EnGraft: a multicentre, open-label, randomised, two-arm, superiority study protocol to assess bioavailability and practicability of Envarsus® versus Advagraf™ in liver transplant recipients

Background Graft rejection and chronic CNI toxicity remain obstacles to organ transplant success. Current formulations of tacrolimus, such as Prograf® and Advagraf™, exhibit limitations in terms of pharmacokinetics and tolerability, related in part to suboptimal bioavailability. As dosing non-compliance can result in graft rejection, the once daily formulation of tacrolimus, Advagraf™, was developed (vs 2x/day Prograf®). Benefits of Advagraf™ are counterbalanced by delayed achievement of therapeutic trough levels and need for up to 50% higher doses to maintain Prograf®-equivalent troughs. Envarsus® is also a prolonged-release once-daily tacrolimus formulation, developed using MeltDose™ drug-delivery technology to increase drug bioavailability; improved bioavailability results in low patient drug absorption variability and less pronounced peak-to-trough fluctuations. In phase III de novo kidney transplant studies, Envarsus® proved non-inferior to twice-daily tacrolimus; however, no phase IV studies show superiority of Envarsus® vs Advagraf™ in de novo liver transplant (LTx) recipients. Methods The EnGraft compares bioavailability and tests superiority of Envarsus® (test arm) versus Advagraf™ (comparator arm) in de novo LTx recipients. A total of 268 patients from 15 German transplant centres will be randomised 1:1 within 14 days post-LTx. The primary endpoint is dose-normalised trough level (C/D ratio) measured 12 weeks after randomisation. Secondary endpoints include the number of dose adjustments, time to reach first defined trough level and incidence of graft rejections. Additionally, clinical and laboratory parameters will be assessed over a 3-year period. Discussion C/D ratio is an estimate for tacrolimus bioavailability. Improving bioavailability and increasing C/D ratio using Envarsus could reduce renal dysfunction and other tacrolimus-related toxicities; previous trials have shown that a higher C/D ratio (i.e. slower tacrolimus metabolism) is not only associated with improved renal function but also linked to reduced neurotoxic side effects. A higher C/D ratio could improve clinical outcomes for LTx recipients; EnGraft has begun, with one third of patients recruited by January 2022

The ATGL lipase cooperates with ABHD5 to mobilize lipids for hepatitis C virus assembly.

Lipid droplets are essential cellular organelles for storage of fatty acids and triglycerides. The hepatitis C virus (HCV) translocates several of its proteins onto their surface and uses them for production of infectious progeny. We recently reported that the lipid droplet-associated α/β hydrolase domain-containing protein 5 (ABHD5/CGI-58) participates in HCV assembly by mobilizing lipid droplet-associated lipids. However, ABHD5 itself has no lipase activity and it remained unclear how ABHD5 mediates lipolysis critical for HCV assembly. Here, we identify adipose triglyceride lipase (ATGL) as ABHD5 effector and new host factor involved in the hepatic lipid droplet degradation as well as in HCV and lipoprotein morphogenesis. Modulation of ATGL protein expression and lipase activity controlled lipid droplet lipolysis and virus production. ABHD4 is a paralog of ABHD5 unable to activate ATGL or support HCV assembly and lipid droplet lipolysis. Grafting ABHD5 residues critical for activation of ATGL onto ABHD4 restored the interaction between lipase and co-lipase and bestowed the pro-viral and lipolytic functions onto the engineered protein. Congruently, mutation of the predicted ABHD5 protein interface to ATGL ablated ABHD5 functions in lipid droplet lipolysis and HCV assembly. Interestingly, minor alleles of ABHD5 and ATGL associated with neutral lipid storage diseases in human, are also impaired in lipid droplet lipolysis and their pro-viral functions. Collectively, these results show that ABHD5 cooperates with ATGL to mobilize triglycerides for HCV infectious virus production. Moreover, viral manipulation of lipid droplet homeostasis via the ABHD5-ATGL axis, akin to natural genetic variation in these proteins, emerges as a possible mechanism by which chronic HCV infection causes liver steatosis

Efficient acute and chronic infection of stem cell-derived hepatocytes by hepatitis C virus.

Transcriptional profiling revealed that HLCs constitutively express messenger RNA of RLRs, and members of the IFN pathway. Moreover, HLCs upregulated IFNs and canonical interferon-regulated genes (IRGs) upon transfection with the double-stranded RNA mimic poly(I:C). Infection of HLCs with Jc1-HCVcc produced only limited viral progeny. In contrast, infection with p100, a Jc1-derived virus population with enhanced replication fitness and partial resistance to IFN, resulted in robust yet transient viraemia. Viral titres declined concomitant with a peak of IRG induction. Addition of ruxolitinib, a JAK/STAT inhibitor, permitted chronic infection and raised p100 infectious virus titres to 1×105 FFU/mL. IRGs expression profiling in infected HLCs revealed a landscape of HCV-dependent transcriptional changes similar to HCV-infected primary human hepatocytes, but distinct from Huh-7.5 cells. Withdrawal of ruxolitinib restored innate immune responses and resulted in HCV clearance

Risk-Adjusted Analysis of Relevant Outcome Drivers for Patients after More Than Two Kidney Transplants

Renal transplantation is the treatment of choice for patients suffering end-stage renal disease, but as the long-term renal allograft survival is limited, most transplant recipients will face graft loss and will be considered for a retransplantation. The goal of this study was to evaluate the patient and graft survival of the 61 renal transplant recipients after second or subsequent renal transplantation, transplanted in our institution between 1990 and 2010, and to identify risk factors related to inferior outcomes. Actuarial patient survival was 98.3%, 94.8%, and 88.2% after one, three, and five years, respectively. Actuarial graft survival was 86.8%, 80%, and 78.1% after one, three, and five years, respectively. Risk-adjusted analysis revealed that only age at the time of last transplantation had a significant influence on patient survival, whereas graft survival was influenced by multiple immunological and surgical factors, such as the number of HLA mismatches, the type of immunosuppression, the number of surgical complications, need of reoperation, primary graft nonfunction, and acute rejection episodes. In conclusion, third and subsequent renal transplantation constitute a valid therapeutic option, but inferior outcomes should be expected among elderly patients, hyperimmunized recipients, and recipients with multiple operations at the site of last renal transplantation

Extended hepatic metastasectomy for renal cell carcinoma—new aspects in times of targeted therapy: a single-center experience over three decades

Purpose!#!Despite the introduction of novel targeted therapies on patients with renal cell carcinoma, syn- and metachronous metastases (including hepatic lesions) are observed frequently and significantly influence patient survival. With introduction of targeted therapies as an effective alternative to surgery, therapeutical strategies in stage IV disease must be reevaluated.!##!Methods!#!This is a retrospective analysis of 40 patients undergoing hepatic resection of histologically confirmed RCC metastases at our institution between April 1993 and April 2017.!##!Results!#!The interval between nephrectomy for renal cell carcinoma and hepatic metastasectomy was 44.0 months (3.3-278.5). Liver resections of different extents were performed, including multivisceral resections. The median follow-up was 37.8 months (0.5-286.5). Tumor recurrence after resection of hepatic metastases occurred in 19 patients resulting in a median disease-free survival of 16.2 months (0.7-265.1) and a median overall survival of 37.8 months (0.5-286.5). Multivariable analysis identified multivisceral resection as an independent risk factor for disease-free and overall survival (p = 0.043 and p = 0.001, respectively). A longer interval between nephrectomy and hepatic metastasectomy was identified as an independent significant protective factor for overall survival (p < 0.001). Patients undergoing metastasectomy after introduction of sunitinib in Europe in 2006 (n = 15) showed a significantly longer overall survival (45.2 (9.1-111.0) versus 27.5 (0.5-286.52) months in the preceding era; p = 0.038).!##!Conclusion!#!Hepatic metastasectomy, including major and extended resections, on patients with metastasized renal cell carcinoma can be performed safely and may facilitate long-term survival. Due to significant morbidity and increased mortality, multivisceral resections must be weighed against other options, such as targeted therapy

The importance of MHC class II in allogeneic bone marrow transplantation and chimerism-based solid organ tolerance in a rat model.

Mixed hematopoietic chimerism enables donor-specific tolerance for solid organ grafts. This study evaluated the influence of different serological major histocompatibility complex disparities on chimerism development, graft-versus-host disease incidence and subsequently on solid organ tolerance in a rat model. For bone marrow transplantation conditioning total body irradiation was titrated using 10, 8 or 6 Gray. Bone marrow transplantation was performed across following major histocompatibility complex mismatched barriers: complete disparity, MHC class II, MHC class I or non-MHC mismatch. Recipients were clinically monitored for graft-versus-host disease and analyzed for chimerism using flow cytometry. After a reconstitution of 100 days, composition of peripheral leukocytes was determined. Mixed chimeras were challenged with heart grafts from allogeneic donor strains to define the impact of donor MHC class disparities on solid organ tolerance on the basis of stable chimerism. After myeloablation with 10 Gray of total body irradiation, chimerism after bone marrow transplantation was induced independent of MHC disparity. MHC class II disparity increased the incidence of graft-versus-host disease and reduced induction of stable chimerism upon myelosuppressive total body irradiation with 8 and 6 Gray, respectively. Stable mixed chimeras showed tolerance towards heart grafts from donors with MHC matched to either bone marrow donors or recipients. Isolated matching of MHC class II with bone marrow donors likewise led to stable tolerance as opposed to matching of MHC class I. In summary, MHC class II disparity was critically associated with the onset of graft-versus host disease and was identified as obstacle for successful development of chimerism after bone marrow transplantation and subsequent donor-specific solid organ tolerance

Characterization of RNA Sensing Pathways in Hepatoma Cell Lines and Primary Human Hepatocytes.

The liver is targeted by several human pathogenic RNA viruses for viral replication and dissemination; despite this, the extent of innate immune sensing of RNA viruses by human hepatocytes is insufficiently understood to date. In particular, for highly human tropic viruses such as hepatitis C virus, cell culture models are needed to study immune sensing. However, several human hepatoma cell lines have impaired RNA sensing pathways and fail to mimic innate immune responses in the human liver. Here we compare the RNA sensing properties of six human hepatoma cell lines, namely Huh-6, Huh-7, HepG2, HepG2-HFL, Hep3B, and HepaRG, with primary human hepatocytes. We show that primary liver cells sense RNA through retinoic acid-inducible gene I (RIG-I) like receptor (RLR) and Toll-like receptor 3 (TLR3) pathways. Of the tested cell lines, Hep3B cells most closely mimicked the RLR and TLR3 mediated sensing in primary hepatocytes. This was shown by the expression of RLRs and TLR3 as well as the expression and release of bioactive interferon in primary hepatocytes and Hep3B cells. Our work shows that Hep3B cells partially mimic RNA sensing in primary hepatocytes and thus can serve as in vitro model to study innate immunity to RNA viruses in hepatocytes

ABHD5/CGI-58, the Chanarin-Dorfman Syndrome Protein, Mobilises Lipid Stores for Hepatitis C Virus Production

Hepatitis C virus (HCV) particles closely mimic human very-low-density lipoproteins (VLDL) to evade humoral immunity and to facilitate cell entry. However, the principles that govern HCV association with VLDL components are poorly defined. Using an siRNA screen, we identified ABHD5 (α/β hydrolase domain containing protein 5, also known as CGI-58) as a new host factor promoting both virus assembly and release. ABHD5 associated with lipid droplets and triggered their hydrolysis. Importantly, ABHD5 Chanarin-Dorfman syndrome mutants responsible for a rare lipid storage disorder in humans were mislocalised, and unable to consume lipid droplets or support HCV production. Additional ABHD5 mutagenesis revealed a novel tribasic motif that does not influence subcellular localization but determines both ABHD5 lipolytic and proviral properties. These results indicate that HCV taps into the lipid droplet triglyceride reservoir usurping ABHD5 lipase cofactor function. They also suggest that the resulting lipid flux, normally devoted to VLDL synthesis, also participates in the assembly and release of the HCV lipo-viro-particle. Altogether, our study provides the first association between the Chanarin-Dorfman syndrome protein and an infectious disease and sheds light on the hepatic manifestations of this rare genetic disorder as well as on HCV morphogenesis

Hepatitis C Virus Strain-Dependent Usage of Apolipoprotein E Modulates Assembly Efficiency and Specific Infectivity of Secreted Virions.

Hepatitis C virus (HCV) is extraordinarily diverse and uses entry factors in a strain-specific manner. Virus particles associate with lipoproteins, and apolipoprotein E (ApoE) is critical for HCV assembly and infectivity. However, whether ApoE dependency is common to all HCV genotypes remains unknown. Therefore, we compared the roles of ApoE utilizing 10 virus strains from genotypes 1 through 7. ApoA and ApoC also support HCV assembly, so they may contribute to virus production in a strain-dependent fashion. Transcriptome sequencing (RNA-seq) revealed abundant coexpression of ApoE, ApoB, ApoA1, ApoA2, ApoC1, ApoC2, and ApoC3 in primary hepatocytes and in Huh-7.5 cells. Virus production was examined in Huh-7.5 cells with and without ApoE expression and in 293T cells where individual apolipoproteins (ApoE1, -E2, -E3, -A1, -A2, -C1, and -C3) were provided in trans All strains were strictly ApoE dependent. However, ApoE involvement in virus production was strain and cell type specific, because some HCV strains poorly produced infectious virus in ApoE-expressing 293T cells and because ApoE knockout differentially affected virus production of HCV strains in Huh-7.5 cells. ApoE allelic isoforms (ApoE2, -E3, and -E4) complemented virus production of HCV strains to comparable degrees. All tested strains assembled infectious progeny with ApoE in preference to other exchangeable apolipoproteins (ApoA1, -A2, -C1, and -C3). The specific infectivity of HCV particles was similar for 293T- and Huh-7.5-derived particles for most strains; however, it differed by more than 100-fold in some viruses. Collectively, this study reveals strain-dependent and host cell-dependent use of ApoE during HCV assembly. These differences relate to the efficacy of virus production and also to the properties of released virus particles and therefore govern viral fitness at the level of assembly and cell entry.IMPORTANCE Chronic HCV infections are a major cause of liver disease. HCV is highly variable, and strain-specific determinants modulate the response to antiviral therapy, the natural course of infection, and cell entry factor usage. Here we explored whether host factor dependency of HCV in particle assembly is modulated by strain-dependent viral properties. We showed that all examined HCV strains, which represent all seven known genotypes, rely on ApoE expression for assembly of infectious progeny. However, the degree of ApoE dependence is modulated in a strain-specific and cell type-dependent manner. This indicates that HCV strains differ in their assembly properties and host factor usage during assembly of infectious progeny. Importantly, these differences relate not only to the efficiency of virus production and release but also to the infectiousness of virus particles. Thus, strain-dependent features of HCV modulate ApoE usage, with implications for virus fitness at the level of assembly and cell entry