Facile preparation of 6-chloro-9-amino-2-hydroxyacridine, a urinary metabolite of quinacrine and quinacrine mustard.

6-Chloro-9-amino-2-hydroxyacridine was found to be a metabolite of both quinacrine and the antimalarial alkylating agent quinacrine mustard. Its structure was confirmed by a one-step reaction of quinacrine with 48 percent hydrobromic acid. The presence of this compound as a metabolite of quinacrine mustard suggests a possible in vivo activation mechanism for its antitumor activity and a pharmacological basis for its toxicity to the liver. In vitro experiments showed that this new compound does react with chromosomes and, therefore, can be both a useful chromosome stain and an intercalating agent.

5'-nucleotide phosphodiesterase activity of floxuridine-resistant mouse glioma.

In tissue culture experiments, cells derived from glioma 26, a transplantable tumor of C57B1/6 mice, were sensitive to both floxuridine (5-fluorodeoxyuridine) and 5-fluorodeoxyuridine-5'-(5-iodo-3-indolyl)phosphate, an enzyme-mediated drug activated by 5'-nucleotide phosphodiesterase. When these compounds were tested on the tumor in animals at a level of 5 mg/kg for 5 days, tumor growth was inhibited approximately 20% by both compounds. When higher levels of 5-fluorodeoxyuridine, 100 mg/kg four times weekly throughout the lifespan of the mouse, were given, the tumor, although inhibited at first, developed resistance and continued to grow until it killed the animal. Phosphodiesterase levels in the tumor rose as the tumor grew. On the other hand, thymidine kinase levels dropped as anticipated from the known 5-fluorodeoxyuridine-resistant hepatoma tissue culture data. This enzyme pattern was maintained in transplantable mouse glioma lines established from the resistant tumors. One of these lines, tested at a level of 5 mg/kg for 5 days, showed no response to 5-fluorodeoxyuridine but was still sensitive to 5-fluorodeoxyuridine-5'-(5-iodo-3-indolyl) phosphate. These experiments, therefore, offer a model system and a rationale for the design and study of more compounds that could be activated by the enzyme phosphodiesterase. Such compounds might be used alternatively when resistance to 5-fluorodeoxyuridine develops, a common clinical experience in the use of this anticancer drug.

Fluorescent neoglycoproteins: antibody-aminodextran-phycobiliprotein conjugates.

New, highly amino-substituted dextran or aminodextran (hereafter denoted Amdex) of various sizes between about 20 and 1000 kDa molecular mass and degrees of amino-substitution between 7 and 40% were prepared and characterized by elemental analyses and polyacrylamide gel electrophoresis. These aminodextrans together with others commercially available were shown by static light scattering, viscosity, and refractive index measurements to adopt a globular structure in aqueous salt solutions. Antibody and fluorescent protein dye, phycoerythrin, or its tandems with cyanin 5. 1 and TEXAS RED, were covalently conjugated to the aminodextrans. The conjugates contained multiple dye molecules and were shown by dynamic light scattering and scanning electron microscopy to assume either globular structure or aggregates thereof. Streptavidin could be substituted for antibody to prepare streptavidin-aminodextran-PE conjugates, which were then used with biotinylated antibody to label subpopulations of white blood cells. The conjugates yielded up to 20-fold amplification of fluorescence intensity over direct antibody-dye conjugates in labeling white blood cells for flow cytometry.

Direct analysis of viral-specific CD8+ T cells with soluble HLA-A2/Tax11-19 tetramer complexes in patients with human T cell lymphotropic virus-associated myelopathy.

Human T cell lymphotropic virus-I (HTLV-I)-associated myelopathy is a slowly progressive neurologic disease characterized by inflammatory infiltrates in the central nervous system accompanied by clonal expansion of HTLV-I-reactive CD8+ T-cells. In patients carrying the HLA-A2 allele, the immune response is primarily directed to the Tax11-19 peptide. The frequency, activation state, and TCR usage of HLA-A2/Tax11-19 binding T cells in patients with HTLV-I-associated myelopathy was determined using MHC class I tetramers loaded with the Tax11-19 peptide. Circulating Tax11-19-reactive T cells were found at very high frequencies, approaching 1:10 circulating CD8+ T cells. T cells binding HLA-A2/Tax11-19 consisted of heterogeneous populations expressing different chemokine receptors and the IL-2R beta-chain but not the IL-2R alpha-chain. Additionally, Tax11-19-reactive CD8+ T cells used one predominant TCR Vbeta-chain for the recognition of the HLA-A2/Tax11-19 complex. These data provide direct evidence for high frequencies of circulating Tax11-19-reactive CD8+ T cells in patients with HTLV-I-associated myelopathy.