Huey Kuan Tan


Background Alcohol-related liver disease (ArLD) contributes to approximately 1% of deaths worldwide and impacts on both economy and healthcare system. Alcohol related hepatitis (AH) - phenomenon of ArLD which is characterised by overactive inflammation is associated with 30% mortality rate at 90 days. Glucocorticoids (GC) are commonly used to treat AH. However, more than 1 in 3 patients fail to respond to GC therapy. Within the GC responders, the efficacy is variable due to individual glucocorticoid resistance. Improving our comprehension of GC non-responsiveness/ resistance is critical for early stratification of these patients and development of alternative therapies. This piece of research aimed to interrogate the role of oxidative stress and compare healthy volunteers (HVs) with AH survivors and non-survivors. These data aimed to assess if oxidative stress measurement can be used as a candidate prognostic biomarker in AH. The effects of oxidative stress on the activities of histone (a protein complex that provides structural support for a chromosome), histone gene expression and phosphoinositide 3-kinase delta (PI3Kδ) signalling were also examined, and followed by a trial of potential therapies to re-sensitize cellular response to GC. Methods Patients admitted to University Hospitals Plymouth NHS Trust who were diagnosed with AH were recruited. Their peripheral bloods were obtained and segregated into T cells and monocytes. Both cell subtypes were defined by their distinctive surface markers CD3+ and CD14+. 2'-7'dichlorofluorescin diacetate (DCFH-DA) staining which fluoresces on oxidation, was used to directly measure level of oxidative stress. Simultaneously, ex vivo exposure to tert-butylhydroperoxide (TBH) to stimulate maximal intracellular oxidative stress acts as positive control. Histone deacetylases (HDAC), histone acetyltransferases (HAT) and antioxidants expression were assessed by qPCR whereas functional activities of class I HDAC, HAT, and PI3Kδ were assessed using ELISA. Results In the flow cytometry experiments, percentage maximal oxidative stress in AH patients was significantly higher than HV in CD14+ monocytes. Among the AH patients, percentage maximal oxidative stress of non-survivors were higher than survivors and close to maximal level. Within the HDAC genes, HDAC 2 and 8 gene expression were significantly upregulated in the survivor group. Further, the functional activities of class 1 HDACs, HATs and PI3Kδ were significantly downregulated in patients with AH in contrast to HVs; similarly in AH non-survivors when compared to survivors. Lastly, GC responsiveness were assessed with BrdU Lymphocyte Incorporation Steroid Sensitivity (BLISS) assay in cell cultures which were treated with Zinc, N-acetylcysteine, sodium butyrate and pan-HDAC inhibitor (Vorinostat). There was no statistically significant change in the in vitro GC responsiveness with these treatments. Conclusion Overall, this thesis presents novel data and highlights that both T cell and monocyte subsets of patients who did not survive AH, have consistently expressed near to maximal levels of oxidative stress. Therefore, oxidative stress can be used as a supplementary prognostic marker for AH. The significantly different expressions of HDAC and HAT genes and the functional activities of class 1 HDACs, HATs and PI3Kδ in AH patients suggest inflammation driven epigenetic modifications in AH. The work presented in this thesis represents a preliminary step forward in understanding the presence and implications of oxidative stress on epigenetic regulation of inflammatory pathways in AH. Further validation with large in-vitro human tissue models and clinical trials are warranted to investigate how these influence immune dysfunction in AH.

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