12-OH-17,18-Epoxyeicosatetraenoic acid alleviates eosinophilic airway inflammation in murine lungs.

BACKGROUND: Asthma is characterized by airway inflammation and obstruction with eosinophil infiltration into the airway. Arachidonic acid, an omega-6 fatty acid, is metabolized into cysteinyl leukotriene with pro-inflammatory properties for allergic inflammation, whereas the omega-3 fatty acid eicosapentaenoic acid (EPA) and its downstream metabolites are known to have anti-inflammatory effects. In this study, we investigated the mechanism underlying the counter-regulatory roles of EPA in inflamed lungs. METHODS: Male C57BL6 mice were sensitized and challenged by ovalbumin (OVA). After EPA treatment, we evaluated the cell count of Bronchoalveolar lavage fluid (BALF), mRNA expressions in the lungs by q-PCR, and the amounts of lipid mediators by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based lipidomics. We investigated the effect of the metabolite of EPA by in vivo and in vitro studies. RESULTS: Eicosapentaenoic acid treatment reduced the accumulation of eosinophils in the airway and decreased mRNA expression of selected inflammatory mediators in the lung. Lipidomics clarified the metabolomic profile in the lungs. Among EPA-derived metabolites, 12-hydroxy-17,18-epoxyeicosatetraenoic acid (12-OH-17,18-EpETE) was identified as one of the major biosynthesized molecules; the production of this molecule was amplified by EPA administration and allergic inflammation. Intravenous administration of 12-OH-17,18-EpETE attenuated airway eosinophilic inflammation through downregulation of C-C chemokine motif 11 (CCL11) mRNA expression in the lungs. In vitro, this molecule also inhibited the release of CCL11 from human airway epithelial cells stimulated with interleukin-4. CONCLUSION: These results demonstrated that EPA alleviated airway eosinophilic inflammation through its conversion into bioactive metabolites. Additionally, our results suggest that 12-OH-17,18-EpETE is a potential therapeutic target for the management of asthma.

[Role of natural killer cells in the antimetastatic effect of defibrinogenation with batroxobin].

The antimetastatic effects of defibrinogenation with batroxobin were investigated in normal mice and in mice with depressed or activated natural killer (NK) cell activity. Batroxobin inhibited the formation of lung metastases after intravenous inoculation of the F10 subline of B16 melanoma. Inhibition of NK activity by treatment of mice with anti-asialo GM1 antibody abrogated the antimetastatic effects of Batroxobin. Conversely, augmentation of NK cell activity by poly I:C plus treatment with batroxobin produced additive antimetastatic effects. Studies on the mechanism of interaction between Batroxobin and NK cells revealed that Batroxobin treatment did not affect splenic NK activity in vitro. From these data, it was found that the antimetastatic effects of batroxobin are dependent on the level of NK activity in the host.

[Analysis of drug sensitivity by successive colony-forming assay].

It is difficult to predict the adequate doses of effective drugs which must be administered in cancer chemotherapy. We have developed a successive colony-forming assay in order to analyze changes of drug sensitivity in human cancer cell lines, and it is used as a model of cancer chemotherapy. Presently, widely used methods include the clonog ceni assay as in vitro sensitivity test established by Salmon and Hamberger et al., for revealing effective drugs. However, formation of a second colony using initial colony-forming cells has not yet been performed. We therefore analyzed the changes occurring in drug sensitivity in 2nd and 3rd colony-forming assays. Using a human lung adenocarcinoma cell line PC-9 (established by Hayata at the Surgical Department in Tokyo Medical School), we measured the changes in sensitivity to CDDP and its derivatives. After picking up the colony-forming cells by micropipette and culturing them in culture medium, we tried to incubate them for 1 hour with the anticancer drug, and then replate them in soft agar. This technique is called "2nd colony-forming assay". We were able to carry this out three times. The results obtained showed that the anti-tumor effect of 254S was the same as that of CDDP, whereas that of CBDCA was less than either of them. Although these tendencies were noticeable even in the 2nd colony-forming assay, the sensitivities to CDDP and its derivatives were diminished in the 3rd colony-forming assay.