Serum N-Glycomics Stratifies Bacteremic Patients Infected with Different Pathogens.

Bacteremia-i.e., the presence of pathogens in the blood stream-is associated with long-term morbidity and is a potential precursor condition to life-threatening sepsis. Timely detection of bacteremia is therefore critical to reduce patient mortality, but existing methods lack precision, speed, and sensitivity to effectively stratify bacteremic patients. Herein, we tested the potential of quantitative serum N-glycomics performed using porous graphitized carbon liquid chromatography tandem mass spectrometry to stratify bacteremic patients infected with Escherichia coli (n = 11), Staphylococcus aureus (n = 11), Pseudomonas aeruginosa (n = 5), and Streptococcus viridans (n = 5) from healthy donors (n = 39). In total, 62 N-glycan isomers spanning 41 glycan compositions primarily comprising complex-type core fucosylated, bisecting N-acetylglucosamine (GlcNAc), and α2,3-/α2,6-sialylated structures were profiled across all samples using label-free quantitation. Excitingly, unsupervised hierarchical clustering and principal component analysis of the serum N-glycome data accurately separated the patient groups. P. aeruginosa-infected patients displayed prominent N-glycome aberrations involving elevated levels of fucosylation and bisecting GlcNAcylation and reduced sialylation relative to other bacteremic patients. Notably, receiver operating characteristic analyses demonstrated that a single N-glycan isomer could effectively stratify each of the four bacteremic patient groups from the healthy donors (area under the curve 0.93-1.00). Thus, the serum N-glycome represents a new hitherto unexplored class of potential diagnostic markers for bloodstream infections.

Pyrexia and acidosis act independently of neutrophil elastase reactive center loop cleavage to effect cortisol release from corticosteroid-binding globulin.

Corticosteroid-binding globulin (CBG) transports cortisol and other steroids. High-affinity CBG (haCBG) undergoes proteolysis of the reactive center loop (RCL) by neutrophil elastase (NE) altering conformation to low-affinity CBG (laCBG). Elevated temperature reduces CBG:cortisol binding affinity. Surface plasmon resonance was used to determine binding profiles of 19 steroids to haCBG and laCBG at 25, 37, and 39°C mimicking pyrexia and pH 7.4 and 7.0 mimicking acidosis, pathophysiological conditions relevant to sepsis. An expected 4-8-fold reduction in affinity for cortisol, cortisone, corticosterone, 11-deoxycortisol, progesterone, 17-hydroxyprogesterone, and prednisolone occurred with NE-mediated haCBG-to-laCBG conversion. CBG:cortisol binding affinity was further reduced 3.5-fold at 39°C relative to 37°C, binding affinity was also reduced by acidosis for both haCBG and laCBG. Using a conformational antibody generated against the RCL, we confirmed RCL antibody binding was eliminated by NE cleavage, but preserved in pyrexia and acidosis. Molecular modeling studies performed at 40°C confirmed a critical role for Trp371, positioned within the steroid-binding pocket, in ligand binding. These studies demonstrated CBG binding affinity to range of steroids is ligand specific and is reduced with NE-mediated haCBG-to-laCBG transition. Reduced CBG:cortisol binding occurs with increased temperature and in acidosis. Increased flexibility of the Trp371 side chain is proposed in the thermo-coupling mechanism of cortisol release. The synergy of NE cleavage, pyrexia, and acidosis on CBG:cortisol binding may serve to enhance cortisol delivery to the interstitial space in inflammation.

High binding site occupancy of corticosteroid-binding globulin by progesterone increases fetal free cortisol concentrations.

OBJECTIVE: Corticosteroid-binding globulin (CBG) binds and transports cortisol in the circulation in high cortisol-binding affinity (haCBG) and low affinity (laCBG) forms, the latter resulting from enzyme cleavage to target cortisol delivery at sites of inflammation. CBG also has substantial progesterone binding affinity, 3-fold less than cortisol. Progesterone and cortisol are important in the maintenance of pregnancy and in fetal development, respectively. The interactions of cortisol, progesterone and CBG affinity forms have not been studied together. We examined the interaction between progesterone and cortisol with CBG during fetal development. STUDY DESIGN: A retrospective cohort analysis of 351 neonates born between January and December 2012 at the Women's and Children's Hospital, Adelaide, South Australia. Cord blood serum samples were collected immediately following delivery. Clinical data was provided by hospital records. Total cortisol, free cortisol, total progesterone, total CBG and haCBG were measured by immunoassay. RESULTS: Cord blood total and free cortisol, and progesterone concentrations increased with gestational age. Cord blood progesterone concentrations were 100-fold luteal and 10-fold those in late pregnancy maternal circulation. The proportion of haCBG to total CBG was similar to that in healthy non-pregnant adults. However, free cortisol comprised approximately 15% of total cortisol, 3-fold higher than that in adults. CONCLUSION: In a manner unique to fetal life, very high progesterone concentrations are capable of elevating free cortisol concentrations through competition with cortisol at CBG's hormone binding site, without altered binding affinity through CBG cleavage or altered CBG hormone-binding affinity. High circulating fetal progesterone concentrations compete for CBG binding with cortisol, leading to a 3-fold increase in the free cortisol fraction in cord blood. Higher free-to-bound cortisol may alter fetal cortisol distribution facilitating cortisol's roles such as neurodevelopment in concert with dehydroepiandrosterone (sulfate) and lung maturation, or support cortisol action at times of low ambient cortisol. This mechanism may underlie the known association between cortisol, progesterone and CBG, and be relevant principally in the fetal circulation due to the high progesterone concentrations encountered.

Total and high-affinity corticosteroid-binding globulin depletion in septic shock is associated with mortality.

CONTEXT: Corticosteroid-binding globulin (CBG) and albumin transport circulating cortisol. Cleavage of high-affinity CBG (haCBG) by neutrophil elastase at inflammatory sites causes cortisol release into tissues, facilitating immunomodulatory effects. OBJECTIVE: To determine whether depletion of haCBG is related to mortality in septic shock. DESIGN: A single-center prospective observational cohort study of patients recruited with critical illness or septic shock, using serum samples collected at 0, 8, 24, 48 and 72 hours. Serum total and haCBG, and total and free cortisol were assayed directly. Glucocorticoid treatment was an exclusion criterion. Mortality was assessed at 28 days from Intensive Care Unit admission. RESULTS: Thirty septic shock (SS) and 42 nonseptic critical illness (CI) patients provided 195 serum samples. SS/CI patients had lower total CBG, haCBG and low-affinity CBG (laCBG) than controls. Total CBG and haCBG were significantly lower in septic shock patients who died than in those that survived (P < 0.009, P = 0.021, respectively). Total and free cortisol were higher in septic than nonseptic individuals. Free/total cortisol fractions were higher in those with low haCBG as observed in septic shock. However, cortisol levels were not associated with mortality. Albumin levels fell in sepsis but were not related to mortality. CONCLUSIONS: Low circulating haCBG concentrations are associated with mortality in septic shock. These results are consistent with an important physiological role for haCBG in cortisol tissue delivery in septic shock.

Differential Effects of Estrogen on Corticosteroid-Binding Globulin Forms Suggests Reduced Cleavage in Pregnancy.

Corticosteroid-binding globulin (CBG) is secreted as high-affinity CBG (haCBG), which may be cleaved by tissue proteases to low-affinity CBG (laCBG), releasing free cortisol. Pregnancy and the estrogen-based combined oral contraceptive pill (COCP) increase CBG concentrations twofold to threefold. The relative effects of these two hyperestrogenic states on the CBG affinity forms are unknown. We performed an observational study in 30 pregnant women, 27 COCP takers and 23 controls. We analyzed circulating total CBG, haCBG, laCBG, and free and total cortisol concentrations. In pregnancy, total CBG and haCBG were increased compared to controls (both P < 0.0001); however, laCBG concentrations were similar. In COCP takers, total CBG and haCBG were increased [802 ± 41 vs compared to controls (both P < 0.0001)], but laCBG was also increased (P = 0.03). Pregnancy and use of COCP were associated with a comparable rise in haCBG, but laCBG was lower in pregnancy (P < 0.0001). These results were consistent with an estrogen-mediated increase in CBG synthesis in both hyperestrogenemic states but with reduced CBG cleavage in pregnancy relative to the COCP, perhaps due to pregnancy-induced CBG glycosylation. Speculatively, increased circulating haCBG concentrations in pregnancy may provide an increased reservoir of CBG-bound cortisol to prepare for the risk of puerperal infection or allow for cortisol binding in the face of competition from increased circulating progesterone concentrations.

Corticosteroid-binding globulin: acute and chronic inflammation.

INTRODUCTION: Corticosteroid-binding globulin (CBG) is the principal transport protein for cortisol binding 80% in a 1:1 ratio. Since its discovery in 1958, CBG's primary function has been considered to be cortisol transport within the circulation. More recent data indicate a cortisol tissue delivery function, particularly at inflammatory sites. CBG's structure as a non-inhibitory serine protease inhibitor allows allosteric structural change after reactive central loop (RCL) cleavage by neutrophil elastase (NE) and RCL insertion into CBG's protein core. Transition from the high to low affinity CBG form reduces cortisol-binding. Areas covered: In acute systemic inflammation, high affinity CBG (haCBG) is depleted proportionate to sepsis severity, with lowest levels seen in non-survivors. Conversely, in chronic inflammation, CBG cleavage is paradoxically reduced in proportion to disease severity, implying impaired targeted delivery of cortisol. CBG's structure allows thermosensitive release of bound cortisol, by reversible partial insertion of the RCL and loosening of CBG:cortisol binding. Recent studies indicate a significant frequency of function-altering single nucleotide polymorphisms of the SERPINA6 gene which may be important in population risk of inflammatory disease. Expert commentary: Further exploration of CBG in inflammatory disease may offer new avenues for treatment based on the model of optimal cortisol tissue delivery.

Corticosteroid-binding globulin cleavage may be pathogen-dependent in bloodstream infection.

OBJECTIVE: The process of enzymatic cleavage of high- to low-affinity corticosteroid-binding globulin (haCBG to laCBG) by neutrophil elastase leads to local tissue release of cortisol. Recently Pseudomonas aeruginosa was shown to instigate CBG cleavage with release of free cortisol in vitro. Hence, CBG cleavage with release of anti-inflammatory cortisol in infection may be pathogen-dependent. Our objective was to determine whether haCBG and laCBG levels are altered in infected patients compared with controls, and whether these alterations were particular to causative bacteria. DESIGN: An observational, cross-sectional study at a public pathology institution and tertiary hospital in Adelaide, South Australia. METHODS: 100 positive blood culture samples and 100 healthy control samples were analysed for serum total CBG, haCBG, laCBG, total and free cortisol, leukocyte and neutrophil count, C-reactive protein and Pitt severity score. RESULTS: Patients with infection had lower serum total CBG, haCBG and laCBG, all P<0.0001, than healthy controls. This was true in patients with and without a systemic inflammatory response and in those with culture-positive and culture-negative infections. Pseudomonas aeruginosa infection was associated with the lowest total and laCBG levels of the pathogen groups despite having the lowest inflammatory markers. CONCLUSIONS: There was evidence of CBG cleavage in early infection both in patients with and without systemic inflammation and regardless of culture status. Pseudomonas infection appeared to enhance cleavage. This observation, along with cleavage in severe neutropenia suggests mechanisms other than neutrophil elastase may be involved in CBG cleavage and local tissue cortisol release in infection.

Reduced corticosteroid-binding globulin cleavage in active rheumatoid arthritis.

OBJECTIVE: Corticosteroid-binding globulin (CBG), the cortisol transport protein, is cleaved from high-affinity (haCBG) to low-affinity (laCBG) CBG at sites of inflammation releasing bioavailable, anti-inflammatory cortisol. Rheumatoid arthritis (RA) is a glucocorticoid-responsive disorder, with paradoxically normal cortisol levels despite elevated inflammatory mediators. Our objective was to determine whether CBG cleavage relates to RA disease activity. We hypothesized that impaired CBG cleavage may limit delivery of free cortisol to inflamed joints in RA. DESIGN: Prospective, cross-sectional observational study. SETTING AND PARTICIPANTS: Fifty-three patients with RA recruited from a Rheumatology outpatient clinic at a tertiary referral centre in Adelaide, Australia, and 73 healthy controls. MEASUREMENTS: Total CBG, haCBG and laCBG, total, free and salivary cortisol, inflammatory markers including interleukin-6 soluble receptor (IL-6sR) and macrophage migration inhibitory factor and clinical measures of disease activity. RESULTS: Among patients with RA, a wide range of disease activity scores was observed (DAS28: range 1·2-6·4). laCBG was lower in patients with RA (mean ± SEM); 153 ± 9, compared with healthy controls; 191 ± 8 nmol/l, P = 0·003. Levels of total and haCBG were higher in patients with more severe RA disease activity. Free and total cortisol, free cortisol:IL-6sR ratio and total cortisol:IL-6sR ratio correlated negatively with disease activity. CONCLUSIONS: These results suggest that patients with RA have reduced CBG cleavage compared to healthy controls and that cleavage is reduced further with higher RA disease activity. Hence, impaired CBG-mediated delivery of endogenous cortisol may perpetuate chronic inflammation in RA.

Corticosteroid-binding globulin cleavage is paradoxically reduced in alpha-1 antitrypsin deficiency: Implications for cortisol homeostasis.

High-affinity corticosteroid-binding globulin (haCBG) is cleaved by neutrophil elastase (NE) resulting in permanent transition to the low cortisol-binding affinity form (laCBG), thereby increasing cortisol availability at inflammatory sites. Alpha-1 antitrypsin (AAT) is the major inhibitor of NE. AAT deficiency (AATD) predisposes patients to early-onset emphysema due to increased proteolytic destruction from the inherent proteinase-antiproteinase imbalance. We hypothesized that AATD may result in increased CBG cleavage in vivo. We collected demographic data and blood samples from 10 patients with AATD and 28 healthy controls measuring total CBG and haCBG levels by parallel in-house ELISAs, as well as AAT, total and free cortisol levels. haCBG was higher (median [range]); 329 [210-551] vs. 250 [175-365] nmol/L; P<0.005, and laCBG lower; 174 [68-229] vs. 220 [119-348] nmol/L; P=0.016 in the AATD group, compared with controls. The ratio of haCBG:total CBG was also higher in AATD; 72 [53-83] vs. 54 [41-72] %; P=0.0001). There was a negative correlation between haCBG:total CBG and AAT levels (P<0.05, R=-0.64). Paradoxically, proteolytic cleavage of CBG was reduced in AATD, despite the recognized increase in NE activity. This implies that NE activity is not the mechanism for systemic CBG cleavage in basal, low inflammatory conditions. Relatively low levels of laCBG may have implications for cortisol action in AATD.

Low-dose hydrocortisone replacement improves wellbeing and pain tolerance in chronic pain patients with opioid-induced hypocortisolemic responses. A pilot randomized, placebo-controlled trial.

UNLABELLED: Long-term opioid therapy has been associated with low cortisol levels due to central suppression of the hypothalamic-pituitary-adrenal axis. The implications of hypocortisolism on wellbeing have not been established. Our aim was to determine whether intervention with physiologic glucocorticoid replacement therapy improves wellbeing and analgesic responses in patients with chronic non-cancer pain on long-term opioid therapy with mild cortisol deficiency. We performed a pilot randomized, double-blind, placebo-controlled crossover study of oral hydrocortisone replacement therapy in 17 patients recruited from a Pain Clinic at a single tertiary center in Adelaide, Australia. Patients were receiving long-term opioid therapy (≥ 20 mg morphine equivalents per day for ≥ 4 weeks) for chronic non-cancer pain with mild hypocortisolism, as defined by a plasma cortisol response ≤ 350 nmol/L at 60 min following a cold pressor test. The crossover intervention included 28-day treatment with either 10mg/m(2)/day of oral hydrocortisone in three divided doses or placebo. Improvement in wellbeing was assessed using Version 2 of the Short Form-36 (SF-36v2), Brief Pain Inventory-Short Form, and Addison's disease quality of life questionnaires; improvement in analgesic response was assessed using cold pressor threshold and tolerance times. Following treatment with hydrocortisone, the bodily pain (P=0.042) and vitality (P=0.013) subscales of the SF-36v2 were significantly better than scores following treatment with placebo. There was also an improvement in pain interference on general activity (P=0.035), mood (P=0.03) and work (P=0.04) following hydrocortisone compared with placebo. This is the first randomized, double-blind placebo-controlled trial of glucocorticoid replacement in opioid users with chronic non-cancer pain and mild hypocortisolism. Our data suggest that physiologic hydrocortisone replacement produces improvements in vitality and pain experiences in this cohort compared with placebo. TRIAL REGISTRATION: Therapeutic Goods Administration Clinical Trials Notification Scheme (Drugs), Trial Number 2012/0476.

Psychosis and cardiovascular disease: is diet the missing link?

OBJECTIVE: To explore the diets of people living with psychotic disorders, and to compare their dietary composition to the general population. METHOD: 184 people with psychotic disorders in Adelaide, South Australia completed a food frequency questionnaire. Physical information and mental health status were collected. Outcome measures included energy and macronutrient intake; fish, sodium, fruit and vegetable intake; micro-nutrient intake; body mass index; waist circumference; and diagnoses of diabetes and hypertension. The RDI of nutrients was derived from Australian Government publications. Comparison dietary data was obtained from surveys carried out by the Australian Bureau of Statistics. RESULTS: The majority of participants were overweight or obese (78%) and 77.5% met the criteria for at-risk waist circumference; and 58% of participants consumed salt and saturated fat in excess of the RDI. Most did not achieve the RDI for fruits and vegetables (97.8%), fibre (88.6%), fish (61.4%), magnesium (73.4%) or folate (86.4%). Women with psychosis had significantly higher intakes of vitamins and minerals compared to women in the general population. Men and women with psychosis consumed more daily total fat, saturated fat and sodium compared to adults in the Australian population, but lower fibre and vitamin E than their male and female counterparts. CONCLUSION: People with psychosis, especially women, report poor dietary choices including increased energy and fat intake, heightening their risk for cardiovascular disease. Women with psychosis report higher intake of vitamins and minerals than women in the general population. Whilst dietary intake contributes to obesity in psychosis, other factors including antipsychotic agents, decreased physical activity and smoking add to the cardiovascular risk.

Continuous subcutaneous hydrocortisone infusion therapy in Addison's disease: a randomized, placebo-controlled clinical trial.

CONTEXT: Patients with Addison's disease (AD) report impaired subjective health status (SHS). Since cortisol exhibits a robust circadian cycle that entrains other biological clocks, impaired SHS may be due to the noncircadian cortisol profile achieved with conventional glucocorticoid replacement. Continuous subcutaneous hydrocortisone infusion (CSHI) reproduces a circadian cortisol profile, but its effects on SHS have not been objectively evaluated. OBJECTIVE: The aim of this study was to determine the effect of CSHI on SHS in AD. SETTING AND DESIGN: This was a multicentre, double-blind, placebo-controlled trial of CSHI vs oral glucocorticoid therapy. Participants received in random order 4 weeks of: CSHI and oral placebo, and subcutaneous placebo and oral hydrocortisone, separated by a 2-week washout period. SHS was assessed using the Short-Form 36 (SF-36), General Health Questionnaire (GHQ-28), Fatigue Scale (FS), Gastrointestinal Symptom Rating Scale (GSRS); and Addison's Quality of Life Questionnaire (AddiQoL). Participants were asked their (blinded) treatment preference. Twenty-four hour urine free cortisol (UFC) and diurnal salivary cortisol collections compared cortisol exposure during each treatment. RESULTS: Ten participants completed the study. Baseline SHS scores (mean ± SE) were consistent with mild impairment: SF-36 physical component summary 48.4 (± 2.4), mental component summary 53.3 (± 3.0); GHQ-28 18.1 (± 3.3); GSRS 3.7 (± 1.6), and AddiQoL 94.7 (± 3.7). FS was similar to other AD cohorts 13.5 (± 1.0) (P = 0.82). UFC between treatments was not different (P = 0.87). The salivary cortisol at 0800 h was higher during CSHI (P = 0.03), but not at any other time points measured. There was no difference between the treatments in the SHS assessments. Five participants preferred CSHI, four oral hydrocortisone, and one was uncertain. CONCLUSIONS: Biochemical measurements indicate similar cortisol exposure during each treatment period, although a more circadian pattern was evident during CSHI. CSHI does not improve SHS in AD with good baseline SHS. This casts some doubt on the potential benefit of circadian cortisol delivery on SHS in AD.