α‐Galactosylceramide activates antitumor immunity against liver tumor

Aim: α‐Galactosylceramide (α‐GalCer) has been attracting attention as a novel approach to treat metastatic liver cancer. We investigated the detailed process of activating liver dendritic cells (DC) and immune cells after α‐GalCer treatment in the mouse liver tumor model. Methods: BALB/c mice bearing CMS4 liver tumor (p53 peptide‐expressing tumor) were treated by α‐GalCer. We evaluated the activation of liver DC and immune cells after α‐GalCer treatment. Interferon (IFN)‐γ enzyme‐linked immunosorbent spot (ELISPOT) assay was performed to detect p53 peptide‐specific cytotoxic T lymphocytes (CTL). To assess the impact of systemic acquired immunity by α‐GalCer treatment, 28 days after liver tumor treatment, CMS4 cells or Colon26 cells were re‐challenged s.c. Results: The liver weights of α‐GalCer‐treated mice were significantly lighter than those of vehicle‐treated mice. Depletion experiments revealed that natural killer (NK) cells were essential for the antitumor effect of α‐GalCer. α‐GalCer treatment significantly increased the population of DC and NK cells in the liver. The expressions of co‐stimulatory molecules on liver DC significantly increased with the peak at 1 day after α‐GalCer administration. IFN‐γ ELISPOT assay demonstrated that p53 peptide‐specific CTL was generated efficiently in α‐GalCer‐treated mice. ⁵¹Cr‐release assay revealed that CD8⁺, not CD4⁺, CTL against CMS4 cells were generated in α‐GalCer‐treated mice. The mice that had been protected from CMS4 liver tumor by α‐GalCer injection became resistant against s.c. CMS4 re‐challenge, but not against Colon26 re‐challenge. Conclusion: These results demonstrated the therapeutic potential of α‐GalCer against liver cancer through activating liver DC and immune cells in the liver.     

Induction of interferon‐λ contributes to Toll‐like receptor‐3‐activated hepatic stellate cell–mediated hepatitis C virus inhibition in hepatocytes

There is limited information about the role of hepatic stellate cells (HSC) in liver innate immunity against hepatitis C virus (HCV). We thus examined whether HSC can produce antiviral factors that inhibit HCV replication in human hepatocytes. HSC expressed functional Toll‐like receptor 3 (TLR‐3), which could be activated by its ligand, polyinosine‐polycytidylic acid (poly I:C), leading to the induction of interferon‐λ (IFN‐λ) at both mRNA and protein levels. TLR‐3 signalling of HSC also induced the expression of IFN regulatory factor 7 (IRF‐7), a key regulator of IFN signalling pathway. When HCV JFH‐1‐infected Huh7 cells were co‐cultured with HSC activated with poly I:C or incubated in media conditioned with supernatant (SN) from poly I:C‐activated HSC, HCV replication was significantly suppressed. This HSC SN action on HCV inhibition was mediated through IFN‐λ, which was evidenced by the observation that antibody to IFN‐λ receptors could neutralize the HSC‐mediated anti‐HCV effect. The role of IFN‐λ in HSC‐mediated anti‐HCV activity is further supported by the observation that HSC SN treatment induced the expression of IRF‐7 and IFN‐stimulated genes (ISGs), OAS‐1 and MxA in HCV‐infected Huh7 cells. These observations indicate that HSC may be a key regulatory bystander, participating in liver innate immunity against HCV infection using an IFN‐λ‐dependent mechanism.     

Effect of genipin on cholestasis induced by estradiol‐17β‐glucuronide and lithocholate‐3‐O‐glucuornide in rats

Aim: Genipin is reported to stimulate the insertion of multidrug resistance protein 2 (Mrp2) in the bile canalicular membrane, thereby causing choleresis by the increased the biliary excretion of glutathione, which has been considered to be a substrate of Mrp2. In the present study, we examined the effect of genipin on cholestasis induced by estradiol‐17β‐glucuronide and lithocholate‐3‐O‐glucuronide, Mrp2 substrates, in rats. Further, the effect of genipin on the biliary excretion of substrates of P‐glycoprotein (P‐gp), vinblastine and erythromycin, was also studied. Methods: The effect of genipin infusion at the rate of 0.5 µmol/min/100 g on cholestasis induced by estradiol‐17β‐glucuronide (0.075 µmol/min/100 g for 20 min) and lithocholate‐3‐O‐glucuronide (0.15 µmol/min/100 g for 40 min) was studied. The effect of genipin infusion on the biliary excretion of a tracer dose of vinblastine and erythromycin infused at the rate of 0.1 µmol/min/100 g was also studied. Results: Genipin relieved estradiol‐17β‐glucuronide‐induced cholestasis, and cumulative biliary estradiol‐17β‐glucuronide excretion for 120 min was increased from 50 ± 20%–81 ± 20% dose. In contrast, genipin had no effect on lithocholate‐3‐O‐glucuronide‐induced cholestasis. Biliary excretion of a tracer dose of vinblastine and the maximum biliary excretion of erythromycin were significantly decreased by genipin. Conclusions: Genipin protected estradiol‐17β‐glucuronide‐induced cholestasis. The mechanism of the protection of cholestasis by genipin is unknown, but it is speculated to be due to a conformational change of P‐gp by genipin, in addition to the stimulation of Mrp2 insertion into the bile canaliculi.     

Structure‐oriented review of 14‐3‐3 protein isoforms in geriatric neuroscience

This review focuses on the possible relevance of 14‐3‐3 proteins in geriatric neuroscience. 14‐3‐3 proteins are mainly localized in the synapses and neuronal cytoplasm. These proteins regulate intracellular signal cascades for differentiation, development, growth, apoptosis and survival. Seven isoforms have so far been identified in mammals. The binding motifs and potential functions of 14‐3‐3 proteins are now recognized to have a wide range of functional relevance. First, we provide a brief summary of the molecular structure and multiple functions of 14‐3‐3 proteins. Second, we review the involvement of 14‐3‐3 proteins in common diseases of geriatric neurology, such as Alzheimer's disease and tauopathies, Parkinson's disease and α‐synucleinopathies, Huntington's disease and polyglutamine diseases, Creutzfeldt–Jakob disease and prion diseases, cerebral infarction, and atherosclerosis. Finally, we discuss the immunohistochemical localization of 14‐3‐3 proteins and its isoforms during the postnatal development of rat brains as a basis for understanding adult neurogenesis. The elucidation of the isoform‐dependent functions of 14‐3‐3 proteins with regard to brain development might be promising for the future development of novel therapeutic interventions for common diseases of geriatric neurology. Geriatr Gerontol Int 2012; ??: ??–??.     

Clinical significance of half‐lives of tumor markers α‐fetoprotein and des‐γ‐carboxy prothrombin after hepatectomy for hepatocellular carcinoma

Aim

The prognostic significance of the half‐lives (HLs) of α‐fetoprotein (AFP) and des‐γ‐carboxy prothrombin (DCP) in patients undergoing hepatectomy for hepatocellular carcinoma (HCC) is unclear. We evaluated the HLs of AFP and DCP in a cohort of such patients.

Methods

This study included data on 202 patients with HCC who underwent curative hepatectomy and had preoperative AFP concentrations ≥100 ng/mL or DCP ≥200 mAU/mL. We calculated the HLs of AFP and DCP from their values just before and 1 month after hepatectomy. We identified three groups: a normalization group, tumor marker concentrations within normal range 1 month post‐hepatectomy; a long group, HL of AFP ≥7 days or DCP ≥4 days; and a short group, remaining patients. We evaluated associations between HL and prognosis.

Results

Three‐year recurrence‐free survival (RFS) in the normalization (n = 70), short (n = 71), and long groups (n = 61) was 41.3%, 46.0%, and 16.8%, respectively (P = 0.002). Five‐year overall survival (OS) of normalization, short, and long groups was 72.6, 70.6 and 43.8%, respectively (P = 0.002). Multivariate analysis revealed that long HL is an independent risk factor for poor RFS (hazard ratio [HR] 2.21, P = 0.0006) and poor OS (HR 2.70, P = 0.004). The extrahepatic recurrence rate was 21.3% (13/61) in the long group, which is higher than in the normalization group (8.6%, 6/70) (P = 0.04) and short group (9.9%, 7/71) (P = 0.07).

Conclusion

Post‐hepatectomy HLs of AFP and DCP are predictors of long‐term outcome in patients with HCC.     

2‐Methoxyestradiol ameliorates glucose tolerance with the increase in β‐cell mass in db/db mice

Aims/Introduction: 2‐Methoxyestradiol (2ME) is an estradiol metabolite with little estrogenic activity. Previous data identified its anti‐carcinogenic properties and possible cardiovascular benefits. However, its effect on diabetes mellitus has not been fully elucidated. The aim of the present study was to determine the effects of 2ME on glucose metabolism in the diabetic state. Materials and Methods: To evaluate the effects of 2ME, pellets of two different doses of the drug were implanted into female db/db mice at the age of 5 weeks. Intraperitoneal glucose tolerance test and insulin tolerance test were carried out at the age of 8 weeks. The pancreas was harvested for morphological analysis and β‐cell function at the age of 9 weeks. Results: 2ME improved random blood glucose levels and glucose tolerance with increases in insulin levels during an intraperitoneal glucose tolerance test. Insulin sensitivity judged by an insulin tolerance test was comparable in the low‐ and high‐dose 2ME groups and the control group. Although glucose‐stimulated insulin secretion in isolated islets was comparable among the three groups, β‐cell mass in 2ME‐treated groups was higher than the control group. In the 2ME‐treated groups, the number of Ki67‐positive cells in islets was higher, whereas the number of cleaved caspase‐3‐positive cells was comparable with the control. Conclusions: 2ME ameliorates glucose tolerance by promoting the proliferation of β‐cell mass in db/db mice. Our data suggests its potential clinical usefulness as a disease‐modifying drug for type 2 diabetes mellitus. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2010.00087.x, 2011)     

Decreased Expression of Thrombomodulin in Endothelial Cells by Fibroblast Growth Factor‐23/α‐Klotho

Chronic kidney disease (CKD) has been known to be a state of excessive fibroblast growth factor‐23 (FGF23) and α‐Klotho deficiency. Patients undergoing hemodialysis have an increased mortality risk associated with cardiovascular disease and endothelial dysfunction. The mechanism responsible for the relationship of FGF23 to endothelial damage in these patients has been unclear. On the other hands, increasing evidences have demonstrated that thrombomodulin (TM) plays an important role in the endothelial barrier. Here, we report the suppression of membrane TM, in a dose‐dependent manner, in human umbilical vein endothelial cells after FGF23 and FGF23/α‐Klotho stimulation. In addition, the levels of soluble TM, which reflect endothelial cell injury, were much higher in cell supernatants after FGF23 and FGF23/α‐Klotho stimulation than in the control supernatant. This study indicates a possible mechanism by which excessive levels of FGF23 are involved in endothelial TM disruption, which has been implicated as a potential cardiovascular risk factor in patients with CKD, especially in HD patients.     

Flt3 ligand treatment modulates parasitemia during infection with rodent malaria parasites via MyD88‐ and IFN‐γ‐dependent mechanisms

We previously showed that treatment of mice with the Flt3 ligand (Flt3L) prevents development of lethal experimental cerebral malaria and inhibits parasitemia during Plasmodium berghei ANKA (PbA) infection. In this study, we investigated the mechanisms underlying the reduction of parasitemia in Flt3L‐treated mice. Studies using gene knockout mice and antibody treatment indicated that the anti‐parasitemia effect of Flt3L was mediated by innate immune system and was dependent on MyD88, IFN‐γ, IL‐12 and natural killer (NK) cells. The number of NK cells and their ability to produce IFN‐γ was enhanced in Flt3L‐treated mice. Phagocytic activity of splenocytes was increased in Flt3L‐treated mice after PbA infection when compared with that in untreated mice, and this activity was mainly mediated by the accumulation of F4/80^midCD11b⁺ cells in the spleen. In both MyD88^?/? and IFN‐γ^?/? mice, the proportion of F4/80^midCD11b⁺ cells was not increased in the spleen of Flt3L‐treated mice after infection. These correlations suggest that NK cells produce IFN‐γ in Flt3L‐treated mice, and accumulation of F4/80^midCD11b⁺ cells in the spleen is promoted by an IFN‐γ ‐dependent manner, culminating in the inhibition of parasitemia. These findings imply that Flt3L promotes effective innate immunity against malaria infection mediated by interplay among varieties of innate immune cells.     

Elevated concentrations of 15‐deoxy‐Δ12,14‐prostaglandin J2 in chronic liver disease propose therapeutic trials with peroxisome proliferator activated receptor γ‐inducing drugs

Background/Aims: Current knowledge confers a crucial role to connective tissue growth factor (CTGF/CCN2) in hepatic fibrogenesis. Hepatocytes are likely to be the major cellular source of CTGF in the liver in which CTGF is sensitively upregulated by TGF‐β. Recently, we demonstrated that the methylxanthine derivate caffeine leads to an upregulation of peroxisome proliferator activated receptor γ (PPARγ) expression in hepatocytes, thus sensitizing these cells to the well‐known inhibitory effect of 15‐deoxy‐Δ^12,14‐prostaglandin J₂ (15‐d‐PGJ₂) on CTGF expression. However, upregulation of the receptor alone is not sufficient per se; its physiological ligand 15‐d‐PGJ₂ is required to exert an inhibitory effect on transforming growth factor‐β (TGF‐β) target genes such as CTGF. Methods: This study compared serum concentrations of 15‐d‐PGJ₂ in Caucasian patients with fibrotic liver diseases (n=289), Caucasian controls (n=136) and Caucasian non‐liver disease (NLD) sick (n=307), as well as of Chinese patients with hepatocellular carcinoma (HCC) (n=43) and Chinese healthy controls (n=63) in order to characterize their suitability for therapeutic approaches with PPARγ‐inducing (i.e. CTGF inhibitory) drugs such as caffeine. Results: The presented data showed that Caucasian patients with ongoing hepatic fibrogenesis (mean 6.2±5.9 μg/L) displayed strikingly higher serum concentrations of 15‐d‐PGJ₂ than healthy probands (mean 2.3±1.0) and Caucasian patients with NLD (mean 2.7±1.4 μg/L). Similar results were found in Chinese patients with fully developed HCC (mean 1.3±0.7 μg/L) compared with Chinese healthy controls (mean 0.4±0.2 μg/L). Conclusions: In conclusion, our data thus proposed an increased suitability of these patient groups for therapeutic approaches with drugs inducing PPARγ expression, such as methylxanthine derivates.