Phosphatidylinositol 4-Kinase III Alpha Governs Cytoskeletal Organization for Invasiveness of Liver Cancer Cells.

BACKGROUND & AIMS: High expression of phosphatidylinositol 4-kinase III alpha (PI4KIIIα) correlates with poor survival rates in patients with hepatocellular carcinoma. In addition, hepatitis C virus (HCV) infections activate PI4KIIIα and contribute to hepatocellular carcinoma progression. We aimed at mechanistically understanding the impact of PI4KIIIα on the progression of liver cancer and the potential contribution of HCV in this process. METHODS: Several hepatic cell culture and mouse models were used to study the functional importance of PI4KIIIα on liver pathogenesis. Antibody arrays, gene silencing, and PI4KIIIα-specific inhibitor were applied to identify the involved signaling pathways. The contribution of HCV was examined by using HCV infection or overexpression of its nonstructural protein. RESULTS: High PI4KIIIα expression and/or activity induced cytoskeletal rearrangements via increased phosphorylation of paxillin and cofilin. This led to morphologic alterations and higher migratory and invasive properties of liver cancer cells. We further identified the liver-specific lipid kinase phosphatidylinositol 3-kinase C2 domain-containing subunit gamma (PIK3C2γ) working downstream of PI4KIIIα in regulation of the cytoskeleton. PIK3C2γ generates plasma membrane phosphatidylinositol 3,4-bisphosphate-enriched, invadopodia-like structures that regulate cytoskeletal reorganization by promoting Akt2 phosphorylation. CONCLUSIONS: PI4KIIIα regulates cytoskeleton organization via PIK3C2γ/Akt2/paxillin-cofilin to favor migration and invasion of liver cancer cells. These findings provide mechanistic insight into the contribution of PI4KIIIα and HCV to the progression of liver cancer and identify promising targets for therapeutic intervention.

Unraveling the dynamics of hepatitis C virus adaptive mutations and their impact on antiviral responses in primary human hepatocytes.

Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture-adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants that underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establish persistence. IMPORTANCE: Hepatitis C virus (HCV) infection remains a global health burden with 58 million people currently chronically infected. However, the detailed molecular mechanisms that underly persistence are incompletely defined. We utilized a long-term cell culture-adapted HCV, exhibiting enhanced replicative fitness in different human liver cell lines, in order to identify molecular principles by which HCV optimizes its replication fitness. Our experimental data revealed that cell culture adaptive mutations confer changes in the host response and usage of various host factors. The latter allows functional flexibility at different stages of the viral replication cycle. However, increased replicative fitness resulted in an increased activation of the innate immune system, which likely poses boundary for functional variation in authentic hepatocytes, explaining the observed attenuation of the adapted virus population in primary hepatocytes.

The matrix metalloproteinase ADAM10 supports hepatitis C virus entry and cell-to-cell spread via its sheddase activity.

Hepatitis C virus (HCV) exploits the four entry factors CD81, scavenger receptor class B type I (SR-BI, also known as SCARB1), occludin, and claudin-1 as well as the co-factor epidermal growth factor receptor (EGFR) to infect human hepatocytes. Here, we report that the disintegrin and matrix metalloproteinase 10 (ADAM10) associates with CD81, SR-BI, and EGFR and acts as HCV host factor. Pharmacological inhibition, siRNA-mediated silencing and genetic ablation of ADAM10 reduced HCV infection. ADAM10 was dispensable for HCV replication but supported HCV entry and cell-to-cell spread. Substrates of the ADAM10 sheddase including epidermal growth factor (EGF) and E-cadherin, which activate EGFR family members, rescued HCV infection of ADAM10 knockout cells. ADAM10 did not influence infection with other enveloped RNA viruses such as alphaviruses and a common cold coronavirus. Collectively, our study reveals a critical role for the sheddase ADAM10 as a HCV host factor, contributing to EGFR family member transactivation and as a consequence to HCV uptake.

A ribavirin-induced ORF2 single-nucleotide variant produces defective hepatitis E virus particles with immune decoy function.

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and is the leading cause of enterically transmitted viral hepatitis worldwide. Ribavirin (RBV) is currently the only treatment option for many patients; however, cases of treatment failures or posttreatment relapses have been frequently reported. RBV therapy was shown to be associated with an increase in HEV genome heterogeneity and the emergence of distinct HEV variants. In this study, we analyzed the impact of eight patient-derived open reading frame 2 (ORF2) single-nucleotide variants (SNVs), which occurred under RBV treatment, on the replication cycle and pathogenesis of HEV. The parental HEV strain and seven ORF2 variants showed comparable levels of RNA replication in human hepatoma cells and primary human hepatocytes. However, a P79S ORF2 variant demonstrated reduced RNA copy numbers released in the supernatant and an impairment in the production of infectious particles. Biophysical and biochemical characterization revealed that this SNV caused defective, smaller HEV particles with a loss of infectiousness. Furthermore, the P79S variant displayed an altered subcellular distribution of the ORF2 protein and was able to interfere with antibody-mediated neutralization of HEV in a competition assay. In conclusion, an SNV in the HEV ORF2 could be identified that resulted in altered virus particles that were noninfectious in vitro and in vivo, but could potentially serve as immune decoys. These findings provide insights in understanding the biology of circulating HEV variants and may guide development of personalized antiviral strategies in the future.

Transplanting old organs promotes senescence in young recipients.

In clinical organ transplantation, donor and recipient ages may differ substantially. Old donor organs accumulate senescent cells that have the capacity to induce senescence in naïve cells. We hypothesized that the engraftment of old organs may induce senescence in younger recipients, promoting age-related pathologies. When performing isogeneic cardiac transplants between age-mismatched C57BL/6 old donor (18 months) mice and young and middle-aged C57BL/6 (3- or 12- month-old) recipients , we observed augmented frequencies of senescent cells in draining lymph nodes, adipose tissue, livers, and hindlimb muscles 30 days after transplantation. These observations went along with compromised physical performance and impaired spatial learning and memory abilities. Systemic levels of the senescence-associated secretory phenotype factors, including mitochondrial DNA (mt-DNA), were elevated in recipients. Of mechanistic relevance, injections of mt-DNA phenocopied effects of age-mismatched organ transplantation on accelerating aging. Single treatment of old donor animals with senolytics prior to transplantation attenuated mt-DNA release and improved physical capacities in young recipients. Collectively, we show that transplanting older organs induces senescence in transplant recipients, resulting in compromised physical and cognitive capacities. Depleting senescent cells with senolytics, in turn, represents a promising approach to improve outcomes of older organs.

EGF receptor modulates HEV entry in human hepatocytes.

BACKGROUND AND AIMS: Being the most common cause of acute viral hepatitis with >20 million cases per year and 70,000 deaths annually, HEV presents a long-neglected and underinvestigated health burden. Although the entry process of viral particles is an attractive target for pharmacological intervention, druggable host factors to restrict HEV entry have not been identified so far. APPROACH AND RESULTS: Here we identify the EGF receptor (EGFR) as a novel host factor for HEV and reveal the significance of EGFR for the HEV entry process. By utilizing RNAi, chemical modulation with Food and Drug Administration-approved drugs, and ectopic expression of EGFR, we revealed that EGFR is critical for HEV infection without affecting HEV RNA replication or assembly of progeny virus. We further unveiled that EGFR itself and its ligand-binding domain, rather than its signaling function, is responsible for the proviral effect. Modulation of EGF expression in HepaRG cells and primary human hepatocytes affected HEV infection. CONCLUSIONS: Taken together, our study provides novel insights into the life cycle of HEV and identified EGFR as a possible target for future antiviral strategies against HEV.

The Human Liver-Expressed Lectin CD302 Restricts Hepatitis C Virus Infection.

C-type lectin domain-containing proteins (CTLDcps) shape host responses to pathogens and infectious disease outcomes. Previously, we identified the murine CTLDcp Cd302 as restriction factor, limiting hepatitis C virus (HCV) infection of murine hepatocytes. In this study, we investigated in detail the human orthologue's ability to restrict HCV infection in human liver cells. CD302 overexpression in Huh-7.5 cells potently inhibited infection of diverse HCV chimeras representing seven genotypes. Transcriptional profiling revealed abundant CD302 mRNA expression in human hepatocytes, the natural cellular target of HCV. Knockdown of endogenously expressed CD302 modestly enhanced HCV infection of Huh-7.5 cells and primary human hepatocytes. Functional analysis of naturally occurring CD302 transcript variants and engineered CD302 mutants showed that the C-type lectin-like domain (CTLD) is essential for HCV restriction, whereas the cytoplasmic domain (CPD) is dispensable. Coding single nucleotide polymorphisms occurring in human populations and mapping to different domains of CD302 did not influence the capacity of CD302 to restrict HCV. Assessment of the anti-HCV phenotype at different life cycle stages indicated that CD302 preferentially targets the viral entry step. In contrast to the murine orthologue, overexpression of human CD302 did not modulate downstream expression of nuclear receptor-controlled genes. Ectopic CD302 expression restricted infection of liver tropic hepatitis E virus (HEV), while it did not affect infection rates of two respiratory viruses, including respiratory syncytial virus (RSV) and the alpha coronavirus HVCoV-229E. Together, these findings suggest that CD302 contributes to liver cell-intrinsic defense against HCV and might mediate broader antiviral defenses against additional hepatotropic viruses. IMPORTANCE The liver represents an immunoprivileged organ characterized by enhanced resistance to immune responses. However, the importance of liver cell-endogenous, noncytolytic innate immune responses in pathogen control is not well defined. Although the role of myeloid cell-expressed CTLDcps in host responses to viruses has been characterized in detail, we have little information about their potential functions in the liver and their relevance for immune responses in this organ. Human hepatocytes endogenously express the CTLDcp CD302. Here, we provide evidence that CD302 limits HCV infection of human liver cells, likely by inhibiting a viral cell entry step. We confirm that the dominant liver-expressed transcript variant, as well as naturally occurring coding variants of CD302, maintain the capacity to restrict HCV. We further show that the CTLD of the protein is critical for the anti-HCV activity and that overexpressed CD302 limits HEV infection. Thus, CD302 likely contributes to human liver-intrinsic antiviral defenses.

Hepatitis D virus infection, innate immune response and antiviral treatments in stem cell-derived hepatocytes.

BACKGROUND: Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) are a valuable model to investigate host-pathogen interactions of hepatitis viruses in a mature and authentic environment. Here, we investigate the susceptibility of HLCs to the hepatitis delta virus (HDV). METHODS: We differentiated hPSC into HLCs, and inoculated them with infectious HDV produced in Huh7NTCP . HDV infection and cellular response was monitored by RTqPCR and immunostaining. RESULTS: Cells undergoing hepatic differentiation become susceptible to HDV after acquiring expression of the viral receptor Na+ -taurocholate co-transporting polypeptide (NTCP) during hepatic specification. Inoculation of HLCs with HDV leads to detection of intracellular HDV RNA and accumulation of the HDV antigen in the cells. Upon infection, the HLCs mounted an innate immune response based on induction of the interferons IFNB and L, and upregulation of interferon-stimulated genes. The intensity of this immune response positively correlated with the level of viral replication and was dependant on both the JAK/STAT and NFκB pathway activation. Importantly, this innate immune response did not inhibit HDV replication. However, pre-treatment of the HLCs with IFNα2b reduced viral infection, suggesting that ISGs may limit early stages of infection. Myrcludex efficiently abrogated infection and blocked innate immune activation. Lonafarnib treatment of HDV mono infected HLCs on the other hand led to exacerbated viral replication and innate immune response. CONCLUSION: The HDV in vitro mono-infection model represents a new tool to study HDV replication, its host-pathogen interactions and evaluate new antiviral drugs in cells displaying mature hepatic functions.

Preoperative hemoglobin levels, extended resections and the body mass index influence survival after pancreaticoduodenectomy.

PURPOSE: The negative influence of perioperative transfusion of packed red blood cells on the prognosis of various malignancies is the focus of recent research interest. The development of a propensity score for the prediction of perioperative transfusion of packed red blood cells (pRBCs) and the identification of independent risk factors for survival, that can either be known prior to or during surgery in patients undergoing pancreaticoduodenectomy for pancreatic head cancer are the two objectives of this study. METHODS: Logistic regression analyses and Cox regression modeling were used to identify independent risk factors for perioperative transfusion of pRBCs and to determine individual risk factors for patient survival. A total of 101 adult patients who underwent surgery between 01/01/2016 and 12/31/2020 were investigated in a single-center retrospective analysis. RESULTS: Preoperative hemoglobin levels (OR: 0.472, 95%-CI: 0.312-0.663, p < 0.001) and extended resections (OR: 4.720, 95%-CI: 1.819-13.296, p = 0.001) were identified as independent risk factors for perioperative transfusion of pRBCs, enabling the prediction of pRBC transfusion with high sensitivity and specificity (AUROC: 0.790). The logit of the derived propensity model for the transfusion of pRBCs (HR: 9.231, 95%CI: 3.083-28.118, p < 0.001) and preoperative Body Mass Index (BMI) (HR, 0.925; 95%-CI: 0.870-0.981, p = 0.008) were independent risk factors for survival. CONCLUSIONS: Low preoperative hemoglobin levels, low BMI values, and extended resections are significant risk factors for survival that can be known and thus potentially be influenced prior to or during surgery. Patient blood management programs and prehabilitation programs should strive to increase preoperative hemoglobin levels and improve preoperative malnutrition.

Robust hepatitis E virus infection and transcriptional response in human hepatocytes.

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and the leading cause for acute viral hepatitis worldwide. The virus is classified as a member of the genus Orthohepevirus A within the Hepeviridae family. Due to the absence of a robust cell culture model for HEV infection, the analysis of the viral life cycle, the development of effective antivirals and a vaccine is severely limited. In this study, we established a protocol based on the HEV genotype 3 p6 (Kernow C-1) and the human hepatoma cell lines HepG2 and HepG2/C3A with different media conditions to produce intracellular HEV cell culture-derived particles (HEVcc) with viral titers between 105 and 106 FFU/mL. Viral titers could be further enhanced by an HEV variant harboring a mutation in the RNA-dependent RNA polymerase. These HEVcc particles were characterized in density gradients and allowed the trans-complementation of subgenomic reporter HEV replicons. In addition, in vitro produced intracellular-derived particles were infectious in liver-humanized mice with high RNA copy numbers detectable in serum and feces. Efficient infection of primary human and swine hepatocytes using the developed protocol could be observed and was inhibited by ribavirin. Finally, RNA sequencing studies of HEV-infected primary human hepatocytes demonstrated a temporally structured transcriptional defense response. In conclusion, this robust cell culture model of HEV infection provides a powerful tool for studying viral-host interactions that should facilitate the discovery of antiviral drugs for this important zoonotic pathogen.

Outcome and safety of a surveillance biopsy guided personalized immunosuppression program after liver transplantation.

Graft survival beyond year 1 has not changed after orthotopic liver transplantation (OLT) over the last decades. Likewise, OLT causes comorbidities such as infection, renal impairment and cancer. We evaluated our single-center real-world individualized immunosuppression program after OLT, based on 211 baseline surveillance biopsies (svLbx) without any procedural complications. Patients were classified as low, intermediate and high rejection risk based on graft injury in svLbx and anti-HLA donor-specific antibodies. While 32% of patients had minimal histological inflammation, 57% showed histological inflammation and 23% advanced fibrosis (>F2), which was not predicted by lab parameters. IS was modified in 79% of patients after svLbx. After immunosuppression reduction in 69 patients, only 5 patients showed ALT elevations and three of these patients had a biopsy-proven acute rejection, two of them related to lethal comorbidities. The rate of liver enzyme elevation including rejection was not significantly increased compared to a svLbx control cohort prior to the initiation of our structured program. Immunosuppression reduction led to significantly better kidney function compared to this control cohort. In conclusion, a biopsy guided personalized immunosuppression protocol after OLT can identify patients requiring lower immunosuppression or patients with graft injury in which IS should not be further reduced.

Initial HCV infection of adult hepatocytes triggers a temporally structured transcriptional program containing diverse pro- and anti-viral elements.

Transcriptional profiling provides global snapshots of virus-mediated cellular reprogramming, which can simultaneously encompass pro- and antiviral components. To determine early transcriptional signatures associated with HCV infection of authentic target cells, we performed ex vivo infections of adult primary human hepatocytes (PHHs) from seven donors. Longitudinal sampling identified minimal gene dysregulation at six hours post infection (hpi). In contrast, at 72 hpi, massive increases in the breadth and magnitude of HCV-induced gene dysregulation were apparent, affecting gene classes associated with diverse biological processes. Comparison with HCV-induced transcriptional dysregulation in Huh-7.5 cells identified limited overlap between the two systems. Of note, in PHHs, HCV infection initiated broad upregulation of canonical interferon (IFN)-mediated defense programs, limiting viral RNA replication and abrogating virion release. We further find that constitutive expression of IRF1 in PHHs maintains a steady-state antiviral program in the absence of infection, which can additionally reduce HCV RNA translation and replication. We also detected infection-induced downregulation of ∼90 genes encoding components of the EIF2 translation initiation complex and ribosomal subunits in PHHs, consistent with a signature of translational shutoff. As HCV polyprotein translation occurs independently of the EIF2 complex, this process is likely pro-viral: only translation initiation of host transcripts is arrested. The combination of antiviral intrinsic and inducible immunity, balanced against pro-viral programs, including translational arrest, maintains HCV replication at a low-level in PHHs. This may ultimately keep HCV under the radar of extra-hepatocyte immune surveillance while initial infection is established, promoting tolerance, preventing clearance and facilitating progression to chronicity.IMPORTANCEAcute HCV infections are often asymptomatic and therefore frequently undiagnosed. We endeavored to recreate this understudied phase of HCV infection using explanted PHHs and monitored host responses to initial infection. We detected temporally distinct virus-induced perturbations in the transcriptional landscape, which were initially narrow but massively amplified in breadth and magnitude over time. At 72 hpi, we detected dysregulation of diverse gene programs, concurrently promoting both virus clearance and virus persistence. On the one hand, baseline expression of IRF1 combined with infection-induced upregulation of IFN-mediated effector genes suppresses virus propagation. On the other, we detect transcriptional signatures of host translational inhibition, which likely reduces processing of IFN-regulated gene transcripts and facilitates virus survival. Together, our data provide important insights into constitutive and virus-induced transcriptional programs in PHHs, and identifies simultaneous antagonistic dysregulation of pro-and anti-viral programs which may facilitate host tolerance and promote viral persistence.

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/ß 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.

Interleukin-18 and High-Mobility-Group-Protein B1 are Early and Sensitive Indicators for Cell Damage During Normothermic Machine Perfusion after Prolonged Cold Ischemic Storage of Porcine Liver Grafts.

In the era of organ machine perfusion, experimental models to optimize reconditioning of (marginal) liver grafts are needed. Although the relevance of cytokine signatures in liver transplantation has been analyzed previously, the significance of molecular monitoring during normothermic machine perfusion (NMP) remains elusive. Therefore, we developed a porcine model of cold ischemic liver graft injury after prolonged static cold storage (SCS) and subsequent NMP: Livers obtained from ten minipigs underwent NMP for 6 h directly after procurement (control group) or after 20 h of SCS. Grafts after prolonged SCS showed significantly elevated AST, ALT, GLDH and GGT perfusate concentrations, and reduced lactate clearance. Bile analyses revealed reduced bile production, reduced bicarbonate and elevated glucose concentrations after prolonged SCS. Cytokine analyses of graft perfusate simultaneously demonstrated an increase of pro-inflammatory cytokines such as Interleukin-1α, Interleukin-2, and particularly Interleukin-18. The latter was the only significantly elevated cytokine compared to controls, peaking as early as 2 h after reperfusion (11,012 ng/ml vs. 1,493 ng/ml; p = 0.029). Also, concentrations of High-Mobility-Group-Protein B1 were significantly elevated after 2 h of reperfusion (706.00 ng/ml vs. 148.20 ng/ml; p < 0.001) and showed positive correlations with AST (r 2 = 0.846) and GLDH (r 2 = 0.918) levels. Molecular analyses during reconditioning of liver grafts provide insights into the degree of inflammation and cell damage and could thereby facilitate future interventions during NMP reducing acute and chronic graft injury.

Correction of heat-induced susceptibility changes in respiratory-triggered 2D-PRF-based thermometry for monitoring of magnetic resonance-guided hepatic microwave ablation in a human-like in vivo porcine model.

PURPOSE: To develop and evaluate susceptibility corrected 2D proton resonance frequency (PRF)-based magnetic resonance (MR)-thermometry for the accurate assessment of the ablation zone of hepatic microwave ablation (MWA). METHODS AND MATERIALS: Twelve hepatic MWA were performed in five LEWE minipigs with human-like fissure-free liver. Temperature maps during ablation of PRF-based MR-thermometry were corrected by modeling heat induced susceptibility changes. Ablation zones were determined using cumulative equivalent minutes at 43 °C (CEM43) as tissue damage model. T1 weighted (w) post-ablation contrast-enhanced (CE) MR-imaging and manually segmented postmortem histology were used for validation. The agreement of uncorrected (raw) and susceptibility corrected (corr) MR-thermometry with T1w post-ablation CE MR-imaging and histology was evaluated. The Wilcoxon-signed rank test and Bland-Altman analysis were applied. RESULTS: With the susceptibility corrected MR-thermometry a significantly increased dice coefficient (raw: 77% vs. corr: 83%, p < 0.01) and sensitivity (raw: 72% vs. corr: 82%, p < 0.01) was found for the comparison to T1w-CE imaging as well as histopathology (dice coefficients: raw: 76% vs. corr: 79%, p < 0.001; sensitivity: raw: 72% vs. corr: 74%, p < 0.001). While major axis length was significantly increased (7.1 mm, p < 0.001) and minor axis length significantly decreased (2.2 mm, p < 0.001) in uncorrected MR-thermometry compared to T1w-CE MR-imaging, no significant bias was found after susceptibility correction. CONCLUSION: Using susceptibility corrected 2D PRF-based MR-thermometry to predict the ablation zones of hepatic MWA provided a good agreement in comparison to T1w post-ablation CE MR-imaging and histopathology.

Impact of perioperative blood transfusions on postoperative renal function and survival after resection of colorectal liver metastases.

BACKGROUND AND AIMS: Recent studies focusing on thoracic surgery suggest postoperative kidney injury depending on the amount of perioperative blood transfusions. Data investigating similar effects after resection of colorectal liver metastases (CRLM) are not available. Aim of this study was therefore to evaluate the influence of perioperative blood transfusions on postoperative renal function and survival after resection of CRLM. METHODS: Seven hundred twenty-seven cases of liver resection for CRLM were retrospectively analyzed. Renal function was measured via estimated glomerular filtration rate (eGFR) and a postoperative decline of ≥ 10% was considered substantial. Potential influences on postoperative kidney function were assessed using univariable and multivariable logistic regression analyses. Cox-regression analyses were performed to estimate the impact on overall survival (OS). RESULTS: Preoperative impaired kidney function (p = 0.001, OR 2.477) and transfusion of > 2 units of packed red blood cells (PRBC) (p = 0.046; OR 1.638) were independently associated with an increased risk for ≥ 10% loss of renal function. Neither a pre-existing renal impairment, nor the additional loss of renal function were associated with reduced survival. Chemotherapies in the context of primary colorectal cancer treatment (p = 0.002), age > 70 years at liver resection (p = 0.005), number (p = 0.001), and size of metastases > 50 mm (p = 0.018), duration of resection > 120 min (p = 0.006) and transfusions of > 2 units of PRBC (p = 0.039) showed a negative independent influence on OS. CONCLUSION: The results demonstrate a negative impact of perioperative blood transfusions on the postoperative renal function and OS. Hence, efforts to reduce blood transfusions should be intensified.

Therapeutic HNF4A mRNA attenuates liver fibrosis in a preclinical model.

BACKGROUND & AIMS: Therapeutic targeting of injuries that require transient restoration of proteins by mRNA delivery is an attractive approach that, until recently, has remained poorly explored. In this study, we examined the therapeutic utility of mRNA delivery for liver fibrosis and cirrhosis. Specifically, we aimed to demonstrate the therapeutic efficacy of human hepatocyte nuclear factor alpha (HNF4A) mRNA in mouse models of fibrosis and cirrhosis. METHODS: We investigated restoration of hepatocyte functions by HNF4A mRNA transfection in vitro, and analyzed the attenuation of liver fibrosis and cirrhosis in multiple mouse models, by delivering hepatocyte-targeted biodegradable lipid nanoparticles (LNPs) encapsulating HNF4A mRNA. To identify potential mechanisms of action, we performed microarray-based gene expression profiling, single-cell RNA sequencing, and chromatin immunoprecipitation. We used primary liver cells and human liver buds for additional functional validation. RESULTS: Expression of HNF4A mRNA led to restoration of the metabolic activity of fibrotic primary murine and human hepatocytes in vitro. Repeated in vivo delivery of LNP-encapsulated HNF4A mRNA induced a robust inhibition of fibrogenesis in 4 independent mouse models of hepatotoxin- and cholestasis-induced liver fibrosis. Mechanistically, we discovered that paraoxonase 1 is a direct target of HNF4A and it contributes to HNF4A-mediated attenuation of liver fibrosis via modulation of liver macrophages and hepatic stellate cells. CONCLUSION: Collectively, our findings provide the first direct preclinical evidence of the applicability of HNF4A mRNA therapeutics for the treatment of fibrosis in the liver. LAY SUMMARY: Liver fibrosis and cirrhosis remain unmet medical needs and contribute to high mortality worldwide. Herein, we take advantage of a promising therapeutic approach to treat liver fibrosis and cirrhosis. We demonstrate that restoration of a key gene, HNF4A, via mRNA encapsulated in lipid nanoparticles decreased injury in multiple mouse models of fibrosis and cirrhosis. Our study provides proof-of-concept that mRNA therapy is a promising strategy for reversing liver fibrosis and cirrhosis.

Distinct Immune Imprints of Post-Liver Transplantation Hepatitis C Persist Despite Viral Clearance.

Recurrence or de novo infection of hepatitis C virus (HCV) after liver transplantation (LT) has been associated with progressive graft hepatitis that can be improved by treatment with novel direct-acting antivirals. Cases of rejection episodes have been described during and after HCV treatment. The evolution of innate and adaptive immune response during and after cure of HCV LT is unknown. We studied 74 protein biomarkers in the plasma of LT patients receiving antiviral therapy. In addition, deep immune phenotyping of both the myeloid and lymphoid immune cell subsets in peripheral blood mononuclear cells was performed. We found that LT patients with active HCV infection displayed distinct alterations of inflammatory protein biomarkers, such as C-X-Cmotif chemokine 10 (CXCL10), caspase 8, C-C motif chemokine 20 (CCL20), CCL19, interferon γ, CUB domain-containing protein 1 (CDCP1), interleukin (IL)-18R1, CXCL11, CCL3, IL8, IL12B, tumor necrosis factor-beta, CXCL6, osteoprotegerin, IL10, fms-related tyrosine kinase 3 ligand, hepatocyte growth factor, urokinase-type plasminogen activator, neurotrophin-3, CCL4, IL6, tumornecrosis factor receptor superfamily member 9, programmed death ligand 1, IL18, and monocyte chemotactic protein 1, and enrichment of peripheral immune cell subsets unlike patients without HCV infection who received transplants. Interestingly, patients who cleared HCV after LT did not normalize the altered inflammatory milieu nor did the peripheral immune cell subsets normalize to what would be seen in the absence of HCV recurrence. Overall, these data indicate that HCV-specific imprints on inflammatory analytes and immune cell subsets after LT are not completely normalized by therapy-induced HCV elimination. This is in line with the clinical observation that cure of HCV after LT did not trigger rejection episodes in many patients.

Surgical complications in pediatric kidney transplantation-Incidence, risk factors, and effects on graft survival: A retrospective single-center study.

The field of pediatric kidney transplantation remains challenging due to an ongoing lack of size-matched grafts and anatomical peculiarities. In the current study, we investigated the incidence of surgical complications in pediatric recipients, with a focus on risk factors and effects on graft outcome. We retrospectively reviewed all 2386 kidney transplantations at our institution from January 2005 until December 2018. Of these, 221 transplants were performed in pediatric recipients, defined as under the age of 18 years. Donor-recipient body surface area ratios were calculated to evaluate the effects of size mismatching. Regression analyses were performed to identify independent risk factors for surgical complications and graft survival, respectively. Perioperative surgical complications requiring revision were observed in 34 (15.4%) cases. Leading cause for revision were vascular complications such as thrombosis or stenosis (n = 15 [6.8%]), which were significantly more frequent in case of young donors, (ie, donor age <6 years; OR: 4.281; CI-95%:1.385-13.226; P = .012), previous nephrectomy (OR: 3.407; CI-95%:1.019-11.387; P = .046), and en-bloc grafts (OR: 4.923; CI-95%:1.355-17.884; P = .015), followed by postoperative hemorrhage (n = 10 [4.5%]), ureteral complications (n = 8 [3.6%]), and lymphoceles (n = 7 [3.2%]). Median follow-up was 84.13 (0.92-175.72) months. One-, 5-, and 10-year graft survival rates were 97.1%, 88.9%, and 65.1%, respectively. Except for vascular complications (HR: 4.727; CI-95%:1.363-16.394; P = .014), none of the analyzed surgical morbidities significantly influenced graft survival. In conclusion, pediatric kidney transplantation achieves excellent long-term results. However, meticulous surgical technique and continuous postoperative monitoring are imperative for early detection and treatment of imminent vascular complications, especially in case of young donors and en-bloc grafts.

[Machine Perfusion for Liver Transplantation - What is Possible and Where Do We Stand in Germany? Review of the Literature and Results of a National Survey]. / Maschinenperfusion in der Lebertransplantation ­ was ist möglich und wo stehen wir in Deutschland? Übersicht der Literatur und Ergebnisse einer nationalen Umfrage.

Machine perfusion of donor livers is currently regarded as the most important innovation in transplant surgery to address the continuing shortage of organs in liver transplantation. Hypothermic machine perfusion (HMP) is safe to use and appears to reduce the risk of biliary complications and improve the long-term survival of transplanted organs following preservation by cold static storage - even in donors after cardiac death. A potential functional test of donor organs during HMP uses flavin mononucleotide and is still under clinical investigation. Normothermic machine perfusion (NMP) has a greater risk of technical problems, but functional testing using conventional laboratory parameters during NMP allows significant expansion of the donor pool, even though no prospective randomised study has been able to demonstrate a survival advantage for transplanted organs after NMP. In addition, the preservation time of the donor organs can be significantly extended with the help of NMP, which is particularly advantageous for complex recipient operations and/or logistics. Both methods could be applied for various scenarios in transplantation medicine - theoretically also in combination. The majority of German transplant centres regard machine perfusion as an important innovation and already actively perform perfusions or are in preparation for doing so. However, the overall practical experience in Germany is still relatively low, with only 2 centres having performed more than 20 perfusions. In the coming years, multi-centre efforts to conduct clinical trials and to develop national guidelines on machine perfusion will therefore be indispensable in order to define the potential of these technological developments objectively and to exploit it optimally for the field of transplantation medicine.