Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis.

Plasma low-density lipoprotein (LDL) is primarily cleared by LDL receptor (LDLR). LDLR can be proteolytically cleaved to release its soluble ectodomain (sLDLR) into extracellular milieu. However, the proteinase responsible for LDLR cleavage is unknown. Here we report that membrane type 1-matrix metalloproteinase (MT1-MMP) co-immunoprecipitates and co-localizes with LDLR and promotes LDLR cleavage. Plasma sLDLR and cholesterol levels are reduced while hepatic LDLR is increased in mice lacking hepatic MT1-MMP. Opposite effects are observed when MT1-MMP is overexpressed. MT1-MMP overexpression significantly increases atherosclerotic lesions, while MT1-MMP knockdown significantly reduces cholesteryl ester accumulation in the aortas of apolipoprotein E (apoE) knockout mice. Furthermore, sLDLR is associated with apoB and apoE-containing lipoproteins in mouse and human plasma. Plasma levels of sLDLR are significantly increased in subjects with high plasma LDL cholesterol levels. Thus, we demonstrate that MT1-MMP promotes ectodomain shedding of hepatic LDLR, thereby regulating plasma cholesterol levels and the development of atherosclerosis.

Mice with Chimeric Human Livers and Their Applications.

The complete life cycle of the hepatitis C virus (HCV) can be recapitulated in vivo using immunodeficient mice that have had their livers extensively repopulated with human hepatocytes. These human liver chimeric mouse models have enabled the study of many aspects of the HCV life cycle, including antiviral interventions that have helped to shape the curative landscape that is available today. The first human liver chimeric mouse model capable of supporting the HCV life cycle was generated in SCID-uPA mice. Although other human liver chimeric mouse models have since been developed, the SCID-uPA mouse model remains one of the most robust in vivo systems available for HCV studies. This chapter reviews development, validation and application of the SCID-uPA mouse model, and discusses their potential application for studying other liver-centric diseases and pathogens and for the design and testing of vaccine candidates for the eradication of HCV.

Metabolism of a Phenylarsenical in Human Hepatic Cells and Identification of a New Arsenic Metabolite.

Environmental contamination and human consumption of chickens could result in potential exposure to Roxarsone (3-nitro-4-hydroxyphenylarsonic acid), an organic arsenical that has been used as a chicken feed additive in many countries. However, little is known about the metabolism of Roxarsone in humans. The objective of this research was to investigate the metabolism of Roxarsone in human liver cells and to identify new arsenic metabolites of toxicological significance. Human primary hepatocytes and hepatocellular carcinoma HepG2 cells were treated with 20 or 100 µM Roxarsone. Arsenic species were characterized using a strategy of complementary chromatography and mass spectrometry. The results showed that Roxarsone was metabolized to more than 10 arsenic species in human hepatic cells. A new metabolite was identified as a thiolated Roxarsone. The 24 h IC50 values of thiolated Roxarsone for A549 lung cancer cells and T24 bladder cancer cells were 380 ± 80 and 42 ± 10 µM, respectively, more toxic than Roxarsone, whose 24 h IC50 values for A549 and T24 were 9300 ± 1600 and 6800 ± 740 µM, respectively. The identification and toxicological studies of the new arsenic metabolite are useful for understanding the fate of arsenic species and assessing the potential impact of human exposure to Roxarsone.

Protein O-fucosylation in Plasmodium falciparum ensures efficient infection of mosquito and vertebrate hosts.

O-glycosylation of the Plasmodium sporozoite surface proteins CSP and TRAP was recently identified, but the role of this modification in the parasite life cycle and its relevance to vaccine design remain unclear. Here, we identify the Plasmodium protein O-fucosyltransferase (POFUT2) responsible for O-glycosylating CSP and TRAP. Genetic disruption of POFUT2 in Plasmodium falciparum results in ookinetes that are attenuated for colonizing the mosquito midgut, an essential step in malaria transmission. Some POFUT2-deficient parasites mature into salivary gland sporozoites although they are impaired for gliding motility, cell traversal, hepatocyte invasion, and production of exoerythrocytic forms in humanized chimeric liver mice. These defects can be attributed to destabilization and incorrect trafficking of proteins bearing thrombospondin repeats (TSRs). Therefore, POFUT2 plays a similar role in malaria parasites to that in metazoans: it ensures the trafficking of Plasmodium TSR proteins as part of a non-canonical glycosylation-dependent endoplasmic reticulum protein quality control mechanism.The role of O-glycosylation in the malaria life cycle is largely unknown. Here, the authors identify a Plasmodium protein O-fucosyltransferase and show that it is important for normal trafficking of a subset of surface proteins, particularly CSP and TRAP, and efficient infection of mosquito and vertebrate hosts.

AMA1 and MAEBL are important for Plasmodium falciparum sporozoite infection of the liver.

The malaria sporozoite injected by a mosquito migrates to the liver by traversing host cells. The sporozoite also traverses hepatocytes before invading a terminal hepatocyte and developing into exoerythrocytic forms. Hepatocyte infection is critical for parasite development into merozoites that infect erythrocytes, and the sporozoite is thus an important target for antimalarial intervention. Here, we investigated two abundant sporozoite proteins of the most virulent malaria parasite Plasmodium falciparum and show that they play important roles during cell traversal and invasion of human hepatocytes. Incubation of P. falciparum sporozoites with R1 peptide, an inhibitor of apical merozoite antigen 1 (AMA1) that blocks merozoite invasion of erythrocytes, strongly reduced cell traversal activity. Consistent with its inhibitory effect on merozoites, R1 peptide also reduced sporozoite entry into human hepatocytes. The strong but incomplete inhibition prompted us to study the AMA-like protein, merozoite apical erythrocyte-binding ligand (MAEBL). MAEBL-deficient P. falciparum sporozoites were severely attenuated for cell traversal activity and hepatocyte entry in vitro and for liver infection in humanized chimeric liver mice. This study shows that AMA1 and MAEBL are important for P. falciparum sporozoites to perform typical functions necessary for infection of human hepatocytes. These two proteins therefore have important roles during infection at distinct points in the life cycle, including the blood, mosquito, and liver stages.

Cell Traversal Activity Is Important for Plasmodium falciparum Liver Infection in Humanized Mice.

Malaria sporozoites are deposited into the skin by mosquitoes and infect hepatocytes. The molecular basis of how Plasmodium falciparum sporozoites migrate through host cells is poorly understood, and direct evidence of its importance in vivo is lacking. Here, we generated traversal-deficient sporozoites by genetic disruption of sporozoite microneme protein essential for cell traversal (PfSPECT) or perforin-like protein 1 (PfPLP1). Loss of either gene did not affect P. falciparum growth in erythrocytes, in contrast with a previous report that PfPLP1 is essential for merozoite egress. However, although traversal-deficient sporozoites could invade hepatocytes in vitro, they could not establish normal liver infection in humanized mice. This is in contrast with NF54 sporozoites, which infected the humanized mice and developed into exoerythrocytic forms. This study demonstrates that SPECT and perforin-like protein 1 (PLP1) are critical for transcellular migration by P. falciparum sporozoites and demonstrates the importance of cell traversal for liver infection by this human pathogen.

A positive feedback loop involving the Wnt/ß-catenin/MYC/Sox2 axis defines a highly tumorigenic cell subpopulation in ALK-positive anaplastic large cell lymphoma.

BACKGROUND: We have previously described the existence of two phenotypically distinct cell subsets in ALK-positive anaplastic large cell lymphoma (ALK + ALCL) based on their differential responsiveness to a Sox2 reporter (SRR2), with reporter-responsive (RR) cells being more tumorigenic and chemoresistant than reporter-unresponsive (RU) cells. However, the regulator(s) of RU/RR dichotomy are not identified. In this study, we aim to delineate the key regulator(s) of RU/RR dichotomy. METHODS: JASPER motif match analysis was used to identify the putative factors binding to SRR2 sequence. SRR2 probe pull-down assay and quantitate real-time PCR were performed to analyze the regulation of Sox2 transcriptional activity by MYC. Methylcellulose colony formation assay, chemoresistance to doxorubicin and mouse xenograft study were performed to investigate the biological functions of MYC. PCR array and western blotting were executed to study related signaling pathways that regulate MYC expression. Immunofluorescence and immunohistochemistry assay were initiated to evaluate the expression of MYC and its correlation with its regulator by chi-square test analysis in human primary tumor cells. RESULTS: We identified MYC as a potential regulator of RU/RR dichotomy. In support of its role, MYC was highly expressed in RR cells compared to RU cells, and inhibition of MYC substantially decreased the Sox2/SRR2 binding, Sox2 transcriptional activity, chemoresistance, and methylcellulose colony formation. In contrast, enforced expression of MYC in RU cells conferred the RR phenotype. The Wnt/ß-catenin pathway, a positive regulator of MYC, was highly active in RR but not RU cells. While inhibition of this pathway in RR cells substantially decreased MYC expression and SRR2 reporter activity, experimental activation of this pathway led to the opposite effects in RU cells. Collectively, our results support a model in which a positive feedback loop involving Wnt/ß-catenin/MYC and Sox2 contributes to the RR phenotype. In a mouse xenograft model, RU cells stably transfected with MYC showed upregulation of the Wnt/ß-catenin/MYC/Sox2 axis and increased tumorigenecity. Correlating with these findings, there was a significant correlation between the expression of active ß-catenin and MYC in ALK + ALCL primary tumor cells. CONCLUSIONS: A positive feedback loop involving the Wnt/ß-catenin/MYC/Sox2 axis defines a highly tumorigenic cell subset in ALK + ALCL.

Hepatitis C Virus-Induced Degradation of Cell Death-Inducing DFFA-Like Effector B Leads to Hepatic Lipid Dysregulation.

UNLABELLED: Individuals chronically infected with hepatitis C virus (HCV) commonly exhibit hepatic intracellular lipid accumulation, termed steatosis. HCV infection perturbs host lipid metabolism through both cellular and virus-induced mechanisms, with the viral core protein playing an important role in steatosis development. We have recently identified a liver protein, the cell death-inducing DFFA-like effector B (CIDEB), as an HCV entry host dependence factor that is downregulated by HCV infection in a cell culture model. In this study, we investigated the biological significance and molecular mechanism of this downregulation. HCV infection in a mouse model downregulated CIDEB in the liver tissue, and knockout of the CIDEB gene in a hepatoma cell line results in multiple aspects of lipid dysregulation that can contribute to hepatic steatosis, including reduced triglyceride secretion, lower lipidation of very-low-density lipoproteins, and increased lipid droplet (LD) stability. The potential link between CIDEB downregulation and steatosis is further supported by the requirement of the HCV core and its LD localization for CIDEB downregulation, which utilize a proteolytic cleavage event that is independent of the cellular proteasomal degradation of CIDEB. IMPORTANCE: Our data demonstrate that HCV infection of human hepatocytesin vitroandin vivoresults in CIDEB downregulation via a proteolytic cleavage event. Reduction of CIDEB protein levels by HCV or gene editing, in turn, leads to multiple aspects of lipid dysregulation, including LD stabilization. Consequently, CIDEB downregulation may contribute to HCV-induced hepatic steatosis.

Tissue Specific Effects of Dietary Carbohydrates and Obesity on ChREBPα and ChREBPß Expression.

Carbohydrate response element binding protein (ChREBP) regulates insulin-independent de novo lipogenesis. Recently, a novel ChREBPß isoform was identified. The purpose of the current study was to define the effect of dietary carbohydrates (CHO) and obesity on the transcriptional activity of ChREBP isoforms and their respective target genes. Mice were subjected to fasting-refeeding of high-CHO diets. In all three CHO-refeeding groups, mice failed to induce ChREBPα, yet ChREBPß increased 10- to 20-fold. High-fat fed mice increased hepatic ChREBPß mRNA expression compared to chow-fed along with increased protein expression. To better assess the independent effect of fructose on ChREBPα/ß activity, HepG2 cells were treated with fructose ± a fructose-1,6-bisphosphatase inhibitor to suppress gluconeogenesis. Fructose treatment in the absence of gluconeogenesis resulted in increased ChREBP activity. To confirm the existence of ChREBPß in human tissue, primary hepatocytes were incubated with high-glucose and the expression of ChREBPα and -ß was determined. As with the animal models, glucose induced ChREBPß expression while ChREBPα was decreased. Taken together, ChREBPß is more responsive to changes in dietary CHO availability than the -α isoform. Diet-induced obesity increases basal expression of ChREBPß, which may increase the risk of developing hepatic steatosis, and fructose-induced activation is independent of gluconeogenesis.

Oxidative Stress Attenuates Lipid Synthesis and Increases Mitochondrial Fatty Acid Oxidation in Hepatoma Cells Infected with Hepatitis C Virus.

Cytopathic effects are currently believed to contribute to hepatitis C virus (HCV)-induced liver injury and are readily observed in Huh7.5 cells infected with the JFH-1 HCV strain, manifesting as apoptosis highly correlated with growth arrest. Reactive oxygen species, which are induced by HCV infection, have recently emerged as activators of AMP-activated protein kinase. The net effect is ATP conservation via on/off switching of metabolic pathways that produce/consume ATP. Depending on the scenario, this can have either pro-survival or pro-apoptotic effects. We demonstrate reactive oxygen species-mediated activation of AMP-activated kinase in Huh7.5 cells during HCV (JFH-1)-induced growth arrest. Metabolic labeling experiments provided direct evidence that lipid synthesis is attenuated, and ß-oxidation is enhanced in these cells. A striking increase in nuclear peroxisome proliferator-activated receptor α, which plays a dominant role in the expression of ß-oxidation genes after ligand-induced activation, was also observed, and we provide evidence that peroxisome proliferator-activated receptor α is constitutively activated in these cells. The combination of attenuated lipid synthesis and enhanced ß-oxidation is not conducive to lipid accumulation, yet cellular lipids still accumulated during this stage of infection. Notably, the serum in the culture media was the only available source for polyunsaturated fatty acids, which were elevated (2-fold) in the infected cells, implicating altered lipid import/export pathways in these cells. This study also provided the first in vivo evidence for enhanced ß-oxidation during HCV infection because HCV-infected SCID/Alb-uPA mice accumulated higher plasma ketones while fasting than did control mice. Overall, this study highlights the reprogramming of hepatocellular lipid metabolism and bioenergetics during HCV infection, which are predicted to impact both the HCV life cycle and pathogenesis.

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.

Triple negative breast cancers comprise a highly tumorigenic cell subpopulation detectable by its high responsiveness to a Sox2 regulatory region 2 (SRR2) reporter.

We have recently described a novel phenotypic dichotomy within estrogen receptor-positive breast cancer cells; the cell subset responsive to a Sox2 regulatory region (SRR2) reporter (RR cells) are significantly more tumorigenic than the reporter unresponsive (RU) cells. Here, we report that a similar phenomenon also exists in triple negative breast cancer (TNBC), with RR cells more tumorigenic than RU cells. First, examination of all 3 TNBC cell lines stably infected with the SRR2 reporter revealed the presence of a cell subset exhibiting reporter activity. Second, RU and RR cells purified by flow cytometry showed that RR cells expressed higher levels of CD44, generated more spheres in a limiting dilution mammosphere formation assay, and formed larger and more complex structures in Matrigel. Third, within the CD44(High)/CD24- tumor-initiating cell population derived from MDA-MB-231, RR cells were significantly more tumorigenic than RU cells in an in vivo SCID/Beige xenograft mouse model. Examination of 4 TNBC tumors from patients also revealed the presence of a RR cell subset, ranging from 1.1-3.8%. To conclude, we described a novel phenotypic heterogeneity within TNBC, and the SRR2 reporter responsiveness is a useful marker for identifying a highly tumorigenic cell subset within the CD44(High)/CD24-tumor-initiating cell population.

Generation of improved mouse models for the study of hepatitis C virus.

Approximately 3% of the world׳s population suffers from chronic infections with hepatitis C virus (HCV). Although current treatment regimes are capable of effectively eradicating HCV infection from these patients, the cost of these combinations of direct-acting antivirals are prohibitive. Approximately 80% of untreated chronic HCV carriers will be at high risk for developing severe liver disease, including fibrosis, cirrhosis, and hepatocellular carcinoma. A vaccine is urgently needed to lessen this global burden. Besides humans, HCV infection can be experimentally transmitted to chimpanzees, and this is the best model for studies of HCV infection and related innate and adaptive immune responses. Although the chimpanzee model yielded valuable insight, limited availability, high cost and ethical considerations limit their utility. The only small animal models of robust HCV infection are highly immunodeficient mice with human chimeric livers. However, these mice cannot be used to study adaptive immune responses and therefore a more relevant animal model is needed to assist in vaccine development. Novel strains of immunodeficient mice have been developed that allow for the engraftment of human hepatopoietic stem cells, as well as functional human lymphoid cells and tissues, effectively creating human immune systems in otherwise immunodeficient mice. These humanized mice are rapidly emerging as pre-clinical bridges for numerous pathogens that, like HCV, only cause infectious disease in humans. This review highlights the potential these new models have for changing the current landscape for HCV research and vaccine development.

Characterization of arsenic hepatobiliary transport using sandwich-cultured human hepatocytes.

Arsenic is a proven human carcinogen and is associated with a myriad of other adverse health effects. This metalloid is methylated in human liver to monomethylarsonic acid (MMA(V)), monomethylarsonous acid (MMA(III)), dimethylarsinic acid (DMA(V)), and dimethylarsinous acid (DMA(III)) and eliminated predominantly in urine. Hepatic basolateral transport of arsenic species is ultimately critical for urinary elimination; however, these pathways are not fully elucidated in humans. A potentially important human hepatic basolateral transporter is the ATP-binding cassette (ABC) transporter multidrug resistance protein 4 (MRP4/ABCC4) that in vitro is a high-affinity transporter of DMA(V) and the diglutathione conjugate of MMA(III) [MMA(GS)(2)]. In rats, the related canalicular transporter Mrp2/Abcc2 is required for biliary excretion of arsenic as As(GS)(3) and MMA(GS)(2). The current study used sandwich cultured human hepatocytes (SCHH) as a physiological model of human arsenic hepatobiliary transport. Arsenic efflux was detected only across the basolateral membrane for 9 out of 14 SCHH preparations, 5 had both basolateral and canalicular efflux. Basolateral transport of arsenic was temperature- and GSH-dependent and inhibited by the MRP inhibitor MK-571. Canalicular efflux was completely lost after GSH depletion suggesting MRP2-dependence. Treatment of SCHH with As(III) (0.1-1 µM) dose-dependently increased MRP2 and MRP4 levels, but not MRP1, MRP6, or aquaglyceroporin 9. Treatment of SCHH with oltipraz (Nrf2 activator) increased MRP4 levels and basolateral efflux of arsenic. In contrast, oltipraz increased MRP2 levels without increasing biliary excretion. These results suggest arsenic basolateral transport prevails over biliary excretion and is mediated at least in part by MRPs, most likely including MRP4.

Additive effect of sirolimus and anti-death receptor 5 agonistic antibody against hepatocellular carcinoma.

BACKGROUND & AIMS: Despite careful patient selection, hepatocellular carcinoma (HCC) recurs in 10-20% of cases after liver transplantation, and the use of potent adjuvant anticancer drugs would be welcome. The aim of this study was to evaluate the efficiency of a combined therapy of rapamycin (sirolimus) and anti-death receptor (DR)5 monoclonal antibody (mAb) on HCC. METHODS: We first assessed the side effects of anti-DR5 mAb administration in vivo by giving various doses of anti-DR5 mAb. Cell proliferation assays were then performed using mouse Hepa1-6 cells or human Huh7 cells to quantify the relative cell viability under various concentrations of sirolimus, anti-DR5 mAb or a combination. Next, one million Hepa1-6 cells were transplanted into C.B17-SCID-beige mice subcutaneously, and four groups were created: (1) untreated, (2) anti-DR5 mAb alone, (3) sirolimus alone and (4) anti-DR5 mAb + sirolimus. RESULTS: Anti-DR5 mAb (200 and 300 µg/day) induced liver dysfunction with partial necrosis of the liver, but 100 µg/day was well tolerated with transaminitis, but normal bilirubin and only minor histological liver damage. In vitro, anti-DR5 mAb lysed Hepa1-6 and Huh7 cells in a dose-dependent manner, and combinations of sirolimus and anti-DR5 mAb demonstrated an additive effect. In vivo studies demonstrated that tumour sizes were significantly smaller in the combined therapy group than in the monotherapy groups. CONCLUSIONS: Combining sirolimus and low-dose anti-DR5 mAb has a significant effect against HCC. This strategy represents a potential novel approach for the management of HCC.

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.

AIM: 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. METHODS: One million viable human hepatocytes, purified from human livers, were injected into the spleens of severe combined immunodeficient/albumin linked-urokinase type plasminogen activator transgenic mice. A clinical strain of HCMV was inoculated in mice with confirmed human hepatocyte engraftment or in non-chimeric controls. Infection was monitored through HCMV titers in the plasma. Mice were administrated ganciclovir (50 mg/kg per day, i.p.) beginning at 2 days post-HCMV inoculation, or human liver natural killer (NK) cells (20 × 10(6) cells/mouse, i.v.) 1 day prior to HCMV inoculation. RESULTS: Chimeric mice that received HCMV showed high plasma titers of HCMV DNA on days 1 and 6 that became undetectable by day 11 post-inoculation. In contrast, non-transplanted mice had only residual plasma inoculum detection at day 1 and no detectable viremia thereafter. The levels of HCMV DNA were reduced by ganciclovir treatment or by human liver NK cell adoptive transfer, while HCMV-infected chimeric mice that were not treated sustained viremia during the follow up. CONCLUSION: Human liver chimeric mice provide an in vivo model for the study of acute HCMV infection of 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 alpha1-antitrypsin, was improved with shorter warm ischemia time. Hepatocytes isolated from older donors (>or=60 years) had lower viability and posttransplant engraftment (P

Therapeutic efficacy of human hepatocyte transplantation in a SCID/uPA mouse model with inducible liver disease.

BACKGROUND: Severe Combined Immune Deficient (SCID)/Urokinase-type Plasminogen Activator (uPA) mice undergo liver failure and are useful hosts for the propagation of transplanted human hepatocytes (HH) which must compete with recipient-derived hepatocytes for replacement of the diseased liver parenchyma. While partial replacement by HH has proven useful for studies with Hepatitis C virus, complete replacement of SCID/uPA mouse liver by HH has never been achieved and limits the broader application of these mice for other areas of biomedical research. The herpes simplex virus type-1 thymidine kinase (HSVtk)/ganciclovir (GCV) system is a powerful tool for cell-specific ablation in transgenic animals. The aim of this study was to selectively eliminate murine-derived parenchymal liver cells from humanized SCID/uPA mouse liver in order to achieve mice with completely humanized liver parenchyma. Thus, we reproduced the HSVtk (vTK)/GCV system of hepatic failure in SCID/uPA mice. METHODOLOGY/PRINCIPAL FINDINGS: In vitro experiments demonstrated efficient killing of vTK expressing hepatoma cells after GCV treatment. For in vivo experiments, expression of vTK was targeted to the livers of FVB/N and SCID/uPA mice. Hepatic sensitivity to GCV was first established in FVB/N mice since these mice do not undergo liver failure inherent to SCID/uPA mice. Hepatic vTK expression was found to be an integral component of GCV-induced pathologic and biochemical alterations and caused death due to liver dysfunction in vTK transgenic FVB/N and non-transplanted SCID/uPA mice. In SCID/uPA mice with humanized liver, vTK/GCV caused death despite extensive replacement of the mouse liver parenchyma with HH (ranging from 32-87%). Surprisingly, vTK/GCV-dependent apoptosis and mitochondrial aberrations were also localized to bystander vTK-negative HH. CONCLUSIONS/SIGNIFICANCE: Extensive replacement of mouse liver parenchyma by HH does not provide a secure therapeutic advantage against vTK/GCV-induced cytotoxicity targeted to residual mouse hepatocytes. Functional support by engrafted HH may be secured by strategies aimed at limiting this bystander effect.

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 (125)I-iodo-2'-deoxyuridine incorporation assay. Low serum human alpha-1 antitrypsin (hAAT, <10 microg/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 microg/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(-/-)gammac(-/-)/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.