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Biomedical subjects

Francesco Negro

Publications and source records attributed to Francesco Negro.

At least 19 recordsLinked to original sources
PubMed2006

In vitro antiviral activity of SCH446211 (SCH6), a novel inhibitor of the hepatitis C virus NS3 serine protease.

BACKGROUND: Current hepatitis C virus (HCV) therapies may cure approximately 60% of infections. They are often contraindicated or poorly tolerated, underscoring the need for safer and more effective drugs. A novel, alpha-ketoamide-derived, substrate-based inhibitor of the HCV serine protease (SCH446211) was developed. Compared with earlier reported inhibitors of similar chemical class, it has a P1'-P2' extension which provides extended interaction with the protease active site. The aim of this study was to evaluate the in vitro antiviral activity of SCH446211. METHODS: Binding constant of SCH446211 to HCV NS3 protease was measured with the chromogenic substrate in vitro cleavage assay. Cell-based activity of SCH446211 was evaluated in replicon cells, which are Huh-7 hepatoma cells stably transfected with a subgenomic HCV RNA as reported previously. After 72 h of incubation with SCH446211, viral transcription and protein expression were measured by real-time RT-PCR (TaqMan), quantitative in situ hybridization, immunoblot and indirect immunofluorescence. RESULTS: The binding constant of SCH446211 to HCV NS3 protease was 3.8 +/- 0.4 nM. HCV replication and protein expression were inhibited by SCH446211 in replicon cells as consistently shown by four techniques. In particular, based on quantitative real-time RT-PCR measurements, the IC50 and IC90 of SCH446211 were estimated to be 40 +/- 20 and 100 +/- 20 nM (n = 17), respectively. Long-term culture of replicon cells with SCH446211 reduced replicon RNA to <0.1 copy per cell. SCH446211 did not show cellular toxicity at concentrations up to 50 microM. CONCLUSIONS: SCH446211 is a potent inhibitor of HCV protease in vitro. Its extended interaction with the HCV NS3 protease active site is associated with potent in vitro antiviral activity. This observation is potentially a useful guide for development of future potent inhibitors against HCV NS3 protease.

Antiviral Agents↗
PubMed2006

Mechanisms and significance of liver steatosis in hepatitis C virus infection.

The pathogenesis of liver damage associated with chronic hepatitis C virus (HCV) infection is thought to be largely immunomediated. However, some frequent histopathological features, such as steatosis, suggest a direct cytopathic effect of HCV. The direct responsibility of HCV in the pathogenesis of steatosis is shown by: (1) the association with HCV genotype 3 infection, suggesting that some viral sequences are involved in the intracellular accumulation of lipids; (2) the correlation between severity of steatosis and HCV replication levels; (3) association between response to treatment and disappearance of steatosis. Experimental studies have shown that the nucleocapsid protein of HCV (core protein) is capable and sufficient to induce lipid accumulation in hepatocytes. Moreover, the observation that chronic hepatitis C patients have reduced serum levels of ApoB suggests an interference with the very-low density lipoprotein (VLDL) assembly, although other mechanisms are possible. In patients with sustained virological response induced by antiviral therapy, such levels are normalized. Other observations suggest that the pathogenesis of steatosis in chronic hepatitis C is not solely due to HCV. The origin of the mild steatosis observed in most patients may be metabolic, since its severity correlates with body mass index and insulin resistance. Most studies have shown a correlation between presence and/or severity of steatosis and fibrosis stage, but it is unclear whether this effect is direct or mediated by the associated insulin resistance, increased susceptibility to apoptosis, or by inflammatory cytokines. Finally, steatosis negatively influences the rate of response to antiviral treatment, as confirmed by large clinical trials. Management of steatosis in chronic hepatitis C requires knowledge of its pathogenesis and may involve both life-style changes and pharmacological interventions, although the latter remain largely experimental.

Antiviral Agents↗
PubMed2006

Interferon (IFN)-gamma-inducible protein-10: association with histological results, viral kinetics, and outcome during treatment with pegylated IFN-alpha 2a and ribavirin for chronic hepatitis C virus infection.

BACKGROUND: We investigated associations between interferon (IFN)-gamma-inducible protein (IP)-10 and liver histological results, viral kinetic response, and treatment outcome in patients infected with hepatitis C virus (HCV) genotypes 1-4. METHODS: Plasma IP-10 was monitored before, during, and after treatment with pegylated IFN- alpha 2a and ribavirin in 265 HCV-infected patients. RESULTS: In univariate analyses, a low baseline IP-10 level was significantly associated with low baseline viral load, rapid viral response (RVR), a sustained viral response (SVR), body mass index <25 kg/m2, and less-pronounced fibrosis, inflammation, and steatosis (for HCV genotypes other than 3). When the results of the univariate analyses were included in multivariate analyses, a low plasma IP-10 level, low baseline viral load, and genotype 2 or 3 infection were independent predictors of an RVR and SVR. IP-10 levels decreased 6 weeks into treatment and remained low in patients with an SVR. By contrast, plasma levels of IP-10 rebounded in patients who had detectable HCV RNA after the completion of treatment. Using cutoff IP-10 levels of 150 and 600 pg/mL for predicting an SVR in patients infected with HCV genotype 1 yielded a specificity and sensitivity of 81% and 95%, respectively. CONCLUSION: Baseline IP-10 levels are predictive of the response to HCV treatment.

Adult↗
PubMed2006

Optimized vaccination regimen linked to exhaustive screening approaches identifies 2 novel HLA-B7 restricted epitopes within hepatitis C virus NS3 protein.

Broad immune responses, in particular specific for the NS3 protein and mediated by both CD8+ and CD4+T lymphocytes, are thought to play a critical role in the control of hepatitis C virus (HCV) infection. In this study, we searched for novel HLA-B*0702 NS3 restricted epitopes following an optimized NS3NS4 immunization protocol in transgenic mice expressing HLA-B*0702 molecule. Combining predicted and overlapping peptides, we identified two novel epitopes, WPA10 (aa 1111-1120) and LSP10 (aa 1153-1162), which triggered significant IFN-gamma-producing T cell frequencies and high CTL responses. Both epitopes were shown to be immunogenic when used as synthetic peptides to immunize mice. The relevance of these epitopes to humans was demonstrated, as both were able in vitro to recall specific IFN-gamma and IL10-producing cells from peripheral blood mononuclear cells of HCV infected patients. Such epitopes enlarge the pool of NS3-specific CD8+T cell epitopes available to perform immunomonitoring of HCV infection and to develop vaccines.

Alleles↗
PubMed2006

Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras.

Chronic liver disease caused by infection with hepatitis C virus (HCV) is an important global health problem that currently affects 170 million people. A major impediment in HCV research and drug development has been the lack of culture systems supporting virus production. This obstacle was recently overcome by using JFH1-based full-length genomes that allow production of viruses infectious both in vitro and in vivo. Although this improvement was important, because of the restriction to the JFH1 isolate and a single chimera consisting of J6CF and JFH1-derived sequences, broadly based comparative studies between different HCV strains were not possible. Therefore, in this study we created a series of further chimeric genomes allowing production of infectious genotype (GT) 1a, 1b, 2a, and 3a particles. With the exception of the GT3a/JFH1 chimera, efficient virus production was obtained when the genome fragments were fused via a site located right after the first transmembrane domain of NS2. The most efficient construct is a GT2a/2a chimera consisting of J6CF- and JFH1-derived sequences connected via this junction. This hybrid, designated Jc1, yielded infectious titers 100- to 1,000-fold higher than the parental isolate and all other chimeras, suggesting that determinants within the structural proteins govern kinetic and efficiency of virus assembly and release. Finally, we describe an E1-specific antiserum capable of neutralizing infectivity of all HCV chimeras.

Antibodies, Monoclonal↗
PubMed2006

IP-10 predicts viral response and therapeutic outcome in difficult-to-treat patients with HCV genotype 1 infection.

Plasma from 173 patients with HCV genotype 1 infection was analyzed for IP-10 levels prior to treatment with pegylated interferon-alpha-2a and ribavirin. Significantly lower IP-10 levels were observed in patients achieving a rapid viral response (RVR) (P < .0001), even in those with body mass index (BMI) > or = 25 kg/m2 (P = .004) and with baseline viral load > or = 2 million IU/mL (P = .001). Similarly, significantly lower IP-10 levels were observed in patients obtaining a sustained viral response (SVR) (P = .0002), including those having higher BMI (P < .05), higher viral load (P = .0005), and both higher BMI and viral load (P < .03). In multivariate logistic regression analyses, a low IP-10 value was independently predictive of both RVR and SVR. A baseline cutoff IP-10 value of 600 pg/mL yielded a negative predictive value (NPV) of 79% (19/24) for all genotype 1-infected patients, which was comparable with that observed using a reduction in HCV-RNA by at least 2 logs after 12 weeks of therapy (NPV 86%; 19/22); by combining the two, 30 of 38 patients (NPV 79%) potentially could have been spared unnecessary therapy. In patients having both higher BMI and viral load, cut-off levels of 150 and 600 pg/mL yielded a positive predictive value (PPV) of 71% and NPV of 100%, respectively. In conclusion, pretreatment IP-10 levels predict RVR and SVR in patients infected with HCV genotype 1, even in those with higher BMI and viral load. A substantial proportion of the latter patients may achieve SVR in spite of unfavorable baseline characteristics if their pretreatment IP-10 level is low. Thus, pretreatment IP-10 analysis may prove helpful in decision-making regarding pharmaceutical intervention.

Adult↗
PubMed2006

Relationship between steatosis, inflammation, and fibrosis in chronic hepatitis C: a meta-analysis of individual patient data.

BACKGROUND & AIMS: Steatosis is a frequent histologic finding in chronic hepatitis C (CHC), but it is unclear whether steatosis is an independent predictor for liver fibrosis. We evaluated the association between steatosis and fibrosis and their common correlates in persons with CHC and in subgroup analyses according to hepatitis C virus (HCV) genotype and body mass index. METHODS: We conducted a meta-analysis on individual data from 3068 patients with histologically confirmed CHC recruited from 10 clinical centers in Italy, Switzerland, France, Australia, and the United States. RESULTS: Steatosis was present in 1561 patients (50.9%) and fibrosis in 2688 (87.6%). HCV genotype was 1 in 1694 cases (55.2%), 2 in 563 (18.4%), 3 in 669 (21.8%), and 4 in 142 (4.6%). By stepwise logistic regression, steatosis was associated independently with genotype 3, the presence of fibrosis, diabetes, hepatic inflammation, ongoing alcohol abuse, higher body mass index, and older age. Fibrosis was associated independently with inflammatory activity, steatosis, male sex, and older age, whereas HCV genotype 2 was associated with reduced fibrosis. In the subgroup analyses, the association between steatosis and fibrosis invariably was dependent on a simultaneous association between steatosis and hepatic inflammation. CONCLUSIONS: In this large and geographically different group of CHC patients, steatosis is confirmed as significantly and independently associated with fibrosis in CHC. Hepatic inflammation may mediate fibrogenesis in patients with liver steatosis. Control of metabolic factors (such as overweight, via lifestyle adjustments) appears important in the management of CHC.

Adult↗
PubMed2005

Real-time multiplex PCR assay to quantify hepatitis C virus RNA in peripheral blood mononuclear cells.

Ultrasensitive methods to measure very low levels of hepatitis C virus (HCV) RNA in biological samples may have diagnostic and prognostic significance and be useful to evaluate the response to antiviral treatment. A sensitive assay to quantify HCV RNA in peripheral blood mononuclear cells (PBMCs) was developed and validated using the iCycler iQ Detection System (Bio-Rad) coupled with TaqMan chemistry. HCV was co-amplified with the endogenous control glyceraldehyde-3-phosphate dehydrogenase in a multiplex reaction. Calculated PCR amplification efficiencies for both target and control genes were used in a mathematical model for relative quantitation of HCV RNA. A linear relationship between input RNA and C(T) values over 6 log dilutions was observed for both HCV- and GAPDH-specific products (R(2) > or = 0.99). As few as 1.5 IU/reaction could be detected, with high accuracy (CV < or= 3.94%) and reproducibility (CV < or = 2.20%). Quantitation of HCV RNA levels ranging from 10(3) to 10(7) IU/ml as measured in 47 plasma samples was highly correlated with values obtained by the COBAS Amplicor HCV Monitor test, v2.0 (Roche) (R(2) = 0.977). In conclusion, this assay provides an excellent tool to determine accurately HCV kinetics in PBMCs during antiviral therapy and to assess the long-term significance of different patterns of response to treatment.

5' Untranslated Regions↗
PubMed2005

[Dermatitis associated with the treatment of chronic hepatitis C].

The introduction of ribavirin to interferon-based treatment regimens for chronic hepatitis C has led to an increased incidence of cutaneous adverse events, which concern up to 25% of treated patients. Generalized pruritus, skin xerosis, and a papulo-microvesicular eruption with a predilection for the limb convexities and sites exposed to friction are typical clinical features. Standard treatment with topical corticosteroids and emollients is generally effective, and interruption of combination treatment can usually be avoided. Awareness for hepatitis C-combination-treatment-associated dermatitis as an entity with constant clinical features that is responsive to topical treatment will improve patient management and compliance. An etiologic link to ribavirin-mediated immunomodulation remains to be investigated.

Antiviral Agents↗
PubMed2005

Barriers to interferon-alpha therapy are higher in intravenous drug users than in other patients with acute hepatitis C.

BACKGROUND/AIMS: Treatment with interferon-alpha (IFN-alpha) may eradicate HCV in most acute hepatitis C patients, thus preventing chronic hepatitis and avoiding less efficacious combination therapy. METHODS: In a prospective study, we evaluated the impact of barriers to successful start and completion of treatment of acute and subacute (<12 months from infection) hepatitis C with pegylated IFN-alpha2b, 1.5 microg/kg, QW, for 24 weeks. RESULTS: Out of 27 patients (22 were active intravenous drug users [IVDU]), 5 cleared HCV spontaneously. Antiviral treatment was indicated in 22 patients: six refused therapy for fear of side effects, whereas two others were lost to observation. Eight patients completed the treatment or received >80% of the scheduled drug: seven (88%) were sustained virological responders 24 weeks after the end of treatment. Six patients (all IVDU) stopped prematurely due to side effects: only one had a sustained virological response. Based on an intent-to-treat analysis, and considering all 14 patients in whom at least one dose of drug was administered, only 8 (57%) became sustained virological responders. CONCLUSIONS: Treatment of acute hepatitis C with pegylated IFN-alpha is highly beneficial, but its effectiveness is affected by a poor rate of acceptance and/or adherence to currently available regimens, especially in IVDU and women.

Acute Disease↗
PubMed2005

An in vitro model of hepatitis C virus genotype 3a-associated triglycerides accumulation.

BACKGROUND/AIMS: The hepatitis C virus (HCV) induces lipid accumulation in vitro and in vivo. Although clinical observations are consistent with a direct effect of HCV genotype 3a on lipid metabolism, experimental systems have focused on the expression of HCV proteins of genotype 1. To extend these observations, we established an in vitro model expressing the HCV core of different genotypes. METHODS: The HCV core protein from patients with severe (genotype 3a) or no (genotypes 1b, 2a, 3h, 4h and 5a) liver steatosis was expressed in Huh7 cells. Core protein expression (by immunohistochemistry and immunoblot) and triglycerides accumulation (by Oil Red O stain and enzymatic measurement) were evaluated 48h after transfection. RESULTS: Although triglyceride accumulation occurred with genotypes 1b, 3a and 3h, the genotype 3a core protein expression resulted in the highest level of accumulation (i.e. about 3-fold with respect to 1b, and 2-fold with respect to 3h). This effect was not related to core protein expression levels and was abolished by culturing cells in lipid-free medium. CONCLUSIONS: Consistent with observations in chronic hepatitis C patients, the in vitro expression of HCV genotype 3a core protein is the ideal candidate model for studying the mechanisms of HCV-associated steatosis.

Adult↗
PubMed2005

International, multicenter, randomized, controlled study comparing dynamically individualized versus standard treatment in patients with chronic hepatitis C.

BACKGROUND/AIMS: The aim of this study was to increase virologic response rates by individualized treatment according to the early virologic response. METHODS: Serum HCV-RNA was frequently quantified in patients with chronic hepatitis C (n=270) treated with peginterferon alfa-2a (180 microg/week) and ribavirin (1000-1200 mg/day). After 6 weeks patients were classified as rapid (RVR), slow (SPR), flat (FPR), or null responders (NUR) and randomized within each viral kinetic class to continue therapy either with an individualized or standard regimen. Individualized therapy comprised peginterferon monotherapy (48 weeks) or shorter combination therapy (24 weeks) for RVR, triple therapy with histamine (1 mg/day) (48 weeks) or prolonged combination therapy (72 weeks) for SPR, triple therapy for FPR, and high-dose peginterferon (360 microg/week) plus ribavirin for NUR patients. RESULTS: Patients were categorized as RVR (n=171), SPR (n=65), FPR (n=10), or NUR (n=22). Overall end-of-treatment and sustained virologic response rates were 77 and 60% in the individualized and 77 and 66% in the standard treatment arm, respectively. Histamine in addition to peginterferon and ribavirin and high-dose peginterferon plus ribavirin did not improve virologic response rates in patients with FPR and NUR, respectively. CONCLUSIONS: An improvement in virologic efficacy was not achieved with the available individualized treatment options.

Adult↗
PubMed2004

Ribavirin/interferon-alpha sequential treatment of recurrent hepatitis C after liver transplantation.

Hepatitis C virus (HCV) infection invariably recurs after liver transplantation (LT), and sequels of chronic hepatitis of the graft are a significant cause of morbidity and mortality. In an uncontrolled trial, 31 patients with histologically confirmed hepatitis C after LT received, sequentially, ribavirin (10 mg/kg body weight q.d.) for 12 weeks, followed by ribavirin at the same dose q.d. plus interferon-alpha (IFN-alpha) [3 million units three times a week (3 MU TIW)] for another 48 weeks. Based on an intent-to-treat analysis, the percentages of patients with undetectable HCV RNA in their serum were 0%, 38.7% and 45.2% after 12, 36 and 60 weeks of therapy, respectively. A sustained virological response, as defined by undetectable serum HCV RNA 24 weeks after the end of treatment, was observed in 9/31 patients (29%). Sustained responders had a significant improvement of their liver inflammatory activity score (P=0.025), but not of their liver fibrosis score. The chances of sustained virological response correlated with the length of treatment, but not with the HCV genotype or baseline HCV RNA level. In conclusion, patients with recurrent hepatitis C after LT might benefit from ribavirin/IFN-alpha therapy, provided that the treatment is tolerated for a sufficient duration of time.

Adult↗
PubMed2004

Antigenic relevance of F protein in chronic hepatitis C virus infection.

The hepatitis C virus (HCV) F protein is a recently described, frameshift product of HCV core encoding sequence of genotype 1a. Its function and antigenic properties are unknown. Using enzyme-linked immunosorbent assay, we assessed the prevalence of anti-F antibodies in 154 patients chronically infected with HCV, 65 patients with other liver diseases, and 121 healthy controls. For this purpose, we expressed a highly purified HCV F recombinant protein from HCV genotype 1a in Escherichia coli. Because the F protein shares the 10 first amino acids with the core protein, the anti-HCV F response was also assessed by a F recombinant protein deleted of its 10 first amino acids [Delta(1-10)-F]. Ninety-six (62%) of the 154 HCV serum samples reacted with the complete F recombinant protein, whereas 39 (25%) showed a weaker anti-Delta(1-10)F reactivity and 150 (97%) had anti-core antibodies. No reactivity against F, Delta(1-10)F, or core was detected in any of the controls. To exclude a potential cross-reaction of anti-F antibodies with anti-core antibodies, a specific enzyme-linked immunosorbent assay was performed for anti-core antibodies. The specificity of anti-F antibodies was confirmed using an F synthetic peptide. The prevalence of anti-F antibodies did not correlate with HCV RNA serum level, genotype, or stage of liver disease. Sequence analysis from 8 anti-F-positive and 5 anti-F-negative serum samples did not reveal any particular difference potentially accounting for their respective anti-F responses. In conclusion, the F protein elicits specific antibodies in 62% of individuals chronically infected with HCV; such anti-F response does not seem to be affected by the F sequence heterogeneity.

Adult↗