Steroid responsiveness in alcohol-associated hepatitis is linked to glucocorticoid metabolism, mitochondrial repair, and heat shock proteins.

BACKGROUND: Alcohol-associated hepatitis (AH) is one of the clinical presentations of alcohol-associated liver disease. AH has poor prognosis, and corticosteroids remain the mainstay of drug therapy. However, ~40% of patients do not respond to this treatment, and the mechanisms underlying the altered response to corticosteroids are not understood. The current study aimed to identify changes in hepatic protein expression associated with responsiveness to corticosteroids and prognosis in patients with AH. METHODS: Patients with AH were enrolled based on the National Institute on Alcohol Abuse and Alcoholism inclusion criteria for acute AH and further confirmed by a diagnostic liver biopsy. Proteomic analysis was conducted on liver samples acquired from patients with AH grouped as nonresponders (AH-NR, n = 7) and responders (AH-R, n = 14) to corticosteroids, and nonalcohol-associated liver disease controls (n = 10). The definition of responders was based on the clinical prognostic model, the Lille Score, where a score < 0.45 classified patients as AH-R and a score > 0.45 as AH-NR. Primary outcomes used to assess steroid response were Lille Score (eg, improved liver function) and survival at 24 weeks. RESULTS: Reduced levels of the glucocorticoid receptor and its transcriptional co-activator, glucocorticoid modulatory element-binding protein 2, were observed in the hepatic proteome of AH-NR versus AH-R. The corticosteroid metabolizing enzyme, 11-beta-hydroxysteroid dehydrogenase 1, was increased in AH-NR versus AH-R along with elevated mitochondrial DNA repair enzymes, while several proteins of the heat shock pathway were reduced. Analysis of differentially expressed proteins in AH-NR who survived 24 weeks relative to AH-NR nonsurvivors revealed several protein expression changes, including increased levels of acute phase proteins, elevated coagulation factors, and reduced mast cell markers. CONCLUSIONS: This study identified hepatic proteomic changes that may predict responsiveness to corticosteroids and mortality in patients with AH.

Kaempferol Ameliorated Alcoholic Hepatitis through Improving Intestinal Barrier Function by Targeting miRNA-155 Signaling.

INTRODUCTION: The aim of this study was to investigate the effect and mechanism of kaempferol on alcoholic steatohepatitis. METHODS: C57BL/6 N mice were utilized to establish Binge-on-Chronic alcohol exposure mice model. Kaempferol was given as the interventional drug to chronic alcohol-fed mice for 6 weeks to assess its effects. In vitro, intestinal epithelial Caco-2 cells were stimulated by alcohol, and miRNA-155 mimics were used to further study the effect of kaempferol to miRNA-155 signaling in intestinal epithelial cells. HE staining and oil red O staining were used to observe the liver and intestinal tissue damage in each group of mice, and ALT, AST, IL-1ß, and TNF-α were detected by kits; lipopolysaccharide (LPS) expression was detected by ELISA kit, and the expression of IL-1ß and TNF-α was assessed by qRT-PCR; Western blot was utilized to assess the excessive inflammatory response of liver and colon tissue and the related signaling pathway activation. RESULTS: Kaempferol treatment significantly improved pathological changes such as steatosis and vacuolated lesions in liver tissue of the alcohol diet model group, and reduced serum ALT and AST enzyme activities and liver tissue interleukin-1ß and tumor necrosis factor-α mRNA expression levels. Kaempferol significantly reduced the expression of miRNA-155 in the intestinal tissue of alcohol-fed mice, significantly increased their cytokine suppressor signaling 1 (SOCS1) protein expression, inhibited the activation of nuclear factor kappa-B and significantly increased the production of the intestinal tight junction proteins occludin and zonula occludens-1. More importantly, kaempferol significantly reduced serum LPS levels in alcoholic steatohepatitis mice. In vitro experiments showed that compared with the control group, kaempferol significantly inhibited the expression level of miRNA-155 in Caco-2 cells under ethanol exposure, decreased the activation of nuclear factor kappa-B, led to an increase in the expression of SOCS1 protein, and increased the production level of occludin protein in Caco-2 cells under the effect of alcohol. In contrast, overexpression of miRNA-155 significantly decreased occludin and SOCS1 protein production and increased nuclear factor kappa-B activation levels in Caco-2 cells, and the administration of kaempferol significantly inhibited this effect. CONCLUSION: Kaempferol improved the stability of gut barrier function to ameliorate hepatic injury induced by alcohol intake through enhancing occludin protein expression, by targeting miR-155 to inhibit the excessive inflammatory response in the intestine.

[Principles of treatment of different forms of alcoholic liver disease: A review].

The review considers the principles of treatment of various forms of alcoholic liver disease from the point of view of the evidence base and clinical recommendations. The main therapy for severe alcoholic hepatitis is systemic glucocorticosteroids, their effect on survival is increased by the addition of antioxidants (N-acetylcysteine, ademethionine). The effect of ademetionine on the life expectancy of patients with alcoholic cirrhosis of Child-Pugh class A and B has been proven. The treatment of patients with mild forms of alcoholic liver disease is not well developed, and the evidence base for most of the drugs used is modest.

Effect of Prophylactic Antibiotics on Mortality in Severe Alcohol-Related Hepatitis: A Randomized Clinical Trial.

Importance: The benefits of prophylactic antibiotics for hospitalized patients with severe alcohol-related hepatitis are unclear. Objective: To determine the efficacy of amoxicillin-clavulanate, compared with placebo, on mortality in patients hospitalized with severe alcohol-related hepatitis and treated with prednisolone. Design, Setting, and Participants: Multicenter, randomized, double-blind clinical trial among patients with biopsy-proven severe alcohol-related hepatitis (Maddrey function score ≥32 and Model for End-stage Liver Disease [MELD] score ≥21) from June 13, 2015, to May 24, 2019, in 25 centers in France and Belgium. All patients were followed up for 180 days. Final follow-up occurred on November 19, 2019. Intervention: Patients were randomly assigned (1:1 allocation) to receive prednisolone combined with amoxicillin-clavulanate (n = 145) or prednisolone combined with placebo (n = 147). Main Outcome and Measures: The primary outcome was all-cause mortality at 60 days. Secondary outcomes were all-cause mortality at 90 and 180 days; incidence of infection, incidence of hepatorenal syndrome, and proportion of participants with a MELD score less than 17 at 60 days; and proportion of patients with a Lille score less than 0.45 at 7 days. Results: Among 292 randomized patients (mean age, 52.8 [SD, 9.2] years; 80 [27.4%] women) 284 (97%) were analyzed. There was no significant difference in 60-day mortality between participants randomized to amoxicillin-clavulanate and those randomized to placebo (17.3% in the amoxicillin-clavulanate group and 21.3% in the placebo group [P = .33]; between-group difference, -4.7% [95% CI, -14.0% to 4.7%]; hazard ratio, 0.77 [95% CI, 0.45-1.31]). Infection rates at 60 days were significantly lower in the amoxicillin-clavulanate group (29.7% vs 41.5%; mean difference, -11.8% [95% CI, -23.0% to -0.7%]; subhazard ratio, 0.62; [95% CI, 0.41-0.91]; P = .02). There were no significant differences in any of the remaining 3 secondary outcomes. The most common serious adverse events were related to liver failure (25 in the amoxicillin-clavulanate group and 20 in the placebo group), infections (23 in the amoxicillin-clavulanate group and 46 in the placebo group), and gastrointestinal disorders (15 in the amoxicillin-clavulanate group and 21 in the placebo group). Conclusion and Relevance: In patients hospitalized with severe alcohol-related hepatitis, amoxicillin-clavulanate combined with prednisolone did not improve 2-month survival compared with prednisolone alone. These results do not support prophylactic antibiotics to improve survival in patients hospitalized with severe alcohol-related hepatitis. Trial Registration: ClinicalTrials.gov Identifier: NCT02281929.

Subclinical versus advanced forms of alcohol-related liver disease: Need for early detection.

Alcohol-related liver disease (ALD) consists of a wide spectrum of clinical manifestations and pathological features, ranging from asymptomatic patients to decompensated cirrhosis and hepatocellular carcinoma. Patients with heavy alcohol intake and advanced fibrosis often develop a subacute form of liver failure called alcohol-induced hepatitis (AH). Globally, most patients with ALD are identified at late stages of the disease, limiting therapeutic interventions. Thus, there is a need for early detection of ALD patients, which is lacking in most countries. The identification of alcohol misuse is hampered by the existence of alcohol underreporting by many patients. There are useful biomarkers that can detect recent alcohol use. Moreover, there are several non-invasive techniques to assess the presence of advanced fibrosis among patients with alcohol misuse, which could identify patients at high risk of liver related events or early death. In this review, we discuss differences between early stages of ALD and AH as the cornerstone of advanced forms. A global overview of epidemiological, anthropometric, clinical, analytical, histological, and molecular differences is summarized in this article. We propose that campaigns aimed at identifying patients with subclinical forms can prevent the development of life-threatening forms.

Liver regeneration as treatment target for severe alcoholic hepatitis.

Severe alcoholic hepatitis (AH) is a distinct entity in the spectrum of alcohol-related liver disease, with limited treatment options and high mortality. Supportive medical care with corticosteroids in selected patients is the only currently available treatment option, often with poor outcomes. Based on the insights into the pathogenetic mechanisms of AH, which are mostly obtained from animal studies, several new treatment options are being explored. Studies have implicated impaired and deranged liver regeneration processes as one of the culprit mechanisms and a potential therapeutic target. Acknowledging evidence for the beneficial effects of granulocyte colony-stimulating factor (G-CSF) on liver regeneration and immunomodulation in animal models, several human studies investigated its role in the treatment of advanced alcohol-related liver disease and AH. Contrary to the previously published studies suggesting benefits of G-CSF in the outcomes of patients with severe AH, these effects were not confirmed by a recently published multicenter randomized trial, suggesting that other options should rather be pursued. Stem cell transplantation represents another option for improving liver regeneration, but evidence for its efficacy in patients with severe AH and advanced alcohol-related liver disease is still very scarce and unconvincing, with established lack of efficacy in patients with compensated cirrhosis. In this review, we summarize the current knowledge on the pathogenesis and experimental therapies targeting liver regeneration. The lack of high-quality studies and evidence is a major obstacle in further treatment development. New insights into the pathogenesis of not only liver injury, but also liver regeneration processes are mandatory for the development of new treatment options. A reliable experimental model of the pathogenesis of AH and processes involved in liver recovery is still missing, and data obtained from animal studies are essential for future research.

Granulocyte colony-stimulating factor attenuates liver damage by M2 macrophage polarization and hepatocyte proliferation in alcoholic hepatitis in mice.

Massive inflammation and liver failure are main contributors to the high mortality in alcohol-associated hepatitis (AH). In recent clinical trials, granulocyte colony-stimulating factor (G-CSF) therapy improved liver function and survival in patients with AH. However, the mechanisms of G-CSF-mediated beneficial effects in AH remain elusive. In this study, we evaluated effects of in vivo G-CSF administration, using a mouse model of AH. G-CSF treatment significantly reduced liver damage in alcohol-fed mice even though it increased the numbers of liver-infiltrating immune cells, including neutrophils and inflammatory monocytes. Moreover, G-CSF promoted macrophage polarization toward an M2-like phenotype and increased hepatocyte proliferation, which was indicated by an increased Ki67-positive signal colocalized with hepatocyte nuclear factor 4 alpha (HNF-4α) and cyclin D1 expression in hepatocytes. We found that G-CSF increased G-CSF receptor expression and resulted in reduced levels of phosphorylated ß-catenin in hepatocytes. In the presence of an additional pathogen-associated molecule, lipopolysaccharide (LPS), which is significantly increased in the circulation and liver of patients with AH, the G-CSF-induced hepatoprotective effects were abolished in alcohol-fed mice. We still observed increased Ki67-positive signals in alcohol-fed mice following G-CSF treatment; however, Ki67 and HNF-4α did not colocalize in LPS-challenged mice. Conclusion: G-CSF treatment increases immune cell populations, particularly neutrophil counts, and promotes M2-like macrophage differentiation in the liver. More importantly, G-CSF treatment reduces alcohol-induced liver injury and promotes hepatocyte proliferation in alcohol-fed mice. These data provide new insights into the understanding of mechanisms mediated by G-CSF and its therapeutic effects in AH.

Serum keratin 19 (CYFRA21-1) is a prognostic biomarker in severe alcoholic hepatitis.

BACKGROUND AND AIMS: Up to 30% of patients with severe alcoholic hepatitis (sAH) die within 3 months of presentation. The degree of ductular reaction, characterized by accumulation of biliary and liver progenitor cells, confers a poor prognosis. Keratin fragments are established serological surrogates of liver injury and keratin 19 (K19) is a histological marker of the ductular reaction. We assessed the relationship between serum K19 levels (viz. CYFRA21-1), histology and outcome in patients with sAH. METHODS: Serum CYFRA21-1 was quantified in pre-treatment serum samples from 824 patients enrolled in the STOPAH trial. The cohort was randomly divided into two groups to test mortality associations; histological analyses were performed using the 87 cases with suitable samples. RESULTS: CYFRA21-1 levels were elevated in sAH and strongly predicted alcoholic steatohepatitis (ASH) on biopsy (area under the receiver operated characteristic [AUROC] 0.785 [95% Confidence Interval 0.602-0.967]) and 90-day survival (AUROC 0.684/0.693). The predictive ability of CYFRA21-1 was comparable with the model of end-stage liver disease (MELD) score and was independently associated with survival in multivariable analysis. CYFRA21-1 moderately correlated with hepatocellular injury markers M30/M65 but displayed a more robust predictive ability. A combination of MELD and CYFRA21-1 conferred a modest improvement in the AUROC value (0.731/0.743). CONCLUSIONS: In sAH serum, CYFRA21-1 levels associate with the presence of ASH on biopsy and independently predict 90-day survival. As a single marker performance is comparable to established scoring systems. Therefore, CYFRA21-1, which is available in many clinical laboratories, may become a useful component of prognostic models.

Adenosine receptor A2B mediates alcoholic hepatitis by regulating cAMP levels and the NF-KB pathway.

Alcoholic hepatitis is a serious form of liver damage. Inflammation is a key factor in alcoholic hepatitis and plays a key role in the progression of alcoholic liver disease. Adenosine receptor A2B (A2BAR) is a member of the adenosine receptor family and generally considered to be a negative regulator of the inflammatory response. We found that A2BAR was the most highly expressed adenosine receptor in ETOH-fed mouse liver tissue and was also highly expressed in primary Kupffer cells and ETOH-induced RAW264.7 cells. In addition, injection of BAY 60-6583 stimulated A2BAR, induced upregulation of the expression levels of cAMP, and reduced ETOH-induced steatosis and inflammation in mice. At the same time, knockdown of A2BAR in vitro increased the inflammatory response in RAW264.7 cells triggered by ETOH. After knockdown of A2BAR in vitro, the release of the inflammatory cytokines IL-6, IL-1ß and TNF-α was increased. After overexpression of A2BAR in vitro, the cAMP level was significantly increased, PKA expression was increased, the expression of phosphorylated proteins in the NF-kB signal transduction pathway was significantly affected, and the expression of the key phosphorylated protein p-P65 was decreased. However, after the simultaneous overexpression of A2BAR and inhibition of PKA, the expression of the key phosphorylated protein p-P65 was still significantly decreased. In addition, after the expression of A2BAR increased or decreased in RAW264.7 cells, AML-12 cells were cultured in the supernatant of RAW264.7 cells stimulated by ETOH, and the apoptosis rate was significantly changed by flow cytometry. These results suggest that A2BAR can reduce alcoholic steatohepatitis by upregulating cAMP levels and negatively regulating the NF-kB pathway. Overall, these findings suggest the significance of A2BAR-mediated inflammation in alcoholic liver disease.

Plasma-Signature-Model for End-Stage Liver Disease Score to Predict Survival in Severe Alcoholic Hepatitis.

BACKGROUND & AIMS: Severe alcoholic hepatitis (AH) is a highly lethal condition and it is still a challenge to predict the outcome. We previously identified and validated a composite score of hepatic 123-gene prognostic signature and the model for end-stage liver disease (MELD) score: gene signature-MELD. However, the need for liver biopsy limits its clinical application. Therefore, we aimed to identify a plasma protein-based surrogate of the gene signature and independently validate its prognostic capability. METHODS: All patients were diagnosed with severe AH at Cliniques universitaires de Bruxelles Hôpital Erasme (Brussels, Belgium), and the plasma samples were collected at admission before any treatment. The primary outcome was death or liver transplantation within 90 days. Using our computational pipeline, named translation of tissue expression to secretome (TexSEC), a hepatic-transcriptome-based prognostic signature was converted to a plasma-based secretome signature, which was optimized in 50 patients by comparing their hepatic molecular dysregulation status and combining it with the MELD score. The composite score was validated independently in 57 patients. RESULTS: The TexSEC and optimization process identified a 6-plasma-protein panel as a surrogate for the 123-gene signature. A composite score with the MELD score, the plasma-signature (ps)-MELD score, was created by using the coefficients of the gene signature-MELD equation. In the validation cohort, the high-risk ps-MELD (n = 23; 40%) was associated significantly with death or liver transplantation within 90 days (adjusted hazard ratio, 4.57; 95% CI, 2.15-9.30; P < .001). The ps-MELD score showed a stable, high prognostic association (time-dependent area under receiver operating characteristics curve, >0.80) and was well calibrated over time; it consistently outperformed existing clinical scores as indicated by various model performance indices. CONCLUSIONS: The high-risk ps-MELD score was associated with short-term survival in patients with severe AH.

IL-1 Signal Inhibition In Alcoholic Hepatitis (ISAIAH): a study protocol for a multicentre, randomised, placebo-controlled trial to explore the potential benefits of canakinumab in the treatment of alcoholic hepatitis.

BACKGROUND: Alcohol consumption causes a spectrum of liver abnormalities and leads to over 3 million deaths per year. Alcoholic hepatitis (AH) is a florid presentation of alcoholic liver disease characterized by liver failure in the context of recent and heavy alcohol consumption. The aim of this study is to explore the potential benefits of the IL-1ß antibody, canakinumab, in the treatment of AH. METHODS: This is a multicentre, double-blind, randomised placebo-controlled trial. Participants will be diagnosed with AH using clinical criteria. Liver biopsy will then confirm that all histological features of AH are present. Up to 58 participants will be recruited into two groups from 15 centres in the UK. Patients will receive an infusion of Canakinumab or matched placebo by random 1:1 allocation. The primary outcome is the difference between groups in the proportion of patients demonstrating histological improvement and will compare histological appearances at baseline with appearances at 28 days to assign a category of "improved" or "not improved". Patients with evidence of ongoing disease activity will receive a second infusion of canakinumab or placebo. Participants will be followed up for 90 days. Secondary outcomes include mortality and change in MELD score at 90 days. DISCUSSION: This phase II study will explore the benefits of the IL-1ß antibody, canakinumab, in the treatment of AH to provide proof of concept that inhibition of IL-1ß signalling may improve histology and survival for patients with AH. TRIAL REGISTRATION: EudraCT 2017-003724-79 . Prospectively registered on 13 April 2018.

Telmisartan alleviates alcohol-induced liver injury by activation of PPAR-γ/ Nrf-2 crosstalk in mice.

Excessive consumption of alcohol may induce severe liver damage, in part via oxidative stress and inflammatory responses, which implicates these processes as potential therapeutic approaches. Prior literature has shown that Telmisartan (TEL) may provide protective effects, presumably mediated by its anti-oxidant and anti-inflammatory activities. The purpose of this study was to determine TEL's hepatoprotective effects and to identify its possible curative mechanisms in alcoholic liver disease. A mouse chronic alcohol plus binge feedings model was used in the current study for induction of alcoholic liver disease (ALD). Our results showed that TEL (10 mg/kg/day) has the ability to reduce serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP). TEL also increased the activity of superoxide dismutase (SOD) and glutathione (GSH) with concomitant reduction of nitric oxide (NO) malonaldehyde (MDA) in the liver homogenate. Moreover, TEL downregulated nuclear factor kappa B (NF-κB) expression and decreased liver content of interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α). These anti-inflammatory and anti-oxidant activities were associated with a significant increase in the expression of nuclear factor erythroid 2-related factor 2 (Nrf-2), peroxisome proliferator-activated receptors -γ (PPAR-γ), and heme oxygenase-1 (Hmox-1). In conclusion, TEL's hepatoprotective effects against ALD may be attributable to its anti-inflammatory and anti-oxidant activities which may be in part via the modulation of PPAR-γ/ Nrf-2/ NF-κB crosstalk.

No Effect in Alcoholic Hepatitis of Gut-Selective, Broad-Spectrum Antibiotics on Bacterial Translocation or Hepatic and Systemic Inflammation.

INTRODUCTION: In alcoholic hepatitis (AH), translocation of gut bacteria may drive hepatic macrophage activation and systemic inflammation. We investigated the effect of oral non-absorbable, broad-spectrum antibiotic treatment on bacterial translocation and liver and systemic inflammation in AH. METHODS: We consecutively recruited 31 patients with AH. Fourteen were given vancomycin 500 mg, gentamycin 40 mg, and meropenem 500 mg once daily for 7 days. Seventeen patients were a reference group receiving standard-of-care. Circulating markers of bacterial translocation and inflammation were measured at baseline, by day 7 and 90. Gut bacteriome profiling was performed before the intervention and at day 7. RESULTS: At study entry, blood lipopolysaccharide-binding protein was multifold higher than normal, remained unchanged at day 7, but decreased at day 90 (P < 0.001) with no difference between the study groups. The macrophage activation markers sCD163 and sCD206 showed the same pattern (P < 0.001, day 90), still without group differences. The systemic inflammation markers tumor necrosis factor-alpha, interleukin (IL)-6, IL-8, and IL-10 showed similar dynamics without group differences. There was no difference in 90-day mortality (total of 6 deaths) between the groups. The remnant gut bacteriome was markedly diversified by the intervention with growth of bacterial species rare for human flora. DISCUSSION: In patients with AH, gut-targeted antibiotic treatment does not change markers of bacterial translocation and liver and systemic inflammation. This suggests that bacterial translocation is less important once the inflammatory process is established or that bacteriome reduction is less important than composition.

Development of Saikosaponin D Liposome Nanocarrier with Increased Hepatoprotective Effect Against Alcoholic Hepatitis Mice.

The mortality rate of ethanol induced liver disease has substantially raised to alert level with an increasing use of alcohol, but development of definite hepatoprotective drug is still challenging. The efficacy of Saikosaponin D, one of the natural herbal medicine has been studied in different diseases. Nonetheless, its clinical application is restricted by poor bioavailability, stability and solubility. This study sought to develop a Saikosaponin D loaded liposome via thin film hydration method. The surface morphology, encapsulation efficiency and drug loading capacity were detected with transmission electron microscopy and HPLC, in vitro dissolution was via dialysis method, but efficacy and safety evaluation was through pharmacokinetics, while the assessment of hepatoprotective activity on alcohol induced acute hepatitis mice models was conducted. The optimized liposomes showed significant greater therapeutic effect on liver, through decreased serum levels of alanine transaminase (ALT) and aspartate transaminase (AST), malondialdehyde (MDA), tumor necrosis factor alpha (TNF-α), total cholesterol (TC) and triglyceride (TG) in liver homogenate. In contrast, levels of glutathione peroxidase (GSH-Px) and total superoxide dismutase (T-SOD) were increased significantly. Pathological study exhibited remarkable alteration of hepatitis liver architecture to almost normal state after administration of Saikosaponin D liposome. The increased hepatoprotective effect of Saikosaponin D liposome was observed during the attenuation of alcoholic hepatitis in mice, which might be ascribable to the anti-oxidative and anti-inflammatory properties of the drug. This study provides a theoretical basis for developing advanced system of Saikosaponin D delivery for the promotion of the therapeutic effects of the liposome against various kinds of diseases.

In Severe Alcoholic Hepatitis, Serum Keratin-18 Fragments Are Diagnostic, Prognostic, and Theragnostic Biomarkers.

INTRODUCTION: Up to 40% of patients with severe alcoholic hepatitis (AH) die within 6 months of presentation, making prompt diagnosis and appropriate treatment essential. We determined the associations between serum keratin-18 (K18) and histological features, prognosis, and differential response to prednisolone in patients with severe AH. METHODS: Total (K18-M65) and caspase-cleaved K18 (K18-M30) were quantified in pretreatment sera from 824 patients enrolled in the Steroids or Pentoxifylline for Alcoholic Hepatitis trial (87 with suitable histological samples) and disease controls. RESULTS: K18 fragments were markedly elevated in severe AH and strongly predicted steatohepatitis (alcoholic steatohepatitis) on biopsy (area under receiver operating characteristics: 0.787 and 0.807). Application of published thresholds to predict alcoholic steatohepatitis would have rendered biopsy unnecessary in 84% of all AH cases. K18-M30 and M65 were associated with 90-day mortality, independent of age and Model for End-stage Liver Disease score in untreated patients. The association for K18-M65 was independent of both age and Model for End-stage Liver Disease in prednisolone-treated patients. Modelling of the effect of prednisolone on 90-day mortality as a function of pretreatment serum K18 levels indicated benefit in those with high serum levels of K18-M30. At low pretreatment serum K18 levels, prednisolone was potentially harmful. A threshold of K18-M30 5 kIU/L predicted therapeutic benefit from prednisolone above this level (odds ratio: 0.433, 95% confidence interval: 0.19-0.95, P = 0.0398), but not below (odds ratio: 1.271, 95% confidence interval: 0.88-1.84, P = 0.199). Restricting prednisolone usage to the former group would have reduced exposure by 87%. DISCUSSION: In a large cohort of patients with severe AH, serum K18 strongly correlated with histological severity, independently associated with 90-day mortality, and predicted response to prednisolone therapy. Quantification of serum K18 levels could assist in clinical decision-making.

Design and rationale of a multicenter defeat alcoholic steatohepatitis trial: (DASH) randomized clinical trial to treat alcohol-associated hepatitis.

BACKGROUND/AIMS: Despite high mortality of alcohol-associated hepatitis, there has been limited advancement in treatment strategies. Defeat Alcoholic Steatohepatitis (DASH) is a multicenter, randomized, double-blind controlled trial whose primary objective was to evaluate the safety and efficacy of a novel combination of 3 drugs targeting different perturbations in AH. METHODS: Severe AH was diagnosed by liver biopsy or clinical and biochemical criteria and model for end stage liver disease (MELD) score ≥ 20 stratified by MELD scores (20-25 and ≥ 26) and randomized to a combination of an interleukin receptor 1 antagonist, Anakinra(100 mg daily for 14 days) to suppress acute inflammation, pentoxifylline (400 mg three times a day for 28 days) to prevent hepatorenal syndrome, and zinc sulfate (220 mg orally once daily for 6 months) or the standard of care therapy including methylprednisolone 32 mg orally once daily for 28 days. The primary efficacy outcome was the unadjusted log-rank test of the Kaplan-Meier survival estimates for the two treatment groups at 180 days. RESULTS: Between July 2012 to March 2018, 500 subjects with severe AH were screened of which 104 subjects were enrolled with MELD score of 25.6 ± 3.2 (20.0-35.0) in the investigational arm and 25.8 ± 4.5 (20.0-40.0) in the standard of care arm. Causes of screen failures included not meeting eligibility criteria (n = 347), declining to participate (n = 39), and other reasons (n = 10). CONCLUSIONS: Data from the DASH consortium studies will determine if a combination of drugs targeting multiple mechanisms of injury in the severe AH will improve clinical outcomes.

A Combination of N-Acetylcysteine and Prednisone Has No Benefit Over Prednisone Alone in Severe Alcoholic Hepatitis: A Retrospective Analysis.

INTRODUCTION: In this study, we assessed whether there were any survival advantages with a combination treatment of intravenous N-acetylcysteine (NAC) and prednisone over prednisone alone in those with severe alcoholic hepatitis [discriminant function (DF) ≥ 32]. PATIENTS AND METHODS: Between January 1, 2013, and February 28, 2019, we identified 68 patients (mean age 47.2 years ± 10.1, 57% women, 65% cirrhosis, MELD score 28.1 ± 6.6) with alcoholic hepatitis, and of those, 21 (31%) received prednisone and 47 (69%) received prednisone + NAC. Lille score ≥ 0.45 was considered a poor response. Renal insufficiency was defined as GFR < 60 ml/min/1.73m2 calculated on two separate occasions. RESULTS: DF (74.2 ± 33.6 vs. 56.9 ± 15.9, p = 0.09) was similar, but MELD (29.2 ± 6.3 vs. 25.5 ± 6.4, p = 0.03) scores were higher in the combination group. The overall 30-day and 90-day mortality was 13.2% (9/68) and 20.6% (14/68), respectively. Women were more likely (OR 4.86, 95% CI 1.62-14.59) to respond to treatment based on Lille score compared to men, but the type of treatment regimen had no effect on Lille score (OR 0.84, 95% CI 0.25-2.78). Treatment regimen had no effect on both adjusted and unadjusted survivals. Multivariate analysis, after adjusting for confounding variables, confirmed these observations. DF + renal insufficiency had the highest AUROC (0.86) to predict mortality. CONCLUSION: The combination treatment of NAC + prednisone is not better than prednisone alone in patients with severe alcoholic hepatitis.

Ex Vivo T Cell Cytokine Expression Predicts Survival in Patients with Severe Alcoholic Hepatitis.

Alcoholic hepatitis (AH) is an acute inflammatory liver condition with high early mortality rate. Steroids improve short-term survival but nonresponders have the worst outcomes. There is a clinical need to identify these high-risk individuals at the time of presentation. T cells are implicated in AH and steroid responsiveness. We measured ;ex vivo T cell cytokine expression as a candidate biomarker of outcomes in patients with AH. Consecutive patients (bilirubin >80 µmol/L and ratio of aspartate aminotransferase to alanine aminotransferase >1.5 who were heavy alcohol consumers with discriminant function [DF] ≥32), were recruited from University Hospitals Plymouth NHS Trust. T cells were obtained and stimulated ;ex vivo. Cytokine expression levels were determined by flow cytometry and protein multiplex analysis. Twenty-three patients were recruited (10 male; median age 51 years; baseline DF 67; 30% 90-day mortality). Compared to T cells from nonsurvivors at day 90, T cells from survivors had higher baseline baseline intracellular interleukin (IL)-10:IL-17A ratio (0.43 vs 1.20, p=0.02). Multiplex protein analysis identified interferon γ (IFNγ) and tumor necrosis factor-α (TNF-α) as independent predictors of 90-day mortality (p=0.04, p=0.01, respectively). The ratio of IFNγ to TNF-α was predictive of 90-day mortality (1.4 vs 0.2, p=0.03). These data demonstrate the potential utility of T cell cytokine release assays performed on pretreatment blood samples as biomarkers of survival in patients with severe AH. Our key findings were that intracellular IL-10:IL-17A and IFNγ:TNF-α in culture supernatants were predictors of 90-day mortality. This offers the promise of developing T cell-based diagnostic tools for risk stratification.