MicroRNA-Mediated Suppression of Oncolytic Adenovirus Replication in Human Liver

MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3′ untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5) in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.     

Chitosan Modification of Adenovirus to Modify Transfection Efficiency in Bovine Corneal Epithelial Cells

Background

The purpose of this study is to modulate the transfection efficiency of adenovirus (Ad) on the cornea by the covalent attachment of chitosan on adenoviral capsids via a thioether linkage between chitosan modified with 2-iminothiolane and Ad cross-linked with N-[γ-maleimidobutyryloxy]succinimide ester (GMBS).

Methodology/Principal Findings

Modified Ad was obtained by reaction with the heterobifunctional crosslinking reagent, GMBS, producing maleimide-modified Ad (Ad-GMBS). Then, the chitosan-SH was conjugated to Ad-GMBS via a thioether bond at different ratios of Ad to GMBS to chitosan-SH. The sizes and zeta potentials of unmodified Ad and chitosan-modified Ads were measured, and the morphologies of the virus particles were observed under transmission electron microscope. Primary cultures of bovine corneal epithelial cells were transfected with Ads and chitosan-modified Ads in the absence or presence of anti-adenovirus antibodies. Chitosan modification did not significantly change the particle size of Ad, but the surface charge of Ad increased significantly from −24.3 mV to nearly neutral. Furthermore, primary cultures of bovine corneal epithelial cells were transfected with Ad or chitosan-modified Ad in the absence or presence of anti-Ad antibodies. The transfection efficiency was attenuated gradually with increasing amounts of GMBS. However, incorporation of chitosan partly restored transfection activity and rendered the modified antibody resistant to antibody neutralization.

Conclusions/Significance

Chitosan can provide a platform for chemical modification of Ad, which offers potential for further in vivo applications.     

Pre-Clinical Development of a Recombinant,Replication-Competent Adenovirus Serotype 4 Vector Vaccine Expressing HIV-1 Envelope 1086 Clade C

Background

There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV-1 infection or limits in vivo viral replication. The objective of these studies is to develop a replication-competent, vaccine vector based on the adenovirus serotype 4 (Ad4) virus expressing HIV-1 envelope (Env) 1086 clade C glycoprotein. Ad4 recombinant vectors expressing Env gp160 (Ad4Env160), Env gp140 (Ad4Env140), and Env gp120 (Ad4Env120) were evaluated.

Methods

The recombinant Ad4 vectors were generated with a full deletion of the E3 region of Ad4 to accommodate the env gene sequences. The vaccine candidates were assessed in vitro following infection of A549 cells for Env-specific protein expression and for posttranslational transport to the cell surface as monitored by the binding of broadly neutralizing antibodies (bNAbs). The capacity of the Ad4Env vaccines to induce humoral immunity was evaluated in rabbits for Env gp140 and V1V2-specific binding antibodies, and HIV-1 pseudovirus neutralization. Mice immunized with the Ad4Env160 vaccine were assessed for IFNγ T cell responses specific for overlapping Env peptide sets.

Results

Robust Env protein expression was confirmed by western blot analysis and recognition of cell surface Env gp160 by multiple bNAbs. Ad4Env vaccines induced humoral immune responses in rabbits that recognized Env 1086 gp140 and V1V2 polypeptide sequences derived from 1086 clade C, A244 clade AE, and gp70 V1V2 CASE A2 clade B fusion protein. The immune sera efficiently neutralized tier 1 clade C pseudovirus MW965.26 and neutralized the homologous and heterologous tier 2 pseudoviruses to a lesser extent. Env-specific T cell responses were also induced in mice following Ad4Env160 vector immunization.

Conclusions

The Ad4Env vaccine vectors express high levels of Env glycoprotein and induce both Env-specific humoral and cellular immunity thus supporting further development of this new Ad4 HIV-1 Env vaccine platform in Phase 1 clinical trials.     

Down-regulation of microRNA-26a promotes mouse hepatocyte proliferation during liver regeneration

Background

Inadequate liver regeneration (LR) is still an unsolved problem in major liver resection and small-for-size syndrome post-living donor liver transplantation. A number of microRNAs have been shown to play important roles in cell proliferation. Herein, we investigated the role of miR-26a as a pivotal regulator of hepatocyte proliferation in LR.

Methodology/Principal Findings

Adult male C57BL/6J mice, undergoing 70% partial hepatectomy (PH), were treated with Ad5-anti-miR-26a-LUC or Ad5-miR-26a-LUC or Ad5-LUC vector via portal vein. The animals were subjected to in vivo bioluminescence imaging. Serum and liver samples were collected to test liver function, calculate liver-to-body weight ratio (LBWR), document hepatocyte proliferation (Ki-67 staining), and investigate potential targeted gene expression of miR-26a by quantitative real-time PCR and Western blot. The miR-26a level declined during LR after 70% PH. Down-regulation of miR-26a by anti-miR-26a expression led to enhanced proliferation of hepatocytes, and both LBWR and hepatocyte proliferation (Ki-67⁺ cells %) showed an increased tendency, while liver damage, indicated by aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total bilirubin (T-Bil), was reduced. Furthermore, CCND2 and CCNE2, as possible targeted genes of miR-26a, were up-regulated. In addition, miR-26a over-expression showed converse results.

Conclusions/Significance

MiR-26a plays crucial role in regulating the proliferative phase of LR, probably by repressing expressions of cell cycle proteins CCND2 and CCNE2. The current study reveals a novel miRNA-mediated regulation pattern during the proliferative phase of LR.     

Truncated Active Human Matrix Metalloproteinase-8 Delivered by a Chimeric Adenovirus-Hepatitis B Virus Vector Ameliorates Rat Liver Cirrhosis

Background

Liver cirrhosis is a potentially life-threatening disease caused by progressive displacement of functional hepatocytes by fibrous tissue. The underlying fibrosis is often driven by chronic infection with hepatitis B virus (HBV). Matrix metalloproteinases including MMP-8 are crucial for excess collagen degradation. In a rat model of liver cirrhosis, MMP-8 delivery by an adenovirus (Ad) vector achieved significant amelioration of fibrosis but application of Ad vectors in humans is subject to various issues, including a lack of intrinsic liver specificity.

Methods

HBV is highly liver-specific and its principal suitability as liver-specific gene transfer vector is established. HBV vectors have a limited insertion capacity and are replication-defective. Conversely, in an HBV infected cell vector replication may be rescued in trans by the resident virus, allowing conditional vector amplification and spreading. Capitalizing on a resident pathogen to help in its elimination and/or in treating its pathogenic consequences would provide a novel strategy. However, resident HBV may also reduce susceptibility to HBV vector superinfection. Thus a size-compatible truncated MMP-8 (tMMP8) gene was cloned into an HBV vector which was then used to generate a chimeric Ad-HBV shuttle vector that is not subject to superinfection exclusion. Rats with thioacetamide-induced liver cirrhosis were injected with the chimera to evaluate therapeutic efficacy.

Results

Our data demonstrate that infectious HBV vector particles can be obtained via trans-complementation by wild-type virus, and that the tMMP8 HBV vector can efficiently be shuttled by an Ad vector into cirrhotic rat livers. There it exerted a comparable beneficial effect on fibrosis and hepatocyte proliferation markers as a conventional full-length MMP-8Ad vector.

Conclusions

Though the rat cirrhosis model does not allow assessing in vivo HBV vector amplification these results advocate the further development of Ad-HBV vectors for liver-specific gene therapy, including and perhaps particularly for HBV-related disease.     

A Strategy for Adenovirus Vector Targeting with a Secreted Single Chain Antibody

Background

Successful gene therapy will require targeted delivery vectors capable of self-directed localization. In this regard, the use of antibodies or single chain antibody fragments (scFv) in conjunction with adenovirus (Ad) vectors remains an attractive means to achieve cell-specific targeting. However, a longstanding barrier to the development of Ad vectors with genetically incorporated scFvs has been the biosynthetic incompatibility between Ad capsid proteins and antibody-derived species. Specifically, scFv require posttranslational modifications not available to Ad capsid proteins due to their cytoplasmic routing during protein synthesis and virion assembly.

Methodology/Principal Findings

We have therefore sought to develop scFv-targeted Ad vectors using a secreted scFv that undergoes the requisite posttranslational modifications and is trafficked for secretion. Formation of the scFv-targeted Ad vector is achieved via highly specific association of the Ad virion and a targeting scFv employing synthetic leucine zipper-like dimerization domains (zippers) that have been optimized for structural compatibility with the Ad capsid and for association with the secreted scFv. Our results show that zipper-containing Ad fiber molecules trimerize and incorporate into mature virions and that zippers can be genetically fused to scFv without ablating target recognition. Most importantly, we show that zipper-tagged virions and scFv provide target-specific gene transfer.

Conclusions/Significance

This work describes a new approach to produce targeted Ad vectors using a secreted scFv molecule, thereby avoiding the problem of structural and biosynthetic incompatibility between Ad and a complex targeting ligand. This approach may facilitate Ad targeting using a wide variety of targeting ligands directed towards a variety of cellular receptors.     

Production of helper-dependent adenovirus vector relies on helper virus structure and complementing

Background

The helper‐dependent (HD) adenoviral (Ad) vector relies on a helper virus to provide viral proteins for vector amplification. HD‐Ad vectors can significantly increase therapeutic gene expression and improve safety. However, the yield of an HD‐Ad vector is generally lower than that of an E1‐deleted first‐generation vector, likely due to the alterations in viral E3 or packaging regions of a helper virus that attenuate its replication and complementing for an HD‐Ad vector.

Methods

To study this question and improve HD‐Ad vector production, we have generated four different helper viruses with a wild‐type or deleted E3 region, and with a relocated loxP. We have also constructed a first‐generation vector with a wild‐type E3 region and without the loxP site. We compared the replication of these viruses in Cre‐positive and ‐negative cells and studied their complementing for HD‐Ad vector production.

Results

Viruses with deleted E3 formed smaller plaques and produced lower titer compared with viruses containing the E3 region. The site where a loxP is inserted can also affect virus replication. Higher yield of HD‐Ad vector was obtained when a helper virus with wild‐type E3 was used. We also showed that deletion of the packaging signal in a helper virus through loxP/Cre interaction decreased the viral DNA complementing ability.

Conclusions

Although the E3 region is not essential for adenovirus replication in vivo, deletion of this region attenuates virus replication. Production of HD‐Ad vector can be further improved by modifications in helper virus structure. Copyright © 2002 John Wiley & Sons, Ltd.

     

Micro-RNA profiling in human serum reveals compartment-specific roles of miR-571 and miR-652 in liver cirrhosis

Background and Aims

Micro-RNAs (miRNAs) have recently emerged as crucial modulators of molecular processes involved in chronic liver diseases. The few miRNAs with previously proposed roles in liver cirrhosis were identified in screening approaches on liver parenchyma, mostly in rodent models. Therefore, in the present study we performed a systematic screening approach in order to identify miRNAs with altered levels in the serum of patients with chronic liver disease and liver cirrhosis.

Methods

We performed a systematic, array-based miRNA expression analysis on serum samples from patients with liver cirrhosis. In functional experiments we evaluated the relationship between alterations of miRNA serum levels and their role in distinct cellular compartments involved in hepatic cirrhosis.

Results

The array analysis and the subsequent confirmation by qPCR in a larger patient cohort identified significant alterations in serum levels of miR-513-3p, miR-571 and miR-652, three previously uncharacterized miRNAs, in patients with alcoholic or hepatitis C induced liver cirrhosis. Of these, miR-571 serum levels closely correlated with disease stages, thus revealing potential as a novel biomarker for hepatic cirrhosis. Further analysis revealed that up-regulation of miR-571 in serum reflected a concordant regulation in cirrhotic liver tissue. In isolated primary human liver cells, miR-571 was up-regulated in human hepatocytes and hepatic stellate cells in response to the pro-fibrogenic cytokine TGF-β. In contrast, alterations in serum levels of miR-652 were stage-independent, reflecting a concordant down-regulation of this miRNA in circulating monocytes of patients with liver cirrhosis, which was inducible by proinflammatory stimuli like bacterial lipopolysaccharide.

Conclusion

Alterations of miR571 and miR-652 serum levels in patients with chronic liver disease reflect their putative roles in the mediation of fibrogenic and inflammatory processes in distinct cellular compartments involved in the pathogenesis of liver cirrhosis.     

DNA Prime/Adenovirus Boost Malaria Vaccine Encoding P. falciparum CSP and AMA1 Induces Sterile Protection Associated with Cell-Mediated Immunity

Background

Gene-based vaccination using prime/boost regimens protects animals and humans against malaria, inducing cell-mediated responses that in animal models target liver stage malaria parasites. We tested a DNA prime/adenovirus boost malaria vaccine in a Phase 1 clinical trial with controlled human malaria infection.

Methodology/Principal Findings

The vaccine regimen was three monthly doses of two DNA plasmids (DNA) followed four months later by a single boost with two non-replicating human serotype 5 adenovirus vectors (Ad). The constructs encoded genes expressing P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1). The regimen was safe and well-tolerated, with mostly mild adverse events that occurred at the site of injection. Only one AE (diarrhea), possibly related to immunization, was severe (Grade 3), preventing daily activities. Four weeks after the Ad boost, 15 study subjects were challenged with P. falciparum sporozoites by mosquito bite, and four (27%) were sterilely protected. Antibody responses by ELISA rose after Ad boost but were low (CSP geometric mean titer 210, range 44–817; AMA1 geometric mean micrograms/milliliter 11.9, range 1.5–102) and were not associated with protection. Ex vivo IFN-γ ELISpot responses after Ad boost were modest (CSP geometric mean spot forming cells/million peripheral blood mononuclear cells 86, range 13–408; AMA1 348, range 88–1270) and were highest in three protected subjects. ELISpot responses to AMA1 were significantly associated with protection (p = 0.019). Flow cytometry identified predominant IFN-γ mono-secreting CD8+ T cell responses in three protected subjects. No subjects with high pre-existing anti-Ad5 neutralizing antibodies were protected but the association was not statistically significant.

Significance

The DNA/Ad regimen provided the highest sterile immunity achieved against malaria following immunization with a gene-based subunit vaccine (27%). Protection was associated with cell-mediated immunity to AMA1, with CSP probably contributing. Substituting a low seroprevalence vector for Ad5 and supplementing CSP/AMA1 with additional antigens may improve protection.

Trial Registration

ClinicalTrials.gov{"type":"clinical-trial","attrs":{"text":"NCT00870987","term_id":"NCT00870987"}}NCT00870987.     

Non-coding keratin variants associate with liver fibrosis progression in patients with hemochromatosis

Background

Keratins 8 and 18 (K8/K18) are intermediate filament proteins that protect the liver from various forms of injury. Exonic K8/K18 variants associate with adverse outcome in acute liver failure and with liver fibrosis progression in patients with chronic hepatitis C infection or primary biliary cirrhosis. Given the association of K8/K18 variants with end-stage liver disease and progression in several chronic liver disorders, we studied the importance of keratin variants in patients with hemochromatosis.

Methods

The entire K8/K18 exonic regions were analyzed in 162 hemochromatosis patients carrying homozygous C282Y HFE (hemochromatosis gene) mutations. 234 liver-healthy subjects were used as controls. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Previously-generated transgenic mice overexpressing K8 G62C were studied for their susceptibility to iron overload. Susceptibility to iron toxicity of primary hepatocytes that express K8 wild-type and G62C was also assessed.

Results

We identified amino-acid-altering keratin heterozygous variants in 10 of 162 hemochromatosis patients (6.2%) and non-coding heterozygous variants in 6 additional patients (3.7%). Two novel K8 variants (Q169E/R275W) were found. K8 R341H was the most common amino-acid altering variant (4 patients), and exclusively associated with an intronic KRT8 IVS7+10delC deletion. Intronic, but not amino-acid-altering variants associated with the development of liver fibrosis. In mice, or ex vivo, the K8 G62C variant did not affect iron-accumulation in response to iron-rich diet or the extent of iron-induced hepatocellular injury.

Conclusion

In patients with hemochromatosis, intronic but not exonic K8/K18 variants associate with liver fibrosis development.     

Interleukin-1 receptor antagonist modulates the early phase of liver regeneration after partial hepatectomy in mice

Background

Cytokine administration is a potential therapy for acute liver failure by reducing inflammatory responses and favour hepatocyte regeneration. The aim of this study was to evaluate the role of interleukin-1 receptor antagonist (IL-1ra) during liver regeneration and to study the effect of a recombinant human IL-1ra on liver regeneration.

Methods

We performed 70%-hepatectomy in wild type (WT) mice, IL-1ra knock-out (KO) mice and in WT mice treated by anakinra. We analyzed liver regeneration at regular intervals by measuring the blood levels of cytokines, the hepatocyte proliferation by bromodeoxyuridin (BrdU) incorporation, proliferating cell nuclear antigen (PCNA) and Cyclin D1 expression. The effect of anakinra on hepatocyte proliferation was also tested in vitro using human hepatocytes.

Results

At 24h and at 48h after hepatectomy, IL-1ra KO mice had significantly higher levels of pro-inflammatory cytokines (IL-6, IL-1β and MCP-1) and a reduced and delayed hepatocyte proliferation measured by BrdU incorporation, PCNA and Cyclin D1 protein levels, when compared to WT mice. IGFBP-1 and C/EBPβ expression was significantly decreased in IL-1ra KO compared to WT mice. WT mice treated with anakinra showed significantly decreased levels of IL-6 and significantly higher hepatocyte proliferation at 24h compared to untreated WT mice. In vitro, primary human hepatocytes treated with anakinra showed significantly higher proliferation at 24h compared to hepatocytes without treatment.

Conclusion

IL1ra modulates the early phase of liver regeneration by decreasing the inflammatory stress and accelerating the entry of hepatocytes in proliferation. IL1ra might be a therapeutic target to improve hepatocyte proliferation.     

Adenovirus-5-vectored P. falciparum vaccine expressing CSP and AMA1. Part B: safety, immunogenicity and protective efficacy of the CSP component

Background

A protective malaria vaccine will likely need to elicit both cell-mediated and antibody responses. As adenovirus vaccine vectors induce both these responses in humans, a Phase 1/2a clinical trial was conducted to evaluate the efficacy of an adenovirus serotype 5-vectored malaria vaccine against sporozoite challenge.

Methodology/Principal Findings

NMRC-MV-Ad-PfC is an adenovirus vector encoding the Plasmodium falciparum 3D7 circumsporozoite protein (CSP). It is one component of a two-component vaccine NMRC-M3V-Ad-PfCA consisting of one adenovector encoding CSP and one encoding apical membrane antigen-1 (AMA1) that was evaluated for safety and immunogenicity in an earlier study (see companion paper, Sedegah et al). Fourteen Ad5 seropositive or negative adults received two doses of NMRC-MV-Ad-PfC sixteen weeks apart, at particle units per dose. The vaccine was safe and well tolerated. All volunteers developed positive ELISpot responses by 28 days after the first immunization (geometric mean 272 spot forming cells/million[sfc/m]) that declined during the following 16 weeks and increased after the second dose to levels that in most cases were less than the initial peak (geometric mean 119 sfc/m). CD8+ predominated over CD4+ responses, as in the first clinical trial. Antibody responses were poor and like ELISpot responses increased after the second immunization but did not exceed the initial peak. Pre-existing neutralizing antibodies (NAb) to Ad5 did not affect the immunogenicity of the first dose, but the fold increase in NAb induced by the first dose was significantly associated with poorer antibody responses after the second dose, while ELISpot responses remained unaffected. When challenged by the bite of P. falciparum-infected mosquitoes, two of 11 volunteers showed a delay in the time to patency compared to infectivity controls, but no volunteers were sterilely protected.

Significance

The NMRC-MV-Ad-PfC vaccine expressing CSP was safe and well tolerated given as two doses, but did not provide sterile protection.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00392015","term_id":"NCT00392015"}}NCT00392015     

Ocular Localization and Transduction by Adenoviral Vectors Are Serotype-Dependent and Can Be Modified by Inclusion of RGD Fiber Modifications

Purpose

To evaluate localization and transgene expression from adenoviral vector of serotypes 5, 35, and 28, ± an RGD motif in the fiber following intravitreal or subretinal administration.

Methods

Ocular transduction by adenoviral vector serotypes ± RGD was studied in the eyes of mice receiving an intravitreous or subretinal injection. Each serotype expressed a CMV-GFP expression cassette and histological sections of eyes were examined. Transgene expression levels were examined using luciferase (Luc) regulated by the CMV promoter.

Results

GFP localization studies revealed that serotypes 5 and 28 given intravitreously transduced corneal endothelial, trabecular, and iris cells. Intravitreous delivery of the unmodified Ad35 serotype transduced only trabecular meshwork cells, but, the modification of the RGD motif into the fiber of the Ad35 viral vector base expanded transduction to corneal endothelial and iris cells. Incorporation of the RGD motif into the fiber knob with deletion of RGD from the penton base did not affect the transduction ability of the Ad5 vector base. Subretinal studies showed that RGD in the Ad5 knob shifted transduction from RPE cells to photoreceptor cells. Using a CMV-Luc expression cassette, intravitreous delivery of all the tested vectors, such as Ad5-, Ad35- and Ad28- resulted in an initial rapid induction of luciferase activity that thereafter declined. Subretinal administration of vectors showed a marked difference in transgene activity. Ad35-Luc gene expression peaked at 7 days and remained elevated for 6 months. Ad28-Luc expression was high after 1 day and remained sustained for one month.

Conclusions

Different adenoviral vector serotypes ± modifications transduce different cells within the eye. Transgene expression can be brief or extended and is serotype and delivery route dependent. Thus, adenoviral vectors provide a versatile platform for the delivery of therapeutic agents for ocular diseases.     

The Protective Effect of Intrasplenic Transplantation of Ad-IL-18BP/IL-4 Gene-Modified Fetal Hepatocytes on ConA-Induced Hepatitis in Mice

Background

Concanavalin A (ConA)-induced hepatitis is an experimental murine model mirroring the pathology of human autoimmune hepatitis.

Aim

To investigate the effects of intrasplenically transplanted fetal hepatocytes (BNL.CL2) transfected with recombinant adenovirus vector expressing the IL-18 binding protein (IL-18BP) and IL-4 fusion protein on ConA-induced hepatitis in mice.

Methods

Ad-IL-18BP/IL-4 was used to infect BNL.CL2 cells. IL-4 and IL-18BP fusion protein expression were detected by ELISA and Western blotting. BNL.CL2 cells infected with Ad-IL-18BP/IL-4 were intrasplenically transplanted into mice. After 10 days, mice were injected with ConA (15 mg/kg), and sacrificed 18 hours later. Liver injury was assessed by serum transaminase and liver histology. TNF-α, IL-18, IL-4, IL-10, IL-12p70 and monocyte-chemoattracting protein (MCP)-1 levels in serum and liver homogenates were detected by ELISA. Signaling molecules in liver homogenates were analyzed by Western blotting.

Results

Ad-IL-18BP/IL-4 effectively expressed the IL-18BP/IL-4 fusion protein for more than 14 days in BNL.CL12 cells. Treatment of mice with Ad-IL-18BP/IL-4-BNL.CL2 before ConA injection significantly reduced the elevated plasma levels of transaminases compared with ConA control groups. TNF-α, IL-18, IL-12p70 and MCP-1 levels in serum and liver homogenates from mice transplanted with Ad-IL-18BP/IL-4-BNL.CL2 were lower and IL-4 and IL-10 levels were higher than control groups. Phosphorylation levels of NF-κB p65, AKT, p38 and JNK1/2 in liver homogenates were markedly suppressed by Ad-IL-18BP/IL-4.

Conclusions

Ad-IL-18BP/IL-4 was effectively transfected into mouse BNL.CL2 cells. Intrasplenic transplantation of Ad-IL-18BP/IL-4-BNL.CL12 cells alleviated the severity of inflammation in ConA-induced experimental hepatitis and provides a useful basis for the targeted gene therapy of liver disease.     

Single Liver Lobe Repopulation with Wildtype Hepatocytes Using Regional Hepatic Irradiation Cures Jaundice in Gunn Rats

Background and Aims

Preparative hepatic irradiation (HIR), together with mitotic stimulation of hepatocytes, permits extensive hepatic repopulation by transplanted hepatocytes in rats and mice. However, whole liver HIR is associated with radiation-induced liver disease (RILD), which limits its potential therapeutic application. In clinical experience, restricting HIR to a fraction of the liver reduces the susceptibility to RILD. Here we test the hypothesis that repopulation of selected liver lobes by regional HIR should be sufficient to correct some inherited metabolic disorders.

Methods

Hepatocytes (10⁷) isolated from wildtype F344 rats or Wistar-RHA rats were engrafted into the livers of congeneic dipeptidylpeptidase IV deficient (DPPIV⁻) rats or uridinediphosphoglucuronateglucuronosyltransferase-1A1-deficient jaundiced Gunn rats respectively by intrasplenic injection 24 hr after HIR (50 Gy) targeted to the median lobe, or median plus left liver lobes. An adenovector expressing hepatocyte growth factor (10¹¹ particles) was injected intravenously 24 hr after transplantation.

Results

Three months after hepatocyte transplantation in DPPIV⁻ rats, 30–60% of the recipient hepatocytes were replaced by donor cells in the irradiated lobe, but not in the nonirradiated lobes. In Gunn rats receiving median lobe HIR, serum bilirubin declined from pretreatment levels of 5.17±0.78 mg/dl to 0.96±0.30 mg/dl in 8 weeks and remained at this level throughout the 16 week observation period. A similar effect was observed in the group, receiving median plus left lobe irradiation.

Conclusions

As little as 20% repopulation of 30% of the liver volume was sufficient to correct hyperbilirubinemia in Gunn rats, highlighting the potential of regiospecific HIR in hepatocyte transplantation-based therapy of inherited metabolic liver diseases.