The benefit of therapeutic granulocyte transfusions in neutropenic individuals remains controversial--most debate centering on whether sufficient numbers of cells can be collected to be useful. Separated granulocytes continuously generate toxic oxygen metabolites when incubated in vitro, even without purposeful stimulation. These metabolites can injure the generating cells themselves as well as other apposing neighboring cells. In this proposal, I plan to validate the notion that by either: (a) inhibiting production of, or (b) scavenging released toxic oxygen species, PMNs of higher functional capability can be prepared for transfusion. For the former, I have recently shown that the iron-containing granulocyte lysosomal constituent, lactoferrin, is critical to granulocyte-mediated target cell damage through its enhancement of hydroxyl radical production; thus, I will use the powerful iron chelator, deferroximine, as well as anti-lactoferrin antibody to inhibit toxic oxygen species production by collected PMNs. For the latter, in addition to traditional O2 radical scavengers such as superoxide dismutase, catalase, thiourea and methionine, I shall particularly investigate the red cell as a more physiologic scavenger. Intact RBCs protect cultured endothelial cells from granulocyte-mediated destruction and protect whole animals from lethal pulmonary injury during extreme hyperoxia exposure; renewable reduced glutathione is the critical red cell scavenger. Functional capability of leukapheresed granulocytes variously manipulated as noted will be assayed in vitro (chemotaxis, lysosomal enzyme release, phagocytosis, adherence to cultured endothelium, metabolic burst) and possibly in vivo (Indium-111 survival and accumulation of skin inflammatory sites). A second and analogous part of these studies will examine whether granulocyte toxic oxygen metabolites injure neighboring platelets during their collection and storage. Several examples of granulocyte/platelet interaction have been examined--all implying that granulocyte products, particularly toxic oxygen species, can generate platelet release phenomena. In this proposal I will attempt to prepare """"""""quieter,"""""""" and thus more functional platelets by inhibiting production of and/or scavenging granulocyte oxygen radicals during collection by the methods outlined above. In vitro (aggregation, granule product release, endothelial culture adhesion, etc.) and in vivo (survival and recovery of variously manipulated platelet products) platelet function will be assessed.
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