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Background & Aims: Patients with alcoholic hepatitis and a modified Maddrey's discriminant function (mDF), SCOPUS: ar.j, info:eu-repo/semantics/published

Histologically assessed hepatocyte ballooning is a key feature discriminating non-alcoholic steatohepatitis (NASH) from steatosis (NAFL). Reliable identification underpins patient inclusion in clinical trials and serves as a key regulatory-approved surrogate endpoint for drug efficacy. High inter/intra-observer variation in ballooning measured using the NASH CRN semi-quantitative score has been reported yet no actionable solutions have been proposed.A focused evaluation of hepatocyte ballooning recognition was conducted. Digitized slides were evaluated by 9 internationally recognized expert liver pathologists on 2 separate occasions: each pathologist independently marked every ballooned hepatocyte and later provided an overall non-NASH NAFL/NASH assessment. Interobserver variation was assessed and a 'concordance atlas' of ballooned hepatocytes generated to train second harmonic generation/two-photon excitation fluorescence imaging-based artificial intelligence (AI).The Fleiss kappa statistic for overall interobserver agreement for presence/absence of ballooning was 0.197 (95% CI 0.094-0.300), rising to 0.362 (0.258-0.465) with a ≥5-cell threshold. However, the intraclass correlation coefficient for consistency was higher (0.718 [0.511-0.900]), indicating 'moderate' agreement on ballooning burden. 133 ballooned cells were identified using a ≥5/9 majority to train AI ballooning detection (AI-pathologist pairwise concordance 19-42%, comparable to inter-pathologist pairwise concordance of between 8-75%). AI quantified change in ballooned cell burden in response to therapy in a separate slide set.The substantial divergence in hepatocyte ballooning identified amongst expert hepatopathologists suggests that ballooning is a spectrum, too subjective for its presence or complete absence to be unequivocally determined as a trial endpoint. A concordance atlas may be used to train AI assistive technologies to reproducibly quantify ballooned hepatocytes that standardize assessment of therapeutic efficacy. This atlas serves as a reference standard for ongoing work to refine how ballooning is classified by both pathologists and AI.For the first time, we show that, even amongst expert hepatopathologists, there is poor agreement regarding the number of ballooned hepatocytes seen on the same digitized histology images. This has important implications as the presence of ballooning is needed to establish the diagnosis of non-alcoholic steatohepatitis (NASH), and its unequivocal absence is one of the key requirements to show 'NASH resolution' to support drug efficacy in clinical trials. Artificial intelligence-based approaches may provide a more reliable way to assess the range of injury recorded as "hepatocyte ballooning".

Background & Aims: The SALVE Histopathology Group (SHG) developed and validated a grading and staging system for the clinical and full histological spectrum of alcohol-related liver disease (ALD) and evaluated its prognostic utility in a multinational cohort of 445 patients. Methods: SALVE grade was described by semiquantitative scores for steatosis, activity (hepatocellular injury and lobular neutrophils) and cholestasis. The histological diagnosis of steatohepatitis due to ALD (histological ASH, hASH) was based on the presence of hepatocellular ballooning and lobular neutrophils. Fibrosis staging was adapted from the Clinical Research Network staging system for non-alcoholic fatty liver disease and the Laennec staging system and reflects the pattern and extent of ALD fibrosis. There are 7 SALVE fibrosis stages (SFS) ranging from no fibrosis to severe cirrhosis. Results: Interobserver κ-value for each grading and staging parameter was >0.6. In the whole study cohort, long-term outcome was associated with activity grade and cholestasis, as well as cirrhosis with very broad septa (severe cirrhosis) (p, SCOPUS: ar.j, info:eu-repo/semantics/published

Summary The harmful use of alcohol has been estimated to cause approximately 3.3 million deaths every year, corresponding to nearly 6% of all deaths globally. Therefore, the effective management and treatment of alcoholic liver disease is a pertinent public health issue. In the following Clinical Practice Guidelines, the latest data on the treatment and management of alcohol-related liver disease will be reviewed and up to date recommendations for clinical management will be provided.

Histological fibrosis stage is one of the most important prognostic factors in compensated and decompensated alcohol-related liver disease (ALD). Morphological assessment of fibrosis is useful for patient stratification, enabling individualised management, and for evaluation of treatment effects in clinical studies. In contrast to most chronic liver diseases where fibrosis is portal-based, fatty liver disease (FLD) of alcoholic or non-alcoholic aetiology (NAFLD) is associated with a centrilobular pattern of injury which leads to perivenular fibrosis and/or pericellular fibrosis. Progression of FLD drives expansive pericellular fibrosis, linking vascular structures and paving the way for the development of cirrhosis. At the cirrhotic stage, ongoing tissue damage leads to increasing fibrosis severity due to parenchymal loss and proliferation of fibrous scars. Histologic fibrosis staging systems have been devised, based on topography and the extent of fibrosis, for most chronic liver diseases. The utility of histological staging is reflected in different risks associated with individual fibrosis stages which cannot be reliably distinguished by non-invasive fibrosis assessment. In contrast to NAFLD, ALD-specific staging systems that enable the standardised prognostication required for clinical management and trials are lacking. Although morphological similarities between NAFLD and ALD exist, differences in clinical and histological features may substantially limit the utility of established NAFLD-specific staging systems for prognostication in ALD. This review summarises morphological features of fibrosis in ALD and compares them to other chronic liver diseases, particularly NAFLD. ALD-related fibrosis is examined in the context of pathogenetic mechanisms of fibrosis progression, regression and clinical settings that need to be considered in future prognostically relevant ALD staging approaches.

Background & Aims The earliest characteristic alterations of the liver pathology in Wilson disease (WD) include steatosis, which is sometimes indistinguishable from non-alcoholic fatty liver disease (NAFLD). Steatosis in WD may reflect copper-induced mitochondrial dysfunction. A genetic polymorphism in rs738409, in the patatin-like phospholipase domain-containing 3 gene ( PNPLA3 ), is strongly associated with appearance of in NAFLD. This study evaluated the role of PNPLA3 and hepatic copper content for development of steatosis in patients with WD. Methods Liver biopsies obtained at diagnosis and the PNPLA3 genotype were analyzed in 98 Caucasian patients with WD (male: 52 [53.1%]; mean age: 27.6years [CI 95%: 24.8–30.4, range: 5.8–61.5]). Steatosis was graded as percentage of lipid containing hepatocytes by an expert hepatopathologist unaware of the results of genetic testing. Results Moderate/severe steatosis (>33% of hepatocytes) was observed in 28 patients (pediatric: n=13/26 [50.0%], adult: n=15/72 [20.8%]; p =0.01). Forty-six patients (46.9%; pediatric: n=7, adult: n=39; p =0.022) had cirrhosis. Multivariate logistic regression identified PNPLA3 G allele (OR: 2.469, CI 95%: 1.203–5.068; p =0.014) and pediatric age (OR: 4.348; 1.577–11.905; p =0.004) as independent variables associated with moderate/severe steatosis. In contrast, hepatic copper content did not impact on moderate/severe steatosis (OR: 1.000, CI 95%: 1.000–1.001; p =0.297). Conclusions Steatosis is common in WD and the PNPLA3 G allele contributes to its pathogenesis. The role of hepatic copper concentration and ATP7B mutations in steatosis development deserve further investigations.

Background & Aims Mice exposed to the hepatocellular carcinogen diethylnitrosamine at 2weeks of age have a high risk of developing primary liver tumors later in life. Previous studies have demonstrated that diethylnitrosamine-treated mice have increased tumor burden when fed an obesigenic "Western" diet rich in lard fat and sugar. However, the role of dietary fats vs. sugars in the promotion of liver cancer is poorly understood. The aim of this study was to determine how altering dietary fats vs. sugars affects tumor burden in the diethylnitrosamine model. Methods C57BL/6N mice were treated with diethylnitrosamine at 2weeks of age and, from 6 to 32weeks of age, fed one of five diets that differed in fat and sugar content, including normal chow, ketogenic, and Western diets. Results Mice fed sugar-rich diets had the greatest tumor burden irrespective of dietary fat content. In contrast, mice fed a high-fat low-sugar diet had the least tumor burden despite obesity and glucose intolerance. When evaluated as independent variables, tumor burden was positively correlated with hepatic fat accumulation, postprandial insulin, and liver IL-6, and inversely correlated with serum adiponectin. In contrast, tumor burden did not correlate with adiposity, fasting insulin, or glucose intolerance. Furthermore, mice fed high sugar diets had lower liver expression of p21 and cleaved caspase-3 compared to mice fed low sugar diets. Conclusions These data indicate that dietary sugar intake contributes to liver tumor burden independent of excess adiposity or insulin resistance in mice treated with diethylnitrosamine.

Background & Aims This study's aim was to assess the histological and metabolic effects of n-3 polyunsaturated fatty acids (PUFAs) vs. placebo while adjusting for the impact of age and weight change in NASH patients. (ClinicalTrials.gov: NCT00681408). Methods Forty-one subjects with non-cirrhotic NASH were enrolled, and 34 completed the study. 17 received n-3 fish oil 3000mg/day and 17 received placebo daily for 1year with typical counselling on caloric intake and physical activity for all subjects. Results N-3- and placebo-treated groups showed no significant difference for the primary end point of NASH activity score (NAS) reduction ⩾2points without fibrosis progression after adjustment for known covariates (n-3, 4/17 (23.5%); placebo, 3/17, (17.6%), p =0.99). Among subjects with increased or stable weight, n-3 subjects showed a larger decrease in liver fat content by MRI than placebo-treated subjects ( p =0.014 for 2nd quartile, p =0.003 for 3rd quartile of weight change). N-3 treatment showed significant fat reduction on the paired analysis of image-assisted fat morphometry regardless of weight loss or gain. Exercise capacity remained markedly reduced in all subjects. No independent effects on markers of hepatocyte injury or insulin sensitivity indices were observed. Conclusion N-3 PUFAs at 3000mg/day for one year did not lead to an improvement in the primary outcome of histological activity in NASH patients (⩾2point NAS reduction). N-3 led to reduced liver fat by multiple measures. Other metabolic effects were not seen, although no detrimental effects were apparent. Whether longer duration, higher dose, or different composition of n-3 therapy would lead to additional benefits is uncertain.

Background & Aims The lysyl oxidase-like protein 2 (LOXL2) promotes stabilization of the extracellular matrix, chemotaxis, cell growth and cell mobility. We aimed to (i) identify stimuli of LOXL2 in cholangiopathies, (ii) characterize the effects of LOXL2 on biliary epithelial cells’ (BECs) barrier function, (iii) compare LOXL2 expression in primary sclerosing cholangitis (PSC), primary biliary cholangitis, and disease controls, and (iv) to determine LOXL2 expression and its cellular sources in four mouse models of cholangiopathies. Methods Cultured murine BECs were challenged with well-known triggers of cellular senescence, hypoxia, phospholipid-deficient Abcb4−/− mouse bile and chenodeoxycholic acid and investigated for LOXL2, SNAIL1 and E-cadherin expression and transepithelial electrical resistance with and without LOX-inhibition. In vivo, LOXL2 expression was studied in PSC livers, and controls and mouse models. We compared LOXL2 serum levels in patients with PSC, secondary SC, primary biliary cholangitis, and controls. Results Cellular senescence, hypoxia, Abcb4−/− bile and chenodeoxycholic acid induced LOXL2 and SNAIL1 expression, repressed E-cadherin expression, and significantly reduced transepithelial electrical resistance in BECs. Notably, all of the pathological changes could be recovered via pharmacological LOX-inhibition. Mouse models showed induced LOXL2 expression in the portal region and in association with ductular reaction. LOXL2 serum levels were significantly elevated in patients with cholangiopathies. In PSC, LOXL2 expression was located to characteristic periductal onion skin-type fibrosis, ductular reaction, Kupffer cells, and fibrotic septa. Importantly, in PSC, LOXL2 overexpression was paralleled by E-cadherin loss in BECs from medium-sized bile ducts. Conclusions Reactive BECs produce LOXL2, resulting in increased tight junction permeability, which can be ameliorated by pharmacological LOX-inhibition in vitro. Reactive BECs, portal myofibroblasts, and Kupffer cells are the main sources of LOXL2 in cholangiopathies. Lay summary In this study, we investigate the role of lysyl oxidase-like protein 2 (LOXL2), an enzyme pivotal in the development of organ fibrosis, in the pathogenesis of cholangiopathies (diseases of bile ducts), such as primary sclerosing cholangitis. We found LOXL2 to be expressed in association with bile duct epithelial injury and uncovered mechanisms for its upregulation and the subsequent effects in vitro and in vivo. Our findings support testing of anti-LOXL2 treatment strategies for patients with primary sclerosing cholangitis.

Background & Aims Hepcidin is the central regulator of iron homeostasis and altered hepcidin signalling results in both hereditary and acquired iron overload. While the association between iron overload and development of end-stage liver disease is well established, the underlying mechanisms are largely unknown. To improve that, we analysed hepcidin knockout (KO) mice as a model of iron overload-associated liver disease. Methods Hepcidin wild type (WT) and KO mice fed with 3% carbonyl iron-containing diet starting at one month of age were compared to age-matched animals kept on standard chow. Liver histology and serum parameters were used to assess the extent of liver injury and fibrosis. Iron distribution was determined by subcellular fractionation and electron microscopy. Results Among mice kept on iron-rich diet, 6months old hepcidin KO mice ( vs. WT) displayed profound hepatic iron overload (3186±411 vs. 1045±159μg/mg tissue, p p Conclusions Hepcidin KO mice represent a novel model of iron overload-related liver diseases and implicate lysosomal injury as a crucial event in iron toxicity.

Background/Aims Hepatocyte "ballooning" is an often used but ill defined term in liver pathology to designate a special form of liver cell degeneration associated with cell swelling and enlargement found particularly in steatohepatitis. Alterations of the intermediate filament cytoskeleton of the hepatocyte may contribute to the pathogenesis of this microscopic change. Ballooning degeneration is considered a hallmark of steatohepatitis, but enlarged hepatocytes may also be observed in a variety of other acute and chronic liver diseases. Methods The intermediate filament cytoskeleton was investigated using keratin 8 and 18 immunohistochemistry in liver diseases associated with enlarged or ballooned hepatocytes. Results Keratin 8/18 immunostaining was drastically reduced or lost in the cytoplasm of ballooned hepatocytes in alcoholic and non-alcoholic steatohepatitis, chronic cholestatic conditions, ischemia/reperfusion injury and in ballooned hepatocytes in chronic hepatitis C cases with concurrent steatohepatitis. In contrast, substantial decrease or loss of keratin 8/18 immunostaining was not noted in cases of acute hepatitis, giant cell hepatitis, chronic hepatitis B, or autoimmune hepatitis. Conclusions Loss of keratin 8/18 immunostaining can serve as an objective marker of a specific type of ballooning degeneration of hepatocytes. Oxidative stress may be a common denominator in the pathogenesis of keratin filament alterations in these conditions.