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Background: Bladder transitional cell carcinoma greatly threatens human health all over the world. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) shows a strong apoptosis-inducing effect on a variety of cancer cells including bladder cancer. However, adenovirus-mediated TRAIL expression still showed cytotoxicity to normal cells mainly due to lack of tumor specificity. Methods: To solve the problem, we applied miRNA response elements (MREs) of miR-1, miR-133 and miR-218 to confer TRAIL expression with specificity to bladder cancer cells. Results: Expression of miR-1, miR-133 and miR-218 was greatly decreased in bladder cancer than normal bladder tissue. Luciferase assay showed that application of the 3 MREs was able to restrain exogenous gene expression to within bladder cancer cells. Subsequently, we constructed a recombinant adenovirus with TRAIL expression regulated by MREs of miR-1, miR-133 and miR-218, namely Ad-TRAIL-MRE-1-133-218. qPCR, immunoblotting and ELISA assays demonstrated that Ad-TRAIL-MRE-1-133-218 expressed in bladder cancer cells, rather than normal bladder cells. The differential TRAIL expression also led to selective apoptosis-inducing and growth-inhibiting effect of Ad-TRAIL-MRE-1-133-218 on bladder cancers. Finally, bladder cancer xenograft in mouse models further confirmed that Ad-TRAIL-MRE-1-133-218 effectively suppressed the growth of bladder cancers. Conclusions: Collectively, we demonstrated that MREs-based TRAIL delivery into bladder cancer cells was feasible and efficient for cancer gene therapy. [ABSTRACT FROM AUTHOR]

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The mouse incisor is a valuable but under-utilized model organ for studying the behavior of adult stem cells. This remarkable tooth grows continuously throughout the animal's lifetime and houses two distinct epithelial stem cell niches called the labial and lingual cervical loop (laCL and liCL, respectively). These stem cells produce progeny that undergo a series of well-defined differentiation events en route to becoming enamel-producing ameloblasts. During this differentiation process, the progeny move out of the stem cell niche and migrate toward the distal tip of the tooth. Although the molecular pathways involved in tooth development are well documented, little is known about the roles of miRNAs in this process. We used microarray technology to compare the expression of miRNAs in three regions of the adult mouse incisor: the laCL, liCL, and ameloblasts. We identified 26 and 35 differentially expressed miRNAs from laCL/liCL and laCL/ameloblast comparisons, respectively. Out of 10 miRNAs selected for validation by qPCR, all transcripts were confirmed to be differentially expressed. In situ hybridization and target prediction analyses further supported the reliability of our microarray results. These studies point to miRNAs that likely play a role in the renewal and differentiation of adult stem cells during stem cell-fueled incisor growth. [ABSTRACT FROM AUTHOR]

Background: Certain murine leukemia viruses (MLVs) are capable of inducing progressive spongiform motor neuron disease in susceptible mice upon infection of the central nervous system (CNS). The major CNS parenchymal target of these neurovirulent retroviruses (NVs) are the microglia, whose infection is largely coincident with neuropathological changes. Despite this close association, the role of microglial infection in disease induction is still unknown. In this paper, we investigate the interaction of the highly virulent MLV, FrCasE, with microglia ex vivo to evaluate whether infection induces specific changes that could account for neurodegeneration. Specifically, we compared microglia infected with FrCasE, a related non-neurovirulent virus (NN) F43/Fr57E, or mock-infected, both at a basic virological level, and at the level of cellular gene expression using quantitative real time RT-PCR (qRT-PCR) and Afffymetrix 430A mouse gene chips. Results: Basic virological comparison of NN, NV, and mock-infected microglia in culture did not reveal differences in virus expression that provided insight into neuropathogenesis. Therefore, microglial analysis was extended to ER stress gene induction based on previous experiments demonstrating ER stress induction in NV-infected mouse brains and cultured fibroblasts. Analysis of message levels for the ER stress genes BiP (grp78), CHOP (Gadd153), calreticulin, and grp58 in cultured microglia, and BiP and CHOP in microglia enriched fractions from infected mouse brains, indicated that FrCasE infection did not induce these ER stress genes either in vitro or in vivo. To broadly identify physiological changes resulting from NV infection of microglia in vitro, we undertook a gene array screen of more than 14,000 well-characterized murine genes and expressed sequence tags (ESTs). This analysis revealed only a small set of gene expression changes between infected and uninfected cells (<18). Remarkably, gene array comparison of NN- and NVinfected microglia revealed only 3 apparent gene expression differences. Validation experiments for these genes by Taqman real-time RT-PCR indicated that only single Ig IL-1 receptor related protein (SIGIRR) transcript was consistently altered in culture; however, SIGIRR changes were not observed in enriched microglial fractions from infected brains. Conclusion: The results from this study indicate that infection of microglia by the highly neurovirulent virus, FrCasE, does not induce overt physiological changes in this cell type when assessed ex vivo. In particular, NV does not induce microglial ER stress and thus, FrCasE-associated CNS ER stress likely results from NV interactions with another cell type or from neurodegeneration directly. The lack of NV-induced microglial gene expression changes suggests that FrCasE either affects properties unique to microglia in situ, alters the expression of microglial genes not represented in this survey, or affects microglial cellular processes at a post-transcriptional level. Alternatively, NV-infected microglia may simply serve as an unaffected conduit for persistent dissemination of virus to other neural cells where they produce acute neuropathogenic effects. [ABSTRACT FROM AUTHOR]

: Aims Lipopolysaccharide (LPS) induces cardiomyocyte caspase-3 activation and proinflammatory factors, in particular tumour necrosis factor-alpha (TNF-α) production, both of which contribute to myocardial dysfunction during sepsis. The present study was to investigate the roles of calpain/calpastatin system in cardiomyocyte caspase-3 activation, TNF-α expression, and myocardial dysfunction during LPS stimulation. : Methods and results In cultured adult rat cardiomyocytes, LPS (1 µg/mL) induced calpain and caspase-3 activity, and up-regulated TNF-α expression. These effects of LPS were abrogated by over-expression of calpastatin, an endogenous calpain inhibitor, transfection of calpain-1 siRNA, or various pharmacological calpain inhibitors. Furthermore, blocking gp91phox-NADPH oxidase prevented calpain and caspase-3 activation and decreased TNF-α expression in LPS-stimulated cardiomyocytes. To investigate the role of calpastatin in endotoxaemia, transgenic mice with calpastatin over-expression (CAST-Tg) and wild-type mice were treated with LPS (4 mg/kg, i.p.) or saline in the presence of calpain inhibitor-III (10 mg/kg, i.p.) for 4 h, and their heart function was measured with a Langendorff system. Over-expression of calpastatin significantly attenuated myocardial dysfunction (P < 0.05). Consistently, calpain activity, caspase-3 activity, and TNF-α expression were also reduced in CAST-Tg and calpain inhibitor-III compared with wild-type and vehicle-treated hearts, respectively. : Conclusion gp91phox-NADPH oxidase-mediated calpain-1 activation induces caspase-3 activation and TNF-α expression in cardiomyocytes during LPS stimulation. Over-expression of calpastatin inhibits calpain activation and improves myocardial function in endotoxaemia. The present study suggests that targeting calpain/calpastatin system may be a potential therapeutic intervention for septic hearts. [ABSTRACT FROM AUTHOR]

IL-22 is a cytokine that regulates tissue homeostasis at barrier surfaces. A variety of IL-22-producing cell types is known, but identification on the single-cell level remains difficult. Therefore, we generated a fate reporter mouse that would allow the identification of IL-22-producing cells and their fate mapping in vivo. To trace IL-22-expressing cells, a sequence encoding Cre recombinase was cloned into the Il22 locus, and IL22Cre mice were crossed with reporter mice expressing enhanced yellow fluorescence protein (eYFP) under control of the endogenous Rosa26 promoter. In IL22CreR26ReYFP mice, the fluorescent reporter permanently labels cells that have switched on Il22 expression, irrespective of cytokine production. Despite a degree of underreporting, eYFP expression was detectable in nonimmune mice and restricted to group 3 innate lymphoid cells (ILC3) in the gut and γδ T cells in skin or lung. Upon skin challenge with imiquimod, eYFP+ γδ and CD4 T cells expanded in the skin. Infection with Citrobacter rodentium initially was controlled by ILC3, followed by expansion of eYFP+ CD4 T cells, which were induced in innate lymphoid follicles in the colon. No eYFP expression was detected in small intestinal Th17 cells, and they did not expand in the immune response. Colonic eYFP+ CD4 T cells exhibited plasticity during infection with expression of additional cytokines, in contrast to ILC3, which remained largely stable. Single-cell quantitative PCR analysis of eYFP+ CD4 T cells confirmed their heterogeneity, suggesting that IL-22 expression is not confined to particular subsets or a dedicated Th22 subset. [ABSTRACT FROM AUTHOR]