Role of Hepatocyte Senescence in the Activation of Hepatic Stellate Cells and Liver Fibrosis Progression.

Hepatocyte senescence is associated with liver fibrosis. However, the possibility of a direct, causal relation between hepatocyte senescence and hepatic stellate cell (HSC) activation was the subject of this study. Liver biopsy specimens obtained from 50 patients with non-alcoholic fatty liver disease and a spectrum of liver fibrosis stages were stained for p16, αSMA, and picrosirius red (PSR). Primary human HSCs were cultured in conditioned media derived from senescent or control HepG2 cells. Expression of inflammatory and fibrogenic genes in HSCs cultured in conditioned media were studied using RT-PCR. ELISAs were undertaken to measure factors known to activate HSCs in the conditioned media from senescent and control HepG2 cells and serum samples from healthy volunteers or patients with biopsy-proven cirrhosis. There was a strong association between proportion of senescent hepatocytes and hepatic stellate cell activation. Both proportion of hepatocyte senescence and hepatic stellate cell activation were closely associated with fibrosis stage. Inflammatory and fibrogenic genes were up-regulated significantly in HSCs cultured in conditioned media from senescent HepG2 cells compared with control HepG2 cells. PDGF levels were significantly higher in the conditioned media from senescent hepatocytes than control HepG2-conditioned media, and in serum samples from patients with cirrhosis than healthy volunteers. In conclusion, this 'proof of concept' study revealed activation of human HSCs by media from senescent HepG2 cells, indicating direct involvement of factors secreted by senescent hepatocytes in liver fibrosis.

The Alpha-1 Antitrypsin Polymer Load Correlates With Hepatocyte Senescence, Fibrosis Stage and Liver-Related Mortality.

BACKGROUND: Alpha-1 antitrypsin deficiency (AATD) is an important, inherited cause of chronic liver disease. Marked variation in fibrosis stages in patients with homozygous deficiency and those factors that determine whether heterozygous carriers develop liver fibrosis, remain unexplained. Murine studies implicate polymerized alpha-1 antitrypsin (AAT) within hepatocytes as pathogenic. AIMS AND METHODS: The relationship between the quantity of polymerized AAT within hepatocytes (polymer load), stage of hepatic fibrosis and liver-related clinical outcomes (death, evolution to hepatocellular carcinoma, or need for liver transplantation) were investigated using liver tissue from 92 patients at first presentation with either homozygous or heterozygous AATD. Further tissue-based studies were undertaken to determine if polymerized AAT was associated with failure of cell cycle progression, accelerated aging or hepatocyte senescence by immunohistochemical analysis. RESULTS: The AAT polymer load correlated closely with hepatic fibrosis stage and long-term clinical outcome, independent of homozygous or heterozygous status. AAT polymers within hepatocytes correlated closely with failure of cell cycle progression assessed using cell cycle phase markers, accelerated aging manifest as shortened telomeres and other markers consistent with hepatocyte senescence manifest as the presence of nuclear p21 expression and enlarged nuclei. The proportion of p21 positive hepatocytes or hepatocytes with enlarged nuclei correlated with hepatic fibrosis stage and the long-term clinical outcome. CONCLUSION: These data suggest that accumulation of AAT polymers within hepatocytes drives senescence. Quantitation of both the AAT polymer load or hepatocyte senescence markers correlated with hepatic fibrosis stage and the long-term clinical outcome. Either or both could be considered markers of disease severity and treatment response in clinical trials.

Reduced circulating BMP10 and BMP9 and elevated endoglin are associated with disease severity, decompensation and pulmonary vascular syndromes in patients with cirrhosis.

BACKGROUND: BMP9, originating from the liver, and BMP10 are circulating BMPs that preserve vascular endothelial integrity. We assessed BMP9, BMP10 and soluble endoglin (sEng) levels and their relationships to liver disease severity and associated pulmonary vascular syndromes in a cohort of well-characterised liver disease patients. METHODS: Plasma samples from patients with liver disease (n = 83) and non-disease controls (n = 21) were assayed for BMP9, BMP10 and sEng. Levels were also assessed in a separate cohort of controls (n = 27) and PoPH patients (n = 8). Expression of mRNA and immunohistochemical staining was undertaken in liver biopsy specimens. Plasma BMP activity was assessed using an endothelial cell bioassay. FINDINGS: Plasma BMP9 and BMP10 levels were normal in patients with compensated cirrhosis or fibrosis without cirrhosis, but markedly reduced in patients with decompensated cirrhosis, including those with hepatopulmonary syndrome (HPS) or portopulmonary hypertension (PoPH). Liver biopsy specimens revealed reduced mRNA expression and immunostaining for these ligands. Patient plasma samples with reduced BMP9 and BMP10 levels exhibited low BMP activity that was restored with exogenous BMP9. Endoglin mRNA expression was increased in cirrhotic livers and elevated circulating sEng levels in PoPH and HPS patients suggested increased endothelial sEng shedding in these syndromes. INTERPRETATION: Plasma BMP9 and BMP10 levels are reduced in decompensated cirrhosis, leading to reduced circulating BMP activity on the vascular endothelium. The pulmonary complications of cirrhosis, PoPH and HPS, are associated with markedly reduced BMP9 and BMP10 and increased sEng levels, suggesting that supplementation with exogenous ligands might be a therapeutic approach for PoPH and HPS.

Unacceptable failures: the final report of the Lancet Commission into liver disease in the UK.

This final report of the Lancet Commission into liver disease in the UK stresses the continuing increase in burden of liver disease from excess alcohol consumption and obesity, with high levels of hospital admissions which are worsening in deprived areas. Only with comprehensive food and alcohol strategies based on fiscal and regulatory measures (including a minimum unit price for alcohol, the alcohol duty escalator, and an extension of the sugar levy on food content) can the disease burden be curtailed. Following introduction of minimum unit pricing in Scotland, alcohol sales fell by 3%, with the greatest effect on heavy drinkers of low-cost alcohol products. We also discuss the major contribution of obesity and alcohol to the ten most common cancers as well as measures outlined by the departing Chief Medical Officer to combat rising levels of obesity-the highest of any country in the west. Mortality of severely ill patients with liver disease in district general hospitals is unacceptably high, indicating the need to develop a masterplan for improving hospital care. We propose a plan based around specialist hospital centres that are linked to district general hospitals by operational delivery networks. This plan has received strong backing from the British Association for Study of the Liver and British Society of Gastroenterology, but is held up at NHS England. The value of so-called day-case care bundles to reduce high hospital readmission rates with greater care in the community is described, along with examples of locally derived schemes for the early detection of disease and, in particular, schemes to allow general practitioners to refer patients directly for elastography assessment. New funding arrangements for general practitioners will be required if these proposals are to be taken up more widely around the country. Understanding of the harm to health from lifestyle causes among the general population is low, with a poor knowledge of alcohol consumption and dietary guidelines. The Lancet Commission has serious doubts about whether the initiatives described in the Prevention Green Paper, with the onus placed on the individual based on the use of information technology and the latest in behavioural science, will be effective. We call for greater coordination between official and non-official bodies that have highlighted the unacceptable disease burden from liver disease in England in order to present a single, strong voice to the higher echelons of government.

Pretreatment prediction of response to ursodeoxycholic acid in primary biliary cholangitis: development and validation of the UDCA Response Score.

BACKGROUND: Treatment guidelines recommend a stepwise approach to primary biliary cholangitis: all patients begin treatment with ursodeoxycholic acid (UDCA) monotherapy and those with an inadequate biochemical response after 12 months are subsequently considered for second-line therapies. However, as a result, patients at the highest risk can wait the longest for effective treatment. We determined whether UDCA response can be accurately predicted using pretreatment clinical parameters. METHODS: We did logistic regression analysis of pretreatment variables in a discovery cohort of patients in the UK with primary biliary cholangitis to derive the best-fitting model of UDCA response, defined as alkaline phosphatase less than 1·67 times the upper limit of normal (ULN), measured after 12 months of treatment with UDCA. We validated the model in an external cohort of patients with primary biliary cholangitis and treated with UDCA in Italy. Additionally, we assessed correlations between model predictions and key histological features, such as biliary injury and fibrosis, on liver biopsy samples. FINDINGS: 2703 participants diagnosed with primary biliary cholangitis between Jan 1, 1998, and May 31, 2015, were included in the UK-PBC cohort for derivation of the model. The following pretreatment parameters were associated with lower probability of UDCA response: higher alkaline phosphatase concentration (p<0·0001), higher total bilirubin concentration (p=0·0003), lower aminotransferase concentration (p=0·0012), younger age (p<0·0001), longer interval from diagnosis to the start of UDCA treatment (treatment time lag, p<0·0001), and worsening of alkaline phosphatase concentration from diagnosis (p<0·0001). Based on these variables, we derived a predictive score of UDCA response. In the external validation cohort, 460 patients diagnosed with primary biliary cholangitis were treated with UDCA, with follow-up data until May 31, 2016. In this validation cohort, the area under the receiver operating characteristic curve for the score was 0·83 (95% CI 0·79-0·87). In 20 liver biopsy samples from patients with primary biliary cholangitis, the UDCA response score was associated with ductular reaction (r=-0·556, p=0·0130) and intermediate hepatocytes (probability of response was 0·90 if intermediate hepatocytes were absent vs 0·51 if present). INTERPRETATION: We have derived and externally validated a model based on pretreatment variables that accurately predicts UDCA response. Association with histological features provides face validity. This model provides a basis to explore alternative approaches to treatment stratification in patients with primary biliary cholangitis. FUNDING: UK Medical Research Council and University of Milan-Bicocca.

In vivo imaging of hepatic neutrophil migration in severe alcoholic hepatitis with 111In-radiolabelled leucocytes.

The study's aim was to image severe alcoholic hepatitis (SAH) using 111In-labelled leucocytes with two objectives in mind: firstly for non-invasive diagnosis and secondly to provide a platform for experimental therapies aiming to inhibit intrahepatic neutrophil migration. 111In-leucocyte scintigraphy was performed 30 min and 24 h post-injection in 19 patients with SAH, 14 abstinent patients with alcohol-related cirrhosis and 11 normal controls. Eleven with SAH and seven with cirrhosis also had 99mTc-nanocolloid scintigraphy. Change in hepatic 111In radioactivity was expressed as decay-corrected 24 h:30 min count ratio and, in SAH, compared with histological grading of steatohepatitis and expression of granulocyte marker, CD15. Hepatic microautoradiography on biopsy specimens obtained 24 h post-injection of 111In-leucocytes was performed in one patient. Median 24 h:30 min hepatic 111In activity ratio was higher in SAH (2.5 (interquartile range (IQR): 1.7-4.0) compared with cirrhotics and normal controls (1.0 (0.8-1.1) and 0.8 (0.7-0.9) respectively, P<0.0001). In SAH, it correlated with CD15 expression (r = 0.62, P=0.023) and was higher in marked compared with mild/moderate steatohepatitis (4.0 (3.0-4.6) compared with 1.8 (1.5-2.6), P=0.006). Hepatic-to-splenic 99mTc count rate ratio was reduced in SAH (0.5 (0.4-1.4)) compared with cirrhotics (2.3( 0.6-3.0)) and three historic normal controls (4.2 (3.8-5.0); P=0.003), consistent with impaired hepatic reticuloendothelial function. Scintigraphic findings in SAH included prominent lung radioactivity at 30 min, likely the result of neutrophil primimg. Microautoradiography demonstrated cell-associated 111In in areas of parenchymal neutrophil infiltration. In conclusion, 111In-leucocyte scintigraphy can non-invasively diagnose SAH and could provide a platform for evaluation of novel treatments aiming to inhibit intrahepatic neutrophil migration.

Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids.

The treatment of common bile duct (CBD) disorders, such as biliary atresia or ischemic strictures, is restricted by the lack of biliary tissue from healthy donors suitable for surgical reconstruction. Here we report a new method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs) for regenerative medicine applications. The resulting ECOs closely resemble primary cholangiocytes in terms of their transcriptomic profile and functional properties. We explore the regenerative potential of these organoids in vivo and demonstrate that ECOs self-organize into bile duct-like tubes expressing biliary markers following transplantation under the kidney capsule of immunocompromised mice. In addition, when seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary characteristics. The resulting bioengineered tissue can reconstruct the gallbladder wall and repair the biliary epithelium following transplantation into a mouse model of injury. Furthermore, bioengineered artificial ducts can replace the native CBD, with no evidence of cholestasis or occlusion of the lumen. In conclusion, ECOs can successfully reconstruct the biliary tree, providing proof of principle for organ regeneration using human primary cholangiocytes expanded in vitro.

Senescence in chronic liver disease: Is the future in aging?

Cellular senescence is a fundamental, complex mechanism with an important protective role present from embryogenesis to late life across all species. It limits the proliferative potential of damaged cells thus protecting against malignant change, but at the expense of substantial alterations to the microenvironment and tissue homeostasis, driving inflammation, fibrosis and paradoxically, malignant disease if the process is sustained. Cellular senescence has attracted considerable recent interest with recognition of pathways linking aging, malignancy and insulin resistance and the current focus on therapeutic interventions to extend health-span. There are major implications for hepatology in the field of fibrosis and cancer, where cellular senescence of hepatocytes, cholangiocytes, stellate cells and immune cells has been implicated in chronic liver disease progression. This review focuses on cellular senescence in chronic liver disease and explores therapeutic opportunities.

Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation.

The study of biliary disease has been constrained by a lack of primary human cholangiocytes. Here we present an efficient, serum-free protocol for directed differentiation of human induced pluripotent stem cells into cholangiocyte-like cells (CLCs). CLCs show functional characteristics of cholangiocytes, including bile acids transfer, alkaline phosphatase activity, γ-glutamyl-transpeptidase activity and physiological responses to secretin, somatostatin and vascular endothelial growth factor. We use CLCs to model in vitro key features of Alagille syndrome, polycystic liver disease and cystic fibrosis (CF)-associated cholangiopathy. Furthermore, we use CLCs generated from healthy individuals and patients with polycystic liver disease to reproduce the effects of the drugs verapamil and octreotide, and we show that the experimental CF drug VX809 rescues the disease phenotype of CF cholangiopathy in vitro. Our differentiation protocol will facilitate the study of biological mechanisms controlling biliary development, as well as disease modeling and drug screening.

Chronic Hepatitis B Virus Infection: The Relation between Hepatitis B Antigen Expression, Telomere Length, Senescence, Inflammation and Fibrosis.

BACKGROUND: Chronic Hepatitis B virus (HBV) infection can lead to the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. We hypothesized that HBV might accelerate hepatocyte ageing and investigated the effect of HBV on hepatocyte cell cycle state and biological age. We also investigated the relation between inflammation, fibrosis and cell cycle phase. METHODS: Liver samples from patients with chronic HBV (n = 91), normal liver (n = 55) and regenerating liver (n = 15) were studied. Immunohistochemistry for cell cycle phase markers and HBV antigens was used to determine host cell cycle phase. Hepatocyte-specific telomere length was evaluated by quantitative fluorescent in-situ hybridization (Q-FISH) in conjunction with hepatocyte nuclear area and HBV antigen expression. The effects of induced cell cycle arrest and induced cellular senescence on HBV production were assessed in vitro. RESULTS: 13.7% hepatocytes in chronic HBV had entered cell cycle, but expression of markers for S, G2 and M phase was low compared with regenerating liver. Hepatocyte p21 expression was increased (10.9%) in chronic HBV and correlated with liver fibrosis. Mean telomere length was reduced in chronic HBV compared to normal. However, within HBV-affected livers, hepatocytes expressing HBV antigens had longer telomeres. Telomere length declined and hepatocyte nuclear size increased as HBV core antigen (HBcAg) expression shifted from the nucleus to cytoplasm. Nuclear co-expression of HBcAg and p21 was not observed. Cell cycle arrest induced in vitro was associated with increased HBV production, in contrast to in vitro induction of cellular senescence, which had no effect. CONCLUSION: Chronic HBV infection was associated with hepatocyte G1 cell cycle arrest and accelerated hepatocyte ageing, implying that HBV induced cellular senescence. However, HBV replication was confined to biologically younger hepatocytes. Changes in the cellular location of HBcAg may be related to the onset of cellular senescence.

Selective insulin resistance in hepatocyte senescence.

Insulin resistance has been described in association with chronic liver disease for decades. Hepatocyte senescence has been demonstrated in chronic liver disease and as many as 80% of hepatocytes show a senescent phenotype in advanced liver disease. The aim of this study was to understand the role of hepatocyte senescence in the development of insulin resistance. Senescence was induced in HepG2 cells via oxidative stress. The insulin metabolic pathway was studied in control and senescent cells following insulin stimulation. GLUT2 and GLUT4 expressions were studied in HepG2 cells and human liver tissue. Further, GLUT2 and GLUT4 expressions were studied in three independent chronic liver disease cohorts. Signalling impairment distal to Akt in phosphorylation of AS160 and FoxO1 was evident in senescent HepG2 cells. Persistent nuclear localisation of FoxO1 was demonstrated in senescent cells despite insulin stimulation. Increased GLUT4 and decreased GLUT2 expressions were evident in senescent cells, human cirrhotic liver tissue and publically available liver disease datasets. Changes in GLUT expressions were associated with a poor clinical prognosis. In conclusion, selective insulin resistance is evident in senescent HepG2 cells and changes in GLUT expressions can be used as surrogate markers of hepatocyte senescence.

The senescent hepatocyte gene signature in chronic liver disease.

Hepatocyte senescence is associated closely with fibrosis stage and an adverse outcome in chronic liver disease, but it is uncertain whether there is a causal relation with clinical manifestations of chronic liver disease, which was the subject of this study of the senescent hepatocyte gene signature. Senescence was induced in HepG2 cells using sub-lethal concentrations of H2O2. Gene expression of control and senescent HepG2 cells were studied. Comparison was made with patients with cirrhosis and three public microarray datasets. H2O2-treated HepG2 cells demonstrated characteristic cellular senescence. There was differential expression of 354 genes in senescence. Up-regulated genes in HepG2 senescence were also up regulated in patients with cirrhosis. The senescent hepatocyte gene signature distinguished liver disease from normal by unsupervised clustering in the public chronic liver disease microarray datasets, with enrichment of the senescence gene signature in all three datasets. The senescent hepatocyte gene signature included changes in cell cycle regulation, morphology, inflammation, signal transduction, metabolism and stellate cell activation, which alongside impaired synthetic function in senescence in vitro were consistent with manifestations of clinical liver disease, suggesting a close relation between hepatocyte senescence and manifestations of chronic liver disease including fibrosis and impaired synthetic function.

Hepatocyte expression of the senescence marker p21 is linked to fibrosis and an adverse liver-related outcome in alcohol-related liver disease.

BACKGROUND AND AIM: Alcohol-related liver disease (ALD) remains a leading cause of liver-related morbidity and mortality. Age, fibrosis stage, MELD score and continued alcohol consumption predict outcome in everyday clinical practice. In previous studies increased hepatocyte nuclear area and hepatocyte expression of p21, both markers of senescence, were associated with increased fibrosis stage and a poor outcome in non-alcohol-related fatty liver disease, while increased hepatocyte nuclear area was related to liver dysfunction in ALD cirrhosis. This study, therefore, investigated the pattern of hepatocyte cell cycle phase distribution and hepatocyte p21 expression in relation to outcome in ALD. METHODS: Liver sections from two cohorts were studied. The first comprised 42 patients across the full spectrum of ALD. The second cohort comprised 77 patients with ALD cirrhosis. Immunohistochemistry assessed hepatocyte expression of cell cycle phase markers and p21. Regenerating liver (n=12) and "normal" liver sections (n=5) served as positive and negative controls, respectively. RESULTS: In the first cohort there was little cell cycle progression beyond G1/S phase and increased hepatocyte p21 expression (p<0.0001), which correlated independently with fibrosis stage (p=0.005) and an adverse liver-related outcome (p=0.03). In the second cohort, both hepatocyte p21 expression (p<0.001) and MELD score (p=0.006) were associated independently with an adverse liver-related outcome; this association was stronger with hepatocyte p21 expression (AUROC 0.74; p=0.0002) than with MELD score (AUROC 0.59; p=0.13). Further, hepatocyte p21 expression co-localised with increased hepatic stellate cell activation. CONCLUSIONS: The findings are consistent with impaired cell cycle progression beyond the G1/S phase in ALD. The striking independent associations between increased hepatocyte p21 expression and both fibrosis stage and an adverse liver-related outcome in both cohorts suggests hepatocyte senescence plays an important role in ALD. Measuring hepatocyte p21 expression is simple and cheap and in this series was a useful measure of long-term prognosis in ALD.

Age-related macular degeneration and modification of systemic complement factor H production through liver transplantation.

PURPOSE: To investigate whether modification of liver complement factor H (CFH) production, by alteration of liver CFH Y402H genotype through liver transplantation (LT), influences the development of age-related macular degeneration (AMD). DESIGN: Multicenter, cross-sectional study. PARTICIPANTS: We recruited 223 Western European patients ≥ 55 years old who had undergone LT ≥ 5 years previously. METHODS: We determined AMD status using a standard grading system. Recipient CFH Y402H genotype was obtained from DNA extracted from recipient blood samples. Donor CFH Y402H genotype was inferred from recipient plasma CFH Y402H protein allotype, measured using enzyme-linked immunosorbent assays. This approach was verified by genotyping donor tissue from a subgroup of patients. Systemic complement activity was ascertained by measuring levels of plasma complement proteins using an enzyme-linked immunosorbent assay, including substrates (C3, C4), activation products (C3a, C4a, and terminal complement complex), and regulators (total CFH, C1 inhibitor). MAIN OUTCOME MEASURES: We evaluated AMD status and recipient and donor CFH Y402H genotype. RESULTS: In LT patients, AMD was associated with recipient CFH Y402H genotype (P = 0.036; odds ratio [OR], 1.6; 95% confidence interval [CI], 1.0-2.4) but not with donor CFH Y402H genotype (P = 0.626), after controlling for age, sex, smoking status, and body mass index. Recipient plasma CFH Y402H protein allotype predicted donor CFH Y402H genotype with 100% accuracy (n = 49). Plasma complement protein or activation product levels were similar in LT patients with and without AMD. Compared with previously reported prevalence figures (Rotterdam Study), LT patients demonstrated a high prevalence of both AMD (64.6% vs 37.1%; OR, 3.09; P<0.001) and the CFH Y402H sequence variation (41.9% vs 36.2%; OR, 1.27; P = 0.014). CONCLUSIONS: Presence of AMD is not associated with modification of hepatic CFH production. In addition, AMD is not associated with systemic complement activity in LT patients. These findings suggest that local intraocular complement activity is of greater importance in AMD pathogenesis. The high AMD prevalence observed in LT patients may be associated with the increased frequency of the CFH Y402H sequence variation. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any materials discussed in this article.

Prevalence and risk factors for liver involvement in individuals with PiZZ-related lung disease.

RATIONALE: α1-Antitrypsin deficiency is one of the most common heritable human diseases, predisposing to liver and lung injury. Significant heterogeneity in phenotypic expression is well documented, but less is known of the prevalence, severity, and correlates of chronic liver disease among individuals presenting with lung disease. OBJECTIVES: To determine the frequency of and risk factors for severe liver fibrosis and cirrhosis among individuals with PiZZ-related lung disease. METHODS: A well-characterized cohort of 57 PiZZ adults attending a tertiary referral respiratory clinic was screened prospectively for clinical, laboratory, radiologic, and (when appropriate) histologic evidence of chronic liver disease. MEASUREMENTS AND MAIN RESULTS: Thirty-six (63.2%) of 57 had a history or clinical findings suggestive of liver disease; or had one or more abnormalities of liver function, or liver ultrasound, and 24 of these underwent liver biopsy. Ten (17.5%) had evidence of severe fibrosis or cirrhosis and were more likely to have higher body mass index (P = 0.04), alanine transaminase (P = 0.0001), alkaline phosphatase (P = 0.0009), prothrombin time (P = 0.0005), and maximal vital capacity (VCmax) (P = 0.04); lower platelet count (P = 0.007); abnormal liver echogenicity (P < 0.001); and splenomegaly (P = 0.001) at ultrasound. Screening with liver ultrasound provided a sensitivity and negative predictive value for severe fibrosis or cirrhosis of 100%, as were the specificity and positive predictive value for platelet count less than or equal to 174,000 per mm(3) and splenomegaly. Among individuals undergoing liver biopsy, fibrosis stage correlated with increasing VCmax (P = 0.02) and % predicted VCmax (P = 0.05), and decreasing residual volume/total lung capacity (TLC) (P = 0.02) and % predicted residual volume/TLC (P = 0.05). CONCLUSIONS: Significant chronic liver disease is common in PiZZ individuals with lung disease and can be screened effectively by a combination of conventional tests of liver function, platelet count, and liver ultrasound.

Induced pluripotent stem cells: from Nobel Prizes to clinical applications.

Advances in basic hepatology have been constrained for many years by the inability to culture primary hepatocytes in vitro, until just over five years ago when the scientific playing field was changed beyond recognition with the demonstration that human skin fibroblasts could be reprogrammed to resemble embryonic cells. The reprogrammed cells, known as induced pluripotent stem cells (iPSCs), were then shown to have the capacity to re-differentiate into almost any human cell type, including hepatocytes. The unlimited number and isogenic nature of the cells that can be generated from tiny fragments of tissue have massive implications for the study of human liver diseases in vitro. Of more immediate clinical importance were recent data demonstrating precision gene therapy on patient specific iPSCs, which opens up the real and exciting possibility of autologous hepatocyte transplantation as a substitute for allogeneic whole liver transplantation, which has been an effective approach to end-stage liver disease, but one that has now been outstripped by demand. In this review, we describe the historical development, current technology and potential clinical applications of induced pluripotency, concluding with a perspective on possible future directions in this dynamic field.

γ-H2AX+CD8+ T lymphocytes cannot respond to IFN-α, IL-2 or IL-6 in chronic hepatitis C virus infection.

BACKGROUND & AIMS: Age is the dominant prognostic factor influencing the natural history of hepatitis C virus (HCV) infection and treatment response. Accelerated lymphocyte telomere shortening in HCV infection correlates with adverse clinical outcomes. Critical telomere shortening generates double-stranded DNA breaks (DSB) inducing the DNA damage response, leading to replicative senescence. The phenotype and function of CD8+ T lymphocytes and the in vitro response to IFN-α in relation to the DNA damage response were investigated in patients with chronic HCV infection. METHODS: CD8+ T lymphocytes with DSB were identified by expression of γ-H2AX (Ser-139) in 134 HCV-exposed subjects and 27 controls. Telomere length was determined by flow-FISH; cytokine expression by intracellular cytokine staining; in vitro responses to IFN-α, IL-2 or IL-6 by phospho-STAT1 (Y701) or phospho-STAT5 (Y694) expression. RESULTS: The proportion of circulating CD8+γ-H2AX+ T lymphocytes rose with increasing fibrosis stage (p=0.0023). CD8+γ-H2AX+ T lymphocytes were enriched in liver compared to blood (p=0.03). CD8+γ-H2AX+ T lymphocytes demonstrated increased IFN-γ (p=0.02) and reduced IL-2 expression (p=0.02). CD8+γ-H2AX+ T lymphocytes failed to phosphorylate STAT1 in response to IFN-α compared to unfractionated CD8+ T lymphocytes (p <0.0001). More widespread failure of Jak/Stat signalling in CD8+γ-H2AX+ T lymphocytes was suggested by impaired phosphorylation of STAT1 with IL-6 (p=0.002) and STAT5 with IL-2 (p=0.0039) compared to unfractionated CD8+ T-lymphocytes. CONCLUSIONS: In chronic HCV infection, CD8+γ-H2AX+ T lymphocytes are highly differentiated with shortened telomeres, are more frequent within the liver, are associated with severe fibrosis and fail to activate Jak/Stat pathways in response to IFN-α, IL-2 or IL-6, perhaps explaining treatment failure in those with severe fibrosis.

A comparison of MR elastography and 31P MR spectroscopy with histological staging of liver fibrosis.

OBJECTIVES: Conventional imaging techniques are insensitive to liver fibrosis. This study assesses the diagnostic accuracy of MR elastography (MRE) stiffness values and the ratio of phosphomonoesters (PME)/phosphodiesters (PDE) measured using (31)P spectroscopy against histological fibrosis staging. METHODS: The local research ethics committee approved this prospective, blinded study. A total of 77 consecutive patients (55 male, aged 49 ± 11.5 years) with a clinical suspicion of liver fibrosis underwent an MR examination with a liver biopsy later the same day. Patients underwent MRE and (31)P spectroscopy on a 1.5 T whole body system. The liver biopsies were staged using an Ishak score for chronic hepatitis or a modified NAS fibrosis score for fatty liver disease. RESULTS: MRE increased with and was positively associated with fibrosis stage (Spearman's rank = 0.622, P < 0.001). PME/PDE was not associated with fibrosis stage (Spearman's rank = -0.041, p = 0.741). Area under receiver operating curves for MRE stiffness values were high (range 0.75-0.97). The diagnostic utility of PME/PDE was no better than chance (range 0.44-0.58). CONCLUSIONS: MRE-estimated liver stiffness increases with fibrosis stage and is able to dichotomise fibrosis stage groupings. We did not find a relationship between (31)P MR spectroscopy and fibrosis stage. KEY POINTS: Magnetic resonance elastography (MRE) and MR spectroscopy can both assess the liver. MRE is superior to ( 31 ) P MR spectroscopy in staging hepatic fibrosis. MRE is able to dichotomise liver fibrosis stage groupings. Gradient-echo MRE may be problematic in genetic haemochromatosis.

Fine mapping and replication of genetic risk loci in primary sclerosing cholangitis.

BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by progressive inflammation and fibrosis of the bile ducts eventually leading to biliary cirrhosis. Recent genetic studies in PSC have identified associations at 2q13, 2q35, 3p21, 4q27, 13q31 and suggestive association at 10p15. The aim of this study was to further characterize and refine the genetic architecture of PSC. METHODS: We analyzed previously reported associated SNPs at four of these non-HLA loci and 59 SNPs tagging the IL-2/IL-21 (4q27) and IL2RA (10p15) loci in 992 UK PSC cases and 5162 healthy UK controls. RESULTS: The most associated SNPs identified were rs3197999 (3p21 (MST1), p = 1.9 × 10⁻6, OR(A vs G) = 1.28, 95% CI (1.16-1.42)); rs4147359 (10p15 (IL2RA), p = 2.6 × 10⁻4, OR(A vs G) = 1.20, 95% CI (1.09-1.33)) and rs12511287 (4q27 (IL-2/IL-21), p = 3.0 × 10⁻4, OR(A vs T) = 1.21, 95% CI (1.09-1.35)). In addition, we performed a meta-analysis for selected SNPs using published summary statistics from recent studies. We observed genome-wide significance for rs3197999 (3p21 (MST1), P (combined) = 3.8 × 10⁻¹²) and rs4147359 (10p15 (IL2RA), P (combined) = 1.5 × 10⁻8). CONCLUSION: We have for the first time confirmed the association of PSC with genetic variants at 10p15 (IL2RA) locus at genome-wide significance and replicated the associations at MST1 and IL-2/IL-21 loci in a large homogeneous UK population. These results strongly implicate the role of IL-2/IL2RA pathway in PSC and provide further confirmation of MST1 association.