The development of neutrophil transfusion therapy as a supportive care measure for acute, severe neutropenia has been limited by the problems of collecting enough cells and maintaining their viability until they can be transfused to a needy recipient. The goal of this proposal is to overcome these obstacles by using the hematopoietic growth factor, granulocyte colony- stimulating factor, to permit collection of large numbers of neutrophils from normal donors and the use of one or several growth factors and pro- inflammatory cytokines to prolong the in vitro survival of the collected cells.
Aim 1 studies the effects of various growth factors and pro- inflammatory cytokines, singly and in combination, and will study the effects of these agents to prolong in vitro survival of neutrophils from normal subjects, and neutrophils from subjects treated with G-CSF. The principal methods for assessing neutrophil function will be luminol and lucigenin-enhanced chemiluminescence and bactericidal activity against staphylococcus areus; other methods will supplement these tests. Cell viability will be assessed chiefly by trypan dye exclusion and DNA fragmentation. FACS analysis of cell surface expression of integrins and selectins currently deemed necessary for neutrophil circulation and migration will also be investigated.
Under Aim 2, the investigators will study the optimal conditions for the collection of neutrophils from normal subjects stimulated with G-CSF, evaluating the separation force, depth of collection into the interface of the leukocytes and erythrocytes during centrifugation, the rate of product collection, and the use of pentastarch as a sedimentation agent.
Under Aim 3, the investigators will study the in vivo kinetics of neutrophils collected under the most favorable conditions, utilizing tritiated diisopropylfluorophosphate labeling to measure blood kinetics and the accumulations of neutrophils in a skin chamber and oral secretions. The in vivo kinetics of cells after periods of storage for 24 to 72 hours also will be studied. In the latter part of the research period, the investigators plan patient studies, labeling neutrophils optimally collected from normal donors, and carefully measuring the in vivo capacity of fresh and stored cells to circulate and to migrate to sites of inflammation.
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