Woo-Chan Son

Human Monoclonal Antibodies against Glucagon Receptor Improve Glucose Homeostasis by Suppression of Hepatic Glucose Output in Diet-Induced Obese Mice

Aim Glucagon is an essential regulator of hepatic glucose production (HGP), which provides an alternative therapeutic target for managing type 2 diabetes with glucagon antagonists. We studied the effect of a novel human monoclonal antibody against glucagon receptor (GCGR), NPB112, on glucose homeostasis in diet-induced obese (DIO) mice. Methods The glucose-lowering efficacy and safety of NPB112 were investigated in DIO mice with human GCGR for 11 weeks, and a hyperinsulinemic-euglycemic clamp study was conducted to measure HGP. Results Single intraperitoneal injection of NPB112 with 5 mg/kg effectively decreased blood glucose levels in DIO mice for 5 days. A significant reduction in blood glucose was observed in DIO mice treated with NPB112 at a dose ≥5 mg/kg for 6 weeks, and its glucose-lowering effect was dose-dependent. Long-term administration of NPB112 also caused a mild 29% elevation in glucagon level, which was returned to the normal range after discontinuation of treatment. The clamp study showed that DIO mice injected with NPB112 at 5 mg/kg were more insulin sensitive than control mice, indicating amelioration of insulin resistance by treatment with NPB112. DIO mice treated with NPB112 showed a significant improvement in the ability of insulin to suppress HGP, showing a 33% suppression (from 8.3 mg/kg/min to 5.6 mg/kg/min) compared to the 2% suppression (from 9.8 mg/kg/min to 9.6 mg/kg/min) in control mice. In addition, no hypoglycemia or adverse effect was observed during the treatment. Conclusions A novel human monoclonal GCGR antibody, NPB112, effectively lowered the glucose level in diabetic animal models with mild and reversible hyperglucagonemia. Suppression of excess HGP with NPB112 may be a promising therapeutic modality for the treatment of type 2 diabetes.

Time-course changes in the expression levels of miR-122, -155, and -21 as markers of liver cell damage, inflammation, and regeneration in acetaminophen-induced liver injury in rats

Drug-induced liver injury (DILI) is a significant threat to patient health and a major concern during drug development. Recently, multiple circulating microRNAs (miRNAs) have been reported to be potential biomarkers for DILI. To adapt and validate miRNAs for clinical use, we investigated the time-course changes in miR-122 expression levels in an acetaminophen-induced liver injury model in rats. In addition, miR-155 and miR-21 were evaluated as makers of inflammation and regeneration, respectively, to characterize liver status. Our results revealed that miR-122 is an early and sensitive biomarker of hepatocellular injury at a stage when alanine transaminase, aspartate transaminase, and total bilirubin were not detectable. However, no significant differences in the expression levels of other miRNAs (miR-155 and -21) were observed between treatment and vehicle groups. Collectively, these time-course changes in the expression levels of miRNAs may be useful as markers for clinical decision-making, in the diagnosis and treatment of DILI.

Standardized Salvia miltiorrhiza Extract Suppresses Hepatic Stellate Cell Activation and Attenuates Steatohepatitis Induced by a Methionine-Choline Deficient Diet in Mice

The aim of this study was to examine the effect of standardized extract of Salvia miltiorrhiza (SME) on gene and protein expression of non-alcoholic steatohepatitis (NASH)-related factors in activated human hepatic stellate cells (HSC), and in mice with steatohepatitis induced by a methionine-choline deficient (MCD) diet. Male C57BL/6J mice were placed on an MCD or control diet for 8 weeks and SME (0, 0.1, 0.5 and 1 mg/kg body weight) was administered orally every other day for 4 or 6 weeks. HSCs from the LX-2 cell line were treated with transforming growth factor β-1 (TGF-β1) or TGF-β1 plus SME (0.1–10 μg/mL). To investigate the effect of SME on reactive oxygen species (ROS)-induced condition, LX-2 cells were treated with hydrogen peroxide (H2O2) or H2O2plus SME (0.1–100 μg/mL). MCD administration for 12 weeks increased mRNA expression of tumor necrosis factor (TNF-α), TGF-β1, interleukin-1β (IL-1β), C-reactive protein (CRP), α-smooth muscle actin (α-SMA), type I collagen, matrix metalloproteinase-2 (MMP-2) and MMP-9. TGF-β1-induced LX-2 cells exhibited similar gene expression patterns. SME treatment significantly reduced the mRNA and protein expression of NASH-related factors in the mouse model and HSCs. Histopathological liver analysis showed improved non-alcoholic fatty liver disease (NAFLD) activity and fibrosis score in SME-treated mice. The in vivo studies showed that SME had a significant effect at low doses. These results suggest that SME might be a potential therapeutic candidate for NAFLD treatment.

Antiatherosclerotic effects of the novel angiotensin receptor antagonist Fimasartan on plaque progression and stability in a rabbit model: a double-blind placebo-controlled trial.

Objectives: To evaluate the effect of the novel angiotensin receptor blocker Fimasartan on the development of atherosclerosis and plaque stabilization in an animal model. Methods: Twenty-four rabbits received an aortic balloon injury from 30 cm to a level just above the aortic valve to the iliac bifurcation using 3 Fr Fogarty catheters on third day of the experiment, followed by a 1% cholesterol diet for 8 weeks. The rabbits were randomized to receive placebo or 3 or 6 mg·kg−1·d−1 Fimasartan. The study was double blinded. The rabbits started receiving their medications 2 days before the aortic balloon injury and treatment continued. Atherosclerosis burden was determined by calculating the intima–media ratio of the infrarenal portion of the aorta because the bulk of the atherosclerotic burden was limited to the infrarenal region. The frequency of plaque disruption with thrombosis and the proportions of the plaques that were occupied by macrophages, smooth muscle cells, and collagen were determined. Results: Relative to the placebo group, the Fimasartan-treated rabbits had less atherosclerosis [intima–media ratio (mean ± SEM) of 1.14 ± 0.21 vs. 1.51 ± 0.26, P = 0.005], fewer disrupted plaques with thrombi (3 of 16 vs. 5 of 8, P = 0.047), lower proportion of macrophages (17.5% ± 2.5% vs. 26% ± 3.5%, P = 0.03), higher proportion of smooth muscle cells (43.5% ± 8.3% vs. 11.9% ± 2.1%, P = 0.001), and higher proportion of collagen (34.3% ± 6.4% vs. 19.7% ± 2.1%, P = 0.02). Conclusions: These results show that the newly developed angiotensin receptor blocker, Fimasartan, attenuated atherosclerosis progression and reduced macrophage accumulation in the rabbit aortic plaques.

Pancreatic cancer induced by in vivo electroporation-enhanced sleeping beauty transposon gene delivery system in mouse.

Objective The aim of this study was to establish a pancreatic tumor model of mouse using the electroporation-enhanced Sleeping Beauty (SB) transposon system. Methods The SB transposon system was used in conjunction with electroporation to deliver oncogenes, c-Myc and HRAS, and shRNA against p53 into the mouse pancreas to induce tumors. Oncogenes (c-Myc and HRAS) and shRNA against p53 gene were directly injected into the pancreas of the mouse along with in vivo electroporation applied on the injection site. The tumors were identified grossly and confirmed using animal positron emission tomographic imaging. The tumors were then characterized using histological and immunohistochemical techniques. The expression of the targeted genes (c-Myc, HRAS, and p53) was analyzed by a real-time quantitative polymerase chain reaction. Results Pancreatic tumors were successfully induced. The tumor phenotype was a sarcomatoid carcinoma, which was verified through immunohistochemistry. Some cysts or duct-like structures suggested to be metaplastic acinar cells were visible in the induced tumor. Conclusions The SB transposon enhanced with electroporation can readily generate pancreatic tumors in the mice, and thus, this model serves as a valuable resource for the mouse models of pancreatic cancer.

Sleeping Beauty transposon system harboring HRAS, c-Myc and shp53 induces sarcomatoid carcinomas in mouse skin

The Sleeping Beauty transposon system is used as a tool for insertional mutagenesis and oncogenesis. However, little is known about the exact histological phenotype of the tumors induced. Thus, we used immunohistochemical markers to enable histological identification of the type of tumor induced by subcutaneous injection of the HRAS, c-Myc and shp53 oncogenes in female C57BL/6 mice. The tumor was removed when it reached 100 mm3 in volume. Subsequently, we used 13 immunohistochemical markers to histologically identify the tumor type. The results suggested that the morphology of the tumor was similar to that of sarcomatoid carcinoma.

Preclinical evaluation of multi antigenic HCV DNA vaccine for the prevention of Hepatitis C virus infection

Direct-acting antiviral treatment for hepatitis C virus (HCV) infection is costly and does not protect from re-infection. For human and chimpanzees, recovery from acute HCV infection correlates with host CD4+ and CD8+ T cell responses. DNA plasmids targeting the HCV non-structural antigens NS3, NS4, and NS5, were previously reported to induce robust and sustained T cell responses in mice and primates. These plasmids were combined with a plasmid encoding cytokine IL-28B, together named as VGX-6150. The dose-dependent T cell response and safety of VGX-6150 administered intramuscularly and followed by electroporation was assessed in mice. Immune responses plateaued at 20 μg/dose with IL-28B demonstrating significant immunoadjuvant activity. Mice administered VGX-6150 at 40, 400, and 800 μg given either as a single injection or as 14 injections given bi-weekly over 26 weeks showed no vaccine related changes in any clinical parameter compared to placebo recipients. There was no evidence of VGX-6150 accumulation at the injection site or in any organ 1 month following the 14th vaccination. Based on these studies, the approximate lethal dose (ALD) exceeds 800 μg/dose and the NOAEL was 800 μg/dose in mouse. In conclusion, VGX-6150 appears safe and a promising preventive vaccine candidate for HCV infection.

Development of a Mouse Model of Prostate Cancer Using the Sleeping Beauty Transposon and Electroporation

The Sleeping Beauty (SB) transposon system is non-viral and uses insertional mutagenesis, resulting in the permanent expression of transferred genes. Although the SB transposon is a useful method for establishing a mouse tumor model, there has been difficulty in using this method to generate tumors in the prostate. In the present study, electroporation was used to enhance the transfection efficiency of the SB transposon. To generate tumors, three constructs (a c-Myc expression cassette, a HRAS (HRas proto-oncogene, GTPase) expression cassette and a shRNA against p53) contained within the SB transposon plasmids were directly injected into the prostate. Electroporation was conducted on the injection site after the injection of the DNA plasmid. Following the tumorigenesis, the tumors were monitored by animal PET imaging and identified by gross observation. After this, the tumors were characterized by using histological and immunohistochemical techniques. The expression of the targeted genes was analyzed by Real-Time qRT-PCR. All mice subjected to the injection were found to have prostate tumors, which was supported by PSA immunohistochemistry. To our knowledge, this is the first demonstration of tumor induction in the mouse prostate using the electroporation-enhanced SB transposon system in combination with c-Myc, HRAS and p53. This model serves as a valuable resource for the future development of SB-induced mouse models of cancer.

Evaluation of Circulating MicroRNA Biomarkers in the Acute Pancreatic Injury Dog Model

This study aimed to evaluate the usefulness of four microRNAs (miRNAs) in an acute pancreatic injury dog model. Acute pancreatitis was induced by infusion of cerulein for 2 h (7.5 μg/kg/h). The levels of well-known miRNAs, microRNA-216a (miR-216a) and microRNA-375 (miR-375), and new candidates microRNA-551b (miR-551b), and microRNA-7 (miR-7), were measured at 0, 0.5, 1, 2, 6, 12, and 24 h with serum amylase and lipase, and histopathological examination was performed. Among the four miRNAs, miR-216a and miR-375, and serum enzymes were significantly increased by cerulein treatment. The expression levels of miRNAs and serum enzymes peaked at 2–6 h with a similar pattern; however, the overall increases in miR-216a and miR-375 levels were much higher than those of the serum enzyme biomarkers. Increased levels of miR-216a and miR-375 were most highly correlated to the degree of individual histopathological injuries of the pancreas, and showed much greater dynamic response than serum enzyme biomarkers. Twenty-four-hour time-course analysis in this study revealed time-dependent changes of miRNA expression levels, from initial increase to decrease by predose level in acute pancreatitis. Our findings demonstrate that, in dogs, miR-216a and miR-375 have the potential to sensitively detect pancreatitis and reflect well the degree of pancreatic injury, whereas miR-551b and miR-7 do not.

Renal oncocytoma in a cat with chronic renal failure

Case summary A 9-year-old male neutered domestic shorthair cat presented with anorexia. Ultrasonography showed an irregularly shaped hypoechoic mass in the cranial pole of the right kidney. Ultrasound-guided fine-needle aspiration of the renal mass was performed. Cytology revealed moderate cellularity smears composed of epithelial cell clusters, which consisted of an exclusive population of oncocytic cells seen in sheets and papillary clusters along with abundant single cells. A moderate-to-abundant amount of densely stained granular cytoplasm with round nuclei and indistinct nucleoli was seen. The cytological diagnosis was renal oncocytic neoplasm. CT and surgical resection revealed a firm tan mass in the right kidney. A final diagnosis of renal oncocytoma was made on the basis of histology, immunohistochemical staining profile (positive for cytokeratin, and negative for chromogranin A, neuron-specific enolase and vimentin) of neoplastic cells, together with the electronic microscopy results. Relevance and novel information We believe that this is the first report of the cytological features of feline renal oncocytoma.