Zhang L, Lewin S, He Y, Lin HH, Jin X, Tuttleton S, Skulsky E, Karanicolas R, Spiegel H, Borkowsky W, Kostrikis L, Ho DD; Conference on Retroviruses and Opportunistic Infections.
Program Abstr 6th Conf Retrovir Oppor Infect Conf Retrovir Oppor Infect 6th 1999 Chic Ill. 1999 Jan 31-Feb 4; 6th: 205 (abstract no. LB1b).
Aaron Diamond AIDS Research Center, Rockefeller University.
A majority of thymocytes destined to be alphabeta T cells will first delete the delta-locus from the alpha-locus of TCR by a specific gene rearrangement, joining deltaRec to psiJalpha to produce an 89Kb TREC. This excisional DNA product, termed alpha1 circle, serves as a marker for RTE since such circles are rapidly diluted as thymocytes divide. We thus designed a real-time PCR assay to quantify alpha1 circles, utilizing a molecular beacon that fluoresces upon hybridization to the amplified target. This assay is sensitive (5-10 copies), specific (negative in immortalized T cell lines, non-lymphoid tissues, & PBMC from kids with DiGeorge or SCID), accurate (cv of approximately 10% down to approximately 10 copies), dynamic (7 logs), and easy to perform (80 tests in <4 hours). When PBMC are sorted into subpopulations, alpha1 circles are found largely in CD4 and CD8 naive T cells, although some (55-fold less) could be detected in memory T cells. None was found in B cells or monocytes. Thymic tissue, as expected, contains high circle numbers (approximately 20,000/10(6) cells). To assess the number of RTE in blood as a function of age, we applied this assay to >400 normal persons age 0 to 95. In the first 15 years of life, there is a relative stability of alpha1 circles in PBMC (median of approximately 25,000 with 90% interval of 8,000-100,000/10(6) cells). Between ages 15 and 20, there is a sharp decrease of >1 log. After age 21, such circles remain relatively stable (with perhaps a slight decrease) at a median level of approximately 700 circles/10(6) cells (90% interval of 100-3,000 circles/10(6) cells). With any age category, there is a considerable normal spread of approximately 1 log. Important implications of our new findings for human lymphocyte biology will be discussed, along with the application of this technique to study HIV pathogenesis and treatment (see accompanying abstract).
Publication Types:
Keywords:
- Acquired Immunodeficiency Syndrome
- Biological Assay
- Cell Movement
- DNA Primers
- HIV Infections
- HIV Seropositivity
- Humans
- Lymphoid Tissue
- Polymerase Chain Reaction
- Receptors, Antigen, T-Cell
- T-Lymphocytes
- Thymus Gland
- analysis
- blood
Other ID:
UI: 102195488
From Meeting Abstracts