The release of soluble neurotoxins and growth promoting factors by activated macrophages/microglia has been reported following chronic HIV infection and various types of brain injury. A neuronal cultured cell bioassay using neonatal rat has been refined for quantitative measurements. Glutamate response has been used as a model NMDA agonist and MK801 as a receptor antagonist. The isolation and characterization of soluble factors isolated from conditioned media from activated human peripheral blood monocytes or HIV-infected macrophages is being monitored with this bioassay. Extracts of conditioned media have been separated into component classes (lipids vs. non-lipids, low versus high molecular weight). Neuronal toxicity has been demonstrated in multiple batches of conditioned media. The toxic activity is characteristic of media from HIV-infected and LPS stimulated macrophages, not from uninfected or LPS-stimulated controls. Activity is highest in a lipid-free fraction containing substances less than 3000 Da. The neurotoxicity is trypsin insensitive. Mass spectrometry is being used to characterize the components in neurotoxic fractions. In a separate project, the CNS fraction of the excitoxic amino acid quinolinate that derives from kynurenine metabolism is being determined by pharmacokinetic studies. Stable isotope labeled L-kynurenine has been prepared and infused into gerbils. The enrichment of peripheral vs. central circulating kynurenine is being measured in control and immune stimulated gerbil brains in order to determine the origin of the enhanced quinolinate concentration in animals with CNS inflammation. - macrophage, HIV, microglia, mass spectrometry, kynurenine, stable isotopes
