Since HIV 1 Tat induces neuronal death via production of TNF a, our results show that the secreted sTNFR Fc protein was able to significantly reduce HIV 1 Tat mediated neurotoxicity by neutralizing TNF a. Next, the possibility of blocking the neurotoxicity mediated by HIV 1 gp120, or the synergistic neurotoxi city by new gp120 and Tat, was evaluated. As shown in Figure 8D, when HTB 11 cells were treated with 100 ng mL gp120 for three days, cell viability in cultures Inhibitors,Modulators,Libraries co treated with conditioned media from the sTNFR Fc vec tor transduced cells was significantly higher than that in cultures co treated with conditioned media from the Fc vector transduced Inhibitors,Modulators,Libraries cells, or in cultures co treated with conditioned media from the non transduced cells.

The secreted sTNFR Fc protein also pro tected HTB 11 target cells from the combined treatment of HIV 1 gp120 and Tat. In cultures treated with 100 ng mL gp120 plus 250 ng mL Tat, cell viability was dra matically increased in cultures that were co treated Inhibitors,Modulators,Libraries with conditioned media from sTNFR Fc vector transduced cells, compared to cultures co treated with conditioned media from the Fc vector transduced or non transduced cells. These results were confirmed when a higher concentration of gp120 was used in similar experiments. Discussion This study employed a lentiviral vector to deliver and express a soluble TNF receptor decoy in microglial and neuronal cells, as a means of protecting cells from TNF mediated cytotoxicity. The results indicated that both microglial and neuronal cells could be efficiently trans duced with Inhibitors,Modulators,Libraries the sTNFR Fc expressing vectors, and the transduced cells showed long term and stable expression and secretion of sTNFR Fc.

With respect to the expres sion levels of sTNFR Fc in these Inhibitors,Modulators,Libraries two cell types, quanti tative analysis of vector mediated gene transfer revealed that transduction of the neuroblastoma cells was considerably more efficient than that of the microglial cells. Although MG132 FDA the transduction efficiency of the micro glial cells was increased to 100% after a second round of transduction, the level of sTNFR Fc secretion from the transduced cells was still much less than that of the transduced neuronal cells. A potential explanation for this difference in protein expression levels is that HTB 11 cells may have a higher integrated vector copy num ber of the vector than CHME 5 cells. This is consistent with previous observations that neural cells are more readily transduced by HIV 1 based vectors than cells of myeloid lineage such as macrophages. Alternatively, it is possible that there may be an intrinsic difference in the ability of the two cell types to produce and secrete sTNFR Fc.