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Yoichi Nishii,1 Kazuki Furuhashi,1 Saki Nakamura,1 Miho Nishio,1 Yuki Nakamura,1 Kengo Ushiro,1 Kentaro Ito,1 Tadashi Sakaguchi,1 Yuta Suzuki,1 Kentaro Fujiwara,1 Taro Yasuma,2 Tetsu Kobayashi,3 Corina D’Alessandro-Gabazza,2 Esteban C Gabazza,2 Osamu Taguchi,1 Osamu Hataji1 1Respiratory Center, Matsusaka Municipal Hospital, Matsusaka, Mie, Japan; 2Department of Immunology, Faculty and Graduate School of Medicine, Mie University, Tsu-city, Mie, Japan; 3Department of Pulmonary and Critical Care Medicine, Faculty and Graduate School of Medicine, Mie University, Tsu-city, Mie, JapanCorrespondence: Esteban C GabazzaDepartment of Immunology, Mie University Graduate School of Medicine, Edobashi 2-174, Tsu-city, Mie, 514-8507, JapanTel +81 59 231 5017Fax +81 59 231 5225Email gabazza@doc.medic.mie-u.ac.jpIntroduction: Many patients with nontuberculous mycobacteria pulmonary disease are asymptomatic. The disease diagnosis is confirmed in only a small proportion of patients with radiological findings suspicious for nontuberculous mycobacteria pulmonary disease. Thus, many patients remained undiagnosed. Here, we evaluated the diagnostic value of digital polymerase chain reaction (PCR) in nontuberculous mycobacteria pulmonary disease.Methods: We prospectively evaluated 123 patients with radiological findings suspicious for nontuberculous mycobacteria pulmonary disease. Digital PCR was performed using bronchial lavage fluid, sputum, saliva, blood, and urine.Results: The culture of bronchial washing fluid was positive for nontuberculous mycobacteria in 53 patients and negative in 70. The positive detection rate of nontuberculous mycobacteria by digital PCR in patients with positive culture (n = 53) was as follows: bronchial lavage fluid 100%, sputum 62.9%, saliva 41.5%, blood 7.5%, and urine 3.8%. All patients with two or more positive partitions for nontuberculous mycobacteria in the digital PCR of bronchial lavage fluid showed nontuberculous mycobacteria growth in the bronchial lavage fluid culture. The digital PCR analysis of the bronchial lavage fluid showed a high sensitivity (100%), specificity (85.7%), positive predictive value (84.1%), negative predictive value (100%), and a high concordance rate (91.9%) with the bronchial lavage fluid culture results. In addition, the culture of bronchial lavage fluid was positive for nontuberculous mycobacteria in patients with two or more positive partitions in the digital PCR of sputum and saliva with a combined positive predictive value of 81.1%.Conclusion: Digital PCR analysis of nontuberculous mycobacteria in bronchial lavage fluid shows a high concordance rate with the bronchial lavage fluid culture results and a high positive predictive value using both sputum and saliva, suggesting the potential usefulness of dPCR for diagnosis of nontuberculous mycobacteria pulmonary disease in clinical practice.Keywords: digital PCR, nontuberculous mycobacteria pulmonary disease, diagnosis, bronchial lavage fluid culture, bronchoscopy

Akihiro Uchida,1,* Taro Yasuma,1,2,* Atsuro Takeshita,1,2,* Masaaki Toda,2 Yuko Okano,1,2 Kota Nishihama,1 Corina N D’Alessandro-Gabazza,2 Valeria Fridman D’Alessandro,2 Chisa Inoue,1 Takehiro Takagi,3 Hiroyuki Mukaiyama,4 Norio Takagi,4 Katsumi Shimizu,4 Yutaka Yano,1 Esteban C Gabazza2 1Department of Diabetes and Endocrinology, Mie University Faculty and Graduate School of Medicine, Tsu, Mie, Japan; 2Department of Immunology, Mie University Faculty and Graduate School of Medicine, Tsu, Mie, Japan; 3Iwasaki Hospital, Tsu, Mie, Japan; 4Tanisake Corporation, Gifu, Japan*These authors contributed equally to this workCorrespondence: Esteban C GabazzaDepartment of Immunology, Mie University School of Medicine, Edobashi 2-174, Tsu-City, Mie, 514-8507, JapanTel +81 59 231 5037Fax +81 59 231 5225Email gabazza@doc.medic.mie-u.ac.jpIntroduction: Diabetes mellitus is a serious threat to public health worldwide. It causes a substantial economic burden, mental and physical disabilities, poor quality of life, and high mortality. Limonite is formed when iron-rich materials from the underground emerge and oxidized on the ground surface. It is currently used to purify contaminated water, absorption of irritant gases, and improve livestock breeding. Limonite can change the composition of environmental microbial communities. In the present study, we evaluated whether limonite can ameliorate glucose metabolism abnormalities by remodeling the gut microbiome.Methods: The investigation was performed using mouse models of streptozotocin-induced diabetes mellitus and high-calorie diet-induced metabolic syndrome.Results: Oral limonite supplement was associated with significant body weight recovery, reduced glycemia with improved insulin secretion, increased number of regulatory T cells, and abundant beneficial gut microbial populations in mice with diabetes mellitus compared to control. Similarly, mice with obesity fed with limonite supplements had significantly reduced body weight, insulin resistance, steatohepatitis, and systemic inflammatory response with significant gut microbiome remodeling.Conclusion: This study demonstrates that limonite supplement ameliorates abnormal glucose metabolism in diabetes mellitus and obesity. Gut microbiome remodeling, inhibition of inflammatory cytokines, and the host immune response regulation may explain the limonite’s beneficial activity under pathological conditions in vivo.Keywords: insulin resistance, limonite, diabetes, streptozotocin, metabolic syndrome, gut microbiome

Takahiro Matsumoto,1,2* Yuki Matsushima,1* Masaaki Toda,1 Ziaurahman Roeen,1 Corina N D'Alessandro-Gabazza,1,5 Josephine A Hinneh,1 Etsuko Harada,1,3 Taro Yasuma,4 Yutaka Yano,4 Masahito Urawa,1,5 Tetsu Kobayashi,5 Osamu Taguchi,5 Esteban C Gabazza1 1Department of Immunology, Mie University Graduate School of Medicine, Tsu, Mie Prefecture, 2BONAC Corporation, BIO Factory 4F, Fukuoka, 3Iwade Research Institute of Mycology, 4Department of Endocrinology, Diabetes and Metabolism, 5Department of Pulmonary and Critical Care Medicine, Mie University Graduate School of Medicine, Tsu, Mie Prefecture, Japan *These authors contributed equally to this work Background: Previous studies have demonstrated the beneficial activity of activated protein C in allergic diseases including bronchial asthma and rhinitis. However, the exact mechanism of action of activated protein C in allergies is unclear. In this study, we hypothesized that pharmacological doses of activated protein C can modulate allergic inflammation by inhibiting dendritic cells. Materials and methods: Dendritic cells were prepared using murine bone marrow progenitor cells and human peripheral monocytes. Bronchial asthma was induced in mice that received intratracheal instillation of ovalbumin-pulsed dendritic cells. Results: Activated protein C significantly increased the differentiation of tolerogenic plasmacytoid dendritic cells and the secretion of type I interferons, but it significantly reduced lipopolysaccharide-mediated maturation and the secretion of inflammatory cytokines in myeloid dendritic cells. Activated protein C also inhibited maturation and the secretion of inflammatory cytokines in monocyte-derived dendritic cells. Activated protein C-treated dendritic cells were less effective when differentiating naïve CD4 T-cells from Th1 or Th2 cells, and the cellular effect of activated protein C was mediated by its receptors. Mice that received adoptive transfer of activated protein C-treated ovalbumin-pulsed dendritic cells had significantly less airway hyperresponsiveness, significantly decreased lung concentrations of Th1 and Th2 cytokines, and less plasma concentration of immunoglobulin E when compared to control mice. Conclusion: These results suggest that dendritic cells mediate the immunosuppressive effect of activated protein C during allergic inflammation. Keywords: allergy, dendritic cells, coagulation, protein C pathway