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.

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.

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.

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.

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.

Prevention of hepatitis C virus infection using a broad cross-neutralizing monoclonal antibody (AR4A) and epigallocatechin gallate.

The anti-hepatitis C virus (HCV) activity of a novel monoclonal antibody (mAb; AR4A) and epigallocatechin gallate (EGCG) were studied in vitro using a HCV cell culture system and in vivo using a humanized liver mouse model capable of supporting HCV replication. Alone, both exhibit reliable cross-genotype HCV inhibition in vitro, and combination therapy completely prevented HCV infection. In vitro AR4A mAb (alone and combined with EGCG) robustly protects against the establishment of HCV genotype 1a infection. EGCG alone fails to reliably protect against an HCV challenge. In conclusion, AR4A mAb represents a safe and efficacious broadly neutralizing antibody against HCV applicable to strategies to safely prevent HCV reinfection following liver transplantation, and it lends further support to the concept of HCV vaccine development. The poor bioavailability of EGCG limits HCV antiviral activity in vitro.

Characterization of lipid and lipoprotein metabolism in primary human hepatocytes.

Primary rodent hepatocytes and hepatoma cell lines are commonly used as model systems to elucidate and study potential drug targets for metabolic diseases such as obesity and atherosclerosis. However, if therapies are to be developed, it is essential that our knowledge gained from these systems is translatable to that of human. Here, we have characterized lipid and lipoprotein metabolism in primary human hepatocytes for comparison to rodent primary hepatocytes and human hepatoma cell lines. Primary human hepatocytes were maintained in collagen coated dishes in confluent monolayers for up to 3 days. We found primary human hepatocytes were viable, underwent lipid synthesis, and were able to secret lipoproteins up to 3 days in culture. Furthermore, the lipoprotein profile secreted by primary human hepatocytes was comparable to that found in human plasma; this contrasts with primary rodent hepatocytes and human hepatoma cells. We also investigated the pharmacological effects of nicotinic acid (niacin, NA), a potent dyslipidemic drug, on hepatic lipid synthesis and lipoprotein secretion. We found NA increased the expression of ATP-binding cassette transporter A1 in primary human hepatocytes, which may potentially explain how NA increases plasma high-density lipoproteins in humans. In conclusion, primary human hepatocytes are a more relevant model to study lipid synthesis and lipoprotein secretion than hepatoma cells or rodent primary hepatocyte models. Further research needs to be done to maintain liver specific functions of primary human hepatocytes in prolonged cultures for these cells to be a viable model.

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.

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

BACKGROUND: Sox2, 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. METHODS: In-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. RESULTS: We 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. CONCLUSION: Sox2 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.

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.

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.

Impact of calcineurin inhibitors with or without interferon on hepatitis C virus titers in a chimeric mouse model of hepatitis C virus infection.

Cyclosporine A (CSA) has potent effects against hepatitis C virus (HCV) in vitro, but its clinical efficacy after liver transplantation (LT) is uncertain. We evaluated the impact of CSA and tacrolimus (TAC) with or without concomitant interferon (IFN) therapy on serum HCV titers in a chimeric mouse model of HCV infection. Six groups of HCV-infected mice received only the vehicle, IFN, CSA, CSA and IFN, TAC, or TAC and IFN for 4 weeks. The quantitative HCV polymerase chain reaction levels were determined after 1, 2, and 4 weeks of drug administration. There were no significant differences in the HCV titers after 4 weeks of treatment between the non-IFN-treated groups (log HCV titers: 3.5 ± 0.3 for the vehicle group, 4.4 ± 0.6 for the CSA group, and 4.3 ± 0.4 for the TAC group, P = 0.3). Although IFN had a consistent effect of reducing HCV titers across the groups, there was no significant impact of CSA on HCV levels when it was used alone or in combination with IFN at any time point. The 4-week HCV titers were as follows: 3.2 ± 0.3 for the IFN group, 4.7 ± 0.4 for the CSA/IFN group, and 4.0 ± 0.5 for the TAC/IFN group (P = 0.07). The CSA/IFN and TAC/IFN groups did not differ significantly (P = 0.6). Six of the 7 animals in the IFN group (85.7%) had an HCV titer decline ≥ 1 log, whereas in the test groups (CSA/IFN and TAC/IFN), 6 of 9 animals (66.7%) achieved a 1-log decline in the HCV titer (P = 1). Using this animal model, we could find no evidence supporting the routine use of CSA after LT in HCV-infected patients.

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.

Lipoprotein profiles in SCID/uPA mice transplanted with human hepatocytes become human-like and correlate with HCV infection success.

Although multiple determinants for hepatitis C virus (HCV) infection are known, it remains partly unclear what determines the human specificity of HCV infection. Presumably, the presence of appropriate entry receptors is essential, and this may explain why HCV is unable to infect nonhuman hepatocytes. However, using mice with chimeric human livers, we show in this study that the presence of human hepatocytes, and therefore human entry receptors, is not sufficient for HCV infection. In successfully transplanted SCID/Alb-uPA mice, infection with HCV is reliable only when ∼70-80% of the liver consists of human hepatocytes. We show that chimeric mice, which are hard to infect with HCV, have significant groups of human hepatocytes that are readily infected with hepatitis B virus. Thus it is unlikely that the lack of infection with HCV can simply be attributed to low hepatocyte numbers. We investigated whether the humanization of lipoprotein profiles is positively associated with infection success. We show that the lipoprotein profiles of chimeric mice become more human-like at high levels of engraftment of human hepatocytes. This and expression of markers of human lipoprotein biosynthesis, human apolipoprotein B (ApoB) and cholesterol ester transfer protein (CETP), show a strong positive correlation with successful infection. Association of HCV in the blood of chimeric mice to ApoB-containing lipoproteins is comparable to association of HCV in patient serum and provides further support for a critical role for ApoB-containing lipoproteins in the infectious cycle of HCV. Our data suggest that the weakest link in the HCV infection chain does not appear to be the presence of human hepatocytes per se. We believe that HCV infection also depends on the presence of sufficient levels of human lipoproteins.

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

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.

UNLABELLED: 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 (IC(50)) values of 0.01 to 0.14 microM for genotype 1, with half maximal effective concentration (EC(50)s) 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.

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.

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.

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.

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.