Medical Research Council Grant Research Articles

Communicating with patients and the public about COVID-19 vaccine safety: recommendations from the Collaboration on Social Science and Immunisation.

Communicating with patients and the public about COVID-19 vaccine safety: recommendations from the Collaboration on Social Science and Immunisation.

Use of Neuromuscular Blocking Agents in Mechanically Ventilated Patients with COVID-19: A Propensity Score Analysis

Background: Neuromuscular blocking agents (NMBA) have been previously used in patients with acute respiratory distress syndrome (ARDS). It is unknown if NMBA are useful in COVID-19 patients who require invasive mechanical ventilation (IMV).Methods: We investigated use of NMBA in COVID-19 patients on IMV from February 1 to November 24, 2020, in 147 hospitals across 6 continents, comprising the COVID-19 Critical Care Consortium. We performed propensity score (PS) matched Cox proportional hazards analysis to appraise the impact of NMBA use for ≥2 days, continuously or discontinuously (treatment), vs. no use of NMBA or only upon commencement of IMV (control) on 28-day intensive care unit (ICU) mortality.Findings: 1548 (72%) patients received any NMBA therapy; 1165 (54%) of patients were stratified in the treatment group, with a median (IQR) time from ICU admission to commencement of NMBA therapy of 0 (0-2) days. The median (IQR) duration of NMBA therapy was 3 (2-6) days (N=1548). Upon commencement of IMV, patients who received NMBA therapy had a lower mean (±SD) PaO2/FiO2 (139±75 vs 157±93; PInterpretation: Use of NMBA is common in COVID-19 patients on IMV and associated with a 2.2-fold increase in risk of 28-day mortality. Until further randomised evidence is available, NMBA should be applied cautiously in routine clinical practice.Funding Statement: University of Queensland, Wesley Medical Research, The Prince Charles Hospital Foundation, The Health Research Board of Ireland; Biomedicine international training research programme for excellent clinician-scientists; European Union’s research and innovation programme (Horizon 2020); la Caixa Foundation. Finally, Carol Hodgson is funded by a National Health and Medical Research Council Grant.Declaration of Interests: Dr. Li Bassi received research support from Fisher & Paykel outside the submitted work. Dr. Dalton has consulting from Innovative ECMO Concepts, Abiomed and Instrumentation Labs , none which affect the current work. Dr. Brodie receives research support from ALung Technologies and he has been on the medical advisory boards for Baxter, Abiomed, Xenios and Hemovent. Dr. Fan reports personal fees from ALung Technologies, Baxter, Fresenius Medical Care, Getinge, and MC3Cardiopulmonary outside the submitted work. Dr. Laffey reports consulting fees from Baxter and Cala Medical, both outside the submitted work. Dr Nichol is supported by a health Research Board of Ireland award (CTN-2014-012). Dr. Fraser receives research support from Fisher & Paykel outside the submitted work. Remaining authors do not have any competing interest to declare. Ethics Approval Statement: Participating hospitals obtained local ethics committee approval and a waiver of informed consent was granted in all cases.De-identified patient data were collected and stored via the REDCap (Vanderbilt/NIH/NCATS UL1 TR000445 v.10.0.23) electronic data capture tool, hosted at the University of Oxford, United Kingdom and University of Queensland.

Simultaneous visualization of left atrial fibrosis and epicardial adipose tissue using 3D Dixon late gadolinium enhancement cardiovascular magnetic resonance

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swedish Medical Research Council (Grant Number: 2018-02779) Swedish Heart and Lung Foundation (Grant Number: 20170440) Background Cardiovascular magnetic resonance (CMR) is a powerful tool to identify structural changes of the left atrium (LA). LA fibrosis can be quantified using late gadolinium enhancement (LGE) and is a major risk factor for progression of atrial fibrillation (AF). Epicardial adipose tissue (EAT) around the LA can also be assessed using CMR and is associated with incidence and severity of AF. The theory that EAT may induce LA wall inflammation and thus promoting LA fibrosis highlights the need for assessment of the interrelation of the spatial distribution of these two AF risk factors. However, previous CMR studies have used separate pulse sequences to visualize LA fibrosis and EAT. Purpose To develop and evaluate a novel 3D Dixon water-fat separated LGE (Dixon-LGE) pulse sequence for visualization and quantification of LA fibrosis and EAT. Methods 19 subjects were prospectively included and scanned on a 1.5T scanner (mean age 54 ± 20 years; 9 female): 10 patients with a history of AF, and 9 patients without any known cardiovascular disease (NCD). The 3D Dixon-LGE scan was performed 20 minutes after Gd contrast injection. Dixon reconstructed water images were used for LGE quantification while the fat images were used to quantify EAT. Segmentations of the LA myocardium and EAT were performed manually. The image intensity ratio (IIR) method was used for fibrosis quantification, by normalizing the intensity of the LA wall by the mean value of the blood pool intensity and classifying fibrosis as pixels with normalized intensity more than 1.2. For comparison with the automated IIR method, fibrosis was manually segmented in the LGE images as ground truth (GT). Results Example images from an AF and NCD patient are shown in the figure. Heart rate was higher (84 ± 14 vs. 65 ± 14 bpm, P = 0.007), and BMI was higher (28 ± 3 vs. 24 ± 3 kg/m², P = 0.014) in the AF group compared to NCD. AF had also larger LA end-systolic volume index (83 ± 16 vs. 38 ± 9 mL/m², P < 0.001) and larger LA EAT volume index (7.1 ± 4.1 vs. 3.2 ± 2.2 mL/m², P = 0.022) compared to NCD. Further, fibrosis as percentage of LA surface area was larger 14.9 ± 18.4 vs. 3.2 ± 2.5 %, P = 0.028 in AF compared to NCD. The interclass correlation coefficient (ICC) for inter-observer and intra-observer measurements of LA volume were 0.99 (95% CI, 0.97%–0.99%) and 0.99 (95% CI, 0.99%–0.99%), respectively, of LA EAT volume were 0.95 (95% CI, 0.87%–0.98%) and 0.97 (95% CI, 0.89%–0.99%), respectively, and of LA fibrosis, IIR method, were 0.98 (95% CI, 0.94%–0.99%) and 0.99 (95% CI, 0.98%–0.99%), respectively. The IIR method had a very high level of agreement with the GT segmentation, ICC = 0.96 (95% CI, 0.78%-0.99%). Conclusions LA fibrosis and EAT can be visualized simultaneously by using a single 3D LGE-Dixon pulse sequence. This new method has a high level of intra- and inter observer repeatability, can reduce the scan time and allows the analysis of spatial correlation of these AF risk factors. Abstract Figure. Dixon-LGE acquisition and quantification

Development of Humoral and Cellular Immunological Memory Against SARS-CoV-2 Despite B-Cell Depleting Treatment in Multiple Sclerosis

B-cell depleting therapies are widely used as immunomodulating agents for autoimmune diseases such as multiple sclerosis. The possible impact of B-cell depletion on development of humoral and cellular immunity to viruses and specifically SARS-CoV-2 has raised concerns with the COVID-19 pandemic. We here determined humoral and cellular responses in participants of an observational trial comprising several multiple sclerosis disease modulatory drugs (COMBAT-MS; NCT03193866). Patients on B-cell depleting treatment, who previously had COVID-19-like symptoms, developed anti-SARS-CoV-2 antibodies and/or T-cell memory irrespective of their B-cell depletion status. Furthermore, antibody titers were similar to those observed with other treatments or controls, and anti-SARS-CoV-2 T-cells displayed functional similarity to controls producing IFN-γ and TNF. These results inform on the role of B- and T-cells in SARS-CoV-2 immunity and provide evidence that B-cell depleting therapy does not substantially abrogate SARS-CoV-2 immunological memory, in turn suggesting a low risk for reinfection and potentially responsiveness to vaccination.Funding: The COMBAT-MS study is funded through a Patient-Centered Outcomes Research Institute (PCORI) Award (MS-1511-33196), modified to include the sub-study reported herein. Additional funding was obtained from the Swedish Medical Research council (grant no. 2017-03054), Swedish Brain Foundation, NEURO Sweden, Tetra Laval, Region Stockholm, Knut and Alice Wallenberg foundation, Erling-Persson family foundation and Petrus och Augusta Hedlunds Stiftelse.Conflict of Interest: Tomas Olsson has received unrestricted grants for extended multiplesclerosis studies in relation to COVID-19 from Biogen and Merck. Not related to this manuscript, T. O. has received unrestricted grants, advisory board/ lectures from Biogen, Merck, Novartis, and Sanofi and F. P. has received research grants from Genzyme, Merck KGaA and UCB, and fees for serving as Chair of DMC in clinical trials with Parexel. All other authors declare no competing interests.Ethical Approval: Study procedures were conducted under the following ethical permits approved by the Swedish ethical review authority; COMBAT-MS: 2017/32-31/4; STOPMSII: 2009/2107-31/2; 2020 00052, with written informed consent from participants.

Global Distribution of Sex Differences in the Incidence and Prevalence of Any Dementia, Alzheimer's Disease, and Vascular Dementia: A Systematic Review and Meta-Analysis

Background: We estimated global incidence of Alzheimer’s disease (AD), Vascular dementia (VaD), and Any dementia reported evaluated whether country level socio-demographic factors could explain variation in gender differences in dementia prevalence and incidence. Methods: We conducted a systematic review with meta-analysis to obtain estimates of incidence and prevalence of AD, VaD and Any dementia using random effects meta-analysis. We included population-based studies with clinical diagnoses or validated dementia screening instruments. We excluded studies lacking a valid clinical diagnoses, population-based sampling, or gender stratified estimates. We used meta-regression to evaluate how country specific factors of life-expectancy, education, and gender differences in development, unemployment and inequality indices influenced estimates. Findings: Our searches identified 205 eligible studies from 8731 articles, representing 998 187 participants across 43 countries. Global incidence (95% CI) of Any dementia/1000 person-years for men at age 60-64, 66-69, 70-74, 75-79, 80-84, 85-89 and 90+ was 2·5 (1·9-3·2), 5·1 (4·0-6·5). 10·1 (7·9-12·8), 19·7 (15·5-24·9), 34·0 (26·9, 43·0), 59·6 (47·3, 74·8), and 84·5 (67·5, 105·4) respectively. For women the corresponding figures were 1·8 (1·3-2·5), 3·4 (2·5-4·7), 9·1 (6·6-12·4), 23·7 (17·3-32·4), 46·44 (34·1, 62·9), 84·54 (62·8, 112·9), and 142·2 (107·4, 185·9). There was no difference in incident Any dementia, or for AD or VaD, or prevalence of Any dementia, in any age group except for the 90+, where it was higher in women. AD was more prevalent in women at most ages, and VaD was only more prevalent in men aged 65-69. Meta-regression revealed that gender differences in Any dementia prevalence, may be explained by the gender differences in life expectancy and education. Interpretation: Globally, there are no gender differences in dementia incidence except in the 90+ age-group. Greater focus on the role gender inequality and life expectancy play as a potential explanations of gender differences in dementia and AD prevalence is needed. Registration Details: The review was registered with the International Prospective Register of Systematic Reviews (PROSPERO, CRD42017053915) and reported in accordance with the PRISMA Statement (Preferred Reporting Items for Systematic Reviews and Meta-Analyses). Funding Information: ARC Laureate Fellowship FL190100011, National Health and Medical Research Council (NHMRC) Grants 1100579, 1102694. NHMRC Dementia Centre for Research Collaboration Declaration of Interests: The authors have no conflicts of interest.

Clinical and Virological Features of SARS-CoV-2 Variants of Concern: A Retrospective Cohort Study Comparing B.1.1.7 (Alpha), B.1.315 (Beta), and B.1.617.2 (Delta)

Background: The impact of SARS-CoV-2 variants of concern (VOCs) on disease severity is unclear. In this retrospective cohort study, we compared outcomes of patients infected with B.1.1.7, B.1.351, and B.1.617.2 with those with wild-type strains from early 2020.Methods: National surveillance data from 1-January-2021 to 22-May-2021 were obtained from the Ministry of Health, and outcomes in relation to VOC were explored. Detailed patient level data from all SARS-CoV-2 patients with VOC infection admitted to our centre between 20-December-2020 and 12-May-2021 were analysed. Outcomes were compared with a cohort of 846 patients admitted January-April 2020.Findings: There were 838 VOC infections in Singapore in the study period. After adjusting for age and gender, B.1.617.2 infection was associated with higher odds of oxygen requirement, ICU admission, or death (adjusted odds ratio (aOR) 4·90, [95% CI 1·43-30·78]. 157 patients with VOCs were admitted to our centre. After adjusting for age, gender, comorbidities, and vaccination, aOR for pneumonia with B.1.617.2 was 1·88 [95% CI 0·95-3·76]) compared with wild-type. B.1.617.2 was associated with significantly lower PCR Ct values and significantly longer duration of Ct value ≤30 (estimated median duration 18 days for B.1.617.2, 13 days for wild-type). Vaccine breakthrough cases were less severe.Interpretation: There was a signal toward increased severity associated with B.1.617.2. The association of B.1.617.2 with lower Ct value and longer viral shedding provides a potential mechanism for increased transmissibility. These findings provide a strong impetus for the rapid implementation of vaccination programmes.Funding Information: National Medical Research Council grants COVID19RF-001 and COVID19RF-008.Declaration of Interests: BEY reports personal fees from Roche and Sanofi, outside the submitted work. All other authors declare no competing interests.Ethics Approval Statement: Informed consent for retrospective data collection was waived as approved by the institutional review board (NHG-DSRB reference number 2020/01122).

Mapping clinical outcomes to generic preference-based outcome measures: development and comparison of methods.

Cost-effectiveness analysis using quality-adjusted life-years as the measure of health benefit is commonly used to aid decision-makers. Clinical studies often do not include preference-based measures that allow the calculation of quality-adjusted life-years, or the data are insufficient. 'Mapping' can bridge this evidence gap; it entails estimating the relationship between outcomes measured in clinical studies and the required preference-based measures using a different data set. However, many methods for mapping yield biased results, distorting cost-effectiveness estimates. Develop existing and new methods for mapping; test their performance in case studies spanning different preference-based measures; and develop methods for mapping between preference-based measures. Fifteen data sets for mapping from non-preference-based measures to preference-based measures for patients with head injury, breast cancer, asthma, heart disease, knee surgery and varicose veins were used. Four preference-based measures were covered: the EuroQoL-5 Dimensions, three-level version (n = 11), EuroQoL-5 Dimensions, five-level version (n = 2), Short Form questionnaire-6 Dimensions (n = 1) and Health Utility Index Mark 3 (n = 1). Sample sizes ranged from 852 to 136,327. For mapping between generic preference-based measures, data from FORWARD, the National Databank for Rheumatic Diseases (which includes the EuroQoL-5 Dimensions, three-level version, and EuroQoL-5 Dimensions, five-level version, in its 2011 wave), were used. Mixture-model-based approaches for direct mapping, in which the dependent variable is the health utility value, including adaptations of methods developed to model the EuroQoL-5 Dimensions, three-level version, and beta regression mixtures, were developed, as were indirect methods, in which responses to the descriptive systems are modelled, for consistent multidirectional mapping between preference-based measures. A highly flexible approach was designed, using copulas to specify the bivariate distribution of each pair of EuroQoL-5 Dimensions, three-level version, and EuroQoL-5 Dimensions, five-level version, responses. A range of criteria for assessing model performance is proposed. Theoretically, linear regression is inappropriate for mapping. Case studies confirm this. Flexible, direct mapping methods, based on different variants of mixture models with appropriate underlying distributions, perform very well for all preference-based measures. The precise form is important. Case studies show that a minimum of three components are required. Covariates representing disease severity are required as predictors of component membership. Beta-based mixtures perform similarly to the bespoke mixture approaches but necessitate detailed consideration of the number and location of probability masses. The flexible, bi-directional indirect approach performs well for testing differences between preference-based measures. Case studies drew heavily on EuroQoL-5 Dimensions. Indirect methods could not be undertaken for several case studies because of a lack of coverage. These methods will often be unfeasible for preference-based measures with complex descriptive systems. Mapping requires appropriate methods to yield reliable results. Evidence shows that widely used methods such as linear regression are inappropriate. More flexible methods developed specifically for mapping show that close-fitting results can be achieved. Approaches based on mixture models are appropriate for all preference-based measures. Some features are universally required (such as the minimum number of components) but others must be assessed on a case-by-case basis (such as the location and number of probability mass points). Further research is recommended on (1) the use of the monotonicity concept, (2) the mismatch of trial and mapping distributions and measurement error and (3) the development of indirect methods drawing on methods developed for mapping between preference-based measures. This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 24, No. 34. See the NIHR Journals Library website for further project information. This project was also funded by a Medical Research Council grant (MR/L022575/1).

Re-evaluating and recalibrating predictors of bacterial infection in children with cancer and febrile neutropenia.

BackgroundNumerous paediatric febrile neutropenia (FN) clinical decision rules (CDRs) have been derived. Validation studies show reduced performance in external settings. We evaluated the association between variables common across published FN CDRs and bacterial infection and recalibrated existing CDRs using these data.MethodsProspective data from the Australian-PICNICC study which enrolled 858 FN episodes in children with cancer were used. Variables shown to be significant predictors of infection or adverse outcome in >1 CDR were analysed using multivariable logistic regression. Recalibration included re-evaluation of beta-coefficients (logistic model) or recursive-partition analysis (tree-based models).FindingsTwenty-five unique variables were identified across 17 FN CDRs. Fourteen were included in >1 CDR and 10 were analysed in our dataset. On univariate analysis, location, temperature, hypotension, rigors, severely unwell and decreasing platelets, white cell count, neutrophil count and monocyte count were significantly associated with bacterial infection. On multivariable analysis, decreasing platelets, increasing temperature and the appearance of being clinically unwell remained significantly associated. Five rules were recalibrated. Across all rules, recalibration increased the AUC-ROC and low-risk yield as compared to non-recalibrated data. For the SPOG-adverse event CDR, recalibration also increased sensitivity and specificity and external validation showed reproducibility.InterpretationDegree of marrow suppression (low platelets), features of inflammation (temperature) and clinical judgement (severely unwell) have been consistently shown to predict infection in children with FN. Recalibration of existing CDRs is a novel way to improve diagnostic performance of CDRs and maintain relevance over time.FundingNational Health and Medical Research Council Grant (APP1104527).

Associations of childhood adiposity with menstrual irregularity and polycystic ovary syndrome in adulthood: the Childhood Determinants of Adult Health Study and the Bogalusa Heart Study.

Is high adiposity in childhood associated with menstrual irregularity and polycystic ovary syndrome (PCOS) in later life? Overall, greater childhood BMI was associated with menstrual irregularity, and greater childhood BMI and waist/height ratio (WHtR) in white but not black participants were associated with PCOS in adulthood. Increased childhood BMI has been associated with irregular menstrual cycles and PCOS symptoms in adulthood in two longitudinal population-based studies, but no study has reported on associations with childhood abdominal obesity. Few studies have investigated whether there are racial differences in the associations of adiposity with PCOS though there has been some suggestion that associations with high BMI may be stronger in white girls than in black girls. The study included 1516 participants (aged 26-41years) from the Australian Childhood Determinants of Adult Health study (CDAH) and 1247 participants (aged 26-57years) from the biracial USA Babies substudy of the Bogalusa Heart Study (BBS) who were aged 7-15years at baseline. At follow-up, questions were asked about menstruation (current for CDAH or before age 40years for BBS), ever having had a diagnosis of PCOS and symptoms of PCOS. In CDAH, a single childhood visit was conducted in 1985. In BBS, multiple childhood visits occurred from 1973 to 2000 and race was reported (59% white; 41% black). In childhood, overweight and obesity were defined by international age-sex-specific standards for BMI and WHtR was considered as an indicator of abdominal obesity. Multilevel mixed-effects Poisson regression estimated relative risks (RRs) adjusting for childhood age, highest parental and own education and age at menarche. The prevalence of childhood obesity was 1.1% in CDAH and 7.5% in BBS. At follow-up, menstrual irregularity was reported by 16.7% of CDAH and 24.5% of BBS participants. The prevalence of PCOS was 7.4% in CDAH and 8.0% in BBS participants. In CDAH, childhood obesity was associated with menstrual irregularity (RR = 2.84, 95% CI: 1.63-4.96) and PCOS (RR = 4.05, 95% CI: 1.10-14.83) in adulthood. With each 0.01 unit increase in childhood WHtR there was a 6% (95% CI: 1-11%) greater likelihood of PCOS. Overall, in BBS, childhood obesity was associated with increased risk of menstrual irregularity (RR = 1.44, 95% CI: 1.08-1.92) in adulthood. Significant interaction effects between race and childhood adiposity were detected in associations with PCOS. In BBS white participants, childhood obesity was associated with PCOS (RR = 2.93, 95% CI: 1.65-5.22) and a 0.01 unit increase in childhood WHtR was associated with an 11% (95% CI: 5-17%) greater likelihood of PCOS in adulthood. In BBS black participants, no statistically significant associations of childhood adiposity measures with PCOS were observed. The classification of menstrual irregularity and PCOS was based on self-report by questionnaire, which may have led to misclassification of these outcomes. However, despite the limitations of the study, the prevalence of menstrual irregularity and PCOS in the two cohorts was consistent with the literature. While the study samples at baseline were population-based, loss to follow-up means the generalizability of the findings is uncertain. Greater childhood adiposity indicates a higher risk of menstrual irregularity and PCOS in adulthood. Whether this is causal or an early indicator of underlying hormonal or metabolic disorders needs clarification. The stronger associations of adiposity with PCOS in white than black participants suggest that there are racial differences in childhood adiposity predisposing to the development of PCOS and other environmental or genetic factors are also important. The CDAH study was supported by grants from the Australian National Health and Medical Research Council (grants 211316, 544923 and 1128373). The Bogalusa Heart Study is supported by US National Institutes of Health grants R01HD069587, AG16592, HL121230, HD032194 and P50HL015103. No competing interests existed.

Retinoid X Receptor Modulators Transcriptionally Regulate Human Aldehyde Oxidase Expression

Aldehyde oxidase‐1 (AOX1) is an oxidative drug‐metabolizing enzyme. Retinoid X receptor (RXR) is a nuclear receptor that elicits various physiological functions and is an obligate partner in specific cell signaling pathways. It regulates gene expression by forming a homodimer or heterodimer with another nuclear receptor, such as retinoic acid receptor (RAR) or peroxisome proliferator‐activated receptor (PPAR). Given the central role of RXR in controlling gene expression, we hypothesized that RXR modulators regulate AOX1 expression in cultured MCF‐7 human breast adenocarcinoma cells. 9‐Cis‐retinoic acid (10 μM), a pan‐RXR and RAR agonist, increased AOX1 mRNA expression at 24, 48, 72, 96, and 120 h post‐treatment, and a plateau was achieved at 96 h. To determine the effect of RXR activation on AOX1 expression, we compared the effect of several RXR‐selective modulators and 9‐cis‐retinoic acid. Non‐cytotoxic concentrations (as assessed by lactate dehydrogenase assay) of LG100268, CD3254, LG100754, SR11237, bexarotene, and 9‐cis‐retinoic acid increased AOX1 mRNA level in a concentration‐dependent manner with EC50 values of 4, 6, 12, 39, 42, and 334 nM, and Emax values of 5, 4, 9, 5, 15, and 9‐fold, respectively. The EC50 values of bexarotene and 9‐cis‐retinoic acid were less than the plasma concentrations of bexarotene (3 μM) and 9‐cis‐retinoic acid (0.5 μM) achieved in cancer patients administered either of these drugs. In contrast to other RXR activators, LG101506, which has no effect on RXR‐RXR homodimer or RXR‐RAR heterodimer and is a selective RXR‐PPAR agonist, decreased AOX1 expression with an EC50 of 1 μM and Emax of 94% decrease. Pretreatment of cells with actinomycin D (a RNA synthesis inhibitor) abolished AOX1 induction by 9‐cis‐retinoic acid, bexarotene, CD3254, SR11237, LG100268, and LG100754, indicating that the induction occurred by a transcriptional mechanism. RXRα mRNA was 6‐fold greater than RXRβ mRNA in MCF‐7 cells. To evaluate the role of RXR isoforms in AOX1 induction, genetic knock‐down approach was used. Transfection of cells with RXRα or RXRβ siRNA decreased the specific RXR mRNA level by 70–80%. RXRα siRNA attenuated AOX1 induction by 9‐cis‐retinoic acid, bexarotene, CD3254, SR11237, LG100268, and LG100754 by 55–70%, whereas RXRβ siRNA attenuated AOX1 induction by only 15–30%. To determine whether RXR transactivates AOX1 promoter, a reporter gene assay was conducted. 9‐Cis‐retinoic acid and selective RXR activators increased luciferase activity in cells transfected with an AOX1 (−1467 to +10) promoter plasmid, indicating that RXR transactivated this region of the promoter. In conclusion, RXR is the first nuclear receptor that transcriptionally regulates AOX1 expression. RXR activators differentially regulate AOX1 expression and this occurs primarily by activating the RXRα isoform. Given that LG100268 is an agonist of RXR homodimer and has no effect on RXR‐RAR heterodimer, whereas LG100754 is a RXR homodimer antagonist and a RXR‐RAR and RXR‐PPAR heterodimer agonist, the results suggest a role of both RXR homodimer and heterodimers in AOX1 induction. Our novel findings suggest interaction between RXR modulators and AOX1‐mediated drug metabolism and toxicity.Support or Funding InformationSingapore Ministry of Education Academic Research Fund Tier 1 and Ministry of Health National Medical Research Council Grants

Regulation of Human Aldehyde Oxidase Expression by Estrogen Receptor Agonists

Human aldehyde oxidase‐1 (AOX1) is a cytosolic molybdo‐flavoenzyme of importance in drug metabolism and toxicity. However, it is not known how the expression of this gene is transcriptionally regulated. Estrogen receptor (ER), which is a nuclear receptor, plays important roles in physiological functions and is implicated in hormonal cancers, but it also regulates the expression of hepatic drug‐metabolizing enzymes. Conflicting microarray data show that AOX1 expression was increased or decreased in breast cancer compared to healthy tissues. We investigated whether ER agonists regulate AOX1 gene expression in a human cell culture model. As analyzed by real‐time polymerase chain reaction assays, MCF‐7 human breast adenocarcinoma cells expressed AOX1 and the basal AOX1 level varied with different types of fetal bovine serum (FBS). 17β‐Estradiol decreased AOX1 mRNA level in ER‐positive MCF‐7 cells, and this decrease was greatest in cells cultured in 10% charcoal‐stripped FBS. Time‐course experiments indicated that 17β‐estradiol decreased AOX1 expression in a time‐dependent manner with a maximum effect occurring after 72 h treatment. Non‐toxic concentrations (as assessed by a cell viability assay) of endogenous estrogens (estrone, 17β‐estradiol, estriol, and estetrol), synthetic estrogens (ethinylestradiol), and phytoestrogens (daidzein and genistein) decreased the expression of AOX1 (by 40–76%) and other known 17β‐estradiol‐regulated genes (SMAD3 and HBP1) and induced known ER‐regulated genes (GREB1 and TFF1) in MCF‐7 cells. Concentration‐response experiment indicated that estrone, 17β‐estradiol, estriol, and estetrol decreased AOX1 expression with EC50 value of 22.5 pM, 1.4 pM, 9.1 pM, and 513.9 pM, respectively, which are physiologically‐relevant concentrations in humans, suggesting that physiological levels of estrogens suppress AOX1 expression. By comparison, ethinylestradiol, daidzein, and genistein decreased AOX1 level with EC50 value of 1.3 pM, 94.6 nM, and 19.4 nM, respectively, which are clinically‐relevant concentrations after administration of the drug or phytoestrogen supplements. The EC50 values for AOX1 suppression and ERα activation by the agonists were well‐correlated. Non‐toxic concentrations of fulvestrant, a ER antagonist, negated both AOX1 suppression and TFF1 induction by estrogens, indicating that ER regulates AOX1 gene. ERα was the major ER isoform expressed in MCF‐7 cells, whereas ERβ was low or negligible, indicating that AOX1 suppression by ER agonists occurred via ERα, but not ERβ, activation in MCF‐7 cells. Suppression of AOX1 by estrogens did not occur in ER‐negative HepG2 human hepatocellular carcinoma cells. In conclusion, ethinylestradiol and 17β‐estradiol are the most potent AOX1 suppressors, followed by estriol, estrone, estetrol, genistein, and daidzein, and the suppression of AOX1 occurs by ERα activation. This is the first study showing hormonal regulation of AOX1 expression and provide insights into potential drug‐chemical interactions. Our findings also imply that interindividual variations in estrogen levels may lead to differences in AOX1 expression and AOX1‐mediated drug metabolism and toxicity.Support or Funding InformationSingapore Ministry of Education Academic Research Fund Tier 1 and Ministry of Health National Medical Research Council Grants

Exploration of Associations Between Deliberate Self-Poisoning and Psychiatric Disorders in Rural Sri Lanka: A Case Control Study

Background: Psychiatric disorders are important predictors of self-harm behaviour. The present study was carried out to determine associations between DSM-IV TR Axis- I & II disorders and deliberate self poisoning (DSP) in a Sri Lankan rural agricultural district. Methods: Patients who had attempted DSP, who resided within the district were selected randomly for the study. Both the cases and age, sex and residential area matched controls were assessed for DSM-IV TR Axis- I & II disorders based on the Structured Clinical Interview for DSM-IV-TR Axis I and II Disorders (SCID I & II) conducted by a specialist psychiatrist. Findings: Cases consisted of 207 (47.3%) males and 231 (52.7%) females. TR axis-I and/or TR axis-II psychiatric diagnoses was found in 89 (25.6%) of cases and 14 (3.2%) controls. Cases with a TR axis-I diagnosis were older (32 and 19 years), p =30 years and having an alcohol use disorder carried a 21 time excess risk for DSP. A fivefold excess risk for DSP was found among 10-19 years old females with borderline personality traits. Interpretations: Depressive disorder and alcohol related disorders were significantly associated with older participants who had attempted DSP. The prevalence of psychiatric disorders among DSP in rural Sri Lanka is significantly lower than reported rates in western countries. Health and research priorities to reduce self-harm in Sri Lanka will also need to address non-psychiatric issues. Trial Registration: (Sri Lanka Clinical Trial Registry No. SLCTR/2010/008). Funding Statement: The study was supported by two Australian National Health and Medical Research Council grants: NHMRC 630650 and NHMRC Fellowship 1059542 (https://www.nhmrc.gov.au/). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: Ethics approval for “A clustered RCT of educational interventions on treatment of patients with acute poisoning in rural Asian hospitals” was obtained from Ethical Review Committee of the Faculty of Medicine, University of Peradeniya. Ethical approval for the sub study was obtained from the human ethics review committee of the Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka.

Increasing the Clinical Psychiatric Knowledge Base About Pathogenic Copy Number Variation.

Specific copy number variants (CNVs) have been robustly associated with intellectual disability, autism, and schizophrenia. Most of the literature focus has been on documenting the existence of these phenomena. There are few data to guide therapeutic choices for these “orphan” diseases. We call for systematic and longitudinal case reports which, if carefully conducted, may provide crucial initial knowledge to guide therapeutics. We provide a step-by-step overview, a tailored set of consensus criteria for high-quality case reports, and a specific set of learning resources.

Profiling the expression and function of oestrogen receptor isoform ER46 in human endometrial tissues and uterine natural killer cells.

STUDY QUESTIONDoes the oestrogen receptor isoform, ER46, contribute to regulation of endometrial function?SUMMARY ANSWERER46 is expressed in endometrial tissues, is the predominant ER isoform in first trimester decidua and is localised to the cell membrane of uterine natural killer (uNK) cells where activation of ER46 increases cell motility.WHAT IS KNOWN ALREADYOestrogens acting via their cognate receptors are essential regulators of endometrial function and play key roles in establishment of pregnancy. ER46 is a 46-kDa truncated isoform of full length ERα (ER66, encoded by ESR1) that contains both ligand- and DNA-binding domains. Expression of ER46 in the human endometrium has not been investigated previously. ER46 is located at the cell membrane of peripheral blood leukocytes and mediates rapid responses to oestrogens. uNK cells are a phenotypically distinct (CD56brightCD16−) population of tissue-resident immune cells that regulate vascular remodelling within the endometrium and decidua. We have shown that oestrogens stimulate rapid increases in uNK cell motility. Previous characterisation of uNK cells suggests they are ER66-negative, but expression of ER46 has not been characterised. We hypothesise that uNK cells express ER46 and that rapid responses to oestrogens are mediated via this receptor.STUDY DESIGN, SIZE, DURATIONThis laboratory-based study used primary human endometrial (n = 24) and decidual tissue biopsies (n = 30) as well as uNK cells which were freshly isolated from first trimester human decidua (n = 18).PARTICIPANTS/MATERIALS, SETTING, METHODSPrimary human endometrial and first trimester decidual tissue biopsies were collected using methods approved by the local institutional ethics committee (LREC/05/51104/12 and LREC/10/51402/59). The expression of ERs (ER66, ER46 and ERβ) was assessed by quantitative PCR, western blot and immunohistochemistry. uNK cells were isolated from first-trimester human decidua by magnetic bead sorting. Cell motility of uNK cells was measured by live cell imaging: cells were treated with 17β-oestradiol conjugated to bovine serum albumin (E2-BSA, 10 nM equivalent), the ERβ-selective agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; 10 nM) or dimethylsulphoxide vehicle control.MAIN RESULTS AND THE ROLE OF CHANCEER46 was detected in proliferative and secretory phase tissues by western blot and was the predominant ER isoform in first-trimester decidua samples. Immunohistochemistry revealed that ER46 was co-localised with ER66 in cell nuclei during the proliferative phase but detected in both the cytoplasm and cell membrane of stromal cells in the secretory phase and in decidua. Triple immunofluorescence staining of decidua tissues identified expression of ER46 in the cell membrane of CD56-positive uNK cells which were otherwise ER66-negative. Profiling of isolated uNK cells confirmed expression of ER46 by quantitative PCR and western blot and localised ER46 protein to the cell membrane by immunocytochemistry. Functional analysis of isolated uNK cells using live cell imaging demonstrated that activation of ER46 with E2-BSA significantly increased uNK cell motility.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONExpression pattern in endometrial tissue was only determined using samples from proliferative and secretory phases. Assessment of first trimester decidua samples was from a range of gestational ages, which may have precluded insights into gestation-specific changes in these tissues. Our results are based on in vitro responses of primary human cells and we cannot be certain that similar mechanisms occur in situ.WIDER IMPLICATIONS OF THE FINDINGSE2 is an essential regulator of reproductive competence. This study provides the first evidence for expression of ER46 in the human endometrium and decidua of early pregnancy. We describe a mechanism for regulating the function of human uNK cells via expression of ER46 and demonstrate that selective targeting with E2-BSA regulates uNK cell motility. These novel findings identify a role for ER46 in the human endometrium and provide unique insight into the importance of membrane-initiated signalling in modulating the impact of E2 on uNK cell function in women. Given the importance of uNK cells to regulating vascular remodelling in early pregnancy and the potential for selective targeting of ER46, this may be an attractive future therapeutic target in the treatment of reproductive disorders.STUDY FUNDING/COMPETING INTEREST(S)These studies were supported by Medical Research Council (MRC) Programme Grants G1100356/1 and MR/N024524/1 to PTKS. H.O.D.C. was supported by MRC grant G1002033. The authors declare no competing interests related to the published work.

The ethics approval process for multisite research studies in Australia: changes sought by the Australian Genomics initiative.

The Australian Genomics Health Alliance is funded by a National Health and Medical Research Council grant (Grant Reference No. 1113531) and the Australian Government’s Medical Research Future Fund

Inclusion health in UK-relevant policy reviews of health inequalities: a thematic, multilevel, governance document analysis

BackgroundSome practitioners and researchers have advocated for greater attention to extreme health disadvantages associated with experiences of social exclusion (eg, homelessness, substance use, or imprisonment), which are not captured by conventional measures of socioeconomic position. It is therefore timely to examine whether such so-called inclusion health concerns feature in health inequalities policy in the UK. We analysed how flagship reviews on health inequalities described, explained, and made recommendations relating to these experiences. MethodsOur purposive sample comprised the five most recent health inequalities policy reviews from different levels of governance in the UK (ie, devolved, national, and international), spanning 2008–14. We undertook thematic analysis using the framework method. The results of line-by-line coding were summarised into framework matrices using NVivo 11 and interpreted using mind-mapping, cross-comparison, and discussion among the project team. FindingsDocuments differed in which experiences were featured and who was constructed as “excluded” or “vulnerable”. Documents proposed various explanations for health inequalities associated with these experiences, from micro (eg, individual behaviours) through meso factors (eg, access to services), to macro forces (eg, globalisation and climate change). Concepts of the life course and so-called intergenerational transmission were also important explanatory themes. Although recommendations spanned all levels from individual to structural, unifying themes included partnership working; early years intervention; and access to health care. Acknowledgement of intersectional aspects of inequalities was largely limited to descriptions and explanations and was less often reflected in recommendations. InterpretationUnderstanding how health inequalities are framed is important, because such framings reflect and shape what the problem is perceived to be, what solutions are proposed, and how success is defined. Although these flagship policy reviews of health inequalities did acknowledge some inclusion health experiences, they used concepts of inclusion, vulnerability, and disadvantage ambiguously. Focus on health care was disproportionate in both explanations and recommendations. To address potential tensions between different framings of health inequalities, policy making in this area would benefit from a clear conceptual framework encompassing diverse forms of social stratification, advantage, and disadvantage. FundingEJT, SVK, and FP are funded by the Medical Research Council grant MC_UU_12017/13 and by the Chief Scientist Office grant SPHSU13. EJT, SVK, and HT are funded by the Medical Research Council grant MC_UU_12017/15 and by the Chief Scientist Office grant SPHSU15. In addition, EJT is funded by a Chief Scientist Office Clinical Academic Fellowship (CAF/17/11) and SVK by an NRS Scottish Senior Clinical Fellowship (SCAF/15/02).

Tissue stiffness at the human maternal-fetal interface.

STUDY QUESTIONWhat is the stiffness (elastic modulus) of human nonpregnant secretory phase endometrium, first trimester decidua, and placenta?SUMMARY ANSWERThe stiffness of decidua basalis, the site of placental invasion, was an order of magnitude higher at 103 Pa compared to 102 Pa for decidua parietalis, nonpregnant endometrium and placenta.WHAT IS KNOWN ALREADYMechanical forces have profound effects on cell behavior, regulating both cell differentiation and migration. Despite their importance, very little is known about their effects on blastocyst implantation and trophoblast migration during placental development because of the lack of mechanical characterization at the human maternal–fetal interface.STUDY DESIGN, SIZE, DURATIONAn observational study was conducted to measure the stiffness of ex vivo samples of human nonpregnant secretory endometrium (N = 5) and first trimester decidua basalis (N = 6), decidua parietalis (N = 5), and placenta (N = 5). The stiffness of the artificial extracellular matrix (ECM), Matrigel®, commonly used to study migration of extravillous trophoblast (EVT) in three dimensions and to culture endometrial and placental organoids, was also determined (N = 5).PARTICIPANTS/MATERIALS, SETTING, METHODSAtomic force microscopy was used to perform ex vivo direct measurements to determine the stiffness of fresh tissue samples. Decidua was stained by immunohistochemistry (IHC) for HLA-G+ EVT to confirm whether samples were decidua basalis or decidua parietalis. Endometrium was stained with hematoxylin and eosin to confirm the presence of luminal epithelium. Single-cell RNA sequencing data were analyzed to determine expression of ECM transcripts by decidual and placental cells. Fibrillin 1, a protein identified by these data, was stained by IHC in decidua basalis.MAIN RESULTS AND THE ROLE OF CHANCEWe observed that decidua basalis was significantly stiffer than decidua parietalis, at 1250 and 171 Pa, respectively (P < 0.05). The stiffness of decidua parietalis was similar to nonpregnant endometrium and placental tissue (250 and 232 Pa, respectively). These findings suggest that it is the presence of invading EVT that is driving the increase in stiffness in decidua basalis. The stiffness of Matrigel® was found to be 331 Pa, significantly lower than decidua basalis (P < 0.05).LARGE SCALE DATAN/ALIMITATIONS, REASONS FOR CAUTIONTissue stiffness was derived by ex vivo measurements on blocks of fresh tissue in the absence of blood flow. The nonpregnant endometrium samples were obtained from women undergoing treatment for infertility. These may not reflect the stiffness of endometrium from normal fertile women.WIDER IMPLICATIONS OF THE FINDINGSThese results provide direct measurements of tissue stiffness during the window of implantation and first trimester of human pregnancy. They serve as a basis of future studies exploring the impact of mechanics on embryo implantation and development of the placenta. The findings provide important baseline data to inform matrix stiffness requirements when developing in vitro models of trophoblast stem cell development and migration that more closely resemble the decidua in vivo.STUDY FUNDING/COMPETING INTEREST(S)This work was supported by the Centre for Trophoblast Research, the Wellcome Trust (090108/Z/09/Z, 085992/Z/08/Z), the Medical Research Council (MR/P001092/1), the European Research Council (772426), an Engineering and Physical Sciences Research Council Doctoral Training Award (1354760), a UK Medical Research Council and Sackler Foundation Doctoral Training Grant (RG70550) and a Wellcome Trust Doctoral Studentship (215226/Z/19/Z).

536P - A prospective study of diffusion-weighted magnetic resonance imaging for predicting outcome following chemoradiotherapy, in squamous cell carcinomas of the anus

BackgroundAnal Squamous Cell Carcinoma (ASCC) is radically treated by chemoradiotherapy (CRT). The use of DW-MRI as a predictive marker of outcome would enable early individualisation of treatment. We aimed to quantify the changes in mean apparent-diffusion-coefficient (ADCMean) between a DW-MRI at diagnosis and on fraction 8-10 of CRT as a biomarker for cellularity, and correlate these with outcome. MethodsThis prospective study recruited patients with ASCC between October 2014 and November 2017. DW-MRI was performed at diagnosis and after fraction 8-10 of radical CRT. A region of interest (ROI) was delineated for all primary tumours and any lymph nodes >2cm on high-resolution T2 –weighted images and propagated to the ADC map. A pre-defined cut-off for percentage change in ADCMean (ΔADC) was set at<20%. Complete response (CR) was assessed 3 months following completion of CRT. Routine clinical follow up data from patients were collected from NHS electronic systems. ResultsTwenty-three of 29 recruited patients underwent paired DW-MRI scans. The median (range) tumour volume was 13.6 cm3 (2.8 cm3 to 84.9 cm3). The median ΔADCMean between scans was 20.7% (95% CI: 12.7%, 34.1%). Nine of 23 (43%) patients had a change of ADCMean <20%. Two patients failed to achieve a CR, with ΔADCMean of 14.6% and 20.3%, respectively. On routine follow up, 2 further patients had relapsed, with ΔADCMean of -1.2% and 6.8%. ConclusionsIn a subset of patients, <20% ΔADCMean on DW-MRI between diagnosis and day 8-10 of CRT was observed. Four patients in the study demonstrated persistent or recurrent disease, all of whom demonstrated an ADC on or below the pre-specified cut-off. Further investigation of the predictive merit of DW-MRI change, and the optimum cut-off is needed in larger cohorts. Clinical trial identificationNCT02145416. Legal entity responsible for the studyUniversity of Oxford. FundingCRUK EPSRC Cancer Imaging Centre in Oxford, the CRUK Oxford Centre and the NIHR.M Hawkins is supported by Medical Research Council grant MC_UU_00001/2. DisclosureR. Muirhead: Honoraria (self), Fee for lecture: Boehringer Ingelheim. V. Goh: Research grant / Funding (self): Siemens. All other authors have declared no conflicts of interest.

A randomized 3-way crossover study indicates that high-protein feeding induces de novo lipogenesis in healthy humans.

BACKGROUNDDietary changes have led to the growing prevalence of type 2 diabetes and nonalcoholic fatty liver disease. A hallmark of both disorders is hepatic lipid accumulation, derived in part from increased de novo lipogenesis. Despite the popularity of high-protein diets for weight loss, the effect of dietary protein on de novo lipogenesis is poorly studied. We aimed to characterize the effect of dietary protein on de novo lipid synthesis.METHODSWe use a 3-way crossover interventional study in healthy males to determine the effect of high-protein feeding on de novo lipogenesis, combined with in vitro models to determine the lipogenic effects of specific amino acids. The primary outcome was a change in de novo lipogenesis–associated triglycerides in response to protein feeding.RESULTSWe demonstrate that high-protein feeding, rich in glutamate, increases de novo lipogenesis–associated triglycerides in plasma (1.5-fold compared with control; P < 0.0001) and liver-derived very low-density lipoprotein particles (1.8-fold; P < 0.0001) in samples from human subjects (n = 9 per group). In hepatocytes, we show that glutamate-derived carbon is incorporated into triglycerides via palmitate. In addition, supplementation with glutamate, glutamine, and leucine, but not lysine, increased triglyceride synthesis and decreased glucose uptake. Glutamate, glutamine, and leucine increased activation of protein kinase B, suggesting that induction of de novo lipogenesis occurs via the insulin signaling cascade.CONCLUSIONThese findings provide mechanistic insight into how select amino acids induce de novo lipogenesis and insulin resistance, suggesting that high-protein feeding to tackle diabetes and obesity requires greater consideration.FUNDINGThe research was supported by UK Medical Research Council grants MR/P011705/1, MC_UP_A090_1006 and MR/P01836X/1. JLG is supported by the Imperial Biomedical Research Centre, National Institute for Health Research (NIHR).

Central Command Increases Muscle Sympathetic Nerve Activity More to Contracting than Non‐Contracting Muscle during Unloaded Rhythmic Exercise in Humans

We have previously shown that the increase in muscle sympathetic nerve activity (MSNA) to contracting muscle during sustained isometric exercise is due primarily to central command, and that the contracting muscle does not exhibit a metaboreflex. Here, we tested the hypothesis that MSNA increases to the contracting muscle also during rhythmic isotonic exercise, in which muscle metabolites will not accumulate because the contraction is performed without external load. MSNA was recorded from the common peroneal nerve in 10 participants and negative‐going sympathetic spikes extracted during 50 cycles of sinusoidal (0.15 Hz) isotonic dorsiflexions of the ipsilateral or contralateral ankle. Electromyographic activity (EMG) was recorded from the tibialis anterior muscle on both sides. Cross‐correlation analysis between the MSNA and EMG revealed a marked cyclic modulation of MSNA to the contracting (ipsilateral) muscle. This modulation, in which MSNA increased during the contraction phase, was three times greater than that to the non‐contracting muscle (modulation index = 27.4 ± 3.2 vs 9.2 ± 1.5 %; P&lt;0.001). There were no differences in either the intensity or magnitude of modulation of EMG during ipsilateral and contralateral contractions. We conclude that, in addition to static isometric exercise, central command increases MSNA to the contracting muscle during rhythmic isotonic exercise.Support or Funding InformationThis work was supported by the National Health &amp; Medical Research Council (Grants 1029782 and 1100040).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.