Normothermic perfusion provides a means to rescue steatotic liver grafts including by pharmacological defatting. In this study, we tested the potential of new drug combinations to trigger defatting in three human culture models, primary hepatocytes with induced steatosis or isolated from steatotic liver, and precision-cut liver slices (PCLS) of steatotic liver. Forskolin, L-carnitine and a PPARα agonist, all were combined with rapamycin, an immunosuppressant that induces autophagy, in a D-FAT cocktail. D-FAT was tested alone or in combination with necrosulfonamide, an inhibitor of mixed lineage kinase domain-like involved in necroptosis. Within 24 hours in all three models, D-FAT induced a decrease in triglyceride content by 30%, attributable to an up-regulation of genes involved in free fatty acid β-oxidation and autophagy, and a down-regulation of those involved in lipogenesis. Defatting was accompanied by a decrease in endoplasmic reticulum stress and in the production of reactive oxygen species. The addition of necrosulfonamide increased the efficacy of defatting by 8%-12% in PCLS, with a trend towards increased autophagy. In conclusion, culture models notably PCLS are insightful to design strategies of liver graft rescue. Defatting can be rapidly achieved by combinations of drugs targeting mitochondrial oxidative metabolism, macro-autophagy, and lipogenesis.

Chronic hepatitis C is a major cause of liver fibrosis and cirrhosis. It is generally accepted that inflammation that occurs in response to hepatocyte infection by the hepatitis C virus (HCV) is the main mechanism that triggers myofibroblast differentiation and stimulation in chronic hepatitis C. The aim of this study was to determine if HCV might infect human liver myofibroblasts (HLMF) and directly stimulate their fibrogenic activities. We evaluated the expression of the viral entry receptors, levels of HCV-RNA and HCV-protein and the expression of fibrosis markers in HLMF by using quantitative PCR, western blot and immunofluorescence analyses. Pseudoparticles (HCVpp) and cell culture–derived HCV (HCVcc) were used to study the ability of HLMF to support viral entry, replication and fibrosis induction. We showed that HLMF expressed all known molecules of the HCV receptor complex, i.e. CD81, LDL-R, scavenger receptor-BI, claudin-1 and occludin. These cells were also permissive to HCVpp entry. Inoculation with HCVcc caused short-term infection of these cells, as shown by their content in positive- and negative-strand HCV RNA, in core and NS3 viral proteins, and by their release of core protein levels in the culture supernatants. HCV infection stimulated myofibroblastic differentiation, proliferation and collagen production in these cells. In addition, evidence of in vivo infection was provided by the detection of positive- and negative-strand HCV RNA in preparations of HLMF obtained from HCV-infected patients. These findings indicate that HCV infection of HLMF can occur and trigger extracellular matrix overproduction, thereby contributing to the development of HCV-related liver fibrosis.

The ongoing organ shortage has forced transplant teams to develop alternate sources of liver grafts. In this setting, ex-situ machine perfusion has rapidly developed as a promising tool to assess viability and improve the function of organs from extended criteria donors, including fatty liver grafts. In particular, normothermic machine perfusion represents a powerful tool to test a liver in full 37 °C metabolism and add pharmacological corrections whenever needed. In this context, many pharmacological agents and therapeutics have been tested to induce liver defatting on normothermic machine perfusion with promising results even on human organs. This systematic review makes a comprehensive synthesis on existing pharmacological therapies for liver defatting, with special focus on normothermic liver machine perfusion as an experimental ex-vivo translational model.

Background: The role of regulatory T cells (Tregs) is now well established in the progression of hepatocellular carcinoma (HCC) linked to Hepatitis C virus (HCV) infection. However, nothing is known about the potential interplay between Tregs and HCV. In this pilot study, we have investigated the ability of Tregs to hang HCV on and the subsequent effect on their suppressive function and phenotype. Moreover, we have evaluated how HCV could promote the recruitment of Tregs by infected primary human hepatocytes. Methods: Tregs of healthy donors were incubated with JFH-1/HCVcc. Viral inoculation was assessed using adapted assays (RT-qPCR, Flow Citometry (FACS) and Western Blot (WB). Expression of Tregs phenotypic (CD4, CD25, CD127 and Foxp3) and functional (IL-10, GZMB, TGF-β1 and IL-2) markers was monitored by RT-qPCR, FACS and ELISA. Suppressive activity was validated by suppressive assays. Tregs recruitment by infected primary hepatic cells was evaluated using Boyden Chamber. Results: Tregs express the classical HCV receptors (CD81, CLDN1 and LDLR) and some co-receptors (CD5). HCV inoculation significantly increases the suppressive phenotype and activity of Tregs, and raises their anergy by inducing an unexpected IL-2 production. Moreover, HCV infection induces the expression of chemokines (CCL17, CXCL16, and CCL20) by primary hepatic human hepatocytes and chemokine receptors (CCR4, CXCR6 and CCR6) by Tregs. Finally, infected hepatocytes have a significantly higher potential to recruit Tregs in a seemingly CCL20-dependent manner. Conclusions: Direct interaction between HCV and Tregs represents a newly defined mechanism that could potentiate HCV immune evasion and favor intratumoral recruitment contributing to HCC progression.

The heregulin-1ß/HER3-driven pathway is implicated in several epithelial malignancies and its blockade is currently undergoing clinical investigation. Paradoxically, the status and the regulation of this pathway is poorly known in hepatocellular carcinoma (HCC). Using 85 HCC obtained after tumour resection, heregulin-1ß and HER3 expression was evaluated by real-time RT-PCR, ELISA and/or immunohistochemistry. Statistics were performed to analyze associations between gene expression and clinicopathological parameters. The effects of insulin on the heregulin-1ß/HER3 pathway was investigated in four HCC cell lines. HER3 mRNA was upregulated in 52 % of tumours, while heregulin-1ß mRNA was downregulated in 82 %. Hepatitis B and C viral infections were respectively associated with high and low HER3 mRNA expression. No association was seen between neither HER3 or heregulin-1ß mRNA and prognostic factors, survival or recurrence. Immunohistochemistry showed predominant cytoplasmic staining of HER3 in tumours but the staining was nonreproducible. HER3 mRNA and protein levels were not correlated in liver tissues. In HCC cells, insulin promoted HER3 proteasomal degradation and inhibited heregulin-1ß stimulation of cell migration. HER3 and insulin receptor co-immunoprecipitated in these cells. The loss of insulin receptor expression by RNA interference sensitized cells to heregulin-1ß-induced AKT phosphorylation. Autocrine heregulin-1ß loop is uncommon in HCC and HER3 mRNA expression is differentially influenced by hepatitis viruses. Insulin is a negative regulator of HER3 protein expression and function in HCC cells. Altogether these data may explain why HER3 and heregulin-1ß expression have no prognostic value and suggest that HCC patients are unlikely to derive benefit from HER3-targeted monotherapies.

Photodynamic therapy (PDT) is a two-stage treatment relying on cytotoxicity induced by photoexcitation of a nontoxic dye, called photosensitizer (PS). Using 5-aminolevulinic acid (5-ALA), the pro-drug of PS protoporphyrin IX, we investigated the impact of PDT on hepatocellular carcinoma (HCC). Optimal 5-ALA PDT dose was determined on three HCC cell lines by analyzing cell death after treatment with varying doses. HCC-patient-derived tumor hepatocytes and healthy donor liver myofibroblasts were treated with optimal 5-ALA PDT doses. The proliferation of cancer cells and healthy donor immune cells cultured with 5-ALA-PDT-treated conditioned media was analyzed. Finally, therapy efficacy on humanized SCID mice model of HCC was investigated. 5-ALA PDT induced a dose-dependent decrease in viability, with an up-to-four-fold reduction in viability of patient tumor hepatocytes. The 5-ALA PDT treated conditioned media induced immune cell clonal expansion. 5-ALA PDT has no impact on myofibroblasts in terms of viability, while their activation decreased cancer cell proliferation and reduced the tumor growth rate of the in vivo model. For the first time, 5-ALA PDT has been validated on primary patient tumor hepatocytes and donor healthy liver myofibroblasts. 5-ALA PDT may be an effective anti-HCC therapy, which might induce an anti-tumor immune response.