Details of a Researcher - Sato, Toshiro

Papers - Sato, Toshiro

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Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5 + stem cell

Yui Shiro, Nakamura Tetsuya, Sato Toshiro, Nemoto Yasuhiro, Mizutani Tomohiro, Zheng Xiu, Ichinose Shizuko, Nagaishi Takashi, Okamoto Ryuichi, Tsuchiya Kiichiro, Clevers Hans, Watanabe Mamoru

Nature Medicine 18 ( 4 ) 618 - 623 2012.04

Research paper (scientific journal), Joint Work, ISSN 1078-8956

Adult stem-cell therapy holds promise for the treatment of gastrointestinal diseases. Here we describe methods for long-term expansion of colonic stem cells positive for leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5 ⁺ cells) in culture. To test the transplantability of these cells, we reintroduced cultured GFP ⁺ colon organoids into superficially damaged mouse colon. The transplanted donor cells readily integrated into the mouse colon, covering the area that lacked epithelium as a result of the introduced damage in recipient mice. At 4 weeks after transplantation, the donor-derived cells constituted a single-layered epithelium, which formed self-renewing crypts that were functionally and histologically normal. Moreover, we observed long-term (>6 months) engraftment with transplantation of organoids derived from a single Lgr5 ⁺ colon stem cell after extensive in vitro expansion. These data show the feasibility of colon stem-cell therapy based on the in vitro expansion of a single adult colonic stem cell.

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Cdx2 determines the fate of postnatal intestinal endoderm

Stringer Emma J., Duluc Isabelle, Saandi Thoueiba, Davidson Irwin, Bialecka Monika, Sato Toshiro, Barker Nick, Clevers Hans, Pritchard Catrin A., Winton Doug J., Wright Nicholas A., Freund Jean Noel, Deschamps Jacqueline, Beck Felix

Development 139 ( 3 ) 465 - 474 2012.02

Research paper (scientific journal), Joint Work, ISSN 0950-1991

Knock out of intestinal Cdx2 produces different effects depending upon the developmental stage at which this occurs. Early in development it produces histologically ordered stomach mucosa in the midgut. Conditional inactivation of Cdx2 in adult intestinal epithelium, as well as specifically in the Lgr5-positive stem cells, of adult mice allows long-term survival of the animals but fails to produce this phenotype. Instead, the endodermal cells exhibit cell-autonomous expression of gastric genes in an intestinal setting that is not accompanied by mesodermal expression of Barx1, which is necessary for gastric morphogenesis. Cdx2- negative endodermal cells also fail to express Sox2, a marker of gastric morphogenesis. Maturation of the stem cell niche thus appears to be associated with loss of ability to express positional information cues that are required for normal stomach development. Cdx2-negative intestinal crypts produce subsurface cystic vesicles, whereas untargeted crypts hypertrophy to later replace the surface epithelium. These observations are supported by studies involving inactivation of Cdx2 in intestinal crypts cultured in vitro. This abolishes their ability to form long-term growing intestinal organoids that differentiate into intestinal phenotypes. We conclude that expression of Cdx2 is essential for differentiation of gut stem cells into any of the intestinal cell types, but they maintain a degree of cell-autonomous plasticity that allows them to switch on a variety of gastric genes.

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Controlled gene expression in primary Lgr5 organoid cultures

Koo Bon Kyoung, Stange Daniel E., Sato Toshiro, Karthaus Wouter, Farin Henner F., Huch Meritxell, Van Es Johan H., Clevers Hans

Nature Methods 9 ( 1 ) 81 - 83 2012.01

Research paper (scientific journal), Joint Work, ISSN 1548-7091

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Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium

Sato Toshiro, Stange Daniel E., Ferrante Marc, Vries Robert G J, Van Es Johan H., Van Den Brink Stieneke, Van Houdt Winan J., Pronk Apollo, Van Gorp Joost, Siersema Peter D., Clevers Hans

Gastroenterology 141 ( 5 ) 1762 - 1772 2011.11

Research paper (scientific journal), Joint Work, ISSN 0016-5085

Background & Aims: We previously established long-term culture conditions under which single crypts or stem cells derived from mouse small intestine expand over long periods. The expanding crypts undergo multiple crypt fission events, simultaneously generating villus-like epithelial domains that contain all differentiated types of cells. We have adapted the culture conditions to grow similar epithelial organoids from mouse colon and human small intestine and colon. Methods: Based on the mouse small intestinal culture system, we optimized the mouse and human colon culture systems. Results: Addition of Wnt3A to the combination of growth factors applied to mouse colon crypts allowed them to expand indefinitely. Addition of nicotinamide, along with a small molecule inhibitor of Alk and an inhibitor of p38, were required for long-term culture of human small intestine and colon tissues. The culture system also allowed growth of mouse Apc-deficient adenomas, human colorectal cancer cells, and human metaplastic epithelia from regions of Barrett's esophagus. Conclusions: We developed a technology that can be used to study infected, inflammatory, or neoplastic tissues from the human gastrointestinal tract. These tools might have applications in regenerative biology through ex vivo expansion of the intestinal epithelia. Studies of these cultures indicate that there is no inherent restriction in the replicative potential of adult stem cells (or a Hayflick limit) ex vivo.

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Isolation and in vitro expansion of human colonic stem cells

Jung Peter, Sato Toshiro, Merlos-Suárez Anna, Barriga Francisco M., Iglesias Mar, Rossell David, Auer Herbert, Gallardo Mercedes, Blasco Maria A., Sancho Elena, Clevers Hans, Batlle Eduard

Nature Medicine 17 ( 10 ) 1225 - 1227 2011.10

Research paper (scientific journal), Joint Work, ISSN 1078-8956

Here we describe the isolation of stem cells of the human colonic epithelium. Differential cell surface abundance of ephrin type-B receptor 2 (EPHB2) allows the purification of different cell types from human colon mucosa biopsies. The highest EPHB2 surface levels correspond to epithelial colonic cells with the longest telomeres and elevated expression of intestinal stem cell (ISC) marker genes. Moreover, using culturing conditions that recreate the ISC niche, a substantial proportion of EPHB2-high cells can be expanded in vitro as an undifferentiated and multipotent population.

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Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts

Sato Toshiro, Van Es Johan H., Snippert Hugo J., Stange Daniel E., Vries Robert G., Van Den Born Maaike, Barker Nick, Shroyer Noah F., Van De Wetering Marc, Clevers Hans

Nature 469 ( 7330 ) 415 - 418 2011.01

Research paper (scientific journal), ISSN 0028-0836

Homeostasis of self-renewing small intestinal crypts results from neutral competition between Lgr5 stem cells, which are small cycling cells located at crypt bottoms. Lgr5 stem cells are interspersed between terminally differentiated Paneth cells that are known to produce bactericidal products such as lysozyme and cryptdins/defensins. Single Lgr5-expressing stem cells can be cultured to form long-lived, self-organizing crypt-villus organoids in the absence of non-epithelial niche cells. Here we find a close physical association of Lgr5 stem cells with Paneth cells in mice, both in vivo and in vitro. CD24
⁺ Paneth cells express EGF, TGF-γ ±, Wnt3 and the Notch ligand Dll4, all essential signals for stem-cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells markedly improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitant loss of Lgr5 stem cells. In colon crypts, CD24
⁺ cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell.

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Lgr5+ve Stem Cells Drive Self-Renewal in the Stomach and Build Long-Lived Gastric Units In Vitro

Barker Nick, Huch Meritxell, Kujala Pekka, van de Wetering Marc, Snippert Hugo J., van Es Johan H., Sato Toshiro, Stange Daniel E., Begthel Harry, van den Born Maaike, Danenberg Esther, van den Brink Stieneke, Korving Jeroen, Abo Arie, Peters Peter J., Wright Nick, Poulsom Richard, Clevers Hans

Cell Stem Cell 6 ( 1 ) 25 - 36 2010.01

Research paper (scientific journal), Joint Work, ISSN 1934-5909

The study of gastric epithelial homeostasis and cancer has been hampered by the lack of stem cell markers and in vitro culture methods. The Wnt target gene Lgr5 marks stem cells in the small intestine, colon, and hair follicle. Here, we investigated Lgr5 expression in the stomach and assessed the stem cell potential of the Lgr5^+ve cells by using in vivo lineage tracing. In neonatal stomach, Lgr5 was expressed at the base of prospective corpus and pyloric glands, whereas expression in the adult was predominantly restricted to the base of mature pyloric glands. Lineage tracing revealed these Lgr5^+ve cells to be self-renewing, multipotent stem cells responsible for the long-term renewal of the gastric epithelium. With an in vitro culture system, single Lgr5^+ve cells efficiently generated long-lived organoids resembling mature pyloric epithelium. The Lgr5 stem cell marker and culture method described here will be invaluable tools for accelerating research into gastric epithelial renewal, inflammation/infection, and cancer.

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Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells

Snippert Hugo J., van der Flier Laurens G., Sato Toshiro, van Es Johan H., van den Born Maaike, Kroon-Veenboer Carla, Barker Nick, Klein Allon M., van Rheenen Jacco, Simons Benjamin D., Clevers Hans

Cell 143 ( 1 ) 134 - 144 2010

Research paper (scientific journal), Joint Work, ISSN 0092-8674

Intestinal stem cells, characterized by high Lgr5 expression, reside between Paneth cells at the small intestinal crypt base and divide every day. We have carried out fate mapping of individual stem cells by generating a multicolor Cre-reporter. As a population, Lgr5^hi stem cells persist life-long, yet crypts drift toward clonality within a period of 1-6 months. We have collected short- and long-term clonal tracing data of individual Lgr5^hi cells. These reveal that most Lgr5^hi cell divisions occur symmetrically and do not support a model in which two daughter cells resulting from an Lgr5^hi cell division adopt divergent fates (i.e., one Lgr5^hi cell and one transit-amplifying [TA] cell per division). The cellular dynamics are consistent with a model in which the resident stem cells double their numbers each day and stochastically adopt stem or TA fates. Quantitative analysis shows that stem cell turnover follows a pattern of neutral drift dynamics.

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Th1/Th17 Immune Response Is Induced by Mesenteric Lymph Node Dendritic Cells in Crohn's Disease

Sakuraba Atsushi, Sato Toshiro, Kamada Nobuhiko, Kitazume Mina, Sugita Akira, Hibi Toshifumi

Gastroenterology 137 ( 5 ) 1736 - 1745 2009.11

Research paper (scientific journal), Joint Work, ISSN 0016-5085

Background & Aims: Dendritic cells (DCs) possess the most potent ability to induce acquired immunity. However, their involvement in the pathogenesis of Crohn's disease (CD) has not yet been determined. We aimed to establish the immune status of mesenteric lymph nodes, the major gut-associated lymphoid tissue, and isolated DCs and determine their involvement in the pathogenesis of CD. Methods: CD4 ⁺ T cells and DCs were isolated from mesenteric lymph nodes of CD, ulcerative colitis, and normal control. The immune status of CD4 ⁺ T cells was analyzed by cytokine production and transcriptional profile. Surface phenotype of DCs was analyzed by flow cytometry. Cytokine production by myeloid DCs was analyzed by real-time polymerase chain reaction and exogenous bacterial stimulation. Immune stimulating activity of DCs was determined by mixed lymphocyte reaction. Results: In CD, mesenteric lymph node CD4 ⁺ T cells produced higher amounts of interferon-γ and interleukin (IL)-17 compared with ulcerative colitis and normal control, and this was dictated by increased T-bet and retinoic acid-related orphan receptor-γ expression. Three subtypes of DCs, myeloid DC, plasmacytoid DC, and mature DC, were identified in all groups. When stimulated with exogenous bacterial derivative, myeloid DCs from CD produced a higher amount of IL-23 and a lower amount of IL-10. Myeloid DCs from CD induced stronger T helper cell (Th)1 immune response in mixed lymphocyte reaction compared with those from ulcerative colitis and normal control. Conclusions: Our findings revealed that mesenteric lymph node is the key pathogenic location of CD elicited by the unique cytokine milieu produced by DCs leading to a dysregulated Th1/Th17 immune response.

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Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche

Sato Toshiro, Vries Robert G., Snippert Hugo J., Van De Wetering Marc, Barker Nick, Stange Daniel E., Van Es Johan H., Abo Arie, Kujala Pekka, Peters Peter J., Clevers Hans

Nature 459 ( 7244 ) 262 - 265 2009.05

Research paper (scientific journal), Joint Work, ISSN 0028-0836

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently demonstrated the presence of about six cycling Lgr5 + stem cells at the bottoms of small-intestinal crypts. Here we describe the establishment of long-term culture conditions under which single crypts undergo multiple crypt fission events, while simultanously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted Lgr5 + stem cells can also initiate these crypt-villus organoids. Tracing experiments indicate that the Lgr5 + stem-cell hierarchy is maintained in organoids. We conclude that intestinal crypt-villus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche.

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Granulocyte and monocyte adsorption apheresis therapy modulates monocyte-derived dendritic cell function in patients with ulcerative colitis

Iwakami Yuko, Sakuraba Atsushi, Sato Toshiro, Takada Yasuhiro, Izumiya Motoko, Ichikawa Hitoshi, Hibi Toshifumi

Therapeutic Apheresis and Dialysis 13 ( 2 ) 138 - 146 2009.04

Research paper (scientific journal), Joint Work, ISSN 1744-9979

The aim of this study was to elucidate the molecular mechanisms responsible for the therapeutic effects of granulocyte and monocyte adsorption apheresis (GMA). We investigated the alterations in circulating monocyte subsets and monocyte-derived dendritic cell (moDC) function after GMA therapy in ulcerative colitis (UC) patients. Eighteen patients with UC were enrolled: 14 patients were responders, and 4 patients were non-responders. Peripheral venous blood was obtained within 5 min before and 5 min after GMA therapy. Flow cytometric analysis for monocyte markers (CD14/CD16) was then performed. Monocyte-derived dendritic cells were obtained and alterations in their phenotype were analyzed by flow cytometry. Their function was also analyzed in a mixed lymphocyte reaction assay between allo-naïve T lymphocytes. Flow cytometric analysis for intracellular interferon (IFN)-γ (T-helper 1 cells) and interleukin (IL)-4 (T-helper 2 cells) was then performed for the stimulated T lymphocytes. In patients who responded to GMA, the average numbers of monocytes, especially CD16⁺ monocytes, were significantly decreased after therapy (P < 0.05). In responders, post-GMA moDCs expressed significantly lower CD80 and B7-DC, which are one of the stimulation and maturation markers of dendritic cells, compared to pre-GMA moDCs. CD83, CD86 and human leukocyte antigen-DR also showed a tendency to decrease. In responders, naïve T lymphocytes stimulated with post-GMA moDCs produced significantly less IFN-γ and IL-4 compared to those stimulated with pre-GMA moDCs. The results of our study show that some of the immunosuppressive effects of GMA therapy may be associated with the modulation of monocyte subsets and moDC function.

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Unique CD14⁺ intestinal macrophages contribute to the pathogenesis of Crohn disease via IL-23/IFN-γ axis

Kamada Nobuhiko, Hisamatsu Tadakazu, Okamoto Susumu, Chinen Hiroshi, Kobayashi Taku, Sato Toshiro, Sakuraba Atsushi, Kitazume Mina T., Sugita Akira, Koganei Kazutaka, Akagawa Kiyoko S., Hibi Toshifumi

Journal of Clinical Investigation 118 ( 6 ) 2269 - 2280 2008.06

Research paper (scientific journal), Joint Work, ISSN 0021-9738

Intestinal macrophages play a central role in regulation of immune responses against commensal bacteria. In general, intestinal macrophages lack the expression of innate-immune receptor CD14 and do not produce proinflammatory cytokines against commensal bacteria. In this study, we identified what we believe to be a unique macrophage subset in human intestine. This subset expressed both macrophage (CD14, CD33, CD68) and DC markers (CD205, CD209) and produced larger amounts of proinflammatory cytokines, such as IL-23, TNF-α, and IL-6, than typical intestinal resident macrophages (CD14 ^-CD33⁺ macrophages). In patients with Crohn disease (CD), the number of these CD14+ macrophages were significantly increased compared with normal control subjects. In addition to increased numbers of cells, these cells also produced larger amounts of IL-23 and TNF-α compared with those in normal controls or patients with ulcerative colitis. In addition, the CD14 ⁺ macrophages contributed to IFN-γ production rather than IL-17 production by lamina propria mononuclear cells (LPMCs) dependent on IL-23 and TNF-α. Furthermore, the IFN-γ produced by LPMCs triggered further abnormal macrophage differentiation with an IL-23-hyperproducing phenotype. Collectively, these data suggest that this IL-23/IFN-γ-positive feedback loop induced by abnormal intestinal macrophages contributes to the pathogenesis of chronic intestinal inflammation in patients with CD.

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Current View

Scoville David H., Sato Toshiro, He Xi C., Li Linheng

Gastroenterology 134 ( 3 ) 849 - 864 2008.03

Research paper (scientific journal), Joint Work, ISSN 0016-5085

Studies using mice have yielded significant amounts of information regarding signaling pathways, such as Wnt, bone morphogenic protein, PtdIns(3,4,5) kinase, and Notch, involved in intestinal development and homeostasis, including stem cell regulation and lineage specification and maturation. However, attempts to model signals definitively that control intestinal stem cells have been difficult because of a long-standing and recently reenergized debate surrounding their location. Although crypt-based columnar cells have been recently shown to display self-renewal and multipotential capacity, a large body of evidence supports long-term label-retaining cells, located on average at the +4 position just above the Paneth cells, as putative stem cells. Herein, we propose that both these cell types represent true intestinal stem cells maintained in different states (quiescent vs actively cycling), presumably via interactions with different microenvironments. Finally, we review current findings regarding the roles of Wnt, bone morphogenic protein, PtdIns(3,4,5) kinase, and Notch pathways within the intestine.

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A pilot open-labeled prospective randomized study between weekly and intensive treatment of granulocyte and monocyte adsorption apheresis for active ulcerative colitis

Sakuraba Atsushi, Sato Toshiro, Naganuma Makoto, Morohoshi Yuichi, Matsuoka Katsuyoshi, Inoue Nagamu, Takaishi Hiromasa, Ogata Haruhiko, Iwao Yasushi, Hibi Toshifumi

Journal of Gastroenterology 43 ( 1 ) 51 - 56 2008.01

Research paper (scientific journal), Joint Work, ISSN 0944-1174

Background. Recently, granulocyte and monocyte adsorption apheresis (GMA) has been shown to be effective for active ulcerative colitis (UC). Its original weekly treatment schedule is effective in about 70% of active UC. However, it takes about 3-4 weeks to achieve remission, and the efficacy of a more frequent treatment schedule has not been elucidated yet. We performed a pilot open-labeled prospective, randomized, controlled study comparing weekly and an intensive treatment schedule with three treatment sessions per week in the first 2 weeks. Methods. Thirty active UC patients with moderate disease activity were prospectively and randomly assigned to receive the original or the intensive treatment schedule for a total of ten sessions. The proportion of the patients achieving remission and the time to achieve remission among them was compared between the two groups. The incidences of adverse effects were also compared between the two groups. Results. The rate of inducing remission in the original and intensive treatment group was 66.7% and 80%, respectively (P = 0.25, NS). The time to achieve remission was 27.2 days in the original group and 10.7 days in the intensive group (P = 0.04). Adverse effects were observed in two patients in each groups (NS). Conclusions. Intensive treatment with GMAis an efficacious and safe treatment for active UC. Because it induces rapid remission, it may be a more ideal treatment regimen than the conventional weekly treatment.

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Lamina Propria c-kit⁺ Immune Precursors Reside in Human Adult Intestine and Differentiate Into Natural Killer Cells

Chinen Hiroshi, Matsuoka Katsuyoshi, Sato Toshiro, Kamada Nobuhiko, Okamoto Susumu, Hisamatsu Tadakazu, Kobayashi Taku, Hasegawa Hirotoshi, Sugita Akira, Kinjo Fukunori, Fujita Jiro, Hibi Toshifumi

Gastroenterology 133 ( 2 ) 559 - 573 2007.08

Research paper (scientific journal), Joint Work, ISSN 0016-5085

Background & Aims: Recent studies have revealed that murine intestinal mucosa contains several kinds of lineage markers (lin)^- c-kit⁺ immune precursor cells. However, immune precursors in the human adult intestine have not been studied extensively. Methods: Lamina propria mononuclear cells and intraepithelial lymphocytes from surgically resected human adult intestine were examined for the surface antigen expression and cytokine profile by immunohistochemistry and flow cytometry. The transcriptional profile of these cells was analyzed by reverse-transcription polymerase chain reaction. The phenotypic and functional characterization of the in vitro differentiating cells from the precursors was examined by flow cytometry. Results: We identified lin^- c-kit⁺ cells scattered throughout lamina propria of the human adult intestine. These intestinal immune precursors expressed CD34, CD38, CD33, interleukin-2Rα, and interleukin-7Rα, and they had much more abundant expression of Id2, PU.1, SpiB1, and lymphotoxin than thymocytes. The lin^- c-kit⁺ immune precursors mainly differentiated into CD56⁺ c-kit^dim cells during in vitro culture. These in vitro differentiating cells corresponded to intestinal natural killer (NK) cells, which had distinct characteristics from their peripheral counterparts, such as CD83 and integrin α_E expression, less cytotoxic activity, and higher interferon-γ production. Furthermore, both c-kit^dim cells and NK cells were increased in lamina propria of Crohn's disease, although there was no change for peripheral blood NK cells. Conclusions: The human intestine may have the unique NK cell differentiation system, which may contribute to maintenance of immune homeostasis in the intestine.

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PTEN-deficient intestinal stem cells initiate intestinal polyposis

He Xi C., Yin Tong, Grindley Justin C., Tian Qiang, Sato Toshiro, Tao W. Andy, Dirisina Raminarao, Porter-Westpfahl Kimberly S., Hembree Mark, Johnson Teri, Wiedemann Leanne M., Barrett Terrence A., Hood Leroy, Wu Hong, Li Linheng

Nature Genetics 39 ( 2 ) 189 - 198 2007.02

Research paper (scientific journal), Joint Work, ISSN 1061-4036

Intestinal polyposis, a precancerous neoplasia, results primarily from an abnormal increase in the number of crypts, which contain intestinal stem cells (ISCs). In mice, widespread deletion of the tumor suppressor Phosphatase and tensin homolog (PTEN) generates hamartomatous intestinal polyps with epithelial and stromal involvement. Using this model, we have established the relationship between stem cells and polyp and tumor formation. PTEN helps govern the proliferation rate and number of ISCs and loss of PTEN results in an excess of ISCs. In PTEN-deficient mice, excess ISCs initiate de novo crypt formation and crypt fission, recapitulating crypt production in fetal and neonatal intestine. The PTEN-Akt pathway probably governs stem cell activation by helping control nuclear localization of the Wnt pathway effector β-catenin. Akt phosphorylates β-catenin at Ser552, resulting in a nuclear-localized form in ISCs. Our observations show that intestinal polyposis is initiated by PTEN-deficient ISCs that undergo excessive proliferation driven by Akt activation and nuclear localization of β-catenin.

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Cytomegalovirus is frequently reactivated and disappears without antiviral agents in ulcerative colitis patients

Matsuoka Katsuyoshi, Iwao Yasushi, Mori Takeshi, Sakuraba Atsushi, Yajima Tomoharu, Hisamatsu Tadakazu, Okamoto Susumu, Morohoshi Yuichi, Izumiya Motoko, Ichikawa Hitoshi, Sato Toshiro, Inoue Nagamu, Ogata Haruhiko, Hibi Toshifumi

American Journal of Gastroenterology 102 ( 2 ) 331 - 337 2007.02

Research paper (scientific journal), Joint Work, ISSN 0002-9270

OBJECTIVE: The clinical significance of cytomegalovirus (CMV) reactivation complicating ulcerative colitis (UC) patients has been uncertain. It has therefore remained undetermined whether or not CMV reactivation should be treated in UC patients under immunosuppression. The aim of the study was to clarify the natural history of CMV reactivation in UC patients. METHODS: Sixty-nine UC patients with moderate to severe activity were enrolled in the study. All of the patients were treated with prednisolone, and/or immunosuppressants such as cyclosporine A. We sequentially monitored CMV reactivation every 2 wk up until 8 wk using the CMV antigenemia (Ag) assay and plasma quantitative real-time polymerase chain reaction (PCR) assay for CMV. RESULTS: Immunoglobulin (Ig) G for CMV was positive in 48 patients (69.6%) and negative in 21 patients (30.4%). CMV was reactivated in 25 patients out of the 48 seropositive patients (52.1%) during the study period. The CMV Ag and PCR values were low and none of the patients showed any evidence of CMV infection on biopsy specimens by hematoxylin and eosin staining. While gancylovir (GCV) was not used except in two patients, clinical outcomes including rates of remission and colectomy were not significantly different among the CMV reactivation-positive, -negative, and CMV IgG negative groups. Furthermore, CMV disappeared without GCV in most of the CMV reactivation-positive patients. CONCLUSIONS: CMV is frequently reactivated in active UC patients; however, it disappears without antiviral agents. Therefore, antiviral therapies should not be necessary for most UC patients with only CMV reactivation as long as CMV Ag values are low.

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Inhibition of neutrophil elastase prevents the development of murine dextran sulfate sodium-induced colitis

Morohoshi Yuichi, Matsuoka Katsuyoshi, Chinen Hiroshi, Kamada Nobuhiko, Sato Toshiro, Hisamatsu Tadakazu, Okamoto Susumu, Inoue Nagamu, Takaishi Hiromasa, Ogata Haruhiko, Iwao Yasushi, Hibi Toshifumi

Journal of Gastroenterology 41 ( 4 ) 318 - 324 2006.04

Research paper (scientific journal), Joint Work, ISSN 0944-1174

Background: Neutrophil elastase (NE) is a major secretory product from activated neutrophils and a major contributor to tissue destruction. However, little is known about the pathogenic contribution of NE to ulcerative colitis (UC). This study was designed to investigate the contribution of NE by measuring NE activity in plasma and colonic mucosal tissue from UC patients and a murine acute colitis model, and to elucidate the therapeutic effect of the NE-specific inhibitor ONO-5046. Methods: The NE enzyme activities in plasma and colonic mucosal tissue from UC patients were directly measured using an enzyme-substrate reaction. Acute colitis was induced in mice by administration of 1.5% dextran sulfate sodium (DSS) for 5 days. DSS-induced colitis mice were then treated with ONO-5046 (50 mg/kg body weight) intraperitoneally twice a day. Results: In UC patients, the NE enzyme activity was significantly elevated in both the plasma and colonic mucosal tissue compared with healthy controls. In DSS-induced colitis mice, the NE enzyme activity increased in parallel with the disease development. ONO-5046 showed therapeutic effects in DSS-treated mice by significantly reducing weight loss and histological score. ONO-5046 suppressed the NE enzyme activities in both plasma and culture supernatant of colonic mucosa from DSS-induced colitis mice. Conclusions: ONO-5046, a specific NE inhibitor, prevented the development of DSS-induced colitis in mice. NE therefore represents a promising target for the treatment of UC patients.

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Abnormally differentiated subsets of intestinal macrophage play a key role in Th1-dominant chronic colitis through excess production of IL-12 and IL-23 in response to bacteria

Kamada Nobuhiko, Hisamatsu Tadakazu, Okamoto Susumu, Sato Toshiro, Matsuoka Katsuyoshi, Arai Kumiko, Nakai Takaaki, Hasegawa Akira, Inoue Nagamu, Watanabe Noriaki, Akagawa Kiyoko S., Hibi Toshifumi

Journal of Immunology 175 ( 10 ) 6900 - 6908 2005.11

Research paper (scientific journal), Joint Work, ISSN 0022-1767

Disorders in enteric bacteria recognition by intestinal macrophages (Mφ) are strongly correlated with the pathogenesis of chronic colitis; however the precise mechanisms remain unclear. The aim of the current study was to elucidate the roles of Mφ in intestinal inflammation by using an IL-10-deficient (IL-10^-/-) mouse colitis model. GM-CSF-induced bone marrow-derived Mφ (GM-Mφ) and M-CSF-induced bone marrow-derived Mφ (M-Mφ) were generated from bone marrow CD11b⁺ cells. M-Mφ from IL-10^-/- mice produced abnormally large amounts of IL-12 and IL-23 upon stimulation with heat-killed whole bacteria Ags, whereas M-Mφ from wild-type (WT) mice produced large amounts of IL-10 but not IL-12 or IL-23. In contrast, IL-12 production by GM-Mφ was not significantly different between WT and IL-10^-/- mice. In ex vivo experiments, cytokine production ability of colonic lamina propria Mφ (CLPMφ) but not splenic Mφ from WT mice was similar to that of M-Mφ, and CLPMφ but not splenic Mφ from IL-10^-/- mice also showed abnormal IL-12p70 hyperproduction upon stimulation with bacteria. Surprisingly, the abnormal IL-12p70 hyperproduction from IL-10^-/- from IL-10^-/- mice was improved by IL-10 supplementation during the differentiation process. These results suggest that CLPMφ and M-Mφ act as anti-inflammatory Mφ and suppress excess inflammation induced by bacteria in WT mice. In IL-10 ^-/- mice, however, such Mφ subsets differentiated into an abnormal phenotype under an IL-10-deficient environment, and bacteria recognition by abnormally differentiated subsets of intestinal Mφ may lead to Th1-dominant colitis via IL-12 and IL-23 hyperproduction. Our data provide new insights into the intestinal Mφ to gut flora relationship in the development of colitis in IL-10^-/- mice.

Osteopontin/Eta-1 upregulated in Crohn's disease regulates the Th1 immune response

Sato T., Nakai T., Tamura N., Okamoto S., Matsuoka K., Sakuraba A., Fukushima T., Uede T., Hibi T.

Gut 54 ( 9 ) 1254 - 1262 2005.09

ISSN 0017-5749

Background and aims: The pathogenesis of Crohn's disease (CD), a chronic inflammatory bowel disease characterised by a Th1 immune response, remains unclear. Osteopontin (OPN) is a phosphoprotein known as an adhesive bone matrix protein. Recent studies have shown that OPN plays an important role in lymphocyte migration, granuloma formation, and interleukin 12 (IL-12) production. The present study investigated expression and the pathophysiological role of OPN in CD. Methods: Plasma OPN concentration was measured by enzyme linked immunosorbent assay. Expression of OPN in human intestinal mucosa was determined using reverse transcription-polymerase chain reaction and western blot, and localisation of OPN was examined by immunohistochemistry. Expression of integrin s, an OPN receptor, on lamina propria mononuclear cells (LPMC) was assessed by flow cytometry. Functional activation of OPN in LPMC was investigated by measuring the production of cytokines. Results: Plasma OPN concentration was significantly higher in patients with CD compared with normal controls or patients with ulcerative colitis (UC). OPN was upregulated in intestinal mucosa from UC and CD patients. OPN producing cells were epithelial or IgG producing plasma cells, or partial macrophages. OPN was detected in areas surrounding granuloma from mucosa in CD. Integrin βs expressing macrophages infiltrated inflamed mucosa in UC and CD; in contrast, there was no expression of integrin βs on intestinal macrophages in normal mucosa. OPN induced production of IL-12 from LPMC in CD but not in normal controls or UC. Conclusions: Increased OPN expression facilitates cytokine production and is closely involved in the Th1 immune response associated with CD.

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