BACKGROUND/AIM:Subjects with metabolic risk factors for non-alcoholic fatty liver disease (NAFLD) are commonly seen by hospital specialists other than gastroenterologists/hepatologists. The aim of this study was to assess the awareness of NAFLD and opinions regarding management among non-hepatologists at two major tertiary hospitals in Brisbane.METHODS:A face-to-face questionnaire assessing current beliefs and practices regarding NAFLD was administered to specialists and specialists-in-training across six specialties (internal medicine, cardiology/cardiac surgery, endocrinology, thoracic medicine, rheumatology and nephrology).RESULTS:One hundred clinicians were surveyed with 99% returning completed questionnaires (>89% questions answered). The majority of respondents (75%) believe the prevalence of NAFLD in the general population to be ≤ 10%, although two-thirds feel that its incidence will rise markedly. The vast majority (>90%) appreciate that traditional cardiovascular risk factors (obesity, hypertriglyceridaemia and diabetes) are risk factors for NAFLD and acknowledge that these are common in non-hepatology patients. Despite this, most believe that NAFLD is uncommon in their own patients (89% indicated a prevalence ≤ 30%). The vast majority (93%) agree that non-alcoholic steatohepatitis (NASH) is associated with increased overall mortality, but 60% also believe that simple steatosis confers increased liver-related mortality. Most (74%) agree that a diagnosis of NASH cannot be made using liver enzymes, but 67% support 6-monthly liver function tests as the most effective way to monitor progression of NAFLD. Most respondents (71%) make no referrals to hepatology for suspected NAFLD.CONCLUSIONS:Non-hepatologists appreciate the seriousness of NAFLD but appear to underestimate its prevalence, especially among their own patients despite known risk factors. Attitudes regarding simple steatosis versus NASH and appropriate monitoring of suspected NAFLD suggest that more can be done to improve the understanding of this disease among non-hepatologists. This has implications for targeting 'at-risk' populations and appropriate referral of patients to hepatology clinics.

Background Liver macrophages are a heterogeneous cell population that produces factors involved in fibrogenesis and matrix turnover, including matrix metalloproteinase (MMP) -9. During liver injury, their close proximity to hepatic progenitor cells and the ductular reaction may enable them to regulate liver repair and fibrosis. Aims To enumerate and characterise liver macrophages in patients with chronic hepatitis C, to determine whether a distinct population of macrophages is associated with the ductular reaction and portal fibrosis. Methods Immunostaining for macrophage markers (CD68, CD163, CCR2), the ductular reaction (keratin-7) and MMP-9 was performed in liver biopsy sections from patients with chronic hepatitis C virus (HCV) (n=85). Results Portal tracts were more densely populated with macrophages (10.5 +/- 0.36 macrophages/HPF) than lobules (7.2 +/- 0.16 macrophages/HPF, P<0.001) and macrophages were found in close proximity to the ductular reaction. 30% of portal and periductal macrophages expressed MMP-9 and these were significantly associated with increasing stage of fibrosis (rs=0.58, 0.68, respectively, both P<0.001). In contrast, MMP-9+ macrophages were largely absent in lobular regions and non-diseased liver. Hepatic MMP-9 mRNA levels and gelatinolytic activity were significantly associated with stage of fibrosis (rs=0.47, rs=0.89, respectively, both P<0.001). Furthermore, a second distinct CCR2+ macrophage population was localised to the centrilobular regions and was predominantly absent from portal and periductal areas. Conclusions These findings demonstrate significant regional differences in macrophage phenotypes, suggesting that there are at least two populations of liver macrophages. We propose that these populations have distinct contributions to the pathogenesis of chronic HCV-related liver disease.

BACKGROUND:A reliable biomarker is required in hepatology clinics for detection and follow-up of heavy alcohol consumption. Carbohydrate-deficient transferrin (CDT) increases with sustained heavy alcohol consumption and is the most specific biomarker of ethanol (EtOH) consumption. Recent introduction of a standardized method for measuring CDT has improved its clinical application. This study was designed to determine whether alcohol-independent factors influence CDT levels in patients with chronic liver disease (CLD).METHODS:The relationship between serum %CDT and self-reported history of alcohol consumption was examined in 254 patients referred for evaluation of liver disease. CDT analysis was performed on serum collected at time of liver biopsy.RESULTS:CDT levels were not affected by severity or etiology of nonalcoholic liver disease. Thirteen of 254 subjects had a %CDT >1.7, predictive of heavy alcohol intake, 6 of whom did not acknowledge heavy drinking. Twelve of these 13 subjects were suspected heavy drinkers on review of their medical records and clinical results. Conversely, not all acknowledged heavy drinkers had %CDT >1.7. Heavy drinkers with a body mass index (BMI) in the overweight or obese range had significantly lower %CDT than lean heavy drinkers. This persisted even when lean body weight was used as an approximation of the EtOH volume of distribution.CONCLUSIONS:An elevated BMI reduces the diagnostic utility of CDT at higher alcohol intake in subjects with CLD using the standardized method. In a hepatology outpatient setting, this assay is likely to be useful to confirm suspicion of heavy drinking in subjects who are not overweight, but cannot reliably identify moderate drinkers or heavy drinkers who are overweight.

Results: Patients with AH had a greater proportion of CD14 + CD16 + monocytes compared with HVs (0.31 v 0.11; p = 0.002) and fewer CD14 lo CD16 + cells (0.01 v 0.03; p = 0.002).In HVs, CD14 + CD16 + monocytes expressed more interleukin (IL)-10 mRNA (3.1 fold difference; p = 0.02) and less TNFa (0.6 fold; p = 0.002) than CD14 + CD16 -monocytes.CD14 lo CD16 + monocytes expressed particularly high levels of TNFa, and CD14 + CD16 + monocytes were less effective than CD14 + CD16 -cells at driving memory T-cell proliferation (14.6% v 28.4%; p = 0.001).Conclusions: Consistent with previous reports in CLD, patients with AH have a greater proportion of CD14 + CD16 + monocytes.Detailed phenotypic analysis of these cells in HVs questions whether CD14 + CD16 + cells are pro-inflammatory as they express more of the anti-inflammatory cytokine IL-10, and are less able to drive memory T-cell proliferation, than CD14 + CD16 -cells.Further studies are being conducted to assess the role of these 3 monocyte subsets in AH.

Infection with hepatitis C virus (HCV) is a major cause of chronic liver disease. Hepatic fibrosis may develop in subjects with chronic HCV infection, culminating in cirrhosis and an increased risk of hepatocellular carcinoma. The rate of development of fibrosis varies substantially between individuals; while it is influenced by a number of demographic and environmental factors, these account for only a small proportion of the variability. There are no clinical markers or tests that predict the rate of fibrosis progression in an individual subject. Thus, there has been increasing interest in the influence of host genetic factors on the rate of disease progression, and whether a genetic signature can be developed to reliably identify individuals at risk of severe disease. Numerous case-control, candidate gene, allele-association studies have examined the relationship between host single nucleotide polymorphisms or other genetic mutations and fibrosis in patients with chronic HCV infection. However, these studies have generally been irreproducible and disappointing. As seen with genetic studies for other diseases, small study cohorts and poor study design have contributed to limited meaningful findings. The successful determination of genetic signatures for fibrosis progression in chronic HCV will require multicenter collaborations using genome-wide association studies, with large, phenotypically well-defined sample sets. While these studies will require a significant financial commitment, a successful outcome offers the potential for personalized therapy and better patient management.

Background Serum hepcidin concentration is potentially affected by inflammation and iron stores in chronic liver disease (CLD), but little is known about the relationship between hepcidin and the degree of hepatic fibrosis. We investigated the potential role of serum hepcidin as a biomarker of advanced liver disease. Methods Serum hepcidin was measured in 332 adults with CLD of varying aetiologies, 45 healthy and 50 non-liver disease patient controls. Liver biopsy data were available for 228 CLD subjects. Results Hepcidin was decreased in CLD patients compared with non-liver disease patient controls (P similar to<similar to 0.0001) but not healthy controls, and was lowest in those with cirrhosis (P similar to<similar to 0.0001). Serum hepcidin correlated with hepatic hepcidin mRNA expression in 91 biopsy samples available for genetic analysis (r similar to=similar to 0.68, P similar to<similar to 0.0001). Hepcidin also correlated positively with serum ferritin concentration, transferrin saturation, ALT, serum albumin and haemoglobin, but negatively with serum bilirubin. The hepcidin:ferritin ratio was significantly lower in CLD subjects compared with healthy and disease controls, and decreased with each increase in the stage of fibrosis and siderosis. The hepcidin:ferritin ratio was associated with progressive fibrosis on linear regression, and a value of less than 0.1 was independently associated with cirrhosis on logistic regression analyses (OR 5.54, P similar to<similar to 0.001). Receiver operating characteristic analysis showed the hepcidin:ferritin ratio was able to distinguish between F0 and F4 stages of fibrosis (area under receiver operating characteristic curve similar to=similar to 0.86). Conclusions The hepcidin:ferritin ratio is reduced in relation to increasing fibrosis in CLD and the use of this ratio may have potential future diagnostic implications as a marker of cirrhosis.

Background We previously demonstrated that hepatic cholesterol loading increases liver injury (serum ALT, cell death, macrophage infi ltration) in high fat (HF)-fed foz/foz (Alms1 mutant) mice with NASH (JGH. 2010; 25: A5). This is associated with NF-κB and c-Jun N-terminal kinase (JNK) activation, and fi brogenesis. We have now investigated whether pharmacological inhibition of cholesterol uptake and/or biosynthesis after the onset of NASH in this model reverses liver injury and infl ammation, and examined the pathways involved. Methods Female foz/foz and WT mice were fed HF diet containing 0.2% cholesterol for 16 wk, after which atorvastatin (20 mg/kg/day, corresponding to human doses adjusted for species differences in statin metabolism) and/or ezetimibe (5 mg/kg/day) was introduced into the diet (vehicle for positive controls); mice were then fed for an additional 8 wk. We determined serum cholesterol and alanine transaminase (ALT) (clinical chemistry, TCH) and hepatic protein expression (western blot) and localisation (IHC). Liver sections stained with H&E were blinded and ranked (by MMY) according to NAFLD activity score. Sirius red-stained sections were subjected to image analysis (ImageJ, NIH, Bethesda) to quantify fi brosis. Results Atorvastatin and/or ezetimibe for 8 wk after NASH onset (at 16 wk) lowered ALT substantially (P < 0.05) vs vehicle-treated foz/foz mice, in association with reduced serum total cholesterol and HDL; liver lipid analyses are in train. Likewise, hepatomegaly was reduced while peri-ovarian adipose wt and serum adiponectin levels increased in drugtreated foz/foz mice. Hepatocellular cell death (by M30 IHC), and expression of nuclear NF-κB p65, JNK, vascular cell adhesion molecule-1 and inter-cellular adhesion molecule-1 were all suppressed in drug treated foz/ foz mice, in addition to hepatic macrophage infi ltration. Drug treatment also lowered serum monocyte chemotactic protein-1 levels in foz/foz mice. Conclusions Inhibition of intestinal/tissue cholesterol uptake/redistribution and/or biosynthesis lowers serum cholesterol and reduces liver injury and infl ammation in foz/foz mice with NASH. These fi ndings support the hypothesis that hepatic cholesterol acts as a lipotoxic mediator of profi brotic liver injury and infl ammation in experimental NASH.

A ductular reaction at the periphery of portal tracts appears to be a common stereotypical response to injury that is observed in a number of chronic liver diseases, including viral hepatitis. It consists of a complex of strings of cholangiocytes, small bile ductules, stroma and inflammatory cells, and occurs at the interface between portal tracts and parenchyma.1 These reactive ductules are postulated to arise from hepatic progenitor cells, small bipotential cells capable of proliferation and differentiation into both hepatocytes and cholangiocytes. This alternative pathway of liver regeneration is activated when mature hepatocytes are unable to proliferate sufficiently to replace lost cells.2 Ductules are rarely seen in normal human liver,3 but increase in number in association with the severity of liver injury. Studies in chronic viral hepatitis, haemochromatosis, and alcoholic and non-alcoholic liver disease have shown a close correlation between progenitor cell expansion and the extent of hepatocellular injury, inflammation or fibrosis, although cause and effect has not been determined.4–7 The study of Svegliati-Baroni and colleagues8 in this issue of Gut ( see page 108 ) demonstrates a significant association between reactive ductular cells, hepatic progenitor cells and stage of fibrosis in patients with chronic hepatitis C virus (HCV) infection, further supporting these earlier studies. Interestingly the current study also …

Interleukin 32 (IL-32) is a recently described proinflammatory cytokine that activates p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kappa B), thereby inducing proinflammatory cytokines such as IL-1 beta and tumor necrosis factor alpha (TNF-alpha). We investigated the role of IL-32 in patients with chronic hepatitis C virus (HCV) infection. Steady-state hepatic messenger RNA (mRNA) levels of IL-32 were determined in a cohort of 90 subjects; anti-IL-32 staining was used in a second cohort of 132 consecutive untreated chronic HCV patients. Correlations with histological features of steatosis, inflammation, and fibrosis were made. In vitro, endogenous IL-32 in monocytes and in the human hepatoma cell line Huh-7.5 were examined. The effects of IL-32-overexpression and IL-32-silencing on HCV replication were studied using HCV luciferase reporter viruses. There were highly significant positive associations between hepatic IL-32 mRNA expression and liver steatosis, inflammation, fibrosis, smooth muscle actin (SMA) area, and serum alanine aminotransferase (ALT) levels. IL-32 protein expression was positively associated with portal inflammation, SMA area, and ALT. In vitro, IL-1 beta and TNF-a significantly induced IL-32 expression in human Huh-7.5 cells. Alone, stimulation with interferon alpha (IFN-alpha) did not induce IL-32 expression in Huh-7.5. However, IFN-alpha exerted a significant additive effect on TNF-alpha-induced but not IL-1 beta-induced IL-32 expression, particularly in CD14(+) monocytes. This effect was dependent both on NF-kappa B and Jak/STAT signaling. Viral infection of Huh-7.5 cells resulted in a significant (11-fold) induction of IL-32 mRNA expression. However, modulation of IL-32 in Huh-7.5 cells by overexpression or silencing did not influence HCV virus replication as determined by luciferase assays. Conclusion: IL-32 is a novel proinflammatory cytokine involved in HCV-associated liver inflammation/fibrosis. IL-32 is expressed by human hepatocytes and hepatoma cells and its expression is regulated by proinflammatory stimuli. (HEPATOLOGY 2011; 53:1819-1829)

AIMTo investigate the influence of macrophages on hepatocyte phenotype and function.METHODSMacrophages were differentiated from THP-1 monocytes via phorbol myristate acetate stimulation and the effects of monocyte or macrophage-conditioned medium on HepG2 mRNA and protein expression determined. The in vivo relevance of these findings was confirmed using liver biopsies from 147 patients with hepatitis C virus (HCV) infection.RESULTSConditioned media from macrophages, but not monocytes, induced a transient morphological change in hepatocytes associated with upregulation of vimentin (7.8 ± 2.5-fold, P = 0.045) and transforming growth factor (TGF)-β1 (2.6 ± 0.2-fold, P < 0.001) and downregulation of epithelial cadherin (1.7 ± 0.02-fold, P = 0.017) mRNA expression. Microarray analysis revealed significant upregulation of lipocalin-2 (17-fold, P < 0.001) and pathways associated with inflammation, and substantial downregulation of pathways related to hepatocyte function. In patients with chronic HCV, real-time polymerase chain reaction and immunohistochemistry confirmed an increase in lipocalin-2 mRNA (F0 1.0 ± 0.3, F1 2.2 ± 0.2, F2 3.0 ± 9.3, F3/4 4.0 ± 0.8, P = 0.003) and protein expression (F1 1.0 ± 0.5, F2 1.3 ± 0.4, F3/4 3.6 ± 0.4, P = 0.014) with increasing liver injury. High performance liquid chromatography-tandem mass spectrometry analysis identified elevated levels of matrix metalloproteinase (MMP)-9 in macrophage-conditioned medium, and a chemical inhibitor of MMP-9 attenuated the change in morphology and mRNA expression of TGF-β1 (2.9 ± 0.2 vs 1.04 ± 0.1, P < 0.001) in macrophage-conditioned media treated HepG2 cells. In patients with chronic HCV infection, hepatic mRNA expression of CD163 (F0 1.0 ± 0.2, F1/2 2.8 ± 0.3, F3/4 5.3 ± 1.0, P = 0.001) and MMP-9 (F0 1.0 ± 0.4, F1/2 2.8 ± 0.3, F3/4 4.1 ± 0.8, P = 0.011) was significantly associated with increasing stage of fibrosis.CONCLUSIONSecreted macrophage products alter the phenotype and function of hepatocytes, with increased expression of inflammatory mediators, suggesting that hepatocytes actively participate in liver injury.

Background and Aim:Hepatitis C virus (HCV) proteins activate the unfolded protein response (UPR) in experimental models. The role of the UPR in the pathogenesis of HCV-induced liver injury has not been determined. Our aim was to investigate the role of the UPR in the pathogenesis of chronic HCV.Methods:Liver biopsy samples from 124 patients with chronic HCV and 24 HCV/HBV-negative subjects with histologically normal liver (NDL) were assessed. The hepatic mRNA expression of components of the UPR was measured by semi-quantitative real-time polymerase chain reaction. Glucose regulated protein (GRP) 78 protein expression was assessed by immunohistochemistry.Results:The expression of GRP78 mRNA and growth arrest and damage inducible protein 34 (GADD34) mRNA was significantly lower in subjects with HCV than NDL (P = 0.007 and P < 0.001, respectively). There was no significant difference in the expression of GRP94 mRNA, spliced X box binding protein 1 (sXBP1) mRNA, C/EBP homologous protein mRNA (CHOP) and ER degradation enhancing alpha-mannosidase-like protein (EDEM) mRNA and GRP78 protein between patients with HCV and NDL. There were no relationships between elements of the UPR and inflammation or fibrosis in patients with HCV.Conclusion:Downstream components of UPR were not activated in patients with chronic HCV. Therefore, the UPR may not play a prominent role in liver injury in patients with chronic HCV infection.

Background Adult non-alcoholic fatty liver disease (NAFLD) involves lobular necroinflammatory activity and fibrosis is typically centrilobular, whereas paediatric NAFLD has predominantly portal fibrosis. The reasons for these differences are unclear. We aimed to determine (a) how centrilobular and portal fibrosis in children relate to histological parameters; and (b) whether atypical fibrosis patterns exist in adults that are unexplained by current fibrogenesis models. Methods Histological features of paediatric (n=38) and adult (n=56) NAFLD were assessed using conventional scoring systems. Keratin-7 immunostaining was used to assess hepatic progenitor cell numbers and the ductular reaction. Centrilobular and portal components of fibrosis were independently scored and fibrosis patterns were classified according to accepted types. Post-treatment (rosiglitazone/gastric banding) biopsies were also examined in adults. Results Twenty-six children (68.4%) had portal-predominant fibrosis, although the typical adult pattern was seen in 11 (28.9%). Portal fibrosis was associated with a ductular reaction (P=0.021) and hepatic progenitor cell expansion (P<0.001), whereas centrilobular fibrosis was associated with lobular inflammation (P=0.026) and ballooning (P=0.001). Before intervention, six adults (10.7%) had atypical fibrosis including 3 (5.4%) with a previously unrecognized pattern of very fine, non-zonal sinusoidal fibrosis. Despite improvements in steatosis and inflammation, more patients developed this unusual pattern after intervention with most having had surgery (9 of 10 adults; P<0.001). Conclusion Differing associations with portal and centrilobular fibrosis in children and atypical fibrosis patterns in adults suggest that multiple fibrogenic pathways exist in NAFLD. This has implications for therapy and understanding pathogenesis.

Over the last few years, the paradigm in hepatology has changed from focusing on a single liver disease to considering concurrent diseases, in particular obesity and related metabolic factors. Obesity has reached epidemic proportions globally and is associated with insulin resistance, steatosis and a low-grade systemic inflammatory state. These metabolic factors have a synergistic role in the natural history and treatment outcomes related to chronic liver disease. This is characterized best in chronic hepatitis C where steatosis and insulin resistance are caused by viral and metabolic effects. Non-alcoholic fatty liver disease and related metabolic abnormalities also exacerbate other diseases, such as alcoholic liver disease and haemochromatosis. In addition, there is growing evidence linking obesity and type 2 diabetes with hepatocellular carcinoma in subjects with chronic viral hepatitis. The pathogenesis of co-morbid disease may be related to increased oxidative stress, inflammatory injury and cell death, along with altered hepatocyte regeneration and repair. Hyperinsulinaemia and other metabolic factors may also have a direct role in the progression of liver injury. Data indicate that weight reduction improves steatosis and inflammation in patients with chronic hepatitis C. This has important clinical and therapeutic implications and suggests that obesity should be actively addressed in the management of patients with other chronic liver diseases.

Introduction Hereditary iron overload associated with mutations in theFerroportin gene produce a dichotomy of phenotypes resulting from eitheran increase or decrease in iron effl ux capacity. In this study we examinedthe molecular basis of iron overload in an Australian family of Vietnameseorigin, characterised the molecular and cellular defect, and correlated thiswith the clinical and pathological phenotype.Methods We analysed the Ferroportin gene by DNA sequencing. Liverbiopsy was performed on two members of the family. Molecular characterisationwas performed by immunofl uorescence microscopy analysis oftransfected liver, kidney and intestinal cell lines. We analysed the ferritinlevels of cells expressing wild-type and mutant ferroportin to defi ne thenature of the molecular defect on iron transport.Results We identifi ed a G to A nucleotide change at position 238 in theFerroportin gene leading to the G80S substitution in family members fromthree different generations. These patients presented with hyperferritinemiain the presence of normal transferrin saturation. Perls’ staining ofliver sections showed a marked presence of iron throughout the parenchyma.Stainable iron was present in Kupffer cells with signifi cant stainingin the periportal hepatocytes. Cellular analysis of the mutant protein indicatesthat this amino acid change does not affect the localisation of theprotein but does affect its ability to transport iron.Conclusions This is the fi rst clinical report of ferroportin disease in afamily of Vietnamese origin and the fi rst report of the G80S ferroportinmutation outside of Southern Europe. We also show that in in vitro studiesthe G80S mutant ferroportin fails to export iron. This is in agreement withthe phenotypic characteristics of patients carrying this mutation which areindicative of the classical form of ferroportin disease which is characterisedby hyperferritinemia with normal transferrin saturation, and ironaccumulation preferentially in macrophages.

Liver macrophages (Kupffer cells) play a key role in the progression of liver injury however their infl uence on hepatocyte function and viability remains unknown. The aim of this study was to investigate whether macrophage-secreted products alter the phenotype of these epithelial cells. Macrophages were differentiated from THP-1 cells (monocyte cell line) via phorbol myristate acetate stimulation. The resulting macrophage-conditionedmedia (MΦCM) was added to two different hepatocyte cell lines, HepG2 and Huh7. Expression of the EMT markers E-cadherin, vimentin, S100A4, collagen, transforming growth factor-β (TGF-β) and α-smooth muscle actin (αSMA) were measured using real-time PCR and immunofl uorescence. Comparisons between untreated and MΦCM-treated cells were analysed using a one-way ANOVA and Tukey’s multiple comparison test. A p value of <0.05 was considered statistically signifi cant. Hepatocytes treated with MΦCM for 24 h acquired mesenchymal-like properties, with reduced E-cadherin (p < 0.05) and enhanced TGF-β (p < 0.001) mRNA expression. Huh7 and HepG2 cells demonstrated partial EMT due to the signifi cant increase in mesenchymal markers, vimentin (p < 0.05) and S100A4 (p < 0.001) respectively. However αSMA andcollagen, two markers associated with complete EMT, showed no signifi - cant change. Interestingly, hepatocyte phenotypic changes were transient as reversal of the mesenchymal-like properties was seen following a further 7 days in culture in normal media. No change in gene expression was observed in hepatocytes treated with conditioned media isolated from undifferentiated THP-1 cells. This in vitro study provides evidence that macrophage-secreted products can induce transient phenotypic changes in hepatocytes that may contribute to fi brogenesis. Importantly, the data raise the possibility that hepatocytes may contribute to TGF-β production during infl ammatory liver injury with recruitment and activation of liver macrophage populations.