Proteomics identifies complement protein signatures in patients with alcohol-associated hepatitis.

Diagnostic challenges continue to impede development of effective therapies for successful management of alcohol-associated hepatitis (AH), creating an unmet need to identify noninvasive biomarkers for AH. In murine models, complement contributes to ethanol-induced liver injury. Therefore, we hypothesized that complement proteins could be rational diagnostic/prognostic biomarkers in AH. Here, we performed a comparative analysis of data derived from human hepatic and serum proteome to identify and characterize complement protein signatures in severe AH (sAH). The quantity of multiple complement proteins was perturbed in liver and serum proteome of patients with sAH. Multiple complement proteins differentiated patients with sAH from those with alcohol cirrhosis (AC) or alcohol use disorder (AUD) and healthy controls (HCs). Serum collectin 11 and C1q binding protein were strongly associated with sAH and exhibited good discriminatory performance among patients with sAH, AC, or AUD and HCs. Furthermore, complement component receptor 1-like protein was negatively associated with pro-inflammatory cytokines. Additionally, lower serum MBL associated serine protease 1 and coagulation factor II independently predicted 90-day mortality. In summary, meta-analysis of proteomic profiles from liver and circulation revealed complement protein signatures of sAH, highlighting a complex perturbation of complement and identifying potential diagnostic and prognostic biomarkers for patients with sAH.

Stigmatizing Language for Alcohol Use Disorder and Liver Disease on Liver Transplant Center Websites.

This cross-sectional study investigates the adoption of recommendations for the use of nonstigmatizing language to describe alcohol use disorder and alcohol-related liver disease among liver transplant centers in the US.

Moderate-high intensity exercise associates with reduced incident alcohol-associated liver disease in high-risk patients.

BACKGROUND: Therapies to prevent alcohol-associated liver disease (ALD) in high-risk patients are needed. AIMS: In this retrospective association study, we examined whether patients with alcohol use disorder (AUD) who reported greater exercise were less likely to develop liver disease. METHODS: In this retrospective cohort study, we used the Mass General Brigham Biobank to investigate the impact of both moderate-high and light-intensity exercise on the development of ALD in patients with AUD, using clinician-provided diagnostic International Classification of Diseases 10 codes. Exercise was evaluated using a questionnaire completed after an AUD diagnosis, and before evidence of liver disease. Cox regressions were used to generate hazard ratios (HRs) for the development of ALD. RESULTS: 1987 patients met inclusion criteria. These patients were followed for an average of 10.7 years. In multivariable analyses, we found that patients that reported at least 2.5 h of moderate-high intensity exercise/week (confidence interval recommendation for exercise) were less likely to develop ALD compared to patients that did not exercise (HR: 0.26, 95%CI: 0.085-0.64, P = 0.007). Indeed, each hour of moderate-high intensity exercise was associated with progressively decreasing odds of developing ALD (HR: 0.76, 95%CI: 0.58-0.91, P = 0.02). Conversely, patients who did not engage in any moderate-high intensity exercise were more likely to develop ALD (HR: 2.76, 95%CI: 1.44-5.40, P = 0.003). CONCLUSIONS: In our cohort, patients with AUD who reported moderate-high intensity exercise showed a lower association with incidence of ALD development than patients who did not exercise.

Integrated hepatology and addiction care for inpatients with alcohol use disorder improves outcomes: a prospective study.

BACKGROUND: Growing literature highlights the need to integrate hepatology and addiction care to improve outcomes for patients with alcohol use disorder and alcohol-associated liver disease. However, prospective data for this approach are lacking. METHODS: We prospectively examined the efficacy of an integrated hepatology and addiction medicine approach on alcohol use and hepatology outcomes in inpatients with alcohol use disorder. FINDINGS: An integrated approach improved the uptake of medical alcohol therapy, hepatic fibrosis screening, and viral hepatitis vaccination compared with a historical control of patients who received addiction medicine care alone. There were no differences in the rates of early alcohol remission. The integration of hepatology and addiction care may improve outcomes in patients with alcohol use disorder.

Serum Fibroblast Growth Factor-21 Discriminates Between Decompensated Alcohol-Associated Cirrhosis and Severe Alcohol-Associated Hepatitis.

INTRODUCTION: We hypothesized that fibroblast growth factor-21 (FGF-21) would be highly expressed in patients with alcohol-associated hepatitis (AH) and could be a novel and biologically relevant predictive biomarker to reliably distinguish severe AH and decompensated alcohol-associated cirrhosis (AC). METHODS: We identified a discovery cohort of 88 subjects with alcohol-associated liver disease (ALD) of varying disease severity from our ALD repository. Our validation cohort consisted of 37 patients with a biopsy-proven diagnosis of AH, AC, or absence of ALD with Model for End-Stage Liver Disease scores ≥10. Serum from both groups during index hospitalization was assayed for FGF-21 by ELISA. We performed receiver operating characteristic analysis and prediction modeling in both cohorts to discriminate between AH and AC in high Model for End-Stage Liver Disease (≥20) patients. RESULTS: In both cohorts, FGF-21 concentrations were highest in subjects with moderate to severe AH compared with those having alcohol use disorder or AC (mean: 2,609 pg/mL, P < 0.0001). The discovery cohort area under the curve of FGF-21 between AH and AC was 0.81 (95% confidence interval: 0.65-0.98, P < 0.01). In the validation cohort, FGF-21 levels were higher in severe AH compared with AC (3,052 vs 1,235 pg/mL, P = 0.03), and the area under the curve was 0.76 (95% confidence interval: 0.56-0.96, P < 0.03). A survival analysis showed that patients with FGF-21 serum levels in the second interquartile had the highest survival compared with all other quartiles. DISCUSSION: FGF-21 performs well as a predictive biomarker to distinguish severe AH from AC and may be helpful in the management and clinical investigation of patients with severe alcohol-associated liver diseases.

Differential Effects of COVID-19 Hospitalization on the Trajectory of Liver Disease Progression.

Background and Aims: Patients with chronic liver disease (CLD) were significantly affected by COVID-19. Despite evidence of acute hepatic injury and increased mortality, the long-term effects of COVID-19 hospitalization on the natural history of CLD patients are unknown. Methods: The Massachusetts General Hospital COVID-19 registry was used to obtain a cohort of CLD patients hospitalized between March 8 and June 3, 2020. The Partners Research Patient Data Registry was used to develop a matched CLD patient control list without COVID-19. Fibrosis-4 index (FIB-4), nonalcoholic fatty liver disease fibrosis score (NFS), and model for end-stage liver disease/Na (MELD-Na) scores were calculated pre-, day of, and 1-year post-discharge from admission. Unpaired t-test was used to compare continuous variables. Results: Fifty-two COVID-19 patients and 92 control patients with CLD were included. Patients with non-cirrhotic CLD who were hospitalized for COVID-19 had an acute rise in FIB-4 on admission with subsequent improvement on one-year follow-up demonstrating no difference in progression of liver disease compared to the controls (P = .87, confidence interval [CI] -0.088 to 0.048). Similar trends were observed in nonalcoholic fatty liver disease patients using NFS (P = .48, CI -0.016 to 0.023). In contrast, patients with cirrhosis experienced rise in MELD-Na postadmission compared to the control cirrhosis group (0.35 vs -0.076/month; P = .04, CI -0.827 to -0.025), suggesting a potential for long-term consequences of COVID-19. Conclusion: Non-cirrhotic CLD patients who survive COVID-19 hospitalization do not appear to have change in FIB-4, NFS scores at one year. However, patients with cirrhosis exhibit increasing MELD-Na one-year post-COVID suggesting a differential effect of acute COVID-19 on the trajectory of established cirrhosis.

Resection of NAFLD/NASH-related Hepatocellular Carcinoma (HCC): Clinical Features and Outcomes Compared with HCC Due to Other Etiologies.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are the leading causes of hepatocellular carcinoma (HCC) worldwide. Limited data exist on surgical outcomes for NAFLD/NASH-related HCC compared with other HCC etiologies. We evaluated differences in clinicopathological characteristics and outcomes of patients undergoing surgical resection for NAFLD/NASH-associated HCC compared with other HCC etiologies. METHODS: Demographic, clinicopathological features, and survival outcomes of patients with surgically resected HCC were collected. NAFLD activity score (NAS) and fibrosis score were assessed by focused pathologic review in a subset of patients. RESULTS: Among 492 patients screened, 260 met eligibility (NAFLD/NASH [n = 110], and other etiologies [n = 150]). Median age at diagnosis was higher in the NAFLD/NASH HCC cohort compared with the other etiologies cohort (66.7 vs. 63.4 years, respectively, P = .005), with an increased percentage of female patients (36% vs. 18%, P = .001). NAFLD/NASH-related tumors were more commonly >5 cm (66.0% vs. 45%, P = .001). There were no significant differences in rates of lymphovascular or perineural invasion, histologic grade, or serum AFP levels. The NAFLD/NASH cohort had lower rates of background liver fibrosis, lower AST and ALT levels, and higher platelet counts (P < .01 for all). Median overall survival (OS) was numerically shorter in NAFLD/NASH vs other etiology groups, however, not statistically significant. CONCLUSIONS: Patients with NAFLD/NASH-related HCC more commonly lacked liver fibrosis and presented with larger HCCs compared with patients with HCC from other etiologies. No differences were seen in rates of other high-risk features or survival. With the caveat of sample size and retrospective analysis, this supports a similar decision-making approach regarding surgical resection for NAFLD/NASH and other etiology-related HCCs.

Safety, tolerability, and effectiveness of anticoagulation and antiplatelet therapy in hereditary hemorrhagic telangiectasia.

BACKGROUND: Antithrombotic therapy (anticoagulation and antiplatelet therapy) is frequently needed in patients with hereditary hemorrhagic telangiectasia (HHT); however, data describing and guiding its use are very limited. OBJECTIVES: To investigate the safety, tolerability, and effectiveness of antithrombotic therapy in HHT in a cohort large enough to compare agents, evaluate for baseline predictors of premature discontinuation, and evaluate hematologic support requirements and healthcare utilization before and after antithrombitc therapy initiation. METHODS: We performed a multicenter observational cohort study characterizing the outcomes of antithrombic therapy in adults with HHT. RESULTS: A total of 119 patients with HHT with 187 discrete antithrombotic therapy episodes were included. Of these, 59 patients (50%) dose-reduced and/or prematurely discontinued therapy (including 52 patients [44%] who discontinued) due to worsened bleeding complications. Initiation at reduced dose intensity had a similar premature discontinuation rate (49%) as initiation at standard dose intensity (43%). In a multivariable logistic model, a history of gastrointestinal bleeding was associated with 3.25-fold odds of discontinuation (p = .001). Hemoglobin was significantly lower (10.8 g/dL vs 12.2 g/dL, p < .001), and the need for hematologic support (intravenous iron and/or red blood cell transfusion) was significantly higher (29 patients vs 12 patients, p = .004) in the 3 months after antithrombotic therapy initiation vs the 3 months before; emergency department visits and hospital admissions due to bleeding also increased. The rates of dose-reduction and/or premature discontinuation were similar regardless of the anticoagulant class (warfarin, 46%; heparin-based, 48%; direct oral anticoagulants, 44%) or with multiple simultaneous agents (44%) but were slightly lower with single-agent antiplatelet therapy (37%). Thromboembolism despite receiving antithrombotic therapy was common (18 patients, 15%) with varying outcomes. CONCLUSION: Antithrombotic therapy is challenging in HHT, resulting in objectively higher morbidity and health care utilization from worsened bleeding. Discontinuation rates approached 50% regardless of the dose intensity at initiation or type of antithrombotic agent used and were higher in patients with a gastrointestinal bleeding history.

Creation of an Inpatient Alcohol Liver Service Improves Early Liver Disease Detection in Patients With Alcohol Use Disorder.

One of the most common and devastating complications of alcohol use disorder (AUD) is the development of alcohol-associated liver disease (ALD).1 Unfortunately, outcomes of patients with ALD are poor, in large part because patients with ALD are diagnosed at a much later stage of disease compared with patients with other causes of liver disease.2 Accordingly, earlier detection is critical in combating the high mortality associated with ALD. In this work, we sought to assess the feasibility and impact of evaluating high-risk patients with no prior documented liver disease for the presence of advanced ALD while admitted to the hospital.

Psychotherapy for Alcohol Use Disorder Is Associated With Reduced Risk of Incident Alcohol-Associated Liver Disease.

BACKGROUND & AIMS: Alcohol-associated liver disease (ALD) is a devastating complication of alcohol use disorder (AUD). Once it develops, ALD is exceedingly difficult to treat; it therefore is critical to identify ways to prevent ALD. By treating the causes of increased alcohol consumption, psychotherapy may offer prophylactic benefit against the development of ALD for patients with AUD. METHODS: In this retrospective cohort study, we used International Classification of Diseases, 9th and 10th revision, codes to identify 9635 patients with AUD in the Mass General Brigham Biobank. The mean follow-up period from AUD diagnosis was 9.2 years. We used Cox regression models to generate hazard ratios (HR) for the development of ALD given the receipt or nonreceipt of psychotherapy, adjusting for a range of other contributors including the receipt of medication-assisted treatment. RESULTS: In our cohort, 60.4% of patients were men, 83.5% of patients were white, the median age was 57.0 years, and 3544 patients (36.8%) received psychotherapy. ALD developed in 1135 patients (11.8%). In multivariable analysis, psychotherapy was associated with a reduced rate of ALD (HR, 0.59; 95% CI, 0.50-0.71; P < .001). This association held for both individual psychotherapy (HR, 0.70; 95% CI, 0.56-0.86; P < .001) and group psychotherapy (HR, 0.76; 95% CI, 0.61-0.94; P = .01). Among patients with cirrhosis, psychotherapy was associated with a lower rate of hepatic decompensation (HR, 0.68; 95% CI, 0.48-0.95; P = .03). CONCLUSIONS: The receipt of psychotherapy in the setting of AUD is associated with reduced incidence and progression of ALD. Given the safety and potential benefit of psychotherapy, clinicians should consider using it to prevent the development of ALD.

The circulating proteomic signature of alcohol-associated liver disease.

Despite being a leading cause of advanced liver disease, alcohol-associated liver disease (ALD) has no effective medical therapies. The circulating proteome, which comprises proteins secreted by different cells and tissues in the context of normal physiological function or in the setting of disease and illness, represents an attractive target for uncovering novel biology related to the pathogenesis of ALD. In this work, we used the aptamer-based SomaScan proteomics platform to quantify the relative concentration of over 1300 proteins in a well-characterized cohort of patients with the spectrum of ALD. We found a distinct circulating proteomic signature that correlated with ALD severity, including over 600 proteins that differed significantly between ALD stages, many of which have not previously been associated with ALD to our knowledge. Notably, certain proteins that were markedly dysregulated in patients with alcohol-associated hepatitis were also altered, to a lesser degree, in patients with subclinical ALD and may represent early biomarkers for disease progression. Taken together, our work highlights the vast and distinct changes in the circulating proteome across the wide spectrum of ALD, identifies potentially novel biomarkers and therapeutic targets, and provides a proteomic resource atlas for ALD researchers and clinicians.

An additional patient with SMAD4-Juvenile Polyposis-Hereditary hemorrhagic telangiectasia and connective tissue abnormalities: SMAD4 loss-of-function and gain-of-function pathogenic variants result in contrasting phenotypes.

Loss-of-function pathogenic variants in somatic and germline cells in SMAD4 may cause cancer and juvenile polyposis-Hereditary Hemorrhagic Telangiectasia (SMAD4-JP-HHT), respectively. In a similar manner, gain-of-function somatic and germline pathogenic variants in SMAD4 can cause various forms of cancer as well as Myhre syndrome. The different SMAD4 molecular mechanisms result in contrasting clinical phenotypes demonstrated by SMAD4-JP-HHT and Myhre syndrome. We report an additional patient with SMAD4-JP-HHT and aortopathy, and expand the phenotype to include severe valvulopathy, cutaneous, ophthalmologic, and musculoskeletal features consistent with an inherited disorder of connective tissue. We compared this 70-year-old man with SMAD4-JP-HHT to 18 additional literature cases, and also compared patients with SMAD4-JP-HHT to those with Myhre syndrome. In contrast to aorta dilation, hypermobility, and loose skin in SMAD4-JP-HHT, Myhre syndrome has aorta hypoplasia, stiff joints, and firm skin representing an intriguing phenotypic contrast, which can be attributed to different molecular mechanisms involving SMAD4. We remind clinicians about the possibility of significant cardiac valvulopathy and aortopathy, as well as connective tissue disease in SMAD4-JP-HHT. Additional patients and longer follow-up will help determine if more intensive surveillance improves care amongst these patients.

Incidence and Progression of Alcohol-Associated Liver Disease After Medical Therapy for Alcohol Use Disorder.

Importance: Alcohol-associated liver disease (ALD) is one of the most devastating complications of alcohol use disorder (AUD), an increasingly prevalent condition. Medical addiction therapy for AUD may play a role in protecting against the development and progression of ALD. Objective: To ascertain whether medical addiction therapy was associated with an altered risk of developing ALD in patients with AUD. Design, Setting, and Participants: This retrospective cohort study used the Mass General Brigham Biobank, an ongoing research initiative that had recruited 127 480 patients between its start in 2010 and August 17, 2021, when data for the present study were retrieved. The mean follow-up duration from AUD diagnosis was 9.2 years. International Statistical Classification of Diseases and Related Health Problems, Tenth Revision diagnosis codes were used to identify ALD and AUD diagnoses. Exposures: Medical addiction therapy was defined as the documented use of disulfiram, acamprosate, naltrexone, gabapentin, topiramate, or baclofen. Patients were considered to be treated if they initiated medical addiction therapy before the relevant outcome. Main Outcomes and Measures: Adjusted odds ratios (aORs) for the development of ALD and hepatic decompensation were calculated and adjusted for multiple risk factors. Results: The cohort comprised 9635 patients with AUD, of whom 5821 were male individuals (60.4%), and the mean (SD) age was 54.8 (16.5) years. A total of 1135 patients (11.8%) had ALD and 3906 patients (40.5%) were treated with medical addiction therapy. In multivariable analyses, medical addiction therapy for AUD was associated with decreased incidence of ALD (aOR, 0.37; 95% CI, 0.31-0.43; P < .001). This association was evident for naltrexone (aOR, 0.67; 95% CI, 0.46-0.95; P = .03), gabapentin (aOR, 0.36; 95% CI, 0.30-0.43; P < .001), topiramate (aOR, 0.47; 95% CI, 0.32-0.66; P < .001), and baclofen (aOR, 0.57; 95% CI, 0.36-0.88; P = .01). In addition, pharmacotherapy for AUD was associated with lower incidence of hepatic decompensation in patients with cirrhosis (aOR, 0.35; 95% CI, 0.23-0.53, P < .001), including naltrexone (aOR, 0.27; 95% CI, 0.10-0.64; P = .005) and gabapentin (aOR, 0.36; 95% CI, 0.23-0.56; P < .001). This association persisted even when medical addiction therapy was initiated only after the diagnosis of cirrhosis (aOR, 0.41; 95% CI, 0.23-0.71; P = .002). Conclusions and Relevance: Results of this study showed that receipt of medical addiction therapy for AUD was associated with reduced incidence and progression of ALD. The associations of individual pharmacotherapy with the outcomes of ALD and hepatic decompensation varied widely.

Substance use disorder is associated with alcohol-associated liver disease in patients with alcohol use disorder.

Background and Aims: Substance use disorder (SUD) commonly associates with alcohol use disorder (AUD), and certain substances have independently been shown to drive liver injury. In this work, we sought to determine if co-existing SUD in patients with AUD associated with the presence of alcohol-associated liver disease (ALD). Methods: We performed a cross-sectional analysis using the Mass General Brigham Biobank to identify patients based on ICD-10 codes. We performed multivariate analyses accounting for a wide range of demographic and clinical variables to evaluate the association between SUD and ALD. We subsequently used the same method to evaluate the association between SUD and hepatic decompensation. Results: We identified 2848 patients with a diagnosis of AUD, 9.0% of which had ALD. 25.2% had a history of SUD. In multivariate analyses, patients with SUD were more frequently diagnosed with ALD compared to those without SUD (OR = 1.95, P = 0.001). Furthermore, the number of concurrent SUDs was positively associated with the diagnosis of ALD (OR: 1.33, P < 0.001). Independent of the presence of other SUDs, opioid use disorder in patients with AUD was associated with ALD (OR = 1.902, P = 0.02). In subsequent analyses, we found that sedative use disorder was associated with hepatic decompensation (OR: 2.068, P = 0.03). Conclusions: In patients with AUD, SUD, and in particular opioid use disorder, was independently associated with the diagnosis of ALD.

Hepatic IRF3 fuels dysglycemia in obesity through direct regulation of Ppp2r1b.

Inflammation has profound but poorly understood effects on metabolism, especially in the context of obesity and nonalcoholic fatty liver disease (NAFLD). Here, we report that hepatic interferon regulatory factor 3 (IRF3) is a direct transcriptional regulator of glucose homeostasis through induction of Ppp2r1b, a component of serine/threonine phosphatase PP2A, and subsequent suppression of glucose production. Global ablation of IRF3 in mice on a high-fat diet protected against both steatosis and dysglycemia, whereas hepatocyte-specific loss of IRF3 affects only dysglycemia. Integration of the IRF3-dependent transcriptome and cistrome in mouse hepatocytes identifies Ppp2r1b as a direct IRF3 target responsible for mediating its metabolic actions on glucose homeostasis. IRF3-mediated induction of Ppp2r1b amplified PP2A activity, with subsequent dephosphorylation of AMPKα and AKT. Furthermore, suppression of hepatic Irf3 expression with antisense oligonucleotides reversed obesity-induced insulin resistance and restored glucose homeostasis in obese mice. Obese humans with NAFLD displayed enhanced activation of liver IRF3, with reversion after bariatric surgery. Hepatic PPP2R1B expression correlated with HgbA1C and was elevated in obese humans with impaired fasting glucose. We therefore identify the hepatic IRF3-PPP2R1B axis as a causal link between obesity-induced inflammation and dysglycemia and suggest an approach for limiting the metabolic dysfunction accompanying obesity-associated NAFLD.

An exploratory analysis of the competing effects of alcohol use and advanced hepatic fibrosis on serum HDL.

While alcohol use has been shown to increase serum HDL, advanced liver disease associates with decreased serum HDL. The combined influence of alcohol consumption and liver fibrosis is poorly defined. In this study, we sought to investigate the competing effects of alcohol use and hepatic fibrosis on serum HDL and to determine if the presence of advanced hepatic fibrosis ablates the reported effect of alcohol consumption on serum HDL. We performed a cross-sectional, exploratory analysis examining the interaction between alcohol use and advanced hepatic fibrosis on serum HDL levels in 10,528 patients from the Partners Biobank. Hepatic fibrosis was assessed using the FIB-4 index. We excluded patients with baseline characteristics that affect serum HDL, independent of alcohol use or the presence or advanced hepatic fibrosis. We observed an incremental correlation between increasing HDL levels and amount of alcohol consumed (P < 0.0001), plateauing in those individuals who drink 1-2 drinks per day, Contrastingly, we found a negative association between the presence of advanced hepatic fibrosis and lower HDL levels, independent of alcohol use (beta coefficient: -0.011075, SEM0.003091, P value: 0.0001). Finally, when comparing subjects with advanced hepatic fibrosis who do not use alcohol to those who do, we observed that alcohol use is associated with increased HDL levels (54.58 mg/dL vs 67.26 mg/dL, p = 0.0009). This HDL-elevating effect of alcohol was more pronounced than that seen in patients without evidence of advanced hepatic fibrosis (60.88 mg/dL vs 67.93 mg/dL, p < 0.0001). Our data suggest that the presence of advanced hepatic fibrosis does not blunt the HDL-elevating effect of alcohol use.

Management of Thrombocytopenia and Coagulopathy in Patients with Chronic Liver Disease Undergoing Therapeutic Endoscopic Interventions.

Management of coagulopathy in patients with advanced liver disease undergoing therapeutic endoscopic procedures is complex. Improvements in the understanding of hemostasis at a physiologic level have highlighted the inaccuracy of currently available clinical tests, like platelet count and prothrombin time, in estimating hemostasis in patients with cirrhosis. With identification of novel factors that contribute to bleeding risk in patients with cirrhosis, there is a dearth of clinical trial data that account for all potentially relevant factors and that examine interventions to reduce bleeding risk. Precise recommendations regarding transfusion strategies based on hemostatic test results in patients with cirrhosis are impractical.

Peptide-based urinary monitoring of fibrotic nonalcoholic steatohepatitis by mass-barcoded activity-based sensors.

Noninvasive detection of nonalcoholic steatohepatitis (NASH), the progressive form of nonalcoholic fatty liver disease, promises to improve patient screening, accelerate drug trials, and reduce health care costs. On the basis of protease dysregulation of the biological pathways of fibrotic NASH, we developed the Glympse Bio Test System (GBTS) for multiplexed quantification of liver protease activity. GBTS-NASH comprises a mixture of 19 mass-barcoded PEGylated peptides that is administered intravenously and senses liver protease activity by releasing mass-barcoded reporters into urine for analysis by mass spectrometry. To identify a protease signature of NASH, transcriptomic analysis of 355 human liver biopsies identified a 13-protease panel that discriminated clinically relevant NASH ≥F2 fibrosis from F0-F1 with high classification accuracy across two independent patient datasets. We screened 159 candidate substrates to identify a panel of 19 peptides that exhibited high activity for our 13-protease panel. In the choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) mouse model, binary classifiers trained on urine samples discriminated fibrotic NASH from simple steatosis and healthy controls across a range of nondisease conditions and indicated disease regression upon diet change [area under receiver operating characteristics (AUROCs) > 0.97]. Using a hepatoprotective triple combination treatment (FXR agonist, ACC and ASK1 inhibitors) in a rat model of NASH, urinary classification distinguished F0-F1 from ≥F2 animals and indicated therapeutic response as early as 1 week on treatment (AUROCs >0.91). Our results support GBTS-NASH to diagnose fibrotic NASH via an infusion of peptides, monitor changes in disease severity, and indicate early treatment response.

Serum HMGB1 associates with liver disease and predicts readmission and mortality in patients with alcohol use disorder.

Identifying the minority of patients with alcohol use disorder (AUD) who develop the wide spectrum of alcohol-associated liver disease (ALD), and risk-stratifying these patients, is of critical importance. High-Mobility Group Box 1 protein (HMGB1) is an alarmin that has been implicated in the pathogenesis of multiple liver diseases. Its use as a biomarker for liver disease in those with AUD has not been studied. In this report, we investigated the association between serum HMGB1 and the presence, severity, and progression of ALD in two well-characterized cohorts of patients with AUD. In our discovery cohort of 80 patients, we found that patients with AUD and ALD exhibited higher serum HMGB1 levels compared to patients with AUD only (p = 0.0002). Additionally, serum HMGB1 levels were positively associated with liver disease severity (p < 0.0001). We found that index serum HMGB1 levels were associated with liver disease progression, defined by an increase in MELD score at 120 days (p = 0.0397). Serum HMGB1 was notable for its diagnostic and prognostic ability; it proved able to distinguish accurately between severe and non-severe forms of ALD in both our discovery cohort (AUC = 0.8199, p = 0.0003) and an independent validation cohort of 74 patients with AUD (AUC = 0.8818, p < 0.0001). Moreover, serum HMGB1 levels effectively predicted both liver-related readmission (AUC = 0.8849, p < 0.0001) and transplantation/death (AUC = 0.8614, p = 0.0002) at 90 days. The predictive potential of HMGB1 was also validated in an independent cohort of patients with AUD. Taken together, our results suggest that serum HMGB1 shows promise as a biologically relevant biomarker for ALD in patients with AUD.