However, to establish the initial feasibility of in vivo siRNA administration, mice overexpressing green fluorescent protein (GFP) were chosen to receive a single intratracheal instillation of a GFPsilencing RNA. What we found was that green fluorescence in the lungs of these animals at 18 h post-instillation was reduced by at least 65% when compared with vehicle-treated GFP mice, while no decrease in fluorescence was seen in other organs outside the lungs (152, 164) . The use of siRNA at these concentrations neither induced substantial activation of type I interferons (165, 166) via activation of TLR-3 (167), TLR-7 (168), TLR-8, TLR-9, and protein kinase-R(PKR) pathways (169) (170) (171) nor via more classic proinflammatory processes (169, 171) . This is in line with reports demonstrating that intravenous delivery of naked siRNA did not induce an interferon response (170) . However, the induction of STAT (signal transducers and activator of transcription)-1 in this context appears to be dose dependent (165) , and thus it cannot be ruled out that different dosages, delivery, or timing of administration might have provided dissimilar results. To gain some insight into the cellular localization of siRNA in the lung, we followed the intrapulmonary deposition of Cy-5 fluorochrome labeled siRNA uptake by confocal immunofluoresence microscopy. Interestingly, labeled siRNA was found to co-localize only with lung epithelial cells counterstained with anti-cytokeratin-18 at 24 h post instillation, but not alveolar macrophages (152) . However, negative results for alveolar macrophages may not preclude the uptake of siRNA by this cell type as one can imagine different kinetics in the various cell types with respect to siRNA uptake and processing. Relative to this, the feasibility of gene silencing in macrophages using siRNA has been described in vitro (172) (173) (174) , however, silencing of typically macrophagederived molecules such as TNF-α and IL-6 during indirect murine lung injury remained unsuccessful in our hands (unpublished observations). Whether this might be attributable to the underlying cell type or perhaps to the degree of upregulation of a certain gene during pathological conditions remains to be elucidated. Irrespective these and the experiments below represent the first studies to demonstrate the feasibility of in vivo lung siRNA as treatment for developing ALI.