Zinc protoporphyrin binding to telomerase complexes and inhibition of telomerase activity.

Zinc protoporphyrin (ZnPP), a naturally occurring metalloprotoporphyrin (MPP), is currently under development as a chemotherapeutic agent although its mechanism is unclear. When tested against other MPPs, ZnPP was the most effective DNA synthesis and cellular proliferation inhibitor while promoting apoptosis in telomerase positive but not telomerase negative cells. Concurrently, ZnPP down-regulated telomerase expression and was the best overall inhibitor of telomerase activity in intact cells and cellular extracts with IC50 and EC50  values of ca 2.5 and 6 µM, respectively. The natural fluorescence properties of ZnPP enabled direct imaging in cellular fractions using non-denaturing agarose gel electrophoresis, western blots, and confocal fluorescence microscopy. ZnPP localized to large cellular complexes (>600 kD) that contained telomerase and dysskerin as confirmed with immunocomplex mobility shift, immunoprecipitation, and immunoblot analyses. Confocal fluorescence studies showed that ZnPP co-localized with telomerase reverse transcriptase (TERT) and telomeres in the nucleus of synchronized S-phase cells. ZnPP also co-localized with TERT in the perinuclear regions of log phase cells but did not co-localize with telomeres on the ends of metaphase chromosomes, a site known to be devoid of telomerase complexes. Overall, these results suggest that ZnPP does not bind to telomeric sequences per se, but alternatively, interacts with other structural components of the telomerase complex to inhibit telomerase activity. In conclusion, ZnPP actively interferes with telomerase activity in neoplastic cells, thus promoting pro-apoptotic and anti-proliferative properties. These data support further development of natural or synthetic protoporphyrins for use as chemotherapeutic agents to augment current treatment protocols for neoplastic disease.

Modification of the third phase in the framework for vertebrate species persistence in urban mosaic environments.

Urbanisation is rapidly transforming natural landscapes with consequences for biodiversity. Little is documented on the response of African wildlife to urbanisation. We reviewed case studies of vertebrate species' responses to urbanisation in KwaZulu-Natal, South Africa to determine trends. Connected habitat mosaics of natural and anthropogenic green spaces are critical for urban wildlife persistence. We present a novel modification to the final of three phases of the framework described by Evans et al. (2010), which documents this sequence for vertebrate species persistence, based on the perspective of our research. Species in suburbia exhibit an initial phase where behavioural and ecological flexibility, life-history traits and phenotypic plasticity either contribute to their success, or they stay at low numbers. Where successful, the next phase is a rapid increase in populations and distribution; anthropogenic food resources and alternate breeding sites are effectively exploited. The modified third phase either continues to spread, plateau or decline.

Metalloprotoporphyrin Inhibition of HCV NS3-4A Protease: Structure-Activity Relationships.

BACKGROUND: Antiviral actions of tetrapyrroles have been described in a number of systems. Our goal was to evaluate antagonism of the HCV NS3-4A protease by a variety of common porphyrins and characterize structure-activity relationships that may be useful for future drug design of HCV and related Flaviviruses. METHODS: Using fluorometric assays, common metalloprotoporphyrins (MPP) all inhibited NS3-4A protease with IC50 values in low micromolar ranges [CoPP (1.4 µM) < ZnPP = MnPP = SnPP < CuPP < FePP (6.5 µM) = protoporphyrin]. RESULTS: Lineweaver-Burk plots confirmed that MPP: NS3 inhibition was basically competitive. All tested MPPs inhibited HCV genotype 1A, 1B, 2A and 3A recombinant proteases with the same fidelity suggesting wide antagonistic capabilities. However, when the MPPs were tested in cellular incubations with HCV replicons only Zn, Fe and free-base protoporphyrin showed comparable EC50 and IC50 values suggesting that there may be critical differences in MPP uptake and intracellular availability. Meso, deutero, and isohematoporphyrin derivatives, with or without metal substitution, all showed less anti-protease and antiviral activities as compared to protoporphyrins, suggesting that the planar, vinyl side chains are important for protease active site binding. MPPs were also active against three common protease mutants (T54A, A156T, and V36M) with equivalent or better IC50 values as compared to wild type enzyme. CONCLUSION: These findings document the versatility of MPPs as antiviral agents with an expanded sensitivity for HCV genotypes and resistance to some common viral mutations. The results also suggest that further study of MPP structure and function will be useful for the development of new antiviral agents.

Ombitasvir/paritaprevir/ritonavir and dasabuvir±ribavirin for chronic HCV infection in US veterans with psychiatric disorders.

Hepatitis C virus (HCV) infections are more common among US veterans receiving care through Veterans Affairs (VA) Medical Centers than among the general population. Historically, HCV therapies had lower efficacy rates in VA patients, possibly due to common comorbidities such as psychiatric disorders and substance abuse. The direct-acting antivirals ombitasvir/paritaprevir/ritonavir and dasabuvir (OBV/PTV/r+DSV)±ribavirin (RBV) are approved in the US for HCV genotype 1 (GT1)-infected adults with or without cirrhosis. This study prospectively evaluated the safety and efficacy of OBV/PTV/r+DSV±RBV in VA patients with HCV GT1 infection. TOPAZ-VA was a phase 3b, open-label trial. Adult US veterans with HCV GT1 infection, without cirrhosis or with compensated cirrhosis, were eligible for enrollment. Patients with GT1a infection received OBV/PTV/r +DSV+RBV for 12 weeks or 24 weeks (for those with cirrhosis); GT1b-infected patients without cirrhosis received OBV/PTV/r +DSV for 12 weeks; those with cirrhosis received OBV/PTV/r +DSV with RBV. The primary endpoint was sustained virologic response at posttreatment week 12 (SVR12); safety was also assessed. Ninety-nine patients were enrolled at 10 sites from May through November 2015. The majority were male (96%), white (60%), and with GT1a infection (68%); 49% reported ongoing psychiatric disorders. Overall, 94% (93/99) achieved SVR12; three patients had a virologic failure. The most common AEs were fatigue (28%), headache (20%), and nausea (15%); six patients discontinued treatment due to AEs. In US veterans with HCV GT1 infection, OBV/PTV/r +DSV±RBV yielded a 94% overall SVR12 rate and was well tolerated. The presence of psychiatric disorders and/or injection drug use did not impact efficacy.

Hepatitis C virus infection inhibits a Src-kinase regulatory phosphatase and reduces T cell activation in vivo.

Among human RNA viruses, hepatitis C virus (HCV) is unusual in that it causes persistent infection in the majority of infected people. To establish persistence, HCV evades host innate and adaptive immune responses by multiple mechanisms. Recent studies identified virus genome-derived small RNAs (vsRNAs) in HCV-infected cells; however, their biological significance during human HCV infection is unknown. One such vsRNA arising from the hepatitis C virus (HCV) E2 coding region impairs T cell receptor (TCR) signaling by reducing expression of a Src-kinase regulatory phosphatase (PTPRE) in vitro. Since TCR signaling is a critical first step in T cell activation, differentiation, and effector function, its inhibition may contribute towards HCV persistence in vivo. The effect of HCV infection on PTPRE expression in vivo has not been examined. Here, we found that PTPRE levels were significantly reduced in liver tissue and peripheral blood mononuclear cells (PBMCs) obtained from HCV-infected humans compared to uninfected controls. Loss of PTPRE expression impaired antigen-specific TCR signaling, and curative HCV therapy restored PTPRE expression in PBMCs; restoring antigen-specific TCR signaling defects. The extent of PTPRE expression correlated with the amount of sequence complementarity between the HCV E2 vsRNA and the PTPRE 3' UTR target sites. Transfection of a hepatocyte cell line with full-length HCV RNA or with synthetic HCV vsRNA duplexes inhibited PTPRE expression, recapitulating the in vivo observation. Together, these data demonstrate that HCV infection reduces PTPRE expression in the liver and PBMCs of infected humans, and suggest that the HCV E2 vsRNA is a novel viral factor that may contribute towards viral persistence.

HCV Induces Telomerase Reverse Transcriptase, Increases Its Catalytic Activity, and Promotes Caspase Degradation in Infected Human Hepatocytes.

INTRODUCTION: Telomerase repairs the telomeric ends of chromosomes and is active in nearly all malignant cells. Hepatitis C virus (HCV) is known to be oncogenic and potential interactions with the telomerase system require further study. We determined the effects of HCV infection on human telomerase reverse transcriptase (TERT) expression and enzyme activity in primary human hepatocytes and continuous cell lines. RESULTS: Primary human hepatocytes and Huh-7.5 hepatoma cells showed early de novo TERT protein expression 2-4 days after infection and these events coincided with increased TERT promoter activation, TERT mRNA, and telomerase activity. Immunoprecipitation studies demonstrated that NS3-4A protease-helicase, in contrast to core or NS5A, specifically bound to the C-terminal region of TERT through interactions between helicase domain 2 and protease sequences. Increased telomerase activity was noted when NS3-4A was transfected into cells, when added to reconstituted mixtures of TERT and telomerase RNA, and when incubated with high molecular weight telomerase 'holoenzyme' complexes. The NS3-4A catalytic effect on telomerase was inhibited with primuline or danoprevir, agents that are known to inhibit NS3 helicase and protease activities respectively. In HCV infected cells, NS3-4A could be specifically recovered with telomerase holoenzyme complexes in contrast to NS5A or core protein. HCV infection also activated the effector caspase 7 which is known to target TERT. Activation coincided with the appearance of lower molecular weight carboxy-terminal fragment(s) of TERT, chiefly sized at 45 kD, which could be inhibited with pancaspase or caspase 7 inhibitors. CONCLUSIONS: HCV infection induces TERT expression and stimulates telomerase activity in addition to triggering Caspase activity that leads to increased TERT degradation. These activities suggest multiple points whereby the virus can influence neoplasia. The NS3-4A protease-helicase can directly bind to TERT, increase telomerase activity, and thus potentially influence telomere repair and host cell neoplastic behavior.

Improved Survival Among all Interferon-α-Treated Patients in HCV-002, a Veterans Affairs Hepatitis C Cohort of 2211 Patients, Despite Increased Cirrhosis Among Nonresponders.

BACKGROUND: As the era of interferon-alpha (IFN)-based therapy for hepatitis C ends, long-term treatment outcomes are now being evaluated. AIM: To more fully understand the natural history of hepatitis C infection by following a multisite cohort of patients. METHODS: Patients with chronic HCV were prospectively enrolled in 1999-2000 from 11 VA medical centers and followed through retrospective medical record review. RESULTS: A total of 2211 patients were followed for an average of 8.5 years after enrollment. Thirty-one percent of patients received HCV antiviral therapy, 15 % with standard IFN/ribavirin only, 16 % with pegylated IFN/ribavirin, and 26.7 % of treated patients achieved sustained virologic response (SVR). Cirrhosis developed in 25.8 % of patients. Treatment nonresponders had a greater than twofold increase in the hazard of cirrhosis and hepatocellular carcinoma, compared to untreated patients, whereas SVR patients were only marginally protected from cirrhosis. Nearly 6 % developed hepatocellular carcinoma, and 27.1 % died during the follow-up period. Treated patients, regardless of response, had a significant survival benefit compared to untreated patients (HR 0.58, CI 0.46-0.72). Improved survival was also associated with college education, younger age, lower levels of alcohol consumption, and longer duration of medical service follow-up-factors typically associated with treatment eligibility. CONCLUSIONS: As more hepatitis C patients are now being assessed for all-oral combination therapy, these results highlight that patient compliance and limiting harmful behaviors contribute a significant proportion of the survival benefit in treated patients and that the long-term clinical benefits of SVR may be less profound than previously reported.

Finally sofosbuvir: an oral anti-HCV drug with wide performance capability.

Chronic Hepatitis C virus (HCV) infection is the leading cause of advanced liver disease worldwide. The virus successfully evades host immune detection and for many years has hampered efforts to find a safe, uncomplicated, and reliable oral antiviral therapy. Initially, interferon and ribavirin therapy was the treatment standard of care, but it offered limited performance across the wide spectrum of HCV disease and was fraught with excessive and often limiting side effects. Sofosbuvir (SOF) is a potent first-in-class nucleoside inhibitor that has recently been approved for treatment of HCV. The drug has low toxicity, a high resistance barrier, and minimal drug interactions with other HCV direct-acting antiviral agents such as protease inhibitors or anti-NS5A agents. SOF is safe and can be used across different viral genotypes, disease stages, and special patient groups, such as those coinfected with human immunodeficiency virus. When used in combination with ribavirin or another direct-acting antiviral agent, SOF has revolutionized the HCV treatment spectrum and set the stage for nearly universal HCV antiviral therapy. More so than any other anti-HCV drug developed to date, SOF offers the widest applicability for all infected patients, and new regimens will be tailored to maximize performance.

Direct-acting antiviral agents and the path to interferon independence.

Chronic infection with hepatitis C virus (HCV) is a major global health problem; there are approximately 120 to 130 million chronic infections worldwide. Since the discovery of HCV 24 years ago, there has been a relentless effort to develop successful antiviral therapies. Studies of interferon-α-based therapies have helped define treatment parameters, and these treatment strategies have cured a substantial percentage of patients. However, interferon-α must be injected; there are problems with tolerability, adherence, and incomplete response in a large percentage of patients. New drug candidates designed to target the virus or the host have recently been introduced at an unprecedented pace. In phase I-III studies, these agents have exceeded expectations and achieved rates of response previously not thought possible. We are, therefore, entering a new era of therapy for HCV infection and interferon independence.

Restoration of type I interferon expression by heme and related tetrapyrroles through inhibition of NS3/4A protease.

BACKGROUND: Tetrapyrrole substrates and products of heme oxygenase are potent inhibitors of hepatitis C virus (HCV) replication. It is not clear whether this occurs through primary induction of type I interferon (IFN), inhibition of viral NS3/4A protease, or a combination of these mechanisms. We studied the antiviral actions of tetrapyrroles and their potential influence on type I IFN induction. METHODS: The effects of tetrapyrrole on NS3/4A protease activity and type I IFN induction were assessed in HCV-permissive cells, replicons, or human embryonic kidney (HEK) 293 cells transfected with NS3/4A protease. Activation of innate immune signaling was determined after transfection of double-strand surrogate nucleic acid antigens or infection with defined sequence HCV cell culture (HCVcc) RNA. RESULTS: Tetrapyrroles failed to directly induce IFN expression at concentrations that inhibited HCV replication and NS3/4A protease activity. However, they potently restored IFN induction after attenuation with NS3/4A protease, a process accompanied by preservation of the adapter protein, mitochondrial antiviral signaling protein, nuclear localization of IFN regulatory factor 3, and augmentation of IFN-stimulated gene products. CONCLUSIONS: Tetrapyrroles do not directly induce IFN, but they dramatically restore type I IFN signaling pathway after attenuation with NS3/4A protease. They show immunomodulatory as well as antiprotease activity and may be useful for treatment of HCV infection.

Heme and HO-1 Inhibition of HCV, HBV, and HIV.

Hepatitis C virus, human immunodeficiency virus, and hepatitis B virus are chronic viral infections that cause considerable morbidity and mortality throughout the world. In the decades following the identification and sequencing of these viruses, in vitro experiments demonstrated that heme oxygenase-1, its oxidative products, and related compounds of the heme oxygenase system inhibit replication of all 3 viruses. The purpose of this review is to critically evaluate and summarize the seminal studies that described and characterized this remarkable behavior. It will also discuss more recent work that discovered the antiviral mechanisms and target sites of these unique antiviral agents. In spite of the fact that these viruses are diverse pathogens with quite profound differences in structure and life cycle, it is significant that heme and related compounds show striking similarity for viral target sites across all three species. Collectively, these findings strongly indicate that we should move forward and develop heme and related tetrapyrroles into versatile antiviral agents that could be used therapeutically in patients with single or multiple viral infections.

Hepatitis C virus infection and hepatic stellate cell activation downregulate miR-29: miR-29 overexpression reduces hepatitis C viral abundance in culture.

BACKGROUND: Chronic hepatitis C virus (HCV)-induced liver fibrosis involves upregulation of transforming growth factor (TGF)-ß and subsequent hepatic stellate cell (HSC) activation. MicroRNAs (miRNAs) regulate HCV infection and HSC activation. METHODS: TaqMan miRNA profiling identified 12 miRNA families differentially expressed between chronically HCV-infected human livers and uninfected controls. To identify pathways affected by miRNAs, we developed a new algorithm (pathway analysis of conserved targets), based on the probability of conserved targeting. RESULTS: This analysis suggested a role for miR-29 during HCV infection. Of interest, miR-29 was downregulated in most HCV-infected patients. miR-29 regulates expression of extracellular matrix proteins. In culture, HCV infection downregulated miR-29, and miR-29 overexpression reduced HCV RNA abundance. miR-29 also appears to play a role in HSCs. Hepatocytes and HSCs contribute similar amounts of miR-29 to whole liver. Both activation of primary HSCs and TGF-ß treatment of immortalized HSCs downregulated miR-29. miR-29 overexpression in LX-2 cells decreased collagen expression and modestly decreased proliferation. miR-29 downregulation by HCV may derepress extracellular matrix synthesis during HSC activation. CONCLUSIONS: HCV infection downregulates miR-29 in hepatocytes and may potentiate collagen synthesis by reducing miR-29 levels in activated HSCs. Treatment with miR-29 mimics in vivo might inhibit HCV while reducing fibrosis.

FibroTest is an independent predictor of virologic response in chronic hepatitis C patients retreated with pegylated interferon alfa-2b and ribavirin in the EPIC³ program.

BACKGROUND & AIMS: EPIC-3 is a prospective, international study that has demonstrated the efficacy of PEG-IFN alfa-2b plus weight-based ribavirin in patients with chronic hepatitis C and significant fibrosis who previously failed any interferon-alfa/ribavirin therapy. The aim of the present study was to assess FibroTest (FT), a validated non-invasive marker of fibrosis in treatment-naive patients, as a possible alternative to biopsy as the baseline predictor of subsequent early virologic (EVR) and sustained virologic response (SVR) in previously treated patients. METHODS: Of 2312 patients enrolled, 1459 had an available baseline FT, biopsy, and complete data. Uni- (UV) and multi-variable (MV) analyses were performed using FT and biopsy. RESULTS: Baseline characteristics were similar as in the overall population; METAVIR stage: 28% F2, 29% F3, and 43% F4, previous relapsers 29%, previous PEG-IFN regimen 41%, high baseline viral load (BVL) 64%. 506 patients (35%) had undetectable HCV-RNA at TW12 (TW12neg), with 58% achieving SVR. The accuracy of FT was similar to that in naive patients: AUROC curve for the diagnosis of F4 vs F2=0.80 (p<0.00001). Five baseline factors were associated (p<0.001) with SVR in UV and MV analyses (odds ratio: UV/MV): fibrosis stage estimated using FT (4.5/5.9) or biopsy (1.5/1.6), genotype 2/3 (4.5/5.1), BVL (1.5/1.3), prior relapse (1.6/1.6), previous treatment with non-PEG-IFN (2.6/2.0). These same factors were associated (p ≤ 0.001) with EVR. Among patients TW12neg, two independent factors remained highly predictive of SVR by MV analysis (p ≤ 0.001): genotype 2/3 (odds ratio=2.9), fibrosis estimated with FT (4.3) or by biopsy (1.5). CONCLUSIONS: FibroTest at baseline is a possible non-invasive alternative to biopsy for the prediction of EVR at 12 weeks and SVR, in patients with previous failures and advanced fibrosis, retreated with PEG-IFN alfa-2b and ribavirin.

Biliverdin inhibits hepatitis C virus nonstructural 3/4A protease activity: mechanism for the antiviral effects of heme oxygenase?

UNLABELLED: Induction of heme oxygenase-1 (HO-1) inhibits hepatitis C virus (HCV) replication. Of the products of the reaction catalyzed by HO-1, iron has been shown to inhibit HCV ribonucleic acid (RNA) polymerase, but little is known about the antiviral activity of biliverdin (BV). Herein, we report that BV inhibits viral replication and viral protein expression in a dose-dependent manner in replicons and cells harboring the infectious J6/JFH construct. Using the SensoLyte 620 HCV Protease Assay with a wide wavelength excitation/emission (591 nm/622 nm) fluorescence energy transfer peptide, we found that both recombinant and endogenous nonstructural 3/4A (NS3/4A) protease from replicon microsomes are potently inhibited by BV. Of the tetrapyrroles tested, BV was the strongest inhibitor of NS3/4A activity, with a median inhibitory concentration (IC(50)) of 9 µM, similar to that of the commercial inhibitor, AnaSpec (Fremont, CA) #25346 (IC(50) 5 µM). Lineweaver-Burk plots indicated mixed competitive and noncompetitive inhibition of the protease by BV. In contrast, the effects of bilirubin (BR) on HCV replication and NS3/4A were much less potent. Because BV is rapidly converted to BR by biliverdin reductase (BVR) intracellularly, the effect of BVR knockdown on BV antiviral activity was assessed. After greater than 80% silencing of BVR, inhibition of viral replication by BV was enhanced. BV also increased the antiviral activity of α-interferon in replicons. CONCLUSION: BV is a potent inhibitor of HCV NS3/4A protease, which likely contributes to the antiviral activity of HO-1. These findings suggest that BV or its derivatives may be useful in future drug therapies targeting the NS3/4A protease.

Correlation between microRNA expression levels and clinical parameters associated with chronic hepatitis C viral infection in humans.

MicroRNAs (miRNAs) are small RNAs that regulate gene expression pathways. Previous studies have shown interactions between hepatitis C virus (HCV) and host miRNAs. We measured miR-122 and miR-21 levels in HCV-infected human liver biopsies relative to uninfected human livers and correlated these with clinical patient data. miR-122 is required for HCV replication in vitro, and miR-21 is involved in cellular proliferation and tumorigenesis. We found that miR-21 expression correlated with viral load, fibrosis and serum liver transaminase levels. miR-122 expression inversely correlated with fibrosis, liver transaminase levels and patient age. miR-21 was induced ∼twofold, and miR-122 was downregulated on infection of cultured cells with the HCV J6/JFH infectious clone, thus establishing a link to HCV. To further examine the relationship between fibrosis and the levels of miR-21 and miR-122, we measured their expression levels in a mouse carbon tetrachloride fibrosis model. As in the HCV-infected patient samples, fibrotic stage positively correlated with miR-21 and negatively correlated with miR-122 levels. Transforming growth factor ß (TGF-ß) is a critical mediator of fibrogenesis. We identified SMAD7 as a novel miR-21 target. SMAD7 is a negative regulator of TGF-ß signaling, and its expression is induced by TGF-ß. To confirm the relationship between miR-21 and the TGF-ß signaling pathway, we measured the effect of miR-21 on a TGF-ß-responsive reporter. We found that miR-21 enhanced TGF-ß signaling, further supporting a relationship between miR-21 and fibrosis. We suggest a model in which miR-21 targeting of SMAD7 could increase TGF-ß signaling, leading to increased fibrogenesis.

Portosystemic encephalopathy due to mesoiliac shunt in a patient without cirrhosis.

Hepatic encephalopathy most commonly occurs in patients with cirrhosis and end-stage liver disease, however, the disorder can also occur in the presence of intrahepatic or extrahepatic shunts when the intrahepatic circulation is effectively bypassed. The majority of extrahepatic shunts described to date develop between a mesenteric vein and inferior vena cava. Herein we report a novel case of a superior mesenteric vein to left internal iliac vein shunt that led to hepatic encephalopathy in a 57-year-old woman with no apparent underlying liver disorder. The patient presented with confusion, disorientation, and hyperammonemia. Workup for parenchymal liver disease was negative and liver biopsy findings did not show significant liver disease. Magnetic resonance imaging revealed a serpiginous 1-cm-wide shunt that diverted superior mesenteric vein blood from the portal confluence to the left internal iliac vein. Surgical closure of the shunt led to marked improvement of the patient with the resolution of hepatic encephalopathy. This report is the first description of a portosystemic shunt, likely congenital, linking these 2 vessels resulting in clinically significant hepatic encephalopathy. The findings emphasize that abdominal and pelvic imaging should be considered in patients with signs of hepatic encephalopathy that have none to minimal hepatic disease.

Hepatitis C virus core protein enhances Telomerase activity in Huh7 cells.

Hepatitis C is an oncogenic virus although the mechanisms responsible for this behavior are not clear. We studied the effects of hepatitis C virus (HCV) core protein expression on Telomerase, an enzyme closely associated with cellular immortalization and neoplasia. The aim of this study was to investigate the effects of HCV core protein on the regulation of Telomerase activity in human hepatoma cells. Regulation and expression of human Telomerase reverse transcriptase (TERT) was compared in Huh7 cells stably transfected with HCV core protein or cells expressing vector alone. Telomerase activity was measured using Quantitative Telomerase Detection (QTD) and telomere length was measured by fluorescence in situ hybridization (FISH). Transient transfection and luciferase assay were used to evaluate TERT promoter activity. Telomerase activity was increased twofold in Huh7 cells expressing HCV core protein compared to controls (P < 0.01). This was accompanied by a 1.4-fold increase of TERT mRNA and 1.9-fold increase in TERT protein (P < 0.01 in either case). Cellular fractionation and immunocytochemical studies showed increased localization of TERT in the nucleus of core-expressing cells as compared to controls. FISH assay confirmed that telomeres of HCV core-expressing Huh7 cells were relatively longer than those of control cells (0.22 + 0.05 vs. 0.12 + 0.03, P < 0.01). TERT promoter activity was enhanced about 30% in HCV core-expressing Huh7 cells compared to control cells (P < 0.02). HCV core protein is associated with increased Telomerase activity in hepatoma cells. These findings suggest that enhancement of Telomerase activity by HCV core protein may contribute to the oncogenicity of HCV.

Peginterferon alfa-2b and ribavirin: effective in patients with hepatitis C who failed interferon alfa/ribavirin therapy.

BACKGROUND & AIMS: Treatment with peginterferon alfa and ribavirin produces a sustained virologic response (SVR) in approximately 60% of hepatitis C virus (HCV)-infected patients. Alternate options are needed for patients who relapse or do not respond to therapy. METHODS: This prospective, international, multicenter, open-label study evaluated efficacy and safety of peginterferon alfa-2b (1.5 microg/kg/wk) plus weight-based ribavirin (800-1400 mg/day) in 2333 chronic HCV-infected patients with significant fibrosis/cirrhosis whose previous interferon alfa/ribavirin therapy failed. Patients with undetectable HCV-RNA at treatment week (TW) 12 received 48 weeks of therapy; patients with detectable HCV-RNA at TW12 could enter maintenance studies at TW18; 188 patients with low/detectable HCV-RNA at TW12 continued therapy at the investigator's request. RESULTS: Overall, 22% of the patients attained SVR (56% with undetectable HCV-RNA and 12% with low/detectable HCV-RNA at TW12). SVR was better in relapsers (38%) than nonresponders (14%), regardless of previous treatment, and in patients previously treated with interferon-alfa/ribavirin (25%) than peginterferon alfa-ribavirin (17%). Predictors of response in patients with undetectable HCV-RNA at TW12 were genotype (2/3 vs 1, respectively; odds ratio [OR] 2.4; P < .0001), fibrosis score (F2 vs F4; OR, 2.2; F3 vs F4; OR, 1.7; P < .0001), and baseline viral load (< or =600,000 vs >600,000 IU/mL; OR, 1.4; P = .0223). These factors plus previous treatment and response were overall predictors of SVR. Safety was similar among fibrosis groups. CONCLUSIONS: Peginterferon alfa-2b plus weight-based ribavirin is effective and safe in patients who failed interferon alfa/ribavirin therapy. Genotype, baseline viral load, and fibrosis stage were predictors of response.