Search

Momoko Miyakawa,1 Shutaro Kubo,1 Hirotsugu Oda,1 Noriko Motoki,2 Masaru Mizuki,2 Teruomi Tsukahara,2 Miyuki Tanaka,1 Koji Yamauchi,1 Fumiaki Abe,1 Tetsuo Nomiyama2 1Food Ingredients and Technology Institute, R&D Division, Morinaga Milk Industry Co., Ltd, Zama, Kanagawa, Japan; 2Department of Preventive Medicine and Public Health, Shinshu University School of Medicine, Matsumoto, Nagano, JapanCorrespondence: Momoko MiyakawaFood Ingredients and Technology Institute, R&D Division, Morinaga Milk Industry Co., Ltd., 1-83, 5-Chome, Higashihara, Zama-City, Kanagawa-Pref. 252-8583, JapanTel +81 46252 3045Fax +81 46252 3017Email mo-miyakawa@morinagamilk.co.jpPurpose: To investigate preliminarily the effect of lactoferrin (LF)-fortified formula on sleep conditions in children.Study Design: A preliminary, randomized, double-blind, placebo-controlled trial.Methods: Healthy children between the ages of 12 and 32 months who attended nursery schools in Japan were divided into two groups and assigned a placebo or LF (48 mg/day)-fortified formula. Children’s sleep conditions were investigated before and after the 13-week intervention using the Japanese Sleep Questionnaire for Preschoolers (JSQ-P).Results: Altogether, 109 participants were randomized. Eight participants were eliminated due to lost to follow-up, withdrawal of consent, and ineligibility, with 101 participants (placebo, n = 48; LF, n = 53) included in the full analysis set (FAS) and used for analysis. Wake-up time, bedtime, and nighttime sleep were comparable between the two groups before and after intervention. The change in total JSQ-P T scores tended to improve in the LF group (placebo vs LF: 0.5 ± 6.5 vs − 1.9 ± 6.1, p = 0.074), in particular, morning symptoms significantly improved (grumpy in the morning, hard to wake-up, and hard to get out of bed) (placebo vs LF: 0.8 ± 6.2 vs − 1.9 ± 6.2, p = 0.028). A better trend was also observed in the LF group regarding restless legs syndrome (RLS)-motor (rubs feet at night and touches feet at night) (placebo vs LF: 2.3 ± 10.7 vs − 0.6 ± 13.5, p = 0.083) and insufficient sleep (stays up more than one hour later the day before a holiday and wakes up more than one hour later on a holiday) (placebo vs LF: 0.1 ± 9.8 vs − 1.7 ± 8.8, p = 0.095). No adverse drug reactions were found.Conclusion: LF intake may improve sleep condition, especially morning symptoms in children above one year of age.Keywords: lactoferrin, sleep, growing-up formula, children

Hirotsugu Oda,1 Momoko Miyakawa,1 Masaru Mizuki,2 Yuka Misawa,3 Teruomi Tsukahara,2 Miyuki Tanaka,1 Koji Yamauchi,1 Fumiaki Abe,1 Tetsuo Nomiyama2 1Food Ingredients and Technology Institute, R&D Division, Morinaga Milk Industry Co., Ltd, Zama, Kanagawa 252-8583, Japan; 2Department of Preventive Medicine and Public Health, Shinshu University School of Medicine, Matsumoto, Nagano 390-8621, Japan; 3Department of Rehabilitation, Nagano Children’s Hospital, Nagano 399-8288, JapanCorrespondence: Hirotsugu OdaFood Ingredients and Technology Institute, R&D Division, Morinaga Milk Industry Co., Ltd., 5-1-83, Zama, Kanagawa 252-8583, JapanTel +81 46 252 3045Fax +81 46 252 3017Email h-oda@morinagamilk.co.jpObjective: To investigate the effects of lactoferrin (LF) on subjective skin conditions in winter.Design: A preliminary, randomized, double-blinded, placebo-controlled trial.Setting and subjects: Healthy adults in Japan.Interventions: Intake of placebo, 200 mg, or 600 mg of LF for 12 weeks in winter.Endpoints: Changes in the scores of subjective skin conditions.Results: Three hundred and forty-six subjects were randomized. Nine subjects (placebo, n=0; 200 mg, n=5; 600 mg, n=4) withdrew consent, and 7 subjects (placebo, n=4; 200 mg, n=2; 600 mg, n=1) were lost to follow-up, resulting in 330 for a full analysis set.Outcomes: Changes in the scores of moisture were greater in the 600 mg group than in the placebo group. Changes in the scores of moisture were greater in the 200 mg and 600 mg groups, and of texture were greater in the 600 mg group than in the placebo group in female subjects.Conclusion: Intake of LF may improve moisture or texture of skin in winter.Keywords: lactoferrin, winter, skin, moisture, texture

Chika Kuroda,1,2,* Katsuya Ueda,1,3,* Hisao Haniu,1,3–5 Haruka Ishida,1,4 Satomi Okano,1,4 Takashi Takizawa,5 Atsushi Sobajima,5 Takayuki Kamanaka,5 Kazushige Yoshida,5 Masanori Okamoto,5 Tamotsu Tsukahara,6 Yoshikazu Matsuda,7 Kaoru Aoki,8 Hiroyuki Kato,5 Naoto Saito1,3–5 1Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, Asahi, Matsumoto, Nagano, Japan; 2Department of Orthopaedic Surgery, Graduate School of Medicine, Shinshu University, Asahi, Matsumoto, Nagano, Japan; 3Department of Biomedical Engineering, Graduate School of Science and Technology, Shinshu University, Asahi, Matsumoto, Nagano, Japan; 4Department of Biomedical Engineering, Graduate School of Medicine, Science and Technology, Shinshu University, Asahi, Matsumoto, Nagano, Japan; 5Department of Orthopaedic Surgery, Shinshu University School of Medicine, Asahi, Matsumoto, Nagano, Japan; 6Department of Molecular Pharmacology and Neuroscience, Nagasaki University Graduate School of Biomedical Sciences, Bunkyo-machi, Nagasaki, Japan; 7Clinical Pharmacology Educational Center, Nihon Pharmaceutical University, Komuro, Ina-machi, Saitama, Japan; 8Physical Therapy Division, School of Health Sciences, Shinshu University, Asahi, Matsumoto, Nagano, Japan *These authors contributed equally to this work Introduction: Carbon nanotubes (CNTs) have various shapes, including needle-like shapes and curled shapes, and the cytotoxicity and carcinogenicity of CNTs differ depending on their shapes and surface modifications. However, the biological responses induced by CNTs and related mechanisms according to the dispersion state of CNTs have not been extensively studied.Materials and methods: We prepared multiwalled CNTs (MWCNTs) showing different dispersions and evaluated these MWCNTs in RAW264 cells to determine cytotoxicity, cellular uptake, and immune responses. Furthermore, RAW264 cells were also used to compare the cellular uptake and cytotoxicity of fibrous MWCNTs and spherical carbon nanohorns (CNHs) exhibiting the same degree of dispersion.Results: Our analysis showed that the cellular uptake, localization, and inflammatory responses of MWCNTs differed depending on the dispersion state. Moreover, there were differences in uptake between MWCNTs and CNHs, even showing the same degree of dispersion. These findings suggested that receptors related to cytotoxicity and immune responses differed depending on the aggregated state of MWCNTs and surface modification with a dispersant. Furthermore, our results suggested that the receptors recognized by the cells differed depending on the particle shape.Conclusion: Therefore, to apply MWCNTs as a biomaterial, it is important to determine the carcinogenicity and toxicity of the CNTs and to examine different biological responses induced by varying shapes, dispersion states, and surface modifications of particles. Keywords: multiwalled carbon nanotubes, aggregation, carbon nanohorns, cytotoxicity, immune response, cellular uptake

Hisao Haniu,1,2 Naoto Saito,2,3 Yoshikazu Matsuda,4 Tamotsu Tsukahara,5 Yuki Usui,1,6,7 Kayo Maruyama,2,3 Seiji Takanashi,1 Kaoru Aoki,1 Shinsuke Kobayashi,1 Hiroki Nomura,1 Manabu Tanaka,1 Masanori Okamoto,1 Hiroyuki Kato1 1Department of Orthopaedic Surgery, Shinshu University School of Medicine, Nagano, Japan; 2Insutitute for Biomedical Sciences, Shinshu University, Nagano, Japan; 3Department of Applied Physical Therapy, Shinshu University School of Health Sciences, Nagano, Japan; 4Clinical Pharmacology Educational Center, Nihon Pharmaceutical University, Saitama, Japan; 5Department of Hematology and Immunology, Kanazawa Medical University, Ishikawa, Japan; 6Research Center for Exotic Nanocarbons, Shinshu University, Nagano, Japan; 7Aizawa Hospital, Sports Medicine Center, Nagano, Japan Abstract: This study aimed to investigate the influence of the shape and size of multi-walled carbon nanotubes (MWCNTs) and cup-stacked carbon nanotubes (CSCNTs) on biological responses in vitro. Three types of MWCNTs – VGCF®-X, VGCF®-S, and VGCF® (vapor grown carbon fibers; with diameters of 15, 80, and 150 nm, respectively) – and three CSCNTs of different lengths (CS-L, 20–80 µm; CS-S, 0.5–20 µm; and CS-M, of intermediate length) were tested. Human bronchial epithelial (BEAS-2B) and malignant pleural mesothelioma cells were exposed to the CNTs (1–50 µg/mL), and cell viability, permeability, uptake, total reactive oxygen species/superoxide production, and intracellular acidity were measured. CSCNTs were less toxic than MWCNTs in both cell types over a 24-hour exposure period. The cytotoxicity of endocytosed MWCNTs varied according to cell type/size, while that of CSCNTs depended on tube length irrespective of cell type. CNT diameter and length influenced cell aggregation and injury extent. Intracellular acidity increased independently of lysosomal activity along with the number of vacuoles in BEAS-2B cells exposed for 24 hours to either CNT (concentration, 10 µg/mL). However, total reactive oxygen species/superoxide generation did not contribute to cytotoxicity. The results demonstrate that CSCNTs could be suitable for biological applications and that CNT shape and size can have differential effects depending on cell type, which can be exploited in the development of highly specialized, biocompatible CNTs. Keywords: multi-walled carbon nanotube, cup-stacked carbon nanotube, cytotoxicity, in vitro, intracellular acidity

Hisao Haniu1, Naoto Saito2, Yoshikazu Matsuda3, Yoong-Ahm Kim4, Ki Chul Park1, Tamotsu Tsukahara5, Yuki Usui6, Kaoru Aoki7, Masayuki Shimizu7, Nobuhide Ogihara7, Kazuo Hara7, Seiji Takanashi7, Masanori Okamoto7, Norio Ishigaki7, Koichi Nakamura7, Hiroyuki Kato71Institute of Carbon Science and Technology, Shinshu University, Matsumoto, Nagano, Japan; 2Department of Applied Physical Therapy, Shinshu University School of Health Sciences, Matsumoto, Nagano, Japan; 3Clinical Pharmacology Educational Center, Nihon Pharmaceutical University, Ina-machi, Saitama, Japan; 4Faculty of Engineering, Shinshu University, Nagano-shi, Nagano, Japan; 5Department of Integrative Physiology and Bio-System Control, Shinshu University School of Medicine, Matsumoto-shi, Nagano, Japan; 6Research Center for Exotic Nanocarbons, Shinshu University, Matsumoto, Nagano, Japan; 7Department of Orthopedic Surgery, Shinshu University School of Medicine, Matsumoto, Nagano, JapanAbstract: Although there have been many reports about the cytotoxicity of multi-walled carbon nanotubes (MWCNTs), the results are still controversial. To investigate one possible reason, the authors investigated the influence of MWCNT dispersants on cellular uptake and cytotoxicity. Cytotoxicity was examined (measured by alamarBlue® assay), as well as intracellular MWCNT concentration and cytokine secretion (measured by flow cytometry) in human bronchial epithelial cells (BEAS-2B) exposed to a type of highly purified MWCNT vapor grown carbon fiber (VGCF®, Showa Denko Kabushiki-gaisha, Tokyo, Japan) in three different dispersants (gelatin, carboxylmethyl cellulose, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine). The authors also researched the relationship between the intracellular concentration of MWCNTs and cytotoxicity by using two cell lines, BEAS-2B and MESO-1 human malignant pleural mesothelioma cells. The intracellular concentration of VGCF was different for each of the three dispersants, and the levels of cytotoxicity and inflammatory response were correlated with the intracellular concentration of VGCF. A relationship between the intracellular concentration of VGCF and cytotoxic effects was observed in both cell lines. The results indicate that dispersants affect VGCF uptake into cells and that cytotoxicity depends on the intracellular concentration of VGCF, not on the exposed dosage. Thus, toxicity appears to depend on exposure time, even at low VGCF concentrations, because VGCF is biopersistent.Keywords: multi-walled carbon nanotube, cytotoxicity, intracellular concentration, dispersant, cytokine secretion

Hisao Haniu1, Naoto Saito2, Yoshikazu Matsuda3, Yoong-Ahm Kim4, Ki Chul Park1, Tamotsu Tsukahara5, Yuki Usui6, Kaoru Aoki7, Masayuki Shimizu7, Nobuhide Ogihara7, Kazuo Hara7, Seiji Takanashi7, Masanori Okamoto7, Norio Ishigaki7, Koichi Nakamura7, Hiroyuki Kato71Institute of Carbon Science and Technology, Shinshu University, Matsumoto, Nagano, Japan; 2Department of Applied Physical Therapy, Shinshu University School of Health Sciences, Matsumoto, Nagano, Japan; 3Clinical Pharmacology Educational Center, Nihon Pharmaceutical University, Ina-machi, Saitama, Japan; 4Faculty of Engineering, Shinshu University, Nagano-shi, Nagano, Japan; 5Department of Integrative Physiology and Bio-System Control, Shinshu University School of Medicine, Matsumoto-shi, Nagano, Japan; 6Research Center for Exotic Nanocarbons, Shinshu University, Matsumoto, Nagano, Japan; 7Department of Orthopaedic Surgery, Shinshu University School of Medicine, Matsumoto, Nagano, JapanAbstract: We examined differences in cellular responses to multi-walled carbon nanotubes (MWCNTs) using malignant pleural mesothelioma cells (MESO-1), bronchial epithelial cells (BEAS-2B), neuroblastoma cells (IMR-32), and monoblastic cells (THP-1), before and after differentiation. MESO-1, BEAS-2B and differentiated THP-1 cells actively endocytosed MWCNTs, resulting in cytotoxicity with lysosomal injury. However, cytotoxicity did not occur in IMR-32 or undifferentiated THP-1 cells. Both differentiated and undifferentiated THP-1 cells exhibited an inflammatory response. Carbon blacks were endocytosed by the same cell types without lysosomal damage and caused cytokine secretion, but they did not cause cytotoxicity. These results indicate that the cytotoxicity of MWCNTs requires not only cellular uptake but also lysosomal injury. Furthermore, it seems that membrane permeability or cytokine secretion without cytotoxicity results from several active mechanisms. Clarification of the cellular recognition mechanism for MWCNTs is important for developing safer MWCNTs.Keywords: multi-walled carbon nanotubes, cytotoxicity, endocytosis, cytokine secretion, reactive oxygen species

Hisao Haniu1, Tamotsu Tsukahara2, Yoshikazu Matsuda3, Yuki Usui4, Kaoru Aoki5, Masayuki Shimizu5, Nobuhide Ogihara5, Kazuo Hara5, Seiji Takanashi5, Masanori Okamoto5, Norio Ishigaki5, Koichi Nakamura5, Hiroyuki Kato5, Naoto Saito6 1Institute of Carbon Science and Technology, 2Department of Integrative Physiology and Bio-System Control, Shinshu University, Matsumoto, Nagano, 3Clinical Pharmacology Educational Center, Nihon Pharmaceutical University, Ina-machi, Saitama, 4Research Center for Exotic Nanocarbons, 5Department of Orthopaedic Surgery, 6Department of Applied Physical Therapy, Shinshu University School of Health Sciences, Shinshu University, Matsumoto, Nagano, Japan Background: In the present study, we investigated whether DJ-1 could serve as a biomarker for assessing the biocompatibility of multiwalled carbon nanotubes (MWCNTs), using the highly purified carbon nanotube, HTT2800. Methods: Using Western blot analysis, we determined DJ-1 protein levels in two different types of cells (one capable and the other incapable of HTT2800 endocytosis). Using quantitative real-time polymerase chain reaction, we also investigated the ability of purified nanotubes to alter DJ-1 mRNA levels. Results: We demonstrated that the DJ-1 protein concentration was reduced, regardless of the cytotoxic activity of intracellular HTT2800. Furthermore, HTT2800 decreased the DJ-1 mRNA levels in a dose-dependent manner. This decrease in DJ-1 mRNA levels was not observed in the case of Sumi black or cup-stacked carbon nanotubes. Conclusion: These data indicate that modification of DJ-1 expression is caused by the cell response to MWCNTs. We conclude that DJ-1 is a promising candidate biomarker for the development of biocompatible MWCNTs. Keywords: multiwalled carbon nanotubes, DJ-1 protein, Western blot, quantitative real-time polymerase chain reaction