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Title: The role of extracellular histones in critical illness and their application in acute pancreatitis
Author: Liu, T.
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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Histones are basic nuclear proteins that bind to genomic DNA. There are five subclasses: core histones H2A, H2B, H3 and H4, and linker histone H1. Release of extracellular histones has been shown in a range of critical illness animal models and human patients including sepsis and acute pancreatitis. Extracellular histones act as damage-associated molecular pattern molecules (DAMPs) on parenchymal epithelial and endothelial cells, cardiomyocytes, immune cells (neutrophils, monocytes, macrophages, dendritic cells and lymphocytes) and platelets to activate Toll-like receptors (TLRs; TLR2 and TLR4) and/or NLR Family Pyrin Domain Containing 3 (NLPR3) inflammasome as well as induce calcium influx, proinflammatory cytokine production, thrombin generation and cell death. Anti-histone strategies such as anti-histone antibodies or pharmacological neutralisers alleviate histone-induced toxicity. To progress the translational relevance of histones in circulation, this thesis explores the development of xMAP technology based assay for measuring relevant toxic histones and also the significance of histone determination in animal models and patients with acute pancreatitis. Truncated histones were generated by recombinant DNA technology and the purity was 75.6-95.1%, except for histone H2A C-terminal (35.5%). Anti-histone single chain variable fragment (ahscFv) was also generated (purity > 95%) and shown to bind to histone subclasses by Western blot and IAsys resonant biosensor. Fluorescein isothiocyanate (FITC)-full length and FITC-truncated histones (H1.1 C-, H2A N-, H3.1 N-terminal) bound to the cell membrane and induced calcium influx in EA.hy926 cells, while other truncated histones did not. ahscFv significantly prevented histone-induced cell death. To measure all toxic histones in one assay, Luminex xMAP multiplex technology was developed. Standard curves of both singleplex and multiplex assays were developed and used to measure the levels of circulating histones in plasma of patients with severe trauma, sepsis and pancreatitis. However, the recovery ratio in spiked plasma was low and the assay failed to detect histones in patient plasma which were detectable by Western blot. Release of circulating histones were investigated in mice with acute pancreatitis induced by either 4 or 12 injection of caerulein (50 µg/kg/h; CER-AP) or by infusion of pancreatic duct with sodium taurocholate (3.5%, 1 ml/kg; NaTC-AP). Four and 12 injections of caerulein resulted in oedematous and necrotising CER-AP, respectively, with marked systemic inflammation and multiple organ injury observed in the necrotising CER-AP. NaTC-AP caused more diffuse pancreatic necrosis, systemic inflammation and multiple organ injury. Circulating histones, as measured by Western blots, were elevated early with further increases in necrotising CER-AP (peak > 100 µg/ml) and NaTC-AP (peak > 140 µg/ml) as disease progressed but not in oedematous CER-AP which were comparable to saline controls. The levels of circulating histones were significantly associated with pancreatic necrosis and multiple organ injury parameters. Circulating histones were then measured from healthy volunteers and a consecutive cohort of acute pancreatitis patients admitted to Royal Liverpool University Hospital (RLUH) within 48 h of disease onset. The predictive values of circulating histones for persistent organ failure (POF), major infection and mortality were compared with biochemical markers and clinical scores. A total of 236 patients (mild 156, moderate 57, severe 23 as per Revised Atlanta Classification) and 47 heathy volunteers were included. The median histone level in severe acute pancreatitis was 18.8 µg/ml (interquartile range: 5.9-33.8), significantly higher than mild 1.1 µg/ml (0.6-2.1) or moderate 1.3 µg/ml (0.5-2.8) category which was comparable with heathy volunteers 1.0 µg/ml (0.5-1.6). The area under the receiver-operating characteristic (AUC) curve of histones for predicting POF and mortality was 0.92 (95% confidence interval [CI]: 0.85-0.99) and 0.96 (0.92-1.00) respectively, which was as or more accurate than tested biomarkers or clinical scores. For infected pancreatic necrosis and/or sepsis, the AUC of histones was 0.78 (0.62-0.94). Histones did not predict or correlate with local pancreatic complications and transient organ failure, but negatively correlated with leucocyte cell viability (r = -0.511, P < 0.01). A study of consecutive acute pancreatitis patients with primary admission to RLUH (n = 260, blood sampling < 24 h) or referred (n = 52) from other hospitals and healthy controls (n = 47) were recruited. Referred patients had POF > 48 h (blood sampling < 24 h of admission to ICU of the RLUH then daily for one week) within 3 weeks of disease onset. Histones, cytokines and routine biochemical markers were measured. Multivariable analyses determined associations between circulating histone levels and variables. There were 235 patients in Group 1 (no POF), 25 in Group 2 (POF < 24 h) and 52 in Group 3 (POF > 48 h). Circulating histones were significantly correlated with tested proinflammatory cytokines, clinical severity scores and individual organ injury parameters. Circulating histones were significantly more elevated in Group 3 compared to Group 2 but both were higher than in Group 1 or healthy volunteers. Multivariable analyses revealed that it was POF, but not pancreatic necrosis or other variables that most significantly associated with elevated circulating histones (odds ratio: 98.1, 95% CI: 14.4-669.0, P < 0.001).
Supervisor: Toh, C. H. ; Wang, G. ; Welters, I. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral