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The hepatitis C virus (HCV) core gene is more conserved at the nucleic acid level than is necessary to preserve the sequence of the core protein, suggesting that it contains information for additional functions. We used a battery of anticore antibodies to test the hypothesis that the core gene directs the synthesis of core protein isoforms. Infectious viruses, replicons, and RNA transcripts expressed a p8 minicore containing the C-terminal portion of the p21 core protein and lacking the N-terminal portion. An interferon resistance mutation, U271A, which creates an AUG at codon 91, upregulated p8 expression in Con1 replicons, suggesting that p8 is produced by an internal initiation event and that 91-AUG is the preferred, but not the required, initiation codon. Synthesis of p8 was independent of p21, as shown by the abundant production of p8 from transcripts containing an UAG stop codon that blocked p21 production. Three infectious viruses, JFH-1 (2a core ), J6/JFH (2a core ), and H77/JFH (1a core ), and a bicistronic construct, Bi-H77/JFH, all expressed both p8 and larger isoforms. The family of minicores ranges in size from 8 to 14 kDa. All lack the N-terminal portion of the p21 core. In conclusion, the core gene contains an internal signal that stimulates the initiation of protein synthesis at or near codon 91, leading to the production of p8. Infectious viruses of both genotype 1 and 2 HCV express a family of larger isoforms, in addition to p8. Minicores lack significant portions of the RNA binding domain of p21 core. Studies are under way to determine their functions.

Chemokine interactions with their receptors have been implicated in hepatic stellate cell (HSC) activation. The hepatic expression of CXCR4 messenger RNA is increased in hepatitis C cirrhotic livers and plasma levels of its endogenous ligand, stromal cell-derived factor-1alpha (SDF-1alpha), correlate with increased fibrosis in these patients. The expression of CXCR4 by HSCs has not been reported. We therefore examined whether HSCs express CXCR4 in vivo and in vitro and explored whether SDF-1alpha/CXCR4 receptor engagement promotes HSC activation, fibrogenesis, and proliferation. The hepatic protein expression of both CXCR4 and SDF-1alpha is increased in hepatitis C cirrhotic livers and immunoflourescent and immunohistochemical staining confirms that HSCs express CXCR4 in vivo. Immortalized human stellate cells as well as primary human HSCs express CXCR4, and cell surface receptor expression increases with progressive culture-induced activation. Treatment of stellate cells with recombinant SDF-1alpha increases expression of alpha-smooth muscle actin and collagen I and stimulates a dose-dependent increase in HSC proliferation. Inhibitor studies suggest that SDF-1alpha/CXCR4-dependent extracellular signal-regulated kinase 1/2 and Akt phosphorylation mediate effects on collagen I expression and stellate cell proliferation.HSCs express CXCR4 receptor in vivo and in vitro. CXCR4 receptor activation by SDF-1alpha is profibrogenic through its effects on HSC activation, fibrogenesis, and proliferation. Extracellular signal-regulated kinase 1/2 and phosphoinositide 3-kinase pathways mediate SDF-1alpha-induced effects on HSC expression of collagen I and proliferation. The availability of small molecule inhibitors of CXCR4 make this receptor an appealing target for antifibrotic approaches.

Gene expression profiling allows the level of activity of thousands of genes to be monitored simultaneously. Profiling is often carried out on specialized chips or slides, which have microarrays of gene targets at predetermined addresses. In the immediate future, microarrays promise to yield new insights into hepatitis C virus (HCV) pathogenesis and to produce 'signatures' that can be used in molecular diagnostics. In the longer-term, they may aid the development of serological tests by identifying genes encoding secretory proteins produced by HCV-infected livers, and they may suggest new avenues for disease intervention by detecting genes whose products are retained in the infected liver.

Lidocaine effects were studied at the single channel level on batrachotoxin-activated eel electroplax (muscle-derived) and on rat brain sodium channels in planar lipid bilayers to investigate whether these effects were the same on structurally different sodium channels. Lidocaine blocked the open state of brain channels with the same voltage dependence, but with 15-times as high a potency as muscle-derived channels. In brain channels, but not muscle-derived ones, the level of the open channel block showed periods of relief. Lidocaine at microM concentrations stabilized the highest conductance state in both channel types and at mM concentrations stabilized subconductance-like states in electroplax, but not in brain channels. In both channel types, lidocaine increased the lifetime and rate of entry to a long-nonconducting state. Since both channel types were studied under identical lipid and ionic conditions, the observed functional differences in the lidocaine action (effects, potency) must reflect channel structural differences.

Many anti-HCV antibodies are available, but more are needed for research and clinical applications. This study examines whether ascitic fluid from cirrhotic patients could be a source of reagent-grade antibodies. Ascitic fluid from 29 HCV patients was screened by ELISA for anti-HCV antibodies against three viral proteins: core, NS4B, and NS5A. Significant patient-to-patient variability in anti-HCV antibody titers was observed. Total ascitic fluid IgG purified by Protein-A chromatography reacted with HCV proteins in immunoblots, cell extracts, and replicon-expressing cells. Affinity-purification using synthetic peptides as bait allowed the preparation of cross-genotypic antibodies directed against pre-selected regions of HCV core, NS4B, and NS5A proteins. The performance of the polyclonal antibodies was comparable to that of monoclonal antibodies. Anti-NS4B antibody preparations reacted with genotype 1a, 1b, and 2a NS4B proteins in immunoblots and allowed NS4B to be localized in replicon-expressing cells. Summary: Ascitic fluid is an abundant source of human polyclonal cross-genotypic antibodies that can be used as an alternative to blood. This study shows the utility of selectively-purifying human polyclonal antibodies from ascitic fluid. Affinity purification allows antibodies to be selected that are comparable to monoclonal antibodies in their ability to react with targeted regions of viral proteins.

Hepatitis C virus (HCV) causes chronic infection in ~3% of the world's population. A search for HCV in brain was prompted by complaints of fatigue and cognitive dysfunction made by patients with HCV infection [1-7]. Specific cognitive deficits and nuclear magnetic resonance abnormalities have been reported in HCV-infected patients [1, 4, 8-10]. These observations, along with increasing evidence of HCV infection and replication in peripheral blood mononuclear cells (PBMCs) [11-16], suggest that the brain may also be a compartment for extrahepatic HCV replication. To date,HCVRNAhas been amplified and sequenced from brain tissue of a limited number of subjects [17-21]. Detection of antigenomic HCV RNA has been reported in various brain regions [19]. The population of brain HCV contains sequence variants that are absent in serum. Both the presence of antigenomic HCV RNA and the presence of brain-specific variants suggest that HCV infection and replication may occur in the brain. Despite the importance of sequence analysis for investigations of brain HCV, no study has examined the suitability of postmortem (PM) material for sequence analysis, although, of necessity, such studies are almost universally performed on PM material. We developed a bioinformatic method to assess the quality of RNA templates from PM tissue. This method demonstrated that brain HCV RNA is a suitable template for sequence analysis. Sequence analysis was then conducted on portions of the 5' untranslated region (UTR) and the E1 (envelope 1) gene. Results of both direct sequencing and quasispecies analysis support the hypothesis that HCV replicates and evolves within the brain.

Hepatitis C virus (HCV) has been detected in the brain tissues of 10 individuals reported to date; it is unclear what clinical factors are associated with this, and with what frequency it occurs. Accordingly, a pilot analysis utilizing reverse transcriptase-polymerase chain reaction (RT- PCR) to detect and sequence HCV in premortem plasma and postmortem brain and liver from 20 human immunodeficiency virus (HIV)-infected and 10 HIV-naive individuals was undertaken. RNA encoding the first 126 amino acids of the HCV E1 envelope protein and the majority of the E1 signal sequence was analyzed in parallel with an 80-base-long segment of the 5' untranslated region (UTR). Liver HCV was detected only in subjects with premortem HCV viremia (10 HIV-infected and 3 HIV-naive). Brain HCV was detected in 6/10 HCV/HIV-coinfected and 1/3 HCV-monoinfected subjects. In the setting of HIV, the magnitude of plasma HCV load did not correlate with the presence of brain HCV. However, coinfected patients with brain HCV were more often off antiretroviral therapy and tended to have higher plasma HIV loads than those with HCV restricted to liver. Furthermore, premortem cerebrospinal fluid (CSF) analysis revealed that HCV/HIV-coinfected patients with brain HCV had detectable CSF HIV, whereas those without brain HCV had undetectable CSF HIV loads (P = .0205). Neuropsychologic tests showed a trend for hierarchical impairment of abstraction/executive functioning in HIV/HCV coinfection, with mean T scores for HIV monoinfected patients 43.2 (7.3), for liver-only HCV 39.5 (9.0), and for those with HCV in brain and liver 33.2 (5.1) (P = .0927). Predominant brain HCV sequences did not match those of the plasma or liver in 4 of the 6 coinfected patients analyzed. We conclude that in the setting of HIV/HCV coinfection, brain HCV is a common phenomenon unrelated to the magnitude of HCV viremia, but related to active HIV disease and detectable CSF HIV. Furthermore, there is sequence evidence of brain compartmentalization. Differences in abstraction/executive function of HCV/HIV coinfected patients compared to HIV monoinfected warrant further studies to determine if neuropsychiatric effects are predicated upon brain infection.

This study tested the hypothesis that hepatitis C virus (HCV) RNA and core antigen levels rise more rapidly after liver transplantation (LT) in recipients of grafts from living donors (LD) versus deceased donors (DD). Eleven consecutive LD and 15 DD recipients were followed prospectively. Before LT, median HCV RNA levels were similar: 5.42 (LDLT) and 5.07 (DDLT) log10 IU/mL (P = NS). During the first 7 hours after LT a trend toward a greater HCV RNA decrease in LDLT patients was seen, although they received fewer blood replacement products during surgery. HCV RNA levels rose more rapidly in LDLT patients between days 1 and 3 (P = .0059) and were higher in this group on days 2, 3, 4, and 5. Core antigen levels were significantly higher in LDLT patients on days 3 and 5, although they were similar before LT (P = NS). Alanine aminotransferase (ALT) values were higher among LDLT patients from 8 to 14 days and from 4 to 24 months. Two-year graft and patient survival were 73% for LDLT patients and 80% for DDLT patients (P = NS). In conclusion, viral load rose more rapidly in LD recipients and reached higher levels shortly after surgery. Greater ALT elevations were evident in the LDLT group, but survival rates were similar. The trend toward a greater initial viral load decrease in patients with LD grafts and the significantly sharper increase suggest that the liver plays a predominant role in both HCV clearance and replication. (HEPATOLOGY 2005;42:1420–1428.)

The hepatitis C virus (HCV) has an alternate reading frame (ARF) that overlaps the core protein gene. The overlapping reading frame distinguishes HCV from all of its known viral relatives, with the possible exception of GB virus B (GBV-B). The ARF is expressed during natural HCV infections and stimulates specific immune responses. Like several essential genes in other viruses (e.g., the human immunodeficiency virus polymerase) the ARF lacks an in-frame AUG start codon, suggesting that its expression involves unusual translation-level events. In vitro studies indicate that ribosomal frameshifting may be one of several processes that can lead to translation of the ARF. Frameshifting yields chimeric proteins that have segments encoded in the core gene covalently attached to amino acids encoded in the ARF. A consistent nomenclature for the ARF's protein products has yet to be established. We propose that all proteins that contain amino acids encoded in the + 1 ARF be called alternate reading frame proteins (ARFPs) and that specific ARFPs, such as the ARFP/F-protein, the double-frameshift protein, and the short form of core + 1, be designated as follows: ARFP/F (ARFP/F-protein), ARFP/DF (double-frameshift), and ARFP/S (short form of core+1). The roles of ARFPs in the HCV life cycle are not yet known. There is a significant possibility that ARFPs may be responsible for some of the effects attributed to the core protein, given that most studies seeking to define the function of the core protein have employed materials likely to contain a combination of the core protein and ARFPs. The observed effects of the core protein include the induction of liver cancer, transformation of cells, and alterations of immune responses. This article reviews the discovery of ARF, describes the RNA structural elements involved in core/ARF gene expression, discusses possible functions of ARFPs, and considers the potential usefulness of ARFPs in vaccines. The HCV ARF is the focus of a new and rapidly expanding area of research, and the results of many ongoing studies are currently available in abstract form only. The preliminary nature of investigations that have not yet been reviewed by peers is noted in the text. Objectives: Upon completion of this article, the reader will be familiar with the discovery of ARF, the RNA structural elements involved in core/ARF gene expression, the possible functions of ARFPs, and the potential usefulness of ARFPs in vaccines. Accreditation: Tufts University School of Medicine (TUSM) is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. Credit: TUSM designates this educational activity fora maximum of 1 Category 1 credit toward the AMA Physicians Recognition Award. Each physician should claim only those credits that he/she actually spent in the educational activity.

Hepatitis C virus (HCV) and HCV core protein are hypothesized to induce hepatic oxidative stress and exacerbate injury caused by other toxins such as ethanol that induce the cytochrome P450 enzyme, CYP2E1. In the current study, the effects of HCV core protein [sequence genotype 1b, (nt 342-915)] on parameters indicative of oxidative stress were evaluated in HepG2 cells stably over expressing CYP2E1 (E47), or vector controls (C34). Stable (>10 passages) expression of HCV core protein and CYP2E1 was confirmed in clonal cell lines at the level of mRNA and immunoreactive protein. Prooxidant production, as determined by cellular oxidation of dichlorodihydrofluorescin and dihydroethidium (HE), was increased by expression of HCV core protein in the presence or absence of CYP2E1. Depletion of glutathione (GSH) with buthionine sulfoximine (BSO) enhanced prooxidant production in both C34 and E47 cells. In addition, prooxidant production was greater in BSO-treated cells expressing HCV core protein, and this effect was further enhanced in cells expressing both HCV core and CYP2E1. The CYP2E1 inhibitor, 4-methylpyrazole, could suppress increased prooxidant production in E47 cells. Finally, cells co-expressing both CYP2E1 and HCV core protein showed significantly decreased viability following GSH depletion. These studies show simultaneous expression of HCV core protein and CYP2E1 increases parameters indicative of oxidative stress as well as sensitization to cell injury induced by GSH depletion. These results support the hypothesis that enhanced injury in hepatocytes over expressing both HCV core protein and CYP2E1 is mediated by increases in oxidative stress.

The neonatal pharmacology of neuromuscular drugs was studied in vivo in newborn rats and in vitro in neonatal phrenic nerve-hemidiaphragm preparations. Drugs used to probe neuromuscular development in rat neonates were physostigmine, edrophonium, neostigmine, 4-aminopyridine, d-tubocurarine (dTc), and succinylcholine. The prejunctional actions of these drugs were monitored in relation to neonatal age by the appearance of stimulus-evoked repetitive discharge initiated by motor nerve endings and the occurrence and magnitude of the resulting enhancement of twitch tension. The occurrence and incidence of drug-induced fasciculations also served to track the development of functional motor nerve endings. Each of these prejunctional actions was inoperative until the third neonatal week, indicative of incomplete motor nerve development. In contrast, 4-aminopyridine, a nonanticholinesterase, evoked these prejunctional actions in 1-wk-old rat neonates. Neostigmine and edrophonium antagonized dTc as early as the first week; presumably, postsynaptic maturation had reached a functional level. 4-Aminopyridine also antagonized dTc at week 1. Rat neonates showed resistance to dTc blockade when tested by neonatal phrenic nerve-hemidiaphragm preparations in vitro. Relationships between age and 85%-95% transmission block declined to the adult level by week 5. This result indicates that in rat neonates, pharmacodynamic rather than pharmacokinetic mechanisms predominate in the development of responsiveness to dTc.

Drug interactions between ethanol and pentobarbital and ethanol and chlordiazepoxide were investigated utilizing mice. At the peak of oral ethanol (0-4 g/kg), either sodium pentobarbital (1-120 mg/kg) or chlordiazepoxide hydrochloride (2-400 mg/kg) was given intraperitoneally. Blood concentrations of ethanol, pentobarbital, chlordiazepoxide, and its pharmacologically active major metabolites were monitored utilizing either gas chromatography or high performance liquid chromatography. Lethality and loss-of-righting reflex were measured as indexes of behavioral drug interactions. It was evident from the isobolographic plot that the interactions between ethanol and pentobarbital and ethanol and chlordiazepoxide were more than additive. Interaction between ethanol and pentobarbital was greater than that between ethanol and chlordiazepoxide. Furthermore, with increasing ethanol pretreatment the shift in dose-response curves for the loss-of-righting reflex was affected more than the shift in dose-response curves for lethality. Blood concentration monitoring of each drug indicated that the rate of biotransformation of pentobarbital was significantly decreased; sequential biotransformation of chlordiazepoxide was also altered, resulting in a large accumulation of demethylchlordiazepoxide in the blood.