Therapeutic Efficacy of Human Hepatocyte Transplantation in a SCID/uPA Mouse Model with Inducible Liver Disease

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.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.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

Reductions in circulating levels of IL-16, IL-7 and VEGF-A in myalgic encephalomyelitis/chronic fatigue syndrome

Recently, differences in the levels of various chemokines and cytokines were reported in patients with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) as compared with controls. Moreover, the analyte profile differed between chronic ME/CFS patients of long duration versus patients with disease of less than 3 years. In the current study, we measured the plasma levels of 34 cytokines, chemokines and growth factors in 100 chronic ME/CFS patients of long duration and in 79 gender and age-matched controls. We observed highly significant reductions in the concentration of circulating interleukin (IL)-16, IL-7, and Vascular Endothelial Growth Factor A (VEGF-A) in ME/CFS patients. All three biomarkers were significantly correlated in a multivariate cluster analysis. In addition, we identified significant reductions in the concentrations of fractalkine (CX3CL1) and monokine-induced-by-IFN-γ (MIG; CXCL9) along with increases in the concentrations of eotaxin 2 (CCL24) in ME/CFS patients. Our data recapitulates previous data from another USA ME/CFS cohort in which circulating levels of IL-7 were reduced. Also, a reduced level of VEGF-A was reported previously in sera of patients with Gulf War Illness as well as in cerebral spinal fluid samples from a different cohort of USA ME/CFS patients. To our knowledge, we are the first to test for levels of IL-16 in ME/CFS patients. In combination with previous data, our work suggests that the clustered reduction of IL-7, IL-16 and VEGF-A may have physiological relevance to ME/CFS disease. This profile is ME/CFS-specific since measurement of the same analytes present in chronic infectious and autoimmune liver diseases, where persistent fatigue is also a major symptom, failed to demonstrate the same changes. Further studies of other ME/CFS and overlapping disease cohorts are warranted in future

Investigating the antiviral role of cell death-inducing DFF45-like effector B in HCV replication

Cell-death-inducing DFF45-like effector B (CIDEB) is an apoptotic host factor, which was recently found to also regulate hepatic lipid homeostasis. Herein we delineate the relevance of these dual roles of CIDEB in apoptosis and lipid metabolism in the context of hepatitis C virus (HCV) replication. We demonstrate that HCV upregulates CIDEB expression in human serum differentiated hepatoma cells. CIDEB overexpression inhibits HCV replication in HCV replicon expressing Huh7.5 cells, while small interfering RNA knockdown of CIDEB expression in human serum differentiated hepatoma cells promotes HCV replication and secretion of viral proteins. Furthermore, we characterize a CIDEB mutant, KRRA, which is deficient in lipid droplet clustering and fusion and demonstrate that CIDEB-mediated inhibition of HCV is independent of the protein's lipid droplet fusogenic role. Our results suggest that higher levels of CIDEB expression, which favour an apoptotic role for the host factor, inhibit HCV. Collectively, our data demonstrate that CIDEB can act as an anti-HCV host factor and contribute to altered triglyceride homeostasis. Here, we illustrate that hepatitis C virus (HCV) replication activates the expression of the liver abundant host factor CIDEB with known regulatory roles in hepatic lipid homeostasis and apoptosis. We demonstrate that siRNA mediated knockdown CIDEB overexpression increase HCV replication and CIDEB knockdown inhibits HCV replication and this inhibition is independent of CIDEB's role in lipid droplet clustering and fusion.Peer reviewed: YesNRC publication: Ye

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 ( ≥ 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

Differential expression of interferon-lambda receptor 1 splice variants determines the magnitude of the antiviral response induced by interferon-lambda 3 in human immune cells.

Type III interferons (IFN-lambdas(λ)) are important cytokines that inhibit viruses and modulate immune responses by acting through a unique IFN-λR1/IL-10RB heterodimeric receptor. Until now, the primary antiviral function of IFN-λs has been proposed to be at anatomical barrier sites. Here, we examine the regulation of IFN-λR1 expression and measure the downstream effects of IFN-λ3 stimulation in primary human blood immune cells, compared with lung or liver epithelial cells. IFN-λ3 directly bound and upregulated IFN-stimulated gene (ISG) expression in freshly purified human B cells and CD8+ T cells, but not monocytes, neutrophils, natural killer cells, and CD4+ T cells. Despite similar IFNLR1 transcript levels in B cells and lung epithelial cells, lung epithelial cells bound more IFN-λ3, which resulted in a 50-fold greater ISG induction when compared to B cells. The reduced response of B cells could be explained by higher expression of the soluble variant of IFN-λR1 (sIFN-λR1), which significantly reduced ISG induction when added with IFN-λ3 to peripheral blood mononuclear cells or liver epithelial cells. T-cell receptor stimulation potently, and specifically, upregulated membrane-bound IFNLR1 expression in CD4+ T cells, leading to greater antiviral gene induction, and inhibition of human immunodeficiency virus type 1 infection. Collectively, our data demonstrate IFN-λ3 directly interacts with the human adaptive immune system, unlike what has been previously shown in published mouse models, and that type III IFNs could be potentially utilized to suppress both mucosal and blood-borne viral infections

Detailed Computational Study of the Active Site of the Hepatitis C Viral RNA Polymerase to Aid Novel Drug Design

The hepatitis C virus (HCV) RNA polymerase, NS5B, is a leading target for novel and selective HCV drug design. The enzyme has been the subject of intensive drug discovery aimed at developing direct acting antiviral (DAA) agents that inhibit its activity and hence prevent the virus from replicating its genome. In this study, we focus on one class of NS5B inhibitors, namely nucleos­(t)­ide mimetics. Forty-one distinct nucleotide structures have been modeled within the active site of NS5B for the six major HCV genotypes. Our comprehensive modeling protocol employed 287 different molecular dynamics simulations combined with the molecular mechanics/Poisson–Boltzmann surface area (MM-PBSA) methodology to rank and analyze these structures for all genotypes. The binding interactions of the individual compounds have been investigated and reduced to the atomic level. The present study significantly refines our understanding of the mode of action of NS5B-nucleotide-inhibitors, identifies the key structural elements necessary for their activity, and implements the tools for ranking the potential of additional much needed novel inhibitors of NS5B

GCV induced histopathological changes.

<p><b>A.</b> vTK+FVB/N mice that did not receive GCV showed normal liver histology. <b>B–C.</b> vTK+FVB/N mice that received 50 mg/kg GCV showed increased cytoplasmic and nuclear enlargement with increased acidophilic bodies (B, arrowhead), apoptotic bodies (B, arrow), and areas of confluent necrosis (C, arrows) (A–C:Hematoxylin and eosin, original magnification ×200). <b>D.</b> TUNEL immunostaining showing increased apoptotic nuclei (arrows) in livers post GCV (Immunoperoxidase, original magnification ×200).</p

Impact of vTK/GCV on non transplanted SCID/uPA mouse liver.

<p>Gross Appearance (upper panels) and H&E staining (lower panels) of livers from non-transplanted and age-matched vTK+SCID/uPA mice (3 months old) treated with 0, 25, 50 and 100 mg/kg GCV (i.p. every 48 h for 10 days). RN, regenerative nodules; PL, pale liver.</p

Histology of chimeric SCID/uPA liver at 12 weeks old.

<p>Engrafted human hepatocytes (HH, brown nuclei) in paraffin embedded liver sections were distinguished from mouse hepatocytes (MH, blue nuclei) and other murine constituents (M, blue nuclei) by hybridization with fluoresceinated Alu probe (i, left). Serial sections were analyzed by PAS staining (right) without (ii) or with (iii) prior diastase treatment. Ceroid macrophages (iii, arrowheads). (Original magnification ×50).</p