Biology subjects

Gilligan, L. C.

Publications and source records attributed to Gilligan, L. C..

3 recordsLinked to original sources

bioRxiv2023

A cardiotonic steroid multiplex method using ultra-high-performance liquid chromatography-tandem mass spectrometry

Background and AimsDigoxin, a cardiotonic steroid (CTS), is commonly prescribed for patients with atrial fibrillation and heart failure. Endogenous CTS have been implicated in cardiovascular disease pathogenesis and can interact with digoxin. We developed an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for the quantification of eleven CTS. Materials and MethodsIsotopically labelled internal standards were added to samples, followed by protein precipitation and solid-phase extraction. Steroids were separated using an Acquity uPLC chromatography system with a Waters CORTECS T3 column (1.6 m 2.1x50 mm) and quantification performed on a Waters TQ-XS mass spectrometer using electrospray ionisation in positive ion mode. Separation used a methanol/ water elution system containing 0.1% formic acid and post-column infusion of lithium chloride. ResultsRun time was 13.5 minutes. Lower limits of quantification ranged from 0.025 to 0.1 ng/ml. Serum recovery ranged from 40.0-98.6% with matrix effects from -22.9% to 7.6%. Plasma recovery ranged from 27.4-83.9% and matrix effects were -27.6-39.1%. Accuracy and precision at three concentrations were within ideal range (<15%) for seven CTS and <20% for the others. ConclusionThis validated UHPLC-MS/MS method provides a comprehensive assessment profiling 11 cardiotonic steroids, offering a sensitive and specific tool for clinical and pre-clinical investigations. HighlightsO_LIMass spectrometry method simultaneously quantifies multiple cardiotonic steroids C_LIO_LIAccurate and specific measurement of clinically relevant digoxin concentration C_LIO_LIMethod validated for measurement of cardiotonic steroids in serum and plasma C_LIO_LIPost-column infusion of lithium chloride substantially improves sensitivity C_LI Research fundingThis work was funded by the British Heart Foundation (PG/17/55/33087, FS/PhD/22/29309, FS/19/12/34204, RG/17/15/33106 to DP, Accelerator Award AA/18/2/34218 to Institute of Cardiovascular Sciences), Wellcome Trust (Seed Award Grant 109604/Z/15/Z to DP) and Department of Clinical Laboratory Sciences, Faculty of Applied medical Sciences, University of Hail. PK was partially supported by European Union AFFECT-AF (grant agreement 847770), and MAESTRIA (grant agreement 965286), British Heart Foundation (PG/17/30/32961; PG/20/22/35093; AA/18/2/34218), German Centre for Cardiovascular Research supported by the German Ministry of Education and Research (DZHK), Deutsche Forschungsgemeinschaft (Ki 509167694), and Leducq Foundation. The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication. Financial disclosuresProf. Kotecha reports grants from the National Institute for Health Research (NIHR CDF-2015-08-074 RATE-AF; NIHR130280 DaRe2THINK; NIHR132974 D2T-NeuroVascular; NIHR203326 Biomedical Research Centre), the British Heart Foundation (PG/17/55/33087, AA/18/2/34218 and FS/CDRF/21/21032), the EU/EFPIA Innovative Medicines Initiative (BigData@Heart 116074), EU Horizon (HYPERMARKER 101095480), UK National Health Service -Data for R&D-Subnational Secure Data Environment programme, UK Dept. for Business, Energy & Industrial Strategy Regulators Pioneer Fund, the Cook & Wolstenholme Charitable Trust, and the European Society of Cardiology supported by educational grants from Boehringer Ingelheim/BMS-Pfizer Alliance/Bayer/Daiichi Sankyo/Boston Scientific, the NIHR/University of Oxford Biomedical Research Centre and British Heart Foundation/University of Birmingham Accelerator Award (STEEER-AF). In addition, he has received research grants and advisory board fees from Bayer, Amomed and Protherics Medicines Development; all outside the submitted work. PK received research support for basic, translational, and clinical research projects from European Union, British Heart Foundation, Leducq Foundation, Medical Research Council (UK), the Deutsche Forschungsgemeinschaft (DFG) and German Centre for Cardiovascular Research, from several drug and device companies active in atrial fibrillation, and has received honoraria from several such companies in the past, but not in the last three years. PK is listed as inventor on two issued patents held by University of Hamburg (Atrial Fibrillation Therapy WO 2015140571, Markers for Atrial Fibrillation WO 2016012783). Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/561354v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@180bd42org.highwire.dtl.DTLVardef@109441corg.highwire.dtl.DTLVardef@1568cd9org.highwire.dtl.DTLVardef@1390caf_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2023

Gonadal androgens are associated with decreased type I interferon production by pDCs and increased IgG titres to BNT162b2 following co-vaccination with live attenuated influenza vaccine in adolescents

mRNA vaccine technologies introduced following the SARS-CoV-2 pandemic have highlighted the need to better understand the interaction of adjuvants and the early innate immune response. Interferon type I (IFN-I) is an integral part of this early innate response and can prime several components of the adaptive immune response. Females are widely reported to respond better than males to seasonal tri- and quad-valent influenza vaccines. Plasmacytoid dendritic cells (pDCs) are the primary cell type responsible for IFN-I production and female pDCs produce more IFN-I than male pDCs since the upstream receptor TLR7 is encoded by the X-chromosome and is biallelically expressed by up to 30% of female immune cells. Additionally, the TLR7 promoter contains putative androgen response elements and androgens have been reported to suppress pDC IFN-I in-vitro. Unexpectedly, therefore, we recently observed that male adolescents mount stronger antibody responses to the Pfizer BNT162b2 mRNA vaccine than female adolescents after controlling for natural SARS-CoV-2 infection. We here examined pDC behaviour in this cohort to determine the impact of IFN-I on anti-Spike and anti-receptor-binding domain titres to BNT162b2. Through LASSO modelling we determined that serum free testosterone was associated with reduced pDC IFN-I but, contrary to the well-described immunosuppressive role for androgens, the more potent androgen dihydrotestosterone was associated with increased IgG titres to BNT162b2. Also unexpectedly, we observed that co-vaccination with live-attenuated influenza vaccine boosted the magnitude of IgG responses to BNT162b2. Together these data support a model where systemic IFN-I increased vaccine-mediated immune responses, but for vaccines with intracellular stages, modulation of the local IFN-I response may alter antigen longevity and consequently vaccine-driven immunity. Author SummaryType I interferons (IFN-I) are potent antiviral proteins which play a central role in activating the immune response and driving inflammation. IFN-I is predominantly produced by plasmacytoid dendritic cells (pDCs) and female pDCs produce more IFN-I than male pDCs. Consequently, females typically generate stronger antibody responses to vaccines such as seasonal influenza vaccines. In addition, females typically suffer more serious adverse events from vaccines. However, we recently reported in a study of adolescents that males generate stronger antibody responses to the SARS-CoV-2 mRNA vaccine BNT162b2 than females. Here we examine the IFN-I response of pDCs in adolescents co-/vaccinated with BNT162b2 and live-attenuated influenza vaccine (LAIV). We find that male sex hormones reduce pDC IFN-I but are associated with increased BNT162b2 antibody titres. We also observe that LAIV boosts BNT162b2 antibody titres through possible bystander activation of immune cells. These findings are consistent with a reportedly higher incidence of adverse events among males associated with this vaccine. Together these data suggest that IFN-I production typically enhances vaccine-specific immune responses but for new mRNA vaccines such as BNT162b2, that are modified to reduce innate immunogenicity, localised dampening of the IFN-I response in vaccinated tissue by male sex hormones may further delay the clearance of the vaccine, increasing vaccine antigen exposure and allowing time for a stronger antibody response.

immunology↗

bioRxiv2023

Inhibition of the glucocorticoid-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 drives concurrent 11-oxygenated androgen excess

Aldo-keto reductase 1C3 (AKR1C3) is a key enzyme in the activation of both classic and 11-oxygenated androgens. In adipose tissue, AKR1C3 is co-expressed with 11{beta}-hydroxysteroid dehydrogenase type 1 (HSD11B1), which catalyses the local activation of glucocorticoids but also the inactivation of 11-oxygenated androgens, and thus has the potential to counteract AKR1C3. Using a combination of in vitro assays and in silico modelling we show that HSD11B1 attenuates the biosynthesis of the potent 11-oxygenated androgen, 11-ketotestosterone, by AKR1C3. Employing ex vivo incubations of human female adipose tissue samples we show that inhibition of HSD11B1 results in the increased peripheral biosynthesis of 11-ketotestosterone. Moreover, circulating 11KT increased 2-3 fold in individuals with type 2 diabetes after receiving the selective oral HSD11B1 inhibitor AZD4017 for 35 days, thus confirming that HSD11B1 inhibition results in systemic increases in 11KT concentrations. Our findings show that HSD11B1 protects against excess 11KT production by adipose tissue, a finding of particular significance when considering the evidence for adverse metabolic effects of androgens in women. Therefore, when targeting glucocorticoid activation by HSD11B1 inhibitor treatment in women, the consequently increased generation of 11-ketotestosterone may offset beneficial effects of decreased glucocorticoid activation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=135 HEIGHT=200 SRC="FIGDIR/small/543687v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@f68f0forg.highwire.dtl.DTLVardef@12a5070org.highwire.dtl.DTLVardef@107231borg.highwire.dtl.DTLVardef@191ca58_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗