Asthma is one of the most common immunological diseases and is characterized by airway hyperresponsiveness (AHR), mucus overproduction, and airway eosinophilia. Although mouse models have provided insight into the mechanisms by which type-2 cytokines induce asthmatic airway inflammation, differences between the rodent and human immune systems hamper efforts to improve understanding of human allergic diseases. In this study, we aim to establish a preclinical animal model of asthmatic airway inflammation using humanized IL-3/GM-CSF or IL-3/GM-CSF/IL-5 Tg NOD/Shi- scid -IL2rγ ^null (NOG) mice and investigate the roles of human type-2 immune responses in the asthmatic mice. Several important characteristics of asthma — such as AHR, goblet cell hyperplasia, T cell infiltration, IL-13 production, and periostin secretion — were induced in IL-3/GM-CSF Tg mice by intratracheally administered human IL-33. In addition to these characteristics, human eosinophilic inflammation was observed in IL-3/GM-CSF/IL-5 Tg mice. The asthmatic mechanisms of the humanized mice were driven by activation of human Th2 and mast cells by IL-33 stimulation. Furthermore, treatment of the humanized mice with an anti–human IL-13 antibody significantly suppressed these characteristics. Therefore, the humanized mice may enhance our understanding of the pathophysiology of allergic disorders and facilitate the preclinical development of new therapeutics for IL-33–mediated type-2 inflammation in asthma.

To avoid microbial contamination risk, vinyl film isolators are generally used in animal microbiome experiments involving germ-free (GF) mice and/or gnotobiotic (GB) mice. However, it can take several months to gain expertise in operating the isolator competently. Furthermore, sterilization and sterility testing, which are essential for isolator preparation, can take more than 20 days. Hence, we built an experimental rearing environment that combines an individual ventilation cage system and a bioBUBBLE clean room enclosure to easily set up an experimental animal microbiome environment for animal facilities. In this work, a three-step evaluation was conducted. First, we examined whether GF mice can be maintained in this rearing environment without bacterial contamination. Next, we examined whether GF and GB mice can be maintained without cross-contamination in one individual ventilation cage rack. Finally, we tested whether GF mice can be maintained in a biological safety cabinet controlled by negative pressure. In our series of experiments, no microbial contamination occurred over more than 3 months. These results indicated that our rearing system that combines the individual ventilation cage and bioBUBBLE systems can be used not only for experiments with GF mice but also for Biosafety Level 2 experiments that handle bacteria. Our system can mitigate various disadvantages of using vinyl film isolators. In conclusion, we established an experimental method with improved working time and efficiency compared with those of the previous vinyl isolator method.

Chimeric NOD/Shi-Scid IL2 receptor gamma-null mice expressing the HSVtk mutant 30 with humanized livers (humanized-liver mice) are valuable models for predicting drug metabolism in humans. However, carboxylesterase 1c (Ces1c) secreted from residual mouse hepatocytes occasionally prevents the precise evaluation of human drug metabolism. To address this issue, we established a Ces1c knockout (KO) humanized-liver mouse model, characterized its plasma enzymatic activity, and investigated its usefulness in predicting drug metabolism in humans. Ces1c KO mice were successfully generated by deleting exon 5 of Ces1c, which encodes the amino acid residue Ser221 in the catalytic triad. The hydrolytic activities of irinotecan, oseltamivir, and temocapril in plasma were significantly lower in Ces1c KO mice than in normal mice, both untransplanted and transplanted with human hepatocytes, similar to those observed in humans. Drug-metabolizing activities in plasma and liver microsomes were comparable between Ces1c KO humanized-liver mice and humans. Following intravenous administration of irinotecan, the plasma concentration of the hydrolytic metabolite 7-ethyl-10-hydroxycamptothecin was significantly reduced in Ces1c KO humanized-liver mice compared to that in normal humanized-liver mice. Furthermore, the plasma concentrations of cytochrome P450 3A-mediated oxidative metabolites 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin and 7-ethyl-10-(4-amino-1-piperidino)carbonyloxycamptothecin were higher in normal and Ces1c KO humanized-liver mice than in wild-type and Ces1c KO mice. The area under the curve values of irinotecan-related compounds after intravenous administration showed the strongest correlation between humans and Ces1c KO humanized-liver mice. These results demonstrate that Ces1c KO humanized-liver mice are a highly suitable model for the accurate prediction of CES1-mediated drug metabolism in humans. SIGNIFICANT STATEMENT: Carboxylesterase 1c (Ces1c) knockout humanized-liver mice that lacked plasma Ces1c were generated. This model reproduced plasma hydrolytic enzyme activity of irinotecan, oseltamivir, and temocapril observed in humans. Ces1c knockout humanized-liver mice can improve the accuracy of predicting human drug metabolism.