41 research outputs found

    Essential roles of DC-derived IL-15 as a mediator of inflammatory responses in vivo

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    Interleukin (IL)-15 is expressed in a variety of inflammatory diseases. However, the contribution of dendritic cell (DC)–derived IL-15 to the development of diseases is uncertain. Using established models of Propionibacterium acnes (P. acnes)– and zymosan-induced liver inflammation, we observed granuloma formation in the livers of wild-type (WT) and RAG-2−/− mice but not in those of IL-15−/− mice. We demonstrate that this is likely caused by an impaired sequential induction of IL-12, IFN-γ, and chemokines necessary for monocyte migration. Likewise, lethal endotoxin shock was not induced in P. acnes– and zymosan-primed IL-15−/− mice or in WT mice treated with a new IL-15–neutralizing antibody. In both systems, proinflammatory cytokine production was impaired. Surprisingly, neither granuloma formation, lethal endotoxin shock, nor IL-15 production was induced in mice deficient for DCs, and adoptive transfer of WT but not IL-15−/− DCs restored the disease development in IL-15−/− mice. Collectively, these data indicate the importance of DC-derived IL-15 as a mediator of inflammatory responses in vivo

    Cell surface markers of functional phenotypic corneal endothelial cells. Invest Ophthalmol Vis Sci 2014

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    PURPOSE. Cultured human corneal endothelial cells (HCECs) are anticipated to serve as an alternative to donor corneas for the treatment of corneal endothelial dysfunction. However, corneal endothelial cells (CECs) tend to exhibit fibroblastic transformation, thereby losing their functional phenotype when cultured. The purpose of this study was to investigate the usefulness of surface markers of CECs displaying fibroblastic phenotypes as a means of cell characterization. METHODS. The expression levels of 242 cell surface antigens were screened in cultured human and monkey CECs using flow cytometry. An expression intensity ratio of nonfibroblastic/ fibroblastic CECs > 2 and of fibroblastic/nonfibroblastic CECs > 2 were selected as indicating nonfibroblastic and fibroblastic markers, respectively. Nonfibroblastic and fibroblastic CECs were mixed, and CD73-positive and -negative cells were sorted using flow cytometry and further cultured. The functional phenotype of the sorted cells was evaluated according to morphology and the expression of function-related (Na þ /K þ -ATPase and ZO-1) and fibroblastic (type I collagen and fibronectin) markers. RESULTS. Flow cytometry analysis demonstrated that CD98, CD166, and CD340 are elevated in HCECs of nonfibroblastic phenotype, while CD9, CD49e, CD44, and CD73 are markers of fibroblastic phenotype HCECs. The CECs that sorted as CD73-negative exhibited normal hexagonal morphology and expressed functional markers, whereas CECs that sorted as CD73-positive exhibited the fibroblastic phenotype. CONCLUSIONS. These markers will be useful for quality control to characterize the phenotype of cells destined for tissue engineering-based therapy. In addition, this selection protocol will provide a novel method for purification of functional cells

    Osteopontin as a Mediator of NKT Cell Function in T Cell-Mediated Liver Diseases

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    AbstractBoth osteopontin (OPN) and natural killer T (NKT) cells play a role in the development of immunological disorders. We examined a functional link between OPN and NKT cells. Concanavalin A (Con A)-induced hepatitis is a well-characterized murine model of T cell-mediated liver diseases. Here, we show that NKT cells secrete OPN, which augments NKT cell activation and triggers neutrophil infiltration and activation. Thus, OPN- and NKT cell-deficient mice were refractory to Con A-induced hepatitis. In addition, a neutralizing antibody specific for a cryptic epitope of OPN, exposed by thrombin cleavage, ameliorated hepatitis. These findings identify NKT cell-derived OPN as a novel target for the treatment of inflammatory liver diseases

    コウシュヨウセイタトウルイノセイカガクテキケンキュウ

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    京都大学0048新制・論文博士工学博士乙第2560号論工博第703号新制||工||282(附属図書館)4116UT51-49-E188(主査)教授 松浦 輝男, 教授 福井 三郎, 教授 庄野 達哉学位規則第5条第2項該当Kyoto UniversityDA

    Gene Signature-Based Development of ELISA Assays for Reproducible Qualification of Cultured Human Corneal Endothelial Cells

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    Citation: Ueno M, Asada K, Toda M, et al. Gene signature-based development of ELISA assays for reproducible qualification of cultured human corneal endothelial cells. Invest Ophthalmol Vis Sci. 2016;57:4295-4305. DOI:10.1167/iovs.16-19806 PURPOSE. To develop a method to qualify the function of cultured human corneal endothelial cells (cHCECs) applicable for clinical settings. METHODS. The diversified gene and microRNA (miRNA) signatures in HCECs from a variety of tissue donors were confirmed by three-dimensional (3D) gene human miRNA profiling. These were compared with those of more than 20 cHCECs distinct in their cell morphology or culture lots. Candidate genes were selected after quantitative (q)RT-PCR validation, and gene products were assayed by ELISA. After three additional screening steps, final candidate cytokines for qualification were selected. RESULTS. Gene and miRNA signatures among distinct cHCEC lots were greatly diversified compared with those among fresh tissues from different age donors. By comparing more than 20 lots of cultures, 32 candidate genes were assigned to be seemingly linked to distinct cHCEC morphologic features. The validation of candidate genes by qRT-PCR revealed the genes, either upregulated or downregulated, corresponding to morphologic variances in cHCECs (e.g., epithelial-mesenchymal transition or cell senescence). Further adding the ELISA results by BioPlex Human Cytokine 27-Plex Panel, 11 candidate cytokines suitable to qualify cHCEC function were selected. In consideration of the presence of these cytokines in the anterior chamber, IL-8, tissue inhibitors of metalloproteinases 1 (TIMP-1), monocyte chemotactic protein-1 (MCP-1), and platelet-derived growth factor-BB (PDGF-BB) were ultimately selected and applied in practice for the qualification of cHCECs actually used in our clinical cell-injection studies. CONCLUSIONS. The specified cytokines properly discriminating the functional features of cHCECs indicates a correlation between profiling signatures and cell morphology. Keywords: gene signature, miRNA, cultured corneal endothelial cells, cytokines T o date, most researchers conceptualize cultured human corneal endothelial cells (cHCECs) only from the aspect that they are derived from corneal endothelium tissue, and disregard details pertaining to the refinement of the biochemical features. In fact, cHCECs contain subpopulations (SPs) that are heterogeneous in their morphology and in their surface markers. 1-3 Studies, either directly or indirectly, including those from Joyce et al., 1 of the presence of frequent chromosomal aneuploidy in cHCECs. Cultures HCECs have an inclination toward cell state transition (CST) into a senescence phenotype, epithelial-mesenchymal transition (EMT), and fibroblastic cell morphology. These findings indicate the necessity of qualifying the features of heterogeneous cHCECs by means of biochemical terms in order to distinguish vulnerable transformed cHCECs 2 and CST (i.e., in order to clarify in detail the fine and distinct characteristics among cHCECs). It is well known that the proliferative potential of HCECs is limited, Trials pertaining to the in vitro expansion of cHCECs without CST and karyotype aneuploidy have been hampered by iovs.arvojournals.org j ISSN: 1552-5783 4295 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License

    Inhibition of TGF-β signaling enables human corneal endothelial cell expansion in vitro for use in regenerative medicine.

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    Corneal endothelial dysfunctions occurring in patients with Fuchs' endothelial corneal dystrophy, pseudoexfoliation syndrome, corneal endotheliitis, and surgically induced corneal endothelial damage cause blindness due to the loss of endothelial function that maintains corneal transparency. Transplantation of cultivated corneal endothelial cells (CECs) has been researched to repair endothelial dysfunction in animal models, though the in vitro expansion of human CECs (HCECs) is a pivotal practical issue. In this study we established an optimum condition for the cultivation of HCECs. When exposed to culture conditions, both primate and human CECs showed two distinct phenotypes: contact-inhibited polygonal monolayer and fibroblastic phenotypes. The use of SB431542, a selective inhibitor of the transforming growth factor-beta (TGF-β) receptor, counteracted the fibroblastic phenotypes to the normal contact-inhibited monolayer, and these polygonal cells maintained endothelial physiological functions. Expression of ZO-1 and Na(+)/K(+)-ATPase maintained their subcellular localization at the plasma membrane. Furthermore, expression of type I collagen and fibronectin was greatly reduced. This present study may prove to be the substantial protocol to provide the efficient in vitro expansion of HCECs with an inhibitor to the TGF-β receptor, and may ultimately provide clinicians with a new therapeutic modality in regenerative medicine for the treatment of corneal endothelial dysfunctions

    Enhancement on primate corneal endothelial cell survival in vitro by a ROCK inhibitor. Invest Ophthalmol Vis Sci.

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    PURPOSE. The transplantation of cultivated corneal endothelial cells (CECs) has gained attention recently for the treatment of patients with corneal endothelial dysfunction. However, an efficient culturing technique for human (H)CECs has yet to be properly established. The present study was conducted to investigate the applicability of the Rho kinase (ROCK) inhibitor Y-27632 in promoting cultivation of cynomolgus monkey (M)CECs. METHODS. MCECs of cynomolgus monkeys were cultured in a medium containing 10 M Y-27632. The number of viable cells adherent to culture plates were monitored by a luminescent cell-viability assay and colony growth was detected by toluidine blue staining. Proliferating cells were detected by Ki67 expression using flow cytometry and a BrdU-labeling assay for immunocytochemistry. Annexin V-positive apoptotic cells were analyzed by flow cytometry. RESULTS. The number of viable cultivate
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