Inactivation of viruses in blood products requires that the method employed display selectivity in its action for viral elements while not affecting the biological entity of interest. Several methods have been developed for the treatment of human plasma or products derived from human plasma. An effective technique for the treatment of the cellular components of blood has been lacking, in part due to the inability to develop agents capable of selectively targeting viral agents in the milieu of cellular material. In this paper, we examine the behavior of a group of viral sensitizers designed to be added to cellular samples and be activated upon exposure to UVA light. Upon activation, these agents are capable of disrupting nucleic acids of the virus in a manner that renders them inactive for proliferation. The selectivity observed in this inactivation is determined by the chemical structure of the sensitizer, which can be varied to increase viral killing capacity while diminishing collateral damage to cellular and protein constituents.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is a potent vasoactive ether lipid produced by activated blood cells and endothelial cells. Vascular smooth muscle cells partially convert exogenous PAF to 1-O-alkyl-2-acetyl-sn-glycerol (AAG), a biologically active diacylglycerol analogue. AAG is formed rapidly (less than 15 s) after exposure of the smooth muscle cells and does not appear to be a substrate for diacylglycerol kinase in these cells. Although most of the compound is metabolized to 1-O-alkyl-sn-glycerol, a small quantity remains as AAG for greater than or equal to 6 h. AAG inhibits phorbol ester binding, and it is as effective an activator of protein kinase C as diolein in an in vitro assay. Furthermore, AAG and PAF produce the same pattern of effects on smooth muscle cell proliferation. These observations suggest that at least some of the actions of PAF in vascular smooth muscle may be mediated through the formation of AAG, a stable, bioactive metabolite that appears to function as a diacylglycerol analogue.

A rapid increase in arachidonic acid incorporation into phosphatidylinositol (PI) occurred following exposure of cultured porcine pulmonary artery smooth muscle cells to calcium ionophore A23187. This response was specific for PI and phosphatidic acid; none of the other phosphoglycerides showed any increase in arachidonic acid incorporation. The incorporation of [3H]inositol also was increased, indicating that complete synthesis of PI rather than only fatty acylation occurred in response to the ionophore. The presence of omega-3 fatty acids, especially eicosapentaenoic acid (EPA), reduced arachidonic acid but not inositol incorporation into PI. Stimulated incorporation of EPA also occurred under these conditions, suggesting that EPA replaces arachidonic acid in the newly synthesized pool of PI. Although much less arachidonic acid was incorporated into the polyphosphoinositides following exposure to the ionophore, arachidonic acid incorporation into these phosphorylated derivatives also decreased when EPA was present. These findings suggest that when omega-3 fatty acids are available, less arachidonic acid is channeled into the inositol phospholipids of activated smooth muscle cells because of replacement by EPA. This may represent a mechanism whereby omega-3 fatty acids, especially EPA, can accumulate in the metabolically active pools of inositol phospholipids and thereby possibly influence the properties or responsiveness of vascular smooth muscle.

Amino or polyamino derivatives of naphthalene (N-H), anthracene (A-H) and 8-alkoxypsoralen (PSR-H) were prepared along with their monobrominated analogs (N-Br, A-Br and PSR-Br). The ammonium salts of these compounds are all water soluble and bind strongly to calf thymus DNA and to lambda phage, a double-helical DNA, protein-coated virus. Binding of the sensitizer to DNA occurs, presumably by a mixture of hydrophobic, intercalative and electrostatic interactions. Relative binding constants to calf thymus DNA and to lambda phage were measured by the ethidium bromide fluorescence quenching assay. In general the brominated analogs bind more tightly to calf thymus DNA and to the virus than to the nonhalogenated analogs. It is demonstrated that the brominated aromatics are much more effective at inactivating lambda phage upon photoactivation (lambda approximately 310 or 350 nm) than are their nonbrominated analogs. At identical sensitizer concentrations (by weight) and light flux N-Br, A-Br, and PSR-Br produce 5-6 more logs of viral inactivation than their nonbrominated counterparts (N-H, A-H and PSR-H, respectively). The bromine effect may originate from light-induced electron transfer and subsequent cleavage of the C-Br bond of the sensitizer radical anion bonds to form aryl radicals. Singlet oxygen cannot be responsible for the viral inactivation because the brominated sensitizers are equally effective in the presence and absence of oxygen. Dithiothreitol does not protect lambda phage from light-induced inactivation by the brominated sensitizer thereby demonstrating that the photogenerated reactive intermediates responsible for the effect are complexed to the virus and are not generated free in solution.

The formation and metabolism of 1-O-alkyl-2-acetyl-sn-glycerol (AAG), a protein kinase C (PKC) activator formed from platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3- phosphocholine; PAF), was studied in HL-60 cells to determine whether differentiation may influence this process. HL-60 cells differentiated to macrophages (HL-60/M phi) with a phorbol ester convert added [3H]PAF to AAG; 22% of the incorporated radioactivity is converted to AAG within 15s. By contrast, neither undifferentiated HL-60 cells (HL-60/U) nor HL-60 cells differentiated to granulocytes (HL-60/GN) with retinoic acid produce AAG from PAF. The HL-60/M phi rapidly convert radiolabeled AAG to 1-O-alkyl-sn-glycerol and, subsequently, to two other unidentified metabolites. However, some apparently unmodified AAG persists in the cell lipids for at least 6 h. The HL-60 subtypes which do not convert PAF to AAG can nevertheless catabolize AAG; HL-60/U and HL-60/GN produce alkylglycerol and the other AAG metabolites. These findings demonstrate that differentiation can alter the processing of PAF in a human leukocyte cell line. Furthermore, they suggest that PAF may produce at least some of its biological effects in macrophages by conversion to AAG.