The hepatic parenchyma-specific contrast by Sonazoid is due to distribution of the microbubbles in Kupffer cells.

Laninamivir octanoate (LO) is an octanoyl ester prodrug of the neuraminidase inhibitor laninamivir. After inhaled administration, LO exhibits clinical efficacy for both treatment and prophylaxis of influenza virus infection, resulting from hydrolytic bioactivation into its pharmacologically active metabolite laninamivir in the pulmonary tissue. In this study, we focused on the identification of LOhydrolyzing enzymes from human pulmonary tissue extract using proteomic correlation profiling-a technology integration of traditional biochemistry and proteomics. In a single elution step by gelfiltration chromatography, LO-hydrolyzing activity was separated into two distinct peaks, designated as peak I and peak II. By mass spectrometry, 1160 and 1003 proteins were identified and quantitated for peak I and peak II, respectively, and enzyme candidates were ranked based on the correlation coefficient between the enzyme activity and the proteomic profiles. Among proteins with a high correlation value, S-formylglutathione hydrolase (esterase D; ESD) and acyl-protein thioesterase 1 (APT1) were selected as the most likely candidates for peak I and peak II, respectively, which was confirmed by LO-hydrolyzing activity of recombinant proteins. In the case of peak II, LO-hydrolyzing activity was completely inhibited by treatment with a specific APT1 inhibitor, palmostatin B. Moreover, immunohistochemical analysis revealed that both enzymes were mainly localized in the pulmonary epithelia, a primary site of influenza virus infection. These findings demonstrate that ESD and APT1 are key enzymes responsible for the bioactivation of LO in human pulmonary tissue.

The potential of newly formulated fluorescent-labeled liposomes for the intravital staining of Kupffer cells was evaluated in rats. Fluorescently labeled phosphatidylcholine (PC) was incorporated into liposomes consisting of PC and phosphatidylserine. After intravenous injection, Kupffer cells in the rat liver were intravitally stained and were clearly delineated under the fluorescence image of both confocal laser scanning microscopy and in vivo microscopy. Specificity of the staining was confirmed by immunohistochemistry using the anti-rat macrophage antibody Ki-M2R, which suggested that the liposomes were selectively entrapped by the hepatic reticuloendothelial system. A time-course study revealed that the suitable observation window was between 16 and 24 h after the injection. Phagocytic activity of Kupffer cells after the administration of liposomes was examined by measuring the amount of hepatic uptake of intravenously administered fluorescent microspheres; no detrimental influence of the liposomes on the phagocytic activity was observed. Additionally, no histopathologic changes were found in the livers from liposome-treated rats. Therefore, the fluorescent-labeled liposomes appear to be a useful research tool for labeling Kupffer cells for in vivo microscopic observation of the liver.

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