Modulation of human airway smooth muscle biology by human adipocytes.

BACKGROUND: Asthma and obesity, two growing epidemics worldwide, may share an underlying causal relationship. Airway hyperresponsiveness (AHR), a defining component of asthma, has been documented in both 'obese' animal models and non-asthmatic obese individuals. However, there is a paucity of evidence that obesity-derived factors directly affect human airway smooth muscles (ASM). METHODS: Experiments were designed with primary ASM and adipocytes isolated from the same human tissue explants (n = 6). The modulatory effects of human adipocytes extracted from subcutaneous (extrathoracic) and visceral (intrathoracic) depots, on ASM biology was examined with respect to proliferation, migration, contractility and pro-inflammatory cytokine synthesis. RESULTS: Adipocyte-conditioned media as well as myocyte-adipocyte co-cultures failed to show any significant changes in the proliferative or migrational properties of the ASM. Adipocyte-conditioned media also had no effect on the contractility or relaxation of bovine tracheal muscle strips. In contrast, there was a moderate yet significant increase of IL-6 and eotaxin release by ASM incubated with adipocyte-conditioned media (P = 0.0035 and P = 0.0067, vs. control, respectively), thereby further consolidating the altered inflammatory state reported for both diseases. CONCLUSION: We report, for the first time, that adipocytes from either subcutaneous or visceral depots can trigger an inflammatory state in the ASM, with negligible modulatory effects on hyperplasia, hypertrophy or contractile properties.

Factors Associated With Participation in an Emergency Department-Based Take-Home Naloxone Program for At-Risk Opioid Users.

STUDY OBJECTIVE: Although the World Health Organization recommends take-home naloxone to address the increasing global burden of opioid-related deaths, few emergency departments (EDs) offer a take-home naloxone program. We seek to determine the take-home naloxone acceptance rate among ED patients at high risk of opioid overdose and to examine factors associated with acceptance. METHODS: At a single urban ED, consecutive eligible patients at risk of opioid overdose were invited to complete a survey about opioid use, overdose experience, and take-home naloxone awareness, and then offered take-home naloxone. The primary outcome was acceptance of take-home naloxone, including the kit and standardized patient training. Univariate and multivariable logistic analyses were used to evaluate factors associated with acceptance. RESULTS: Of 241 eligible patients approached, 201 (83.4%) completed the questionnaire. Three-quarters of respondents used injection drugs, 37% were women, and 26% identified as "Indigenous." Of 201 respondents, 137 (68.2%; 95% confidence interval [CI] 61.7% to 74.7%) accepted take-home naloxone. Multivariable analysis revealed that factors associated with take-home naloxone acceptance included witnessing overdose in others (odds ratio [OR] 4.77; 95% CI 2.25 to 10.09), concern about own overdose death (OR 3.71; 95% CI 1.34 to 10.23), female sex (OR 2.50; 95% CI 1.21 to 5.17), and injection drug use (OR 2.22; 95% CI 1.06 to 4.67). CONCLUSION: A two-thirds ED take-home naloxone acceptance rate in patients using opioids should encourage all EDs to dispense take-home naloxone. ED-based take-home naloxone programs have the potential to improve access to take-home naloxone and awareness in individuals most vulnerable to overdoses.

PPAR ligands decrease human airway smooth muscle cell migration and extracellular matrix synthesis.

Airway smooth muscle cells produce extracellular matrix proteins, which in turn can promote smooth muscle survival, proliferation and migration. Currently available therapies have little effect on airway smooth muscle matrix production and migration. Peroxisome proliferator-activated receptor (PPAR) ligands are reported to decrease migration and matrix production in various cell lines. In this study, we examined the effect of PPAR ligands on human airway smooth muscle (HASM) matrix production and migration. PPAR expression was examined by RT-PCR and Western blotting. Endogenous PPAR activity was examined by transfecting cells with a PPAR response element-luciferase reporter plasmid. We observed that HASM cells express PPARα, ß and γ. A six-fold induction of luciferase activity was observed by stimulating cells with a pan-agonist, indicating endogenous PPAR activity. The PPAR ligands ciglitazone, 15-deoxy-Δ12,14-prostaglandin J(2) and WY-14643 decreased migration towards platelet-derived growth factor receptor. This was not mediated by inhibiting Akt phosphorylation or promoting PTEN activity, but partly through cyclooxygenase-2 induction and prostaglandin E(2) production that increased cyclic AMP levels in the cells. All three ligands also caused an inhibition of collagen and fibronectin secretion by cultured smooth muscle cells. We conclude that PPAR ligands decrease HASM migration and matrix production and are, therefore, potentially useful for modulating airway remodelling.

Enhancing Knowledge and Attitudes Regarding Opioid Use Disorder Among Private Primary Care Clinics: A Quality Improvement Project.

ABSTRACT: Opioid use disorder (OUD) continues to impact communities worldwide. British Columbia specifically declared a public health emergency in April 2016. It is known that patients with OUD often experience barriers in access to care, including limited knowledge and training among providers, as well as persisting stigma in the medical community. The Doctor of Nursing Practice quality improvement project sought to provide barrier-targeted OUD education while using multiple effective teaching methods, such as test-enhanced learning, to family nurse practitioners (FNPs) working among private primary care clinics to assess the impact on knowledge and attitudes. In review of an experience survey, zero participants had received prior education on OUD (N = 7). The Drug and Drug Problems Perceptions Questionnaire was used to assess attitudes. In review of the data, attitudes before receiving education (Mdn = 74) improved after receiving barrier-targeted education (Mdn = 66), W = 0, p < .05. Knowledge was tested at three time points. After a review of unique identifiers, four participant tests were successfully linked. It was found that knowledge after receiving education (M = 7.75, Mdn = 7.5) improved in comparison with baseline knowledge (M = 6, Mdn = 6) and further improved after a 1-month time frame (M = 8.5, Mdn = 8.5). Although the project was limited by sample size, providing education to FNPs who have not received prior education on OUD, and using modalities such as test-enhanced learning, showed a favorable impact on knowledge and attitudes. In light of the opioid epidemic, nursing leaders must continue to actively engage practicing FNPs and students with OUD education. FNPs are well positioned to be champions in this area and may mobilize teams to overcome barriers among private primary care clinics and increase access to care.

Advancing virtual primary care for people with opioid use disorder (VPC OUD): a mixed-methods study protocol.

INTRODUCTION: The emergence of COVID-19 introduced a dual public health emergency in British Columbia, which was already in the fourth year of its opioid-related overdose crisis. The public health response to COVID-19 must explicitly consider the unique needs of, and impacts on, communities experiencing marginalisation including people with opioid use disorder (PWOUD). The broad move to virtual forms of primary care, for example, may result in changes to healthcare access, delivery of opioid agonist therapies or fluctuations in co-occurring health problems that are prevalent in this population. The goal of this mixed-methods study is to characterise changes to primary care access and patient outcomes following the rapid introduction of virtual care for PWOUD. METHODS AND ANALYSIS: We will use a fully integrated mixed-methods design comprised of three components: (a) qualitative interviews with family physicians and PWOUD to document experiences with delivering and accessing virtual visits, respectively; (b) quantitative analysis of linked, population-based administrative data to describe the uptake of virtual care, its impact on access to services and downstream outcomes for PWOUD; and (c) facilitated deliberative dialogues to co-create educational resources for family physicians, PWOUD and policymakers that promote equitable access to high-quality virtual primary care for this population. ETHICS AND DISSEMINATION: Approval for this study has been granted by Research Ethics British Columbia. We will convene PWOUD and family physicians for deliberative dialogues to co-create educational materials and policy recommendations based on our findings. We will also disseminate findings via traditional academic outputs such as conferences and peer-reviewed publications.

Molecular motors: how to make models that can be used to convey the concept of molecular ratchets and thermal capture.

A wide variety of cellular processes use molecular motors, including processive motors that move along some form of track (e.g., myosin with actin, kinesin or dynein with tubulin) and polymerases that move along a template (e.g., DNA and RNA polymerases, ribosomes). In trying to understand how these molecular motors actually move, many apply their understanding of how man-made motors work: the latter use some form of energy to exert a force or torque on its load. However, quite a different mechanism has been proposed to possibly account for the movement of molecular motors. Rather than hydrolyzing ATP to push or pull their load, they might use their own thermal vibrational energy as well as that of their load and their environment to move the load, capturing those movements that occur along a desired vector or axis and resisting others; ATP hydrolysis is required to make backward movements impossible. This intriguing thermal capture or Brownian ratchet model is relatively more difficult to convey to students. In this report, we describe several teaching aids that are very easily constructed using widely available household materials to convey the concept of a molecular ratchet.

Predicting Toxicity and Response to Pembrolizumab Through Germline Genomic HLA Class 1 Analysis.

Background: Human leukocyte antigen class 1 (HLA-1)-dependent immune activity is linked to autoimmune diseases. HLA-1-dependent CD8+ T cells are required for immune checkpoint blockade antitumor activity. It is unknown if HLA-1 genotype is predictive of toxicity to immune checkpoint blockade. Methods: Patients with advanced solid tumors stratified into 5 cohorts received single agent pembrolizumab (anti-programmed cell death-1) 200 mg intravenously every 3 weeks in an investigator-initiated phase II trial (Investigator-Initiated Phase II Study of Pembrolizumab Immunological Response Evaluation study, NCT02644369). Germline whole-exome sequencing of peripheral blood mononuclear cells was performed using the Illumina HiSeq2500 platform. HLA-1 haplotypes were predicted from whole-exome sequencing using HLAminer and HLAVBSeq. Heterozygosity of HLA-A, -B, and -C, individual HLA-1 alleles, and HLA haplotype dimorphism at positions -21 M and -21 T of the HLA-A and -B leader sequence were analyzed as predictors of toxicity defined as grade 2 or greater immune-related adverse events and clinical benefit defined as complete or partial response, or stable disease for 6 or more cycles of pembrolizumab. Statistical significance tests were 2-sided. Results: In the overall cohort of 101 patients, the frequency of toxicity and clinical benefit from pembrolizumab was 22.8% and 25.7%, respectively. There was no association between any of the HLA-1 loci or alleles with toxicity. HLA-C heterozygosity had an association with decreased clinical benefit relative to HLA-C homozygosity when controlling for cohort (odds ratio = 0.28, 95% confidence interval = 0.09 to 0.91, P = .04). HLA-A and -B haplotype -21 M/T dimorphism and heterozygosity of HLA-A, -B, and -C were not predictive of outcomes. Conclusions: HLA-C heterozygosity may predict decreased response to pembrolizumab. Prospective validation is required.

Centromeric cohesion failure invokes a conserved choreography of chromosomal mis-segregations in pancreatic neuroendocrine tumor.

BACKGROUND: Pancreatic neuroendocrine tumors (PANETs) are rare, slow growing cancers that often present with local and distant metastasis upon detection. PANETS contain distinct karyotypes, epigenetic dysregulation, and recurrent mutations in MEN1, ATRX, and DAXX (MAD+); however, the molecular basis of disease progression remains uncharacterized. METHODS: We evaluated associations between aneuploidy and the MAD+ mutational state of 532 PANETs from 11 published genomic studies and 19 new cases using a combination of exome, targeted panel, shallow WGS, or RNA-seq. We mapped the molecular timing of MAD+ PANET progression using cellular fractions corrected for inferred tumor content. RESULTS: In 287 PANETs with mutational data, MAD+ tumors always exhibited a highly recurrent signature of loss of heterozygosity (LOH) and copy-number alterations affecting 11 chromosomes, typically followed by genome doubling upon metastasis. These LOH chromosomes substantially overlap with those that undergo non-random mis-segregation due to ectopic CENP-A localization to flanking centromeric regions in DAXX-depleted cell lines. Using expression data from 122 PANETs, we found decreased gene expression in the regions immediately adjacent to the centromere in MAD+ PANETs. Using 43 PANETs from AACR GENIE, we inferred this signature to be preceded by mutations in MEN1, ATRX, and DAXX. We conducted a meta-analysis on 226 PANETs from 8 CGH studies to show an association of this signature with metastatic incidence. Our study shows that MAD+ tumors are a genetically diverse and aggressive subtype of PANETs that display extensive chromosomal loss after MAD+ mutation, which is followed by genome doubling. CONCLUSIONS: We propose an evolutionary model for a subset of aggressive PANETs that is initiated by mutation of MEN1, ATRX, and DAXX, resulting in defects in centromere cohesion from ectopic CENP-A deposition that leads to selective loss of chromosomes and the LOH phenotype seen in late-stage metastatic PANETs. These insights aid in disease risk stratification and nominate potential therapeutic vulnerabilities to treat this disease.

Latency and interval therapy affect the evolution in metastatic colorectal cancer.

While comparison of primary tumor and metastases has highlighted genomic heterogeneity in colorectal cancer (CRC), previous studies have focused on a single metastatic site or limited genomic testing. Combining data from whole exome and ultra-deep targeted sequencing, we explored possible evolutionary trajectories beyond the status of these mutations, particularly among patient-matched metastatic tumors. Our findings confirm the persistence of known clinically-relevant mutations (e.g., those of RAS family of oncogenes) in CRC primary and metastases, yet reveal that latency and interval systemic therapy affect the course of evolutionary events within metastatic lesions. Specifically, our analysis of patient-matched primary and multiple metastatic lesions, developed over time, showed a similar genetic composition for liver metastatic tumors, which were 21-months apart. This genetic makeup was different from those identified in lung metastases developed before manifestation of the second liver metastasis. These results underscore the role of latency in the evolutionary path of metastatic CRC and may have implications for future treatment options.

Development and Evaluation of the Online Addiction Medicine Certificate: Free Novel Program in a Canadian Setting.

BACKGROUND: Despite the enormous bur-den of disease attributable to drug and alcohol addiction, there remain major challenges in implementing evi-dence-based addiction care and treatment modalities. This is partly because of a persistent lack of accessible, specialized training in addiction medicine. In response, a new online certificate in addiction medicine has been established in Vancouver, Canada, free of charge to participants globally. OBJECTIVE: The objective of this study was to evaluate and examine changes in knowledge acquisition among health care professionals before and after the completion of an online certificate in addiction medicine. METHODS: Learners enrolled in a 17-module certificate program and completed pre- and postknowledge tests using online multiple-choice questionnaires. Knowledge acquisition was then evaluated using a repeated measures t test of mean test scores before and after the online course. Following the certificate completion, a subset of learners completed the online course evaluation form. RESULTS: Of the total 6985 participants who registered for the online course between May 15, 2017 and February 22, 2018, 3466 (49.62%) completed the online pretest questionnaire. A total of 1010 participants completed the full course, achieving the required 70% scores. TThe participants self-reported working in a broad range of health-related fields, including nursing (n=371), medicine (n=92), counseling or social work (n=69), community health (n=44), and pharmacy (n=34). The median graduation year was 2010 (n=363, interquartile range 2002-2015). Knowledge of the addiction medicine increased significantly postcertificate (mean difference 28.21; 95% CI 27.32 to 29.10; P<.001). Physicians scored significantly higher on the pretest than any other health discipline, whereas the greatest improvement in scores was seen in the counseling professions and community outreach. CONCLUSIONS: This free, online, open-access certificate in addiction medicine appeared to improve knowledge of learners from a variety of disciplines and backgrounds. Scaling up low threshold learning opportunities may further advance addiction medicine training, thereby helping to narrow the evidence-to-practice gap.

Yes, not now, or never: an analysis of reasons for refusing or accepting emergency department-based take-home naloxone.

OBJECTIVE: Take-home naloxone (THN) reduces deaths from opioid overdose. To increase THN distribution to at-risk emergency department (ED) patients, we explored reasons for patients' refusing or accepting THN. METHODS: In an urban teaching hospital ED, we identified high opioid overdose risk patients according to pre-specified criteria. We offered eligible patients THN and participation in researcher-administered surveys, which inquired about reasons to refuse or accept THN and about THN dispensing location preferences. We analyzed refusal and acceptance reasons in open-ended responses, grouped reasons into categories (absolute versus conditional refusals,) then searched for associations between patient characteristics and reasons. RESULTS: Of 247 patients offered THN, 193 (78.1%) provided reasons for their decision. Of those included, 69 (35.2%) were female, 91 (47.2%) were under age 40, 61 (31.6%) were homeless, 144 (74.6%) reported injection drug use (IDU), and 131 (67.9%) accepted THN. Of 62 patients refusing THN, 19 (30.7%) felt "not at risk" for overdose, while 28 (45.2%) gave conditional refusal reasons: "too sick," "in a rush," or preference to get THN elsewhere. Non-IDU was associated with stating "not at risk," while IDU, homelessness, and age under 40 were associated with conditional refusals. Among acceptances, 86 (65.7%) mentioned saving others as a reason. Most respondents preferred other dispensing locations beside the ED, whether or not they accepted ED THN. CONCLUSION: ED patients refusing THN felt "not at risk" for overdose or felt their ED visit was not the right time or place for THN. Most accepting THN wanted to save others.

An interim report on the investigator-initiated phase 2 study of pembrolizumab immunological response evaluation (INSPIRE).

BACKGROUND: Immune checkpoint inhibitors (ICIs) demonstrate unprecedented efficacy in multiple malignancies; however, the mechanisms of sensitivity and resistance are poorly understood and predictive biomarkers are scarce. INSPIRE is a phase 2 basket study to evaluate the genomic and immune landscapes of peripheral blood and tumors following pembrolizumab treatment. METHODS: Patients with incurable, locally advanced or metastatic solid tumors that have progressed on standard therapy, or for whom no standard therapy exists or standard therapy was not deemed appropriate, received 200 mg pembrolizumab intravenously every three weeks. Blood and tissue samples were collected at baseline, during treatment, and at progression. One core biopsy was used for immunohistochemistry and the remaining cores were pooled and divided for genomic and immune analyses. Univariable analysis of clinical, genomic, and immunophenotyping parameters was conducted to evaluate associations with treatment response in this exploratory analysis. RESULTS: Eighty patients were enrolled from March 21, 2016 to June 1, 2017, and 129 tumor and 382 blood samples were collected. Immune biomarkers were significantly different between the blood and tissue. T cell PD-1 was blocked (≥98%) in the blood of all patients by the third week of treatment. In the tumor, 5/11 (45%) and 11/14 (79%) patients had T cell surface PD-1 occupance at weeks six and nine, respectively. The proportion of genome copy number alterations and abundance of intratumoral 4-1BB+ PD-1+ CD8 T cells at baseline (P < 0.05), and fold-expansion of intratumoral CD8 T cells from baseline to cycle 2-3 (P < 0.05) were associated with treatment response. CONCLUSION: This study provides technical feasibility data for correlative studies. Tissue biopsies provide distinct data from the blood and may predict response to pembrolizumab.

Minimally Invasive Real-Time Detection of Actionable Mutations in Patients With Metastatic Solid Tumors Using Fine-Needle and Liquid Biopsies.

PURPOSE: Fine-needle biopsy (FNB) and liquid biopsy are minimally invasive methods of tumor sampling that provide feasible means to assess tumor genotypes in real time. However, more data are needed to establish the strength of these methods by benchmarking against the current gold standard methods, core-needle biopsy (CNB) or surgical excision of the tumor. PATIENTS AND METHODS: Eligible patients with advanced solid tumors were prospectively recruited. We performed mutation profiling using matched tumor DNA obtained by CNB, FNB and liquid biopsy, and matrix-assisted laser desorption/ionization time-of-flight custom mass-spectrometry or targeted next-generation DNA sequencing. The actionability of detected mutations was determined using the OncoKB Web tool. Agreement between mutations detected in CNBs, FNBs, and circulating tumor DNA (ctDNA) was examined. RESULTS: Forty-one patients underwent tumor biopsy. Thirty CNBs (73%) and 34 FNBs (83%) had sufficient tumor and DNA for mutation profiling. Median DNA yield from CNB and FNB were 775 ng (interquartile range, 240 to 347 4ng) and 649 ng (interquartile range, 180 to1350 ng), respectively. Of 29 CNB/FNB pairs available for comparison, actionable mutation results were concordant in 28 (96%). Six of nine actionable mutations (67%) that were found by CNB, FNB, or both were detectable in ctDNA. Two additional actionable mutations were found exclusively in ctDNA. CONCLUSION: Optimally processed FNB and liquid biopsy can be used routinely for tumor mutation profiling to identify actionable mutations.