1. In collaboration with Dr. Mark Hayes, we continued our studies of the regulation of IL-12 p35 in monocytes and showed that transcription of IL-12 p35 occurs from a consensus TATA-box-like promoter contained within the ORF described for lymphoblastoid cells. Recognition of this promoter in monocytes requires the differentiative effects of interferon-g. We molecularly cloned the p35 gene, described the intron/exon structure, identified the promoter regions for both monocytes and lymphoblastoid cells, and confirmed the transcription start sites using 5' RACE and ribonuclease protection assays. Studies evaluating the function of the p35 signal peptide sequence indicate a novel intracellular trafficking signal governing movement of the nascent polypeptide into the endoplasmic reticulum. Deletional analysis confirms the functionality of this site. 2. We have developed a method for RT-PCR analysis of paraffin-embedded, archived pathology samples. In collaboration with Drs. Giovanna Tosato and Julie Teruya-Feldstein (NCI) we investigated the expression of chemokines in disease states and showed that the chemokine eotaxin is associated with tissue eosinophilia observed in Hodgkin~s Disease and that such tissues also express elevated levels of other chemokines, including Mig, IP-10, RANTES, and MIP-1. We also showed that Mig, a monocyte-inducible chemokine, is associated with vascular damage observed in some Epstein-Barr virus-positive lymphoproliferative disease. 3. In collaboration with the laboratories of Drs. Kathleen Clouse and Eda Bloom we continued our examinations of the functions played by nitric oxide in normal and disease states. We showed that HIV-1-infected macrophages induce expression of the unregulated form of NO synthase, iNOS, in bystander astrocytes and presented evidence that this mechanism may account for the sever neuronal damage observed in neural AIDS. We also showed that natural killer cells express a G-protein regulated form NO synthase (eNOS) which functions to protect them from activation-induced cell death through regulation of TNF- expression. 4. We continued our investigations of the stability of AAV during manufacturing and have constructed two prototype AAV vectors (one containing the IL-2 receptor subunit and the other green fluorescent protein) with the intention of examining the biologic stability of these vectors in models of inflammation in vivo. 5. In collaboration with Dr. Carolyn Wilson and Dr. Papia Banerjee (Biotransplant, Charlestown, MA) we continued our studies of porcine endogenous retrovirus through generation and examination of molecular clones of PoEV for biologic function.
