[Show abstract][Hide abstract] ABSTRACT: Control of the rearrangement and expression of the T cell receptor alpha and delta chains is critical for determining T cell type. The process of delta deletion is a candidate mechanism for maintaining separation of the alpha and delta loci. Mice harboring a transgenic reporter delta deletion construct show alpha/beta T cell lineage-specific use of the transgenic elements. A 48-basepair segment of DNA, termed HPS1A, when deleted from this reporter construct, loses tight lineage-specific rearrangement control of transgenic elements, with abundant rearrangements of transgenic delta-deleting elements now in gamma/delta T cells. Furthermore, HPS1A augments recombination frequency of extrachromosomal substrates in an in vitro recombination assay. DNA binding proteins recognizing HPS1A have been identified and are restricted to early B and T cells, during the time of active rearrangement of endogenous TCR and immunoglobulin loci. These data are consistent with delta deletion playing an important role in maintaining separate TCR alpha and delta loci.
Journal of Experimental Medicine 08/1997; 186(1):91-100. · 13.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytokines are small protein hormones produced during an immune response that are responsible for mediation and regulation of many aspects of immunity. Measurement of cytokines by several different methods has led to a broader understanding of the immune response. This paper describes a sensitive, reproducible, and quantitative RT-PCR assay for the simultaneous measurement of multiple cytokines. The main features of the methodology are: RNA competitors which control for all aspects of the process from RNA extraction, through reverse transcription (RT) and PCR amplification; a general cloning vector, pQPCR1, for building RNA competitors that does not require prior analyte cDNA cloning; and analysis by plate based EIA. This RT-PCR-EIA system is shown to be more sensitive than agarose gel electrophoresis followed by EtBr staining, measuring PCR product in the sub-nanogram range. It also extends the linear dynamic range of detection to a four log fold range of analyte concentration. The assay is reproducible, with coefficients of variation (CVs) in the 10-20% range. Moreover, the cloning vector is designed to accommodate multiple primer templates, thus allowing simultaneous quantitation of many different analytes from a single RT reaction. The described system is versatile and adapts to numerous analytes.
Journal of Immunological Methods 01/1996; 187(2):273-85. · 2.01 Impact Factor