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.

Alcoholic liver disease and hepatitis C:A frequently underestimated combination

Alcoholic liver disease(ALD) and hepatitis C virus(HCV) infection represent, either alone or in combination, more than two thirds of all patients with liver disease in the Western world.This review discusses the epidemiology and combined impact of ALD and HCV on the progres sion of liver disease.ALD and HCV affect the progres sion of liver disease to liver cirrhosis and hepatocellular carcinoma(HCC) in a synergistic manner.Thus, the risk for HCC increases f ive times with a daily alcohol con sumption of 80 g;in the presence of HCV it is increased 20fold, and a combination of both risk factors leads to a more than 100fold risk for HCC development.Alcohol consumption also decreases the response to interferon treatment which is probably due to a lack of compliance than a direct effect on HCV replication.Several molecu lar mechanisms are discussed that could explain the synergistic interaction of alcohol and HCV on disease progression.They include modulation of the immune response and apoptosis, increased oxidative stress via induction of CYP2E1 and the hepatic accumulation of iron.Thus, both HCV and alcohol independently cause hepatic iron accumulation in > 50% of patients probably due to suppression of the liversecreted systemic iron hormone hepcidin.A better understanding of hepcidin regulation could help in developing novel therapeutic approaches to treat the chronic disease in the future.For now, it can be generally concluded that HCVinfect ed patients should abstain from alcohol and alcoholicsshould be encouraged to participate in detoxification programs.

Increased liver stiffness in alcoholic liver disease:Differentiating fibrosis from steatohepatitis

AIM:To test if inflammation also interferes with liver stiffness (LS) assessment in alcoholic liver disease (ALD) and to provide a clinical algorithm for reliable fibrosis assessment in ALD by FibroScan (FS).METHODS:We first performed sequential LS analysis before and after normalization of serum transaminases in a learning cohort of 50 patients with ALD admitted for alcohol detoxification. LS decreased in almost all patients within a mean observation interval of 5.3 d. Six patients (12%) would have been misdiagnosed with F3and F4 fibrosis but LS decreased below critical cut-off values of 8 and 12.5 kPa after normalization of trans-aminases. RESULTS:Of the serum transaminases,the decrease in LS correlated best with the decrease in glutamic oxaloacetic transaminase (GOT). No significant chang-es in LS were observed below GOT levels of 100 U/L. After establishing the association between LS and GOT levels,we applied the rule of GOT < 100 U/L for reliable LS assessment in a second validation cohort of 101 patients with histologically confi rmed ALD. By ex-cluding those patients with GOT > 100 U/L at the time of LS assessment from this cohort,the area under the receiver operating characteristic (AUROC) for cirrhosis detection by FS improved from 0.921 to 0.945 while specificity increased from 80% to 90% at a sensitivity of 96%. A similar AUROC could be obtained for lower F3 fibrosis stage if LS measurements were restricted to patients with GOT < 50 U/L. Histological grading of inflammation did not further improve the diagnostic accuracy of LS.CONCLUSION:Coexisting steatohepatitis markedly increases LS in patients with ALD independent of fibrosis stage. Postponing cirrhosis assessment by FS during alcohol withdrawal until GOT decreases to < 100 U/mL signif icantly improves the diagnostic accuracy.

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

AIM To investigate the influence of PNPLA3 genotype in heavy drinkers on serum markers and liver stiffness(LS) during alcohol withdrawal and its association with histology.METHODS Caucasian 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.RESULTS The 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 < 0.005), ballooning(r = 0.319, P < 0.005) and less with steatosis(r = 0.264, P < 0.05). Mean LS was lowest in CC carriers(13.1 k Pa) as compared to CG and GG carriers(17.6 and 17.2 kPa). Notably, LS primarily correlated with fibrosis stage(r = 0.828, P < 0.005), ballooning(r = 0.516, P < 0.005), steatohepatitis(r = 0.319, P < 0.005) but not with steatosis. After alcohol withdrawal, LS did not change in CC carriers, significantly decreased in CG-carriers from 17.6 to 12.7 kPa but to a lesser extent in GG carriers from 17.6 to 14.5 kPa. This was due to prolonged resolution of inflammation with significantly elevated aspartate transaminase levels after alcohol withdrawal in GG carriers. Non-invasive fibrosis assessment by LS in all patients showed a significantly higher F0 rate as compared to the biopsy cohort(47% 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.CONCLUSION In 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.

Liver stiffness in pregnant women with intrahepatic cholestasis of pregnancy:A case control study

BACKGROUND Intrahepatic cholestasis of pregnancy(ICP)is a rare but severe complication for both the mother and the unborn child.The diagnosis is primarily based on elevated serum levels of bile acids.In a large ICP cohort,we here study in detail liver stiffness(LS)using transient elastography(TE),now widely used to noninvasively screen for liver cirrhosis within minutes.AIM To specifically explore LS in a large cohort of women with ICP compared to a control group with uncomplicated pregnancy.METHODS LS and hepatic steatosis marker controlled attenuation parameter(CAP)were measured in 100 pregnant women with ICP using TE(Fibroscan,Echosens,Paris,France)between 2010 and 2020.In 17 cases,LS could be measured postpartum.450 women before and 38 women after delivery with uncomplicated pregnancy served as control group.Routine laboratory,levels of bile acids and apoptosis marker caspase-cleaved cytokeratin 18 fragment(M30)were also measured.RESULTS Women with ICP had significantly elevated transaminases but normal gammaglutamyl transferase(GGT).Mean LS was significantly increased at 7.3±3.0 kPa compared to the control group at 6.2±2.3 kPa(P<0.0001).Postpartum LS decreased significantly in both groups but was still higher in ICP(5.8±1.7 kPa vs 4.2±0.9 kPa,P<0.0001),respectively.In ICP,LS was highly significantly correlated with levels of bile acids and M30 but not transaminases.No correlation was seen with GGT that even increased significantly after delivery in the ICP group.Bile acids were mostly correlated with the liver apoptosis marker M30,LS and levels of alanine aminotransferase,aspartate aminotransferase,and bilirubin.In multivariate analysis,LS remained the sole parameter that was independently associated with elevated bile acids.CONCLUSION In conclusion,LS is significantly elevated in ICP which is most likely due to toxic bile acid accumulation and hepatocyte apoptosis.In association with conventional laboratory markers,LS provides additional non-invasive information to rapidly identify women at risk for ICP.

Does pressure cause liver cirrhosis? The sinusoidal pressure hypothesis

Independent of their etiology, all chronic liver diseases ultimately lead to liver cirrhosis, which is a majorhealth problem worldwide. The underlying molecular mechanisms are still poorly understood and no efficient treatment strategies are available. This paper introduces the sinusoidal pressure hypothesis(SPH), which identifies an elevated sinusoidal pressure(SP) as cause of fibrosis. SPH has been mainly derived from recent studies on liver stiffness. So far, pressure changes have been exclusively seen as a consequ-ence of cirrhosis. According to the SPH, however, an elevated SP is the major upstream event that initiates fibrosis via biomechanic signaling by stretching of perisinusoidal cells such as hepatic stellate cells or fibroblasts(SPH part?Ⅰ: initiation). Fibrosis progression is determined by the degree and time of elevated SP. The SPH predicts that the degree of extracellular matrix eventually matches SP with critical thresholds > 12 mmH g and > 4 wk. Elevated arterial flow and final arterialization of the cirrhotic liver represents the self-perpetuating key event exposing the low-pressure-organ to pathologically high pressures(SPH part?Ⅱ: perpetuation). It also defines the "point of no return" where fibrosis progression becomes irreversible. The SPH is able to explain the macroscopic changes of cirrhotic livers and the uniform fibrotic response to various etiologies. It also opens up new views on the role of fat and disease mechanisms in other organs. The novel concept will hopefully stimulate the search for new treatment strategies.

Transient micro-elastography:A novel non-invasive approach to measure liver stiffness in mice

AIM:To develop and validate a transient micro-elastography device to measure liver stiffness(LS) in mice.METHODS:A novel transient micro-elastography(TME) device,dedicated to LS measurements in mice with a range of measurement from 1-170 kPa,was developed using an optimized vibration frequency of 300 Hz and a 2 mm piston.The novel probe was validated in a classical fibrosis model(CCl4) and in a transgenic murine model of systemic amyloidosis.RESULTS:TME could be successfully performed in control mice below the xiphoid cartilage,with a mean LS of 4.4 ± 1.3 kPa,a mean success rate of 88%,and an excellent intra-observer agreement(0.98).Treatment with CCl4 over seven weeks drastically increased LS as compared to controls(18.2 ± 3.7 kPa vs 3.6 ± 1.2 kPa).Moreover,fibrosis stage was highly correlated with LS(Spearman coefficient = 0.88,P < 0.01).In the amyloidosis model,much higher LS values were obtained,reaching maximum values of > 150 kPa.LS significantly correlated with the amyloidosis index(0.93,P < 0.0001) and the plasma concentration of mutant hapoA-□(0.62,P < 0.005).CONCLUSION:Here,we have established the first non-invasive approach to measure LS in mice,and have successfully validated it in two murine models of high LS.

Liver stiffness reversibly increases during pregnancy and independently predicts preeclampsia

AIM To study liver stiffness(LS) during pregnancy and its association with complications during pregnancy.METHODS In this observational, diagnostic study, 537 pregnant women were prospectively enrolled at the Department of Obstetrics and Gynecology, University hospital Heidelberg and Salem Medical Center. LS was measured using the Fibroscan device(Echosens, Paris) in all women and in 41 cases 24 h after delivery. Clinical and morphological data were recorded and abdominal ultrasound and standard laboratory tests were performed. No complications were observed in 475 women(controls) while preeclampsia and intrahepatic cholestasis of pregnancy(ICP) developed in 22 and 40 women, respectively.RESULTS In controls, LS increased significantly from initially 4.5 ± 1.2 kPa in the second trimester to 6.0 ± 2.3 kPa(P < 0.001) in the third trimester. In the third trimester, 41% of women had a LS higher than 6 kPa. Elevated LS in controls was significantly correlated with alkaline phosphatase, leukocytes, gestational age and an increase in body weight and body mass index(BMI). In women with pregnancy complications, LS was significantly higher as compared to controls(P < 0.0001). Moreover, in multivariate analysis, LS was an independent predictor for preeclampsia with an odds ratio of 2.05(1.27-3.31) and a cut-off value of 7.6 kPa. In contrast, ICP could not be predicted by LS. Finally, LS rapidly decreased in all women within 24 h after delivery from 7.2 ± 3.3 kPa down to 4.9 ± 2.2 kPa(P < 0.001).CONCLUSION During pregnancy, LS significantly and reversibly increases in the final trimester of pregnant women without complications. In women with preeclampsia, LS is significantly elevated and an independent noninvasive predictor.

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.