PR COMPREHENSIVE CTR FOR HIV DISPARITIES: PSM: LAB CORE

HIV 差异的公关综合点击率：PSM：实验室核心

• 批准号: 7724746
• 负责人: Yasuhiro Yamamura
• 金额: $ 32.03万
• 依托单位: UNIVERSITY OF PUERTO RICO MED SCIENCES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7724746
• 关键词: AIDS/HIV problem; Area; Baltimore; Biohazardous Substance; Biological Assay; Blood; Blood capillaries; Budgets; CD4 Positive T Lymphocytes; Clinical; Clinical Psychology; Color; Computer Retrieval of Information on Scientific Projects Database; DNA; DNA Sequence; DNA Sequence Analysis; Data; Detection; Development; Diagnosis; Doctor of Medicine; Doctor of Philosophy; Educational workshop; Enrollment; Ensure; Enzyme-Linked Immunosorbent Assay; Evaluation; FOLH1 gene; Faculty; Fostering; Funding; Future; Gagging; Gene Expression; Genetic; Genotype; Grant; HIV; HIV diagnosis; HIV drug resistance; Heating; Hepatitis C virus; Housing; Human Papillomavirus; Human Virology; Individual; Institutes; Institution; Laboratories; Maryland; Mental Health; Methods; Monitor; Mutation Analysis; National Institute of Mental Health; Nature; Numbers; Patients; Phase; Plasma; Polymerase Chain Reaction; Preparation; Procedures; Production; Progress Reports; Proteins; Puerto Rico; Reaction; Reporting; Research; Research Infrastructure; Research Personnel; Research Project Grants; Resources; Reverse Transcriptase Polymerase Chain Reaction; Risk; Running; Sampling; Series; Services; Source; Staging; Structure; System; T-Lymphocyte; T-Lymphocyte Subsets; Testing; Time; Training; United States National Institutes of Health; Universities; Update; Validation; Variant; Viral Load result; Western Blotting; base; capillary; chemokine; cost; cytokine; day; immunological status; instrument; programs; prospective; research clinical testing

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Laboratory Core The specific aims of the core laboratory activity remain unchanged except that some additional tests are now available. The laboratory core will provide HIV related testing for clinical evaluation of patients enrolled in one or the other ongoing as well as developing projects under the auspice of the Puerto Rico CCHD-HIV. The following tests are implemented through the core. 1. HIV and HCV ELISA and HIV Western blot analyses. 2. Flow cytometric analysis of CD4 T cell enumeration, intracytoplastic cytokine production by T cell subsets and quantification of extra-cellularly produced cytokines-chemokines. 3. HIV and HCV viral load assessment by Roche Amplicor procedures. 4. HCV Genotyping by the line-probe assay (LIPA). 5. HIV drug resistance mutation analysis (genotyping) by the Bayer (Visible Genetics) TruGene procedure. 6. Flow cytometric analysis of HIV infected T cells. 7. HIV clade (genotype) analysis by DNA sequencing. 8. Human papillomavirus (HPV) genotyping methods have been incorporated into the core service. Progress report (1) The nature of the HIV and HCV diagnosis tests remain the same as reported previously reported: namely, no new diagnosis of HIV or HCV was performed during this period, and all core tests provided were for characterization and staging of HIV infected individuals who had been diagnosed prior to enrollment in the CCHD projects. (2) Updating of HIV and HCV Testing Formats. 2a) During the last project year, Roche HCV test format was switched from the "Amplicor" PCR-ELISA to the "TaqMan" real-time RT-PCR. Amplicor HIV Monitor test format was also switched during the current year period to TaqMan real-time PCR. Both test systems are equipped with Ampli-Prep automated sample preparation workstation, which reduces technical variations as well as the biohazard risk to operators. Updating of technologists on these procedures were also completed during this period 2b)Homebrew procedure for HIV viral load assessment by real-time RT-PCR (HIV gag-probe specific) has been validated. A partial validation data are shown in Figure 1. This validated procedure may be utilized, when a much larger than budgeted amount of tests is required for the projects. It should be considered a viable, less costly alternative to the FDA-approved kit-based procedures that have been offered through the core. 2c)HIV genotyping based on a homebrew DNA sequencing using Visible-Genetics long Run Tower automated DNA sequencers has been validated. This procedure shall significantly reduce the costs of HIV genotyping from the current ca. $250 to $100 or lower. A new, high capacity automated DNA sequencer, ABI 3730 with 48 capillaries was purchased by the PSM for the RCMI and CCHD activities. When fully validated (anticipated in August 2007), it will be possible to complete DNA sequencing in a much shorter time and the costs will also be significantly reduced. For example, HIV genotyping may be provided, if requested, at below $100 for CCHD investigators. 2d)Layout of the DNA/RNA sample preparation room for clinical PCR such as Ampli-Taq and COBAS (Roche) was significantly modified to accommodate the newly implemented the Roche test format change from Amplicor to AmpliTaq. In addition, separate rooms were assigned for template-free room for mastermix preparation, PCR amplification, and DNA sequencing. The last room currently houses 3 VG Long-Run Tower DNA Sequencers and one ABI 3730 48 capillaries DNA Sequencer. Additional cooling units were installed in the DNA sequencing room to accommodate the extra heat generated by the new instrument. Separation of each room for separate procedure minimizes the potential risk of template carry over (contamination) from one room to the other. Laboratory coats for each laboratory are identified by 3 different colors to ensure that no laboratory coat will be moved to another room. Future plans: (1) During the 2007 annual CCHD investigators' retreat in Ponce, certain new developments in expansion of ongoing research projects necessitated the core service of cytokine-chemokine quantification in the blood of patients undergoing evaluation. Assessments of intra-cellular and extra-cellular cytokines/chemokines (including those in the patients' blood/plasma) were requested by investigators for better assessments of individuals' mental health and immunological status. Quantification of cytokines (chemokines) or their gene expression by protein- or genetic-arrays will be standardized during this period to assist further development of such research projects through the CCHD initiative. (2)In order to promote the development of research projects in AIDS/HIV associated mental health areas, a workshop has been organized to facilitate the research infrastructure/project development using the existing Clinical Psychology program of the PSM. The workshop will be held on June 19-20, 2007, at the PSM with the following outside panelists: C. Zorrilla, M.D. (UPR, CCHD PI), S. Loue, Ph.D., J.D. (Case Western Reserve University, Cleveland, OH), L. Temoshok, Ph.D.(Institute of Human Virology, U-Maryland at Baltimore, MD), and D. Stoff, Ph.D. (NIMH). The workshop will, in addition to fostering the development of research projects by junior investigators, develop the mental health core infrastructure which may further contribute to the CCHD activities. The 2 day workshop may help identify potential research projects that may be included in the next phase CCHD structure. (3)It is planned that during the neat program year period, a series of meetings will be organized with the current and prospective CCHD faculties for identifying the emerging needs for the Laboratory Core function. As described above, as projects progress, different technical needs will be identified. Since a new competitive renewal proposal of the CCHD program will begin to be organized during the next year, and a number of projects or project areas that will be included in the next phase program will be identified, the infrastructure of the Laboratory Core also requires adjustments. New technical developments and training of technologists to accommodate the needs will be implemented shortly thereafter. For example, as described above (1), a microarray detection system using chimoluminescence reaction has already been installed in the laboratory. Pilot testings of cytokine-chemokine microarrays are being standardized. The tests may be available shortly after the summer of 2007.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 Laboratory Core The specific aims of the core laboratory activity remain unchanged except that some additional tests are now available. 该实验室核心将为参与波多黎各 CCHD-HIV 资助的一项或另一项正在进行的以及正在开发的项目的患者提供 HIV 相关检测，以进行临床评估。 The following tests are implemented through the core. 1. HIV and HCV ELISA and HIV Western blot analyses. 2. CD4 T 细胞计数、T 细胞亚群产生的胞质内细胞因子以及细胞外产生的细胞因子-趋化因子的定量的流式细胞术分析。 3. HIV and HCV viral load assessment by Roche Amplicor procedures. 4. HCV Genotyping by the line-probe assay (LIPA). 5.采用Bayer (Visible Genetics) TruGene 程序进行HIV 耐药性突变分析（基因分型）。 6. HIV感染的T细胞的流式细胞术分析。 7. 通过 DNA 测序进行 HIV 进化枝（基因型）分析。 8. Human papillomavirus (HPV) genotyping methods have been incorporated into the core service. 进度报告 (1) HIV和HCV诊断测试的性质与之前报告的相同：即在此期间没有进行新的HIV或HCV诊断，提供的所有核心测试都是针对在加入CCHD项目之前已诊断的HIV感染者的特征和分期。 (2) 更新HIV和HCV检测格式。 2a) During the last project year, Roche HCV test format was switched from the "Amplicor" PCR-ELISA to the "TaqMan" real-time RT-PCR.今年期间，Amplicor HIV Monitor 测试格式也转换为 TaqMan 实时 PCR。 两种测试系统均配备 Ampli-Prep 自动化样品制备工作站，可减少技术变化以及操作员的生物危害风险。 Updating of technologists on these procedures were also completed during this period 2b)Homebrew procedure for HIV viral load assessment by real-time RT-PCR (HIV gag-probe specific) has been validated.图 1 显示了部分验证数据。当项目需要的测试量远大于预算数量时，可以使用此验证程序。 It should be considered a viable, less costly alternative to the FDA-approved kit-based procedures that have been offered through the core. 2c) 基于使用 Visible-Genetics 长运行塔自动 DNA 测序仪的自制 DNA 测序的 HIV 基因分型已得到验证。 This procedure shall significantly reduce the costs of HIV genotyping from the current ca. $250 to $100 or lower. A new, high capacity automated DNA sequencer, ABI 3730 with 48 capillaries was purchased by the PSM for the RCMI and CCHD activities.当完全验证后（预计于 2007 年 8 月），将有可能在更短的时间内完成 DNA 测序，并且成本也将显着降低。 例如，如果 CCHD 调查人员提出要求，可以以低于 100 美元的价格提供 HIV 基因分型服务。 2d) 用于临床 PCR 的 DNA/RNA 样品制备室（例如 Ampli-Taq 和 COBAS (Roche)）的布局进行了重大修改，以适应新实施的 Roche 测试格式从 Amplicor 到 AmpliTaq 的更改。 此外，还为无模板室分配了单独的房间，用于 Mastermix 制备、PCR 扩增和 DNA 测序。 最后一个房间目前装有 3 台 VG 长运行塔式 DNA 测序仪和一台 ABI 3730 48 毛细管 DNA 测序仪。 DNA 测序室安装了额外的冷却装置，以容纳新仪器产生的额外热量。 将每个房间分开进行单独的程序可以最大限度地减少模板从一个房间转移到另一个房间的潜在风险。每个实验室的实验室外套都用 3 种不同的颜色进行标识，以确保不会将实验室外套移至另一个房间。 未来计划： (1) 2007 年年度 CCHD 研究人员在庞塞务虚会期间，正在进行的研究项目扩展中的某些新进展需要对接受评估的患者血液中细胞因子-趋化因子定量的核心服务。研究人员要求对细胞内和细胞外细胞因子/趋化因子（包括患者血液/血浆中的细胞因子/趋化因子）进行评估，以便更好地评估个体的心理健康和免疫状态。 在此期间，通过蛋白质或基因阵列对细胞因子（趋化因子）或其基因表达进行的定量将被标准化，以协助通过 CCHD 计划进一步开发此类研究项目。 (2)为了促进艾滋病/艾滋病毒相关心理健康领域研究项目的发展，组织了一次研讨会，以利用PSM现有的临床心理学计划促进研究基础设施/项目的开发。该研讨会将于 2007 年 6 月 19 日至 20 日在 PSM 举行，由以下外部小组成员参加：C. Zorrilla, M.D.（UPR、CCHD PI）、S. Loue, Ph.D.、J.D.（凯斯西储大学，克利夫兰，俄亥俄州）、L. Temoshok, Ph.D.（人类病毒学研究所，马里兰大学巴尔的摩，马里兰州）和 D.斯托夫博士(NIMH). 除了促进初级研究人员开发研究项目外，研讨会还将开发心理健康核心基础设施，这可能会进一步促进 CCHD 的活动。为期 2 天的研讨会可能有助于确定下一阶段 CCHD 结构中可能包含的潜在研究项目。 (3)计划在整洁计划年期间，将与现有和未来的 CCHD 院系组织一系列会议，以确定实验室核心功能的新需求。如上所述，随着项目的进展，将会确定不同的技术需求。由于CCHD计划将于明年开始组织新的竞争性更新提案，并且将确定纳入下一阶段计划的一些项目或项目领域，因此核心实验室的基础设施也需要调整。此后不久将实施新技术开发和技术人员培训以满足需求。例如，如上所述(1)，实验室中已经安装了利用化学发光反应的微阵列检测系统。 细胞因子趋化因子微阵列的试点测试正在标准化。 这些测试可能会在 2007 年夏天之后不久推出。