A Phase I/Randomized Phase II Study to Evaluate the Safety, Pharmacokinetics, and Efficacy of Nintedanib versus Sorafenib in Asian Patients with Advanced Hepatocellular Carcinoma.

BACKGROUND: Nintedanib is an oral, triple angiokinase inhibitor of vascular endothelial growth factor/platelet-derived growth factor/fibroblast growth factor receptors. This randomized, multicenter, open-label, phase I/II study evaluated the safety, pharmacokinetics, maximum tolerated dose (MTD) in terms of dose-limiting toxicities (DLTs), and efficacy of nintedanib versus sorafenib in Asian patients with unresectable advanced hepatocellular carcinoma (HCC). PATIENTS AND METHODS: For the phase I portion, patients were stratified into two groups according to their alanine aminotransferase/aspartate aminotransferase (ALT/AST) and Child-Pugh score at baseline. For phase I, the primary endpoint was determination of the MTD in terms of DLTs. For phase II, patients with a Child-Pugh score of 5-6, an Eastern Cooperative Oncology Group performance score ≤2, and an ALT/AST ≤2× the upper limit of normal were enrolled and randomized 2: 1 to nintedanib 200 mg twice daily (b.i.d.) (the MTD determined in phase I) or sorafenib 400 mg b.i.d. continuously in 28-day cycles until intolerable adverse events (AEs) or disease progression (PD); treatment beyond PD was allowed if clinical benefit was perceived. The primary endpoint for phase II was time to progression (TTP) by central independent review (CIR; by Response Evaluation Criteria in Solid Tumors v1.0); the secondary endpoints included overall survival (OS). All analyses were exploratory. RESULTS: The MTD was 200 mg in both groups. For phase II, 95 patients were randomized to nintedanib (n = 63) or sorafenib (n = 32). For nintedanib and sorafenib, respectively, the median CIR TTP was 2.8 vs. 3.7 months (hazard ratio [HR] = 1.21, 95% confidence interval [CI] 0.73-2.01) and the median OS 10.2 vs. 10.7 months (HR = 0.94, 95% CI 0.59-1.49). Nintedanib-treated patients had fewer grade 3 or higher AEs (56 vs. 84%), serious AEs (46 vs. 56%), and AEs leading to dose reduction (19 vs. 59%) and drug discontinuation (24 vs. 34%). AEs associated more frequently with nintedanib were vomiting and nausea, whereas those associated more frequently with sorafenib were ALT/AST increases, diarrhea, rash, and palmar-plantar erythrodysesthesia syndrome. CONCLUSIONS: Nintedanib showed numerically similar efficacy to sorafenib for CIR TTP and OS in Asian patients with advanced HCC and adequate liver function. AEs were generally manageable.

Adjuvant heparanase inhibitor PI-88 therapy for hepatocellular carcinoma recurrence.

AIM: To demonstrate that administering heparanase inhibitor PI-88 at 160 mg/d is safe and promising in reducing hepatocellular carcinoma (HCC) recurrence for up to 3 year following curative resection. METHODS: A total of 143 patients (83.1% of the 172 participants in the phase II study) participated in the follow-up study. Of these patients, 50 had received no treatment, 48 had received 160 mg/d PI-88, and 45 had received 250 mg/d PI-88 during the phase II trial. Safety parameters and the following efficacy endpoints were investigated: (1) time to recurrence; (2) disease-free survival; and (3) overall survival. RESULTS: PI-88 at 160 mg/d delayed the onset and frequency of HCC recurrence, and provided a clinically significant survival advantage for up to 3 years after treatment compared with those of the control group: (1) the recurrence-free rate increased from 50% to 63%, and (2) time to recurrence at the 36th percentile was postponed by 78%. The efficacy of administering PI-88 at 250 mg/d was confounded by a high dropout rate (11 out of 54 patients). Additionally, subgroup analyses of patients with (1) multiple tumors or a single tumor ≥ 2 cm; and (2) hepatitis B or C revealed that administering PI-88 at 160 mg/d conferred the most significant survival advantage (56.8% improvement in disease-free survival, P = 0.045) for patients with both risk factors for recurrence. CONCLUSION: Administering PI-88 at 160 mg/d is a safe and well-tolerated dosage that may confer significant clinical benefits for patients with HCC.

High-dose hypofractionated X-ray radiotherapy for hepatocellular carcinoma: Tumor responses and toxicities.

Hypofractionated radiotherapy (RT) has been employed to treat hepatocellular carcinoma (HCC). The present study aimed to report the treatment effects, the dose-response associations and the factors that are associated with radiation-induced liver disease (RILD) in a high-dose hypofractionated RT procedure. A total of 40 patients with non-metastatic HCC who underwent RT for local control of irradiated tumors were studied. The treatment technique was that of three-dimensional conformal or intensity-modulated radiation therapy, with a fraction size of 3 Gy and a total dose of 40-66 Gy in 14-23 fractions. The biologically-effective dose (BED) was 52.0-85.8 Gy10 (median, 74.1 Gy10). Tumor regression was observed in 28 patients (70.0%) with a complete response, partial response, stable disease and progressive disease status in 11 (27.5%), 17 (42.5%), five (12.5%) and seven patients (17.5%), respectively. The one-, two- and five-year overall survival (OS) and in-field control (IFC) rates were 60, 40 and 21% and 73, 62 and 56%, respectively. A positive correlation also emerged between the radiation dose and the IFC (P=0.035). Eight of the 40 patients (20%) developed non-classic RILD. A higher Cancer of the Liver Italian Program score was associated with a higher probability of non-classic RILD (P=0.02). The tumor response and IFC rate of HCC following irradiation were significantly dose-dependent. High-dose hypofractionated X-ray RT is a feasible and effective treatment for HCC in patients with good liver function and for those who meet the criteria for a curative attempt.

Sunitinib versus sorafenib in advanced hepatocellular cancer: results of a randomized phase III trial.

PURPOSE: Open-label, phase III trial evaluating whether sunitinib was superior or equivalent to sorafenib in hepatocellular cancer. PATIENTS AND METHODS: Patients were stratified and randomly assigned to receive sunitinib 37.5 mg once per day or sorafenib 400 mg twice per day. Primary end point was overall survival (OS). RESULTS: Early trial termination occurred for futility and safety reasons. A total of 1,074 patients were randomly assigned to the study (sunitinib arm, n = 530; sorafenib arm, n = 544). For sunitinib and sorafenib, respectively, median OS was 7.9 versus 10.2 months (hazard ratio [HR], 1.30; one-sided P = .9990; two-sided P = .0014); median progression-free survival (PFS; 3.6 v 3.0 months; HR, 1.13; one-sided P = .8785; two-sided P = .2286) and time to progression (TTP; 4.1 v 3.8 months; HR, 1.13; one-sided P = .8312; two-sided P = .3082) were comparable. Median OS was similar among Asian (7.7 v 8.8 months; HR, 1.21; one-sided P = .9829) and hepatitis B-infected patients (7.6 v 8.0 months; HR, 1.10; one-sided P = .8286), but was shorter with sunitinib in hepatitis C-infected patients (9.2 v 17.6 months; HR, 1.52; one-sided P = .9835). Sunitinib was associated with more frequent and severe adverse events (AEs) than sorafenib. Common grade 3/4 AEs were thrombocytopenia (29.7%) and neutropenia (25.7%) for sunitinib; hand-foot syndrome (21.2%) for sorafenib. Discontinuations owing to AEs were similar (sunitinib, 13.3%; sorafenib, 12.7%). CONCLUSION: OS with sunitinib was not superior or equivalent but was significantly inferior to sorafenib. OS was comparable in Asian and hepatitis B-infected patients. OS was superior in hepatitis C-infected patients who received sorafenib. Sunitinib-treated patients reported more frequent and severe toxicity.

Brivanib in patients with advanced hepatocellular carcinoma who were intolerant to sorafenib or for whom sorafenib failed: results from the randomized phase III BRISK-PS study.

PURPOSE: Brivanib is a selective dual inhibitor of vascular endothelial growth factor and fibroblast growth factor receptors implicated in tumorigenesis and angiogenesis in hepatocellular carcinoma (HCC). An unmet medical need persists for patients with HCC whose tumors do not respond to sorafenib or who cannot tolerate it. This multicenter, double-blind, randomized, placebo-controlled trial assessed brivanib in patients with HCC who had been treated with sorafenib. PATIENTS AND METHODS: In all, 395 patients with advanced HCC who progressed on/after or were intolerant to sorafenib were randomly assigned (2:1) to receive brivanib 800 mg orally once per day plus best supportive care (BSC) or placebo plus BSC. The primary end point was overall survival (OS). Secondary end points included time to progression (TTP), objective response rate (ORR), and disease control rate based on modified Response Evaluation Criteria in Solid Tumors (mRECIST) and safety. RESULTS: Median OS was 9.4 months for brivanib and 8.2 months for placebo (hazard ratio [HR], 0.89; 95.8% CI, 0.69 to 1.15; P = .3307). Adjusting treatment effect for baseline prognostic factors yielded an OS HR of 0.81 (95% CI, 0.63 to 1.04; P = .1044). Exploratory analyses showed a median time to progression of 4.2 months for brivanib and 2.7 months for placebo (HR, 0.56; 95% CI, 0.42 to 0.76; P < .001), and an mRECIST ORR of 10% for brivanib and 2% for placebo (odds ratio, 5.72). Study discontinuation due to treatment-related adverse events (AEs) occurred in 61 brivanib patients (23%) and nine placebo patients (7%). The most frequent treatment-related grade 3 to 4 AEs for brivanib included hypertension (17%), fatigue (13%), hyponatremia (11%), and decreased appetite (10%). CONCLUSION: In patients with HCC who had been treated with sorafenib, brivanib did not significantly improve OS. The observed benefit in the secondary outcomes of TTP and ORR warrants further investigation.

Clinical and virological characteristics post HBsAg seroclearance in hepatitis B virus carriers with hepatic steatosis versus those without.

BACKGROUND: It has been suggested hepatic steatosis contributes to seroclearance of hepatitis B surface antigen (HBsAg) in chronic hepatitis B virus (HBV) infection. Although HBsAg seroclearance generally confers favorable outcome, hepatic steatosis may account for progressive liver fibrosis and cirrhosis. Further studies are needed to compare clinical and virological characteristics post HBsAg seroclearance between subjects with hepatic steatosis and those without. METHODS: One-hundred and fifty-five HBsAg carriers with HBsAg seroclearance were enrolled. Subjects with moderate-severe hepatic steatosis as diagnosed by ultrasonography were designated as having hepatic steatosis. RESULTS: There were 69 subjects with hepatic steatosis and 86 without. Subjects with hepatic steatosis had significantly higher body mass index (BMI; 27.8 ± 3.5 vs. 23.0 ± 3.1, P < 0.001), were more likely to be male (78.3 vs. 63.9%, P = 0.05), and were significantly younger at HBsAg seroclearance (48.7 ± 8.9 years vs. 53.4 ± 8.9 years, P = 0.001), than those without. The frequency of anti-HBsAg seroconversion (56.5 vs. 59.3%, P = 0.72) and HBV viremia (20.3 vs. 15.1%, P = 0.40) was not significantly different between subjects with and without hepatic steatosis, but the incidence of abnormal AST and ALT was significantly higher in the former (23.2 vs. 0%, P < 0.0001; and 30.4 vs. 0%, P < 0.0001, respectively), and progression to liver cirrhosis tended to be more likely in the former than in the latter (10.1 vs. 3.5%, P = 0.09). CONCLUSIONS: In HBsAg carriers with increased body mass index, hepatic steatosis can accelerate HBsAg seroclearance by approximately 5 years. However, the beneficial effects of HBsAg seroclearance should be balanced against the harmful effects of hepatic steatosis.

Viral load, genotypes, and mutants in hepatitis B virus-related hepatocellular carcinoma: special emphasis on patients with early hepatocellular carcinoma.

BACKGROUND/AIMS: The role of viral factors in the pathogenesis of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is still inconclusive. Whether virological features such as viral load or mutants might change with the progression of HCC remains unknown. A case-control study including patients with early HCC and HBsAg carriers who are presumed to be at the minimal potential of HCC as controls might better identify factors significantly associated with HCC development. METHODS: Virological features were compared between 59 patients with early HCC (a solitary tumor of size ≤ 3 cm) and 101 patients with non-early HCC. A case-control study was performed by comparing 59 patients with early HCC and 1:2 age-matched inactive carriers with persistent normal alanine aminotransferase (ALT) levels. RESULTS: HBV DNA levels, HBV genotypes, and the frequency of precore A1896 and basal core promoter T1762/A1764 mutations showed no significant difference between patients with early HCC and those with non-early HCC. In the case-control study, patients with early HCC had significantly higher HBV DNA levels, and higher frequencies of genotype C HBV and basal core promoter T1762/A1764 mutation, but a similar frequency of precore A1896 mutation. Multiple logistic regression analysis identified HBV DNA levels ≥ 2,000 IU/mL and basal core promoter T1762/A1764 mutation as being independent factors for HCC development. Additionally, there was a synergistic effect between high viral load and basal core promoter T1762/A1764 mutation on HCC development. CONCLUSIONS: Virological features did not change significantly with the progression of HCC. HBV DNA levels ≥ 2,000 IU/mL and basal core promoter T1762/A1764 mutation were two independent viral factors for HCC.

Long-term results of a randomized, observation-controlled, phase III trial of adjuvant interferon Alfa-2b in hepatocellular carcinoma after curative resection.

OBJECTIVE: To investigate the clinical efficacy of adjuvant interferon alfa-2b (IFNα-2b) therapy on recurrence-free survival (RFS) of patients with postoperative viral hepatitis-related hepatocellular carcinoma (HCC). BACKGROUND: Despite most individual trials have failed to meet their primary endpoint, recent pooled-data meta-analyses suggest that adjuvant IFN therapy may significantly reduce the incidence of recurrence in curatively ablated HCC. METHODS: Patients with curative resection of viral hepatitis-related HCC were eligible, and were stratified by underlying viral etiology and randomly allocated to receive either 53 weeks of adjuvant IFNα-2b treatment or observation alone. The primary endpoint of this study was RFS. RESULTS: A total of 268 patients were enrolled with 133 in the IFNα-2b arm and 135 in the control arm. Eighty percent of them were hepatitis B surface antigen seropositive. At a median follow-up of 63.8 months, 154 (57.5%) patients had tumor recurrence and 84 (31.3%) were deceased. The cumulative 5-year recurrence-free and overall survival rates of intent-to-treat cohort were 44.2% and 73.9%, respectively. The median RFS in the IFNα-2b and control arms were 42.2 (95% confidence interval [CI], 28.1-87.1) and 48.6 (95% CI, 25.5 to infinity) months, respectively (P = 0.828, log-rank test). Adjuvant IFNα-2b treatment was associated with a significantly higher incidence of leucopenia and thrombocytopenia. Thirty-four (24.8%) of treated patients required dose reduction, and 5 (3.8%) of these patients subsequently withdrew from therapy because of excessive toxicity. Adjuvant IFNα-2b only temporarily suppressed viral replication during treatment period. CONCLUSIONS: In this study, adjuvant IFNα-2b did not reduce the postoperative recurrence of viral hepatitis-related HCC. More potent antiviral therapy deserves to be explored for this patient population. This study is registered at ClinicalTrials.gov and carries the identifier NCT00149565.

Instantaneous frequency-based ultrasonic temperature estimation during focused ultrasound thermal therapy.

Focused ultrasound thermal therapy relies on temperature monitoring for treatment guidance and assurance of targeting and dose control. One potential approach is to monitor temperature change through ultrasonic-backscattered signal processing. The current approach involves the detection of echo time-shifts based on cross-correlation processing from segmented radiofrequency (RF) data. In this study, we propose a novel ultrasonic temperature-measurement approach that detects changes in instantaneous frequency along the imaging beam direction. Focused ultrasound was used as the heating source, and the 1-D beamformed RF signals provided from an ultrasound imager were used to verify the proposed algorithm for temperature change estimation. For comparison, a conventional cross-correlation technique was also evaluated. Heating experiments testing tissue-mimicking phantoms and ex vivo porcine muscles were conducted. The results showed that temperature can be well estimated by the proposed algorithm in the temperature range, where the relationship of sound speed versus temperature is linear. Compared with the cross-correlation-based algorithm, the proposed new algorithm yields a six-fold increase in computational efficiency, along with comparable contrast-detection ability and precision. This new algorithm may serve as an alternative method for implementing temperature estimation into a clinical ultrasound imager for thermal therapy guidance.

Hepatic inflammation mediated by hepatitis C virus core protein is ameliorated by blocking complement activation.

BACKGROUND: The pathogenesis of inflammation and fibrosis in chronic hepatitis C virus (HCV) infection remains unclear. Transgenic mice with constitutive HCV core over-expression display steatosis only. While the reasons for this are unclear, it may be important that core protein production in these models begins during gestation, in contrast to human hepatitis C virus infection, which occurs post-natally and typically in adults. AIMS: To more realistically model the effect of core protein production in the adult liver, we developed a mouse with conditional expression of HCV core and examined the effect of core protein production in the adult liver. METHODS: Liver biopsy samples from transgenic mice with tetracycline(tet)-regulated conditional core protein expression were evaluated immunohistologically. Microarray analysis of HCV core transgenic mice with steatohepatitis pointed to a role of the complement pathway. This was further explored by blocking complement activation by in vivo administration of CD55 (decay accelerating factor for complement), which inhibits activation of C3. RESULTS: Transgenic mice exhibited low, intermediate, or high HCV core protein expression when fed a permissive diet of standard chow. Aside from hepatic steatosis, hepatic inflammation and fibrosis were seen in mice with intermediate levels of core protein. Microarray analyses of inflamed liver demonstrated activation of both the complement (C3 up-regulation) and coagulation pathways (fibrinogen B up-regulation). Administration of CD55 reduced hepatic inflammation. CONCLUSION: Transgenic mice that conditionally express intermediate HCV core protein develop inflammation, steatosis, and fibrosis. These effects mediated by HCV core are reduced by administration of CD55, a regulator of the complement pathway. The model may be valuable in investigating the pathogenesis of liver inflammation in chronic hepatitis C.

Impact of gender, viral transmission and aging in the prevalence of hepatitis B surface antigen.

BACKGROUND: Age, gender, and perinatal infection are associated with hepatocarcinogenesis. The influence of perinatal transmission in chronic hepatitis B virus infection between genders at different ages is not well documented. METHODS: A consecutive series of individuals who had general check-ups and three groups of relatives of patients with hepatocellular carcinoma were analyzed. Siblings of index cases and children of female index cases represented groups with high perinatal transmission, while children of male index cases represented a low perinatal transmission group. RESULTS: A total of 45,035 individuals who had general check-ups and 14,513 first degree relatives of patients with hepatocellular carcinoma were included. The families of patients with hepatocellular carcinoma included 4,455 siblings of index cases, 7,111 children of male index cases, and 2,947 children of female index cases. The prevalence of hepatitis B surface antigen (HBsAg) was high in groups with high perinatal infection and in men. Gender differences in the prevalence of HBsAg diminished in children of female index cases and siblings of index cases, and in all groups after the age of 60 years. The prevalence of HBsAg declined with increasing age in all groups, with the highest decline in male siblings of index cases ( 1.37% per year) and the lowest in female children of male index cases ( 0.05% per year) in the 35-59 year-old period. Hepatitis C antibody was higher in women (5.7%) than in men (4.0%) in the general check-up group. CONCLUSIONS: Females were less susceptible to become HBsAg carriers if HBV was not transmitted during the perinatal period. The prevalence of HBsAg declined significantly in high perinatal infection groups, implying that neonatal tolerance does not endure.

Long-term outcome of hepatitis B e antigen-negative hepatitis B surface antigen carriers in relation to changes of alanine aminotransferase levels over time.

UNLABELLED: The baseline alanine aminotransferase (ALT) level was reported to have prognostic value in chronic hepatitis B virus (HBV) infection, during which ALT may change over time. Instead of baseline ALT, this study aimed to study the prognostic value of the height of ALT during the course of chronic HBV infection. A total of 4376 asymptomatic hepatitis B e antigen (HBeAg) negative, surface antigen (HBsAg) carriers with baseline ALT less than 2 times the upper limit of normal (ULN) were monitored with ALT measurement and ultrasonography every 3 to 12 month for over 3 years. Maximal ALT levels during follow-up were correlated with long-term outcomes using morbidity and mortality data from hospital records, cancer registration, and national mortality database. Baseline ALT level was normal in 3673 subjects and increased to abnormal level in 1720 (46.8%) during a mean follow-up period of 13.4 +/- 5.2 (3.0-28.7) years. The incidence of liver cirrhosis, hepatocellular carcinoma (HCC), and mortality increased with increasing maximal ALT level during follow-up, especially in those with maximal ALT of at least 2 times ULN, as compared with those who maintained normal ALT. Cox regression analysis indicated that age at entry, sex, and maximal ALT level during follow-up were significant independent factors associated with the development of cirrhosis, HCC, and mortality whereas cirrhosis was also an independent factor for HCC development and mortality. CONCLUSION: Persistently normal ALT was associated with excellent long-term prognosis, whereas increasing ALT levels of at least 2 times ULN during follow-up was associated with increasing morbidity and mortality. ALT of at least 2 times ULN is therefore an appropriate threshold for anti-HBV therapy, whereas those with ALT 1 to 2 times ULN require liver biopsy for decision.

Heparanase inhibitor PI-88 as adjuvant therapy for hepatocellular carcinoma after curative resection: a randomized phase II trial for safety and optimal dosage.

BACKGROUND/AIMS: Hepatocellular carcinoma recurrence after curative treatment adversely influences clinical outcome. It is important to explore adjuvant therapies. This phase II/stage 1 multi-center, randomized trial investigated the safety, optimal dosage and preliminary efficacy of PI-88, a novel heparanase inhibitor, in the setting of post-operative recurrence of HCC according to a Simon's 2-stage design. METHODS: Three groups were included: one untreated arm (Group A) and two PI-88 arms (Group B: 160 mg/day; Group C: 250 mg/day). Treatment groups received PI-88 over nine 4-week treatment cycles, followed by a 12-week treatment-free period. Safety and optimal dosage were assessed. RESULTS: Overall, 172 patients were randomized and 168 were included in the intention-to-treat (ITT) population. Treatment-related adverse effects included cytopenia, injection site hemorrhage, PT prolongation, etc. Four serious adverse events were possibly related to PI-88 treatment. One (1.8%) group B patients and six (10.5%) group C had hepatotoxicity-related withdrawals. Among the ITT population, 29 patients (50%) in Group A, 35 (63%) in Group B, and 22 (41%) in Group C remained recurrence-free at completion. Calculated T(1) value suggested 160 mg/day treatment satisfied the criteria for the next stage of the trial. CONCLUSIONS: PI-88 at 160 mg/day is optimal and safe, and shows preliminary efficacy as an adjunct therapy for post-operative HCC.

The various impacts of the hepatitis C virus core protein upon hepatic oxidative stress after common bile duct ligation and partial hepatectomy.

INTRODUCTION: Although it is uncertain how the hepatitis C virus (HCV) core protein influences hepatic oxidative stress after partial hepatectomy and common bile duct ligation (CBDL) this may be crucial for the prognosis of patients with HCV infection who have undergone hepatic resection, or who have complications due to a biliary tract obstruction. MATERIALS AND METHODS: A group of double transgenic mice (DTM) that express both the tetracycline transactivator (tTA) and the HCV core, with conditional, acute expression of the HCV core in the context of the mature liver were subjected to 43% partial hepatectomy and CBDL. The levels of thioredoxin-1, thiobarbituric acid reactive substances (TBARS), and 4-hydroxynonenal (4-HNE) were evaluated in liver samples taken 3 days after the operations. RESULTS: The DTM had significantly higher TBARS levels than mice that were transgenic for only tTA (i.e. single transgenic mice; STM) and non-transgenic mice (NTM) after a sham laparotomy, CBDL and partial hepatectomy. Of the DTM, the TBARS levels were higher in female mice than in males after a sham laparotomy (P = 0.02) and CBDL (P = 0.0001). 4-HNE staining data were compatible with these results. Furthermore, male DTM exhibited higher levels of thioredoxin-1 than female DTM after sham laparotomy (P = 0.012) and CBDL (P = 0.008). CONCLUSIONS: The HCV core increases hepatic oxidative stress in vivo and female DTM are more vulnerable to the oxidative stress caused by acute core expression with, or without, CBDL. The fact that the female DTM had lower thioredoxin-1 levels may account for this observation.

Acute expression of hepatitis C core protein in adult mouse liver: Mitochondrial stress and apoptosis.

OBJECTIVE: In infection with hepatitis C virus (HCV), spontaneous clearance of the virus occurs in 30-40% of cases. By contrast, in chronic infection, this is rare. The basis for viral clearance in acute disease is unknown. Whereas cellular immune responses have been studied in detail, few data exist on the role of viral structural proteins, such as the core protein. The purpose of this study was to investigate the effects of core produced de novo within adult mouse hepatocytes by using a new transgenic mouse line in which expression of HCV core is regulated by tetracycline (tet-off). MATERIAL AND METHODS: In this work, transgenic mice with conditional HCV core were created, to study the acute expression of HCV core protein in the context of the mature liver. The subcellular distribution of the core, hepatocellular oxidative stress and apoptosis were monitored. RESULTS: Core protein is readily detectable and strongly associated with cytoplasmic lipid vesicles, endoplasmic reticulum and mitochondria. Mitochondrial oxidative stress was evidenced by a reduction in thioredoxin-2 (trx2). Concurrently, caspase-3 activity and TUNEL increased and, over time, the level of core protein in the liver declined. CONCLUSIONS: Mice that are conditionally transgenic for HCV core protein, which is readily detected and morphologically associated with steatosis in individual hepatocytes, were developed. Acute expression of core protein causes mitochondrial stress, as demonstrated by a reduction in trx2 and in the apoptosis of core-positive hepatocytes. We speculate that these events could be involved in the clearance of virus during acute hepatitis C, by both reducing the burden of virus in the liver and effectively priming the immune response.

Aberrant activation of nuclear factor of activated T cell 2 in lamina propria mononuclear cells in ulcerative colitis.

AIM: To investigate the role of nuclear factor of activated T cell 2 (NFAT2), the major NFAT protein in peripheral T cells, in sustained T cell activation and intractable inflammation in human ulcerative colitis (UC). METHODS: We used two-dimensional gel-electrophoresis, immunohistochemistry, double immunohistochemical staining, and confocal microscopy to inspect the expression of NFAT2 in 107, 15, 48 and 5 cases of UC, Crohn's disease (CD), non-specific colitis, and 5 healthy individuals, respectively. RESULTS: Up-regulation with profound nucleo-translocation/activation of NFAT2 of lamina propria mononuclear cells (LPMC) of colonic mucosa was found specifically in the affected colonic mucosa from patients with UC, as compared to CD or NC (P < 0.001, Kruskal-Wallis test). Nucleo-translocation/activation of NFAT2 primarily occurred in CD8+T, but was less prominent in CD4+ T cells or CD20+B cells. It was strongly associated with the disease activity, including endoscopic stage (tau = 0.2145, P = 0.0281) and histologic grade (tau = 0.4167, P < 0.001). CONCLUSION: We disclose for the first time the nucleo-translocation/activation of NFAT2 in lamina propria mononuclear cells in ulcerative colitis. Activation of NFAT2 was specific for ulcerative colitis and highly associated with disease activity. Since activation of NFAT2 is implicated in an auto-regulatory positive feedback loop of sustained T-cell activation and NFAT proteins play key roles in the calcium/calcineurin signaling pathways, our results not only provide new insights into the mechanism for sustained intractable inflammation, but also suggest the calcium-calcineurin/NFAT pathway as a new therapeutic target for ulcerative colitis.

Altered expression patterns of lipid metabolism genes in an animal model of HCV core-related, nonobese, modest hepatic steatosis.

BACKGROUND: Because the gene expression patterns of nonobese hepatic steatosis in affected patients remain unclear, we sought to explore these patterns using an animal model of nonobese hepatic steatosis. METHODS: We developed mice that conditionally express the hepatitis C virus (HCV) core protein regulated by the tetracycline transactivator (tTA). Microarray analyses and reverse-transcription polymerase chain reaction were performed using liver samples of both the double transgenic mice (DTM), which express both the HCV core and tTA, and single transgenic mice (STM), which express tTA alone, at 2 months of age. Functional categories of genes with altered expression were classified using gene ontology programs. Serum glucose, lipid levels, and systemic blood pressure were also measured. RESULTS: Approximately 20-30% of hepatocytes from the DTM were steatotic. No significant differences were observed in the serum glucose, lipid content, or blood pressure levels between the DTM and STM. Gene expression analyses revealed Sterol-regulatory element-binding protein (SREBP) pathway activation and dysregulation of the following genes involved in lipid metabolism: 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1, Apolipoprotein AII, Apolipoprotein CI, acyl-CoA thioesterase I, and fatty acid binding protein 1; in mitochondrial function: solute carrier family 25 member 25 and cytochrome c oxidase subunit II; in immune reaction: complement component 3, lymphocyte antigen 6 complex, locus A, lymphocyte antigen 6 complex, locus C, lymphocyte antigen 6 complex, locus D, and lymphocyte antigen 6 complex, locus E. CONCLUSION: Some genes of lipid metabolism, mitochondrial function, and immune reaction and the SREBP pathway are involved in HCV core-related, nonobese, modest hepatic steatosis.

Gene gun bombardment with DNA-coated gold particles is a potential alternative to hydrodynamics-based transfection for delivering genes into superficial hepatocytes.

Although in vivo nonviral gene delivery to the liver is critical for hepatic gene therapy, there are a number of technical obstacles. Enhanced green fluorescent protein (EGFP)-encoding DNA was coated onto gold particles (gold-DNA), dissolved in phosphate-buffered saline (pure DNA), and prepared as a polymer adjuvant (jetPEI)-galactosidase solution (polymer-DNA). Murine liver transfection was attempted by nonviral approaches, which included hydrodynamics-based transfection (HBT) of pure DNA, transport and transhepatic injection of polymer-DNA, and gene gun bombardment with pure DNA, gold-DNA, and polymer-DNA. Only HBT and gene gun bombardment yielded significant numbers of EGFP(+) hepatocytes. With the exception of the edge of the liver, HBT had a whole-liver transfection rate of 20% under optimized conditions. HBT resulted in marked hepatic infarctions, most prominently at the edge of the liver. For gene gun bombardment, the transfection rate was pressure dependent and limited to 15% for gold-DNA. Triple or quadruple bombardment at 30 psi resulted in a transfection rate comparable to that of a single bombardment at higher pressure, but was associated with minimal scattered hepatic necrosis. The EGFP(+) hepatocytes were located mainly in the superficial layers. We conclude that both HBT and gene gun bombardment yielded efficient murine hepatocyte transfection in vivo. Severe hepatic infarction impedes foreign gene expression in the superficial hepatocytes after HBT. Repeated bombardment with gold-DNA, using an accelerated particle gene gun at 30 psi, is a potential alternative to HBT for delivering genes to superficial hepatocytes in vivo, although gold-related hepatic necrosis is a persistent problem.

Prevalence of and risk factors for gallbladder polyps detected by ultrasonography among healthy Chinese: analysis of 34 669 cases.

BACKGROUND AND AIM: Gallbladder (GB) polyps are tumor or tumor-like projections arising from GB mucosa. Although most polyps are benign, some early GB carcinomas present as polypoid lesions. The diagnosis of GB polyps is relatively easy by ultrasonography. Although numerous studies have investigated GB polyps, few studies have addressed the prevalence of and factors associated with GB polyps for specific ethnic populations. This study analyzes the prevalence and factors associated with GB polyps in a Chinese population who can afford a paid general checkup. METHODS: The prevalence of and risk factors for GB polyps diagnosed by ultrasonography were retrospectively investigated in 34 669 Chinese patients who underwent a general checkup at Chang Gung Memorial Hospital (Taipei, Taiwan) between 2000 and 2003. Demographic, hemogram, serum biochemistry, hepatitis B surface antigen, hepatitis C antibody, and ultrasonography study data was available for all the patients. The correlations between the prevalence of GB polyps and age, sex, body height, body weight, body mass index, hemogram, serum biochemistry, and viral markers were examined for all the patients. RESULTS: Excluding the patients who underwent cholecystectomy, the overall prevalence of GB polyps was 9.5% and highest for middle-aged males. The analyzed risk factors with increased odds ratios (OR) for the development of GB polyps were male sex (OR 0.646, P < 0.0005) and hepatitis B virus surface antigen positivity (OR 1.113, P < 0.0005). Other demographic characteristics and biochemical parameters, including body height, body weight, body mass index, lipid profile, chronic hepatitis C virus infection, and liver function did not correlate with the presence of GB polyps. CONCLUSION: The prevalence of GB polyps among the Chinese in this study is higher than that reported for other populations. Chinese males and other patients with chronic hepatitis B viral infections have a high risk for developing GB polyps.

AFP-L3 in chronic liver diseases with persistent elevation of alpha-fetoprotein.

BACKGROUND: Alpha-fetoprotein (AFP) is an important marker for hepatocellular carcinoma (HCC). However, persistent elevation of AFP is found in patients with chronic liver diseases. The value of AFP-L3, which is more specific than AFP, was examined in such patients. METHODS: We enrolled patients without image-detectable tumor, but with transient AFP value > 900 ng/mL (group A) or with persistent AFP value > 50 ng/mL for longer than 6 months (group B). Forty-one patients with HCC and AFP value > 50 ng/mL were included as the HCC control group (group C). AFP-L3 measurement was done by lectin-affinity electrophoresis coupled with antibody-affinity blotting. The study patients were followed with AFP, liver biochemistry and abdominal ultrasound at 3- to 6-month intervals. Additional studies were done when a tumor was suspected. RESULTS: One of 17 patients in group A and 13 of 39 patients in group B developed HCC within 2 years. When the cutoff value of AFP-L3 ratio was 15%, both the sensitivity and specificity were 71% for prediction of HCC during the next 2 years in all patients. Ninety percent of tumors larger than 5 cm had AFP-L3 > 15%, compared with only 60% for tumors smaller than 2 cm. Three patients in group A had AFP-L3 ratio > 17.5%. One patient developed HCC 10 months later; the other 2 patients were associated with hepatic failure. CONCLUSION: AFP-L3 provides a clue in HCC detection in patients with persistent elevation of AFP. However, AFP-L3 could be highly elevated in severe hepatitis.