Non-invasive diagnosis of alcoholic liver disease

Alcoholic liver disease(ALD)is the most common liver disease in the Western world.For many reasons,it isunderestimated and underdiagnosed.An early diagnosis is absolutely essential since it(1)helps to identify patients at genetic risk for ALD;(2)can trigger efficient abstinence namely in non-addicted patients;and(3)initiate screening programs to prevent life-threateningcomplications such as bleeding from varices,spontaneous bacterial peritonitis or hepatocellular cancer.The two major end points of ALD are alcoholic liver cirrhosis and the rare and clinically-defined alcoholic hepatitis(AH).The prediction and early diagnosis of both entities is still insufficiently solved and usually relies on acombination of laboratory,clinical and imaging findings.It is not widely conceived that conventional screeningtools for ALD such as ultrasound imaging or routine laboratory testing can easily overlook ca.40%of manifest alcoholic liver cirrhosis.Non-invasive methods such as transient elastography(Fibroscan),acoustic radiation force impulse imaging or shear wave elastography have significantly improved the early diagnosis of alcoholiccirrhosis.Present algorithms allow either the exclusion or the exact definition of advanced fibrosis stages in ca.95%of patients.The correct interpretation of liver stiffness requires a timely abdominal ultrasound and actual transaminase levels.Other non-invasive methods such as controlled attenuation parameter,serum levels of M30 or M65,susceptometry or breath tests are under current evaluation to assess the degree of steatosis,apoptosis and iron overload in these patients.Liver biopsy still remains an important option to rule out comorbidities and to confirm the prognosis namely for patients with AH.

Primary liver injury and delayed resolution of liver stiffness after alcohol detoxification in heavy drinkers with the PNPLA3 variant I148M

AIMTo investigate the influence of PNPLA3 genotype in heavy drinkers on serum markers and liver stiffness (LS) during alcohol withdrawal and its association with histology. METHODSCaucasian heavy drinkers (n = 521) with a mean alcohol consumption of 192.1 g/d (median alcohol consumption: 169.0 g/d; 95%CI: 179.0-203.3) were enrolled at the Salem Medical Center, University of Heidelberg. LS was measured by transient elastography (Fibroscan, Echosens SA, Paris, France). LS and serum markers were prospectively studied in these patients with all stages of alcoholic liver disease (steatosis, steatohepatitis, fibrosis) prior and after alcohol detoxification with a mean observation interval of 6.2 ± 3.2 d. A liver biopsy with histological analysis including the Kleiner score was obtained in 80 patients. RESULTSThe PNPLA3 rs738409 genotype distribution for CC, CG and GG was 39.2%, 52.6% and 8.2%. GG genotype primarily correlated with histological steatohepatitis (r = 0.404, P r = 0.319, P r = 0.264, P r = 0.828, P r = 0.516, P r = 0.319, P vs 6%) with 3.8% more CC carriers while 3.7% less were seen in the F4 cirrhosis group. Thus, about 20% of patients with alcoholic liver cirrhosis would be attributable to PNPLA3 G variants. The OR to develop cirrhosis corrected for age, gender and body mass index was 1.295 (95%CI: 0.787-2.131) for CG + GG carriers. CONCLUSIONIn heavy drinkers, PNPLA3 GG primarily correlates with ballooning/steatohepatitis but not steatosis resulting in a delayed inflammation-associated resolution of LS. Consequently, sustained ballooning-associated LS elevation seems to be a potential risk factor for fibrosis progression in PNPLA3 GG carriers.

Direct modulation of hepatocyte hepcidin signaling by iron

BACKGROUND Liver-secreted hepcidin is the systemic master switch of iron homeostasis and decreased levels of hepcidin are considered to cause iron overload not only in hereditary hemochromatosis but also in hemolytic anemia and chronic liver diseases.The regulation of hepcidin is complex and its response to iron is still not completely understood.AIM To study the direct effect of iron on various established hepcidin signaling pathways in hepatoma cells or primary hepatocytes.METHODS Hepcidin mRNA expression was studied by quantitative real-time(qRT)-PCR in the presence of various forms of iron including ferric ammonium citrate(FAC)in hepatoma cells(Huh7),murine primary hepatocytes and an established co-culture model of phorbol myristate acetate-differentiated THP-1 monocytes and Huh7 cells.To analyze hepcidin signaling,the response to bone morphogenetic protein 6(BMP6),interleukin(IL)-6,IL-1β,hypoxia and lipopolysaccharide(LPS)were studied.Hepcidin and small mothers against decapentaplegic 6(SMAD6)mRNA levels were assessed by qRT-PCR and the expression of phosphorylated signal transducer and activator of transcription 3(phospho-STAT3),STAT3,phospho-SMAD1/5/8 and SMAD1 proteins were analyzed by western blot.RESULTS All iron III forms including FAC efficiently blocked hepcidin mRNA expression at non-toxic dosages in Huh7 cells or primary hepatocytes in a time and dosedependent manner(P<0.001;P<0.05).Hepcidin blockage could be efficiently blunted by iron chelators salicylaldehyde isonicotinoyl hydrazone(SIH)and Desferal(P<0.001).FAC also inhibited BMP6,hypoxia,IL-1βand IL-6-mediated hepcidin induction(P<0.001;P<0.001;P<0.05;P<0.001),and FAC also inhibited LPS-mediated hepatic hepcidin induction in co-culture model(P<0.001).Moreover,FAC reduced SMAD6 mRNA and p-SMAD1/5/8 protein expression at basal or upon stimulation by BMP6(P<0.05;P<0.01),and FAC also reduced SMAD6 and p-SMAD1/5/8 expression under hypoxia(P<0.01;P<0.05).However,FAC has no significant effect on p-STAT3 protein expression at basal or upon stimulation by various stimuli.Notably,in the presence of the BMP/SMAD signaling pathway inhibitor LDN193189 Hydrochloride(LDN),FAC was unable to further decrease hepcidin,SMAD6 and p-SMAD1/5/8 expression compared with LDN alone.CONCLUSION Iron directly blocks hepatocellular hepcidin signaling through the BMP/SMAD pathway but independent of STAT3.This mechanism may contribute to continued iron overload in many pathophysiological conditions ultimately causing a vicious cycle of continued hepcidin suppression.