David L.J. Tyrrell

Hepatitis C virus replication in mice with chimeric human livers

Lack of a small animal model of the human hepatitis C virus (HCV) has impeded development of antiviral therapies against this epidemic infection. By transplanting normal human hepatocytes into SCID mice carrying a plasminogen activator transgene (Alb-uPA), we generated mice with chimeric human livers. Homozygosity of Alb-uPA was associated with significantly higher levels of human hepatocyte engraftment, and these mice developed prolonged HCV infections with high viral titers after inoculation with infected human serum. Initial increases in total viral load were up to 1950-fold, with replication confirmed by detection of negative-strand viral RNA in transplanted livers. HCV viral proteins were localized to human hepatocyte nodules, and infection was serially passaged through three generations of mice confirming both synthesis and release of infectious viral particles. These chimeric mice represent the first murine model suitable for studying the human hepatitis C virus in vivo.

TRAIL Inhibits Tumor Growth but Is Nontoxic to Human Hepatocytes in Chimeric Mice

Tumor necrosis factor (TNF) family ligand TNF-α and Fas ligand (FasL) can trigger apoptosis in solid tumors, but their clinical usage has been limited by hepatotoxicity. TNF-related apoptosis-inducing ligand (TRAIL) is a newly identified member of the TNF family, and its clinical application currently is under a similar debate. Here, we report a recombinant soluble form of human TRAIL (114 to 281 amino acids) that induces apoptosis in tumor cells but not human hepatocytes. We first isolated human hepatocytes from patients and showed that the human hepatocytes expressed Fas but no TRAIL death receptor DR4 and little DR5 on the cell surface. Antibody cross-linked FasL, but not TRAIL, triggered apoptosis of the human hepatocytes through cleavage of caspases. We then examined TRAIL hepatotoxicity in severe combined immunodeficient/Alb-uPA chimeric mice harboring human hepatocytes. Intravenous injection of FasL, but not TRAIL, caused apoptotic death of human hepatocytes within the chimeric liver, thus killing the mice. Finally, we showed that repeated intraperitoneal injections of TRAIL inhibited intraperitoneal and subcutaneous tumor growth without inducing apoptosis in human hepatocytes in these chimeric mice. The results indicate that the recombinant soluble human TRAIL has a profound apoptotic effect on tumor cells but is nontoxic to human hepatocytes in vitro and in vivo.

Structural polypeptides of measles virus.

The structural polypeptides of two strains of measles virus grown in Vero cells were analysed in SDS-PAGE slab gels. Six major polypeptides were identified with mol. wt. of 79000, 72000, 60000, 43000, 40000 and 36000. The largest polypeptide was sensitive to trypsin digestion and was the dominant glycosylated polypeptide identified when the virus was grown in medium containing 3H-fucose or 3H-glucosamine or when the virus was treated with galactose oxidase and labelled with 3H-sodium borohydride. It is concluded that the 79000 mol. wt. polypeptide represents the haemagglutinin. Treatment with non-ionic detergent removed this polypeptide and also the 40000 mol. wt. polypeptide from the virus envelope. The 40000 mol. wt. polypeptide is probably associated with haemolysin and cell fusion activities and is analogous to the F1 of paramyxoviruses. A polypeptide of mol. wt. approx. 20000 detected after glycoprotein labelling may represent the F2 of measles virus. The 43000 mol. wt. polypeptide co-migrates with cellular actin and is the only major measles polypeptide that is heavily labelled when the virus is grown on Vero cells prelabelled with 35S-methionine. Thus it may represent cellular actin incorporated into the virus during maturation. The quantity of the 72000 mol. wt. polypeptide relative to the other major polypeptides varied considerably in different virus preparations. The role of the polypeptide could not be defined. By analogy with previously published data the 60000 and 36000 mol. wt. polypeptides are inferred to represent nucleocapsid and membrane proteins, respectively.

Genotypic succession of mutations of the hepatitis B virus polymerase associated with lamivudine resistance

BACKGROUND/AIMS Hepatitis B mutant strains of virus emerging during treatment with the nucleoside analog lamivudine are being increasingly recognized. In the majority of lamivudine-resistant isolates the mutations have been reported to occur within the YMDD motif of the viral polymerase, either as a single mutation M552I or as M552V concomitant with L528M. We analyzed the time course and genetic succession pattern during the emergence of lamivudine resistance. METHODS Seven patients with breakthrough viremia in the setting of chronic hepatitis (n=5) or recurrent HBV after liver transplantation (n=2) were investigated. Pre- and post-breakthrough serum samples were evaluated by single- or second-round PCR amplification and sequencing analysis. RESULTS Genotypic succession of the virus populations was observed to occur from M552I to M552I/L528M (n=2) and from L528M to M552V/L528M (n=1). The double mutations M552I/L528M (n=4) or M552V/L528M (n=2) were found in six out of seven patients, and represented the stable virus populations throughout the follow-up period. Breakthrough viremia was not associated with the single L528M mutation. The mean duration of uninterrupted treatment with lamivudine until breakthrough was 422 days (range 182-642) and was longer in the setting of chronic hepatitis B than in recurrent hepatitis B after liver transplantation. HBV DNA levels after breakthrough were lower than pretreatment levels in the majority of patients with chronic hepatitis but higher after liver transplantation. CONCLUSION Our observations show that the virus populations conferring resistance to lamivudine can evolve from single to double mutations at amino acid 552 and 528 of the HBV polymerase, and that M552I/ L528M or M552V/L528M seem to be the predominant mutations arising during long-term antiviral therapy with lamivudine.

Activity-based Protein Profiling Identifies a Host Enzyme, Carboxylesterase 1, Which Is Differentially Active during Hepatitis C Virus Replication

Hepatitis C virus (HCV) relies on many interactions with host cell proteins for propagation. Successful HCV infection also requires enzymatic activity of host cell enzymes for key post-translational modifications. To identify such enzymes, we have applied activity-based protein profiling to examine the activity of serine hydrolases during HCV replication. Profiling of hydrolases in Huh7 cells replicating HCV identified CES1 (carboxylesterase 1) as a differentially active enzyme. CES1 is an endogenous liver protein involved in processing of triglycerides and cholesterol. We observe that CES1 expression and activity were altered in the presence of HCV. The knockdown of CES1 with siRNA resulted in lower levels of HCV replication, and up-regulation of CES1 was observed to favor HCV propagation, implying an important role for this host cell protein. Experiments in HCV JFH1-infected cells suggest that CES1 facilitates HCV release because less intracellular HCV core protein was observed, whereas HCV titers remained high. CES1 activity was observed to increase the size and density of lipid droplets, which are necessary for the maturation of very low density lipoproteins, one of the likely vehicles for HCV release. In transgenic mice containing human-mouse chimeric livers, HCV infection also correlates with higher levels of endogenous CES1, providing further evidence that CES1 has an important role in HCV propagation.

Structure-Activity Relationship of a New Class of Anti-Hepatitis B Virus Agents

ABSTRACT N-Nonyl-deoxy-galactonojirimycin (N-nonyl-DGJ) has been shown to reduce the amount of hepatitis B virus (HBV) produced by tissue cultures under conditions where cell viability is not affected. We show here that the compound N-nonyl-DGJ was effective against lamivudine-resistant HBV mutants bearing the YMDD motif in the polymerase gene, consistent with the compounds activity being distinct from those of nucleoside inhibitors. To better understand the chemical structures that influence its antiviral activity, a series of imino sugar derivatives were made and tested for their antiviral activity against HBV. This work suggests that the antiviral activity of the alkovirs requires an alkyl chain length of at least eight carbons but that the galactose-based head group can be modified with little or no loss in activity.

In vitro antiviral activities of myristic acid analogs against human immunodeficiency and hepatitis B viruses.

A group of myristic acid analogs, designed as alternative substrates for N-myristoyltransferase (NMT), were evaluated against human immunodeficiency virus (HIV), hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) in vitro. Antiviral potency was increased when S or O was substituted for -CH2- in myristic acid and selectivity was affected by the presence and position of the heteroatoms and phenyl groups. A correlation was established among anti-HIV activity, Log P and Log D7.4 and between anti-HIV activity and carbonyl-heteroatom interatomic distances in the myristoyl analogs. 12-Thioethyldodecanoic acid 6 was moderately active (EC50 = 9.37 microM) against HIV-infected T4-lymphocytes (CEM-SS cell line), and it exhibited in vitro activity (EC50 = 17.8 microM) against HBV-producing 2.2.15 cell cultures derived from a human hepatoblastoma cell line (Hep G2). 12-Methoxydodecanoic acid 1 exhibited in vitro activity (EC50 = 20-30 microM) against hepatitis B in the HBV DNA-transfected 2.2.15 cell line. At a concentration of 10 microg/ml, none of the fatty acids significantly inhibited the replication of DHBV in infected hepatocytes.

Factors affecting hepatocyte isolation, engraftment, and replication in an in vivo model

Human hepatocyte transplantation is an alternative treatment for acute liver failure and liver diseases involving enzyme deficiencies. Although it has been successfully applied in selected recipients, both isolation and transplantation outcomes have the potential to be improved by better donor selection. This study assessed the impact of various donor variables on isolation outcomes (yield and viability) and posttransplant engraftment, using the SCID/Alb‐uPA (severe combined immunodeficient/urokinase type plasminogen activator under the control of an albumin promoter) human liver chimeric mouse model. Human hepatocytes were obtained from 90 human liver donor specimens and were transplanted into 3942 mice. Multivariate analysis revealed improved viability with younger donors (P = 0.038) as well as with shorter warm ischemic time (P = 0.012). Hepatocyte engraftment, assessed by the posttransplant level of serum human α1‐antitrypsin, was improved with shorter warm ischemia time. Hepatocytes isolated from older donors (≥60 years) had lower viability and posttransplant engraftment (P ≤ 0.01). In conclusion, the selection of young donors (<60 years) and rapid liver specimen retrieval, allowing for shorter warm ischemia time, are key determinants for the success of both the isolation of high viability human hepatocytes and their subsequent posttransplantation capacity for engraftment and expansion. Liver Transpl 16:974‐982, 2010.

Synthesis, Transformation Chemistry, and Biological Activity of Guanine Nucleosides and Analoges

Abstract Regiospecific (9-) and regioselective (7-) coupling procedures have been developed with easily prepared guanine derivatives. Acyclovir and 9-(D-pentofuranosyl)guanine nucleosides have been prepared without observed formation of 7-isomers. Guanosine has been functionalized and transformed into a variety of base and/or sugar modified analogues. Potent activity against HIV and a hepadnavirus has been found.

Therapy for Recurrent Herpetic Whitlow

Excerpt To the editor: Herpesvirus infection of the hand was first described in 1909 (1). Since then, its recurrent nature and a high incidence in various populations, such as health care professio...