Peptide microarray-based analysis of antibody responses to SARS-CoV-2 identifies unique epitopes with potential for diagnostic test development.

Humoral immunity to the Severe Adult Respiratory Syndrome (SARS) Coronavirus (CoV)-2 is not fully understood yet but is a crucial factor of immune protection. The possibility of antibody cross-reactivity between SARS-CoV-2 and other human coronaviruses (HCoVs) would have important implications for immune protection but also for the development of specific diagnostic ELISA tests. Using peptide microarrays, n = 24 patient samples and n = 12 control samples were screened for antibodies against the entire SARS-CoV-2 proteome as well as the Spike (S), Nucleocapsid (N), VME1 (V), R1ab, and Protein 3a (AP3A) of the HCoV strains SARS, MERS, OC43, and 229E. While widespread cross-reactivity was revealed across several immunodominant regions of S and N, IgG binding to several SARS-CoV-2-derived peptides provided statistically significant discrimination between COVID-19 patients and controls. Selected target peptides may serve as capture antigens for future, highly COVID-19-specific diagnostic antibody tests.

Paradoxical Relationship Between Body Mass Index and Thyroid Hormone Levels: A Study Using Mendelian Randomization.

CONTEXT: Free T3 (FT3) has been positively associated with body mass index (BMI) in cross-sectional studies in healthy individuals. This is difficult to reconcile with clinical findings in pathological thyroid dysfunction. OBJECTIVE: We aimed to investigate whether childhood adiposity influences FT3 levels. DESIGN: Mendelian randomization using genetic variants robustly associated with BMI. SETTING: Avon Longitudinal Study of Parents and Children, a population-based birth cohort. PARTICIPANTS: A total of 3014 children who had thyroid function measured at age 7, who also underwent dual x-ray absorptiometry scans at ages 9.9 and 15.5 years and have genetic data available. MAIN OUTCOME MEASURES: FT3. RESULTS: Observationally at age 7 years, BMI was positively associated with FT3: ß-standardized (ß-[std]) = 0.12 (95% confidence interval [CI]: 0.08, 0.16), P = 4.02 × 10(-10); whereas FT4 was negatively associated with BMI: ß-(std) = -0.08 (95% CI: -0.12, -0.04), P = 3.00 × 10(-5). These differences persisted after adjustment for age, sex, and early life environment. Genetic analysis indicated 1 allele change in BMI allelic score was associated with a 0.04 (95% CI: 0.03, 0.04) SD increase in BMI (P = 6.41 × 10(-17)). At age 7, a genetically determined increase in BMI of 1.89 kg/m(2) was associated with a 0.22 pmol/L (95% CI: 0.07, 0.36) increase in FT3 (P = .004) but no substantial change in FT4 0.01 mmol/L, (95% CI: -0.37, 0.40), P = .96. CONCLUSION: Our analysis shows that children with a genetically higher BMI had higher FT3 but not FT4 levels, indicating that higher BMI/fat mass has a causal role in increasing FT3 levels. This may explain the paradoxical associations observed in observational analyses. Given rising childhood obesity levels, this relationship merits closer scrutiny.

Whole-genome sequence-based analysis of thyroid function.

Normal thyroid function is essential for health, but its genetic architecture remains poorly understood. Here, for the heritable thyroid traits thyrotropin (TSH) and free thyroxine (FT4), we analyse whole-genome sequence data from the UK10K project (N=2,287). Using additional whole-genome sequence and deeply imputed data sets, we report meta-analysis results for common variants (MAF≥1%) associated with TSH and FT4 (N=16,335). For TSH, we identify a novel variant in SYN2 (MAF=23.5%, P=6.15 × 10(-9)) and a new independent variant in PDE8B (MAF=10.4%, P=5.94 × 10(-14)). For FT4, we report a low-frequency variant near B4GALT6/SLC25A52 (MAF=3.2%, P=1.27 × 10(-9)) tagging a rare TTR variant (MAF=0.4%, P=2.14 × 10(-11)). All common variants explain ≥20% of the variance in TSH and FT4. Analysis of rare variants (MAF<1%) using sequence kernel association testing reveals a novel association with FT4 in NRG1. Our results demonstrate that increased coverage in whole-genome sequence association studies identifies novel variants associated with thyroid function.

Subcutaneous pulsatile glucocorticoid replacement therapy.

The glucocorticoid hormone cortisol is released in pulses resulting in a complex and dynamic ultradian rhythm of plasma cortisol that underlies the classical circadian rhythm. These oscillating levels are also seen at the level of tissues such as the brain and trigger pulses of gene activation and downstream signalling. Different patterns of glucocorticoid presentation (constant vs pulsatile) result not only in different patterns of gene regulation but also in different neuroendocrine and behavioural responses. Current 'optimal' glucocorticoid replacement therapy results in smooth hormone blood levels and does not replicate physiological pulsatile cortisol secretion. Validation of a novel portable pulsatile continuous subcutaneous delivery system in healthy volunteers under dexamethasone and metyrapone suppression. Pulsatile subcutaneous hydrocortisone more closely replicates physiological circadian and ultradian rhythmicity.

Temporal growth and geographic variation in the use of laboratory tests by NHS general practices: using routine data to identify research priorities.

BACKGROUND: Laboratory tests are extensively used for diagnosis and monitoring in UK primary care. Test usage by GPs, and associated costs, have grown substantially in recent years. AIM: This study aimed to quantify temporal growth and geographic variation in utilisation of laboratory tests. DESIGN AND SETTING: Retrospective cohort study using data from general practices in the UK. METHOD: Data from the General Practice Research Database, including patient demographics, clinical details, and laboratory test results, were used to estimate rates of change in utilisation between 2005 and 2009, and identify tests with greatest inter-regional variation, by fitting random-effects Poisson regression models. The study also investigated indications for test requests, using diagnoses and symptoms recorded in the 2 weeks before each test. RESULTS: Around 660 000 tests were recorded in 230 000 person-years of follow-up. Test use increased by 24.2%, from 23 872 to 29 644 tests per 10 000 person-years, between 2005 and 2009. Tests with the largest increases were faecal occult blood (121%) and C-reactive protein (86%). There was substantial geographic variation in test utilisation; GPs in some regions requested tests such as plasma viscosity and cardiac enzymes at a rate more than three times the national average. CONCLUSION: Increases in the use of laboratory tests have substantial resource implications. Rapid increases in particular tests may be supported by evidence-based guidelines, but these are often vague about who should be tested, how often, and for how long. Substantial regional variation in test use may reflect uncertainty about diagnostic accuracy and appropriate indications for the laboratory test. There is a need for further research on the diagnostic accuracy, therapeutic impact, and effect on patient health outcomes of the most rapidly increasing and geographically variable tests.

Rapid glucocorticoid receptor-mediated inhibition of hypothalamic-pituitary-adrenal ultradian activity in healthy males.

A complex dynamic ultradian rhythm underlies the hypothalamic-pituitary-adrenal (HPA) circadian rhythm. We have investigated in normal human male subjects the importance, site of action, and receptor-mediated processes involved in rapid basal corticosteroid feedback and its interaction with corticotrophin releasing hormone (CRH) drive. Pro-opiomelanocortin (POMC), ACTH, and cortisol were measured every 10 min from healthy males during the awakening period or late afternoon using an automated blood sampling system. Mathematical modeling into discrete pulses of activity revealed that intravenous infusion of the synthetic mixed glucocorticoid/mineralocorticoid agonist prednisolone produced rapid inhibition of ACTH and cortisol pulsatility within 30 min in the morning and afternoon. Any pulse that had commenced at the time of injection was unaffected, and subsequent pulsatility was inhibited. Prednisolone also inhibited ACTH and cortisol secretion in response to exogenous CRH stimulation, inferring rapid feedback inhibition at the anterior pituitary. Circulating POMC peptide concentrations were unaffected, suggesting that the rapid corticosteroid inhibitory effect specifically targeted ACTH secretion from pituitary corticotrophs. Prednisolone fast feedback was only reduced by glucocorticoid receptor antagonist pretreatment and not by mineralocorticoid receptor antagonism, suggesting a glucocorticoid receptor-mediated pathway. The intravenous prednisolone suppression test provides a powerful new tool to investigate HPA abnormalities underlying metabolic and psychiatric disease states.

Hypothalamic-pituitary-adrenal axis activation in obstructive sleep apnea: the effect of continuous positive airway pressure therapy.

CONTEXT: Obstructive sleep apnea (OSA) is a common condition with significant cardiovascular and metabolic comorbidity. We hypothesized that these may result from OSA-induced perturbations of endogenous ultradian hypothalamic-pituitary-adrenal axis activity. OBJECTIVE: The aim of the study was to investigate ACTH and cortisol ultradian patterns using an automated, repetitive blood sampling technique. DESIGN: Samples for ACTH and cortisol were collected from 10 patients with moderate to severe OSA under basal conditions, at 10-min intervals over 24 h, at diagnosis and 3 months after compliant continuous positive airway pressure (CPAP) therapy. Multiple-parameter deconvolution estimated specific measures of ACTH and cortisol pulsatile secretion from blood hormone concentrations. RESULTS: Mean total ACTH and cortisol production were elevated pre-CPAP compared to post-CPAP (ACTH, 1459.8 +/- 123.0 vs. 808.1 +/- 97.9 pg/ml, P < 0.001; cortisol, 5748.9 +/- 364.9 vs. 3817.7 +/- 351.7 nmol/liter, P < 0.001) as were mean total pulsatile production (ACTH, 764.1 +/- 86.3 vs. 383.5 +/- 50.0 pg/ml, P = 0.002; cortisol, 4715.9 +/- 253.3 vs. 3227.7 +/- 258.8 nmol/liter, P < 0.001). ACTH and cortisol secretory burst mean half-duration were higher at diagnosis (12.3 +/- 0.7 and 13.5 +/- 0.7 vs. 7.8 +/- 0.4 and 8.4 +/- 0.6 min, respectively, P < 0.001); thus, 95% of each ACTH secretion occurred in 21.0 +/- 1.2 vs. 12.9 +/- 0.8 min post-CPAP (P < 0.001) and for cortisol in 23.0 +/- 1.2 vs. 14.2 +/- 1.1 min post-CPAP (P < 0.001). Approximate entropy (ApEn) revealed greater disorderliness in both ACTH (P = 0.03) and cortisol (P = 0.001) time series pre-CPAP. Forward and reverse cross-ApEn suggested nodal disruption at central and adrenal levels pre-CPAP (P = 0.01). Significantly elevated cortisol responses to a single breath of 35% CO(2) occurred pre-CPAP (P = 0.006). CONCLUSIONS: Untreated compared to treated OSA is associated with marked disturbances in ACTH and cortisol secretory dynamics, resulting in prolonged tissue exposure to disordered, elevated hormone levels.

Plasma apelin levels in obstructive sleep apnea and the effect of continuous positive airway pressure therapy.

Apelin is a peptide hormone with cardiovascular and glucose homeostasis properties, and obstructive sleep apnea (OSA) is complicated by cardiovascular and metabolic comorbidities. Plasma apelin has not been previously assessed in OSA. We investigated the response of plasma apelin to a 2-h 75 g oral glucose tolerance test (OGTT) and the effect of 3 months compliant continuous positive airway pressure (CPAP) therapy in 15 obese males with newly diagnosed OSA. Plasma apelin and serum cortisol were recorded 10 minutely, while serum insulin and glucose were measured 30 minutely. Ten subjects had plasma apelin measured at intervals across a 24-h period to investigate for circadian variation in apelin levels, and this was repeated following 3 months compliant CPAP therapy. Fasting (0.342+/-0.038 vs 0.288+/-0.024 ng/ml, P=0.04), 30 min (0.399+/-0.035 vs 0.312+/-0.036 ng/ml, P=0.007) and 120 min (0.402+/-0.030 vs 0.259+/-0.024 ng/ml, P<0.001) apelin levels were reduced following CPAP. The area under curve for apelin OGTT response was lower post-CPAP (44.1+/-3.3 vs 35.8+/-2.3 ng/ml per min, P<0.001). Mean OGTT apelin levels showed a significant treatment effect (P=0.006) and a time effect (P<0.001), and the effect of time was different pre- versus post-CPAP (P=0.005). No significant variability in apelin levels existed across the 24-h period at diagnosis. Lower levels were evident overnight following treatment (P=0.004). Improvements in insulin and glucose parameters and reduced cortisol levels were found post-CPAP. In summary, untreated OSA was associated with elevated plasma apelin levels, altered apelin secretory dynamics in response to oral glucose and lack of an apparent circadian variability, which was restored following CPAP.

Comparison of two prediction equations with radionuclide glomerular filtration rate: validation in routine use.

BACKGROUND: Recent guidelines recommend automatic reporting of estimated glomerular filtration rate (eGFR) using the abbreviated Modification of Diet in Renal Disease (MDRD) equation with every request for plasma creatinine. We evaluated the performance of the MDRD and Cockcroft-Gault equations in routine use compared with a radionuclide GFR method. METHOD: Data collected on 601 patients aged 16-85 years who had undergone radionuclide GFR and eGFR was calculated using both equations for each patient. Calculations of bias, correlation coefficients and percentage estimates within 30% and 50% of radionuclide GFR were used in comparisons. RESULTS: Overall, there was no significant difference in the performance between the two equations when compared with radionuclide GFR. When all creatinine results <60 micromol/L were adjusted to 60 micromol/L, and when creatinine results <60 micromol/L were excluded, MDRD performed better than the Cockcroft-Gault equation. MDRD was also superior for prediction of radionuclide GFR of <60 mL/min/1.73 m(2) (P=0.014). CONCLUSION: The abbreviated MDRD formula for eGFR is superior to the Cockcroft-Gault equation for prediction of radionuclide GFR of <60 microL/min/1.73 m(2)and is more accurate when creatinine results <60 micromol/L are either corrected to 60 micromol/L or excluded from eGFR calculations.

Toxic effects of BZP-based herbal party pills in humans: a prospective study in Christchurch, New Zealand.

AIM: This study describes patterns of human toxicity related to the use of 1-benzylpiperazine (BZP)-based 'herbal party pills'. METHODS: From 1 April 2005 to 1 September 2005 all presentations associated with party pill use were captured on a prospective data collection form. RESULTS: There were 61 patients who presented on 80 occasions to the Emergency Department of Christchurch Hospital, New Zealand. Patients with adverse effects took an average of 4.5 tablets/capsules. Patients with mild to moderate toxicity experienced symptoms such as insomnia, anxiety, nausea, vomiting, palpitations, dystonia, and urinary retention. Some adverse reactions persisted up to 24 hours after ingestion. Fifteen toxic seizures were recorded. Two patients suffered life-threatening toxicity with status epilepticus and severe respiratory and metabolic acidosis. CONCLUSIONS: Herbal party pills have been sold without regulation since 2000, and are now widely used by young New Zealanders. The principal ingredient of these pills is 1-benzylpiperazine (BZP). They appear to have a narrow safety margin when used recreationally by some humans, possibly because of intrinsic pharmacodynamic properties, self-dosing variability, or genetic polymorphism. Those with seizure disorders or coronary disease should avoid BZP as should those taking prescription sympathomimetics or anticholinergics. Coingestion with MDMA or amphetamine should also be cautioned against. The results of this study indicate that BZP can cause unpredictable and serious toxicity in some individuals. Furthermore, the results of this study should be carefully considered in any discussion on the legal status of piperazine-based party pills.

Metformin, but not leptin, regulates AMP-activated protein kinase in pancreatic islets: impact on glucose-stimulated insulin secretion.

Metformin, a drug widely used in the treatment of type 2 diabetes, has recently been shown to act on skeletal muscle and liver in part through the activation of AMP-activated protein kinase (AMPK). Whether metformin or the satiety factor leptin, which also stimulates AMPK in muscle, regulates this enzyme in pancreatic islets is unknown. We have recently shown that forced increases in AMPK activity inhibit insulin secretion from MIN6 cells (da Silva Xavier G, Leclerc I, Varadi A, Tsuboi T, Moule SK, and Rutter GA. Biochem J 371: 761-774, 2003). Here, we explore whether 1) glucose, metformin, or leptin regulates AMPK activity in isolated islets from rodent and human and 2) whether changes in AMPK activity modulate insulin secretion from human islets. Increases in glucose concentration from 0 to 3 and from 3 to 17 mM inhibited AMPK activity in primary islets from mouse, rat, and human, confirming previous findings in insulinoma cells. Incubation with metformin (0.2-1 mM) activated AMPK in both human islets and MIN6 beta-cells in parallel with an inhibition of insulin secretion, whereas leptin (10-100 nM) was without effect in MIN6 cells. These studies demonstrate that AMPK activity is subject to regulation by both glucose and metformin in pancreatic islets and clonal beta-cells. The inhibitory effects of metformin on insulin secretion may therefore need to be considered with respect to the use of this drug for the treatment of type 2 diabetes.