Marlene Dominguez

A New Scoring System for Prognostic Stratification of Patients With Alcoholic Hepatitis

OBJECTIVES: Prognostic stratification of patients with alcoholic hepatitis (AH) may improve the clinical management and facilitate clinical trials. We aimed at developing a scoring system capable of providing prognostic stratification of patients with AH.METHODS: Patients with biopsy-proven AH were prospectively included between 2000 and 2006. The biochemical, clinical, portal hemodynamic and histological parameters were evaluated. A Cox regression model was used for univariate and multivariate analyses. A predictive score was built using variables obtained at admission identified in the multivariate analysis. The resulting score was validated in an independent prospective cohort.RESULTS: In total, 103 patients with biopsy-proven AH were included in the study cohort. Age, serum bilirubin, serum creatinine, and international normalized ratio (INR) independently predicted 90-day mortality. We generated the Age, serum Bilirubin, INR, and serum Creatinine (ABIC) score: (age × 0.1) + (serum bilirubin × 0.08) + (serum creatinine × 0.3) + (INR × 0.8). The area under the curve (AUC) was 0.82. Using the Kaplan-Meier analysis with the cutoff values of 6.71 and 9.0, we identified patients with low, intermediate, and high risk of death at 90 days (100%, 70%, and 25% of survival rate, respectively). Using the same cutoff values, the ABIC score also stratified patients according to their risk of death at 1 yr. These results were validated by a confirmatory cohort (N = 80).CONCLUSIONS: The ABIC score is a new tool that allows the stratification of risk of death in patients with AH at 90 days and 1 yr. This score can help improve the management of these patients and also help to perform clinical trials.

Hepatic Expression of CXC Chemokines Predicts Portal Hypertension and Survival in Patients With Alcoholic Hepatitis

BACKGROUND & AIMS Alcoholic hepatitis (AH) is characterized by hepatocellular damage, inflammation, and fibrosis. We performed a prospective study to associate hepatic expression of the CXC subfamily of chemokines with histology findings and prognosis of patients with AH. METHODS Liver biopsy samples from 105 patients with AH and 5 normal liver samples (controls) were evaluated for steatosis, inflammation, fibrosis, and cholestasis. Computer-based morphometric analysis assessed the numbers of infiltrating CD3+ T cells and CD15+ cells (neutrophils); terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining was used to quantify apoptosis. Expression of CXC and CC chemokines and selected signaling components were assessed by quantitative reverse-transcription polymerase chain reaction; protein levels of interleukin (IL)-8 and Gro-alpha also were determined by immunohistochemistry. Serum levels of IL-8 and Gro-alpha were measured by enzyme-linked immunosorbent assay. The Cox regression model identified variables associated with mortality. RESULTS Most patients (75%) had severe AH; their 90-day mortality rate was 21.9%. In AH liver samples, expression of the CXC subfamily members IL-8, Gro-alpha, CXCL5, CXCL6, CXCL10, and platelet factor 4 was up-regulated and compared with controls. The CC chemokine CCL2, but not CCL5, also was up-regulated. Higher expression levels of IL-8, CXCL5, Gro-gamma, and CXCL6 were associated with worse prognosis. Expression of CXC components correlated with neutrophil infiltration and the severity of portal hypertension. In the multivariate analysis, IL-8 protein levels were an independent predictor of 90-day mortality. IL-8 and Gro-alpha serum levels did not correlate with prognosis. CONCLUSIONS Hepatic expression of CXC components correlates with prognosis of patients with AH. Reagents that target CXC chemokines might be developed as therapeutics.

Ghrelin attenuates hepatocellular injury and liver fibrogenesis in rodents and influences fibrosis progression in humans.

There are no effective antifibrotic therapies for patients with liver diseases. We performed an experimental and translational study to investigate whether ghrelin, an orexigenic hormone with pleiotropic properties, modulates liver fibrogenesis. Recombinant ghrelin was administered to rats with chronic (bile duct ligation) and acute (carbon tetrachloride) liver injury. Hepatic gene expression was analyzed by way of microarray analysis and quantitative polymerase chain reaction. The hepatic response to chronic injury was also evaluated in wild‐type and ghrelin‐deficient mice. Primary human hepatic stellate cells were used to study the effects of ghrelin in vitro. Ghrelin hepatic gene expression and serum levels were assessed in patients with chronic liver diseases. Ghrelin gene polymorphisms were analyzed in patients with chronic hepatitis C. Recombinant ghrelin treatment reduced the fibrogenic response, decreased liver injury and myofibroblast accumulation, and attenuated the altered gene expression profile in bile duct–ligated rats. Moreover, ghrelin reduced the fibrogenic properties of hepatic stellate cells. Ghrelin also protected rats from acute liver injury and reduced the extent of oxidative stress and inflammation. Ghrelin‐deficient mice developed exacerbated hepatic fibrosis and liver damage after chronic injury. In patients with chronic liver diseases, ghrelin serum levels decreased in those with advanced fibrosis, and ghrelin gene hepatic expression correlated with expression of fibrogenic genes. In patients with chronic hepatitis C, polymorphisms of the ghrelin gene (−994CT and −604GA) influenced the progression of liver fibrosis. Conclusion: Ghrelin exerts antifibrotic effects in the liver and may represent a novel antifibrotic therapy. (HEPATOLOGY 2010;51:974–985.)

Systemic inflammatory response and serum lipopolysaccharide levels predict multiple organ failure and death in alcoholic hepatitis.

Alcoholic hepatitis (AH) frequently progresses to multiple organ failure (MOF) and death. However, the driving factors are largely unknown. At admission, patients with AH often show criteria of systemic inflammatory response syndrome (SIRS) even in the absence of an infection. We hypothesize that the presence of SIRS may predispose to MOF and death. To test this hypothesis, we studied a cohort including 162 patients with biopsy‐proven AH. The presence of SIRS and infections was assessed in all patients, and multivariate analyses identified variables independently associated with MOF and 90‐day mortality. At admission, 32 (19.8%) patients were diagnosed with a bacterial infection, while 75 (46.3%) fulfilled SIRS criteria; 58 patients (35.8%) developed MOF during hospitalization. Short‐term mortality was significantly higher among patients who developed MOF (62.1% versus 3.8%, P < 0.001). The presence of SIRS was a major predictor of MOF (odds ratio = 2.69, P = 0.025) and strongly correlated with mortality. Importantly, the course of patients with SIRS with and without infection was similar in terms of MOF development and short‐term mortality. Finally, we sought to identify serum markers that differentiate SIRS with and without infection. We studied serum levels of high‐sensitivity C‐reactive protein, procalcitonin, and lipopolysaccharide at admission. All of them predicted mortality. Procalcitonin, but not high‐sensitivity C‐reactive protein, serum levels identified those patients with SIRS and infection. Lipopolysaccharide serum levels predicted MOF and the response to prednisolone. Conclusion: In the presence or absence of infections, SIRS is a major determinant of MOF and mortality in AH, and the mechanisms involved in the development of SIRS should be investigated; procalcitonin serum levels can help to identify patients with infection, and lipopolysaccharide levels may help to predict mortality and the response to steroids. (Hepatology 2015;62:762–772)

Transcriptome analysis identifies TNF superfamily receptors as potential therapeutic targets in alcoholic hepatitis

Objective Alcoholic hepatitis (AH) is a severe clinical condition that needs novel therapies. The identification of targets for therapy is hampered by the lack of animal models of advanced AH. The authors performed a translational study through a transcriptome analysis in patients with AH to identify new molecular targets. Design Hepatic gene expression profiling was assessed by DNA microarray in patients with AH (n=15) and normal livers (n=7). Functional analysis was assessed by gene set enrichment analysis. Quantitative PCR was performed in patients with AH (n=40), hepatitis C (n=18), non-alcoholic steatohepatitis (n=20) and in mouse models of acute and chronic liver injury. Protein expression was assessed by immunohistochemistry and western blotting. Results Gene expression analysis showed 207 genes >5-fold differentially expressed in patients with AH and revealed seven pathways differentially regulated including ‘cytokine–cytokine receptor interaction’. Several tumour necrosis factor (TNF) superfamily receptors, but not ligands, were overexpressed in AH. Importantly, Fn14 was the only TNF superfamily receptor exclusively upregulated in AH compared with other liver diseases and correlated with both 90-day mortality and severity of portal hypertension. Fn14 protein expression was detected in areas of fibrogenesis and in a population of hepatocytes. Fn14 expression was increased in experimental models of liver injury and was detected in progenitor cells. Conclusion Translational research revealed that TNF superfamily receptors are overexpressed in AH. Fn14, the receptor for TNF-like weak inducer of apoptosis, is selectively upregulated in patients with AH. TNF superfamily receptors could represent a potential target for therapy.

Effects of losartan on hepatic expression of nonphagocytic NADPH oxidase and fibrogenic genes in patients with chronic hepatitis C

Angiotensin II promotes liver fibrogenesis by stimulating nonphagocytic NADPH oxidase (NOX)-induced oxidative stress. Angiotensin II type 1 (AT1) receptor blockers attenuate experimental liver fibrosis, yet their effects in human liver fibrosis are unknown. We investigated the effects of losartan on hepatic expression of fibrogenic, inflammatory, and NOX genes in patients with chronic hepatitis C (CHC). Fourteen patients with CHC and liver fibrosis received oral losartan (50 mg/day) for 18 mo. Liver biopsies were performed at baseline and after treatment. The degree of inflammation and fibrosis was evaluated by histological analysis (METAVIR). Collagen content was measured by morphometric quantification of Sirius red staining. Overall collagen content and fibrosis stage remained stable in the whole series, yet the fibrosis stage decreased in seven patients. Inflammatory activity improved in seven patients. The effect of losartan on hepatic expression of 31 profibrogenic and inflammatory genes and components of the NOX complex was assessed by quantitative PCR. Losartan treatment was associated with a significant decrease in the expression of several profibrogenic and NOX genes including procollagen alpha1(I) and alpha1(IV), urokinase-type plasminogen activator, metalloproteinase type 2, NOX activator 1 (NOXA-1) and organizer 1 (NOXO-1), and Rac-1. Losartan was well tolerated in all patients and was effective in attenuating the activity of the systemic renin-angiotensin system. No effects on serum liver tests or viral load were observed. We conclude that prolonged administration of losartan, an oral AT1 receptor blocker, is associated with downregulation of NOX components and fibrogenic genes in patients with CHC. Controlled studies are warranted to assess the effect of AT1 receptor blockers in chronic liver injury.

Cigarette smoking exacerbates nonalcoholic fatty liver disease in obese rats

The prevalence of cigarette smoking (CS) is increased among obese subjects, who are susceptible to develop nonalcoholic fatty liver disease (NAFLD). We investigated the hepatic effects of CS in control and obese rats. Control and obese Zucker rats were divided into smokers and nonsmokers (n = 12 per group). Smoker rats were exposed to 2 cigarettes/day, 5 days/week for 4 weeks. The effects of CS were assessed by biochemical analysis, hepatic histological examination, immunohistochemistry, and gene expression analysis. Phosphorylation of AKT and extracellular signal‐regulated kinase (ERK) and quantification of carbonylated proteins were assessed by western blotting. As expected, obese rats showed hypercholesterolemia, insulin resistance, and histological features of NAFLD. Smoking did not modify the lipidic or glucidic serum profiles. Smoking increased alanine aminotransferase serum levels and the degree of liver injury in obese rats, whereas it only induced minor changes in control rats. Importantly, CS increased the histological severity of NAFLD in obese rats. We also explored the potential mechanisms involved in the deleterious effects of CS. Smoking increased the degree of oxidative stress and hepatocellular apoptosis in obese rats, but not in controls. Similarly, smoking increased the hepatic expression of tissue inhibitor of metalloproteinase‐1 and procollagen‐alpha2(I) in obese rats, but not in controls. Finally, smoking regulated ERK and AKT phosphorylation. The deleterious effects of CS were not observed after a short exposure (5 days). Conclusion: CS causes oxidative stress and worsens the severity of NAFLD in obese rats. Further studies should assess whether this finding also occurs in patients with obesity and NAFLD. (HEPATOLOGY 2010.)

Acute Kidney Injury Is an Early Predictor of Mortality for Patients With Alcoholic Hepatitis

BACKGROUND & AIMS Alcoholic hepatitis (AH) is a severe condition with high mortality. To improve therapeutic strategies, it is important to identify factors that affect survival times. The age, bilirubin, international normalized ratio, and creatinine scoring system (also known as the ABIC scoring system) was developed previously to determine the prognosis of patients with AH. We studied effects of acute kidney injury (AKI) on survival of patients with AH. METHODS We retrospectively analyzed data from 103 patients with biopsy-proven AH. AKI was defined as an abrupt reduction (within 48 h) in kidney function that resulted in an absolute increase of at least 0.3 mg/dL (or a 50% increase) in serum levels of creatinine from baseline (the AKI network [AKIN] criteria). RESULTS Twenty-nine patients (28%) developed AKI during hospitalization, with a median time to diagnosis of 3 days. Overall 90-day mortality was 23%, which was significantly higher among patients with AKI than those without (65% vs 7%; P < .0001). The age, bilirubin, international normalized ratio, and creatinine score (P < .0001) and development of AKI (P < .0001) were the most accurate independent predictors of 90-day mortality. The presence of systemic inflammatory response syndrome (P < .0001), serum bilirubin (P = .01), and international normalized ratio at admission (P = .03) were the most accurate predictors of AKI. Importantly, the AKIN criteria were more accurate than traditional criteria for renal failure (serum creatinine >1.5 mg/dL) in predicting 90-day mortality (area under the receiver operating characteristic, 0.83 vs 0.70, respectively; P = .02). CONCLUSIONS Development of AKI reduces survival of patients with AH, in the short term. The AKIN criteria are useful and more accurate than traditional criteria in predicting mortality. Strategies to prevent AKI therefore should be considered in the management of patients with AH.

Atorvastatin attenuates angiotensin II-induced inflammatory actions in the liver.

Statins exert beneficial effects in chronically damaged tissues. Angiotensin II (ANG II) participates in liver fibrogenesis by inducing oxidative stress, inflammation, and transforming growth factor-beta1 (TGF-beta1) expression. We investigate whether atorvastatin modulates ANG II-induced pathogenic effects in the liver. Male Wistar rats were infused with saline or ANG II (100 ng kg(-1) min(-1)) for 4 wk through a subcutaneous osmotic pump. Rats received either vehicle or atorvastatin (5 mg kg(-1) day(-1)) by gavage. ANG II infusion resulted in infiltration of inflammatory cells (CD43 immunostaining), oxidative stress (4-hydroxynonenal), hepatic stellate cells (HSC) activation (smooth muscle alpha-actin), increased intercellular adhesion molecule (ICAM-1), and interleukin-6 hepatic gene expression (quantitative PCR). These effects were markedly blunted in rats receiving atorvastatin. The beneficial effects of atorvastatin were confirmed in an additional model of acute liver injury (carbon tetrachloride administration). We next explored whether the beneficial effects of atorvastatin on ANG II-induced actions are also reproduced at the cellular level. We studied HSC, a cell type with inflammatory and fibrogenic properties. ANG II (10(-8)M) stimulated cell proliferation, proinflammatory actions (NF-kappaB activation, ICAM-1 expression, interleukin-8 secretion) as well as expression of procollagen-alpha(1(I)) and TGF-beta1. All of these effects were reduced in the presence of atorvastatin (10(-7)M). These results indicate that atorvastatin attenuates the pathogenic events induced by ANG II in the liver both in vivo and in vitro. Therefore, statins could have beneficial effects in conditions characterized by hepatic inflammation.

Acidic sphingomyelinase controls hepatic stellate cell activation and in vivo liver fibrogenesis

The mechanisms linking hepatocellular death, hepatic stellate cell (HSC) activation, and liver fibrosis are largely unknown. Here, we investigate whether acidic sphingomyelinase (ASMase), a known regulator of death receptor and stress-induced hepatocyte apoptosis, plays a role in liver fibrogenesis. We show that selective stimulation of ASMase (up to sixfold), but not neutral sphingomyelinase, occurs during the transdifferentiation/activation of primary mouse HSCs into myofibroblast-like cells, coinciding with cathepsin B (CtsB) and D (CtsD) processing. ASMase inhibition or genetic down-regulation by small interfering RNA blunted CtsB/D processing, preventing the activation and proliferation of mouse and human HSCs (LX2 cells). In accordance, HSCs from heterozygous ASMase mice exhibited decreased CtsB/D processing, as well as lower levels of alpha-smooth muscle actin expression and proliferation. Moreover, pharmacological CtsB inhibition reproduced the antagonism of ASMase in preventing the fibrogenic properties of HSCs, without affecting ASMase activity. Interestingly, liver fibrosis induced by bile duct ligation or carbon tetrachloride administration was reduced in heterozygous ASMase mice compared with that in wild-type animals, regardless of their sensitivity to liver injury in either model. To provide further evidence for the ASMase-CtsB pathway in hepatic fibrosis, liver samples from patients with nonalcoholic steatohepatitis were studied. CtsB and ASMase mRNA levels increased eight- and threefold, respectively, in patients compared with healthy controls. These findings illustrate a novel role of ASMase in HSC biology and liver fibrogenesis by regulating its downstream effectors CtsB/D.