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: 7622827
• 负责人: Yasuhiro Yamamura
• 金额: $ 17.26万
• 依托单位: UNIVERSITY OF PUERTO RICO MED SCIENCES
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7622827
• 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.