Activation of extrinsic apoptosis pathway in HCV monoinfected and HIV–HCV coinfected patients,irrespective of liver disease severity

Chronic hepatitis C virus (HCV) infection is associated with increased levels of peripheral T cell apoptosis. We aimed to study whether T cell apoptosis markers indicate pathways that may contribute to clinical progression in HCV monoinfected and HIV–HCV coinfected patients. Activation of the extrinsic apoptosis pathways was measured by levels of death receptor Fas, initiator caspase 8 and effector caspases 3 and 7 activity and Annexin V binding on peripheral CD4 and CD8 T cells of HCV monoinfected and HIV/HCV coinfected patients, as well as healthy controls and HIV-infected, hepatitis B virus-infected and primary biliary cirrhosis disease controls. Association with liver fibrosis was assessed by biopsy or by transient elastography. HCV monoinfected and HIV–HCV coinfected patients displayed enhanced peripheral CD4 and CD8 T cell apoptosis. Caspase 8 activity was highest in HIV–HCV coinfection, without enhanced downstream activity of caspases 3 and 7. Level of peripheral T cell apoptosis was independent of liver fibrosis or other disease parameters in all disease groups. The extrinsic apoptosis pathway is upregulated in HCV monoinfection and HIV–HCV coinfection, but this is independent of liver disease severity.     

Multiple Introduction and Naturally Occuring Drug Resistance of HCV among HIV-Infected Intravenous Drug Users in Yunnan: An Origin of China’s HIV/HCV Epidemics

Background

The human immunodeficiency virus 1 (HIV-1) epidemic in China historically stemmed from intravenous drug users (IDUs) in Yunnan. Due to a shared transmission route, hepatitis C virus (HCV)/HIV-1 co-infection is common. Here, we investigated HCV genetic characteristics and baseline drug resistance among HIV-infected IDUs in Yunnan.

Methods

Blood samples of 432 HIV-1/HCV co-infected IDUs were collected from January to June 2014 in six prefectures of Yunnan Province. Partial E1E2 and NS5B genes were sequenced. Phylogenetic, evolutionary and genotypic drug resistance analyses were performed.

Results

Among the 293 specimens successfully genotyped, seven subtypes were identified, including subtypes 3b (37.9%, 111/293), 3a (21.8%, 64/293), 6n (14.0%, 41/293), 1b (10.6%, 31/293), 1a (8.2%, 24/293), 6a (5.1%, 15/293) and 6u (2.4%, 7/293). The distribution of HCV subtypes was mostly related to geographic location. Subtypes 3b, 3a, and 6n were detected in all six prefectures, however, the other four subtypes were detected only in parts of the six prefectures. Phylogeographic analyses indicated that 6n, 1a and 6u originated in the western prefecture (Dehong) and spread eastward and showed genetic relatedness with those detected in Burmese. However, 6a originated in the southeast prefectures (Honghe and Wenshan) bordering Vietnam and was transmitted westward. These subtypes exhibited different evolutionary rates (between 4.35×10⁻⁴ and 2.38×10⁻³ substitutions site^-1 year^-1) and times of most recent common ancestor (tMRCA, between 1790.3 and 1994.6), suggesting that HCV was multiply introduced into Yunnan. Naturally occurring resistance-associated mutations (C316N, A421V, C445F, I482L, V494A, and V499A) to NS5B polymerase inhibitors were detected in direct-acting antivirals (DAAs)-naïve IDUs.

Conclusion

This work reveals the temporal-spatial distribution of HCV subtypes and baseline HCV drug resistance among HIV-infected IDUs in Yunnan. The findings enhance our understanding of the characteristics and evolution of HCV in IDUs and are valuable for developing HCV prevention and management strategies for this population.     

Immunopathologic differences of Sjögren's syndrome versus sicca syndrome in HCV and HIV infection

A clinical picture of dry eye and dry mouth with the histological counterpart of focal lymphocytic sialoadenitis, usually detected in minor salivary glands, is considered the hallmark of Sjögren's syndrome. The association of sicca complaints and focal sialoadenitis can be also found in a number of other diseases, including some systemic viral infections. Among these conditions, chronic hepatitis C virus infection, associated with mixed cryoglobulinaemia and extra-hepatic manifestations, and HIV infection, particularly in the phase of diffuse interstitial lymphocytic infiltration, may mimic the clinical and histological aspects of Sjögren's syndrome. However, each disorder is characterised by specific, disease-related immunopathological aspects. Besides sicca complaints, the various disorders may also share a number of systemic extra-glandular features and the possible development of mucosa-associated lymphoid tissue lymphomas. This latter event represents in all of these diseases the final result of an antigen-driven chronic stimulation of B lymphocytes.     

Is a 3-month course of treatment with Peg-interferon effective in acute HCV hepatitis?

From February 2002, all the consecutive patients referring to the Department of Infectious Diseases, University of Turin, who were diagnosed as having acute HCV hepatitis were included in a prospective cohort study to evaluate if a 3-month course of Peg-Interferon alpha-2b (1.5 microg/kg once weekly) is effective to decrease the risk of progression to chronic disease. ALT and HCV-RNA measurements were scheduled at week 4 and 12 during treatment, and 24 weeks after the end of therapy. As of April 2003, ten patients were enrolled in the study. As to HCV genotype, seven patients had type 1 and 3 type non-1. At entry, median HCV-RNA level was 129500 (range: 3000-3100000 copies/mL) and six patients were symptomatic. Treatment was given within 20 days (range: 8-30) of the ALT peak. All patients completing 4 weeks (n = 9) and 12 weeks of treatment (n = 7) had undetectable HCV-RNA. Five patients who completed the 24-week follow-up after the end of treatment had a sustained viral response with ALT levels within normal range. Therapy was well tolerated in all patients. Even if our data are not definitive, our results show that once-weekly administration of Peg-interferon alfa-2b in patients with acute HCV infection may be an effective and convenient regimen.     

Synthesis and anti-BVDV activity of novel δ-sultones in vitro: Implications for HCV therapies

In this study we report the synthesis and activity against bovine viral diarrhea virus (BVDV) of a novel series of bicycle δ-sultones containing γ-lactones. BVDV is responsible for major losses in cattle. Some of the synthesized δ-sultones showed pronounced anti-BVDV activity with EC₅₀ values of 0.12–1.0 μM and no significant cytotoxicity. Among them, the ortho bromosubstituted derivative 4f (EC₅₀ = 0.12 μM) showed better antiviral activity than other derivatives and was 10 fold more that of than positive control ribavirin (EC₅₀ = 1.3 μM). BVDV is also considered to be a valuable surrogate for the hepatitis C virus (HCV) in antiviral drug studies. The above results provided a novel candidate for the development of anti-HCV agents.     

Proteomic analysis of hepatitis C virus (HCV) core protein transfection and host regulator PA28γ knockout in HCV pathogenesis: a network-based study

Hepatitis C virus (HCV) causes chronic liver disease worldwide. HCV Core protein (Core) forms the viral capsid and is crucial for HCV pathogenesis and HCV-induced hepatocellular carcinoma, through its interaction with the host factor proteasome activator PA28γ. Here, using BD-PowerBlot high-throughput Western array, we attempt to further investigate HCV pathogenesis by comparing the protein levels in liver samples from Core-transgenic mice with or without the knockout of PA28γ expression (abbreviated PA28γ(-/-)CoreTG and CoreTG, respectively) against the wild-type (WT). The differentially expressed proteins integrated into the human interactome were shown to participate in compact and well-connected cellular networks. Functional analysis of the interaction networks using a newly developed data warehouse system highlighted cellular pathways associated with vesicular transport, immune system, cellular adhesion, and cell growth and death among others that were prominently influenced by Core and PA28γ in HCV infection. Follow-up assays with in vitro HCV cell culture systems validated VTI1A, a vesicular transport associated factor, which was upregulated in CoreTG but not in PA28γ(-/-)CoreTG, as a novel regulator of HCV release but not replication. Our analysis provided novel insights into the Core-PA28γ interplay in HCV pathogenesis and identified potential targets for better anti-HCV therapy and potentially novel biomarkers of HCV infection.     

Naturally Occurring Mutations in the Nonstructural Region 5B of Hepatitis C Virus (HCV) from Treatment-Na?ve Korean Patients Chronically Infected with HCV Genotype 1b

The nonstructural 5B (NS5B) protein of the hepatitis C virus (HCV) with RNA-dependent RNA polymerase (RdRp) activity plays a pivotal role in viral replication. Therefore, monitoring of its naturally occurring mutations is very important for the development of antiviral therapies and vaccines. In the present study, mutations in the partial NS5B gene (492 bp) from 166 quasispecies of 15 genotype-1b (GT) treatment-naïve Korean chronic patients were determined and mutation patterns and frequencies mainly focusing on the T cell epitope regions were evaluated. The mutation frequency within the CD8+ T cell epitopes was significantly higher than those outside the CD8+ T cell epitopes. Of note, the mutation frequency within predicted CD4+ T cell epitopes, a particular mutational hotspot in Korean patients was significantly higher than it was in patients from other areas, suggesting distinctive CD4+ T cell-mediated immune pressure against HCV infection in the Korean population. The mutation frequency in the NS5B region was positively correlated with patients with carrier-stage rather than progressive liver disease (chronic hepatitis, liver cirrhosis and hepatocellular carcinoma). Furthermore, the mutation frequency in four codons (Q309, A333, V338 and Q355) known to be related to the sustained virological response (SVR) and end-of treatment response (ETR) was also significantly higher in Korean patients than in patients from other areas. In conclusion, a high degree of mutation frequency in the HCV GT-1b NS5B region, particularly in the predicted CD4+ T cell epitopes, was found in Korean patients, suggesting the presence of distinctive CD4+ T cell pressure in the Korean population. This provides a likely explanation of why relatively high levels of SVR after a combined therapy of pegylated interferon (PEG-IFN) and ribavirin (RBV) in Korean chronic patients with GT-1b infections are observed.     

IFN-Alpha Receptor-1 Upregulation in PBMC from HCV Na?ve Patients Carrying CC Genotype. Possible Role of IFN-Lambda

Background and Aims

IL-28B gene polymorphisms predict better therapeutic response and spontaneous clearance of HCV. Moreover, higher expression of IFN-lambda has been reported in patients with the rs12979860 CC favourable genotype. The study aim was to establish possible relationships between IL-28B rs12979860 genotypes and expression of IFN-alpha receptor-1 (IFNAR-1) in naïve HCV patients, and to explore the possible role of IFN-lambda.

Methods

IFNAR-1 mRNA levels were measured in PBMC from naïve patients with chronic hepatitis C with different IL-28 genotypes. The ability of IFN-lambda to up-regulate the expression of IFNAR-1 was established in PBMC from healthy donors carrying different IL-28B genotypes.

Results

Lower IFNAR-1 mRNA levels were observed in PBMC from HCV-infected naïve patients as compared to healthy donors. In healthy donors, IFNAR-1 mRNA levels were independent from IL-28B genotype, while in HCV patients, an increasing gradient was observed in TT vs CT vs CC carriers. In the latter group, a direct correlation between IFNAR-1 and endogenous IL-28B expression was observed. Moreover, IFN-lambda up-regulated IFNAR-1 expression in normal PBMC in a time-and dose-dependent manner, with a more effective response in CC vs TT carriers.

Conclusion

Endogenous levels of IFN-lambda may be responsible for partial restoration of IFNAR-1 expression in HCV patients with favourable IL-28 genotype. This, in turn, may confer to CC carriers a response advantage to either endogenous or exogenous IFN-alpha, representing the biological basis for the observed association between CC genotype and favourable outcome of either natural infection (clearance vs chronicization) or IFN therapy.     

献血员血清抗—HCV和HCV RNA检测

本文对宜昌市的２９９９名献血员,５９４名自然人群的血清进行了抗－ＨＣＶ、ＨＣＶＲＮＡ检测,结果表明,献血员中抗－ＨＣＶ阳性２３例,阳性率为０．７７％。其中宜昌、巴东分别为０．７３％和０．７６％,沙市为０．８３％。地区、性别、城乡之间抗－ＨＣＶ阳性率无显著性差异（ｐ＞０．０５）。参照以往文献报导,此阳性率比国内报导的低,与国际的相一致。５９４名自然人群抗－ＨＣＶ结果均为阴性,献血员与自然人群之间抗－ＨＣＶ阳性率差异显著（ｐ＜０．０５）。２０例抗－ＨＣＶ阳性者中检出ＨＣＶ　ＲＮＡ阳性６例,占３０％（６／２０）,占受检献血员的０．２０％（６／２９９９）,比武汉（０．８０％）低４倍,地区之间无显著差异（ｐ＞０．０５）。由此初步确认：宜昌市献血员中丙型病毒性肝炎感染低于国内某些大中城市、宜昌市（含七县二市）巴东县、沙市市是丙型病毒性肝炎低感染区。但存在无症状病毒血症携带者传染源。     

Is interleukin-10 gene polymorphism a predictive marker in HCV infection?

The clinical outcome of hepatitis C virus (HCV) infection varies between individuals - from spontaneous viral clearance and persistence without complication, to chronic hepatitis, cirrhosis and hepatocellular carcinoma. Also patterns of response to interferon-based anti-HCV therapy are different from person to person. This diversity may be affected by host genetic factors, including alterations in genes encoding cytokines. Interleukin-10, as an anti-inflammatory cytokine and immune response modulator, may influence on HCV infection susceptibility as well as spontaneous and treatment-induced HCV eradication. Moreover, it is stated that IL-10 has antifibrotic properties and play a role in progression of liver disease. This review summarized studies on interleukin-10 gene polymorphisms (mainly promoter SNPs at positions -1082(G/A), -819(C/T) and -592(C/A)), which may determine IL-10 production, regarding susceptibility to HCV infection, course of HCV-related liver disease (fibrosis, cirrhosis, hepatocellular carcinoma, ALT abnormalities), spontaneous viral elimination as well as hepatitis C treatment outcomes. Analysis of hereby summarized studies shows that it is difficult to unambiguously determine the importance of IL-10 polymorphism as a predictor of clinical outcome of hepatitis C and response to anti-HCV therapy before its beginning. Thus, future larger studies need to address these issues. Continuation of studies on interleukin-10 polymorphisms as well as identification of other candidate predictive markers in HCV infection has important practical implications and there is a chance that may contribute to reduce the scale of hepatitis C problem.     

Cost-Effectiveness of HBV and HCV Screening Strategies – A Systematic Review of Existing Modelling Techniques

Introduction

Studies evaluating the cost-effectiveness of screening for Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) are generally heterogeneous in terms of risk groups, settings, screening intervention, outcomes and the economic modelling framework. It is therefore difficult to compare cost-effectiveness results between studies. This systematic review aims to summarise and critically assess existing economic models for HBV and HCV in order to identify the main methodological differences in modelling approaches.

Methods

A structured search strategy was developed and a systematic review carried out. A critical assessment of the decision-analytic models was carried out according to the guidelines and framework developed for assessment of decision-analytic models in Health Technology Assessment of health care interventions.

Results

The overall approach to analysing the cost-effectiveness of screening strategies was found to be broadly consistent for HBV and HCV. However, modelling parameters and related structure differed between models, producing different results. More recent publications performed better against a performance matrix, evaluating model components and methodology.

Conclusion

When assessing screening strategies for HBV and HCV infection, the focus should be on more recent studies, which applied the latest treatment regimes, test methods and had better and more complete data on which to base their models. In addition to parameter selection and associated assumptions, careful consideration of dynamic versus static modelling is recommended. Future research may want to focus on these methodological issues. In addition, the ability to evaluate screening strategies for multiple infectious diseases, (HCV and HIV at the same time) might prove important for decision makers.     

Oncostatin M synergistically inhibits HCV RNA replication in combination with interferon-α

Oncostatin M (OSM), a member of the interleukin-6 family, possesses various functions, including hepatocyte differentiation and suppression of melanoma cell growth. Here, we report anti-hepatitis C virus (HCV) activity of OSM as a new function of this cytokine. OSM possessed marked anti-HCV activity (50% effective concentration: 0.71 ng/ml) in an HCV RNA replication cell culture system. The most striking finding is that OSM exhibited synergistic inhibitory activity on interferon (IFN)-α even at a low concentration with weak anti-HCV activity, such as 25 pg/ml. OSM is a candidate anti-HCV reagent and may improve the current IFN therapy for patients with chronic hepatitis C.     

Biotransformation of Digitoxin in Cell Cultures of Capsicum frutescens in the Presence of β-cyclodextrin

Cell suspension cultures of Capsicum frutescens accumulated digoxin, purpureaglycoside A and other unknown derivatives when digitoxin, a cardiac glycoside, was used as a precursor. The feeding of digitoxin complexed with &#103 -cyclodextrin increased the accumulation of digoxin, purpureaglycoside A and other unknown derivatives. Control cultures (without digitoxin) did not produce any of these metabolites. The growth of cells was affected by both digitoxin as well as digitoxin- &#103 -cyclodextrin. The accumulation of purpureaglycoside A and digoxin reached a maximum of 1241 and 374 &#119 g 100 ml &#109 1 culture on the 6th and 2nd day, respectively, which was 3.9 and 4.5 fold higher than cultures treated with digitoxin alone (sampled on the 13th day). The other unknown derivatives formed in digitoxin- &#103 -cyclodextrin fed cultures were 15 times higher than digitoxin alone fed C. frutescens cultures. The addition of glucose to digitoxin- &#103 -cyclodextrin treated cultures increased the accumulation of purpureaglycoside A which reached a maximum of 3589 &#119 g 100 ml &#109 1 culture after 12 h incubation, which was a 2.9 fold increase over cultures treated with digitoxin- &#103 -cyclodextrin alone.     

HIV and Hepatitis C Mortality in Massachusetts, 2002–2011: Spatial Cluster and Trend Analysis of HIV and HCV Using Multiple Cause of Death

Background

Infectious diseases, while associated with a much smaller proportion of deaths than they were 50 years ago, still play a significant role in mortality across the state of Massachusetts. Most analysis of infectious disease mortality in the state only take into account the underlying cause of death, rather than contributing causes of death, which may not capture the full extent of mortality trends for infectious diseases such as HIV and the Hepatitis C virus (HCV).

Methods

In this study we sought to evaluate current trends in infectious disease mortality across the state using a multiple cause of death methodology. We performed a mortality trend analysis, identified spatial clusters of disease using a 5-step geoprocessing approach and examined spatial-temporal clustering trends in infectious disease mortality in Massachusetts from 2002–2011, with a focus on HIV/AIDS and HCV.

Results

Significant clusters of high infectious disease mortality in space and time throughout the state were detected through both spatial and space time cluster analysis. The most significant clusters occurred in Springfield, Worcester, South Boston, the Merrimack Valley, and New Bedford with other smaller clusters detected across the state. Multiple cause of death mortality rates were much higher than underlying cause mortality alone, and significant disparities existed across race and age groups.

Conclusions

We found that our multi-method analyses, which focused on contributing causes of death, were more robust than analyses that focused on underlying cause of death alone. Our results may be used to inform public health resource allocation for infectious disease prevention and treatment programs, provide novel insight into the current state of infectious disease mortality throughout the state, and benefited from approaches that may more accurately document mortality trends.     

Absence of XMRV in Peripheral Blood Mononuclear Cells of ARV-Treatment Na?ve HIV-1 Infected and HIV-1/HCV Coinfected Individuals and Blood Donors

Background

Xenotropic murine leukemia virus-related virus (XMRV) has been found in the prostatic tissue of prostate cancer patients and in the blood of chronic fatigue syndrome patients. However, numerous studies have found little to no trace of XMRV in different human cohorts. Based on evidence suggesting common transmission routes between XMRV and HIV-1, HIV-1 infected individuals may represent a high-risk group for XMRV infection and spread.

Methodology/Principal Findings

DNA was isolated from the peripheral blood mononuclear cells (PBMCs) of 179 HIV-1 infected treatment naïve patients, 86 of which were coinfected with HCV, and 54 healthy blood donors. DNA was screened for XMRV provirus with two sensitive, published PCR assays targeting XMRV gag and env and one sensitive, published nested PCR assay targeting env. Detection of XMRV was confirmed by DNA sequencing. One of the 179 HIV-1 infected patients tested positive for gag by non-nested PCR whereas the two other assays did not detect XMRV in any specimen. All healthy blood donors were negative for XMRV proviral sequences. Sera from 23 HIV-1 infected patients (15 HCV⁺) and 12 healthy donors were screened for the presence of XMRV-reactive antibodies by Western blot. Thirteen sera (57%) from HIV-1⁺ patients and 6 sera (50%) from healthy donors showed reactivity to XMRV-infected cell lysate.

Conclusions/Significance

The virtual absence of XMRV in PBMCs suggests that XMRV is not associated with HIV-1 infected or HIV-1/HCV coinfected patients, or blood donors. Although we noted isolated incidents of serum reactivity to XMRV, we are unable to verify the antibodies as XMRV specific.     

Evaluation of 2′-α-fluorine modified nucleoside phosphonates as potential inhibitors of HCV polymerase

Ribonucleoside phosphonate analogues containing 2′-α-fluoro modifications were synthesized and their potency evaluated against HCV RNA polymerase. The diphosphophosphonate (triphosphate equivalent) adenine and cytidine analogues displayed potent inhibition of the HCV polymerase in the range of 1.9–2.1 μM, but only modest cell-based activity in the HCV replicon. Pro-drugs of the parent nucleoside phosphonates improved the cell-based activity.     

Green fluorescent protein – Tagged HCV non-enveloped capsid like particles: Development of a new tool for tracking HCV core uptake

Circulating ‘free’ non-enveloped Hepatitis C virus (HCV) core protein has been demonstrated in HCV-infected patients, and HCV subgenomes with deletions of the envelope proteins have been previously identified. Initial studies from our laboratory, previously published, indicated that expression of HCV core in insect cells can direct the formation of capsid-like particles lacking the envelope glycoproteins. These protein nanospheres, morphologically similar to natural capsids, were shown to be taken up by human hepatic cells and to produce cell-signalling events. To follow the intracellular fate of these particles we fused the core protein to eGFP. We demonstrate that the chimeric proteins core₁₇₃-eGFP, eGFP-core₁₉₁ and eGFP-core₁₇₃ can be efficiently expressed, self-assembled, and form fluorescent non-enveloped capsid-like particles. By using confocal microscopy and FACS analysis, we provide evidence that the fluorescent nanospheres can not only enter human hepatic cells – the main target of HCV – but also human immune cells such as T and B lymphocytes, as well as human myeloid leukaemia cells differentiated along the monocyte/macrophage-like pathway. The fluorescent particles might thus be used to trace the intracellular trafficking of naked HCV capsids as showed by live microscopy and to further understand their biological significance.