Donna N. Douglas

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.

HCV796: A selective nonstructural protein 5B polymerase inhibitor with potent anti‐hepatitis C virus activity In Vitro, in mice with chimeric human livers, and in humans infected with hepatitis C virus

Anti‐hepatitis C virus (HCV) drug development has been challenged by a lack of experience with inhibitors inclusive of in vitro, animal model, and clinical study. This manuscript outlines activity and correlation across such a spectrum of models and into clinical trials with a novel selective nonstructural protein 5B (NS5B) polymerase inhibitor, HCV796. Enzyme assays yielded median inhibitory concentration (IC50) values of 0.01 to 0.14 μM for genotype 1, with half maximal effective concentration (EC50s) of 5 nM and 9 nM against genotype 1a and 1b replicons. In the chimeric mouse model, a 2.02 ± 0.55 log reduction in HCV titer was seen with monotherapy, whereas a suboptimal dose of 30 mg/kg three times per day in combination with interferon demonstrated a 2.44 log reduction (P = 0.001 versus interferon alone) Clinical outcomes in combination with pegylated interferon and ribavirin have revealed additive efficacy in treatment naïve patients. Abnormal liver function test results were observed in 8% of HCV‐796 patients treated for over 8 weeks, resulting in suspension of further trial activity. Conclusion: The RNA‐dependent RNA polymerase inhibitor HCV796 demonstrated potent anti‐HCV activity consistently through enzyme inhibition assays, subgenomic replicon, and chimeric mouse studies. Strong correlations of outcomes in the mouse model were seen with subsequent clinical trials, including a plateau in dose‐related antiviral activity and additive impact from combination therapy with interferon. These outcomes demonstrate the utility of the range of in vitro and in vivo models now available for anti‐HCV drug development and support the potential utility of polymerase inhibitors in future combination therapies for HCV treatment. (HEPATOLOGY 2009.)

Regulation of the enzymes of hepatic microsomal triacylglycerol lipolysis and re-esterification by the glucocorticoid dexamethasone

Hepatic VLDL (very-low-density lipoprotein) assembly is a complex process that is largely regulated by the provision of lipid for apolipoprotein B assembly. Intracellular stored TAG (triacylglycerol) undergoes an initial lipolysis followed by re-esterification of the lipolytic products to form TAG prior to their incorporation into a VLDL particle. TGH (TAG hydrolase) is a lipase that hydrolyses intracellular TAG within the hepatocyte. We have utilized both dexamethasone-injected mouse and primary hepatocyte models to address whether stimulation of TAG biosynthesis by the synthetic glucocorticoid, dexamethasone, altered hepatic lipolysis and re-esterification and the provision of stored TAG for lipoprotein secretion. Dexamethasone treatment resulted in decreased TGH expression, primarily due to a dexamethasone-induced decrease in TGH mRNA stability. The expression and activities of diacylglycerol acyltransferases 1 and 2 were stimulated by dexamethasone. The combination of reduced intracellular TAG lipolysis and increased TAG biosynthesis contributed to the accumulation of TAG within the livers of dexamethasone-injected mice. The rate of hepatic TAG secretion in dexamethasonetreated mice was maintained at similar levels as in control mice. Our data demonstrate that stimulation of de novo TAG synthesis by dexamethasone increased the proportion of secreted TAG that was derived from de novo sources, while the utilization of stored TAG for secretion was reduced. The results show that, during markedly increased TAG synthesis, some TAGs are diverted from the cytosolic storage pool and are utilized directly for VLDL assembly within the endoplasmic reticulum lumen.

Anti-HCV therapies in chimeric scid-Alb/uPA mice parallel outcomes in human clinical application†

Compounds with in vitro anti‐hepatitis C virus (HCV) activity are often advanced directly into clinical trials with limited or no in vivo efficacy data. This limits prediction of clinical efficacy of compounds in the HCV drug pipeline, and may expose human subjects to unnecessary treatment effects. The scid‐Alb‐uPA mouse supports proliferation of transplanted human hepatocytes and subsequent HCV infection. Cohorts of genotype 1a HCV‐infected mice were treated with interferon α‐2b(IFN‐α), BILN‐2061 (anti‐NS3 protease), or HCV371 (anti‐NS5B polymerase). Mice treated with 1350IU/g/day IFN‐α intramuscularly for 10 to 28 days demonstrated reduced viral titers compared with controls in all five experiments (P < .05, t test); viral titers rebounded after treatment withdrawal. A more pronounced antiviral effect with IFN‐α was seen in genotype 3a–infected mice. Pilot studies with BILN2061 confirmed exposure to 10X replicon EC50 at trough and reduced viral titer over 2 log at 4 days. In a second 7‐day study, mean HCV RNA titers dropped 1.1 log in BILN2061‐treated animals, 0.6 log in IFN‐treated mice, and rose 0.2 log in controls (P = .013, ANOVA). Pre‐existing mutants with partial resistance to BILN2061 were identified by sequencing both the human inoculum and sera from treated mice. The polymerase inhibitor HCV371 yielded a decline in HCV titers of 0.3 log relative to vehicle‐treated controls (P = NS). Performance of all three antiviral regimens in the chimeric mouse model paralleled responses in humans. In conclusion, this system may help selection of lead compounds for advancement into human trials with an increased likelihood of clinical success while broadening the tools available for study of the biology of HCV infection. (HEPATOLOGY 2006;43:1346–1353.)

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.

STAT1 is phosphorylated and downregulated by the oncogenic tyrosine kinase NPM-ALK in ALK-positive anaplastic large-cell lymphoma.

The tumorigenicity of most cases of ALK-positive anaplastic large-cell lymphoma (ALK+ ALCL) is driven by the oncogenic fusion protein NPM-ALK in a STAT3-dependent manner. Because it has been shown that STAT3 can be inhibited by STAT1 in some experimental models, we hypothesized that the STAT1 signaling pathway is defective in ALK+ ALCL, thereby leaving the STAT3 signaling unchecked. Compared with normal T cells, ALK+ ALCL tumors consistently expressed a low level of STAT1. Inhibition of the ubiquitin-proteasome pathway appreciably increased STAT1 expression in ALK+ ALCL cells. Furthermore, we found evidence that NPM-ALK binds to and phosphorylates STAT1, thereby promoting its proteasomal degradation in a STAT3-dependent manner. If restored, STAT1 is functionally intact in ALK+ ALCL cells, because it effectively upregulated interferon-γ, induced apoptosis/cell-cycle arrest, potentiated the inhibitory effects of doxorubicin, and suppressed tumor growth in vivo. STAT1 interfered with the STAT3 signaling by decreasing STAT3 transcriptional activity/DNA binding and its homodimerization. The importance of the STAT1/STAT3 functional interaction was further highlighted by the observation that short interfering RNA knockdown of STAT1 significantly decreased apoptosis induced by STAT3 inhibition. Thus, STAT1 is a tumor suppressor in ALK+ ALCL. Phosphorylation and downregulation of STAT1 by NPM-ALK represent other mechanisms by which this oncogenic tyrosine kinase promotes tumorigenesis.

Sox2 suppresses the invasiveness of breast cancer cells via a mechanism that is dependent on Twist1 and the status of Sox2 transcription activity

BackgroundSox2, an embryonic stem cell marker, is aberrantly expressed in a subset of breast cancer (BC). While the aberrant expression of Sox2 has been shown to significantly correlate with a number of clinicopathologic parameters in BC, its biological significance in BC is incompletely understood.MethodsIn-vitro invasion assay was used to evaluate whether the expression of Sox2 is linked to the invasiveness of MCF7 and ZR751 cells. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and/or Western blots were used to assess if Sox2 modulates the expression of factors known to regulate epithelial mesenchymal transition (EMT), such as Twist1. Chromatin immunoprecipitation (ChIP) was used to assess the binding of Sox2 to the promoter region of Twist1.ResultsWe found that siRNA knockdown of Sox2 expression significantly increased the invasiveness of MCF7 and ZR751 cells. However, when MCF7 cells were separated into two distinct subsets based on their differential responsiveness to the Sox2 reporter, the Sox2-mediated effects on invasiveness was observed only in ‘reporter un-responsive’ cells (RU cells) but not ‘reporter responsive’ cells (RR cells). Correlating with these findings, siRNA knockdown of Sox2 in RU cells, but not RR cells, dramatically increased the expression of Twist1. Accordingly, using ChIP, we found evidence that Sox2 binds to the promoter region of Twist1 in RU cells only. Lastly, siRNA knockdown of Twist1 largely abrogated the regulatory effect of Sox2 on the invasiveness in RU cells, suggesting that the observed Sox2-mediated effects are Twist1-dependent.ConclusionSox2 regulates the invasiveness of BC cells via a mechanism that is dependent on Twist1 and the transcriptional status of Sox2. Our results have further highlighted a new level of biological complexity and heterogeneity of BC cells that may carry significant clinical implications.

Arylacetamide deacetylase: A novel host factor with important roles in the lipolysis of cellular triacylglycerol stores, VLDL assembly and HCV production

BACKGROUND & AIMS Very low density lipoproteins (VLDLs) are triacylglycerol (TG)-rich lipoproteins produced by the human liver. VLDLs derive the majority of their TG cargo from the lipolysis of TG stored in hepatocellular lipid droplets (LDs). Important roles for LDs and the VLDL secretory pathway in the cell culture production of infectious hepatitis C virus (HCV) have been established. We hypothesized that TG lipolysis and VLDL production are impaired during HCV infection so that these cellular processes can be diverted towards HCV production. METHODS We used an HCV permissive cell culture system (JFH-1/HuH7.5 cells) to examine the relationship between TG lipolysis, VLDL assembly, and the HCV lifecycle using standard biochemical approaches. RESULTS Lipolysis of cellular TG and VLDL production were impaired in HCV infected cells during the early peak of viral infection. This was partially explained by an apparent deficiency of a putative TG lipase, arylacetamide deacetylase (AADAC). The re-introduction of AADAC to infected cells restored cellular TG lipolysis, indicating a role for HCV-mediated downregulation of AADAC in this process. Defective lipolysis of cellular TG stores and VLDL production were also observed in HuH7.5 cells stably expressing a short hairpin RNA targeting AADAC expression, proving AADAC deficiency contributes to these defective pathways. Finally, impaired production of HCV was observed with AADAC knockdown cells, demonstrating a role for AADAC in the HCV lifecycle. CONCLUSIONS This insight into the biology of HCV infection and possibly pathogenesis identifies AADAC as a novel and translationally relevant therapeutic target.

Human cytomegalovirus infection in humanized liver chimeric mice

Cytomegalovirus is a common viral pathogen that influences the outcome of organ transplantation. To date, there is no established method to evaluate the effects of human CMV (HCMV) treatments in vivo except for human clinical trials. In the current study, we describe the development of a mouse model that supports the in vivo propagation of HCMV.

Critical role of natural killer cells in the rejection of human hepatocytes after xenotransplantation into immunodeficient mice

The severe combined immunodeficiency/albumin linked‐urokinase type plasminogen activator (SCID/Alb‐uPA) human liver chimeric mouse model has added a new dimension to studies of liver based human diseases and has important potential for study of human hepatic drug metabolism. However, it remains unclear if natural killer (NK) cell in SCID/Alb‐uPA mice has an important negative impact on engraftment and expansion of human hepatocytes after transplantation. Here, we explore the role of mouse NK cells in the rejection of transplanted human hepatocytes in SCID/Alb‐uPA mice. We assessed NK cell activity in vivo, using 125I‐iodo‐2′‐deoxyuridine incorporation assay. Low serum human alpha‐1 antitrypsin (hAAT, <10 μg/ml) recipients, representing graft failure, showed resistance to engraftment of MHC class I knockout marrow (indicating high NK cell activity), while NK cell‐depleted low hAAT recipients and high hAAT (>100 μg/ml) recipients accepted MHC class I knockout marrow, indicating a correlation between low NK cell activity, in vivo, and high level human hepatocyte engraftment. We also showed that higher level engraftment of human hepatocytes was achieved in both NK cell‐depleted SCID/Alb‐uPA mice and Rag2−/−γc−/−/Alb‐uPA (T,B and NK cell deficient) mice compared with untreated SCID/Alb‐uPA mice. These results support a critical role for mouse NK cells in the rejection of human hepatocytes xenotransplanted to immunodeficient mice.