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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Heme oxygenase-1 suppresses hepatitis C virus replication and increases resistance of hepatocytes to oxidant injury.

UNLABELLED: Oxidative injury to hepatocytes occurs as a result of hepatitis C virus (HCV) infection and replication. Modulation of host cell antioxidant enzymes such as heme oxygenase-1 (HO-1) may be useful therapeutically to minimize cellular injury, reduce viral replication, and attenuate liver disease. In this report, we evaluated the effects of HO-1 overexpression on HCV replication and hepatocellular injury. Full-length (FL) (Con1) or nonstructural (NS) replicons (I 389 NS3-3') were transfected with complete human HO-1 sequences or empty vector for control. Cell lines overexpressing HO-1 (twofold to sixfold above basal values) or empty vector were isolated, and their HCV RNA synthesis, pro-oxidant levels, and resistance to oxidative injury were assessed. HO-1 overexpression decreased HCV RNA replication in both FL and NS replicons without affecting cellular growth or DNA synthesis. The attenuation of HCV replication was significantly reversed in both replicon systems with HO-1 small interfering RNA (siRNA) knockdown. Both FL and NS replicons that overexpress HO-1 showed reduced prooxidant levels at baseline and increased resistance to oxidant-induced cytotoxicity. HO-1 induction with hemin also markedly decreased HCV replication in both parental FL and NS replicon cell lines. Conversely, knockdown of HO-1 messenger RNA (mRNA) by siRNA in parental FL or NS replicons did not significantly affect HCV replication, suggesting that less than basal levels of HO-1 had minimal effect on HCV replication. CONCLUSION: Overexpression or induction of HO-1 results in decreased HCV replication as well as protection from oxidative damage. These findings suggest a potential role for HO-1 in antiviral therapy and therapeutic protection against hepatocellular injury in HCV infection.

Increased plasma nitric oxide, L-arginine, and arginase-1 in cirrhotic patients with progressive renal dysfunction.

BACKGROUND AND AIMS: Increased levels of nitric oxide (NO) are hypothesized to contribute to renal dysfunction in patients with decompensated cirrhosis. In this study, we examined whether splanchnic and/or peripheral NO levels and L-arginine (L-Arg) correlate with progressive renal dysfunction in cirrhotics. METHODS: Serum NO metabolites (NOx) and L-Arg were measured in: controls (n = 10); organ donors (n = 12); compensated cirrhotics (n = 17), cirrhotics with ascites (n = 25), refractory ascites (n = 11) or hepatorenal syndrome type II (HRS) (n = 11) and chronic renal failure patients (n = 18). RESULTS: Plasma NOx and L-Arg levels rose progressively with worsening renal function in decompensated cirrhotics. Both NOx and L-Arg levels were highest in patients with HRS (P < 0.001 and P < 0.025, respectively). While there were no differences in NOx levels related to the site of sampling, L-Arg levels were lowest in hepatic venous blood. There were significant relationships of NOx and L-Arg with Model for End-Stage Liver Disease score and Child-Pugh scores (P < 0.04 and P < 0.01, respectively). Multivariate analysis showed a significant relationship between NOx, L-Arg and HRS. CONCLUSION: Worsening renal function in decompensated cirrhosis is accompanied by progressive elevation in plasma NOx and L-Arg. These findings support the hypothesis that NO-mediated vasodilation is probably linked with the mechanism of progressive renal failure in decompensated cirrhotics.

Altered expression of iron regulatory genes in cirrhotic human livers: clues to the cause of hemosiderosis?

Hepatic iron deposition unrelated to hereditary hemochromatosis occurs commonly in cirrhosis but the pathogenesis of this condition is unknown. The aim of this study was to compare the expression of genes involved in the regulation of iron metabolism in cirrhotic (n=22) and control human livers (n=5). Transcripts were quantitated by real-time RT-PCR and protein levels were assessed by western blot. Hepatic iron concentrations (HICs) were measured by a spectrophotometric method. Levels of hepcidin mRNA did not differ between controls and cirrhotic livers; there was a highly significant correlation between hepcidin transcript levels and HIC in the latter group. Ferroportin, divalent metal transporter-1 (DMT1), and ferritin heavy chain mRNA levels were significantly higher in cirrhotic human livers than in controls (P=0.007, 0.039, and 0.025, respectively). By western blot, ferroportin and DMT1 levels were generally diminished in the cirrhotic livers compared to controls; neither correlated with HIC. In contrast, the abundance of ferritin increased with increasing HIC in the cirrhotic livers, whereas transferrin receptor decreased, indicating physiologically appropriate regulation. In conclusion, hepcidin expression appears to be appropriately responsive to iron status in cirrhosis. However, there are complex alterations in DMT1 and ferroportin expression in cirrhotic liver, including decreases in ferroportin and DMT1 at the protein level that may play a role in aberrant regulation of iron metabolism in cirrhosis.

Increased hepatic telomerase activity in a rat model of iron overload: a role for altered thiol redox state?

Telomeres are repeated sequences at chromosome ends that are incompletely replicated during mitosis. Telomere shortening caused by proliferation or oxidative damage culminates in replicative arrest and senescence, which may impair regeneration during chronic liver injury. Whereas the effects of experimental liver injury on telomeres have received little attention, prior studies suggest that telomerase, the enzyme complex that catalyzes the addition of telomeric repeats, is protective in some rodent liver injury models. Thus, the aim of this study was to determine the effects of iron overload on telomere length and telomerase activity in rat liver. Mean telomere lengths were similar in iron-loaded and control livers. However, telomerase activity was increased 3-fold by iron loading, with no change in levels of TERT mRNA or protein. Because thiol redox state has been shown to modulate telomerase activity in vitro, hepatic thiols were assessed. Significant increases in GSH (1.5-fold), cysteine (15-fold), and glutamate cysteine ligase activity (1.5-fold) were observed in iron-loaded livers, whereas telomerase activity was inhibited by treatment with N-ethylmaleimide. This is the first demonstration of increased telomerase activity associated with thiol alterations in vivo. Enhanced telomerase activity may be an important factor contributing to the resistance of rodent liver to iron-induced damage.