Caveolin-1 (Cav-1) is a membrane protein. Recently, it has been reported that secreted Cav-1 induces angiogenesis in inflammatory microenvironment. However, it is unclear that Cav-1 regulates gingival inflammation. Therefore, we investigated the Cav-1 function to periodontal cells. Expression of Cav-1 in human periodontitis tissues was examined pathologically. Secretion of Cav-1 from human gingival fibroblasts (HGFs) or human periodontal ligament cells (HPLFs) treated with IL-1β and TNF-α was examined using Western blotting. Likewise, intracellular signals induced by Cav-1 were examined. Finally, we examined whether the secreted Cav-1 induces production of inflammatory mediators in HGFs using ELISA or qRT-PCR. Pathologically, high expression of Cav-1 was observed in human periodontitis tissues. Cav-1 secretion increased in both cultured HGFs and HPLFs treated with IL-1β and TNF-α. Cav-1 induced phosphorylation of JNK and ERK, but not Stat3 in HGFs. Furthermore, Cav-1 increased proMMP-1 and VEGF secretion in HGFs, and the VEGF secretion was statistically suppressed by JNK inhibitor SP600125, but not ERK inhibitor PD98059. ProMMP-1 secretion was suppressed statistically by both SP600125 and PD98059. In addition, Cav-1 increased significantly MMP-1, -10 and -14 mRNA expressions, whereas no increase of TIMPs mRNA was observed in HGFs treated with Cav-1. These data suggest that secreted Cav-1 derived from periodontal fibroblastic cells enhances inflammation-related several proteases and VEGF secretion in HGFs via MAPKs pathway, resulting in progression of periodontitis through induction of tissue degradation or angiogenesis.
Human malignant pleural mesothelioma (MPM) is highly aggressive, and its prognosis is very poor. For an early diagnosis of MPM and developing new therapeutic strategies against the malignancy, it is necessary to better understand biological characteristics of MPM. In this study, we established two cell lines from pleural effusions derived from patients with MPM. Both cell lines expressed tumor markers of mesothelioma such as mesothelin, podoplanin, WT1, calretinin and keratin 5/6 whereas they did not express either CEA or TTF-1 which are often used as markers of lung adenocarcinoma. The cell lines harboured wild-type TP53, produced hyaluronic acid, and were not infected with SV40. When these two cell lines were cultured under hypoxia (1% O2), they showed particular responses to the hypoxic condition, distinct from those to normoxic condition (21% O2). Namely, the ability to form a colony originating from a single cell (plating efficiency and cloning efficiency) was stimulated under hypoxia in both cell lines. On the other hand, when the assays of cell growth were started at a relatively high cell density, the growth of both cell lines, regardless of anchorage-dependent or -independent, decreased under hypoxia. The differences of their growth between under hypoxia and under normoxia, and those depending on the cell density, may provide useful hints for developing a new strategy for diagnosis or therapy of MPM.
Therapeutic angiogenesis has emerged as one of the most promising therapies for severe ischemic cardiovascular diseases with no optional therapy. Several investigators have reported that transplantation of cultured adipose-derived regenerative cells (cADRCs) to ischemic tissues promotes neovascularization and blood perfusion recovery; however, cell therapy using cultured cells has several restrictions. To resolve this problem, the angiogenic capacity of freshly isolated ADRCs (fADRCs) obtained from Lewis rats was compared with cADRCs, both in vivo and in vitro. Flow cytometric analysis showed that fADRCs contained several cell types such as endothelial progenitor cells and endothelial cells; however, these cells were present in a very small proportion in cADRCs. Transplantation of fADRCs in mice significantly improved blood perfusion, capillary density, and production of several angiogenic factors in transplanted ischemic limbs compared with a saline-injected group, whereas these effects were not observed in the cADRCs-injected group. fADRCs also showed significantly higher expression levels of angiogenic factors than cADRCs in the in vitro study. Furthermore, fADRC stimulated tube formation more remarkably than cADRC in an in vitro tube formation assay. These results suggested that fADRCs have an effective angiogenic capacity, and they would be more valuable as a source for cell-based therapeutic angiogenesis than cADRCs or other stem/progenitor cells.
Internleukin-1 (IL-1) and IL-6 are the most potent proinflammatory cytokines being involved in inflammatory diseases such as periodontitis. The objective of this study was to examine the synergistic effects of IL-1β and IL-6 on gingival inflammation by targeting cultured human gingival fibroblasts (HGFs). HGFs were treated with IL-1β or IL-6/soluble IL-6R (sIL-6R), and total RNA and total cell lysate were collected to examine expression of gp130 known as a signal transducer of IL-6 using qRT-PCR and Western blotting. IL-1β-mediated IL-6 productivity in HGFs was examined using ELISA method. Likewise, after HGFs and THP-1 macrophages were treated with IL-1β, TNF-α and IL-6, sIL-6R productivity was examined. Next, HGFs were treated with IL-6/ sIL-6R after pretreatment of IL-1β, and the intracellular signals were examined using Western blotting. Finally, various mRNA/protein expressions in HGFs treated with IL-6/sIL-6R after pretreatment of IL-1β were examined using qRT-PCR and ELISA method. IL-1β increased significantly both gp130 and IL-6 expression in HGFs. IL-6 increased significantly sIL-6R production in THP-1 macrophages but not HGFs. Co-stimulation with IL-1β and IL-6/sIL-6R induced dramatically the phosphorylation of Stat3, ERK and JNK in HGFs. Interestingly, expression of various inflammation- related molecules such as MMP-1, MCP-1, IL-1ra, bFGF and VEGF were enhanced by co-stimulation with IL-1β and IL-6/sIL-6R in HGFs. Gingival inflammation is regulated by HGFs affected by both IL-1β and IL-6/sIL-6R synergistically through induction of gp130 expression, resulting in progression of periodontitis.
Wilms' tumor gene 1 (WT1) has been proposed as an attractive target for cancer immunotherapy. A natural 9-mer peptide (CYTWNQMNL), which bound to human leukocyte antigen (HLA)-A∗24:02, was identified from among WT1-specific cytotoxic T lymphocyte (CTL) epitopes. This natural WT1 CTL epitope peptide was further modified (CMTWNQMNL) to enhance its binding affinity to HLA-A∗24:02. This modified WT1 CTL epitope peptide was superior to the natural peptide for inducing HLA-A∗24:02-restricted WT1-specific CTLs. Here we induced several WT1 CTLs that reacted with both modified and natural WT1 tetramers from peripheral blood mononuclear cells. Then, T-cell receptor (TCR) genes were isolated from these WT1 CTLs to determine their Vα and Vβ usage. These TCR genes were transduced into human T lymphoma cells to establish a stable cell line, SK37, which expressed a WT1-specific TCR. We confirmed that SK37 cells reacted with both modified and natural WT1 tetramers, which indicated that SK37 cells could be a useful tool for WT1 tetramer reagent quality assurance. One the basis of these findings, we propose that this WT1 tetramer, which was quality-assured using established SK37 cells, will contribute to reliable immunomonitoring of tumor-specific CTL responses of cancer patients who receive WT1-targeted cancer vaccine therapy or TCR-gene therapy.
We have previously shown the duct system in the rat pancreas to consist of two parts: a fine proximal (intercalated) duct and thicker distal (intralobular and interlobular) duct, with the latter part displaying morphological signs indicative of a bicarbonate-rich fluid secretion. In this study the pancreatic duct system in the Japanese monkey Macaca fuscata was observed by scanning electron microscopy after the hydrolytic exposure of cell surfaces as well as by transmission electron microscopy of ultrathin sections. Cellular expression of the water channel aquaporin 1 (AQP1) was also examined immunohistochemically. In contrast to the segmented duct system in the rat, all the duct cells in the monkey pancreas consistently displayed rich mitochondria in the cytoplasm, elaborate interdigitations of cell processes, and an intense immunoreactivity for AQP1 on the apical and basolateral cell membrane to favor active ion transport and osmotic water movement across the epithelium. Both the existence of secretory canaliculi and basal trabeculae in the duct epithelium and randomized localization of primary cilia on the luminal cell surfaces were demonstrated for the first time in monkeys, and the physiological implications of these phenomena are discussed.