Essential oil components of turmeric inhibit hepatic lipidification and liver fibrosis in a diet-induced NASH model rats.

In this study, the fraction extracted from turmeric powder with 50% ethanol and fractionated with n-hexane were administered to diet-induced NASH model rats. NASH model was prepared with SD rats by feeding an originally designed choline-deficient, high-fat, high-fructose (HFF-CD) diet for 10 weeks. To the HFF-CD diet, hexane fraction and 50% ethanol fraction after hexane fractionation were added at 100 mg/kg body weight. 10 weeks later, blood samples and liver were collected for the following parameters: lipid weights, serum ALT, AST, TG, liver TG, TBARS levels, lipid metabolism-related gene expression and histopathological examination of the liver. As the results, the hexane fraction and 50% ethanol fraction showed a decrease in lipid weight, a decrease in hepatic TG, and activation of PPAR-α in the lipid metabolism-related gene test. These results suggest that the hexane fraction of turmeric has an inhibitory effect on fat accumulation in the liver by promoting lipid metabolism in NASH model rats.

Myeloid FoxO1 depletion attenuates hepatic inflammation and prevents nonalcoholic steatohepatitis.

Hepatic inflammation is culpable for the evolution of asymptomatic steatosis to nonalcoholic steatohepatitis (NASH). Hepatic inflammation results from abnormal macrophage activation. We found that FoxO1 links overnutrition to hepatic inflammation by regulating macrophage polarization and activation. FoxO1 was upregulated in hepatic macrophages, correlating with hepatic inflammation, steatosis, and fibrosis in mice and patients with NASH. Myeloid cell conditional FoxO1 knockout skewed macrophage polarization from proinflammatory M1 to the antiinflammatory M2 phenotype, accompanied by a reduction in macrophage infiltration in liver. These effects mitigated overnutrition-induced hepatic inflammation and insulin resistance, contributing to improved hepatic metabolism and increased energy expenditure in myeloid cell FoxO1-knockout mice on a high-fat diet. When fed a NASH-inducing diet, myeloid cell FoxO1-knockout mice were protected from developing NASH, culminating in a reduction in hepatic inflammation, steatosis, and fibrosis. Mechanistically, FoxO1 counteracts Stat6 to skew macrophage polarization from M2 toward the M1 signature to perpetuate hepatic inflammation in NASH. FoxO1 appears to be a pivotal mediator of macrophage activation in response to overnutrition and a therapeutic target for ameliorating hepatic inflammation to stem the disease progression from benign steatosis to NASH.

Administration of mucuna beans (Mucuna pruriences (L.) DC. var. utilis) improves cognition and neuropathology of 3 × Tg-AD mice.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of extracellular amyloid-beta peptides (Aß) resulting in senile plaques and intracellular hyperphosphorylated tau protein resulting in neurofibrillary tangles (NFTs). Mucuna beans (Mucuna pruriences (L.) DC. var. utilis) are unique plants containing 3-9% L-3,4-dihydroxyphenylalanine (L-DOPA). Here we investigated the effect of the administration of Mucuna beans on AD prevention by feeding triple-transgenic mice (3 × Tg-AD mice) with a diet containing Mucuna beans for 13 months. The levels of Aß oligomers and detergent-insoluble phosphorylated tau decreased in the brain of mice fed with Mucuna beans (Mucuna group) compared to those of the Control group. Aß accumulation and phosphorylated tau accumulation in the brain in the Mucuna group were also reduced. In addition, administration of Mucuna beans improved cognitive function. These results suggest that administration of Mucuna beans may have a preventive effect on AD development in 3 × Tg-AD mice.

Morphological and Developmental Traits of the Binucleation of Male Accessory Gland Cells in the Benthic Water Bug, Aphelocheirus vittatus (Hemiptera: Aphelochiridae).

The male accessory glands (MAGs) in insects are pair(s) of internal reproductive organs that produce and secrete the plasma component of seminal fluid. In various insects, MAG size is important for male reproductive success because the fluid provides physiologically active substances and/or nutrients to females to control sperm as well as female reproductive behaviors. Although the MAG epithelial cells in most insect species are standard mononucleate cells, those in some insect taxa are binucleate due to incomplete cytokinesis (e.g., Drosophila [Fallén] [Diptera: Drosophilidae]) or cell fusion (e.g., Cimex [Linnaeus] [Hemiptera: Cimicidae]). In the case of Drosophila, the apicobasal position of the two nuclei relative to the epithelial plane changes from vertical to horizontal after nutrient intake, which allows the volume of the MAG cavity to expand effectively. On the other hand, in the case of Cimex, the positions of the two nuclei do not change apicobasally in response to feeding, but their position relative to the proximodistal axis varies depending on the tubular/spherical organ morphology. Here, we report that the MAG of the benthic water bug Aphelocheirus vittatus (Matsumura) (Hemiptera: Aphelochiridae) shows binucleation in all epithelial cells. Despite the phylogenetically close relationship between Aphelocheirus and Cimex, the MAG cells in Aphelocheirus showed a Drosophila-like apicobasal change in the position of the two nuclei in response to feeding. Furthermore, the cytological processes during binucleation are more similar to those in Drosophila (incomplete cytokinesis) than to those in Cimex (cell fusion). These results indicate that the physiological role and mechanism of binucleation in MAG cells changed during the evolution of Hemiptera.

Depletion of hepatic forkhead box O1 does not affect cholelithiasis in male and female mice.

Cholelithiasis is one of the most prevalent gastroenterological diseases and is characterized by the formation of gallstones in the gallbladder. Both clinical and preclinical data indicate that obesity, along with comorbidity insulin resistance, is a predisposing factor for cholelithiasis. Forkhead box O1 (FoxO1) is a key transcription factor that integrates insulin signaling with hepatic metabolism and becomes deregulated in the insulin-resistant liver, contributing to dyslipidemia in obesity. To gain mechanistic insights into how insulin resistance is linked to cholelithiasis, here we determined FoxO1's role in bile acid homeostasis and its contribution to cholelithiasis. We hypothesized that hepatic FoxO1 deregulation links insulin resistance to impaired bile acid metabolism and cholelithiasis. To address this hypothesis, we used the FoxO1LoxP/LoxP-Albumin-Cre system to generate liver-specific FoxO1-knockout mice. FoxO1-knockout mice and age- and sex-matched WT littermates were fed a lithogenic diet, and bile acid metabolism and gallstone formation were assessed in these animals. We showed that FoxO1 affected bile acid homeostasis by regulating hepatic expression of key enzymes in bile acid synthesis and in biliary cholesterol and phospholipid secretion. Furthermore, FoxO1 inhibited hepatic expression of the bile acid receptor farnesoid X receptor and thereby counteracted hepatic farnesoid X receptor signaling. Nonetheless, hepatic FoxO1 depletion neither affected the onset of gallstone disease nor impacted the disease progression, as FoxO1-knockout and control mice of both sexes had similar gallstone weights and incidence rates. These results argue against the notion that FoxO1 is a link between insulin resistance and cholelithiasis.

Binucleation of male accessory gland cells in the common bed bug Cimex lectularius.

The insect male accessory gland (MAG) is an internal reproductive organ responsible for the synthesis and secretion of seminal fluid components, which play a pivotal role in the male reproductive strategy. In many species of insects, the effective ejaculation of the MAG products is essential for male reproduction. For this purpose, the fruit fly Drosophila has evolved binucleation in the MAG cells, which causes high plasticity of the glandular epithelium, leading to an increase in the volume of seminal fluid that is ejaculated. However, such a binucleation strategy has only been sporadically observed in Dipteran insects, including fruit flies. Here, we report the discovery of binucleation in the MAG of the common bed bug, Cimex lectularius, which belongs to hemimetabolous Hemiptera phylogenetically distant from holometabolous Diptera. In Cimex, the cell morphology and timing of synchrony during binucleation are quite different from those of Drosophila. Additionally, in Drosophila, the position of the two nuclei in the adult stage changes as a result of the mating history or the nutrient conditions; however, it remains stable in Cimex. These differences suggest that binucleation in the Cimex MAG plays a unique role in the male reproductive system that is distinct from that of Drosophila.

Unique Superparamagnetic-like Behavior Observed in Non-π-delocalized Nitroxide Diradical Compounds Showing Discotic Liquid Crystalline Phase.

A unique superparamagnetic-like behavior and a large "positive magneto-LC effect" were observed in the solid phases and the hexagonal columnar (Colh ) liquid crystalline (LC) phase, respectively, of novel achiral non-π-delocalized nitroxide diradical compounds (R,S)-1, which showed polymorphism in the solid phases (solids I and II). The SQUID magnetization measurement revealed that (1) (R,S)-1 containing a small amount of racemic diastereomers (R*,R*)-1 possessed an unusual and large temperature-independent magnetic susceptibility (χTIM >0) component in the original nanocrystalline solid I that was responsible for the observed superparamagnetic-like behavior under low magnetic fields and did not arise from the contamination by extrinsic magnetic metal or metal ion impurities, besides ordinary temperature-dependent paramagnetic susceptibility (χpara >0) and temperature-independent diamagnetic susceptibility (χdia <0) components, (2) a large increase in molar magnetic susceptibility (χM ) (positive magneto-LC effect) that occurred at the solid I-to-liquid crystal transition upon heating was preserved as an additional χTIM increase in the resulting polymorphic nanocrystalline solid II by cooling, and (3) such unique magnetic phenomena were induced by thermal processing for (R,S)-1 or by adding a small amount of (R*,R*)-1 to (R,S)-1 as the impurity.

Sex- and Tissue-Specific Role of Estrogen Sulfotransferase in Energy Homeostasis and Insulin Sensitivity.

Estrogen sulfotransferase catalyzes the sulfoconjugation and deactivation of estrogens. Previously, we showed that loss of Est in male ob/ob mice, but not in female ob/ob mice, exacerbated the diabetic phenotype, but the underlying mechanism was unclear. In this study, we show that transgenic reconstitution of Est in the adipose tissue, but not in the liver, attenuated diabetic phenotype in Est-deficient ob/ob mice (obe mice). Mechanistically, adipose reconstitution of Est in obe mice (oae mice) resulted in reduced local and systemic inflammation, improved insulin sensitivity, and increased energy expenditure. At the molecular level, adipose induction of lipocalin-2 (Lcn2) in oae males may have contributed to the inhibition of inflammation because the level of Lcn2 was negatively associated with tumor necrosis factor (Tnf) α expression, and treatment of differentiated adipocytes with Lcn2 antagonized Tnfα-responsive inhibition of insulin signaling. The metabolic benefit of adipose reconstitution of Est was sex specific, because adipose reconstitution of Est in obe females had little effect. Interestingly, despite their improved metabolic functions, obe male mice with reconstituted Est in their adipose tissue failed to ameliorate the impairment of the structure and function of the pancreatic islets. In summary, our study uncovers a crucial adipose- and male-specific role of Est in maintaining the whole-body energy homeostasis.

Preparation of Robust Metal-Free Magnetic Nanoemulsions Encapsulating Low-Molecular-Weight Nitroxide Radicals and Hydrophobic Drugs Directed Toward MRI-Visible Targeted Delivery.

With a view to developing a theranostic nanomedicine for targeted drug delivery systems visible by magnetic resonance (MR) imaging, robust metal-free magnetic nanoemulsions (mean particle size less than 20 nm) consisting of a biocompatible surfactant and hydrophobic, low molecular weight 2,2,5-trimethyl-5-(4-alkoxy)phenylpyrrolidine-N-oxyl radicals were prepared in pH 7.4 phosphate-buffered saline (PBS). The structure of the nanoemulsions was characterized by electron paramagnetic resonance spectroscopy, and dynamic light scattering and small-angle neutron-scattering measurements. The nanoemulsions showed high colloidal stability, low cytotoxicity, enough reduction resistance to excess ascorbic acid, and sufficient contrast enhancement in the proton longitudinal relaxation time (T1 ) weighted MR images in PBS in vitro (and preliminarily in vivo). Furthermore, the hydrophobic anticancer drug paclitaxel could be encapsulated inside the nanoparticles, and the resulting paclitaxel-loaded nanoemulsions were efficiently incorporated into HeLa cells to suppress cell growth.

APOC3 Protein Is Not a Predisposing Factor for Fat-induced Nonalcoholic Fatty Liver Disease in Mice.

Nonalcoholic fatty liver disease (NAFLD), characterized by excessive fat accumulation in liver, is prevalent in obesity. Genetic factors that link obesity to NAFLD remain obscure. Apolipoprotein C3 (APOC3) is a lipid-binding protein with a pivotal role in triglyceride metabolism. Humans with APOC3 gain-of-function mutations and mice with APOC3 overproduction are associated with hypertriglyceridemia. Nonetheless, it remains controversial whether APOC3 is culpable for diet-induced NAFLD. To address this fundamental issue, we fed APOC3-transgenic and wild-type littermates a high fructose diet or high fat diet, followed by determination of the effect of APOC3 on hepatic lipid metabolism and inflammation and the progression of NAFLD. To gain mechanistic insight into NAFLD, we determined the impact of APOC3 on hepatic triglyceride synthesis and secretion versus fatty acid oxidation. APOC3-transgenic mice were hypertriglyceridemic, culminating in marked elevation of triglycerides, cholesterols, and non-esterified fatty acids in plasma. Despite the prevailing hypertriglyceridemia, APOC3-transgenic mice, relative to wild-type littermates, had similar weight gain and hepatic lipid content without alterations in hepatic expression of key genes involved in triglyceride synthesis and secretion and fatty acid oxidation. APOC3-transgenic and wild-type mice had similar Kupffer cell content without alterations in hepatic expression of pro- and anti-inflammatory cytokines. APOC3 neither exacerbated diet-induced adiposity nor aggravated the degree of steatosis in high fructose or high fat-fed APOC3-transgenic mice. These effects ensued independently of weight gain even after 10-month high fat feeding. We concluded that APOC3, whose dysregulation is liable for hypertriglyceridemia, is not a predisposing factor for linking overnutrition to NAFLD in obesity.

A Nuclear Factor Involved in Transcriptional Regulation of the AREBP Gene.

The AICAR responsive element binding protein (AREBP) suppresses transcription of the gluconeogenic enzyme genes in response to AICAR treatment. Moreover, overexpression of AREBP also suppresses gluconeogenic gene expressions in animals, indicating that AREBP plays an important role in gluconeogenesis. Through a combination of systematic analyses of the AREBP gene promoter and assays for DNA-protein interaction, we identified a nuclear factor involved in tissue-specific expression of AREBP. By targeting this nuclear factor, pharmacological or nutraceutical induction of AREBP gene expression is expected to reduce blood glucose levels in patient with insulin resistance.

Effect of Hypertriglyceridemia on Beta Cell Mass and Function in ApoC3 Transgenic Mice.

Hypertriglyceridemia results from increased production and decreased clearance of triglyceride-rich very low-density lipoproteins, a pathological condition that accounts for heightened risk of ischemic vascular diseases in obesity and type 2 diabetes. Despite its intimate association with insulin resistance, whether hypertriglyceridemia constitutes an independent risk for beta cell dysfunction in diabetes is unknown. Answering this fundamental question is stymied by the fact that hypertriglyceridemia is intertwined with hyperglycemia and insulin resistance in obese and diabetic subjects. To circumvent this limitation, we took advantage of apolipoprotein C3 (ApoC3)-transgenic mice, a model with genetic predisposition to hypertriglyceridemia. We showed that ApoC3-transgenic mice, as opposed to age/sex-matched wild-type littermates, develop hypertriglyceridemia with concomitant elevations in plasma cholesterol and non-esterified fatty acid levels. Anti-insulin and anti-glucagon dual immunohistochemistry in combination with morphometric analysis revealed that ApoC3-transgenic and wild-type littermates had similar beta cell and alpha cell masses as well as islet size and architecture. These effects correlated with similar amplitudes of glucose-stimulated insulin secretion and similar degrees of postprandial glucose excursion in ApoC3-transgenic versus wild-type littermates. Oil Red O histology did not visualize lipid infiltration into islets, correlating with the lack of ectopic triglyceride and cholesterol depositions in the pancreata of ApoC3-transgenic versus wild-type littermates. ApoC3-transgenic mice, despite persistent hypertriglyceridemia, maintained euglycemia under both fed and fasting conditions without manifestation of insulin resistance and fasting hyperinsulinemia. Thus, hypertriglyceridemia per se is not an independent risk factor for beta cell dysfunction in ApoC3 transgenic mice.

FoxO1 Plays an Important Role in Regulating ß-Cell Compensation for Insulin Resistance in Male Mice.

ß-Cell compensation is an essential mechanism by which ß-cells increase insulin secretion for overcoming insulin resistance to maintain euglycemia in obesity. Failure of ß-cells to compensate for insulin resistance contributes to insulin insufficiency and overt diabetes. To understand the mechanism of ß-cell compensation, we characterized the role of forkhead box O1 (FoxO1) in ß-cell compensation in mice under physiological and pathological conditions. FoxO1 is a key transcription factor that serves as a nutrient sensor for integrating insulin signaling to cell metabolism, growth, and proliferation. We showed that FoxO1 improved ß-cell compensation via 3 distinct mechanisms by increasing ß-cell mass, enhancing ß-cell glucose sensing, and augmenting ß-cell antioxidative function. These effects accounted for increased glucose-stimulated insulin secretion and enhanced glucose tolerance in ß-cell-specific FoxO1-transgenic mice. When fed a high-fat diet, ß-cell-specific FoxO1-transgenic mice were protected from developing fat-induced glucose disorder. This effect was attributable to increased ß-cell mass and function. Furthermore, we showed that FoxO1 activity was up-regulated in islets, correlating with the induction of physiological ß-cell compensation in high-fat-induced obese C57BL/6J mice. These data characterize FoxO1 as a pivotal factor for orchestrating physiological adaptation of ß-cell mass and function to overnutrition and obesity.

Preparation, characterization and magnetic behavior of a spin-labelled physical hydrogel containing a chiral cyclic nitroxide radical unit fixed inside the gelator molecule.

An optically active amphiphilic nitroxide radical compound [(S,S,R)-], which contains a paramagnetic (2S,5S)-2,5-dimethyl-2,5-diphenylpyrrolidine-N-oxyl radical group fixed in the inner position together with a hydrophobic long alkyl chain and a hydrophilic (R)-alanine residue in the opposite terminal positions, was found to serve as a low-molecular-weight gelator in H2O to give rise to a spin-labelled physical hydrogel. Characterization of the hydrogel was performed by microscopic (SEM, TEM and AFM) techniques, XRD and SAXS measurements, and IR, UV and CD spectroscopies. The gel-sol transition temperature was determined by EPR spectral line-width (ΔHpp) analysis. Measurement of the temperature dependence of relative paramagnetic susceptibility (χrel) for the hydrogel and sol phases was achieved by means of the double-integration of VT-EPR spectra.

Forkhead Box O6 (FoxO6) Depletion Attenuates Hepatic Gluconeogenesis and Protects against Fat-induced Glucose Disorder in Mice.

Excessive endogenous glucose production contributes to fasting hyperglycemia in diabetes. FoxO6 is a distinct member of the FoxO subfamily. To elucidate the role of FoxO6 in hepatic gluconeogenesis and assess its contribution to the pathogenesis of fasting hyperglycemia in diabetes, we generated FoxO6 knock-out (FoxO6-KO) mice followed by determining the effect of FoxO6 loss-of-function on hepatic gluconeogenesis under physiological and pathological conditions. FoxO6 depletion attenuated hepatic gluconeogenesis and lowered fasting glycemia in FoxO6-KO mice. FoxO6-deficient primary hepatocytes were associated with reduced capacities to produce glucose in response to glucagon. When fed a high fat diet, FoxO6-KO mice exhibited significantly enhanced glucose tolerance and reduced blood glucose levels accompanied by improved insulin sensitivity. These effects correlated with attenuated hepatic gluconeogenesis in FoxO6-KO mice. In contrast, wild-type littermates developed fat-induced glucose intolerance with a concomitant induction of fasting hyperinsulinemia and hyperglycemia. Furthermore, FoxO6-KO mice displayed significantly diminished macrophage infiltration into liver and adipose tissues, correlating with the reduction of macrophage expression of C-C chemokine receptor 2 (CCR2), a factor that is critical for regulating macrophage recruitment in peripheral tissues. Our data indicate that FoxO6 depletion protected against diet-induced glucose intolerance and insulin resistance by attenuating hepatic gluconeogenesis and curbing macrophage infiltration in liver and adipose tissues in mice.

FoxO6 integrates insulin signaling with MTP for regulating VLDL production in the liver.

Excessive production of triglyceride-rich very low-density lipoproteins (VLDL-TG) contributes to hypertriglyceridemia in obesity and type 2 diabetes. To understand the underlying mechanism, we studied hepatic regulation of VLDL-TG production by (forkhead box O6) FoxO6, a forkhead transcription factor that integrates insulin signaling to hepatic metabolism. We showed that transgenic mice expressing a constitutively active FoxO6 allele developed hypertriglyceridemia, culminating in elevated VLDL-TG levels and impaired postprandial TG clearance. This effect resulted in part from increased hepatic VLDL-TG production. We recapitulated these findings in cultured HepG2 cells and human primary hepatocytes, demonstrating that FoxO6 promoted hepatic VLDL-TG secretion. This action correlated with the ability of FoxO6 to stimulate hepatic production of microsomal triglyceride transfer protein (MTP), a molecular chaperone that catalyzes the rate-limiting step in VLDL-TG assembly and secretion. FoxO6 was shown to bind to the MTP promoter and stimulate MTP promoter activity in HepG2 cells. This effect was inhibited by insulin, consistent with the ability of insulin to promote FoxO6 phosphorylation and disable FoxO6 DNA-binding activity. Mutations of the FoxO6 target site within the MTP promoter abrogated FoxO6-mediated induction of MTP promoter activity. Hepatic FoxO6 expression became deregulated in insulin-resistant mice with obesity and type 2 diabetes. FoxO6 inhibition in insulin-resistant liver suppressed hepatic MTP expression and curbed VLDL-TG overproduction, contributing to the amelioration of hypertriglyceridemia in obese and diabetic db/db mice. These results characterize FoxO6 as an important signaling molecule upstream of MTP for regulating hepatic VLDL-TG production.

Low-dose vitamin K2 (MK-4) supplementation for 12 months improves bone metabolism and prevents forearm bone loss in postmenopausal Japanese women.

Menaquinone-4 (MK-4) administered at a pharmacological dosage of 45 mg/day has been used for the treatment of osteoporosis in Japan. However, it is not known whether a lower dose of MK-4 supplementation is beneficial for bone health in healthy postmenopausal women. The aim of this study was to examine the long-term effects of 1.5-mg daily supplementation of MK-4 on the various markers of bone turnover and bone mineral density (BMD). The study was performed as a randomized, double-blind, placebo-controlled trial. The participants (aged 50-65 years) were randomly assigned to one of two groups according to the MK-4 dose received: the placebo-control group (n = 24) and the 1.5-mg MK-4 group (n = 24). The baseline concentrations of undercarboxylated osteocalcin (ucOC) were high in both groups (>5.1 ng/ml). After 6 and 12 months, the serum ucOC concentrations were significantly lower in the MK-4 group than in the control group. In the control group, there was no significant change in serum pentosidine concentrations. However, in the MK-4 group, the concentration of pentosidine at 6 and 12 months was significantly lower than that at baseline. The forearm BMD was significantly lower after 12 months than at 6 months in the control group. However, there was no significant decrease in BMD in the MK-4 group during the study period. These results suggest that low-dose MK-4 supplementation for 6-12 months improved bone quality in the postmenopausal Japanese women by decreasing the serum ucOC and pentosidine concentrations, without any substantial adverse effects.

Role of nuclear localization of PSMB1 in transcriptional activation.

The production of retinol binding protein 4 (RBP4) is higher in adipose tissue in type 2 diabetes. We have found that proteasome subunit beta type 1 (PSMB1) is a transcriptional activator of Rbp4. In the present study, the putative tyrosine phosphorylation site in PSMB1 was mutated to phenylalanine. The mutated form of PSMB1 displayed increased nuclear translocation, resulting in activation of transcription in adipocytes.

[A case of hemosuccus pancreaticus caused by rupture of an aneurysm of the splenic artery].

Hemorrhage through the pancreatic duct into the duodenum, the so-called "hemosuccus pancreaticus", is a rare cause of gastrointestinal bleeding. A 75-year-old man, who was treated with anticoagulation agents for an old myocardial infarction, was admitted to our hospital for sudden tarry stools and abdominal pain. His hemoglobin level slightly dropped to 12.6g/dL. His liver function tests results and the serum amylase level were elevated. A second upper gastrointestinal endoscopy revealed continuous bleeding from the ampulla of Vater. A rupture of an aneurysm of the splenic artery to the pancreatic duct was suggested by abdominal computed tomographic scan, abdominal magnetic resonance imaging, celiac arteriography, and endoscopic ultrasonography. The conservative treatment of stopping the bleeding with anticoagulation agents was successful.

The nanoparticulation by octaarginine-modified liposome improves α-galactosylceramide-mediated antitumor therapy via systemic administration.

Alpha-galactosylceramide (αGC), a lipid antigen present on CD1d molecules, is predicted to have clinical applications as a new class of adjuvant, because αGC strongly activates natural killer T (NKT) cells which produce large amounts of IFN-γ. Here, we incorporated αGC into stearylated octaarginine-modified liposomes (R8-Lip), our original delivery system developed for vaccines, and investigated the effect of nanoparticulation. Unexpectedly, the systemic administered R8-Lip incorporating αGC (αGC/R8-Lip) failed to improve the immune responses mediated by αGC compared with soluble αGC in vivo, although αGC/R8-Lip drastically enhanced αGC presentation on CD1d in antigen presenting cells in vitro. Thus, we optimized the αGC/R8-Lip in vivo to overcome this inverse correlation. In optimization in vivo, we found that size control of liposome and R8-modification were critical for enhancing the production of IFN-γ. The optimization led to the accumulation of αGC/R8-Lip in the spleen and a positive therapeutic effect against highly malignant B16 melanoma cells. The optimized αGC/R8-Lip also enhanced αGC presentation on CD1d in antigen presenting cells and resulted in an expansion in the population of NKT cells. Herein, we show that R8-Lip is a potent delivery system, and size control and R8-modification in liposomal construction are promising techniques for achieving systemic αGC therapy.