The long-term objective of this translational U01 is to define pathophysiologically informed and clinically relevant biomarkers based on the sphingolipidomic cargo of extracellular vesicles (EVs) that will serve for the diagnosis and prognosis of subjects with alcoholic hepatitis (AH). AH is the severest form of alcohol-induced liver disease with high mortality, high health care utilization, and the absence of effective therapies. Health care professionals are limited by the lack of predictive and prognostic biomarkers that could risk-stratify AH patients and individualize their therapy based on biomarker profiles. The current proposal links hepatocyte-derived sphinogolipid cargo on EVs to macrophage-mediated liver inflammation by proposing that sphingolipids on EVs from ethanol-damaged hepatocytes recruit macrophages in to the liver, resulting in liver injury and inflammation. Our preliminary data show that EVs are elevated in AH with the following features: i) EV sphingolipids are elevated in AH; ii) EV sphingolipid levels correlate with clinical parameters such as the international normalized ratio, bilirubin, and MELD score; iii) EVs activate proinflammatory macrophage effector responses via sphingolipid signaling; and iv) Hepatocyte-derived EVs can be detected via a novel nanoplasmon enhanced scattering (nPES) assay. This has led to the central hypothesis that circulating EV sphingolipid cargo is a pathogenically relevant biomarker for the diagnosis and prognosis of AH. Therefore, the goals of this proposal are to: i) develop an EV sphingolipidomic diagnostic signature for AH; ii) Demonstrate the ability of EV sphingolipidomics to predict mortality in AH; iii) Extend EV sphingolipidomics to a ?proof-of-mechanism? assay for the beneficial effects observed in AH patients treated with therapeutic agents proposed in our linked complementary Clinical Trial Pilot U01 (RFA-AA-18-005); and iv) Develop a novel clinical test for the diagnosis of AH utilizing nPES technology to diagnose AH by detecting hepatocyte-derived EVs in plasma microsamples. First, we will determine the diagnostic performance of EV sphingolipids for AH in a single-site training cohort followed by a multi-site validation cohort. Second, we will directly test the hypothesis that EV sphingolipids can predict survival and response to a therapy that is expected to reduce the release of EVs from hepatocytes. Third, we will develop and validate a novel clinical assay for the diagnosis of AH. Thus, this multi-PI U01 grant application will yield a liquid biopsy biomarker for AH diagnosis and prognosis.

Public Health Relevance

Alcoholic hepatitis is a severe illness with high mortality and no effective therapies due in part to the lack of tests that permit early diagnosis. We propose to develop a novel blood test based on the detection of liver- derived extracellular vesicles for the early and rapid diagnosis of alcoholic hepatitis, and we will also test whether these extracellular vesicles can predict which patients are most likely to die from this condition. Therefore, with the proposed studies we hope to identify, characterize, and validate that extracellular vesicles can serve as a clinically relevant biomarker for alcoholic hepatitis which can risk-stratify patients and uncover potentially targetable pathways for treatment.

National Institute of Health (NIH)
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Research Project--Cooperative Agreements (U01)
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Special Emphasis Panel (ZAA1)
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Orosz, Andras
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Mayo Clinic, Rochester
United States
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Greuter, Thomas; Shah, Vijay H (2018) Too Stiff, Too Late . . . Timing Is Everything in Antiangiogenic Treatment of Liver Fibrosis. Hepatology :
Samala, Niharika; Lourens, Spencer G; Shah, Vijay H et al. (2018) Posttraumatic Stress Disorder in Patients with Heavy Alcohol Consumption and Alcoholic Hepatitis. Alcohol Clin Exp Res 42:1933-1938
Liangpunsakul, Suthat; Beaudoin, James J; Shah, Vijay H et al. (2018) Interaction between the patatin-like phospholipase domain-containing protein 3 genotype and coffee drinking and the risk for acute alcoholic hepatitis. Hepatol Commun 2:29-34
Peeraphatdit, Thoetchai Bee; Simonetto, Douglas A; Shah, Vijay H (2018) Exploring new treatment paradigms for alcoholic hepatitis by extrapolating from NASH and cholestasis. J Hepatol 69:275-277
Singal, Ashwani K; Louvet, Alexandre; Shah, Vijay H et al. (2018) Grand Rounds: Alcoholic Hepatitis. J Hepatol 69:534-543
Chirapongsathorn, Sakkarin; Krittanawong, Chayakrit; Enders, Felicity T et al. (2018) Incidence and cost analysis of hospital admission and 30-day readmission among patients with cirrhosis. Hepatol Commun 2:188-198
Puri, Puneet; Liangpunsakul, Suthat; Christensen, Jeffrey E et al. (2018) The circulating microbiome signature and inferred functional metagenomics in alcoholic hepatitis. Hepatology 67:1284-1302
Wang, Ruisi; Ding, Qian; De Assuncao, Thiago M et al. (2017) Hepatic Stellate Cell Selective Disruption of Dynamin-2 GTPase Increases Murine Fibrogenesis through Up-Regulation of Sphingosine-1 Phosphate-Induced Cell Migration. Am J Pathol 187:134-145
Beaudoin, James J; Long, Nanye; Liangpunsakul, Suthat et al. (2017) An exploratory genome-wide analysis of genetic risk for alcoholic hepatitis. Scand J Gastroenterol 52:1263-1269
Simonetto, D A; Shah, V H; Kamath, P S (2017) Improving survival in ACLF: growing evidence for use of G-CSF. Hepatol Int 11:473-475

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