Bone Marrow-Derived Cells Implanted into Radiation-Injured Urinary Bladders Reconstruct Functional Bladder Tissues in Rats

The purpose of this study was to determine whether bone marrow-derived cells implanted into radiation-injured urinary bladders could reconstruct functional bladder tissues. The pelvic region of anesthetized female Sprague-Dawley (SD) rats was irradiated with 2 Gy once a week for 5 weeks. After the last irradiation, the rats were maintained for 2 weeks. Bone marrow cells were harvested from the femurs of donor male green fluorescence protein (GFP)-transfected SD rats and cultured for 7 days. Two weeks after the last radiation exposure, the cultured adherent, proliferating bone marrow-derived cells were implanted into the walls of irradiated urinary bladders. For controls, cell-free solutions were similarly injected. Four weeks after donor cell or control implantations, cystometric, histological, and immunohistochemical investigations were performed. Two weeks after the last irradiation, the smooth muscle layers and nerve fibers of the irradiated urinary bladders were disorganized. The proportions of smooth muscle layer and nerve fiber areas were significantly decreased compared with sham-irradiated urinary bladders. In addition, the remaining smooth muscle cells within the irradiated urinary bladders expressed P4HB, an indicator of collagen synthesis. In the cystometric investigations, the voiding interval of irradiated rats was irregularly prolonged, 7.92 +/- 1.09 min, and the residual volume, 0.13 +/- 0.03 mL, was significantly higher compared with the sham-irradiated rats (5.50 +/- 0.43mL and 0.05 +/- 0.01 mL). After 4 weeks, the smooth muscle layers and nerve fibers in the cell-free control urinary bladders remained similar to the pre-implanted irradiated urinary bladders; however, the cell-implanted urinary bladders contained reconstructed smooth muscle layers and nerve fibers, the proportions of each were significantly higher than those in the cell-free injected controls. The expression of P4HB within the cell-implanted urinary bladders decreased. Some GFP-positive implanted cells differentiated into smooth muscle-and nerve-like cells and became organized into the reconstructed tissues. The voiding interval of the cell-implanted rats, 5.46 +/- 0.33 min, was regular and similar to that of the sham-irradiated rats, and significantly less than that of the cell-free injected controls, 7.39 +/- 0.54 min. The residual volume, 0.04 +/- 0.01 mL, was similar to that of the sham-irradiated rats and significantly decreased compared with that of the cell-free injected controls, 0.15 +/- 0.05 mL. Therefore, the implantation of bone marrow-derived cells is a potentially useful treatment for radiotherapy-induced urinary dysfunctions.ArticleTISSUE ENGINEERING PART A. 18(15-16):1698-1709 (2012)journal articl

IL-15-High-Responder Developing NK Cells Bearing Ly49 Receptors in IL-15(-/-) Mice

信州大学博士（医学）・学位論文・平成24年3月31日授与（甲第910号）・好沢克Originally published in The Journal of Immunology. Yoshizawa, K; Nakajima, S; Notake, T; Miyagawa, SI; Hida, S; Taki, S. 2011. IL-15-High-Responder Developing NK Cells Bearing Ly49 Receptors in IL-15(-/-) Mice . J. Immunol. Vol.187(10): pp5162-pp5169. Copyright (C) 2011 The American Association of Immunologists, Inc.In mice lacking IL-15, NK cell development is arrested at immature stages, providing an opportunity to investigate the earliest developing NK cells that would respond to IL-15. We show in this study that immature NK cells were present in the spleen as well as bone marrow (BM) and contained IL-15-high-responder cells. Thus, mature NK cells were generated more efficiently from IL-15(-/-) than from control donor cells in radiation BM chimeras, and the rate of IL-15-induced cell division in vitro was higher in NK cells in the spleen and BM from IL-5(-/-) mice than in those from wild-type mice. Phenotypically, NK cells developed in IL-15(-/-) mice up to the minor but discrete CD11b(-)CD27(+)DX5(hi)CD51(dull)CD127(dull)CD122(hi) stage, which contained the majority of Ly49G2(+) and D+ NK cells both in the spleen and BM. Even among wild-type splenic NK cells, IL-15-induced proliferation was most prominent in CD11b(-)DX5(hi) cells. Notably, IL-15-mediated preferential expansion (but not conversion from Ly49(-) cells) of Ly49(+) NK cells was observed in vitro only for NK cells in the spleen. These observations indicated the uneven distribution of NK cells of different developing stages with variable IL-15 responsiveness in these lymphoid organs. Immature NK cells in the spleen may contribute, as auxiliaries to those in BM, to the mature NK cell compartment through IL-15-driven extramarrow expansion under steady-state or inflammatory conditions. The Journal of Immunology, 2011, 187: 5162-5169.ArticleJOURNAL OF IMMUNOLOGY. 187(10):5162-5169 (2011)journal articl

Bone Marrow-Derived Cells Implanted into Radiation-Injured Urinary Bladders Reconstruct Functional Bladder Tissues in Rats

The purpose of this study was to determine whether bone marrow-derived cells implanted into radiation-injured urinary bladders could reconstruct functional bladder tissues. The pelvic region of anesthetized female Sprague-Dawley (SD) rats was irradiated with 2 Gy once a week for 5 weeks. After the last irradiation, the rats were maintained for 2 weeks. Bone marrow cells were harvested from the femurs of donor male green fluorescence protein (GFP)-transfected SD rats and cultured for 7 days. Two weeks after the last radiation exposure, the cultured adherent, proliferating bone marrow-derived cells were implanted into the walls of irradiated urinary bladders. For controls, cell-free solutions were similarly injected. Four weeks after donor cell or control implantations, cystometric, histological, and immunohistochemical investigations were performed. Two weeks after the last irradiation, the smooth muscle layers and nerve fibers of the irradiated urinary bladders were disorganized. The proportions of smooth muscle layer and nerve fiber areas were significantly decreased compared with sham-irradiated urinary bladders. In addition, the remaining smooth muscle cells within the irradiated urinary bladders expressed P4HB, an indicator of collagen synthesis. In the cystometric investigations, the voiding interval of irradiated rats was irregularly prolonged, 7.92 +/- 1.09 min, and the residual volume, 0.13 +/- 0.03 mL, was significantly higher compared with the sham-irradiated rats (5.50 +/- 0.43mL and 0.05 +/- 0.01 mL). After 4 weeks, the smooth muscle layers and nerve fibers in the cell-free control urinary bladders remained similar to the pre-implanted irradiated urinary bladders; however, the cell-implanted urinary bladders contained reconstructed smooth muscle layers and nerve fibers, the proportions of each were significantly higher than those in the cell-free injected controls. The expression of P4HB within the cell-implanted urinary bladders decreased. Some GFP-positive implanted cells differentiated into smooth muscle-and nerve-like cells and became organized into the reconstructed tissues. The voiding interval of the cell-implanted rats, 5.46 +/- 0.33 min, was regular and similar to that of the sham-irradiated rats, and significantly less than that of the cell-free injected controls, 7.39 +/- 0.54 min. The residual volume, 0.04 +/- 0.01 mL, was similar to that of the sham-irradiated rats and significantly decreased compared with that of the cell-free injected controls, 0.15 +/- 0.05 mL. Therefore, the implantation of bone marrow-derived cells is a potentially useful treatment for radiotherapy-induced urinary dysfunctions.ArticleTISSUE ENGINEERING PART A. 18(15-16):1698-1709 (2012)journal articl

IL-15 inhibits pre-B cell proliferation by selectively expanding Mac-1+B220+ NK cells

ArticleBIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. 369(4): 1139-1143 (2008)journal articl

IL-15–High-Responder Developing NK Cells Bearing Ly49 Receptors in IL-15 −/−

信州大学博士（医学）・学位論文・平成24年3月31日授与（甲第910号）・好沢克Originally published in The Journal of Immunology. Yoshizawa, K; Nakajima, S; Notake, T; Miyagawa, SI; Hida, S; Taki, S. 2011. IL-15-High-Responder Developing NK Cells Bearing Ly49 Receptors in IL-15(-/-) Mice . J. Immunol. Vol.187(10): pp5162-pp5169. Copyright (C) 2011 The American Association of Immunologists, Inc.In mice lacking IL-15, NK cell development is arrested at immature stages, providing an opportunity to investigate the earliest developing NK cells that would respond to IL-15. We show in this study that immature NK cells were present in the spleen as well as bone marrow (BM) and contained IL-15-high-responder cells. Thus, mature NK cells were generated more efficiently from IL-15(-/-) than from control donor cells in radiation BM chimeras, and the rate of IL-15-induced cell division in vitro was higher in NK cells in the spleen and BM from IL-5(-/-) mice than in those from wild-type mice. Phenotypically, NK cells developed in IL-15(-/-) mice up to the minor but discrete CD11b(-)CD27(+)DX5(hi)CD51(dull)CD127(dull)CD122(hi) stage, which contained the majority of Ly49G2(+) and D+ NK cells both in the spleen and BM. Even among wild-type splenic NK cells, IL-15-induced proliferation was most prominent in CD11b(-)DX5(hi) cells. Notably, IL-15-mediated preferential expansion (but not conversion from Ly49(-) cells) of Ly49(+) NK cells was observed in vitro only for NK cells in the spleen. These observations indicated the uneven distribution of NK cells of different developing stages with variable IL-15 responsiveness in these lymphoid organs. Immature NK cells in the spleen may contribute, as auxiliaries to those in BM, to the mature NK cell compartment through IL-15-driven extramarrow expansion under steady-state or inflammatory conditions. The Journal of Immunology, 2011, 187: 5162-5169.ArticleJOURNAL OF IMMUNOLOGY. 187(10):5162-5169 (2011)journal articl

Target-selective cytosolic delivery of cargo proteins using the VHH-presented OLE-ZIP capsules

In the pursuit of a new generation of protein pharmaceuticals, the efficient delivery of these therapeutics into cells stands out as a crucial challenge. In this study, we have developed a novel approach utilizing protein capsules modified with VHH antibodies as cytosolic carriers for protein pharmaceuticals. For the protein capsule component, we opted for the OLE-ZIP protein capsules, which can be prepared from the amphiphilic two-helix bundled protein OLE-ZIP using the water-in-oil (w/o) emulsion method. The spacious interior of the OLE-ZIP capsules allows for the stable encapsulation of over 200 molecules of protein pharmaceuticals, such as RNase A and Cre recombinase, in one capsule. By presenting the VHH antibody with an affinity for cell-type-specific receptors such as the epidermal growth factor receptor (EGFR) on the capsule surface, we achieved cell-type selective endocytic uptake in A431 cell lines (high expression level of EGFR) over NHDF and MCF-7 cells (normal expression level of EGFR). This selective uptake was followed by the subsequent release of the encapsulated protein pharmaceuticals into the cytosol of the target cells. Unlike our previous version of the OLE-ZIP protein capsules modified with IgG antibodies, cytosolic delivery of pharmaceutical proteins was little impacted by the presence of other IgGs, which are abundant in the bloodstream. This improved characteristic suggests potential advantages for practical applications, including intravenous administration

2‐Deoxy‐d‐glucose induces deglycosylation of proinflammatory cytokine receptors and strongly reduces immunological responses in mouse models of inflammation

博士（医学）乙日本医科大学202