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Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication. In this study, we aimed to further characterize the role of macrophage-derived EVs in immune responses against hepatitis C virus (HCV) and the potential of polyunsaturated fatty acids (PUFAs) to modulate this modality of innate immunity. To this end, EVs were isolated from interferon-stimulated macrophage cultures or from serum of patients with acute or chronic hepatitis C. EVs were characterized by electron microscopy, flow cytometry, RNA-sequencing, and Western blot analysis. The effect of EVs on replication of HCV was assessed in coculture models. Functional analyses were performed to assess the impact of PUFAs on EV-mediated antiviral immunity. We found that macrophages secreted various cytokines shortly after stimulation with type I and II IFN, which orchestrated a fast but short-lasting antiviral state. This rapid innate immune answer was followed by the production of macrophage-derived EVs, which induced a late, but long-lasting inhibitory effect on HCV replication. Of note, exposure of macrophages to PUFAs, which are important regulators of immune responses, dampened EV-mediated antiviral immune responses. Finally, EVs from patients with hepatitis C exhibited long-lasting antiviral activities during IFN therapy as well. The antiviral effect of EVs from Caucasian and Japanese patients differed, which may be explained by different nutritional uptake of PUFAs. In conclusion, our data indicate that macrophage-derived EVs mediate long-lasting inhibitory effects on HCV replication, which may bridge the time until efficient adaptive immune responses are established, and which can be blunted by PUFAs.
Tumor progression largely depends on the presence of alternatively polarized (M2) tumor-associated macrophages (TAMs), whereas the classical M1-polarized macrophages can promote anti-tumorigenic immune responses. Thus, selective inhibition of M2-TAMs is a desirable anti-cancer approach in highly resistant tumor entities such as hepatocellular carcinoma (HCC) or breast cancer. We here examined whether a peptide that selectively binds to and is internalized by in vitro-differentiated murine M2 macrophages as compared to M1 macrophages, termed M2pep, could be used to selectively target TAMs in HCC and breast carcinoma. We confirmed selectivity of M2pep for in vitro M2 polarized macrophages. Upon incubation of suspended mixed 4T1 tumor cells with M2pep, high amounts of the TAMs were found to be associated with M2pep, whereas in mixed tumor cell suspensions from two HCC mouse models, M2pep showed only low-degree binding to TAMs. M2pep also showed low-degree targeting of liver macrophages. This indicates that the TAMs in different tumor entities show different targeting of M2pep and that M2pep is a very promising approach to develop selective M2-TAM-targeting in tumor entities containing M2-TAMs with significant amounts of the so far elusive M2pep receptor(s).
Genetic heterogeneity of primary lesion and metastasis in small intestine neuroendocrine tumors
(2018)
Data on intratumoral heterogeneity of small intestine neuroendocrine tumors (SI-NETs) and related liver metastasis are limited. The aim of this study was to characterize genetic heterogeneity of 5 patients with SI-NETs. Therefore, formalin-fixed, paraffin-embedded tissue samples of primary and metastatic lesions as well as benign liver of five patients with synchronously metastasized, well differentiated SI-NETs were analyzed with whole exome sequencing. For one patient, chip based 850k whole DNA methylome analysis was performed of primary and metastatic tumor tissue as well as control tissue. Thereby, 156 single nucleotide variants (SNVs) in 150 genes were identified and amount of mutations per sample ranged from 9–34 (mean 22). The degree of common (0–94%) and private mutations per sample was strongly varying (6–100%). In all patients, copy number variations (CNV) were found and the degree of intratumoral heterogeneity of CNVs corresponded to SNV analysis. DNA methylation analysis of a patient without common SNVs revealed a large overlap of common methylated CpG sites. In conclusion, SI-NET primary and metastatic lesions show a highly varying degree of intratumoral heterogeneity. Driver events might not be detectable with exome analysis only, and further comprehensive studies including whole genome and epigenetic analyses are warranted.
Background: Definite diagnosis and therapeutic management of cholangiocarcinoma (CCA) remains a challenge. The aim of the current study was to investigate feasibility and potential impact on clinical management of targeted sequencing of intraductal biopsies.
Methods: Intraductal biopsies with suspicious findings from 16 patients with CCA in later clinical course were analyzed with targeted sequencing including tumor and control benign tissue (n = 55 samples). A CCA-specific sequencing panel containing 41 genes was designed and a dual strand targeted enrichment was applied.
Results: Sequencing was successfully performed for all samples. In total, 79 mutations were identified and a mean of 1.7 mutations per tumor sample (range 0–4) as well as 2.3 per biopsy (0–6) were detected and potentially therapeutically relevant genes were identified in 6/16 cases. In 14/18 (78%) biopsies with dysplasia or inconclusive findings at least one mutation was detected. The majority of mutations were found in both surgical specimen and biopsy (68%), while 28% were only present in biopsies in contrast to 4% being only present in the surgical tumor specimen.
Conclusion: Targeted sequencing from intraductal biopsies is feasible and potentially improves the diagnostic yield. A profound genetic heterogeneity in biliary dysplasia needs to be considered in clinical management and warrants further investigation.
Translational impact: The current study is the first to demonstrate the feasibility of sequencing of intraductal biopsies which holds the potential to impact diagnostic and therapeutical management of patients with biliary dysplasia and neoplasia.
Immune-modulating therapy is a promising therapy for patients with cholangiocarcinoma (CCA). Microsatellite instability (MSI) might be a favorable predictor for treatment response, but comprehensive data on the prevalence of MSI in CCA are missing. The aim of the current study was to determine the prevalence of MSI in a German tertiary care hospital. Formalin-fixed paraffin-embedded tissue samples, obtained in the study period from 2007 to 2015 from patients with CCA undergoing surgical resection with curative intention at Johann Wolfgang Goethe University hospital, were examined. All samples were investigated immunohistochemically for the presence of MSI (expression of MLH1, PMS2, MSH2, and MSH6) as well as by pentaplex polymerase chain reaction for five quasimonomorphic mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-22, and NR-24). In total, 102 patients were included, presenting intrahepatic (n = 35, 34.3%), perihilar (n = 42, 41.2%), and distal CCA (n = 25, 24.5%). In the immunohistochemical analysis, no loss of expression of DNA repair enzymes was observed. In the PCR-based analysis, one out of 102 patients was found to be MSI-high and one out of 102 was found to be MSI-low. Thus, MSI seems to appear rarely in CCA in Germany. This should be considered when planning immune-modulating therapy trials for patients with CCA.
Background & Aims: Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are increasingly a cause of cirrhosis and hepatocellular carcinoma globally. This burden is expected to increase as epidemics of obesity, diabetes and metabolic syndrome continue to grow. The goal of this analysis was to use a Markov model to forecast NAFLD disease burden using currently available data.
Methods: A model was used to estimate NAFLD and NASH disease progression in eight countries based on data for adult prevalence of obesity and type 2 diabetes mellitus (DM). Published estimates and expert consensus were used to build and validate the model projections.
Results: If obesity and DM level off in the future, we project a modest growth in total NAFLD cases (0–30%), between 2016–2030, with the highest growth in China as a result of urbanization and the lowest growth in Japan as a result of a shrinking population. However, at the same time, NASH prevalence will increase 15–56%, while liver mortality and advanced liver disease will more than double as a result of an aging/increasing population.
Conclusions: NAFLD and NASH represent a large and growing public health problem and efforts to understand this epidemic and to mitigate the disease burden are needed. If obesity and DM continue to increase at current and historical rates, both NAFLD and NASH prevalence are expected to increase. Since both are reversible, public health campaigns to increase awareness and diagnosis, and to promote diet and exercise can help manage the growth in future disease burden.
Lay summary: Non-alcoholic fatty liver disease and non-alcoholic steatohepatitis can lead to advanced liver disease. Both conditions are becoming increasingly prevalent as the epidemics of obesity and diabetes continue to increase. A mathematical model was built to understand how the disease burden associated with non-alcoholic fatty liver disease and non-alcoholic steatohepatitis will change over time. Results suggest increasing cases of advanced liver disease and liver-related mortality in the coming years.
Background: Patients with chronic hepatitis C virus (HCV) infection and active or previous hepatitis B virus (HBV) are at risk of HBV reactivation (HBV-R) during direct-acting antiviral (DAA) therapy. Recent reports suggest that HBV-R may even occur several months after completion of DAA therapy. The aim of this study was to assess the risk of HBV-R in patients with resolved HBV after successful DAA therapy during long-term follow-up (FU).
Methods: Among 848 patients treated for chronic HCV, all patients with resolved HBV and long-term FU data were eligible for inclusion. Patients were HBV DNA/hepatitis B surface antigen (HBsAg)–negative at the end of therapy (EOT) and were followed for up to 52 weeks thereafter. Patients underwent regular alanine transaminase (ALT) testing, and additional HBV DNA/HBsAg testing was performed at FU week 12, end of FU, and in case of an ALT increase above the upper limit of normal (>ULN).
Results: A total of 108 patients were followed up for a mean (range) of 41.5 (24–52) weeks after EOT. None of the patients experienced reverse HBsAg seroconversion or reappearance of HBV DNA. One patient received a liver transplantation; 1 patient was diagnosed with de novo hepatocellular carcinoma, and 2 patients died. Eighteen patients (16.7%) had increased ALT levels (grade 0/1). Of those, the majority were male (72.2%) and significantly more patients had cirrhosis (66.7% vs 36.2%, P = .015) or received ribavirin as part of their treatment regimen (86.7% vs 46.8%, P = .041). None of these were associated with HBV-R.
Conclusions: Our results indicate that the risk of HBV-R in patients with resolved HBV treated with DAAs for HCV is low during long-term follow-up.
Patients after orthopic liver transplantation (OLT) are at risk of developing graft dysfunction. Sphingolipids (SL’s) have been identified to play a pivotal role in the regulation of hepatocellular apoptosis, inflammation and immunity. We aimed to investigate the serum SL profile in a prospective real-world cohort of post-OLT patients. From October 2015 until July 2016, 149 well-characterized post-OLT patients were analyzed. SL’s were assessed in serum probes via Liquid Chromatography/Tandem Mass Spectrometry. Twenty-nine (20%) patients had a biopsy proven graft rejection with decreased C20-ceramide (Cer) (p = 0.042), C18-dihydroceramide (DHC) (p = 0.022) and C24DHC (p = 0.060) levels. Furthermore, C18DHC (p = 0.044) and C24DHC (p = 0.011) were significantly down-regulated in patients with ischemic type biliary lesions (ITBL; n = 15; 10%). One-hundred and thirty-three patients (89%) have so far received tacrolimus as the main immunosuppressive agent with observed elevations of C14Cer (p = 0.052), C18Cer (p = 0.049) and C18:1Cer (p = 0.024). Hepatocellular carcinoma (HCC) pre-OLT was associated with increases in C24:1Cer (p = 0.024) and C24:1DHC (p = 0.024). In this large prospective cross-sectional study of patients, post-OLT serum levels of (very-)long chain (dihydro-)ceramides associate with graft rejection, ITBL, tacrolimus intake and HCC pre-OLT. Hence, serum SL’s may be indicative of graft complications. Further research is necessary to identify their diverse mechanistic role in regulating immunity and inflammation in patients post-OLT.
Objectives: Sphingolipids (SLs) have been implicated as potent regulators of the hepatitis B virus (HBV) life cycle. We investigated the SL biomarker potential regarding virologic endpoints in a prospective subgroup of patients with HBeAg-negative chronic HBV infection.
Methods: From 2009–2016 98 patients with HBeAg-negative HBV infection were prospectively followed over four years. Clinical, laboratory and imaging data were evaluated annually. SLs were assessed in available serum probes via liquid chromatography coupled to tandem mass spectrometry.
Results: Of those 98 patients, 10 (10.2%) showed HBV reactivation, 13 (13.2%) lost HBsAg and 9 (9.1%) gained status of HBsAg-/HBsAb-coexistence, whereas 66 (67.3%) had no events. Within the four-year analysis sphingosine (p = 0.020), sphinganine (p<0.001), dhS1P (p<0.001), C16DHC (p<0.01) and C20Cer (p<0.001) showed a significant upregulation in patients without virologic events, C18Cer significantly decreased (p<0.001). At baseline decreased S1P-, dhS1P- and C16Cer-levels were observed in patients with upcoming status of HBsAg-/HBsAb-coexistence. S1P and dhS1P levels were elevated HBV genotype D infected patients.
Conclusions: In a prospective cohort of patients with a HBeAg-negative HBV infection, serum SLs associated with the virologic course and HBV genotype D. Further studies are required to elucidate SLs as potential novel predictors of the course of HBeAg-negative HBV infection.
Hepatitis C virus (HCV) infection is associated with alterations in host lipid and insulin signaling cascades, which are partially explained by a dependence of the HCV life cycle on key molecules in these metabolic pathways. Yet, little is known on the role in the HCV life cycle of glycogen synthase kinase 3 (GSK3), one of the most important kinases in cellular metabolism. Therefore, the impact of GSK3 on the HCV life cycle was assessed in human hepatoma cell lines harboring subgenomic genotype 1b and 2a replicons or producing cell culture-derived HCV genotype 2a by exposure to synthetic GSK3 inhibitors, GSK3 gene silencing, overexpression of GSK3 constructs and immunofluorescence analyses. In addition, the role of GSK3 in hepatitis E virus (HEV) replication was investigated to assess virus specificity of the observed findings. We found that both inhibition of GSK3 function by synthetic inhibitors as well as silencing of GSK3β gene expression resulted in a decrease of HCV replication and infectious particle production, whereas silencing of the GSK3α isoform had no relevant effect on the HCV life cycle. Conversely, overexpression of GSK3β resulted in enhanced HCV replication. In contrast, GSK3β had no effect on replication of subgenomic HEV replicon. The pro-viral effect of GSK3β on HCV replication was mediated by supporting expression of microRNA-122 (miR-122), a micro-RNA which is mandatory for wild-type HCV replication, as GSK3 inhibitors suppressed miR-122 levels and as inhibitors of GSK3 had no antiviral effect on a miR-122-independent HCV mutant. In conclusion, we have identified GSK3β is a novel host factor supporting HCV replication by maintaining high levels of hepatic miR-122 expression.