我们非常感谢詹尼Scimone与视频制作的优秀援助。

对感染艾滋病毒的血液样本的处理需要一些重要的建议。 所有血液标本必须在强大的防漏容器运送。采集标本时，必须小心，以避免污染容器的外观和附带任何手续的标本。 所有处理受感染的血液的人必须戴手套。手套必须改变和标本的处理完成后洗净双手。 艾滋病毒感染者的血液样品的处理，必须做到在II级生物危害柜罩。 应采用机械吹打艾滋病。 必须加以限制，其中有别无选择的情况下使用的针头或其他锐器（包括玻璃如移液器或毛细管）。 实验室表面必须与适当的化学消毒剂后溢出的血液净化，工作时活动结束。 必须净化污染的材料使用或重复使用前必须正确处置医疗废物的路线通过。 每一个可能具有传染性的血液或体液（即那些需要的普遍预防措施）的职业暴露事件应被视为医疗紧急干预必须迅速启动才能有效。 该协议要求5天完成。详细的时间表是在图1。 使用图2中所描述的方法，确定细菌种类。 1。样品采集<ol><li> 9毫升全血抽入EDTA的含管。 </li><li>于2000年在室温下10分钟的转速离心管获得等离子体。 </li><li>离子被收集在一个无菌2毫升的Eppendorf管。 </li></ol> 2。血浆样品中的DNA提取<ol><li>充分消毒罩，吸液管和实验所需的材料，以保证无菌。 </li><li>位置至少30分钟，在紫外线灯的所有材料。 </li><li>用消毒液擦拭手套。 </li><li>浸泡在乙醇一些纸巾和引擎盖下。每次小费是被丢弃擦拭湿纸巾吸管。 </li></ol> DNA是使用商业套件制造商的说明（易DNA试剂盒，Invitrogen公司，卡尔斯巴德CA，美国）提取。 <ol start="5"><li> 350μL血浆放置在无菌2毫升Eppendorf管中。 </li><li> 350μL超纯水过滤，作为阴性对照。 </li><li>添加10μL的溶菌酶（1mg/ml的）样品。 </li><li> 30分钟在37 ° C。 </li><li>加入500μL裂解液，轻轻混匀样品。 </li><li> 7分钟的孵育65 ° C。 </li><li>加入氯仿900μL的样品。 </li><li>涡大力直到样品均匀粘稠。 </li><li>添加200μL降水方案和涡大力。 </li><li>离心样品在室温下10分钟的转速在10500分开的阶段，并形成接口。 </li><li>上层水相转移到一个新的离心管中，含有1毫升乙醇100％。 </li><li>在4个样品离心10分钟的转速在10500 ° C。 </li><li>除去乙醇。 </li><li>加入乙醇70％1毫升。 </li><li>离心样品的最高速度为10分钟，在4 ° C。 </li><li>除去乙醇。应与pipettator删除残留的乙醇。 </li><li>在超纯水50μL重悬沉淀。 </li><li>用分光光度计读取DNA的浓度。 </li></ol> 3。 16S rRNA基因的PCR PCR扩增9如前所述。 <ol><li>扩增100μL反应10 10X PCR缓冲液，5μL25 mM的氯化镁，2毫米的总dNTPs 5μL，50微米底漆RW01（AACTGGAGGAAGGTGGGGAT）1μL，1μL50微米底漆DG74液组成的混合物的DNA （AGGAGGTGATCCAACCGCA），34μL的H20和0.5μLTaq DNA聚合酶（AmpliTaq金牌，应用生物系统，福斯特城，加利福尼亚，美国）。 </li><li>为了避免可能造成的污染细菌Taq聚合酶，PCR混合物中的过滤器传输。 </li><li>离心过滤器（单片机，Millipore公司，比尔里卡，马萨诸塞州，美国）为30分钟，在500 RCF在4 ° C。 </li><li>分装在PCR混合物，根据需要放大（正面和负面的PCR控制，样品和提取的水）样本数。 </li><li>使用以下热循环条件:94 ° C为10分钟，每次1分钟的40倍，94 ° C，55 ° C和72 ° C，并在72 ° C 10分钟</li><li>可视化的PCR产物在2％琼脂糖凝胶。使用100 bp的DNA阶梯。 PCR产物的大小约360基点（图3）。 </li></ol> 4。 PCR产物纯化只有PCR阳性标本应得到净化。净化是利用商业Kit中的下列制造商的说明（PureLink PCR扩增microkit，Invitrogen公司，卡尔斯巴德的CA，美国）。 <ol><li>新增4卷结合缓冲液，每1体积的PCR产物中含有异丙醇。 </li><li>传输与一列的结合缓冲液PCR产物。 </li><li>在室温下在10000 RCF离心1分钟。 </li><li>与洗涤缓冲液含有乙醇洗净列。 </li><li>在室温下在10000 RCF离心1分钟。 </li><li>以最大速度离心1分钟在室温下干燥的硅膜，并删除任何残留的洗涤缓冲液，用乙醇。 </li><li>加入10μL超纯水。 </li><li> 1分钟在室温下孵育。 </li><li>最大速度为2分钟，在室温下离心收集纯化的DNA。 </li></ol> 5。克隆溶原性肉汤（LB）板的制备<ol><li>溶于约800毫升的水混合的LB 25克。 </li><li>带来的最终体积至1 L。 </li><li>加入15克Bactoagar。 </li><li>高压灭菌器为20分钟。 </li><li>经过高压灭菌，大力摇动烧瓶中的解决方案混合熔化的琼脂。 </li><li>冷却到50 ° C的解决方案</li><li>添加氨苄青霉素（50微克/毫升）和漩涡，直到完全溶解。 </li><li>倒入深约3毫米的钢板。 </li><li>离开板在室温下。 </li><li>传播的X加仑（40mg/ml）和IPTG每个LB平板上，孵育（100毫米），在37 ° C，直到准备使用。 </li></ol>感受态细胞转化执行转换使用商业Kit中的下列制造商的说明（地形TA克隆试剂盒，Invitrogen公司，卡尔斯巴德的CA，美国）。 <ol><li>准备克隆反应的组合（1-4μL新鲜的PCR产物，1μL盐溶液，如有必要的载体和水1μL）。 </li><li>轻轻混合反应，并在室温下孵育5-10分钟。 </li><li>广场上冰的反应。 </li><li>新增2成一小瓶主管细胞克隆反应液，轻轻混匀。 </li><li>在冰上孵育30分钟。 </li><li>热休克的细胞在30秒42 ° C。 </li><li>立即转移到冰管。 </li><li>添加250μL介质。 </li><li>第tighly管和动摇管水平在37℃1小时。 </li><li> 50μL的蔓延，从每一个预热选择性板改造。 </li><li>过夜孵育在37 ° C。 </li></ol>潜伏期之后，白色和蓝色的菌落平板上发展。白色菌落呈阳性的PCR产物插入蓝色菌落PCR产物插入负。 6。测序分析测序反应<ol><li>混合使用这些试剂:2,5μLDNA，1μL引物（5pmol /μL）1,1μLBigDye（应用生物系统，福斯特城，加利福尼亚，美国）。 </li><li>热循环条件:96 ° C为1分钟10秒，55 ° C的25倍为4分15秒和60℃96℃。 </li></ol>柱纯化<ol><li>对于每一个反应，准备一个MicroSpin列（QIAGEN，米兰，意大利）。 </li><li>反相柱和旋涡混合树脂。 </li><li>柱底部对齐关闭，松开盖子¼圈，并将其放置在离心管列。 </li><li>离心1分钟3200转。 </li><li>列转移到一个干净的离心管。 </li><li>仔细吸管中心列到整个PCR测序反应。 </li><li>离心1分钟3200转。 </li><li>纯化的DNA eluite管（20μL超纯水）。 </li></ol>测序分析<ol><li>纯化样品装入96多孔板（20μL）。 </li><li>加载到音序器板。自动化DNA测序生成四色色谱图显示的测序运行的结果。 </li><li>作为对公共序列数据库的查询输入的核苷酸序列。在NCBI数据库和服务器上执行搜索，基本局部比对搜索工具（BLAST）。 </li><li>只考虑有98-100％的同源性的细菌。 </li></ol> 7。代表性的成果<img src="/files/ftp_upload/2830/2830fig1.jpg" alt="图1" /> 图1。时间轴的过程。 <img src="/files/ftp_upload/2830/2830fig2.jpg" alt="图2" /> 图2整个细菌鉴定程序流程图。 <img src="/files/ftp_upload/2830/2830fig3.jpg" alt="图3" /> 图3显示广泛的16S rRNA基因的PCR产物2％琼脂糖凝胶。 1巷包含一个100bp的DNA梯状条带，2巷包含PCR阳性对照，泳道3显示负PCR控制。巷4显示了一个HIV阳性病人的样本，和5巷中含有水。 6巷SHOWS样品从一个健康的个体和阴性PCR反应; 7巷含有水。只有HIV阳性患者，显示了积极的PCR扩增。作为一个在提取步骤阴性对照用超纯水，表明没有发生污染。 <img src="/files/ftp_upload/2830/2830fig4.jpg" alt="图4" /> 图4。0.7％琼脂糖凝胶电泳显示质粒提取小量程序。 1巷包含1个碱基的DNA梯状条带，2巷包含蓝色的控制殖民地，车道从12月3日含有白色菌落。从蓝色菌落的质粒不包含的PCR产物插入。所有10个白色菌落中含有正确插入质粒。 <img src="/files/ftp_upload/2830/2830fig5.jpg" alt="图5" /> 图5显示了一个例子从一个艾滋病毒抗体阳性的个体血浆中的细菌测序分析。我们的结果表明，在HIV感染的微生物易位涉及到一个polimicrobic菌群，这是不是艾滋病毒阴性者中，建议在控制细菌移位肠道免疫功能的大量失败。

<ol>
	<li>Hooper, L. V., Macpherson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+adaptations+that+maintain+homeostasis+with+the+intestinal+microbiota.">Immune adaptations that maintain homeostasis with the intestinal microbiota.</a> Nat Rev Immunol. 10, 159-169 (2010).</li><li>Macpherson, A. J., Harris, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+commensal+intestinal+bacteria+and+the+immune+system.">Interactions between commensal intestinal bacteria and the immune system.</a> Nat Rev Immunol. 4, 478-485 (2004).</li><li>Kanauchi, O., Mitsuyama, K., Araki, Y., Andoh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+intestinal+flora+in+the+treatment+of+inflammatory+bowel+disease.">Modification of intestinal flora in the treatment of inflammatory bowel disease.</a> Curr Pharm Des. 9, 333-346 (2003).</li><li>Brenchley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+translocation+is+a+cause+of+systemic+immune+activation+in+chronic+HIV+infection.">Microbial translocation is a cause of systemic immune activation in chronic HIV infection.</a> Nat Med. 12, 1365-1371 (2006).</li><li>Brenchley, J. M., Price, D. A., Douek, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+disease:+fallout+from+a+mucosal+catastrophe.">HIV disease: fallout from a mucosal catastrophe.</a> Nat Immunol. 7, 235-239 (2006).</li><li>Jiang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+levels+of+bacterial+DNA+correlate+with+immune+activation+and+the+magnitude+of+immune+restoration+in+persons+with+antiretroviral-treated+HIV+infection.">Plasma levels of bacterial DNA correlate with immune activation and the magnitude of immune restoration in persons with antiretroviral-treated HIV infection.</a> J Infect Dis. 199, 1177-1185 (2009).</li><li>Marchetti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+translocation+is+associated+with+sustained+failure+in+CD4++T-cell+reconstitution+in+HIV-infected+patients+on+long-term+highly+active+antiretroviral+therapy.">Microbial translocation is associated with sustained failure in CD4+ T-cell reconstitution in HIV-infected patients on long-term highly active antiretroviral therapy.</a> AIDS. 22, 2035-2038 (2008).</li><li>Marchetti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Microbial+Translocation+and+Immune+Hyperactivation+in+Disease+Progression+of+HIV++Patients+with+Preserved+CD4+Count+in+the+Absence+of+ART.">Role of Microbial Translocation and Immune Hyperactivation in Disease Progression of HIV+ Patients with Preserved CD4 Count in the Absence of ART.</a> , (2010).</li><li>Greisen, K., Loeffelholz, M., Purohit, A., Leong, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR+primers+and+probes+for+the+16S+rRNA+gene+of+most+species+of+pathogenic+bacteria,+including+bacteria+found+in+cerebrospinal+fluid.">PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid.</a> J Clin Microbiol. 32, 335-351 (1994).</li></ol>

<p class="jove_content"在此，我们显示的PCR /测序协议的特点艾滋病毒感染者外周血中的细菌易位。</p&gt;<p class="jove_content"&gt;最可靠的结果是使用EDTA含管收集血浆时获得。采血后，血浆应通过离心分离2 / 3小时内，以避免溶血和DNA片段退化。第一样本储存在-20 ° C，约5天，然后在-80 ° C应避免多个冻融循环，这可能会导致细菌的DNA损失。</p&gt;<p class="jove_content"&gt;血浆样品应解冻后12小时内使用。</p&gt;<p class="jove_content"&gt...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> Easty - DNA试剂盒</td><td> Invitrogen公司</td><td> K 180001 </td><td></td></tr><tr><td>氯仿</td><td> Sigma - Aldrich公司</td><td> C2432 </td><td></td></tr><tr><td>乙醇</td><td> Sigma - Aldrich公司</td><td> E7203 </td><td></td><tr/><tr><td>超纯娃儿</td><td> Invitrogen公司</td><td> 10977049 </td><td></td></tr><tr><td>溶菌酶</td><td> Fluka公司</td><td> 62970 </td><td> 10微克/毫升Distillated水</td></tr><tr><td> AmpliTaq金牌</td><td>应用生物系统</td><td> 26478701 </td><td></td></tr><tr><td>单片机100 </td><td> Millipore公司</td><td> 42413 </td><td></td></tr><tr><td> PCR引物</td><td> Invitrogen公司</td><td></td><td></td></tr><tr><td>琼脂糖</td><td> Eppendorf公司</td><td> C1343 </td><td></td></tr><tr><td> DNA梯状</td><td> Invitrogen公司</td><td> 15628019 </td><td></td></tr><tr><td> Purelink PCR扩增微套件</td><td> Invitrogen公司</td><td> K310050 </td><td></td></tr><tr><td> LB琼脂，粉末</td><td> Invitrogen公司</td><td> 22700025 </td><td></td></tr><tr><td> Bactoagar </td><td> Invitrogen公司</td><td></td><td></td></tr><tr><td>氨苄青霉素</td><td> Invitrogen公司</td><td> 11593027 </td><td> 10毫克/毫升Distillated水</td></tr><tr><td> X - gal的</td><td> Invitrogen公司</td><td> 15520034 </td><td> 40mg/mL在DMF </td></tr><tr><td>经IPTG </td><td> Invitrogen公司</td><td> 15529019 </td><td> 100MM在Distillated水</td></tr><tr><td>地形助教克隆试剂盒</td><td> Invitrogen公司</td><td> K450002 </td><td></td></tr><tr><td> Purelink快速质粒小量制备试剂盒</td><td> Invitrogen公司</td><td> K210010 </td><td></td></tr><tr><td>大染料</td><td>应用生物系统</td><td> 4337455 </td><td></td></tr><tr><td> DyeEx 2.0自旋套件</td><td> Qiagen公司</td><td> 63204 </td><td></td></tr></tbody></table>

sequencing of bacterial microflora in peripheral blood: our experience with hiv-infected patients

生理健康的胃肠道是殖民地的影响共生微生物的体液免疫和细胞的粘膜免疫系统1,2的发展的种类繁多。 屏蔽的微生物是通过一个强大的黏膜屏障的免疫系统。感染和抗生素被称为改变正常的消化道障碍和居民的细菌组成，这可能会在可能的免疫异常导致3。 艾滋病毒会导致违反与胃肠黏膜免疫和全身血液循环进入细菌的生物制品，如内毒素和细菌的DNA片段，这有助于全身免疫激活4-7泄漏的渐进破坏屏障。在艾滋病毒/艾滋病的免疫病理及治疗 4,8微生物易位是牵连。 我们的目的是表征转位HIV感染患者的外周血中的细菌组成。为了追求我们的目标，我们成立了ribosomial panbacteric 16S基因测序分析PCR反应。 简单地说，艾滋病毒感染者和正常人全血使用。在这些患者中预计，鉴于目前健康的个人正常的肠道动态平衡没有菌群易位。静脉穿刺的全血和血浆分离收集，DNA是从血浆中提取和使用执行范围广泛的PCR反应ribosomial panbacteric 16S 基因 9 。执行下面的PCR产物纯化，克隆和测序分析。

Watch this Scientific Journal Video about Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients at JoVE.com

Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients

我们的实验将展示如何执行转位HIV阳性患者的外周血中的细菌种类的测序分析。

测序外周血中细菌菌群:我们的经验与艾滋病毒感染者

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral ...

sequencing-bacterial-microflora-peripheral-blood-our-experience-with

Research

JoVE Journal

Medicine

12.2K 次观看.  San Paolo Hospital University of Milan, Italy. 我们的实验将展示如何执行转位HIV阳性患者的外周血中的细菌种类的测序分析。

视频: Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing &gt; 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.

Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

Anti-retroviral therapy to treat HIV/AIDS is monitored in South Africa on a large scale. Flow cytometry is combined for haematology (CD45), immunology (CD4) and viral-load linked CD38 assay. Recorded at NHLS-SA laboratories, Johannesburg, these modern methods are cost-efficient with heightened local internal quality control, serving as role-models for resource-limited diagnostics.

艾滋病毒/艾滋病的综合成本有效的实验室监测:非洲的角色模型

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

科研

免疫与感染

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

在艾滋病毒感染者病毒载量低于检测限制标准的临床实验放大和量化HIV - 1 RNA

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

用于下一代测序的近全长 HIV-1 Proviruses 的扩增

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

遗传学

Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Antisense Protein (ASP) RNA Transcripts in Patients by Strand-Specific RT-PCR

In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). In HIV-1, the antisense protein ASP gene is located on the negative strand of env, in the reading frame -2, spanning the junction gp120/gp41. In the sense orientation, the 3&#39; end of the ASP open reading frame overlaps with gp120 hypervariable regions V4 and V5. The study of ASP RNA has been thwarted by a phenomenon known as RT-self-priming, whereby RNA secondary structures have the ability to prime RT in absence of the specific primer, generating non-specific cDNAs. The combined use of high RNA denaturation with biotinylated reverse primers in the RT reaction, together with affinity purification of the cDNA onto streptavidin-coated magnetic beads, has allowed us to selectively amplify ASP RNA in CD4+ T cells derived from individuals infected with HIV-1. Our method is relatively low-cost, simple to perform, highly reliable, and easily reproducible. In this respect, it represents a powerful tool for the study of antisense transcription not only in HIV-1 but also in other biological systems.

Detection of Human Immunodeficiency Virus Type 1 HIV-1 Antisense Protein ASP RNA Transcripts in Patients by Strand-Specific RT-PCR

RNA hairpins and loops can function as primers for reverse transcription (RT) in absence of sequence-specific primers, interfering with the study of overlapping antisense transcripts. We have developed a technique able to identify strand-specific RNA, and we have used it to study HIV-1 antisense protein ASP.

通过斯特兰德特异性RT-PCR检测患者人类免疫缺陷病毒类型1（HIV-1）反义蛋白（ASP）RNA转录本

In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

一个<em&gt;体外</em&gt;模型在HIV合并感染的背景下测量免疫应答疟疾

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

收集，分离，和人类宫颈样品的流式细胞仪分析

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

医学

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

从HIV-1阳性者的血浆分离外来体的

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing bronchoalveolar lavage (BAL) fluid. The BAL fluid is rich in cells and can thus provide a 'snapshot' of the pulmonary immune milieu. CD4 T cells are the best characterized HIV reservoirs, while there is strong evidence to suggest that tissue macrophages, including alveolar macrophages (AMs), also serve as viral reservoirs. However, much is still unknown about the role of AMs in the context of HIV reservoir establishment and maintenance. Therefore, developing a protocol for processing BAL fluid to obtain cells that may be used in virological and immunological assays to characterize and evaluate the cell populations and subsets within the lung is relevant for understanding the role of the lungs as HIV reservoirs. Herein, we describe such a protocol, employing standard techniques such as simple centrifugation and flow cytometry. The CD4 T cells and AMs may then be used for subsequent applications, including immunophenotyping and HIV DNA and RNA quantification.

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

We describe a method for processing bronchoalveolar lavage fluid and matched peripheral blood from chronically HIV-infected individuals on antiretroviral therapy to assess pulmonary HIV reservoirs. These methods result in the acquisition of highly pure CD4 T cells and alveolar macrophages that may subsequently be used for immunophenotyping and HIV DNA/RNA quantifications by ultrasensitive polymerase chain reaction.

用于阿尔韦拉尔巨噬细胞和CD4和T细胞免疫造血和HIV储液评估的支气管性洗浴液和匹配血液的处理

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing ...

Isolation, Transfection, and Culture of Primary Human Monocytes

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4+ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14+ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.

Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.

原人类单核细胞的隔离、转染和文化

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. ...

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.