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The rapidly aging population in industrialized countries comes with an increased incidence of intrahepatic cholangiocarcinoma (iCC) which presents new challenges for oncological treatments especially in elderly patients. Thus, the question arises to what extent the benefit of surgical resections, as the only curative treatment option, outweighs possible perioperative risks in patients ≥ 80 years of age (octogenarians). We therefore retrospectively analyzed 311 patients who underwent resection for iCC at Hannover Medical School between January 1996 and December 2022. In total, there were 11 patients older than 80 years in our collective. Despite similar tumor size, octogenarians underwent comparatively less extensive surgery (54.5% major resections in octogenarians vs. 82.7% in all other patients; p = 0.033) with comparable rates of lymphadenectomy and tumor-free resection margins. Furthermore, we did not observe increased major postoperative morbidity (Clavien-Dindo ≥ IIIa complications: 27.3% vs. 34.3% in all other patients; p = 0.754) or mortality (estimated 1-year OS of 70.7% vs. 72.5% in all other patients, p = 0.099). The length of intensive care unit (ICU) or intermediate care unit (IMC) stay was significantly longer in octogenarians, however, with a comparable length in total hospital stay. The estimated overall survival (OS) did also not differ significantly, although a trend towards reduced long-term survival was observed (14.5 months vs. 28.03 months in all other patients; p = 0.099). In conclusion, primary resection is a justifiable and safe therapeutic option even in octogenarians but requires an even more thorough preoperative patient selection.
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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.
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Factores Despolimerizantes de la Actina , Carcinoma Hepatocelular , Movimiento Celular , Citoesqueleto , Neoplasias Hepáticas , Invasividad Neoplásica , Paxillin , Transducción de Señal , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Humanos , Animales , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Citoesqueleto/metabolismo , Citoesqueleto/patología , Paxillin/metabolismo , Ratones , Factores Despolimerizantes de la Actina/metabolismo , Factores Despolimerizantes de la Actina/genética , Fosforilación , Hepacivirus , Línea Celular Tumoral , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Células Hep G2 , Hepatitis C/patología , Hepatitis C/metabolismo , Hepatitis C/virología , Interferencia de ARNRESUMEN
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.
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Aptitud Genética , Hepacivirus , Hepatocitos , Interacciones Microbiota-Huesped , Inmunidad Innata , Mutación , Humanos , Células Cultivadas , Estrés del Retículo Endoplásmico , Aptitud Genética/genética , Aptitud Genética/inmunología , Hepacivirus/genética , Hepacivirus/crecimiento & desarrollo , Hepacivirus/inmunología , Hepacivirus/fisiología , Hepatitis C/inmunología , Hepatitis C/virología , Hepatocitos/inmunología , Hepatocitos/virología , Interacciones Microbiota-Huesped/inmunología , MicroARNs/metabolismo , Pase Seriado , Respuesta de Proteína Desplegada , Tropismo Viral , Virión/crecimiento & desarrollo , Virión/metabolismo , Replicación Viral/genética , Replicación Viral/inmunologíaRESUMEN
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.
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Senescencia Celular , Trasplante de Órganos , Animales , Ratones , Senoterapéuticos , Ratones Endogámicos C57BL , Trasplante de Órganos/efectos adversos , ADN/farmacología , Envejecimiento/fisiologíaRESUMEN
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 which 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 establishing persistence.
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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.
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Hepacivirus , Hepatitis C , Humanos , Hepacivirus/fisiología , Receptores Depuradores de Clase B/genética , Receptores Depuradores de Clase B/metabolismo , Internalización del Virus , Proteínas Portadoras , Receptores ErbB/metabolismo , Tetraspanina 28/genética , Tetraspanina 28/metabolismo , Proteína ADAM10/genética , Proteína ADAM10/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismoRESUMEN
Ex vivo machine perfusion (EVMP) is an emerging technique for preserving explanted solid organs with primary application in allogeneic organ transplantation. EVMP has been established as an alternative to the standard of care static-cold preservation, allowing for prolonged preservation and real-time monitoring of organ quality while reducing/preventing ischemia-reperfusion injury. Moreover, it has paved the way to involve expanded criteria donors, e.g., after circulatory death, thus expanding the donor organ pool. Ongoing improvements in EVMP protocols, especially expanding the duration of preservation, paved the way for its broader application, in particular for reconditioning and modification of diseased organs and tumor and infection therapies and regenerative approaches. Moreover, implementing EVMP for in vivo-like preclinical studies improving disease modeling raises significant interest, while providing an ideal interface for bioengineering and genetic manipulation. These approaches can be applied not only in an allogeneic and xenogeneic transplant setting but also in an autologous setting, where patients can be on temporary organ support while the diseased organs are treated ex vivo, followed by reimplantation of the cured organ. This review provides a comprehensive overview of the differences and similarities in abdominal (kidney and liver) and thoracic (lung and heart) EVMP, focusing on the organ-specific components and preservation techniques, specifically on the composition of perfusion solutions and their supplements and perfusion temperatures and flow conditions. Novel treatment opportunities beyond organ transplantation and limitations of abdominal and thoracic EVMP are delineated to identify complementary interdisciplinary approaches for the application and development of this technique.
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De novo immune responses are considered major challenges in gene therapy. With the aim to lower innate immune responses directly in cells targeted by adeno-associated virus (AAV) vectors, we equipped the vector capsid with a peptide known to interfere with Toll-like receptor signaling. Specifically, we genetically inserted in each of the 60 AAV2 capsid subunits the myeloid differentiation primary response 88 (MyD88)-derived peptide RDVLPGT, known to block MyD88 dimerization. Inserting the peptide neither interfered with capsid assembly nor with vector production yield. The novel capsid variant, AAV2.MB453, showed superior transduction efficiency compared to AAV2 in human monocyte-derived dendritic cells and in primary human hepatocyte cultures. In line with our hypothesis, AAV2.MB453 and AAV2 differed regarding innate immune response activation in primary human cells, particularly for type I interferons. Furthermore, mice treated with AAV2.MB453 showed significantly reduced CD8+ T cell responses against the transgene product for different administration routes and against the capsid following intramuscular administration. Moreover, humoral responses against the capsid were mitigated as indicated by delayed IgG2a antibody formation and an increased NAb50. To conclude, insertion of the MyD88-derived peptide into the AAV2 capsid improved early steps of host-vector interaction and reduced innate and adaptive immune responses.
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Hepatitis E virus (HEV) usually causes a self-limiting disease, but especially immunocompromised individuals are at risk to develop a chronic and severe course of infection. Janus kinase (JAK) inhibitors (JAKi) are a novel drug class for the treatment of autoimmune inflammatory rheumatic disease (AIRD). As JAKs play a key role in innate immunity, viral infections and reactivations are frequently reported during JAKi treatment in AIRD patients. The aim of this study was to characterize the influence of JAKis on HEV replication. To this end, we evaluated liver enzymes of an AIRD patient under JAKi therapy with hepatitis E. Further, experiments with HEV (Kernow-C1 p6) were performed by infection of primary human hepatocytes (PHHs) followed by immunofluorescence staining of viral markers and transcriptomic analysis. Infection experiments in PHHs displayed an up to 50-fold increase of progeny virus production during JAKi treatment and transcriptomic analysis revealed induction of antiviral programs during infection. Upregulation of interferon-stimulated genes (ISG) was perturbed in the presence of JAKis, concomitant with elevated HEV RNA levels. The obtained results suggest that therapeutic JAK inhibition increases HEV replication by modulating the HEV-triggered immune response. Therefore, JAKi treatment and the occurrence of elevated liver enzymes requires a monitoring of potential HEV infections.
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Virus de la Hepatitis E , Hepatitis E , Humanos , Virus de la Hepatitis E/genética , Quinasas Janus , Interferones/farmacología , Antivirales/farmacología , Antivirales/uso terapéutico , Replicación ViralRESUMEN
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.
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Hepatitis D , Virus de la Hepatitis Delta , Humanos , Virus de la Hepatitis Delta/genética , Antivirales/farmacología , Antivirales/uso terapéutico , Hepatitis D/tratamiento farmacológico , Hepatocitos/metabolismo , Inmunidad Innata , Interferones/uso terapéutico , Células Madre , Replicación Viral , Virus de la Hepatitis B/genéticaRESUMEN
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.
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Transfusión Sanguínea , Pancreaticoduodenectomía , Adulto , Humanos , Índice de Masa Corporal , Pancreaticoduodenectomía/efectos adversos , Estudios Retrospectivos , HemoglobinasRESUMEN
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.
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Virus de la Hepatitis E , Hepatocitos , Humanos , Hepatocitos/metabolismo , Antivirales/farmacología , Receptores ErbB/metabolismo , Interferencia de ARN , Transducción de Señal , Virus de la Hepatitis E/genética , Replicación ViralRESUMEN
The incidence of pediatric liver tumors in general has been rising over the last years and so is the number of children undergoing liver transplantation for this indication. To contribute to the ongoing improvement of pre- and post-transplant care, we aim to describe outcome and risk factors in our patient cohort. We have compared characteristics and outcome for patients transplanted for hepatoblastoma to other liver malignancies in our center between 1983 and 2022 and analysed influential factors on tumor recurrence and mortality using nominal logistic regression analysis. Of 39 children (16 f) who had transplants for liver malignancy, 31 were diagnosed with hepatoblastoma. The proportion of malignant tumors in the transplant cohort rose from 1.9% (1983-1992) to 9.1% in the current decade (p < 0.0001). Hepatoblastoma patients were transplanted at a younger age and were more likely to have tumor extent beyond the liver. Post-transplant bile flow impairment requiring intervention was significantly higher compared to our total cohort (48 vs. 24%, p > 0.0001). Hearing loss was a common side effect of ototoxic chemotherapy in hepatoblastoma patients (48%). The most common maintenance immunosuppression were mTor-inhibitors. Risk factors for tumor recurrence in patients with hepatoblastoma were higher AFP before transplant (AFPpre-LTX), a low ratio of AFPmax to AFPpre-LTX and salvage transplantation. Liver malignancies represent a rising number of indications for liver transplantation in childhood. Primary tumor resection can spare a liver transplant with all its long-term complications, but in case of tumor recurrence, transplantation might have inferior outcome. The rate of acute biopsy-proven rejections and biliary complications in comparison to our total transplant cohort needs further investigations.
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Reliable, easy-to-handle phenotypic screening platforms are needed for the identification of anti-SARS-CoV-2 compounds. Here, we present caspase 3/7 activity as a readout for monitoring the replication of SARS-CoV-2 isolates from different variants, including a remdesivir-resistant strain, and of other coronaviruses in numerous cell culture models, independently of cytopathogenic effect formation. Compared to other models, the Caco-2 subline Caco-2-F03 displayed superior performance. It possesses a stable SARS-CoV-2 susceptibility phenotype and does not produce false-positive hits due to drug-induced phospholipidosis. A proof-of-concept screen of 1,796 kinase inhibitors identified known and novel antiviral drug candidates including inhibitors of phosphoglycerate dehydrogenase (PHGDH), CDC like kinase 1 (CLK-1), and colony stimulating factor 1 receptor (CSF1R). The activity of the PHGDH inhibitor NCT-503 was further increased in combination with the hexokinase II (HK2) inhibitor 2-deoxy-D-glucose, which is in clinical development for COVID-19. In conclusion, caspase 3/7 activity detection in SARS-CoV-2-infected Caco-2-F03 cells provides a simple phenotypic high-throughput screening platform for SARS-CoV-2 drug candidates that reduces false-positive hits.
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In pediatric liver transplantation (pLT), the risk for the manifestation and relevance of intra-abdominal hypertension (IAH) and abdominal compartment syndrome (ACS) is high. This observational study aimed to evaluate the incidence, relevance and risk factors for IAH and ACS by monitoring the intra-abdominal pressure (IAP), macro- and microcirculation (near-infrared spectroscopy (NIRS)), clinical and laboratory status and outcomes of 27 patients (16 female) after pLT (median age at pLT 35 months). Of the patients, 85% developed an elevated IAP, most of them mild. However, 17% achieved IAH° 3, 13% achieved IAH° 4 and 63% developed ACS. A multiple linear regression analysis identified aortal hepatic artery anastomosis and cold ischemia time (CIT) as risk factors for increased IAP and longer CIT and staged abdominal wall closure for ACS. ACS patients had significantly longer mechanical ventilation (p = 0.004) and LOS-PICU (p = 0.003). No significant correlation between NIRS or biliary complications and IAH or ACS could be shown. IAH and ACS after pLT were frequent. NIRS or grade of IAH alone should not be used for monitoring. A longer CIT is an important risk factor for higher IAP and ACS. Therefore, approaches such as the ex vivo machine perfusion of donor organs, reducing CIT effects on them, have great potential. Our study provides important basics for studying such approaches.
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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.
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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.
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Trasplante de Hígado , Animales , Porcinos , Trasplante de Hígado/efectos adversos , Preservación de Órganos , Interleucina-18 , Porcinos Enanos , Perfusión , HígadoRESUMEN
Background & Aims: HBV persistence is maintained by both an episomal covalently closed circular (ccc)DNA reservoir and genomic integration of HBV DNA fragments. While cccDNA transcription is regulated by Cullin4A-DDB1-HBx-mediated degradation of the SMC5/6 complex, HBsAg expression from integrants is largely SMC5/6 independent. Inhibiting neddylation of Cullin-RING ubiquitin ligases impairs degradation of substrates. Herein, we show that targeting neddylation pathway components by small-interfering (si)RNAs or the drug MLN4924 (pevonedistat) suppresses expression of HBV proteins from both cccDNA and integrants. Methods: An siRNA screen targeting secretory pathway regulators and neddylation genes was performed. Activity of MLN4924 was assessed in infection and integration models. Trans-complementation assays were used to study HBx function in cccDNA-driven expression. Results: siRNA screening uncovered neddylation pathway components (Nedd8, Ube2m) that promote HBsAg production post-transcriptionally. Likewise, MLN4924 inhibited production of HBsAg encoded by integrants and reduced intracellular HBsAg levels, independent of HBx. MLN4924 also profoundly inhibited cccDNA transcription in three infection models. Using the HBV inducible cell line HepAD38 as a model, we verified the dual action of MLN4924 on both cccDNA and integrants with sustained suppression of HBV markers during 42 days of treatment. Conclusions: Neddylation is required both for transcription of a cccDNA reservoir and for the genomic integration of viral DNA. Therefore, blocking neddylation might offer an attractive approach towards functional cure of chronic hepatitis B. Lay summary: Current treatments for chronic hepatitis B are rarely able to induce a functional cure. This is partly because of the presence of a pool of circular viral DNA in the host nucleus, as well as viral DNA fragments that are integrated into the host genome. Herein, we show that a host biological pathway called neddylation could play a key role in infection and viral DNA integration. Inhibiting this pathway could hold therapeutic promise for patients with chronic hepatitis B.
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Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft failure, ongoing organ donor shortages, and the increased use of marginal grafts call for the improvement of current concepts, such as the implementation of organ machine perfusion. In order to evaluate new methods of graft reconditioning and modulation, translational models are required. With respect to anatomical and physiological similarities to humans and recent progress in the field of xenotransplantation, pigs have become the main large animal species used in transplantation models. After the initial introduction of a porcine orthotopic liver transplant model by Garnier et al. in 1965, several modifications have been published over the past 60 years. Due to specifies-specific anatomical traits, a veno-venous bypass during the anhepatic phase is regarded as a necessity to reduce intestinal congestion and ischemia resulting in hemodynamic instability and perioperative mortality. However, the implementation of a bypass increases the technical and logistical complexity of the procedure. Furthermore, associated complications such as air embolism, hemorrhage, and the need for a simultaneous splenectomy have been reported previously. In this protocol, we describe a model of porcine orthotopic liver transplantation without the use of a veno-venous bypass. The engraftment of donor livers after static cold storage of 20 h - simulating extended criteria donor conditions - demonstrates that this simplified approach can be performed without significant hemodynamic alterations or intraoperative mortality and with regular uptake of liver function (as defined by bile production and liver-specific CYP1A2 metabolism). The success of this approach is ensured by an optimized surgical technique and a sophisticated anesthesiologic volume and vasopressor management. This model should be of special interest for workgroups focusing on the immediate postoperative course, ischemia-reperfusion injury, associated immunological mechanisms, and the reconditioning of extended criteria donor organs.
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Trasplante de Hígado , Obtención de Tejidos y Órganos , Animales , Humanos , Hígado/cirugía , Trasplante de Hígado/métodos , Perfusión , Porcinos , Donantes de TejidosRESUMEN
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.