Anti-EXOG monoclonal antibody, clone 3G8

Tetrahydrobiopterin is an essential cofactor in the biosynthesis of nitric oxide, but in the presence of O-2, autooxidation of tetrahydrobiopterin leads to the production of superoxide anions and the subsequent chemical inactivation of nitric oxide. As a result, the present experiments were designed to determine the effect of exog enous tetrahydrobiopterin on isolated canine basilar arteries. Rings with and without endothelium were suspended for isometric force recording in modified Krebs-Ringer bicarbonate solution bubbled with 94% O-2-6% CO2 (37 degrees C; pH 7.4). A radioimmunoassay technique was used to measure guanosine 3',5'cyclic monophosphate production (cGMP). Tetrahydrobiopterin (10(-7) to 10(-4) M), but not dihydrobiopterin or biopterin, caused endothelium-dependent contractions. Superoxide dismutase (150 U/ml) abolished tetrahydrobiopterin-induced contractions, but a H2O2 scavenger, catalase (1,200 U/ml), and hydroxyl radical scavengers deferoxamine (10(-4) M) and dimethylsulfoxide (10(-4) to 10(-3) M) did not significantly affect the contractions. A cyclooxygenase inhibitor indomethacin (10(-5) M), significantly reduced the contractile effect of tetrahydrobiopterin. With the prostaglandin H-2/thromboxane A(2) receptor antagonist, SQ-29548 (10(-6) M) present, contractions reversed to relaxations. In rings with endothelium, tetrahydrobiopterin (10(-4) M) significantly decreased the levels of cGMP. These studies suggest that autooxidation of exogenous tetrahydrobiopterin induces endothelium-dependent contractions by 1) chemical inactivation of nitric oxide by superoxide anions and 2) activation of arachidonic acid metabolism via the cyclooxygenase pathway with subsequent release of endoperoxide and/or thromboxane A(2) from endothelial cells.

Exogenous, nonreplicating protein antigens (Ags) are usually taken up by antigen-presenting cells (APCs) via endocytosis or pinocytosis and enter the major histocompatibility complex (MHC) class II processing and presentation pathway. Although exogenous Ags are not processed and presented in the class I pathway by most cells, soluble proteins can enter the class I processing and presentation pathway if they are introduced directly into the cytoplasm of APCs. The purpose of these studies was to determine whether exog enous proteins could be processed and presented to T cells if they were delivered into cells by electroporation. The conditions for electroporation were optimized so that the viability of the electroporated cells was high, and the majority of electroporated cells had protein incorporated. Electroporated B cells not only presented exogenous ovalbumin to CD8(+), class I MHC-restricted T cells but also stimulated CD4(+), class II MHC-restricted T cells. Electroporated cells also primed Ag-specific cytotoxic T lymphocytes (CTLs) in vivo, stimulated CTL precursors in vitro, and served as target cells for lysis by Ag-specific CTLs, indistinguishable from transfected cells. Thus, electropermeabilized cells were structurally intact, and the introduced exogenous protein was processed and presented in association with both class I and class II MHC molecules. This approach is as efficient and reproducible as other techniques of delivering exogenous proteins into the intracellular processing pathways. These studies suggest that electroporation could be employed for the study of cell-mediated immunity to various exogenous proteins.