This proposal focuses on the application of sensitive liquid chromatography-mass spectrometry (LC-MS) platforms for the broad detection and quantification of protein abundances towards the molecular mechanistic study of alcoholic hepatitis (AH). Significant previous work has been performed in the application of proteomic technologies towards the study of liver related diseases, but it has not been applied clinically to investigate AH. Such technologies have the ability to provide an unbiased view of key biofluids, cells, and tissues, where there is limited understanding concerning the mechanistic onset of AH, the molecular signatures of disease progression, and importantly any signatures which can help determine treatment outcomes and assess new treatment regiments. In conjunction with other investigators within the U01 consortium, Battelle, Pacific Northwest National Laboratory (PNNL) proposes to apply both discovery and verification-based proteomic technologies towards the investigation of AH in conjunction with the proposed clinical trial using blood plasma, liver tissue, and isolated circulating blood cells, to determine the differentiating set of expressed and/or secreted proteins for patients undergoing corticosteroid and anti-cytokine treatment. The goals of this study will be to identify and validate key molecular markers which would clarify understanding of the mechanism of AH disease progression and potential treatment effects. Correlation, integration, and functional mapping of the protein-level results with complementary studies performed across the U01 consortium will provide important context to initial findings and interpretations at the systems level. The proposed specific aims are as follows:
Specific Aim 1- Identify proteome differences between alcoholic hepatitis, alcoholic cirrhosis, and normal control populations. We will employ sensitive, quantitative LC-MS analysis of liver tissue and blood plasma to characterize the proteome of each of the three patient and control populations and determine the differential protein signature for subsequent functional mapping, correlation, and verification. These efforts will be primarily linked across transcriptome, histochemistry, and Immunology studies to gain a molecular understanding of AH pathology compared to non-hepatic controls. Additionally, initial proteome characterization of ascites, when available, will be performed to establish a baseline understanding of the protein component of this biofluid, and for subsequent correlation with the proteome of both liver tissue and blood plasma.
Specific Aim 2- Identify proteomic based signatures of response within the prednisolone + mycophenolate and rilonacept treatment clinical trial to examine the longitudinal effect of treatment. High sensitivity LC-MS approaches will be employed to study the proteome response to corticosteroid (prednisolone + mycophenolate) and anti-interleukin-1 (rilonacept) treatment using plasma and isolated blood cell populations. Quantitative information from the identified proteins will be used to statistically determine the profile of proteins that are indicative of disease progression and treatment effect. Correlation of results will be performed across orthogonal measures of patient response to treatment collected in parallel within the U01 consortium which includes transcriptome, Immunology, histology, liver cell injury and clinical responses to treatment. This will facilitate downstream pathway functional mapping providing both a more complete view of treatment response, mechanism of disease, and potential indicators of the patient trajectory.
Specific Aim 3 Verify correlative protein markers in AH based upon U01 integrated data analysis using targeted quantitative proteomics measurements. Sensitive LC-SRM-MS platforms will be used to verify and orthogonally profile proteins of interest previously identified in SA1 and SA2 as well as any potential protein marker candidates based upon orthogonal data integration across the U01 consortium that appears critical for understanding AH pathology, but are supported by only limited quantitative information. Targeted LC-SRM-MS measurements will specifically verify previous findings and in doing so, justify the development of orthogonal detection assays, i.e., ELISA, Westerns, or histological markers.