Torpy, David JamesRankin, WayneMeyer, Emily Jane2022-08-042022-08-042022https://hdl.handle.net/2440/135911Septic shock is a life-threatening organ dysfunction due to a dysregulated host response to pathogenic infection and is clinically defined by persistent hypotension requiring vasopressors to maintain a mean arterial pressure > 65 mmHg despite fluid resuscitation. Septic shock is common and has a mortality of 25–40%. Despite large randomised controlled trials, the benefits of hydrocortisone administration to patients with septic shock remain uncertain. Corticosteroid-binding globulin (CBG) is the principal transport protein for cortisol in the circulation. Neutrophil elastase (NE) cleavage of the reactive centre loop (RCL), irreversibly converts high affinity (ha) CBG to a low cortisol-binding affinity (la) state, providing the targeted release of immunomodulatory cortisol at inflammatory sites. CBG is consumed and production in septic shock is reduced. Serum CBG concentrations fall substantially in proportion to sepsis severity and in survivors levels normalise within days. An acquired CBG deficiency in septic shock may reduce the capacity of cortisol to prevent clinical deterioration thus predicting organ failure and mortality. CBG transports cortisol and other steroids. Development of a novel homogenous ligand bind assay using surface plasmon resonance (SPR) technology allowed for the determination of binding affinities of CBG to cortisol and 18 other steroid ligands at temperatures and acidic conditions that mimic the pathophysiological conditions of septic shock. The most potent binding was recorded for haCBG binding to cortisol (KD 42 nM) and corticosterone (KD 45 nM). An expected 4–8-fold reduction in affinity for cortisol, cortisone, corticosterone, 11- deoxycortisol, progesterone, 17-hydroxyprogesterone and prednisolone occurred with NEmediated haCBG-to-laCBG conversion. CBG:cortisol binding affinity was further reduced 3.5- fold at 39 °C relative to 37 °C; binding affinity was further reduced by acidosis for both haCBG and laCBG. Using a conformational antibody (9G12) generated against the RCL, confirmed RCL antibody binding was eliminated by NE cleavage, but preserved in pyrexia and acidosis. In silico molecular dynamic simulation studies performed at 40 °C identified the thermocoupling mechanism of cortisol release is mediated through the destabilisation of Trp371, a tryptophan residue located within the cortisol-binding pocket, suggesting an additional non-RCL mechanism for the delivery of anti-inflammatory cortisol in pyrexia. CBG has a cortisol tissue delivery role in inflammation, with pyrexia and acidosis reversibly reducing cortisol binding affinity. Taken together the synergy of NE cleavage, pyrexia and acidosis on CBG:cortisol binding may serve to enhance cortisol delivery to the interstitial space in inflammation. Novel measurement of total CBG and haCBG by enzyme-linked immunosorbent assay is at the forefront of CBG research and this thesis is predicated on this methodology. I hypothesised that severe deficiency in CBG (<200 nmol/L [Reference interval 269–641 nmol/L]) may independently predict mortality and is associated with key disease severity markers in patients with septic shock. An initial study of 30 patients with septic shock showed an association between CBG and mortality. Total CBG and haCBG were significantly lower in septic shock patients who died than in those who survived (P < 0.009, P = 0.021, respectively). Total and free cortisol were higher in septic than non-septic individuals, however cortisol levels were not associated with mortality. Albumin levels fell in sepsis but were not related to mortality. Approximately onethird of septic shock patients have admission CBG concentrations below 200 nmol/L. A subsequent large single centre prospective observational study of 135 patients with septic shock revealed the novel finding that CBG deficiency (<200 nmol/L) on Intensive Care Unit (ICU) admission independently predicted ICU mortality (3.2-fold risk ratio) and was associated with the need for ventilator and vasopressor support. Mortality rates in ICU were higher in the CBG <200 nmol/L vs the CBG ≥200 nmol/L group: 32.4% vs 13.9%; Odds ratio (OR), 2.97, (95% confidence interval (CI) 1.19,7.41); P=0.02: 28-days; OR 2.25 (95% CI 0.99,5.11): 90-days; OR 2.21 (95% CI 0.99,4.91). Multivariate analysis revealed 4 factors independently associated with ICU mortality: CBG <200 nmol/L (OR 3.23, 95% CI 1.06,9.88), Acute Physiology and Chronic Health Evaluation II >25 (OR 3.58, 95% CI 1.20,10.68); SOFA liver score (OR 1.98, 95% CI 1.04,3.72); and renal-replacement therapy (OR 6.59, 95% CI 2.17,20.01). CBG concentration was the only directly reversible independent mortality risk factor. Nadir CBG levels were associated with higher SOFA cardiovascular scores, norepinephrine total dose (μg) P<0.01 and duration (days) P<0.01. Plasma cortisol concentrations, serum albumin concentrations and hydrocortisone administration did not relate to ICU mortality. Septic shock is a public health emergency and represents 20% of all global deaths. Standard treatments including fluids, vasopressors and antimicrobials often fail, however despite this there has been no new therapeutic advances in over 20 years. Using SPR technology were able to demonstrate the importance of temperature and pH in CBG:cortisol binding affinity, supporting its role in the delivery of cortisol in states of pyrexia and acidaemia. Our clinical studies in septic shock demonstrate a strong relationship between CBG deficiency and disease severity, cardiac dysfunction and increased mortality. This accords with CBG’s role in targeted delivery of immunomodulatory cortisol to tissues to quell systemic inflammatory processes. Given the 3.2-fold increase in mortality, replenishing CBG may have a large therapeutic effect in those with CBG <200 nmol/L in the treatment of septic shock, avoiding the multiorgan failure cascade that accompanies severe illness and death. Human clinical trials investigating CBG supplementation in septic shock may be of practical benefit and warrants further investigation.enCorticosteroid-binding globulinCBGCortisolSeptic shockSepsisThesis