich</td>
 <td>W302600</td>
 <td></td>
 </tr>
 <tr>
 <td>Tryptic soy agar</td>
 <td>Fisher Scientific</td>
 <td>R01202</td>
 <td></td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Kendro Laboratory Products</td>
 <td>750043077</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe filter</td>
 <td>Millipore</td>
 <td>SLGP033RS</td>
 <td></td>
 </tr>
 </tbody>
</table>

prp as a new approach to prevent infection: preparation and in vitro antimicrobial properties of prp

植入相关感染已成为全球范围内的医疗保健行业越来越具有挑战性由于越来越大的抗药性，抗生素有抗药性的细菌，动物和人类之间传播的，成本高，治疗感染。 在这项研究中，我们公开了一种新的策略，在防止植入物相关感染的基础上的潜在的富血小板血浆（PRP）的抗菌性能，可能是有效的。由于其促进愈合的研究，PRP（生物制品）已经被越来越多地用于临床应用，包括骨科手术，牙周和口腔外科，口腔颌面外科手术，整形外科，运动医学等。 PRP可能是一个先进的替代传统的抗生素治疗，防止植入相关感染。比常规抗生素治疗州市的PRP的使用可能是有利的NCE PRP是不太可能诱导抗生素耐药性和PRP的抗菌和愈合促进性能可能具有协同作用对预防感染。这是众所周知的，病原体和人类细胞竞相种植体表面，和PRP促进愈合的属性可以改善人类的细胞附着，从而减少了感染的几率。此外，PRP是固有的生物相容性，安全，无传染性疾病的风险。 我们的研究中，我们选择了一些临床中常见的骨科感染的细菌菌株和PRP检查是否有对这些细菌的体外抗菌性能。我们已经准备好PRP使用两次离心的做法，让所有的样品中获得相同的血小板浓度。我们已经取得了抗菌剂的研究结果一致，发现PRP具有较强的体外抗菌性能，对细菌，如methi的Cillin的敏感和耐甲氧西林金黄色葡萄球菌，A组链球菌 ， 淋球菌 。因此，使用的PRP可能有潜力以防止感染和昂贵的手术后的植入物相关的感染的治疗，以减少需要。

PRP的可重复使用两次离心的方法（ 图1）的制备。 PRP的发现呈现强（高达100倍减少的CFUs） 在体外的抗菌性能，对耐甲氧西林S.黄色葡萄球菌 （MRSA）（ 图3），这是常见的在世界各地的医院14。同样，，PRP具有较强的抗菌性能，对甲氧西林敏感的S.金黄色葡萄球菌 （MSSA），A组链球菌 ， 淋球菌 。 <p class="jo...

Watch this Scientific Journal Video about PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP at JoVE.com

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

植入相关感染是一个重要的临床并发症。这项研究描述了一种方法，使用富血小板血浆（PRP），以防止植入物相关感染，给出了协议，准备PRP血小板浓度不变，并报告新发现的抗菌性能的PRP和检查等抗菌性能的相关协议<em&gt;在体外。</em

PRP作为一种新的方法，以防止感染的制备及<em在体外</em&gt;抗菌性能的PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

prp-as-new-approach-to-prevent-infection-preparation-vitro

Research

JoVE Journal

Immunology and Infection

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

21.2K 次观看.  West Virginia University. 植入相关感染是一个重要的临床并发症。这项研究描述了一种方法，使用富血小板血浆（PRP），以防止植入物相关感染，给出了协议，准备PRP血小板浓度不变，并报告新发现的抗菌性能的PRP和检查等抗菌性能的相关协议在体外。

视频: PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

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 ...

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

简单而强大在体内和在体外方法研究病毒大会

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

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 ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

监测改变膜极性，膜完整性和细胞内离子浓度<em&gt;肺炎链球菌</em&gt;使用荧光染料

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...