Induction of antigen-specific immunity by mesoporous silica nanoparticles incorporating antigen peptides.

Mesoporous silica nanoparticles (MSNs) are physically and chemically stable inorganic nanomaterials that have been attracting much attention as carriers for drug delivery systems in the field of nanomedicine. In the present study, we investigated the potential of MSN vaccines that incorporate antigen peptides for use in cancer immunotherapy. In vitro experiments demonstrated that fluorescently labeled MSNs accumulated in a line of mouse dendritic cells (DC2.4 cells), where the particles localized to the cytosol. These observations could suggest that MSNs have potential for use in delivering the loaded molecules into antigen-presenting cells, thereby stimulating the host acquired immune system. In vivo experiments demonstrated prolonged survival in mice implanted with ovalbumin (OVA)-expressing lymphoma cells (E.G7-OVA cells) following subcutaneous inoculation with MSNs incorporating OVA antigen peptides. Furthermore, OVA-specific immunoglobulin G antibodies and cytotoxic T lymphocytes were detected in the serum and the spleen cells, respectively, of mice inoculated with an MSN-OVA vaccine, indicating the induction of antigen-specific responses in both the humoral and cellular immune systems. These results suggested that the MSN therapies incorporating antigen peptides may serve as novel vaccines for cancer immunotherapy.

Myc upregulates Ggct, γ-glutamylcyclotransferase to promote development of p53-deficient osteosarcoma.

Osteosarcoma (OS) in humans is characterized by alterations in the TP53 gene. In mice, loss of p53 triggers OS development, for which c-Myc (Myc) oncogenicity is indispensable. However, little is known about which genes are targeted by Myc to promote tumorigenesis. Here, we examined the role of γ-glutamylcyclotransferase (Ggct) which is a component enzyme of the γ-glutamyl cycle essential for glutathione homeostasis, in human and mouse OS development. We found that GGCT is a poor prognostic factor for human OS, and that deletion of Ggct suppresses p53-deficient osteosarcomagenesis in mice. Myc upregulates Ggct directly by binding to the Ggct promoter, and deletion of a Myc binding site therein by genome editing attenuated the tumorigenic potential of p53-deficient OS cells. Taken together, these results show a rationale that GGCT is widely upregulated in cancer cells and solidify its suitability as a target for anticancer drugs.

C/ebpα represses the oncogenic Runx3-Myc axis in p53-deficient osteosarcoma development.

Osteosarcoma (OS) is characterized by TP53 mutations in humans. In mice, loss of p53 triggers OS development, and osteoprogenitor-specific p53-deleted mice are widely used to study the process of osteosarcomagenesis. However, the molecular mechanisms underlying the initiation or progression of OS following or parallel to p53 inactivation remain largely unknown. Here, we examined the role of transcription factors involved in adipogenesis (adipo-TFs) in p53-deficient OS and identified a novel tumor suppressive molecular mechanism mediated by C/ebpα. C/ebpα specifically interacts with Runx3, a p53 deficiency-dependent oncogene, and, in the same manner as p53, decreases the activity of the oncogenic axis of OS, Runx3-Myc, by inhibiting Runx3 DNA binding. The identification of a novel molecular role for C/ebpα in p53-deficient osteosarcomagenesis underscores the importance of the Runx-Myc oncogenic axis as a therapeutic target for OS.

Liver progenitor cells may construct cysts having heterogeneous gene expression of liver-enriched transcription factors in mice with conditional knockout of the Hhex gene.

The deletion of the Hhex (Hematopoietically expressed homeobox) gene causes agenesis of the liver and polycystic liver disease depending on its timing. The present study was undertaken to determine the role of the Hhex gene in not only signaling cascades to cyst and abnormal bile duct formation but also the liver progenitor contribution to cystic development. Liver-specific Hhex knockout mice (Alb-Cre/HhexloxP/loxP) in adult stages were used. Wild-type and conditional knockout (cKO) livers were immunohistologically compared for cell growth, and gene expression of liver functions, biliary markers and cystic markers. In Hhex cKO livers, cyst formation and dilated intrahepatic bile ducts were noted, which resembled the histology of the von Meyenburg complex. Ki67 immunohistochemistry showed that the growth activity in bile ducts and cysts of cKO livers was elevated compared with that of wild-type livers. There were far fewer liver progenitor cells or bile ductule cells around portal veins of cKO livers than in wild-type livers. Several liver-enriched transcription factors, including Foxa1 and Foxa2, were heterogeneously expressed in bile ducts and cysts of cKO livers whereas their expression in wild-type bile ducts was comparatively homogeneous. PC1 and PC2 immunohistochemistry revealed their up-regulation in cysts of cKO livers. These data indicate that Hhex is not only required for proper bile duct morphogenesis, but is also involved in cyst formation through promoted cell growth. Liver progenitor cells may form cysts. Unbalanced expression of liver-enriched transcription factors might be involved in cyst formation. Hhex cKO mice may be a good animal model for hepatic cystic diseases.

Runx3 is required for oncogenic Myc upregulation in p53-deficient osteosarcoma.

Osteosarcoma (OS) in human patients is characterized by genetic alteration of TP53. Osteoprogenitor-specific p53-deleted mice (OS mice) have been widely used to study the process of osteosarcomagenesis. However, the molecular mechanisms responsible for the development of OS upon p53 inactivation remain largely unknown. In this study, we detected prominent RUNX3/Runx3 expression in human and mouse p53-deficient OS. Myc was aberrantly upregulated by Runx3 via mR1, a consensus Runx site in the Myc promoter, in a manner dependent on p53 deficiency. Reduction of the Myc level by disruption of mR1 or Runx3 knockdown decreased the tumorigenicity of p53-deficient OS cells and effectively suppressed OS development in OS mice. Furthermore, Runx inhibitors exerted therapeutic effects on OS mice. Together, these results show that p53 deficiency promotes osteosarcomagenesis in human and mouse by allowing Runx3 to induce oncogenic Myc expression.

The Effects of Brown Algae-Derived Monosaccharide L-Fucose on Lipid Metabolism in C57BL/6J Obese Mice.

Obesity is a global public health problem and a risk factor for several metabolic disorders as well as cancer. In this study, we investigated the effects of L-fucose on lipid metabolism through chronic and acute in vivo experiments in mice. In the chronic test, mice were fed a high-calorie diet (HCD) containing 0.0001%, 0.001%, 0.01%, and 0.1% L-fucose for one month. The L-fucose supplementation inhibited body weight and visceral fat mass gain in HCD-fed mice. The results of the acute test showed that L-fucose increased the ratio of serum high molecular weight adiponectin and enhanced glucose and lipid catabolism. Furthermore, L-fucose also decreased the expression of adipogenic genes (peroxisome proliferator-activated receptor γ and cluster of differentiation 36). In conclusion, this study provides a new approach to combat obesity and the related diseases.

Novel in situ visualisation of rat intestinal absorption of polyphenols via matrix-assisted laser desorption/ionisation mass spectrometry imaging.

Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) is presently used in physiological evaluations for visualisation of targets in organs. In the present study, MALDI-MSI was used as a visualisation technique to investigate the intestinal absorption of polyphenols. Nifedipine/phytic acid-aided MALDI-MSI was performed to visualise theaflavin-3'-O-gallate (TF3'G) and epicatechin-3-O-gallate (ECG) in the rat jejunum for 50-µM, 60-min transport experiments. Non-absorbable TF3'G was successfully visualised at the apical region, whereas absorbable ECG was detected throughout the rat jejunum. MALDI-MSI was also performed to determine the transport routes of the target metabolites. Signals corresponding to TF3'G and ECG in the membranes were diminished following treatment with inhibitors targeting the monocarboxylic acid transporter and organic anion transporting polypeptides. Enhanced visualisation of TF3'G was achieved by inhibiting efflux routes. Our findings demonstrated that the present MALDI-MSI can provide critical spatial informations on intestinal absorption of targets, by which TF3'G and ECG were incorporated into intestinal tissues, followed by efflux back to the apical compartment. In addition, MALDI-MSI analyses suggested that TF3'G was resistant to phase II metabolism during the influx/efflux processes, whereas ECG was susceptible to methylation and sulphation reactions. In conclusion, inhibitor-aided MALDI-MSI could serve as a powerful in situ visualisation technique for verifying intestinal transport routes and investigating the metabolism of penetrants.

Effect of oral theaflavin administration on body weight, fat, and muscle in healthy subjects: a randomized pilot study.

Theaflavins are reddish-colored polyphenols in black tea. To test the efficacy of theaflavin administration on body fat and muscle, we performed a randomized, double-blind, placebo-controlled study and investigated the effect of theaflavins administration on the body composition using of healthy subjects. In this study, 30 male and female Japanese were enrolled and participants were randomly allocated to receive placebo, theaflavin (50 or 100 mg/day), or catechin (400 mg/ml) for 10 weeks. The effects were evaluated using body weight, body fat percentage, subcutaneous fat percentage, and skeletal muscle percentage. Theaflavin administration significantly improved body fat percentage, subcutaneous fat percentage, and skeletal muscle percentage when compared to with the placebo. In contrast, there was no significant difference in all measured outcomes between the catechin and the placebo groups. The results indicate that oral administration of theaflavin had a beneficial effect on body fat and muscle in healthy individuals.

Identification of polypeptides constituting lactophorin by monoclonal antibody to bovine milk lactophorin.

A monoclonal antibody to lactophorin (LP) was prepared by creating hybridoma from mouse myeloma cells and spleen cells from mice immunized with PAS-4 concentrated fraction from bovine milk fat globule membrane. The prepared antibody recognized a polypeptide moiety of LP27, the major component constituting LP, but not a carbohydrate moiety. Immunoblot analysis showed that all polypeptides (LP17, LP20, LP27, LP40, and LP50) constituting LP were recognized by the antibody. The identities of LP20, LP40, and LP50 were verified by N-terminal and internal amino acid sequencing. LP20 contains hydrolysate of LP27 besides LP27 without the O-glycosyl sugar chain. These results suggest that LP40 and LP50 are homo- or heterodimers of LP20 and LP27. This is the first report to the effect that LP was constructed from several forms of polypeptides, derived from LP27.