On the use of multiple imputation to address data missing by design as well as unintended missing data in case-cohort studies with a binary endpoint.

BACKGROUND: Case-cohort studies are conducted within cohort studies, with the defining feature that collection of exposure data is limited to a subset of the cohort, leading to a large proportion of missing data by design. Standard analysis uses inverse probability weighting (IPW) to address this intended missing data, but little research has been conducted into how best to perform analysis when there is also unintended missingness. Multiple imputation (MI) has become a default standard for handling unintended missingness and is typically used in combination with IPW to handle the intended missingness due to the case-control sampling. Alternatively, MI could be used to handle both the intended and unintended missingness. While the performance of an MI-only approach has been investigated in the context of a case-cohort study with a time-to-event outcome, it is unclear how this approach performs with a binary outcome. METHODS: We conducted a simulation study to assess and compare the performance of approaches using only MI, only IPW, and a combination of MI and IPW, for handling intended and unintended missingness in the case-cohort setting. We also applied the approaches to a case study. RESULTS: Our results show that the combined approach is approximately unbiased for estimation of the exposure effect when the sample size is large, and was the least biased with small sample sizes, while MI-only and IPW-only exhibited larger biases in both sample size settings. CONCLUSIONS: These findings suggest that a combined MI/IPW approach should be preferred to handle intended and unintended missing data in case-cohort studies with binary outcomes.

Evaluation of multiple imputation approaches for handling missing covariate information in a case-cohort study with a binary outcome.

BACKGROUND: In case-cohort studies a random subcohort is selected from the inception cohort and acts as the sample of controls for several outcome investigations. Analysis is conducted using only the cases and the subcohort, with inverse probability weighting (IPW) used to account for the unequal sampling probabilities resulting from the study design. Like all epidemiological studies, case-cohort studies are susceptible to missing data. Multiple imputation (MI) has become increasingly popular for addressing missing data in epidemiological studies. It is currently unclear how best to incorporate the weights from a case-cohort analysis in MI procedures used to address missing covariate data. METHOD: A simulation study was conducted with missingness in two covariates, motivated by a case study within the Barwon Infant Study. MI methods considered were: using the outcome, a proxy for weights in the simple case-cohort design considered, as a predictor in the imputation model, with and without exposure and covariate interactions; imputing separately within each weight category; and using a weighted imputation model. These methods were compared to a complete case analysis (CCA) within the context of a standard IPW analysis model estimating either the risk or odds ratio. The strength of associations, missing data mechanism, proportion of observations with incomplete covariate data, and subcohort selection probability varied across the simulation scenarios. Methods were also applied to the case study. RESULTS: There was similar performance in terms of relative bias and precision with all MI methods across the scenarios considered, with expected improvements compared with the CCA. Slight underestimation of the standard error was seen throughout but the nominal level of coverage (95%) was generally achieved. All MI methods showed a similar increase in precision as the subcohort selection probability increased, irrespective of the scenario. A similar pattern of results was seen in the case study. CONCLUSIONS: How weights were incorporated into the imputation model had minimal effect on the performance of MI; this may be due to case-cohort studies only having two weight categories. In this context, inclusion of the outcome in the imputation model was sufficient to account for the unequal sampling probabilities in the analysis model.

Validation of the OWLS, a Screening Tool for Measuring Prescription Opioid Use Disorder in Primary Care.

OBJECTIVE: The OWLS is a screening tool for prescription opioid use disorder designed for use in primary care. This study aimed to confirm the optimal wording, scoring methods, and cutoff for the OWLS. DESIGN AND SETTING: Cross-sectional analysis of an online sample. SUBJECTS: Participants comprised those with chronic noncancer pain who regularly used prescription opioids. METHODS: Eligible participants self-completed an online version of the OWLS prescription opioid use disorder screening tool and the Composite International Diagnostic Interview Substance Abuse module. Receiver operating characteristics were calculated for three scoring methods for the OWLS, and these were compared with DSM-5 classification of any use disorder and moderate to severe opioid use disorder. RESULTS: Among the sample (N = 324), utilizing scoring method (i) (i.e., positive endorsement ≥ response option "a little bit") and a cutoff of 3 increased the percentage of correctly classified participants, with concurrent increases in specificity and decreases in false discovery rate, and false positive rate. CONCLUSION: OWLS utilizing scoring method (i) with a cutoff of 3 was shown to be the optimal version and scoring method of this tool. This represents a time-efficient, simple scoring method, allowing for quick and accurate screening for opioid use disorder to occur.

Validity and Reliability of the Computer-Administered Routine Opioid Outcome Monitoring (ROOM) Tool.

OBJECTIVE: The Routine Opioid Outcome Monitoring (ROOM) tool measures outcomes with opioids using an established framework which includes domains such as pain, mood, opioid use disorder, alcohol use, and constipation. This study aims to validate and establish the test-retest reliability of the computer-administered ROOM tool. DESIGN AND SETTING: Cross-sectional analysis of an online sample. SUBJECTS: Participants comprised those with chronic noncancer pain who regularly used prescription opioids. METHODS: Participants self-completed the online ROOM tool along with other validated measures (validation questionnaire), and those who were agreeable also completed the online test-retest questionnaire approximately two weeks later. Subcomponents of the ROOM tool (i.e., pain, mood, alcohol use, opioid use disorder, and constipation) were validated against longer measures of the same construct using Pearson correlation coefficients. Intraclass correlation coefficients were used to assess the stability of the ROOM tool over time. RESULTS: A total of 324 participants completed the validation questionnaire, of whom 260 also completed the test-retest questionnaire. The opioid use disorder domain showed good sensitivity (73.6) and specificity (75.8) against the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition, any opioid use disorder. All ROOM components showed moderate correlation (r = 0.55-0.73) with their longer counterparts. Test-retest reliability was fair (0.58-0.75), indicating that responses were relatively stable over time. Reliability did vary, however, based on the components being measured and how certain tools were scored. CONCLUSION: The computer-administered ROOM tool is a valid approach for brief monitoring of outcomes with prescribed opioids in primary care settings and appears to be acceptable to people who are using prescribed opioids for chronic pain.

Parental personality and early life ecology: a prospective cohort study from preconception to postpartum.

Personality reliably predicts life outcomes ranging from social and material resources to mental health and interpersonal capacities. However, little is known about the potential intergenerational impact of parent personality prior to offspring conception on family resources and child development across the first thousand days of life. We analysed data from the Victorian Intergenerational Health Cohort Study (665 parents, 1030 infants; est. 1992), a two-generation study with prospective assessment of preconception background factors in parental adolescence, preconception personality traits in young adulthood (agreeableness, conscientiousness, emotional stability, extraversion, and openness), and multiple parental resources and infant characteristics in pregnancy and after the birth of their child. After adjusting for pre-exposure confounders, both maternal and paternal preconception personality traits were associated with numerous parental resources and attributes in pregnancy and postpartum, as well as with infant biobehavioural characteristics. Effect sizes ranged from small to moderate when considering parent personality traits as continuous exposures, and from small to large when considering personality traits as binary exposures. Young adult personality, well before offspring conception, is associated with the perinatal household social and financial context, parental mental health, parenting style and self-efficacy, and temperamental characteristics of offspring. These are pivotal aspects of early life development that ultimately predict a child's long-term health and development.

Identification of 12/15-lipoxygenase as a suppressor of myeloproliferative disease.

Though Abl inhibitors are often successful therapies for the initial stages of chronic myelogenous leukemia (CML), refractory cases highlight the need for novel molecular insights. We demonstrate that mice deficient in the enzyme 12/15-lipoxygenase (12/15-LO) develop a myeloproliferative disorder (MPD) that progresses to transplantable leukemia. Although not associated with dysregulation of Abl, cells isolated from chronic stage 12/15-LO-deficient (Alox15) mice exhibit increased activation of the phosphatidylinositol 3-kinase (PI3-K) pathway, as indicated by enhanced phosphorylation of Akt. Furthermore, the transcription factor interferon consensus sequence binding protein (ICSBP) is hyperphosphorylated and displays decreased nuclear accumulation, translating into increased levels of expression of the oncoprotein Bcl-2. The ICSBP defect, exaggerated levels of Bcl-2, and prolonged leukemic cell survival associated with chronic stage Alox15 MPD are all reversible upon treatment with a PI3-K inhibitor. Remarkably, the evolution of Alox15 MPD to leukemia is associated with additional regulation of ICSBP on an RNA level, highlighting the potential usefulness of the Alox15 model for understanding the transition of CML to crisis. Finally, 12/15-LO expression suppresses the growth of a human CML-derived cell line. These data identify 12/15-LO as an important suppressor of MPD via its role as a critical upstream effector in the regulation of PI3-K-dependent ICSBP phosphorylation.

Longitudinal prediction of periconception alcohol use: a 20-year prospective cohort study across adolescence, young adulthood and pregnancy.

BACKGROUND AND AIMS: Alcohol consumption is common in adolescence and young adulthood and may continue into pregnancy, posing serious risk to early fetal development. We examine the frequency of periconception alcohol use (prior to pregnancy awareness) and the extent to which adolescent and young adult alcohol use prospectively predict periconception use. DESIGN: A longitudinal, population-based study. SETTING: Victoria, Australia. PARTICIPANTS: A total of 289 women in trimester three of pregnancy (age 29-35 years; 388 pregnancies). MEASURES: The main exposures were binge [≥ 4.0 standard drinks (SDs)/day] and frequent (≥ 3 days/week) drinking in adolescence (mean age = 14.9-17.4 years) and young adulthood (mean age 20.7-29.1 years). Outcomes were frequency (≥ 3 days/week, ≥ monthly, never) and quantity (≥ 4.0 SDs, ≥ 0.5 and < 4.0 SDs, none) of periconception drinking. FINDINGS: Alcohol use was common in young adulthood prior to pregnancy (72%) and in the early weeks of pregnancy (76%). The proportions drinking on most days and binge drinking were similar at both points. Reflecting a high degree of continuity in alcohol use behaviours, most women who drank periconceptionally had an earlier history of frequent (77%) and/or binge (85%) drinking throughout the adolescent or young adult years. Young adult binge drinking prospectively predicted periconception drinking quantity [odds ratio (OR) = 3.7, 95% confidence interval (CI) = 1.9-7.4], compared with women with no prior history. Similarly, frequent young adult drinking prospectively predicted frequent periconception drinking (OR = 30.7, 95% CI = 12.3-76.7). CONCLUSIONS: Women who engage in risky (i.e. frequent and binge) drinking in their adolescent and young adult years are more likely to report risky drinking in early pregnancy prior to pregnancy recognition than women with no prior history of risky drinking.

Examining longitudinal associations between self-reported depression, anxiety and stress symptoms and hair cortisol among mothers of young children.

BACKGROUND: Maternal mental health is critically important given its impacts on both women's and children's outcomes. Hair cortisol concentrations (HCC) may provide insight into physiological processes underpinning mental health. This study investigated associations between mothers' self-reported mental health symptoms and their HCC at 1, 2 and 3 years postpartum. METHODS: Longitudinal study of Australian mothers recruited for their experience of adversity in pregnancy ('right@home' trial, N=722). Mental health symptoms were self-reported using the Depression, Anxiety and Stress Scales (DASS). Associations between DASS total and subscale scores and HCC were estimated using linear regression and generalized estimating equation (GEE) models, examining associations: at each age; across all ages (multivariate GEE); and with persistence of high symptom severity. Missing data were addressed using multiple imputation. RESULTS: 546/722 (76%) women provided at least one hair sample (71% at 1, 61% at 2, 49% at 3 years). Associations between DASS total or subscale scores and HCC were not evident across time points. Only dichotomized high depression symptom severity was associated with higher HCC in the GEE models (ß=0.12, p=0.04). There was no evidence of associations between persistence of high DASS symptom severity and HCC at 3 years. LIMITATIONS: The DASS measured self-reported symptoms for the preceding week whereas HCC captured average cortisol over three months. Associations amongst mothers experiencing adversity may not represent patterns in the general population. CONCLUSIONS: Considered in context with existing literature, these findings suggest that HCC provides limited insight into the mental health of mothers experiencing adversity across the early postpartum years.

Cannabis and tobacco use prior to pregnancy and subsequent offspring birth outcomes: a 20-year intergenerational prospective cohort study.

There is increasing evidence that the life-course origins of health and development begin before conception. We examined associations between timing and frequency of preconception cannabis and tobacco use and next generation preterm birth (PTB), low birth weight (LBW) and small for gestational age. 665 participants in a general population cohort were repeatedly assessed on tobacco and cannabis use between ages 14-29 years, before pregnancy. Associations were estimated using logistic regression. Preconception parent (either maternal or paternal) daily cannabis use age 15-17 was associated with sixfold increases in the odds of offspring PTB (aOR 6.65, 95% CI 1.92, 23.09), and offspring LBW (aOR 5.84, 95% CI 1.70-20.08), after adjusting for baseline sociodemographic factors, parent sex, offspring sex, family socioeconomic status, parent mental health at baseline, and concurrent tobacco use. There was little evidence of associations with preconception parental cannabis use at other ages or preconception parental tobacco use. Findings support the hypothesis that the early life origins of growth begin before conception and provide a compelling rationale for prevention of frequent use during adolescence. This is pertinent given liberalisation of cannabis policy.

Preventing postnatal depression: a causal mediation analysis of a 20-year preconception cohort.

Postnatal depression (PND) is common and predicts a range of adverse maternal and offspring outcomes. PND rates are highest among women with persistent mental health problems before pregnancy, and antenatal healthcare provides ideal opportunity to intervene. We examined antenatal perceived social support as a potential intervention target in preventing PND symptoms among women with prior mental health problems. A total of 398 Australian women (600 pregnancies) were assessed repeatedly for mental health problems before pregnancy (ages 14-29 years, 1992-2006), and again during pregnancy, two months postpartum and one year postpartum (2006-2014). Causal mediation analysis found that intervention on perceived antenatal social support has the potential to reduce rates of PND symptoms by up to 3% (from 15 to 12%) in women with persistent preconception symptoms. Supplementary analyses found that the role of low antenatal social support was independent of concurrent antenatal depressive symptoms. Combined, these two factors mediated up to more than half of the association between preconception mental health problems and PND symptoms. Trialling dual interventions on antenatal depressive symptoms and perceived social support represents one promising strategy to prevent PND in women with persistent preconception symptoms. Interventions promoting mental health before pregnancy may yield an even greater reduction in PND symptoms by disrupting a developmental cascade of risks via these and other pathways. This article is part of the theme issue 'Multidisciplinary perspectives on social support and maternal-child health'.

Recent trends in heroin and pharmaceutical opioid-related harms in Victoria, Australia up to 2018.

AIMS: To characterize the trajectory in the years leading up to 2018 in pharmaceutical opioid and heroin morbidity in Victoria, Australia, and to assess the effect on that trajectory of reformulation of oxycodone to a form that could not be easily snorted or injected. DESIGN: Interrupted time-series analyses of population-level data before versus after reformulation of oxycodone, stratified by sex. SETTING: Victoria, Australia. PARTICIPANTS: The population of Victoria aged 12+ years. MEASUREMENTS: Ambulance patient care and emergency department (ED) records were examined using both fixed-code and free-text fields, with each record manually cleaned and checked by trained coders. These were used to derive the output variables providing an index of harm: rates of opioid-related ambulance attendances and ED attendances for pharmaceutical opioids and heroin. The input variable was pre- versus post-oxycodone reformulation. FINDINGS: There were 30 045 opioid-related ambulance attendances from January 2012 to October 2018 (54% heroin-related), and 10 113 ED attendances from July 2008 to June 2018 (39% heroin-related). There was an increase in the rate (events per 100 000 people per year) of all opioid ED attendances from 2008 to 2018 [increase = 0.063; 95% confidence interval (CI) = 0.049, 0.078]. Pharmaceutical opioid ED attendances decreased from 2014 onwards (slope change = -0.083; 95% CI = -0.108, -0.059). Heroin-related ED attendances increased from 2014 to 2018; 11 324 heroin-related ambulance attendances and 1980 ED attendances were observed from April 2014 to June 2018, compared with the respective estimates of 8176, and 1661 had the pre-April 2014 trend continued (ambulance slope change = 0.296, 95% CI = 0.104, 0.489; ED slope change = 0.026, 95% CI = 0.005, 0.046). The inflection point of 2014 coincided with the re-formulation of oxycodone. CONCLUSION: In Victoria, Australia, there appears to have been a trend starting around mid-2014 of increasing heroin-related harm, and a flattening of the increase or a decrease of harms relating to pharmaceutical opioids. These changes may, in part, reflect reformulation of oxycodone to reduce the extent to which it can be injected or snorted.

Comparing rates and characteristics of ambulance attendances related to extramedical use of pharmaceutical opioids in Victoria, Australia from 2013 to 2018.

BACKGROUND AND AIMS: Despite increases in opioid prescribing and related morbidity and mortality, few studies have comprehensively documented harms across opioid types. We examined a population-wide indicator of extramedical pharmaceutical opioid-related harm to determine if the supply-adjusted rates of ambulance presentations, the severity of presentations or other attendance characteristics differed by opioid type. DESIGN: Retrospective observational study of coded ambulance patient care records related to extramedical pharmaceutical opioid use, January 2013 to September 2018. SETTING: Australia CASES: Primary analyses used Victorian data (n = 9823), with available data from other Australian jurisdictions (n = 4338) used to determine generalizability. MEASUREMENTS: We calculated supply-adjusted rates of attendances using Poisson regression, and used multinomial logistic regression to compare demographic, presentation severity, mental health, substance use and other characteristics of attendances associated with seven pharmaceutical opioids. FINDINGS: In Victoria, the highest rates of attendance [per 100 000 oral morphine equivalent mg (OME)] were for codeine (0.273/100 000) and oxycodone (0.113/100 000). The lowest rates were for fentanyl (0.019/100 000) and tapentadol (0.005/100 000). Oxycodone-naloxone rates (0.031/100 000) were lower than for oxycodone as a single ingredient (0.113/100 000). Fentanyl-related attendances were associated with the most severe characteristics, most likely to be an accidental overdose, most likely to have naloxone administered and least likely to be transferred to hospital. In contrast, codeine-related attendances were more likely to involve suicidal thoughts/behaviours, younger females and be transported to hospital. Supply-adjusted attendance rates for individual opioids were stable over time. Victorian states were broadly consistent with non-Victorian states. CONCLUSIONS: In Australia, rates and characteristics of opioid-related harm vary by opioid type. Supply-adjusted ambulance attendance rates appear to be both stable over time and unaffected by large changes in supply.

Parental mental health before and during pregnancy and offspring birth outcomes: A 20-year preconception cohort of maternal and paternal exposure.

BACKGROUND: Preterm birth (PTB) and small for gestational age (SGA) are increasingly prevalent, with major consequences for health and development into later life. There is emerging evidence that some risk processes begin before pregnancy. We report on associations between maternal and paternal common mental disorders (CMD) before and during pregnancy and offspring PTB and SGA. METHODS: 398 women with 609 infants and 267 men with 421 infants were assessed repeatedly for CMD symptoms before pregnancy between age 14 and 29 and during pregnancy. Associations between preconception and antenatal CMD symptoms and offspring gestational age/PTB and size for gestational age/SGA were estimated using linear and Poisson regression. FINDINGS: In men, persistent preconception CMD across adolescence and young adulthood predicted offspring PTB after adjustment for ethnicity, education, BMI and adolescent substance use (adjusted RR 7·0, 95% CI 1·8,26·8), corresponding to a population attributable fraction of 31% of preterm births. In women, antenatal CMD symptoms predicted offspring PTB (adjusted RR 4·4, 95% CI 1·4,14·1). There was little evidence of associations with SGA. INTERPRETATION: This first report of an association between paternal preconception mental health and offspring gestational age, while requiring replication in larger samples, complements earlier work on stress in animals, and further strengthens the case for expanding preconception mental health care to both men and women. FUNDING: National Health and Medical Research Council (Australia), Victorian Health Promotion Foundation, Australian Rotary Health, Colonial Foundation, Perpetual Trustees, Financial Markets Foundation for Children (Australia), Royal Children's Hospital Foundation, Murdoch Children's Research Institute, Australian Research Council.

12/15-lipoxygenase-dependent myeloid production of interleukin-12 is essential for resistance to chronic toxoplasmosis.

Interleukin-12 (IL-12) is critical for resistance to Toxoplasma gondii during both the acute and chronic stages of infection. However, the cellular and molecular pathways that regulate IL-12 production during chronic toxoplasmosis are incompletely defined. We recently discovered that 12/15-lipoxygenase (12/15-LOX), which oxidizes unsaturated lipids in macrophages, is a novel and selective regulator of IL-12 production. We now demonstrate the essential role of this enzyme in the chronic phase of toxoplasmosis. Although 12/15-LOX-deficient mice were resistant to acute T. gondii infection, 80% of 12/15-LOX-deficient mice died during chronic toxoplasmosis, compared to no deaths in wild-type controls. The morbidity of chronically infected 12/15-LOX mice was associated with an increase in brain inflammation and parasite burden. These data suggest that the evolution of the immune response to T. gondii is accompanied by an increasing requirement for 12/15-LOX-mediated signaling. Consistent with this conclusion, 12/15-LOX activity was enhanced during chronic, but not acute, toxoplasmosis. Furthermore, the enhanced susceptibility of 12/15-LOX-deficient mice to chronic toxoplasmosis was associated with reduced production of IL-12 and gamma interferon (IFN-gamma) that was not evident during acute infection. Importantly, ex vivo IFN-gamma production by 12/15-LOX-deficient splenocytes could be rescued by the addition of recombinant IL-12. These data establish that 12/15-LOX is a critical mediator of the chronic type 1 inflammatory response and that immune mediators can be subject to distinct cellular and/or molecular mechanisms of regulation at different stages of inflammation.

CD44 expressed on both bone marrow-derived and non-bone marrow-derived cells promotes atherogenesis in ApoE-deficient mice.

OBJECTIVE: The purpose of this study was to distinguish the contributions of CD44 expressed on bone marrow-derived and non-bone marrow-derived cells to atherosclerosis. METHODS AND RESULTS: Using bone marrow chimeras, we compared the contributions of CD44 expressed on bone marrow-derived cells versus non-bone marrow-derived cells to the vascular inflammation underlying atherosclerosis. We show that CD44 in both bone marrow-derived and non-bone marrow-derived compartments promotes atherosclerosis in apoE-/- mice and mediates macrophage and T cell recruitment to lesions in vivo. We also demonstrate that CD44 on endothelial cells (ECs) as well as on macrophages and T cells enhances leukocyte-endothelial cell adhesion and transendothelial migration in vitro. Furthermore, CD44 on vascular smooth muscle cells (VSMCs) regulates their hyaluronan (HA)-dependent migration. Interestingly, in mice lacking CD44 in both compartments, where we observed the least inflammation, we also observed enhanced fibrous cap formation. CONCLUSIONS: CD44 expressed on bone marrow-derived and non-bone marrow-derived cells both promote atherosclerosis in apoE-deficient mice. Furthermore, CD44 plays a pivotal role in determining the balance between inflammation and fibrosis in atherosclerotic lesions which can impact clinical outcome in humans.

Changes in Australian prescription opioid use following codeine rescheduling: A retrospective study using pharmaceutical benefits data.

PURPOSE: In February 2018, Australia up-scheduled the 'weak' opioid codeine to a prescription only medication. This study aimed to analyse the change in prescribing trends for codeine and other commonly prescribed opioids in Australia following this policy change to determine if removal of over-the-counter codeine resulted in an increase in opioid prescribing. METHODS: Data was obtained through the Australian Government Department of Human Services statistics website, and contained monthly data about subsidised national prescription numbers for codeine, oxycodone, oxycodone-naloxone, tapentadol, tramadol, morphine, and fentanyl, from January 2016 to December 2018. Segmented linear regression accounting for autocorrelation was used to assess the effect of codeine rescheduling on the supply trends of these opioids. RESULTS: Rescheduling codeine to remove over-the-counter (non-prescription) supply does not appear to have had an immediate effect on the prescription rates of codeine, and there is no significant change in these rates in the months following. Analysis of data showed decreasing trends for codeine and most other schedule 8 prescription opioids, with no increase in any prescribed opioids associated with codeine up scheduling. CONCLUSIONS: Despite concerns, substitution of over-the-counter codeine with higher strength prescribed codeine has not been observed at a population level, nor has a shift to other prescribed opioids occurred. Overall, opioid prescribing in Australia has been decreasing since 2016, both for strong and weak opioids.

How prepared are pharmacists to provide over-the-counter naloxone? The role of previous education and new training opportunities.

INTRODUCTION: and Aims: Opioid overdose can be reversed with timely administration of naloxone. In Australia, naloxone was rescheduled from prescription only (S4) to pharmacist only over-the-counter (OTC, S3) in February 2016, increasing access for the general public. A key barrier to naloxone supply by pharmacists is a lack of knowledge, highlighting the role of pharmacist education. Community pharmacists' education, experience, and training preferences related to naloxone provision, overdose, and substance use disorder were examined. METHODS: Online survey data from a national sample of Australian pharmacists on their educational preferences regarding naloxone and overdose prevention, and prior training on substance use disorder (n = 595) was analyzed using bivariate and multivariate regression analysis. Data from qualitative semi-structured telephone interviews with pharmacists about OTC naloxone provision (n = 21) was analyzed using thematic analysis. RESULTS: Most pharmacists (81%, n = 479) were willing to be trained in opioid overdose prevention, with greater willingness to attend training associated with younger age, being female, fewer years of practice, not having attended previous education on substance use disorder, and higher confidence in issues relating to substance use disorder. Qualitative interviews confirmed community pharmacists' willingness to attend training but analysis revealed low awareness, knowledge, and confidence about naloxone and preventing opioid overdose. Most pharmacists preferred online training or webinars. DISCUSSION AND CONCLUSION: Most community pharmacists in Australia are willing to attend training on providing naloxone and preventing opioid overdose. There are opportunities to develop and expand the online presence of training, guidelines, and education materials to facilitate the expanded supply of OTC naloxone.

Comparing rates and characteristics of ambulance attendances related to extramedical use of pharmaceutical opioids in Australia: a protocol for a retrospective observational study.

INTRODUCTION AND AIMS: Extramedical use of, and associated harms with pharmaceutical opioids are common. Analysis of coded ambulance clinical records provides a unique opportunity to examine a national population-level indicator of relative harms. This protocol describes an observational study with three aims: (1) to compare supply adjusted rates of pharmaceutical opioid-related ambulance attendances for buprenorphine, codeine, fentanyl, oxycodone, oxycodone-naloxone, morphine, pethidine, tramadol and tapentadol; (2) to compare presentation characteristics for these commonly used pharmaceutical opioids and (3) to describe the context surrounding ambulance presentations related to oxycodone, a widely used opioid with an established abuse liability, and tapentadol, a more recent 'atypical' opioid on the Australian market, with fewer studies that have directly examined signals of extramedical use. METHOD: Trained coders extract data from clinical records for ambulance presentations relating to extramedical use of commonly used pharmaceutical opioids. These data form the basis of a large, national database that captures alcohol-related and drug-related harms. Supply adjusted rates of presentations will be examined using Poisson regression. Multinomial logistic regression will be used to compare severity and other characteristics of attendances relating to different pharmaceutical opioids. Tapentadol-related and oxycodone-related cases will be qualitatively examined to understand the situationally specific contexts of the ambulance attendances outside of the characteristics captured in routinely coded variables. ETHICS AND DISSEMINATION: Ethics approval related to analysis of ambulance attendance data was obtained from the Eastern Health Human Research Ethics Committee (E122 08-09), with an amendment specific to the qualitative analysis. Findings will be submitted for peer review in 2019. The understanding of risk profiles in real-world settings is of international public health importance. The analysis and publication of findings from this national dataset of clinical records will provide one of the most nuanced analyses to date of relative harms across nine pharmaceutical opioids over a 6-year period.

CD44 regulates vascular gene expression in a proatherogenic environment.

OBJECTIVE: To identify early changes in vascular gene expression mediated by CD44 that promote atherosclerotic disease in apolipoprotein E (apoE)-deficient (apoE-/-) mice. METHODS AND RESULTS: We demonstrate that CD44 is upregulated and functionally activated in aortic arch in the atherogenic environment of apoE-/- mice relative to wild-type (C57BL/6) controls. Moreover, CD44 activation even in apoE-/- mice is selective to lesion-prone regions because neither the thoracic aorta from apoE-/- mice nor the aortic arch of C57BL/6 mice exhibited upregulation of CD44 compared with thoracic aorta of CD57BL/6 mice. Consistent with these observations, gene expression profiling using cDNA microarrays and quantitative polymerase chain reaction revealed that approximately 155 of 19,200 genes analyzed were differentially regulated in the aortic arch, but not in the thoracic aorta, in apoE-/- CD44-/- mice compared with apoE-/- CD44+/+ mice. However, these genes were not regulated by CD44 in the context of a C57BL/6 background, illustrating the selective impact of CD44 on gene expression in a proatherogenic environment. The patterns of differential gene expression implicate CD44 in focal adhesion formation, extracellular matrix deposition, and angiogenesis, processes critical to atherosclerosis. CONCLUSIONS: CD44 is an early mediator of atherogenesis by virtue of its ability to regulate vascular gene expression in response to a proatherogenic environment.

Apolipoprotein E suppresses the type I inflammatory response in vivo.

Apolipoprotein E (apoE) is synthesized in the liver and in macrophages, and it has antiatherogenic properties that are mediated, at least in part, through the regulation of plasma cholesterol homeostasis. Previous data suggest that apoE also has antiinflammatory properties that may contribute to protection against atherosclerosis independent of its role in lipid metabolism. In this study, apoE knockout and C57BL/6 mice were stimulated with low-dose lipopolysaccharide (LPS) and other Toll-like receptor (TLR) agonists. We show that apoE modulates the systemic type I inflammatory response in vivo. The proinflammatory cytokines tumor necrosis factor alpha, interleukin (IL)-6, IL-12, and interferon-gamma were upregulated to a significantly greater extent in apoE-deficient mice than in wild-type mice at both the mRNA and protein levels following administration of LPS. In contrast, hypercholesterolemic low-density lipoprotein receptor/apobec-1 double knockout mice had a similar cytokine response as wild-type mice, eliminating hypercholesterolemia as a cause for the exaggerated cytokine response. Importantly, reconstitution of apoE expression in the liver of apoE-deficient mice normalized the LPS-induced plasma protein levels of IL-12p40. Furthermore, there was selective upregulation of plasma IL-12 in apoE knockout mice by a TLR3 agonist, poly I:C, but not by other TLR agonists, CpG oligonucleotide or Toxoplasma gondii antigen. This implies that apoE selectively regulates TLR4- and TLR3-mediated signaling of IL-12 production. These results indicate that apoE modulates the T helper-1-type immune response in vivo by modulating IL-12 production.