Distinct Roles of α7 nAChRs in Antigen-Presenting Cells and CD4+ T Cells in the Regulation of T Cell Differentiation.

It is now apparent that immune cells express a functional cholinergic system and that α7 nicotinic acetylcholine receptors (α7 nAChRs) are involved in regulating T cell differentiation and the synthesis of antigen-specific antibodies and proinflammatory cytokines. Here, we investigated the specific function α7 nAChRs on T cells and antigen presenting cells (APCs) by testing the effect of GTS-21, a selective α7 nAChR agonist, on differentiation of CD4+ T cells from ovalbumin (OVA)-specific TCR transgenic DO11.10 mice activated with OVA or OVA peptide323-339 (OVAp). GTS-21 suppressed OVA-induced antigen processing-dependent development of CD4+ regulatory T cells (Tregs) and effector T cells (Th1, Th2, and Th17). By contrast, GTS-21 up-regulated OVAp-induced antigen processing-independent development of CD4+ Tregs and effector T cells. GTS-21 also suppressed production of IL-2, IFN-γ, IL-4, IL-17, and IL-6 during OVA-induced activation but, with the exception IL-2, enhanced their production during OVAp-induced activation. In addition, during antigen-nonspecific, APC-independent anti-CD3/CD28 antibody-induced CD4+ polyclonal T cell activation in the presence of respective polarizing cytokines, GTS-21 promoted development of all lineages, which indicates that GTS-21 also acts via α7 nAChRs on T cells. These results suggest 1) that α7 nAChRs on APCs suppress CD4+ T cell activation by interfering with antigen presentation through inhibition of antigen processing; 2) that α7 nAChRs on CD4+ T cells up-regulate development of Tregs and effector T cells; and that α7 nAChR agonists and antagonists could be potentially useful agents for immune response modulation and enhancement.

Visualization and in vivo tracking of the exosomes of murine melanoma B16-BL6 cells in mice after intravenous injection.

The development of exosomes as delivery vehicles requires understanding how and where exogenously administered exosomes are distributed in vivo. In the present study, we designed a fusion protein consisting of Gaussia luciferase and a truncated lactadherin, gLuc-lactadherin, and constructed a plasmid expressing the fusion protein. B16-BL6 murine melanoma cells were transfected with the plasmid, and exosomes released from the cells were collected by ultracentrifugation. Strong luciferase activity was detected in the fraction containing exosomes, indicating their efficient labeling with gLuc-lactadherin. Then, the labeled B16-BL6 exosomes were intravenously injected into mice, and their tissue distribution was evaluated. Pharmacokinetic analysis of the exosome blood concentration-time profile revealed that B16-BL6 exosomes disappeared very quickly from the blood circulation with a half-life of approximately 2min. Little luciferase activity was detected in the serum at 4h after exosome injection, suggesting rapid clearance of B16-BL6 exosomes in vivo. Moreover, sequential in vivo imaging revealed that the B16-BL6 exosome-derived signals distributed first to the liver and then to the lungs. These results indicate that gLuc-lactadherin labeling is useful for tracing exosomes in vivo and that B16-BL6 exosomes are rapidly cleared from the blood circulation after systemic administration.

Age-related effects of fasting on ketone body production during lipolysis in rats.

OBJECTIVE: The age-related effects of fasting on lipolysis, the production of ketone bodies, and plasma insulin levels were studied in male 3-, 8-, and 32-week-old Sprague-Dawley rats. METHODS: The rats were divided into fasting and control groups. The 3-, 8- and 32-week-old rats tolerated fasting for 2, 5, and 12 days, respectively. RESULTS: Fasting markedly reduced the weights of perirenal and periepididymal white adipose tissues in rats in the three age groups. The mean rates of reduction in both these adipose tissue weights during fasting periods were higher in the order of 3 > 8 > 32-week-old rats. Fasting transiently increased plasma free fatty acid (FFA), total ketone body, ß-hydroxybutyrate, and acetoacetate concentrations in the rats in the three age groups. However, plasma FFA, total ketone body, ß-hydroxybutyrate, and acetoacetate concentrations in the 3-week-old rats reached maximal peak within 2 days after the onset of fasting, although these concentrations in the 8- and 32-week-old rats took more than 2 days to reach the maximal peak. By contrast, the augmentation of plasma FFA, total ketone body, ß-hydroxybutyrate, and acetoacetate concentrations in the rats in the three age groups had declined at the end of each experimental period. Thus, the capacity for fat mobilization was associated with tolerance to fasting. Plasma insulin concentrations in the rats in the three age groups were dramatically reduced during fasting periods, although basal levels of insulin were higher in the order of 32 > 8 > 3 week-old rats. CONCLUSION: These results suggest that differences in fat metabolism patterns among rats in the three age groups during prolonged fasting were partly reflected the metabolic turnover rates, plasma insulin levels, and amounts of fat storage.

Positive correlation between the generation of reactive oxygen species and activation/reactivation of transgene expression after hydrodynamic injections into mice.

PURPOSE: Hydrodynamic injection has been shown to reactivate silenced transgene expression in mouse liver. In this study, the roles of inflammatory cytokines and reactive oxygen species (ROS) in the reactivation were examined. METHODS: Production of inflammatory cytokines and ROS by hydrodynamic injection of saline was examined in mice that had received a hydrodynamic injection of a plasmid expressing Gaussia luciferase. The level of reporter gene expression was used as an indicator of the reactivation. The involvement of cytokines and ROS was examined by depleting Kupffer cells or by pre-administration of antioxidants, respectively. RESULTS: A hydrodynamic injection of saline induced a significant production of interleukin (IL)-6. Depleting Kupffer cells using clodronate liposomes markedly reduced the IL-6 production but had no significant effect on the transgene expression. On the other hand, an injection of catalase or N-acetylcysteine significantly inhibited the hydrodynamic injection-induced reactivation of silenced transgene expression. The silenced expression was also reactivated by carbon tetrachloride, an inducer of oxidative stress in the liver, in a dose-dependent manner, and this reactivation was significantly inhibited by catalase. CONCLUSIONS: These findings show a positive correlation between the generation of ROS and the reactivation of silenced transgene expression after hydrodynamic injections.