Hirofumi Uto

Characteristics of Patients With Nonalcoholic Steatohepatitis Who Develop Hepatocellular Carcinoma

BACKGROUND & AIMS Nonalcoholic steatohepatitis (NASH) can progress to hepatocellular carcinoma (HCC). We aimed to characterize the clinical features of NASH patients with HCC. METHODS In a cross-sectional multicenter study in Japan, we examined 87 patients (median age, 72 years; 62% male) with histologically proven NASH who developed HCC. The clinical data were collected at the time HCC was diagnosed. RESULTS Obesity (body mass index ≥25 kg/m(2)), diabetes, dyslipidemia, and hypertension were present in 54 (62%), 51 (59%), 24 (28%), and 47 (55%) patients, respectively. In nontumor liver tissues, the degree of fibrosis was stage 1 in 10 patients (11%), stage 2 in 15 (17%), stage 3 in 18 (21%), and stage 4 (ie, liver cirrhosis) in 44 (51%). The prevalence of cirrhosis was significantly lower among male patients (21 of 54, 39%) compared with female patients (23 of 33, 70%) (P = .008). CONCLUSIONS Most patients with NASH who develop HCC are men; the patients have high rates of obesity, diabetes, and hypertension. Male patients appear to develop HCC at a less advanced stage of liver fibrosis than female patients.

Proanthocyanidin from Blueberry Leaves Suppresses Expression of Subgenomic Hepatitis C Virus RNA

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. While searching for new natural anti-HCV agents in agricultural products, we found a potent inhibitor of HCV RNA expression in extracts of blueberry leaves when examined in an HCV subgenomic replicon cell culture system. This activity was observed in a methanol extract fraction of blueberry leaves and was purified by repeated fractionations in reversed-phase high-performance liquid chromatography. The final purified fraction showed a 63-fold increase in specific activity compared with the initial methanol extracts and was composed only of carbon, hydrogen, and oxygen. Liquid chromatography/mass-ion trap-time of flight analysis and butanol-HCl hydrolysis analysis of the purified fraction revealed that the blueberry leaf-derived inhibitor was proanthocyanidin. Furthermore, structural analysis using acid thiolysis indicated that the mean degree of polymerization of the purified proanthocyanidin was 7.7, consisting predominantly of epicatechin. Proanthocyanidin with a polymerization degree of 8 to 9 showed the greatest potency at inhibiting the expression of subgenomic HCV RNA. Purified proanthocyanidin showed dose-dependent inhibition of expression of the neomycin-resistant gene and the NS-3 protein gene in the HCV subgenome in replicon cells. While characterizing the mechanism by which proanthocyanidin inhibited HCV subgenome expression, we found that heterogeneous nuclear ribonucleoprotein A2/B1 showed affinity to blueberry leaf-derived proanthocyanidin and was indispensable for HCV subgenome expression in replicon cells. These data suggest that proanthocyanidin isolated from blueberry leaves may have potential usefulness as an anti-HCV compound by inhibiting viral replication.

Prevention of hepatitis B virus reactivation in patients receiving immunosuppressive therapy or chemotherapy

With the increasing use of potent immunosuppressive therapy, reactivation of hepatitis B virus (HBV) in endemic regions is becoming a clinical problem requiring special attention. A recent annual nationwide survey clarified that HBV reactivation related to immunosuppressive therapy has been increasing in patients with malignant lymphoma, other hematological malignancies, oncological or rheumatological disease. In the survey, rituximab plus steroid‐containing chemotherapy was identified as a risk factor for HBV reactivation in hepatitis B surface antigen (HBsAg) negative patients with malignant lymphoma. In this setting, HBV reactivation resulted in fatal fulminant hepatitis regardless of the treatment of nucleoside analog. The Intractable Hepatobiliary Disease Study Group and the Study Group for the Standardization of Treatment of Viral Hepatitis Including Cirrhosis jointly developed guidelines for preventing HBV reactivation. The essential features of the guideline are as follows. All patients should be screened for HBsAg by a sensitive method before the start of immunosuppressive therapy. Second, hepatitis B core antigen (HBcAb) and hepatitis B surface antibody (HBsAb) testing should be performed in HBsAg negative patients, especially those receiving intensive immunosuppressive therapy. Prophylaxis with nucleoside analogs is essential for preventing HBV reactivation in HBsAg positive patients. In contrast, HBsAg negative with HBcAb and/or HBsAb positive patients should be monitored monthly for an increase in serum HBV DNA during and 12 months after completion of chemotherapy. Nucleoside analogs should be administrated immediately when HBV DNA becomes positive during this period. This strategy facilitates commencement of nucleoside analogs at an early stage of HBV reactivation and results in prevention of severe hepatitis.

Increased rate of death related to presence of viremia among hepatitis C virus antibody–positive subjects in a community-based cohort study†

The overall mortality of patients infected with hepatitis C virus (HCV) has not been fully elucidated. This study analyzed mortality in subjects positive for antibody to HCV (anti‐HCV) in a community‐based, prospective cohort study conducted in an HCV hyperendemic area of Japan. During a 10‐year period beginning in 1995, 1125 anti–HCV‐seropositive residents of Town C were enrolled into the study and were followed for mortality through 2005. Cause of death was assessed by death certificates. Subjects with detectable HCV core antigen (HCVcAg) or HCV RNA were considered as having hepatitis C viremia and were classified as HCV carriers; subjects who were negative for both HCVcAg and HCV RNA (i.e., viremia‐negative) were considered as having had a prior HCV infection and were classified as HCV noncarriers. Among the anti–HCV‐positive subjects included in the analysis, 758 (67.4%) were HCV carriers, and 367 were noncarriers. A total of 231 deaths occurred in these subjects over a mean follow‐up of 8.2 years: 176 deaths in the HCV carrier group and 55 in the noncarrier group. The overall mortality rate was higher in HCV carriers than in noncarriers, adjusted for age and sex (hazard ratio, 1.53; 95% confidence interval, 1.13‐2.07). Although liver‐related deaths occurred more frequently among the HCV carriers (hazard ratio, 5.94; 95% confidence interval, 2.58‐13.7), the rates of other causes of death did not differ between HCV carriers and noncarriers. Among HCV carriers, a higher level of HCVcAg (≥100 pg/mL) and persistently elevated alanine aminotransferase levels were important predictors of liver‐related mortality. Conclusion: The presence of viremia increases the rate of mortality, primarily due to liver‐related death, among anti–HCV‐seropositive persons in Japan. (HEPATOLOGY 2009.)

Early diagnostic potential for hepatocellular carcinoma using the SELDI ProteinChip system.

Early detection of HCC increases the potential for curative treatment and improves survival. To facilitate early detection of HCC, this study sought to identify novel diagnostic markers of HCC using surface‐enhanced laser desorption ionization time‐of‐flight mass spectrometry (SELDI‐TOF/MS) ProteinChip technology. Serum samples were obtained from 153 patients with or without HCC, all of whom had been diagnosed with HCV‐associated chronic liver disease. To identify proteins associated with HCC, serum samples were analyzed using SELDI‐TOF/MS. We constructed an initial decision tree for the correct diagnosis of HCC using serum samples from patients with (n = 35) and without (n = 44) HCC. Six protein peaks were selected to construct a decision tree using this first group. The efficacy of the decision tree was then assessed using a second group of patients with (n = 29) and without (n = 33) HCC. The sensitivity and specificity of this decision tree for the diagnosis of HCC were 83% and 76%, respectively. For a third group, we analyzed sera from seven patients with HCC obtained before the diagnosis of HCC by ultrasonography (US) and from five patients free of HCC for the past 3 years. Use of these diagnostic markers predicted the diagnosis of HCC in six of these seven patients before HCC was clinically apparent without any false positives. Conclusion: Serum profiling using the SELDI ProteinChip system is useful for the early detection and prediction of HCC in patients with chronic HCV infection. (HEPATOLOGY 2007;45:948–956.)

Fatal Propionic Acidemia in Mice Lacking Propionyl-CoA Carboxylase and Its Rescue by Postnatal, Liver-specific Supplementation via a Transgene

Propionic acidemia (PA) is an inborn error of metabolism caused by the genetic deficiency of propionyl-CoA carboxylase (PCC). By disrupting the α-subunit gene of PCC, we created a mouse model of PA (PCCA−/−), which died in 24–36 h after birth due to accelerated ketoacidosis. A postnatal, liver-specific PCC expression via a transgene in a far lower level than that in wild-type liver, allowed PCCA−/− mice to survive the newborn and early infant periods, preventing a lethal fit of ketoacidosis (SAP+PCCA−/− mice). Interestingly, SAP+PCCA−/− mice, in which the transgene expression increased after the late infant period, continued to grow normally while mice harboring a persistent low level of PCC died in the late infant period due to severe ketoacidosis, clearly suggesting the requirement of increased PCC supplementation in proportion to the animal growth. Based on these results, we propose a two-step strategy to achieve an efficient PA prevention in human patients: a partial PCC supplementation in the liver during the newborn and early infant periods, followed by a larger amount of supplementation in the late infant period.

Hepatocyte growth factor accelerates the proliferation of hepatic oval cells and possibly promotes the differentiation in a 2-acetylaminofluorene/partial hepatectomy model in rats

Background:  Hepatocyte growth factor (HGF) is the primary agent promoting the proliferation of mature hepatocytes. The purpose of the present paper was to clarify the effects of HGF on the proliferation and  differentiation  of  hepatic  oval  cells  using  a  2‐acetylaminofluorene/partial  hepatectomy  (2‐AAF/PH) model in rats.

Human neutrophil peptides 1-3 are useful biomarkers in patients with active ulcerative colitis.

Background: A specific useful biomarker for diagnosing ulcerative colitis (UC) has not yet been described. This study employed proteomics to identify serum protein biomarkers for UC. Methods: Ninety‐four blood samples were isolated from patients and controls (including 48 UC, 22 Crohns disease [CD], 5 colorectal cancer, and 6 infectious colitis patients and 13 healthy subjects). Serum samples were analyzed using the SELDI‐TOF/MS ProteinChip system. After applying the samples to ProteinChip arrays, we assessed differences in the proteomes using Ciphergen ProteinChip software and identified candidate proteins, which were then characterized in immunoassays. Results: Preliminary analysis using the ProteinChip system revealed significant peak‐intensity differences for 27 serum proteins between 11 patients with UC and 7 healthy subjects. Among these proteins, 3 proteins (with mass/charge ratios of approximately 3400) were identified as human neutrophil peptides 1–3 (HNP 1–3). The presence of HNP 1–3 in the patient sera was confirmed using immunoassays. Enzyme‐linked immunosorbent assays demonstrated that the mean plasma concentration of HNP 1–3 was significantly higher in patients with active UC (n = 28) than in patients whose UC was in remission (n = 20) or patients with CD (n = 22), infectious colitis, or healthy subjects, and tended to be higher than in patients with colon cancer. In addition, the plasma concentration of HNP 1–3 in patients that responded to corticosteroids‐based therapy decreased after treatment, whereas it was not changed in nonresponders. Conclusions: HNP 1–3 is a novel biomarker that may be useful for diagnosing patients with active UC and predicting treatment outcomes.

Hepatocyte growth factor facilitates the repair of large colonic ulcers in 2,4,6-trinitrobenzene sulfonic acid-induced colitis in rats

Background: Hepatocyte growth factor (HGF) modulates intestinal epithelial cell proliferation and migration, serving as a critical regulator of intestinal wound healing. The aim of this study was to clarify the effects of administration of recombinant human HGF on colonic mucosal damage in vivo. Methods: Rats were given 7.5 mg of 2,4,6‐trinitrobenzene sulfonic acid (TNBS) per rectum on day 0. On day 5, the degree of TNBS‐induced colitis was evaluated endoscopically, and rats suffering from large ulcers (occupying more than two thirds of the luminal circumference) were treated with intravenous bolus injections of recombinant human HGF (1.0 mg/kg per day) or phosphate‐buffered saline (PBS) for 5 days. Results: Rats with TNBS‐induced colitis given human HGF showed a significant reduction in colonic ulcer coverage and large intestinal shortening compared with those treated with PBS. Administration of recombinant human HGF also stimulated the proliferation of epithelial cells and reduced the inflammatory cell infiltrate. Finally, HGF treatment decreased the myeloperoxidase activity and tumor necrosis factor &agr; levels in the TNBS‐inflamed colon tissues. Conclusions: These results indicate that intravenous injection of HGF accelerates colonic mucosal repair and reduces infiltration of inflammatory cells in rats with TNBS‐induced colitis and suggest that HGF has the potential to be a new therapeutic modality to promote intestinal mucosal repair in patients with inflammatory bowel disease.

Liver regeneration after partial hepatectomy in rat is more impaired in a steatotic liver induced by dietary fructose compared to dietary fat.

Hepatic steatosis (HS) has a negative effect on liver regeneration, but different pathophysiologies of HS may lead to different outcomes. Male Sprague-Dawley rats were fed a high fructose (66% fructose; H-fruc), high fat (54% fat; H-fat), or control chow diet for 4 weeks. Based on hepatic triglyceride content and oil red O staining, HS developed in the H-fruc group, but was less severe compared to the H-fat group. Hepatic mRNA expression levels of fatty acid synthase and fructokinase were increased and those of carnitine palmitoyltransferase-1 and peroxisome proliferator-activated receptor-α were decreased in the H-fruc group compared to the H-fat group. Liver regeneration after 70% partial hepatectomy (PHx) was evaluated by measuring the increase in postoperative liver mass and PCNA-positive hepatocytes, and was impaired in the H-fruc group compared to the H-fat and control groups on days 3 and 7. Serum levels of tumor necrosis factor-α, interleukin-6 and hepatocyte growth factor did not change significantly after PHx. In contrast, serum TGF-β1 levels were slightly but significantly lower in the control group on day 1 and in the H-fat group on day 3 compared to the level in each group on day 0, and then gradually increased. However, the serum TGF-β1 level did not change after PHx in the H-fruc group. These results indicate that impairment of liver regeneration after PHx in HS is related to the cause, rather than the degree, of steatosis. This difference may result from altered metabolic gene expression profiles and potential dysregulation of TGF-β1 expression.