EASL position paper on clinical follow-up after HCV cure.

Following the advent of direct-acting antivirals (DAAs), hepatitis C virus (HCV) infection can be cured in almost all infected patients. This has led to a number of clinical questions regarding the optimal management of the millions of patients cured of HCV. This position statement provides specific guidance on the appropriate follow-up after a sustained virological response in patients without advanced fibrosis, those with compensated advanced chronic liver disease, and those with decompensated cirrhosis. Guidance on hepatocellular carcinoma risk assessment and the management of extrahepatic manifestations of HCV is also provided. Finally, guidance is provided on the monitoring and treatment of reinfection in at-risk patients. The recommendations are based on the best available evidence and are intended to help healthcare professionals involved in the management of patients after treatment for HCV.

Recent treatment advances and practical management of hepatitis D virus.

Hepatitis D virus (HDV), also referred to as hepatitis delta virus, is the smallest virus capable of causing human disease. It is unable to replicate on its own and can only propagate in the presence of hepatitis B virus (HBV). Infection with both HBV and HDV frequently results in more severe disease than HBV alone, with higher instances of cirrhosis, liver failure and hepatocellular carcinoma (HCC). Thus, there is a need for effective treatment for HDV; however, currently approved treatment options are very limited both in terms of their efficacy and availability. This makes the management of HDV a challenge for physicians. In this review, we look at the background, diagnosis and treatment of HDV, informed by our hospital data, to set out the optimal management of HDV; we also explore novel treatment options for this disease.

Metabolic dysfunction and cancer in HCV: Shared pathways and mutual interactions.

HCV hijacks many host metabolic processes in an effort to aid viral replication. The resulting hepatic metabolic dysfunction underpins many of the hepatic and extrahepatic manifestations of chronic hepatitis C (CHC). However, the natural history of CHC is also substantially influenced by the host metabolic status: obesity, insulin resistance and hepatic steatosis are major determinants of CHC progression toward hepatocellular carcinoma (HCC). Direct-acting antivirals (DAAs) have transformed the treatment and natural history of CHC. While DAA therapy effectively eradicates the virus, the long-lasting overlapping metabolic disease can persist, especially in the presence of obesity, increasing the risk of liver disease progression. This review covers the mechanisms by which HCV tunes hepatic and systemic metabolism, highlighting how systemic metabolic disturbance, lipotoxicity and chronic inflammation favour disease progression and a precancerous niche. We also highlight the therapeutic implications of sustained metabolic dysfunction following sustained virologic response as well as considerations for patients who develop HCC on the background of metabolic dysfunction.

Evaluation of the mechanisms of sarcopenia in chronic inflammatory disease: protocol for a prospective cohort study.

BACKGROUND: Several chronic inflammatory diseases co-exist with and accelerate sarcopenia (reduction in muscle strength, function and mass) and negatively impact on both morbidity and mortality. There is currently limited research on the extent of sarcopenia in such conditions, how to accurately assess it and whether there are generic or disease-specific mechanisms driving sarcopenia. Therefore, this study aims to identify potential mechanisms driving sarcopenia within chronic inflammatory disease via a multi-modal approach; in an attempt to help define potential interventions for future use. METHODS: This prospective cohort study will consist of a multi-modal assessment of sarcopenia and its underlying mechanisms. Recruitment will target three chronic inflammatory diseases: chronic liver disease (CLD) (n=50), with a subset of NAFLD (n=20), inflammatory bowel disease (IBD) (n=50) and rheumatoid arthritis (RA) (n=50) both before and after therapeutic intervention. In addition, 20 age and sex matched healthy individuals will be recruited for comparison. Participants will undergo 4 assessment visits at weeks 0, 2, 12 and 24. Visits will consist of the following assessments: blood tests, anthropometrics, functional assessment, quadriceps muscle imaging, actigraphy, quality of life questionnaires, food diary collection and muscle biopsy of the vastus lateralis (at weeks 2 and 24 only). In addition, stool and urine samples will be collected for future microbiome and metabolomics analysis. DISCUSSION: This is the first study to use a multi-modal assessment model to phenotype sarcopenia in these chronic inflammatory diseases. We hope to identify generic as well as disease-specific mechanisms driving sarcopenia. We appreciate that these cohorts do require separate standards of care treatments which limit comparison between groups. ETHICS AND DISSEMINATION: The study is approved by the Health Research Authority - West Midlands Solihull Research Ethics Service Committee Authority (REC reference: 18/WM/0167). Recruitment commenced in January 2019 and will continue until July 2021. The study was halted in March 2020 and again in January 2021 with the COVID-19 pandemic. The findings will be disseminated through peer-reviewed publications and conference presentations. All data will be stored on a secure server. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04734496.

Feasibility, Efficacy, and Safety of Percutaneous Muscle Biopsies in Patients With Chronic Liver Disease.

INTRODUCTION: Sarcopenia is present in many chronic disease states including decompensated end stage liver disease (ESLD) and non-cirrhotic non-alcoholic fatty liver disease (NAFLD). Sarcopenia in ESLD can negatively impact quality of life and increase mortality. Despite this, very little is understood about the mechanisms of sarcopenia in these conditions. One key reason for this is the reluctance to undertake percutaneous muscle biopsies due to the perceived increased risks. ESLD can induce thrombocytopaenia and coagulopathy which significantly increases the risk of bleeding. In addition, patients with either NAFLD or ESLD often have co-morbidities that would require additional care and risk assessment. Thus, the aim of this study was to establish an effective and safe protocol for the implementation of percutaneous muscle biopsies in patients with NAFLD and ESLD. METHODS: A total of 47 patients with ESLD and 9 patients with non-cirrhotic NAFLD were recruited from the Liver Unit, Queen Elizabeth Hospital (Birmingham, United Kingdom). A total of 71 percutaneous vastus lateralis biopsies were attempted over two study visits. A vigorous safety screening occurred prior to and during each visit and a strict protocol was followed to mitigate against complications and risk. RESULTS: A total of 85% of patients consented to the muscle biopsy at either visit (48/56). A total of 9% of consented biopsies could not occur due to medical considerations, including high international normalised ratio (INR) (n = 3) and the use of aspirin (n = 4). Muscle tissue was obtained from 90% of attempts, with a mean average yield (wet weight tissue) of 98.1 ± 52.9 mg. CONCLUSION: Percutaneous muscle biopsies are both feasible and yield sufficient tissue in an ESLD population. The procedure is effective for obtaining muscle tissue whilst also safe, with only one adverse event. This study provides evidence for the successful use of muscle biopsies in this population, even in consideration of disease specific complications, medications, and comorbidities.

Persistent Hepatitis E virus infection across England and Wales 2009-2017: Demography, virology and outcomes.

The first clinical case of persistent HEV infection in England was reported in 2009. We describe the demography, virology and outcomes of patients identified with persistent HEV infection in England and Wales between 2009 and 2017. A series of 94 patients with persistent HEV infection, defined by HEV viraemia of more than 12 weeks, was identified through routine reference laboratory testing. Virology, serology and clinical data were recorded through an approved PHE Enhanced Surveillance System. Sixty-six cases (70.2%) were transplant recipients, 16 (17.0%) had an underlying haematological malignancy without stem cell transplantation, six (6.4%) had advanced HIV infection, five (5.3%) were otherwise immunosuppressed, and one patient (1.1%) had no identified immunosuppression. Retrospective analysis of 46 patients demonstrated a median 38 weeks of viraemia before diagnostic HEV testing. At initial diagnosis, 16 patients (17.0%) had no detectable anti-HEV serological response. Of 65 patients treated with ribavirin monotherapy, 11 (16.9%) suffered virological relapse despite undetectable RNA in plasma or stool at treatment cessation. Persistent HEV infection remains a rare diagnosis, but we demonstrate that a broad range of immunocompromised patients are susceptible. Both lack of awareness and the pauci-symptomatic nature of persistent HEV infection likely contribute to significant delays in diagnosis. Diagnosis should rely on molecular testing since anti-HEV serology is insufficient to exclude persistent HEV infection. Finally, despite treatment with ribavirin, relapses occur even after cessation of detectable faecal shedding of HEV RNA, further emphasising the requirement to demonstrate sustained virological responses to treatment.

The Impact of Direct-acting Antivirals on Overall Mortality and Tumoral Recurrence in Patients With Hepatocellular Carcinoma Listed for Liver Transplantation: An International Multicenter Study.

BACKGROUND: There is a lack of data on the use of direct-acting antivirals (DAA) on the risk of death and tumoral recurrence in patients with hepatitis C virus (HCV) and hepatocellular carcinoma (HCC) listed for liver transplantation (LT). We aimed to assess the impact of antiviral treatment on mortality and HCC recurrence patients with HCC-HCV. METHODS: This was a retrospective multicenter study of patients with HCC-HCV listed for LT from 2005 to 2015. Patients were divided according to the antiviral treatment received after HCC diagnosis: DAA, interferon (IFN), or no antiviral. Intention-to-treat overall survival and HCC recurrence incidence were compared by the Kaplan-Meier method. Multivariable regression analysis was performed to identify risk factors for outcomes. RESULTS: A total of 1012 HCV-HCC patients were listed for LT during the study period. The median follow-up was 4.0 (interquartile range = 2.3-6.7) years. Mortality was 5.6 (95% confidence interval [CI], 4.3-7.2), 13.1 (95% CI, 11.0-15.7), and 6.2 (95% CI, 5.4-7.2) deaths per 100 person-year among patients treated with DAA, IFN, and antiviral naïve, respectively (P < 0.001). Of the 875 HCV-HCC transplant recipients, the 5-year recurrence-free survival was 93.4%, 84.8%, 73.9% for the pre-LT DAA, pre-LT IFN, and antiviral naïve groups, respectively (P < 0.001). After multivariable regression, the use of pre-LT DAA was not associated to risk of recurrence (hazard ratio = 0.44 [95% CI, 0.19-1.00]). Post-LT DAA was not related to increased risk of recurrence (hazard ratio = 0.62 [95% CI, 0.33-1.16]). CONCLUSIONS: In this multicenter intent-to-treat study, DAA therapy was not found to be a risk factor for mortality or HCC recurrence after adjusting for potential confounders.

Toward Systematic Screening for Persistent Hepatitis E Virus Infections in Transplant Patients.

BACKGROUND: Persistent hepatitis E virus genotype 3 (HEV G3) infections affect solid organ transplant (SOT) recipients and hematopoietic stem cell transplant (HSCT) recipients, but the burden in these cohorts in the United Kingdom is unknown. We established an audit to determine the point prevalence of HEV viremia in SOT and HSCT patients in the United Kingdom and compare different testing approaches to inform screening strategies. METHODS: Between January 5, 2016, and September 21, 2016, 3044 patients undergoing therapeutic drug monitoring at a single transplant center were screened for HEV ribonucleic acid (RNA) in minipools. A total of 2822 patients who could be characterized included 2419 SOT patients, 144 HSCT patients and 259 patients with no available transplant history. HEV RNA-positive samples were characterized by serology and genomic phylogeny. HEV antigen (HEV-Ag) testing was performed on RNA-positive samples, 420 RNA-negative samples and 176 RNA-negative blood donor samples. RESULTS: Nineteen of 2822 patients were viremic with G3 HEV giving a prevalence of 0.67%. The median alanine aminotransferase was significantly higher in the HEV viremic patients (P < 0.0001); however, 2 viremic patients had an alanine aminotransferase value within the normal range at the time of screening. The HEV-Ag assay identified 18/19 viremic patients and all those patients with proven viremia longer than 4 weeks. CONCLUSIONS: Transplant recipients in the United Kingdom are at a low but significant risk of HEV infection. HEV-Ag detection could be an alternative to RNA detection where the goal is to identify established persistent HEV infection, particularly where expertise, facilities, or cost prohibit RNA testing.

Daclatasvir combined with sofosbuvir or simeprevir in liver transplant recipients with severe recurrent hepatitis C infection.

Daclatasvir (DCV) is a potent, pangenotypic nonstructural protein 5A inhibitor with demonstrated antiviral efficacy when combined with sofosbuvir (SOF) or simeprevir (SMV) with or without ribavirin (RBV) in patients with chronic hepatitis C virus (HCV) infection. Herein, we report efficacy and safety data for DCV-based all-oral antiviral therapy in liver transplantation (LT) recipients with severe recurrent HCV. DCV at 60 mg/day was administered for up to 24 weeks as part of a compassionate use protocol. The study included 97 LT recipients with a mean age of 59.3 ± 8.2 years; 93% had genotype 1 HCV and 31% had biopsy-proven cirrhosis between the time of LT and the initiation of DCV. The mean Model for End-Stage Liver Disease (MELD) score was 13.0 ± 6.0, and the proportion with Child-Turcotte-Pugh (CTP) A/B/C was 51%/31%/12%, respectively. Mean HCV RNA at DCV initiation was 14.3 × 6 log10 IU/mL, and 37% had severe cholestatic HCV infection. Antiviral regimens were selected by the local investigator and included DCV+SOF (n = 77), DCV+SMV (n = 18), and DCV+SMV+SOF (n = 2); 35% overall received RBV. At the end of treatment (EOT) and 12 weeks after EOT, 88 (91%) and 84 (87%) patients, respectively, were HCV RNA negative or had levels <43 IU/mL. CTP and MELD scores significantly improved between DCV-based treatment initiation and last contact. Three virological breakthroughs and 2 relapses occurred in patients treated with DCV+SMV with or without RBV. None of the 8 patient deaths (6 during and 2 after therapy) were attributed to therapy. In conclusion, DCV-based all-oral antiviral therapy was well tolerated and resulted in a high sustained virological response in LT recipients with severe recurrent HCV infection. Most treated patients experienced stabilization or improvement in their clinical status.

Clinical and laboratory features and natural history of seronegative hepatitis in a nontransplant centre.

BACKGROUND: Seronegative hepatitis is a recognized cause of liver failure requiring transplantation. The aetiology is unknown, but might relate to an unidentified virus or immune dysregulation. There are few data on seronegative hepatitis presenting to nontransplant centres. OBJECTIVES: To describe the clinical/laboratory features and natural history of seronegative hepatitis and compare these with viral/autoimmune hepatitis. METHODS: Cases of seronegative, viral and autoimmune hepatitis were identified from 2080 consecutive patients attending a rapid-access jaundice clinic over a 14-year period. RESULTS: Of 881 patients with hepatocellular jaundice, 27 (3%) had seronegative hepatitis, 44 (5%) autoimmune and 62 (7%) viral hepatitis (acute hepatitis A, B, C and E viruses). Fifteen out of 27 (56%) patients with seronegative hepatitis were male, median age 60 years (range 14-74). Peak bilirubin was 63 µmol/l (range 9-363), alanine aminotransferase 932 IU/l (range 503-3807). Duration of illness was 7 weeks (range 4-12). No patients developed liver failure or had further bouts of hepatitis. One patient developed acute lymphoblastic leukaemia shortly after presentation.There was no difference in age/sex of patients with seronegative hepatitis and those with viral hepatitis. Compared with autoimmune hepatitis (age 65 years, range 15-91), patients with seronegative hepatitis were younger (P=0.002) and more likely to be male (P=0.004). Patients with autoimmune hepatitis were more likely (P<0.0001) to have an albumin less than 35 g/l, international normalized ratio greater than 1.2, raised IgG and positive antinuclear/smooth muscle antibody, compared with patients with seronegative hepatitis. CONCLUSION: Seronegative hepatitis presenting to a nontransplant centre is generally a self-limiting illness. The aetiology is more likely to be viral than autoimmune.

p38α inhibits liver fibrogenesis and consequent hepatocarcinogenesis by curtailing accumulation of reactive oxygen species.

Most hepatocellular carcinomas (HCC) develop in the context of severe liver fibrosis and cirrhosis caused by chronic liver inflammation, which also results in accumulation of reactive oxygen species (ROS). In this study, we examined whether the stress-activated protein kinase p38α (Mapk14) controls ROS metabolism and development of fibrosis and cancer in mice given thioacetamide to induce chronic liver injury. Liver-specific p38α ablation was found to enhance ROS accumulation, which appears to be exerted through the reduced expression of antioxidant protein HSP25 (Hspb1), a mouse homolog of HSP27. Its reexpression in p38α-deficient liver prevents ROS accumulation and thioacetamide-induced fibrosis. p38α deficiency increased expression of SOX2, a marker for cancer stem cells and the liver oncoproteins c-Jun (Jun) and Gankyrin (Psmd10) and led to enhanced thioacetamide-induced hepatocarcinogenesis. The upregulation of SOX2 and c-Jun was prevented by administration of the antioxidant butylated hydroxyanisole. Intriguingly, the risk of human HCC recurrence is positively correlated with ROS accumulation in liver. Thus, p38α and its target HSP25/HSP27 appear to play a conserved and critical hepatoprotective function by curtailing ROS accumulation in liver parenchymal cells engaged in oxidative metabolism of exogenous chemicals. Augmented oxidative stress of liver parenchymal cells may explain the close relationship between liver fibrosis and hepatocarcinogenesis.

Multigenerational epigenetic adaptation of the hepatic wound-healing response.

We investigated whether ancestral liver damage leads to heritable reprogramming of hepatic wound healing in male rats. We found that a history of liver damage corresponds with transmission of an epigenetic suppressive adaptation of the fibrogenic component of wound healing to the male F1 and F2 generations. Underlying this adaptation was less generation of liver myofibroblasts, higher hepatic expression of the antifibrogenic factor peroxisome proliferator-activated receptor γ (PPAR-γ) and lower expression of the profibrogenic factor transforming growth factor ß1 (TGF-ß1) compared to rats without this adaptation. Remodeling of DNA methylation and histone acetylation underpinned these alterations in gene expression. Sperm from rats with liver fibrosis were enriched for the histone variant H2A.Z and trimethylation of histone H3 at Lys27 (H3K27me3) at PPAR-γ chromatin. These modifications to the sperm chromatin were transmittable by adaptive serum transfer from fibrotic rats to naive rats and similar modifications were induced in mesenchymal stem cells exposed to conditioned media from cultured rat or human myofibroblasts. Thus, it is probable that a myofibroblast-secreted soluble factor stimulates heritable epigenetic signatures in sperm so that the resulting offspring better adapt to future fibrogenic hepatic insults. Adding possible relevance to humans, we found that people with mild liver fibrosis have hypomethylation of the PPARG promoter compared to others with severe fibrosis.

The NF-kappaB p50:p50:HDAC-1 repressor complex orchestrates transcriptional inhibition of multiple pro-inflammatory genes.

BACKGROUND & AIMS: The pro-inflammatory functions of NF-kappaB must be tightly regulated to prevent inappropriate tissue damage and remodelling caused by activated inflammatory and wound-healing cells. The p50 subunit of NF-kappaB is emerging as an important repressor of immune and inflammatory responses, but by mechanisms that are poorly defined. This study aims to delineate p50 target genes in activated hepatic stellate cells and to outline mechanisms utilised in their repression. METHODS: Hepatic stellate cells were isolated from nfkb1(p50)-deficient or Wt mice and gene expression compared using microarray. Target genes were verified by qRT-PCR and p50-mediated HDAC-1 recruitment to the target genes demonstrated using chromatin immunoprecipitation. RESULTS: We identify p50 as transcriptional repressor of multiple pro-inflammatory genes including Ccl2, Cxcl10, Gm-csf, and Mmp-13. These genes are over-expressed in nfkb1(p50)-deficient mice suffering from chronic hepatitis and in fibrogenic/inflammatory hepatic stellate cells isolated from nfkb1(-/-) liver. We identify Mmp-13 as a bona-fide target gene for p50 and demonstrate that p50 is required for recruitment of the transcriptional repressor histone deacetylase (HDAC)-1 to kappaB sites in the Mmp-13 promoter. Chromatin immunoprecipitations identified binding of HDAC-1 to specific regulatory regions of the Ccl2, Cxcl10, Gm-csf genes that contain predicted kappaB binding motifs. Recruitment of HDAC-1 to these genes was not observed in nfkb1(-/-) cells suggesting a requirement for p50 in a manner similar to that described for Mmp-13. CONCLUSIONS: Recruitment of HDAC-1 to inflammatory genes provides a widespread mechanism to explain the immunosuppressive properties of p50.

Wound healing and local neuroendocrine regulation in the injured liver.

The hepatic wound-healing response is a complex process involving many different cell types and factors. It leads to the formation of excessive matrix and a fibrotic scar, which ultimately disrupts proper functioning of the liver and establishes cirrhosis. Activated hepatic myofibroblasts, which are derived from cells such as hepatic stellate cells (HSCs), play a key role in this process. Upon chronic liver injury, there is an upregulation in the local neuroendocrine system and it has recently been demonstrated that activated HSCs express specific receptors and respond to different components of this system. Neuroendocrine factors and their receptors participate in a complex network that modulates liver inflammation and wound healing, and controls the development and progression of liver fibrosis. The first part of this review provides an overview of the molecular mechanisms governing hepatic wound healing. In the second section, we explore important components of the hepatic neuroendocrine system and their recently highlighted roles in HSC biology and hepatic fibrogenesis. We discuss the therapeutic interventions that are being developed for use in antifibrotic therapy.

Hepatic nuclear factor kappa B regulates neutrophil recruitment to the injured brain.

Acute brain injury is associated with induction of hepatic chemokine expression, which is an essential element in the subsequent recruitment of leukocytes to the damaged brain. To further understand the significance of the hepatic inflammatory response, we focused on nuclear factor (NF)-kappa B, a pivotal regulator of inflammation. Nondestructive real-time whole-body imaging was undertaken in the 3XNF-kappa B-luciferase mouse to monitor NF-kappa B activation. Acute brain injury induced by intracerebral injection of interleukin-1 provoked rapid activation of hepatic and CNS NF-kappa B, with only minimal changes in other organs. Elevated NF-kappa B in the brain was limited to the site of the lesion, whereas hepatic NF-kappa B was widespread. The function of NF-kappa B in this model was determined by monitoring leukocyte recruitment to the liver and brain of nf kappa b1 mice, which lack the anti-inflammatory p50:p50 NF-kappa B homodimer. Brain injury in the nf kappa b1 mice was associated with increased neutrophil recruitment to the liver and brain compared with wild-type mice, thereby confirming a regulatory role for the NF-kappa B system. To determine the role of hepatic NF-kappa B, it was selectively inhibited by intravenous adenoviral-mediated delivery of an I kappa B alpha super-repressor. This treatment significantly reduced the numbers of neutrophils recruited to the brain. In conclusion, acute brain injury is associated with rapid and robust activation of hepatic NF-kappa B, which is required for efficient mobilization of circulating leukocytes to the brain.

Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production.

Hepatocellular carcinoma (HCC), the most common liver cancer, occurs mainly in men. Similar gender disparity is seen in mice given a chemical carcinogen, diethylnitrosamine (DEN). DEN administration caused greater increases in serum interleukin-6 (IL-6) concentration in males than it did in females. Furthermore, ablation of IL-6 abolished the gender differences in hepatocarcinogenesis in mice. DEN exposure promoted production of IL-6 in Kupffer cells (KCs) in a manner dependent on the Toll-like receptor adaptor protein MyD88, ablation of which also protected male mice from DEN-induced hepatocarcinogenesis. Estrogen inhibited secretion of IL-6 from KCs exposed to necrotic hepatocytes and reduced circulating concentrations of IL-6 in DEN-treated male mice. We propose that estrogen-mediated inhibition of IL-6 production by KCs reduces liver cancer risk in females, and these findings may be used to prevent HCC in males.

Nuclear factor-kappaB and the hepatic inflammation-fibrosis-cancer axis.

Nuclear factor-kappaB (NF-kappaB) is a transcriptional regulator of genes involved in immunity, inflammatory response, cell fate, and function. Recent attention has focused on the pathophysiological role of NF-kappaB in the diseased liver. In vivo studies using rodent models of liver disease and cell-targeted perturbation of NF-kappaB activity have revealed complex and multicellular functions in hepatic inflammation, fibrosis, and the development of hepatocellular carcinoma - a process we have termed the "inflammation-fibrosis-cancer axis". This review summarizes the current state of knowledge and provides insight into the vast complexity of the hepatic NF-kappaB signaling system, which should provide a rich source of new therapeutic targets.