Isolation of amoenamide A and five antipodal prenylated alkaloids from Aspergillus amoenus NRRL 35600.

A new prenylated alkaloid, Amoenamide A (6), was isolated from the fungus Aspergillus amoenus NRRL 35600. Previously, 6 was postulated to be a precursor of Notoamide E4 (21) converted from Notoamide E (16), which was a key precursor of the prenylated indole alkaloids in the fungi of the genus Aspergillus. We previously succeeded in the isolation of two pairs of antipodes, Stephacidin A (1) and Notoamide B (2), from A. amoenus and A. protuberus MF297-2 and expected the presence of other antipodes in the culture of A. amoenus. We here report five new antipodes (7-11) along with a new metabolite (12), which was isolated as a natural compound for the first time, from A. amoenus.

Efficacy and Safety of Sofosbuvir Plus Ribavirin Treatment for Patients with Chronic Hepatitis C Genotype 2.

OBJECTIVES: The efficacy of sofosbuvir plus ribavirin (RBV) treatment for hepatitis C virus (HCV) genotype 2 focusing on virological response was compared with that of pegylated interferon (peg-IFN) plus RBV treatment. Safety of the former focusing on the decline in hemoglobin levels was compared with that of the latter and assessed in terms of age and inosine triphosphatase (ITPA). METHODS: Patients (n = 17) receiving sofosbuvir plus RBV and those (n = 24) receiving peg-IFN plus RBV diagnosed with chronic HCV genotype 2 were enrolled in this study, and the efficacy and safety of both treatments were assessed. RESULTS: Rapid virological response was attained with sofosbuvir plus RBV treatment compared with peg-IFN plus RBV treatment. All patients under sofosbuvir plus RBV treatment achieved end-of-treatment response compared with 70% who sustained viral response under the peg-IFN plus RBV treatment, with the former demonstrating greater virological response. The decline in hemoglobin levels under the former treatment was greater than that under the latter and in patients over 65 years of age with ITPA gene major. CONCLUSION: Efficacy and safety of sofosbuvir plus RBV treatment were clearly demonstrated compared with those of peg-IFN plus RBV treatment. The decline in hemoglobin levels was not related to the discontinuation of the former treatment, irrespective of age or the effect of the ITPA gene.

Coffee Intake and Liver Enzyme Association in Korean Immigrants and Japanese: A Comprehensive Cross-Sectional Study.

OBJECTIVES: Significant inverse association between coffee intake and the levels of liver enzymes has been reported. We demonstrated higher prevalence of metabolic syndrome in Korean immigrants (KIs) than in indigenous Japanese (IJs). The aim of this study was to investigate whether the association between coffee intake and liver enzyme levels was different between the 2 ethnic groups. METHODS: This study is a cross-sectional study including a total of 966 subjects comprising KIs and IJs. The association between the quintiles of coffee intake and dichotomous values of liver enzymes was evaluated by logistic regression analysis in KIs, IJs, a high-risk group (current smokers or alcohol drinkers ≥45 g/day), and a low-risk group (non-smokers and alcohol drinkers <45 g/day). RESULTS: In KIs, a significant inverse association between coffee intake and serum aspartate aminotransferase (AST) levels was observed. In the IJs, a significant inverse association between coffee intake and serum alanine aminotransferase levels was observed. In the high-risk group, a significant inverse association between coffee intake and serum AST and gamma-glutamyltransferase levels was observed. CONCLUSION: No difference was observed between KIs and IJs regarding the association between coffee and liver enzymes. Coffee might inhibit hepatic damage by alcohol drinking and smoking.

Comparison of Daclatasvir and Asunaprevir for Chronic HCV 1b Infection with Telaprevir and Simeprevir plus Peginterferon and Ribavirin, with a Focus on the Prevention of Occurrence and Recurrence of Hepatocellular Carcinoma.

OBJECTIVES: The efficacy of the all-oral administration of daclatasvir and asunaprevir for 24 weeks was compared with that of telaprevir for 12 weeks plus pegylated interferon and ribavirin (PEG-IFN/RBV) for 24 weeks, and that of simeprevir for 12 weeks plus PEG-IFN/RBV for 24 weeks, with a focus on the prevention of occurrence and recurrence of hepatocellular carcinoma (HCC). The levels of alanine aminotransferase (ALT) and α-fetoprotein (AFP) as suppressive markers of HCC were also measured. METHODS: Patients received daclatasvir and asunaprevir (n = 17), simeprevir plus PEG-IFN/RBV (n = 15) and telaprevir plus PEG-IFN/RBV (n = 25). Sustained virological response (SVR) and the mean change in the level of serum ALT, AFP and platelet (PLT) count were compared among the three groups. RESULTS: No difference in SVR was observed in patients given daclatasvir with asunaprevir (SVR4), telaprevir plus PEG-IFN/RBV or simeprevir plus PEG-IFN/RBV (SVR24). Also, no significant difference was observed in the mean change of serum ALT, AFP or PLT count among the three groups. CONCLUSION: The preventive effect of the IFN-free, all-oral regimen of daclatasvir and asunaprevir was observed with a focus on the occurrence and recurrence of HCC, as was IFN-based treatment with telaprevir or simeprevir plus PEG-IFN/RBV.

Recent Advances in the Management of Chronic Hepatitis B Including Suppression of Hepatocellular Carcinoma by Entecavir and Interferon.

At present, for adults with chronic hepatitis B virus (HBV) infection, two new analogues, entecavir (ETV) and tenofovir, are recommended as the first-line therapy by the EASL (European Association for the Study of the Liver), AASLD (American Association for the Study of Liver Diseases), and APASL (Asian Pacific Association for the Study of the Liver) guidelines. The use of pegylated interferon-α (PEG IFN-α) is recommended as the first-line therapy instead of standard IFN-α according to the above 3 guidelines. In this paper, the aim was to assess: (1) the long-term efficacy and safety as well as the resistance to ETV and tenofovir disoproxil fumarate (TDF); (2) the efficacy of PEG IFN-α; (3) the role of combination therapy with IFN plus two analogues, such as lamivudine and ETV; (4) the efficacy and safety of two analogues with cirrhosis, and (5) suppression of hepatocellular carcinoma (HCC) by ETV and IFN treatment. The results are as follows: (1) both ETV and TDF showed long-term efficacy and safety; (2) PEG IFN-α resulted in a greater decline in HBV DNA levels and a higher rate of HBeAg seroconversion; (3) combination therapy with IFN plus two analogues did not elevate the rate of sustained responses; (4) both ETV and TDF showed efficacy and safety with cirrhosis (ETV especially displayed efficacy and safety with decompensated cirrhosis), and (5) suppression of HCC was observed by ETV and IFN.

Usefulness of Cytokeratin-18M65 in Diagnosing Non-Alcoholic Steatohepatitis in Japanese Population.

OBJECTIVE: The aim of this study was to evaluate cytokeratin-18M65 (CK-18M65) for distinguishing between simple steatosis (SS) and non-alcoholic steatohepatitis (NASH) against healthy individuals (HIs) in Japanese population. METHODS: The serum from 24 HIs, 21 patients with SS and 20 patients with NASH were examined. Serum CK-18M65 was measured by enzyme-linked immunosorbent assay. RESULTS: Aspartate aminotransferase was significantly different between NASH patients and HIs with p < 0.0001 (SS patients and HIs: p < 0.0001), as was alanine aminotransferase between NASH patients and HIs with p < 0.0001 (SS patients and HIs: p < 0.0001). Serum CK-18M65 increased in a stepwise fashion in HIs and also in SS and NASH patients. Multivariate logistic regression analysis revealed that NASH could be diagnosed with the use of CK-18M65 alone (p = 0.0285, OR 1.0038, 95% CI 1.0004-1.0073). At the optimal cut-off level of 548 U/l, CK-18M65 had an AUC value of 0.7369, 60.00% sensitivity and 85.70% specificity. In patients with NASH, no significant difference was observed between low fibrosis (Stage 0-1, 794.30 ± 454.41, n = 10) and high fibrosis (Stage 2-3, 809.70 ± 641.43, n = 10; p = 0.5967) and between slight steatosis (<33%, 512.89 ± 229.65, n = 9) and moderate steatosis (≥33%, 655.13 ± 480.78, n = 32) in patients with non-alcoholic fatty liver disease (NAFLD; p = 0.7647) with the use of CK-18M65. CONCLUSION: Serum CK-18M65 distinguished NASH from SS, but could not assess the severity of steatosis in NAFLD patients or the grade of fibrosis in NASH patients in Japanese population.

Characteristics of Hypovascular versus Hypervascular Well-Differentiated Hepatocellular Carcinoma Smaller Than 2 cm - Focus on Tumor Size, Markers and Imaging Detectability.

OBJECTIVES: The characteristics of hypovascular and hypervascular well-differentiated hepatocellular carcinomas (HCCs) were compared in terms of tumor size, tumor markers and detectability by imaging modalities. METHODS: Well-differentiated HCC nodules that are smaller than 2 cm (n = 27) were evaluated in 27 patients using histopathology and divided into 2 groups: hypovascular (n = 10) and hypervascular (n = 17). The diagnostic sensitivity of imaging modalities was then evaluated for efficiency in disclosing tumor size and tumor markers in the 2 types. RESULTS: No difference was observed in tumor size and tumor markers between the 2 types; however, the sensitivity of contrast-enhanced CT, contrast-enhanced ultrasonography and arterioportal angiography was significantly different between the 2 types, whereas that by Gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid enhanced magnetic resonance imaging (Gd-EOB-DTPA MRI) demonstrated no difference. CONCLUSION: Hypovascular HCC could be diagnosed by Gd-EOB-DTPA MRI in the hepatobiliary phase.

Isolation of notoamide S and enantiomeric 6-epi-stephacidin A from the fungus Aspergillus amoenus: biogenetic implications.

Notoamide S has been hypothesized to be a key biosynthetic intermediate for characteristic metabolites stephacidin A, notoamide B, and versicolamide B in Aspergillus sp. but has not yet been isolated. The isolation of notoamide S and an enantiomeric mixture of 6-epi-stephacidin A enriched with the (-)-isomer from Aspergillus amoenus is reported. The presence of (+)-versicolamide B suggests that the fungus possesses only the oxidase, which converts (+)-6-epi-stephacidin A into (+)-Versicolamide B, but not for (-)-6-epi-Stephacidin A.

Factors of response to pegylated interferon/ribavirin combination therapy and mechanism of viral clearance.

OBJECTIVES: This study explores viral factors of the interferon (IFN) and ribavirin (RBV) resistance-determining region (IRRDR), the IFN sensitivity-determining region (ISDR) and the core protein, and host factor interleukin 28B associated with response to pegylated IFN (PEG-IFN) and RBV combination therapy, and the correlation of viral and host factors with IFN-λ1. METHODS: A total of 58 patients underwent PEG-IFN/RBV combination therapy for 48 weeks. The pretreatment factors associated with rapid virological response (RVR) and sustained virological response (SVR) were analyzed. Pretreatment IFN-λ1 serum levels were compared with the viral and host factors. RESULTS: Univariate analysis showed that IRRDR ≥6 and ISDR ≥2 were significant pretreatment predictors of RVR, and multivariate analysis identified IRRDR ≥6 and hemoglobin as significant predictors of SVR. Pretreatment IFN-λ1 was significantly higher in the SVR group than in the non-SVR group and also in the IRRDR ≥6 group than in the IRRDR ≤5 group. CONCLUSIONS: IRRDR ≥6 was the only significant predictor of SVR and was correlated with IFN-λ1. High serum levels of IFN-λ1 may be conducive to effective PEG-IFN/RBV combination therapy because of the immunomodulatory system.

Prediction of response to pegylated interferon/ribavirin combination therapy for chronic hepatitis C genotypes 2a and 2b and high viral load.

OBJECTIVE: We investigated the impact of host genetics represented by the single nucleotide polymorphism (SNP) of the IL28B gene and viral genetic variations within the nonstructural protein 5A (NS5A) [including the interferon (IFN)/ribavirin (RBV) resistance-determining region (IRRDR) and the IFN sensitivity-determining region (ISDR)] on the outcome of pegylated IFN and RBV (PEG-IFN/RBV) treatment. METHODS: Sixty-six patients infected with hepatitis C virus (HCV)-2a or HCV-2b who received PEG-IFN/RBV for 24 weeks were examined. RESULTS: In HCV-2a, the major genotype of IL28B SNP showed a tendency toward association with sustained virological response (SVR) and rapid virological response (RVR), and four or more mutations in IRRDR (IRRDR[2a] ≥4) and one or more mutations in ISDR plus its carboxyl-flanking region (ISDR/+C[2a] ≥1) were significantly associated with SVR and RVR. In HCV-2b, one or more mutations in the N-terminal part of IRRDR (IRRDR/N[2b] ≥1) were significantly associated with RVR. Multivariate analysis identified the major genotype of IL28B SNP and IRRDR[2a] ≥4 as independent predictive factors of SVR in HCV-2a, with IRRDR[2a] ≥4 being more powerful than the IL28B SNP. Also, IRRDR[2a] ≥4 in HCV-2a and IRRDR/N[2b] ≥1 in HCV-2b were significant determiners of RVR. CONCLUSION: The NS5A sequence heterogeneity and IL28B SNP are useful factors to predict the sensitivity to PEG-IFN/RBV therapy in HCV-2a and HCV-2b infections.

Outcome of double-filtration plasmapheresis plus interferon treatment in nonresponders to pegylated interferon plus ribavirin combination therapy.

OBJECTIVES: We assessed the outcome of double-filtration plasmapheresis (DFPP) combined with pegylated interferon (PEG-IFN) and ribavirin (RBV) therapy in patients infected with hepatitis C virus (HCV)-1b whose HCV had not disappeared during PEG-IFN/RBV combination therapy, or who had relapsed after the end of the therapy. Additionally, we investigated factors predictive of sustained virological response (SVR), including host and viral genetic factors, to DFPP plus IFN/RBV therapy. METHODS: A total of 40 patients infected with HCV-1b whose HCV virus had not been eradicated by previous PEG-IFN/RBV therapy were enrolled for treatment by DFPP plus IFN/RBV. Rapid virological response (RVR) and SVR were assessed, and pretreatment factors associated with SVR - the interleukin (IL)28B gene, the IFN/RBV resistance-determining region (IRRDR) and the IFN sensitivity-determining region (ISDR) - were analyzed. RESULTS: Of the 40 patients, 9 (23%) achieved RVR and 10 (25%) achieved SVR. The significant factors associated with SVR were IL28B major and RVR, as assessed by multivariate analysis (p = 0.0182, p = 0.0005). CONCLUSION: Patients whose HCV is not eradicated by previous PEG-IFN/RBV would be good candidates for combined DFPP and IFN/RBV retreatment provided they demonstrate IL28B major and have achieved RVR.

Well-differentiated hepatocellular carcinoma detected as hypovascularity by only CT during hepatic arteriography.

We describe a well-differentiated hepatocellular carcinoma (HCC) with alcohol-related liver cirrhosis in a 69-year-old man. Ultrasonography (US) disclosed a 10 mm hypoechoic nodule in segment 4; Sonazoid contrast-enhanced US and gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI) revealed no defect in either the Kupffer phase or the hepatobiliary phase. Computed tomography during hepatic arteriography (CTHA), however, revealed a hypovascular nodule, but CT during arterial portography showed no perfusion defect. Histological analysis indicated a well-differentiated HCC. Thus, our detection of well-differentiated HCC disclosed by only CTHA attested to the efficiency of this modality, suggesting that it is more sensitive than Gd-EOB-GTPA-enhanced MRI.

Successful retrograde arterial inflow through a muscular branch in a free anterolateral thigh chimeric flap transfer.

In this report, we present a case in which a free anterolateral thigh (ALT) flap was transferred for head and neck reconstruction after oropharyngeal cancer ablation, and a retrograde arterial inflow was used to salvage the flap when the main arterial pedicle showed usual repeated spasms. The flap was raised as a chimera flap comprising a fasciocutaneous flap and a vastus lateralis muscle flap. After reperfusion, the pedicle artery exhibited spasms repeatedly and vascular flow was unstable. Therefore, we performed arterial supercharge. In the distal portion of the muscle flap, a small arterial branch was dissected as a reverse-flow arterial pedicle. The recipient artery was also a retrograde limb of the superior thyroid artery. The flap survived; however, postoperative ultrasonographic echo evaluation revealed that the spastic descending branch of the lateral circumflex femoral artery was obstructed and that the reverse-flow muscular perforator alone nourished the whole flap. In free ALT flap transfer, a small perforator level artery was able to nourish a flap, even in a retrograde manner. Moreover, when the vasculature of the free flap is unstable, retrograde arterial supply to a small perforator can be an option to save the flap transfer.

Correlation between insulin resistance and outcome of pegylated interferon and ribavirin therapy, hepatic steatosis, hepatic fibrosis in chronic hepatitis C-1b and high viral load.

BACKGROUND/AIMS: Insulin resistance (IR) has been reported to be an independent predictor of treatment outcome in chronic hepatitis C patients. METHODS: We analyzed the relationship between IR and the outcome of pegylated interferon and ribavirin (PEG-IFN/RBV) therapy, taking into account host factors of body mass index and histological index, such as rate of fatty change and fibrosis. Japanese patients (n = 30; 19 men and 11 women; median age 60.0 ± 8.7 years) with chronic hepatitis C-1b with a high viral load were treated with PEG-IFN-α2b/RBV for 48 weeks. RESULTS: Sustained virological response (SVR) was seen in 60% (18/30) and non-SVR in 40% (12/30). HOMA-IR (homeostasis model of assessment-insulin resistance index) at the start and at 24 weeks of treatment showed no statistical difference between SVR and non-SVR. Correlation was observed between HOMA-IR and body mass index (r = 0.45, p = 0.013). Among 20 patients, steatosis and fibrosis were assessed by biopsy. Correlation was observed between HOMA-IR and steatosis (r = 0.57, p = 0.0093), whereas no correlation was observed between HOMA-IR and fibrosis. CONCLUSION: A larger prospective study is needed to clarify the role of IR in the outcome of PEG-IFN/RBV combination therapy and hepatic fibrosis in Japanese patients.

Urinary protein fraction in healthy subjects using cellulose acetate membrane electrophoresis followed by colloidal silver staining.

We previously reported a rapid and highly sensitive colloidal silver staining solution suitable for the cellulose acetate membrane. This method was useful for detecting even very small amounts of urinary protein. In the present study, we examined urinary protein fractions in healthy subjects, using cellulose acetate membrane electrophoresis (CAE) with a highly sensitive colloidal silver staining, in an attempt to determine the clinical relevance of urinary protein fractions. Sixty unconcentrated spot urine specimens were analyzed by CAE and calculated by densitometry. All of the samples were separated into five fractions by CAE. The mean +/- 1 SD of the percentage of five fractions was 28.37 +/- 8.51 in albumin, 4.30 +/- 4.19 in alpha1-globulin, 14.41 +/- 6.14 in alpha2-globulin, 19.45 +/- 7.10 in beta-globulin, and 33.46 +/- 8.24 in gamma-globulin. The albumin/globulin (A/G) ratio was 0.41 +/- 0.17. These six items and the concentrations of total protein, albumin, and beta-N-acetyl-D-glucosaminidase (NAG) did not significantly differ between males and females. NAG is the marker of tubulointerstitial nephropathy. The results suggest that there are no gender-dependent differences in the urinary protein fractions of healthy subjects.

Analysis of an expanded width of albumin fraction by cellulose acetate membrane electrophoresis in IgA nephropathy urine before treatment.

Cellulose acetate membrane electrophoresis with colloidal silver stain re-vealed that the width of the albumin fraction in IgA nephropathy (IgAN) urine before treatment was significantly expanded. This phenomenon was not shown in IgAN urine after treatment or in non-IgAN urine. There was a reverse correlation between the width of the albumin fraction and the albumin con-centration in IgAN urine. By immuno-fixation, Tamm-Horsfall protein (THP) was located in the same position as the albumin band in IgAN urine before treatment; however, in the urine of a healthy subject it was located in the same position as alpha(1)-globulin. By ELISA, the THP-albumin complex concentration in IgAN urine before treatment was significantly higher than in the other two diseases. The width of the albumin fraction and the sodium ion concentra-tion of the urine were significantly correlated. The THP/albumin ratio in IgAN urine before treatment was significantly higher than in the other two groups. This suggests that the characteristic expanded width of albumin found by immunofixation indicates a THP-albumin complex, and that the sodium concentration of urine is involved in the formation of this complex.

[Vasodilatory action of FK 409 on bovine retinal arteries--analysis by a microvessel perfusion system].

PURPOSE: We investigated the vasodilatory action of FK 409 on bovine retinal arteries using a microvessel perfusion system in vitro. MATERIALS AND METHODS: Bovine retinal arteries were obtained from bovine eyes. The arteries, 3 mm long and 110 microns in diameter, were attached to the microvessel perfusion system. The arteries were perfused with Tyrode solution containing 10(-6) M U46619, followed by 10(-6) M U46619 and FK 409 (10(-3) M or 10(-4) M). We also perfused the arteries with carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide. The inner diameter of the arteries was measured under a microscope and analyzed by computer. RESULTS: FK 409 prevented contraction of bovine retinal arteries induced by perfusion of 10(-6) M U46619 when the arteries were perfused with 10(-3) to 10(-4) M FK 409. Addition of 100 microM PTIO, an nitric oxide (NO)-specific extinction agent, into the mixture of U46619 and FK 409 hadan antagonistic effect on the contractive action of FK 409. CONCLUSION: These results suggest that FK 409 has a dilatational effect on bovine retinal arteries and that the dilatational action of FK 409 is possibly produced by NO.