Analysis of MicroRNA in Bile Cytologic Samples Is Useful for Detection and Diagnosis of Extrahepatic Cholangiocarcinoma.

OBJECTIVES: This study aimed to develop reliable biomarkers that improve the ability of bile cytology to diagnose cholangiocarcinoma vs benign biliary lesions. METHODS: Many studies indicate that microRNAs (miRNAs) are potential candidates for the early diagnosis of cancer. We analyzed the expression of five tumor-associated miRNAs (miR-31-5p, miR-122-5p, miR-378d, miR-182-5p, and miR-92a-3p) in cytology samples using quantitative reverse transcription polymerase chain reaction. We collected 52 surgically resected tissue samples, 84 cytologic specimens from smears (53 cases of cancer and 31 cases of noncancer), and 40 residual sediments after smearing for routine cytology at Hiroshima University Hospital. RESULTS: The expression of miR-31-5p, miR-378d, and miR-122-5p was significantly higher in cancer tissues than those in normal tissues, while miR-182-5p expression was lower. The expression of miR-31-5p, miR-378d, miR-182-5p, and miR-92a-3p was significantly higher in detached cell samples from smears of cholangiocarcinoma cases than in those from noncancer cases. CONCLUSIONS: These results suggest that the analysis of miRNAs in bile cytologic specimens is a promising auxiliary tool for distinguishing cholangiocarcinoma from benign biliary lesions.

Erratum to: Efficacy of Additional Canagliflozin Administration to Type 2 Diabetes Patients Receiving Insulin Therapy: Examination of Diurnal Glycemic Patterns Using Continuous Glucose Monitoring (CGM).

Unfortunately, the original publication of this paper contained errors in the presentation of Fig. 2.

Efficacy of Additional Canagliflozin Administration to Type 2 Diabetes Patients Receiving Insulin Therapy: Examination of Diurnal Glycemic Patterns Using Continuous Glucose Monitoring (CGM).

INTRODUCTION: The efficacy of administering a sodium-glucose cotransporter 2 inhibitor during insulin therapy has not been established. In this study, we examined its effects based on diurnal glycemic patterns using continuous glucose monitoring (CGM). METHODS: The subjects were 15 patients who had received insulin therapy for 1 year or more. A CGM device was attached to all subjects for 1 week. The administration of canagliflozin at 100 mg was started 4 days after attachment. The mean glucose concentrations, standard deviation (SD), mean amplitude of glycemic excursions (MAGE), mean of daily difference of blood glucose (MODD), and area under the curve (AUC) (≥180, <70 mg h/dL) after the start of administration were compared with the pretreatment values. In addition, we compared changes in the number of insulin units between basal and bolus insulin. Furthermore, we investigated the influence of canagliflozin on oxidative stress markers and cytokines using 8-hydroxy-2'-deoxyguanosine (8-OHdG), tumor necrosis factor-α (TNF-α), and adiponectin as parameters. RESULTS: The mean glucose concentrations decreased from 161.1 to 139.1 mg/dL (P < 0.01). The SD decreased from 36.5 to 29.6 mg/dL (P = 0.05). The MAGE decreased from 89.2 to 77.4 mg/dL (P < 0.01), and the MODD decreased from 34.3 to 25.5 mg/dL (P < 0.05). All parameters showed significant improvements in diurnal changes. AUC of ≥180, i.e., the total area of blood glucose levels at or above 180 on the blood glucose curve of CGM, decreased from 339.1 to 113.6 mg/dL (P < 0.05). AUC of <70, i.e., the total area of blood glucose levels below 70 on the blood glucose curve of CGM, slightly decreased from 1.6 to 0.3 mg/dL (P = 0.08). The total number of basal insulin units decreased from 128 to 76, and that of bolus insulin decreased from 266 to 154; the dose of insulin could be markedly decreased. In addition, the mean 8-OHdG level decreased from 11.4 to 10.8 ng/mg Cre (P < 0.05), and the mean TNF-α level decreased from 2.31 to 1.79 pg/mL (P = 0.10). The mean adiponectin level increased from 5.01 to 5.53 µg/mL (P < 0.05). CONCLUSION: Canagliflozin improved blood glucose changes in type 2 diabetes using insulin. In addition, the results suggest its antioxidant actions. TRIAL REGISTRATION: University Hospital Medical Information Network (UMIN no. 000019429).

The effects of intermittent use of the SGLT-2 inhibitor, dapagliflozin, in overweight patients with type 2 diabetes in Japan: a randomized, crossover, controlled clinical trial.

BACKGROUND: This study examined the effects of short-term administration of the sodium glucose cotransporter 2 (SGLT-2) inhibitor, dapagliflozin, on visceral fat area (VFA) in Japanese patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: In this randomized, crossover, controlled clinical trial, overweight patients with type 2 diabetes were randomized to treatment with 5 mg dapagliflozin for the first (n = 27) or second 12-week study period (n = 29). The parameters evaluated at baseline and after 12 and 24 weeks included blood pressure, hemoglobin A1c (HbA1c), body composition, VFA, and subcutaneous fat area (SFA). RESULTS: In both groups, dapagliflozin administration improved the levels of HbA1c, body weight, blood pressure, total fat mass, and VFA. Cessation of dapagliflozin, however, reversed the improvements in HbA1c, blood pressure, body weight, and SFA levels, whereas reductions in VFA and total fat mass levels were somewhat maintained even after 12 weeks without treatment. CONCLUSIONS: Dapagliflozin led to decreases in VFA and, consequently, body weight after a short treatment period. However, these effects were largely reversed by the cessation of dapagliflozin, suggesting that this agent should be administered continuously to maintain clinical usefulness in overweight patients with type 2 diabetes.

Fixed-dose combination of alogliptin/pioglitazone improves glycemic control in Japanese patients with type 2 diabetes mellitus independent of body mass index.

This study investigated the effects of switching from combination therapy with either alogliptin (Alo) or pioglitazone (Pio) to fixed-dose combination therapy (FDCT) with alogliptin and pioglitazone (Alo-Pio FDCT). The usefulness and efficacy of Alo-Pio FDCT were investigated. A total of 50 outpatients with type 2 diabetes mellitus (T2DM) treated with Alo and 47 outpatients with T2DM treated with Pio were switched to Alo-Pio FDCT, and its efficacy and usefulness were evaluated. Significant improvements were observed in hemoglobinA1c (HbA1c), alanine transaminase (ALT), and γ-glutamyl transpeptidase (GGT) levels after switching to Alo-Pio FDCT for 16 weeks in both groups. Only the group switching from Alo to Alo-Pio FDCT showed significant improvements in high-density lipoprotein cholesterol (HDL) levels and triglyceride levels. In a multivariate logistic regression model of the variation in the change of HbA1c at 16 weeks, ALT and GGT were independent predictors of the change of HbA1c at 16 weeks. In addition, the switch to Alo-Pio FDCT improved glycemic control to a certain degree regardless of BMI. Switching from either Alo or Pio to Alo-PIO FDCT may, unlike monotherapy with a DPP-4 inhibitor, be effective for patients with T2DM regardless of whether they are obese or lean.

Evaluation of a Premixed Insulin Analog Suspension in Japanese People with Type 2 Diabetes and the Clinical Importance of Improved Injection Techniques: A Cross-Sectional Pilot Study.

INTRODUCTION: Patients with type 2 diabetes, who live in Asian countries, often use premixed insulin analogs. However, if these solutions are insufficiently mixed prior to use, patients will receive inaccurate doses of intermediate- and/or short-acting insulin, which could affect diabetes control. This study aimed to determine whether insulin users were correctly resuspending premixed insulin analog solutions prior to use. METHODS: We investigated whether Japanese patients with type 2 diabetes were correctly resuspending their premixed insulin analog solutions by assessing the optical densities (ODs) of the solutions. RESULTS: Among 476 patients who used premixed insulin analogs, we found that the ODs of residual insulin differed significantly from the control values, particularly for high-mix insulin suspensions. CONCLUSION: Our findings suggest that patients should be educated about the importance of properly resuspending these insulin analog solutions prior to use. TRIAL REGISTRATION: University Hospital Medical Information Network (UMIN No. 000022329).

Clinical Implication of Plasma Hydrogen Sulfide Levels in Japanese Patients with Type 2 Diabetes.

Objective The goal of the present study was to investigate the plasma hydrogen sulfide (H2S) levels in patients with type 2 diabetes, as the plasma H2S levels in Japanese patients with type 2 diabetes remain unclear. Methods The plasma H2S levels were measured in 154 outpatients with type 2 diabetes and 66 outpatients without diabetes. All blood samples were collected in the outpatient department from 09:00 to 10:00. The patients had fasted from 21:00 the previous evening and had not consumed alcohol or caffeine or smoked until sample collection. The plasma H2S levels were measured using the methylene blue assay. The plasma H2S levels were determined in triplicate, and the average concentrations were calculated against a calibration curve of sodium sulfide. Results The patients with type 2 diabetes showed a progressive reduction in the plasma H2S levels (45.115.5 M versus 54.026.4 M, p<0.05), which paralleled poor glycemic control. There was a significant correlation between a reduction in the plasma H2S levels and the HbA1c levels (=-0.505, p<0.01), Furthermore, a reduction in the plasma H2S levels was found to be related to a history of cardiovascular diseases in patients with type 2 diabetes (39.913.8 M versus 47.515.9 M, p<0.01). Conclusion Collectively, the plasma H2S levels were reduced in patients with type 2 diabetes, which may have implications in the pathophysiology of cardiovascular disease in diabetic patients. The trial was registered with the University Hospital Medical Information Network (UMIN no. #000020549).

Impact of teneligliptin on oxidative stress and endothelial function in type 2 diabetes patients with chronic kidney disease: a case-control study.

BACKGROUND: The aim of the present study was to elucidate the effect of teneligliptin on oxidative stress and endothelial function in Japanese patients with type 2 diabetes and chronic kidney disease (CKD). METHODS: Forty-five patients with type 2 diabetes and CKD who received sitagliptin for at least 12 months were randomized to either continue sitagliptin (n = 23) or switch to teneligliptin (n = 22) for 24 weeks. The following parameters were evaluated at baseline and after 24 weeks of treatment with continued sitagliptin or teneligliptin: blood pressure, haemoglobin A1c (HbA1c), estimated glomerular filtration rate (eGFR), urinary albumin excretion, endothelial function by reactive hyperaemia index (RHI; EndoPAT(®) system), reactive oxygen metabolites (ROMs) measured by the d-ROMS test, 8-hydroxy-2'-deoxyguanosine, urinary liver-type fatty acid binding protein (L-FABP), and urinary 8-isoprostane. RESULTS: The two groups did not significantly differ with regard to age, male-to-female ratio, duration of diabetes, body mass index, HbA1c, eGFR, or urinary albumin excretion levels at baseline. We found no significant differences in changes of HbA1c, eGFR, or urinary albumin excretion levels between the two groups after 24 weeks of treatment. However, treatment with teneligliptin, but not sitagliptin, significantly improved RHI values and was correlated with the percent changes in RHI and d-ROMs. CONCLUSIONS: The present study demonstrated that teneligliptin, can improve endothelial function and reduce renal and vascular oxidative stress in patients with type 2 diabetes and CKD, independently of reducing albuminuria or improving glucose control. Trial registration UMIN000017180.

Postprandial Reactive Hypoglycemia Treated with a Low-dose Alpha-glucosidase Inhibitor: Voglibose May Suppress Oxidative Stress and Prevent Endothelial Dysfunction.

We encountered a 62-year-old woman who experienced frequent episodes of hypoglycemia. She was diagnosed with postprandial reactive hypoglycemia according to the results of oral glucose and sucrose tolerance tests, having undergone an endocrinological examination and image inspection. The administration of low-dose voglibose, an alpha-glucosidase inhibitor (α-GI), improved the glucose fluctuations and inhibited hypoglycemic symptoms. Voglibose is also known to diminish oxidative stress and maintain endothelial function after hyperglycemia. An α-GI might effectively prevent hypoglycemic symptoms and endothelial dysfunction by suppressing oxidative stress in such cases.

Anti-Hepatitis C Virus Activity of a Crude Extract from Longan (Dimocarpus longan Lour.) Leaves.

Infection with hepatitis C virus (HCV) results in hepatitis C, a disease characterized by chronic infection, cirrhosis, and hepatocellular carcinoma. Currently, the standard therapy is a combination of pegylated interferon-α plus ribavirin with NS3 protease inhibitors. Addition of NS3 protease inhibitors to the standard therapy improves response rates; however, use of NS3 protease inhibitors is also associated with significant adverse effects and an increase in the overall cost of treatment. Therefore, there is a need to develop safe and inexpensive drugs for the treatment of HCV infections. In this study, we examined the antiviral activity of a crude extract from Dimocarpus longan leaves against HCV (genotype 2a strain JFH1). The D. longan crude extract (DL-CE) exhibited anti-HCV activity with a 50% effective concentration (EC50) of 19.4 µg/ml without cytotoxicity. A time-of-addition study demonstrated that DL-CE has anti-HCV activity at both the entry and post-entry steps and markedly blocks the viral entry step through direct virucidal activity with marginal inhibition of virion assembly. Co-treatment of DL-CE with cyclosporine A, an immunosuppressant or telaprevir, an NS3 protease inhibitor, resulted in additive and synergistic antiviral effects, respectively. Our findings suggest that DL-CE may be useful as an add-on therapy candidate for treating HCV infections.

Virocidal activity of Egyptian scorpion venoms against hepatitis C virus.

BACKGROUND: Hepatitis C virus (HCV) is a major global health problem, causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Development of well-tolerated regimens with high cure rates and fewer side effects is still much needed. Recently, natural antimicrobial peptides (AMPs) are attracting more attention as biological compounds and can be a good template to develop therapeutic agents, including antiviral agents against a variety of viruses. Various AMPs have been characterized from the venom of different venomous animals including scorpions. METHODS: The possible antiviral activities of crude venoms obtained from five Egyptian scorpion species (Leiurus quinquestriatus, Androctonus amoreuxi, A. australis, A. bicolor and Scorpio maurus palmatus) were evaluated by a cell culture method using Huh7.5 cells and the J6/JFH1-P47 strain of HCV. Time-of-addition experiments and inactivation of enzymatic activities of the venoms were carried out to determine the characteristics of the anti-HCV activities. RESULTS: S. maurus palmatus and A. australis venoms showed anti-HCV activities, with 50% inhibitory concentrations (IC50) being 6.3 ± 1.6 and 88.3 ± 5.8 µg/ml, respectively. S. maurus palmatus venom (30 µg/ml) impaired HCV infectivity in culture medium, but not inside the cells, through virocidal effect. The anti-HCV activity of this venom was not inhibited by a metalloprotease inhibitor or heating at 60°C. The antiviral activity was directed preferentially against HCV. CONCLUSIONS: S. maurus palmatus venom is considered as a good natural source for characterization and development of novel anti-HCV agents targeting the entry step. To our knowledge, this is the first report describing antiviral activities of Egyptian scorpion venoms against HCV, and may open a new approach towards discovering antiviral compounds derived from scorpion venoms.

Comparison between mechanical stress and bone mineral density in the femur after total hip arthroplasty by using subject-specific finite element analyses.

The mechanism underling bone mineral density (BMD) loss that occurs in the femur after total hip arthroplasty (THA) remains unknown. We compared the equivalent stress and strain energy density (SED) to BMD in the femur after THA using subject-specific finite element analyses. Twenty-four patients who had undergone primary cementless THA were analysed. BMD was measured using dual-energy X-ray absorptiometry (DEXA) at 1 week and 3, 6 and 12 months after THA. Seven regions of interest (ROIs) were defined in accordance with Gruen's system (ROIs 1-7). Computed tomography images of the femurs were acquired pre- and postoperatively, and the images were converted into three-dimensional finite element (FE) models. Equivalent stress and SED were analysed and compared with DEXA data. BMD was maintained 1 year after THA in ROIs 3, 4, 5 and 6, whereas BMD decreased in ROIs 1, 2 and 7. FE analysis revealed that equivalent stress in ROIs 3, 4, 5 and 6 was much higher than that in ROIs 1, 2 and 7. A significant correlation was observed between the rate of changes in BMD and equivalent stress. Reduction of equivalent stress may contribute to decrease in BMD in the femur after THA.

Inhibition of hepatitis C virus replication by chalepin and pseudane IX isolated from Ruta angustifolia leaves.

Hepatitis C virus (HCV) infection is highly prevalent among global populations, with an estimated number of infected patients being 170 million. Approximately 70-80% of patients acutely infected with HCV will progress to chronic liver disease, such as liver cirrhosis and hepatocellular carcinoma, which is a substantial cause of morbidity and mortality worldwide. New therapies for HCV infection have been developed, however, the therapeutic efficacies still need to be improved. Medicinal plants are promising sources for antivirals against HCV. A variety of plants have been tested and proven to be beneficial as antiviral drug candidates against HCV. In this study, we examined extracts, their subfractions and isolated compounds of Ruta angustifolia leaves for antiviral activities against HCV in cell culture. We isolated six compounds, chalepin, scopoletin, γ-fagarine, arborinine, kokusaginine and pseudane IX. Among them, chalepin and pseudane IX showed strong anti-HCV activities with 50% inhibitory concentration (IC50) of 1.7 ± 0.5 and 1.4 ± 0.2 µg/ml, respectively, without apparent cytotoxicity. Their anti-HCV activities were stronger than that of ribavirin (2.8 ± 0.4 µg/ml), which has been widely used for the treatment of HCV infection. Mode-of-action analyses revealed that chalepin and pseudane IX inhibited HCV at the post-entry step and decreased the levels of HCV RNA replication and viral protein synthesis. We also observed that arborinine, kokusaginine and γ-fagarine possessed moderate levels of anti-HCV activities with IC50 values being 6.4 ± 0.7, 6.4 ± 1.6 and 20.4 ± 0.4 µg/ml, respectively, whereas scopoletin did not exert significant anti-HCV activities at 30 µg/ml.

Add-on treatment with teneligliptin ameliorates glucose fluctuations and improves glycemic control index in Japanese patients with type 2 diabetes on insulin therapy.

BACKGROUND: This study investigated whether teneligliptin, a novel dipeptidyl peptidase-4 inhibitor, ameliorated glucose fluctuations in hospitalized Japanese patients with type 2 diabetes receiving insulin therapy, with or without other antidiabetes drugs, and using continuous glucose monitoring (CGM). PATIENTS AND METHODS: Twenty-six patients with type 2 diabetes were admitted for glycemic control. After admission, patients continued to be treated with optimal dietary therapy plus insulin therapy, with or without other antidiabetes drugs, until they achieved stable glycemic control. CGM measurements were made for 7 consecutive days. On Days 1-3, patients received insulin with or without other antidiabetes drugs, and on Days 4-7, teneligliptin 20 mg once daily at breakfast was added to ongoing therapy. Doses of insulin were fixed during the study. Levels of serum glycated albumin (GA), 1,5-anhydro-d-glucitol (1,5-AG), and high-sensitivity C-reactive protein (hsCRP) were measured. RESULTS: Add-on treatment with teneligliptin led to significant improvements in 24-h mean glucose levels, the proportion of time in normoglycemia, mean amplitude of glycemic excursions, and total area under the curve within 2 h after each meal. The proportion of time in hypoglycemia and hsCRP levels did not increase significantly compared with before teneligliptin. Values of 1,5-AG and GA were significantly improved by treatment with teneligliptin. CONCLUSIONS: Addition of teneligliptin to insulin therapy led to a significant improvement in diurnal glycemic control and significant reductions in glucose fluctuations in 24-h periods without increasing hypoglycemia in Japanese patients with type 2 diabetes on insulin therapy, with or without other antidiabetes agents.

Induction of cell-mediated immune responses in mice by DNA vaccines that express hepatitis C virus NS3 mutants lacking serine protease and NTPase/RNA helicase activities.

Effective therapeutic vaccines against virus infection must induce sufficient levels of cell-mediated immune responses against the target viral epitopes and also must avoid concomitant risk factors, such as potential carcinogenic properties. The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) carries a variety of CD4(+) and CD8(+) T cell epitopes, and induces strong HCV-specific T cell responses, which are correlated with viral clearance and resolution of acute HCV infection. On the other hand, NS3 possesses serine protease and nucleoside triphosphatase (NTPase)/RNA helicase activities, which not only play important roles in viral life cycle but also concomitantly interfere with host defense mechanisms by deregulating normal cellular functions. In this study, we constructed a series of DNA vaccines that express NS3 of HCV. To avoid the potential harm of NS3, we introduced mutations to the catalytic triad of the serine protease (H57A, D81A and S139A) and the NTPase/RNA helicase domain (K210N, F444A, R461Q and W501A) to eliminate the enzymatic activities. Immunization of BALB/c mice with each of the DNA vaccine candidates (pNS3[S139A/K210N], pNS3[S139A/F444A], pNS3[S139A/R461Q] and pNS3[S139A/W501A]) that expresses an NS3 mutant lacking both serine protease and NTPase/helicase activities induced T cell immune responses to the degree comparable to that induced by the wild type NS3 and the NS3/4A complex, as demonstrated by interferon-γ production and cytotoxic T lymphocytes activities against NS3. The present study has demonstrated that plasmids expressing NS3 mutants, NS3(S139A/K210N), NS3(S139A/F444A), NS3(S139A/R461Q) and NS3(S139A/W501A), which lack both serine protease and NTPase/RNA helicase activities, would be good candidates for safe and efficient therapeutic DNA vaccines against HCV infection.

Discrepancy between clinical and radiological responses to tocilizumab treatment in patients with systemic-onset juvenile idiopathic arthritis.

OBJECTIVE: Tocilizumab (TCZ), an antiinterleukin-6 receptor monoclonal antibody, is clinically beneficial in patients with systemic-onset juvenile idiopathic arthritis (sJIA). We investigated the clinical and radiological outcomes of TCZ therapy in patients with sJIA. METHODS: We retrospectively evaluated 2 clinical trials (NCT00144599 and NCT00144612) involving 40 patients with sJIA who received intravenous TCZ (8 mg/kg) every 2 weeks. Clinical data and radiographs of the hands and large joints were assessed before and during TCZ treatment. The Poznanski score, modified Larsen scores of the hands and large joints, and Childhood Arthritis Radiographic Score of the Hip (CARSH) were recorded. RESULTS: After a mean duration of 4.5 years of TCZ treatment, clinical data had improved significantly, the mean Poznanski score improved from -1.5 to -1.1, the mean Larsen score of the hands deteriorated from 7.0 to 10.0, the mean Larsen score for the large joints deteriorated from 5.9 to 6.8, and the CARSH worsened from 3.9 to 6.2. The Larsen score for the large joints improved in 11 cases (28%), remained unchanged in 8 cases (20%), and worsened in 21 cases (52%). Matrix metalloproteinase 3 (MMP-3) levels remained significantly higher (278 mg/dl) in patients with worsened Larsen scores than in patients with improved or unchanged scores (65 mg/dl). Logistic regression analysis showed that older age at disease onset was a significant risk factor for radiographic progression. CONCLUSION: The modified Larsen score of the large joints deteriorated in half the patients who had high MMP-3 levels during TCZ treatment and who were significantly older at disease onset.

Antiviral activity of extracts from Morinda citrifolia leaves and chlorophyll catabolites, pheophorbide a and pyropheophorbide a, against hepatitis C virus.

The development of complementary and/or alternative drugs for treatment of hepatitis C virus (HCV) infection is still needed. Antiviral compounds in medicinal plants are potentially good targets to study. Morinda citrifolia is a common plant distributed widely in Indo-Pacific region; its fruits and leaves are food sources and are also used as a treatment in traditional medicine. In this study, using a HCV cell culture system, it was demonstrated that a methanol extract, its n-hexane, and ethyl acetate fractions from M. citrifolia leaves possess anti-HCV activities with 50%-inhibitory concentrations (IC(50)) of 20.6, 6.1, and 6.6 µg/mL, respectively. Bioactivity-guided purification and structural analysis led to isolation and identification of pheophorbide a, the major catabolite of chlorophyll a, as an anti-HCV compound present in the extracts (IC(50) = 0.3 µg/mL). It was also found that pyropheophorbide a possesses anti-HCV activity (IC(50) = 0.2 µg/mL). The 50%-cytotoxic concentrations (CC(50)) of pheophorbide a and pyropheophorbide a were 10.0 and 7.2 µg/mL, respectively, their selectivity indexes being 33 and 36, respectively. On the other hand, chlorophyll a, sodium copper chlorophyllin, and pheophytin a barely, or only marginally, exhibited anti-HCV activities. Time-of-addition analysis revealed that pheophorbide a and pyropheophorbide a act at both entry and the post-entry steps. The present results suggest that pheophorbide a and its related compounds would be good candidates for seed compounds for developing antivirals against HCV.

Anti-hepatitis C virus compounds obtained from Glycyrrhiza uralensis and other Glycyrrhiza species.

Development of complementary and/or alternative drugs for treatment of hepatitis C virus (HCV) infection is still much needed from clinical and economic points of view. Antiviral substances obtained from medicinal plants are potentially good targets to study. Glycyrrhiza uralensis and G. glabra have been commonly used in both traditional and modern medicine. In this study, extracts of G. uralensis roots and their components were examined for anti-HCV activity using an HCV cell culture system. It was found that a methanol extract of G. uralensis roots and its chloroform fraction possess anti-HCV activity with 50%-inhibitory concentrations (IC(50)) of 20.0 and 8.0 µg/mL, respectively. Through bioactivity-guided purification and structural analysis, glycycoumarin, glycyrin, glycyrol and liquiritigenin were isolated and identified as anti-HCV compounds, their IC(50) being 8.8, 7.2, 4.6 and 16.4 µg/mL, respectively. However, glycyrrhizin, the major constituent of G. uralensis, and its monoammonium salt, showed only marginal anti-HCV activity. It was also found that licochalcone A and glabridin, known to be exclusive constituents of G. inflata and G. glabra, respectively, did have anti-HCV activity, their IC(50) being 2.5 and 6.2 µg/mL, respectively. Another chalcone, isoliquiritigenin, also showed anti-HCV activity, with an IC(50) of 3.7 µg/mL. Time-of-addition analysis revealed that all Glycyrrhiza-derived anti-HCV compounds tested in this study act at the post-entry step. In conclusion, the present results suggest that glycycoumarin, glycyrin, glycyrol and liquiritigenin isolated from G. uralensis, as well as isoliquiritigenin, licochalcone A and glabridin, would be good candidates for seed compounds to develop antivirals against HCV.

Antiviral activities of Indonesian medicinal plants in the East Java region against hepatitis C virus.

BACKGROUND: Hepatitis C virus (HCV) is a major cause of liver disease and a potential cause of substantial morbidity and mortality worldwide. The overall prevalence of HCV infection is 2%, representing 120 million people worldwide. Current standard treatment using pegylated interferon and ribavirin is effective in only 50% of the patients infected with HCV genotype 1, and is associated with significant side effects. Therefore, it is still of importance to develop new drugs for treatment of HCV. Antiviral substances obtained from natural products, including medicinal plants, are potentially good targets to study. In this study, we evaluated Indonesian medicinal plants for their anti-HCV activities. METHODS: Ethanol extracts of 21 samples derived from 17 species of medicinal plants explored in the East Java region were tested. Anti-HCV activities were determined by a cell culture method using Huh7.5 cells and HCV strains of 9 different genotypes (1a to 7a, 1b and 2b). RESULTS: Four of the 21 samples tested showed antiviral activities against HCV: Toona sureni leaves (TSL) with 50% inhibitory concentrations (IC50) of 13.9 and 2.0 µg/ml against the HCV J6/JFH1-P47 and -P1 strains, respectively, Melicope latifolia leaves (MLL) with IC50 of 3.5 and 2.1 µg/ml, respectively, Melanolepis multiglandulosa stem (MMS) with IC50 of 17.1 and 6.2 µg/ml, respectively, and Ficus fistulosa leaves (FFL) with IC50 of 15.0 and 5.7 µg/ml, respectively. Time-of-addition experiments revealed that TSL and MLL inhibited both at the entry and post-entry steps while MMS and FFL principally at the entry step. TSL and MLL inhibited all of 11 HCV strains of all the genotypes tested to the same extent. On the other hand, FFL showed significantly weaker inhibitory activities against the HCV genotype 1a strain, and MMS against the HCV strains of genotypes 2b and 7a to a lesser extent, compared to the other HCV genotypes. CONCLUSIONS: Ethanol extracts of TSL, MLL, MMS and FFL showed antiviral activities against all the HCV genotypes tested with the exception that some genotype(s) showed significant resistance to FFL and to MMS to a lesser extent. These plant extracts may be good candidates for the development of anti-HCV drugs.

Hyperparathyroidism caused by a functional parathyroid cyst.

A 67-year-old Japanese woman was admitted to our hospital for malaise and loss of appetite. Relevant biochemical examinations showed definite hypercalcaemia and elevated serum levels of intact parathyroid hormone (PTH). We performed thyroid ultrasonography and CT of the neck, which revealed a cystic lesion in the right lower lobe of the thyroid glands. Ultrasound-guided fine-needle aspiration was performed, and PTH level of the cystic fluid was markedly elevated. Technetium-99m-hexakis 2-methoxyisobutyi isonitrile sesta scintigraphy showed intense ring-shaped accumulation of radioactivity in the wall of the cyst. The patient underwent a right lobectomy to resect the cystic parathyroid adenoma. After surgery, her serum calcium and PTH level returned to normal ranges.