Summary Dendritic cells (DCs) loaded with tumour antigens have been successfully used to induce protective tumour immunity in murine models and human trials. However, it is still unclear which DC administration route elicits a superior therapeutic effect. Herein, we investigated the vaccine efficiency of DC2.4 cells, a murine dendritic cell line, pulsed with ovalbumin (OVA) in the murine E.G7-OVA tumour model after immunization via various routes. After a single vaccination using 1 × 10 6 OVA-pulsed DC2.4 cells, tumour was completely rejected in the intradermally (i.d.; three of four mice), subcutaneously (s.c.; three of four mice), and intraperitoneally (i.p.; one of four mice) immunized groups. Double vaccinations enhanced the anti-tumour effect in all groups except the intravenous (i.v.) group, which failed to achieve complete rejection. The anti-tumour efficacy of each immunization route was correlated with the OVA-specific cytotoxic T lymphocyte (CTL) activity evaluated on day 7 post-vaccination. Furthermore, the accumulation of DC2.4 cells in the regional lymph nodes was detected only in the i.d.-and s.c.-injected groups. These results demonstrate that the administration route of antigen-loaded DCs affects the migration of DCs to lymphoid tissues and the magnitude of antigen-specific CTL response. Furthermore, the immunization route affects vaccine efficiency.

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We have previously determined the chemical structures of two 2-phenylbenzotriazole mutagens (PBTA-1 and PBTA-2) in blue cotton-adsorbed material from the Nishitakase River in Kyoto, Japan. In the present study, further analysis of mutagenic substances in the Nikko River, which flows through Aichi Prefecture in Japan, allowed the isolation of a new mutagen. Material (2.2 g) adsorbed on blue cotton (3 kg) at a site below the sewage plant on the Nikko River was purified by various column chromatographies, and a mutagen (120 microg) accounting for 11% of the total mutagenicity was isolated. On the basis of data from UV, mass, and (1)H NMR spectra of the mutagen, the compound was deduced to be a PBTA-1 analogue. As with PBTA-1, the mutagen was able to be synthesized from the azo dye 2-[(2-bromo-4, 6-dinitrophenyl)azo]-4-methoxy-5-[(2-hydroxyethyl)amino]acetanilide by reduction and chlorination. Since all spectra of the mutagen isolated from the river water were the same as those of the synthesized form, the structure was concluded to be 2-[2-(acetylamino)-4-[(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino -7-bromo-4-chloro-2H-benzotriazole (PBTA-3). PBTA-3 is a potent mutagen, inducing 81 000 and 3 000 000 revertants per microgram of Salmonella typhimurium TA 98 and YG1024 respectively, in the presence of an S9 mix. In addition to its detection in the water of the Nikko River, PBTA-3 was detected in water samples from three other rivers flowing through regions where dyeing industries have been developed. Like PBTA-1 and PBTA-2, PBTA-3 might have also been produced from azo dyes during industrial processes in dyeing factories and/or through treatment at sewage plants.

To clarify the formation of mutagens in the Maillard reaction of glucose and amino acids, 20 amino acids were separately incubated with glucose in the presence or absence of hydroxyl radicals produced by the Fenton reaction. After 1 week at 37 degrees C and pH 7.4, the reaction mixtures of glucose and tryptophan with and without the Fenton reagent showed mutagenicity toward Salmonella typhimurium YG1024 in the presence of a mammalian metabolic system (S9 mix). To identify mutagens in the reaction mixture, blue rayon-adsorbed material from a mixture of glucose, tryptophan, and the Fenton reagent was separated by column chromatography using various solid and mobile phases, and one mutagen, which accounted for 18% of the total mutagenicity of the reaction mixture, was isolated. The chemical structure of the mutagen was determined to be 5-amino-6-hydroxy-8H-benzo[6,7]azepino[5,4,3-de]quinolin-7-one (ABAQ) on the basis of ESI mass, high-resolution APCI mass, (1)H NMR, (13)C NMR, and IR spectral analyses and chemical synthesis of the mutagen. The novel aromatic amine showed high mutagenicity toward S. typhimurium TA98 and YG1024 with S9 mix, inducing 857 revertants of TA98 and 6007 revertants of YG1024/microg, respectively. The mutagenicity of ABAQ was comparable to that of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, which is a mutagenic and carcinogenic hetrocyclic amine in cooked meat and fish formed through the Maillard reaction at high temperature.