Yukari Kuramoto

Mannosylated cationic liposomes/CpG DNA complex for the treatment of hepatic metastasis after intravenous administration in mice

Immunotherapy using immunostimulatory CpG DNA could be a promising new therapeutic approach to combat refractory hepatic metastasis. In this study, we report the use of a conventional cationic liposomes/CpG DNA complex (Bare/CpG DNA lipoplex) and a mannosylated cationic liposomes/CpG DNA complex (Man/CpG DNA lipoplex) for effective inhibition of hepatic metastasis in mice. After intravenous administration of Bare/CpG DNA lipoplex, higher amounts of IL-12 and IFN-gamma were produced in serum or liver compared with naked CpG DNA, and their production was increased further by Man/CpG DNA lipoplex. Then, Bare/CpG DNA lipoplex and Man/CpG DNA lipoplex were administered intravenously to hepatic metastasis model mice, and the numbers of tumor cells (colon26/Luc) were quantitatively assayed. The number of tumor cells in Man/CpG DNA lipoplex-treated mice was same as those in Bare/CpG DNA lipoplex-treated mice. These results suggest that intravenous administration of not only Bare/CpG DNA lipoplex but also Man/CpG DNA lipoplex could be an efficient immunotherapy for hepatic metastasis.

Use of mannosylated cationic liposomes/ immunostimulatory CpG DNA complex for effective inhibition of peritoneal dissemination in mice

Immunotherapy using immunostimulatory CpG DNA could be a promising new therapeutic approach to combat refractory peritoneal dissemination. In the present study, we report the use of a mannosylated cationic liposomes/immunostimulatory CpG DNA complex (Man/CpG DNA lipoplex) for effective inhibition of peritoneal dissemination in mice.

Analysis of in vivo nuclear Factor-κB activation during liver inflammation in mice : Prevention by catalase delivery

Nuclear factor-κB (NF-κB) is a transcription factor that plays crucial roles in inflammation, immunity, cell proliferation, and apoptosis. Until now, there have been few studies of NF-κB activation in whole animals because of experimental difficulties. Here, we show that mice receiving a simple injection of plasmid vectors can be used to examine NF-κB activation in the liver. Two plasmid vectors, pNF-κB-Luc (firefly luciferase gene) and pRL-SV40 (Renilla reniformis luciferase gene), were injected into the tail vein of mice by the hydrodynamics-based procedure, an established method of gene transfer to mouse liver. Then, the ratio of the firefly and R. reniformis luciferase activities (F/R) was used as an indicator of the NF-κB activity in the liver. Injection of thioacetamide or lipopolysaccharide plus d-galactosamine increased the F/R ratio in the liver, and this was significantly (P < 0.001) inhibited by an intravenous injection of catalase derivatives targeting liver nonparenchymal cells. Imaging the firefly luciferase expression in live mice clearly demonstrated that the catalase derivatives efficiently prevented the NF-κB-mediated expression of the firefly luciferase gene. Plasma transaminases and the survival rate of mice supported the findings obtained by the luminescence-based analyses. Thus, this method, which requires no genetic recombination techniques, is highly sensitive to the activation of NF-κB and allows us to continuously examine the activation in live animals. In conclusion, this novel, simple, and sensitive method can be used not only for analyzing the NF-κB activation in the organ under different inflammatory conditions but also for screening drug candidates for the prevention of liver inflammation.

Efficient peritoneal dissemination treatment obtained by an immunostimulatory phosphorothioate-type CpG DNA/cationic liposome complex in mice.

Peritoneal dissemination remains the most difficult type of metastasis to treat, and current systemic chemotherapy or radiotherapy tends to have little effect; therefore, immunotherapy using immunostimulatory CpG DNA could be a promising new therapeutic approach. Recently, we have reported that intraperitoneal administration of phosphodiester (PO) CpG DNA-lipoplex could efficiently inhibit peritoneal dissemination in mice. In this study, chemically modified phosphorothioate (PS)-CpG DNA and natural PO-CpG DNA were complexed with DOTMA/cholesterol cationic liposomes (PS-CpG DNA-lipoplex and PO-CpG DNA-lipoplex) and their antitumor activity was evaluated in a mouse model of peritoneal dissemination. Intraperitoneal administration of the PS-CpG DNA-lipoplex inhibited the proliferation of tumor cells in the greater omentum and the mesentery more efficiently than PO-CpG DNA-lipoplex. PS-CpG DNA-lipoplex induced higher cytokine production from primary cultured mouse peritoneal macrophages, suggesting that the high antitumor activity of the PS-CpG DNA-lipoplex is mediated by a high rate of cytokine production from immunocompetent cells such as macrophages. The serum transaminase levels of mice receiving intraperitoneal PS-CpG DNA-lipoplex treatment were measured to evaluate systemic toxicity, and these were found to be the same as those of untreated mice. These results suggest that intraperitoneal administration of PS-CpG DNA-lipoplex could be efficient immunotherapy for peritoneal dissemination.