To investigate the association between cytokine and adhesion molecule expression and the development of inflammatory cell infiltrates, we have begun in vivo leukocyte trafficking studies. We have labeled cells with the fluorescent cell linker PKH26-GL. This aliphatic fluorescent chromophore localizes within the lipid bilayer of the cell membrane retaining both biologic and proliferative activity and remains stable in vivo for > than 100 days. In preliminary studies we have infused PKH26-labeled autologous PBMCs into endotoxin-treated macaques. These animals were killed at 48 and 72 hours p.i. and tissues collected to examine the distribution of labeled cells. By fluorescence microscopy the labeled cells were localized to lymphoid tissue, lung and brain collected at necropsy. In the lung and brain the PKH26-labeled perivascular cells were associated with diffuse expression of VCAM-1 as demonstrated by combined fluorescence microscopy for PKH26-labeled cells and immunohistochemistry for VCAM-1. To monitor longer term survival of PKH26-labeled PBMCs in SIV-infected monkeys, two animals were infused with 4 x 107 autologous PKH26-labeled PBMCs two times each (seven days between infusions). One animal was killed and examined at 30 days postinfusion and the other had an axillary lymph node biopsy at 90 days postinfusion. Abundant PKH26-labeled cells were readily detected by fluorescence microscopy within peripheral lymph nodes and other organs at 30 days postinfusion. By 90 days postinfusion the numbers of PKH26-labeled cells found in the axillary lymph node were less than the numbers in peripheral nodes at 30 days postinfusion, however, they remained readily detectable. Collectively, these results demonstrate the viability of PKH26-labeled cells over long periods of time in vivo.
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