| Research Abstract |
Although pathogenesis of HTLV-I associated myelopathy has been extensively studied, the mechanism of cell damage constructing central nervous system is still unclear. On the other hand, primary culture procedures of neuron and glial cells are now established and available for a cellular model for viral infections.
In this study, we tried to investigate the mechanisms of in-vitro neuronal and glial cell death as a result of HTLV-I infection.
First of all, we investigated the effect of HTLV-I infected cells such as MT-1, MT-2, HCT-1, W7TM-I and lymphocytes derived from HAM patients against the primary cerebellar granular cells of rats. 1. Supernatants of MT-1 and MT-2 damaged the cerebellar granular cells in dose-dependent manner. The evaluation was performed by MTT assay and fluorescin diacetate assay. Anti-P40tax (tax protein) antibody failed to neutralized this cell damage, suggesting p40tax may affecting expression of cellular factors such as inflammatory cytokines.
Tax gene which is integrated into expression vector was transfected into primary astrocytes, microglia and cerebellar granular cells, respectively. Only few cells can be transfected by lipofectoamine system in each experiment, indicating this method is not available for primary cells.
So, we proceeded to use cell lines to try to determine whether tax-transfected cells can produce cytotoxic factors. Macrophage cell line, U937 supposed to be a model of microglial cells were transfected with tax gene. We disclosed that tax-transfected U937 were over-expressing inducible nitric oxide synthase (iNOS) by semi-quantitative PCR.Direct evidence for over-production of NO was measured by detecting using chemiluminescence and adding Vanadium (III)(FES-450, Scholar-Tee Co., Ltd., Japan). These exaggerated NO production were enhanced by gamma-IFN.This result indicating that microglia/macrophage might damage neuronal cells by exaggerated NO production.
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