PubMed · 9528015
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A Persidis. 1998. Functional antigenics.. https://doi.org/10.1038/nbt0398-305
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A highly sensitive method for detection of antigens on the surface of a single blood cell using thermal lens microscopy is described. Colloidal gold, coated with antibody, was used to stain membrane antigens of leukocytes. Human leukocyte antigens on the lymphocytes and mononuclear leukocytes were observed by new thermal lens microscopy, which involves spectrometry using a laser-induced thermal-lens effect. Antigens of HLA-A, -B, and -C loci on the lymphocytes were identified and quantitated using a single cell. The image of HLA-A, -B, and -C antigen distribution on a mononuclear leukocyte was obtained. Our laser microscope, newly devised for measuring convex surface cells, is a powerful analytical tool for detecting and quantitating localized antigens in a single cell and/or cell-surface-associated molecules.
We have developed a new method that allows detection and isolation of viable, antigen-specific, human T cells from a heterogeneous pool of T cells. We have engineered a self-inactivating retroviral vector containing multiple (3 or 6) nuclear factor of activated T-cell (NFAT)-binding sites, followed by the minimal IL2 promoter and the reporter gene GFP. Jurkat cells, primary T-cell blasts, and T-cell clones were transduced with high efficiency (20%-40%). Stimulation of the transduced cells with phorbol myristate acetate (PMA) and ionomycin resulted in a high level expression of GFP that was maximal after 12 to 14 hours and remained stable for another 12 hours. Activation of T cells carrying the construct containing 6 NFAT-binding sites resulted in the highest mean fluorescence intensity. Cyclosporin-A and FK506 were able to block the activation-dependent GFP expression. Activation of transduced T-cell blasts with the superantigen staphylococcal enterotoxin B or of transduced antigen-specific T-cell clones with cognate antigen resulted in GFP expression. After an overnight stimulation of a heterogeneous T-cell bulk culture with an HLA mismatched stimulator cell (JY), the GFP expressing cells were cloned. As expected, the cloning frequency of the antigen-specific GFP(+) cells was considerably higher than that of the total T-cell population. Most of the T-cell clones were either cytolytic, or proliferative toward JY stimulator cells. Interestingly, we also isolated T-cell clones that were noncytolytic and nonproliferative toward JY cells, but specifically up-regulated GFP after an overnight stimulation with JY. (Blood. 2000;96:459-466)
The c-myc gene is frequently deregulated and overexpressed in human cancers, and strategies designed to inhibit c-myc expression in cancer cells may have considerable therapeutic value. The purpose of the present work was to characterize the antigene and antiproliferative activity of a triple helix-forming oligonucleotide (TFO) targeted to a homopurine-homopyrimidine sequence in the P2 promoter of the c-myc gene. The TFO was synthesized with phosphorothioate (PS) internucleotide linkages to confer resistance to intra- and extracellular nucleases. This property is required of oligonucleotides designed for in vivo testing and therapeutic applications. The PS-TFO was found to form triplex DNA with affinity and specificity comparable with that of the corresponding phosphodiester TFO, as shown by gel mobility shift and footprinting assays. Fluorescence microscopy and polyacrylamide gel analysis showed that the fluorescein-labeled PS-TFO accumulated in nuclei of CEM leukemia cells and remained intact for at least 72 h. Incubation of CEM cells with PS-TFO reduced c-myc RNA and protein levels. A single exposure of leukemia cells to the PS-TFO was sufficient to induce dose-dependent growth inhibitory effects. Growth inhibition correlated with accumulation of cells in S phase and with induction of cell death by apoptosis. The PS-TFO was also effective in other leukemia and lymphoma cell lines. Control oligonucleotides had minimal effects in all assays. These data indicate that the c-myc-targeted PS-TFO is an effective antigene and antiproliferative agent, with potential for testing in vivo as a novel approach to cancer therapy.