TACTICS - Trial of Advanced CT Imaging and Combined Education Support for Drip and Ship: evaluating the effectiveness of an 'implementation intervention' in providing better patient access to reperfusion therapies: protocol for a non-randomised controlled stepped wedge cluster trial in acute stroke.

INTRODUCTION: Stroke reperfusion therapies, comprising intravenous thrombolysis (IVT) and/or endovascular thrombectomy (EVT), are best practice treatments for eligible acute ischemic stroke patients. In Australia, EVT is provided at few, mainly metropolitan, comprehensive stroke centres (CSC). There are significant challenges for Australia's rural and remote populations in accessing EVT, but improved access can be facilitated by a 'drip and ship' approach. TACTICS (Trial of Advanced CT Imaging and Combined Education Support for Drip and Ship) aims to test whether a multicomponent, multidisciplinary implementation intervention can increase the proportion of stroke patients receiving EVT. METHODS AND ANALYSIS: This is a non-randomised controlled, stepped wedge trial involving six clusters across three Australian states. Each cluster comprises one CSC hub and a minimum of three primary stroke centre (PSC) spokes. Hospitals will work in a hub and spoke model of care with access to a multislice CT scanner and CT perfusion image processing software (MIStar, Apollo Medical Imaging). The intervention, underpinned by behavioural theory and technical assistance, will be allocated sequentially, and clusters will move from the preintervention (control) period to the postintervention period. PRIMARY OUTCOME: Proportion of all stroke patients receiving EVT, accounting for clustering. SECONDARY OUTCOMES: Proportion of patients receiving IVT at PSCs, proportion of treated patients (IVT and/or EVT) with good (modified Rankin Scale (mRS) score 0-2) or poor (mRS score 5-6) functional outcomes and European Quality of Life Scale scores 3 months postintervention, proportion of EVT-treated patients with symptomatic haemorrhage, and proportion of reperfusion therapy-treated patients with good versus poor outcome who presented with large vessel occlusion at spokes. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the Hunter New England Human Research Ethics Committee (18/09/19/4.13, HREC/18/HNE/241, 2019/ETH01238). Trial results will be disseminated widely through published manuscripts, conference presentations and at national and international platforms regardless of whether the trial was positive or neutral. TRIAL REGISTRATION NUMBER: ACTRN12619000750189; UTNU1111-1230-4161.

Blockade of High-Fat Diet Proteomic Phenotypes Using Exercise as Prevention or Treatment.

The increasing consumption of high-fat foods combined with a lack of exercise is a major contributor to the burden of obesity in humans. Aerobic exercise such as running is known to provide metabolic benefits, but how the overconsumption of a high-fat diet (HFD) and exercise interact is not well characterized at the molecular level. Here, we examined the plasma proteome in mice for the effects of aerobic exercise as both a treatment and as a preventative regimen for animals on either a HFD or a healthy control diet. This analysis detected large changes in the plasma proteome induced by the HFD, such as increased abundance of SERPINA7, ALDOB, and downregulation of SERPINA1E and complement factor D (CFD; adipsin). Some of these changes were significantly reverted using exercise as a preventative measure but not as a treatment regimen. To determine if either the intensity or duration of exercise influenced the outcome, we compared high-intensity interval training and endurance running. Endurance running slightly outperformed high-intensity interval training exercise, but overall, both provided similar reversion in abundance of plasma proteins modulated by the HFD, including SERPINA7, apolipoprotein E, SERPINA1E, and CFD. Finally, we compared the changes induced by overconsumption of a HFD with previous data from mice fed on an isocaloric high-saturated fatty acid or polyunsaturated fatty acid diet. This identified several common changes, including not only increased apolipoprotein C-II and apolipoprotein E but also highlighted changes specific for overconsumption of a HFD (fructose-bisphosphate aldolase B, SERPINA7, and CFD), saturated fatty acid-based diets (SERPINA1E), or polyunsaturated fatty acid-based diets (haptoglobin). Together, these data highlight the importance of early intervention with exercise to revert HFD-induced phenotypes and suggest some of the molecular mechanisms leading to the changes in the plasma proteome generated by HFD consumption. Web-based interactive visualizations are provided for this dataset (larancelab.com/hfd-exercise), which give insight into diet and exercise phenotypic interactions on the plasma proteome.

Multi-omics Analysis of the Intermittent Fasting Response in Mice Identifies an Unexpected Role for HNF4α.

Every-other-day fasting (EODF) is an effective intervention for the treatment of metabolic disease, including improvements in liver health. But how the liver proteome is reprogrammed by EODF is currently unknown. Here, we use EODF in mice and multi-omics analysis to identify regulated pathways. Many changes in the liver proteome are distinct after EODF and absent after a single fasting bout. Key among these is the simultaneous induction by EODF of de novo lipogenesis and fatty acid oxidation enzymes. Together with activation of oxidative stress defenses, this contributes to the improvements in glucose tolerance and lifespan after EODF. Enrichment analysis shows unexpected downregulation of HNF4α targets by EODF, and we confirm HNF4α inhibition. Suppressed HNF4α targets include bile synthetic enzymes and secreted proteins, such as α1-antitrypsin or inflammatory factors, which reflect EODF phenotypes. Interactive online access is provided to a data resource (https://www.larancelab.com/eodf), which provides a global view of fasting-induced mechanisms in mice.

Sex-specific metabolic responses to 6 hours of fasting during the active phase in young mice.

KEY POINTS: Night time/active phase food restriction for 6 h impaired glucose intolerance in young male and female mice. Females displayed increased capacity for lipogenesis and triglyceride storage in response to a short daily fast. Females had lower fasting insulin levels and an increased potential for utilizing fat for energy through ß-oxidation compared to males. The need for the inclusion of both sexes, and the treatment of sex as an independent variable, is emphasized within the context of this fasting regime. ABSTRACT: There is growing interest in understanding the mechanistic significance and benefits of fasting physiology in combating obesity. Increasing the fasting phase of a normal day can promote restoration and repair mechanisms that occur during the post-absorptive period. Most studies exploring the effect of restricting food access on mitigating obesity have done so with a large bias towards the use of male mice. Here, we disentangle the roles of sex, food intake and food withdrawal in the response to a short-term daily fasting intervention, in which food was removed for 6 h in the dark/active phase of young, 8-week-old mice. We showed that the removal of food during the dark phase impaired glucose tolerance in males and females, possibly due to the circadian disruption induced by this feeding protocol. Although both sexes demonstrated similar patterns of food intake, body composition and various metabolic markers, there were clear sex differences in the magnitude and extent of these responses. While females displayed enhanced capacity for lipogenesis and triglyceride storage, they also had low fasting insulin levels and an increased potential for utilizing available energy sources such as fat for energy through ß-oxidation. Our results highlight the intrinsic biological and metabolic disparities between male and female mice, emphasizing the growing need for the inclusion of both sexes in scientific research. Furthermore, our results illustrate sex-specific metabolic pathways that regulate lipogenesis, obesity and overall metabolic health.

Small-protein Enrichment Assay Enables the Rapid, Unbiased Analysis of Over 100 Low Abundance Factors from Human Plasma.

Unbiased and sensitive quantification of low abundance small proteins in human plasma (e.g. hormones, immune factors, metabolic regulators) remains an unmet need. These small protein factors are typically analyzed individually and using antibodies that can lack specificity. Mass spectrometry (MS)-based proteomics has the potential to address these problems, however the analysis of plasma by MS is plagued by the extremely large dynamic range of this body fluid, with protein abundances spanning at least 13 orders of magnitude. Here we describe an enrichment assay (SPEA), that greatly simplifies the plasma dynamic range problem by enriching small-proteins of 2-10 kDa, enabling the rapid, specific and sensitive quantification of >100 small-protein factors in a single untargeted LC-MS/MS acquisition. Applying this method to perform deep-proteome profiling of human plasma we identify C5ORF46 as a previously uncharacterized human plasma protein. We further demonstrate the reproducibility of our workflow for low abundance protein analysis using a stable-isotope labeled protein standard of insulin spiked into human plasma. SPEA provides the ability to study numerous important hormones in a single rapid assay, which we applied to study the intermittent fasting response and observed several unexpected changes including decreased plasma abundance of the iron homeostasis regulator hepcidin.

Proteomic Analysis of Human Plasma during Intermittent Fasting.

Intermittent fasting (IF) increases lifespan and decreases metabolic disease phenotypes and cancer risk in model organisms, but the health benefits of IF in humans are less clear. Human plasma derived from clinical trials is one of the most difficult sample sets to analyze using mass spectrometry-based proteomics due to the extensive sample preparation required and the need to process many samples to achieve statistical significance. Here, we describe an optimized and accessible device (Spin96) to accommodate up to 96 StageTips, a widely used sample preparation medium enabling efficient and consistent processing of samples prior to LC-MS/MS. We have applied this device to the analysis of human plasma from a clinical trial of IF. In this longitudinal study employing 8-weeks IF, we identified significant abundance differences induced by the IF intervention, including increased apolipoprotein A4 (APOA4) and decreased apolipoprotein C2 (APOC2) and C3 (APOC3). These changes correlated with a significant decrease in plasma triglycerides after the IF intervention. Given that these proteins have a role in regulating apolipoprotein particle metabolism, we propose that IF had a positive effect on lipid metabolism through modulation of HDL particle size and function. In addition, we applied a novel human protein variant database to detect common protein variants across the participants. We show that consistent detection of clinically relevant peptides derived from both alleles of many proteins is possible, including some that are associated with human metabolic phenotypes. Together, these findings illustrate the power of accessible workflows for proteomics analysis of clinical samples to yield significant biological insight.

TRAIL signaling is proinflammatory and proviral in a murine model of rhinovirus 1B infection.

the aim of this study is to elucidate the role of TRAIL during rhinovirus (RV) infection in vivo. Naïve wild-type and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-deficient (Tnfsf10-/-) BALB/c mice were infected intranasally with RV1B. In separate experiments, Tnfsf10-/- mice were sensitized and challenged via the airway route with house dust mite (HDM) to induce allergic airways disease and then challenged with RVIB or UV-RVIB. Airway hyperreactivity (AHR) was invasively assessed as total airways resistance in response to increasing methacholine challenge and inflammation was assessed in bronchoalveolar lavage fluid at multiple time points postinfection. Chemokines were quantified by ELISA of whole lung lysates and viral load was determined by quantitative RT-PCR and tissue culture infective dose (TCID50). Human airway epithelial cells (BEAS2B) were infected with RV1B and stimulated with recombinant TRAIL or neutralizing anti-TRAIL antibodies and viral titer assessed by TCID50 HDM-challenged Tnfsf10-/- mice were protected against RV-induced AHR and had suppressed cellular infiltration in the airways upon RV infection. Chemokine C-X-C-motif ligand 2 (CXCL2) production was suppressed in naïve Tnfsf10-/- mice infected with RV1B, with less RV1B detected 24 h postinfection. This was associated with reduced apoptotic cell death and a reduction of interferon (IFN)-λ2/3 but not IFN-α or IFN-ß. TRAIL stimulation increased, whereas anti-TRAIL antibodies reduced viral replication in RV1B-infected BEAS2B cells in vitro. In conclusion, TRAIL promotes RV-induced AHR, inflammation and RV1B replication, implicating this molecule and its downstream signaling pathways as a possible target for the amelioration of RV1B-induced allergic and nonallergic lung inflammation and AHR.

Toll-like receptor 7 governs interferon and inflammatory responses to rhinovirus and is suppressed by IL-5-induced lung eosinophilia.

BACKGROUND: Asthma exacerbations represent a significant disease burden and are commonly caused by rhinovirus (RV), which is sensed by Toll-like receptors (TLR) such as TLR7. Some asthmatics have impaired interferon (IFN) responses to RV, but the underlying mechanisms of this clinically relevant observation are poorly understood. OBJECTIVES: To investigate the importance of intact TLR7 signalling in vivo during RV exacerbation using mouse models of house dust mite (HDM)-induced allergic airways disease exacerbated by a superimposed RV infection. METHODS: Wild-type and TLR7-deficient (Tlr7(-/-)) BALB/c mice were intranasally sensitised and challenged with HDM prior to infection with RV1B. In some experiments, mice were administered recombinant IFN or adoptively transferred with plasmacytoid dendritic cells (pDC). RESULTS: Allergic Tlr7(-/-) mice displayed impaired IFN release upon RV1B infection, increased virus replication and exaggerated eosinophilic inflammation and airways hyper reactivity. Treatment with exogenous IFN or adoptive transfer of TLR7-competent pDCs blocked these exaggerated inflammatory responses and boosted IFNγ release in the absence of host TLR7 signalling. TLR7 expression in the lungs was suppressed by allergic inflammation and by interleukin (IL)-5-induced eosinophilia in the absence of allergy. Subjects with moderate-to-severe asthma and eosinophilic but not neutrophilic airways inflammation, despite inhaled steroids, showed reduced TLR7 and IFNλ2/3 expression in endobronchial biopsies. Furthermore, TLR7 expression inversely correlated with percentage of sputum eosinophils. CONCLUSIONS: This implicates IL-5-induced airways eosinophilia as a negative regulator of TLR7 expression and antiviral responses, which provides a molecular mechanism underpinning the effect of eosinophil-targeting treatments for the prevention of asthma exacerbations.

TNF-related apoptosis-inducing ligand (TRAIL) regulates midline-1, thymic stromal lymphopoietin, inflammation, and remodeling in experimental eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is an inflammatory disorder of the esophagus defined by eosinophil infiltration and tissue remodeling with resulting symptoms of esophageal dysfunction. TNF-related apoptosis-inducing ligand (TRAIL) promotes inflammation through upregulation of the E3 ubiquitin-ligase midline-1 (MID1), which binds to and deactivates the catalytic subunit of protein phosphatase 2Ac, resulting in increased nuclear factor κB activation. OBJECTIVE: We sought to elucidate the role of TRAIL in EoE. METHODS: We used Aspergillus fumigatus to induce EoE in TRAIL-sufficient (wild-type) and TRAIL-deficient (TRAIL(-/-)) mice and targeted MID1 in the esophagus with small interfering RNA. We also treated mice with recombinant thymic stromal lymphopoietin (TSLP) and TRAIL. RESULTS: TRAIL deficiency and MID1 silencing with small interfering RNA reduced esophageal eosinophil and mast cell numbers and protected against esophageal circumference enlargement, muscularis externa thickening, and collagen deposition. MID1 expression and nuclear factor κB activation were reduced in TRAIL(-/-) mice, whereas protein phosphatase 2Ac levels were increased compared with those seen in wild-type control mice. This was associated with reduced expression of CCL24, CCL11, CCL20, IL-5, IL-13, IL-25, TGFB, and TSLP. Treatment with TSLP reconstituted hallmark features of EoE in TRAIL(-/-) mice and recombinant TRAIL induced esophageal TSLP expression in vivo in the absence of allergen. Post hoc analysis of gene array data demonstrated significant upregulation of TRAIL and MID1 in a cohort of children with EoE compared with that seen in controls. CONCLUSION: TRAIL regulates MID1 and TSLP, inflammation, fibrosis, smooth muscle hypertrophy, and expression of inflammatory effector chemokines and cytokines in experimental EoE.

CCL7 and IRF-7 Mediate Hallmark Inflammatory and IFN Responses following Rhinovirus 1B Infection.

Rhinovirus (RV) infections are common and have the potential to exacerbate asthma. We have determined the lung transcriptome in RV strain 1B-infected naive BALB/c mice (nonallergic) and identified CCL7 and IFN regulatory factor (IRF)-7 among the most upregulated mRNA transcripts in the lung. To investigate their roles we employed anti-CCL7 Abs and an IRF-7-targeting small interfering RNA in vivo. Neutralizing CCL7 or inhibiting IRF-7 limited neutrophil and macrophage influx and IFN responses in nonallergic mice. Neutralizing CCL7 also reduced activation of NF-κB p65 and p50 subunits, as well as airway hyperreactivity (AHR) in nonallergic mice. However, neither NF-κB subunit activation nor AHR was abolished with infection of allergic mice after neutralizing CCL7, despite a reduction in the number of neutrophils, macrophages, and eosinophils. IRF-7 small interfering RNA primarily suppressed IFN-α and IFN-ß levels during infection of allergic mice. Our data highlight a pivotal role of CCL7 and IRF-7 in RV-induced inflammation and IFN responses and link NF-κB signaling to the development of AHR.

MicroRNA-9 regulates steroid-resistant airway hyperresponsiveness by reducing protein phosphatase 2A activity.

BACKGROUND: Steroid-resistant asthma is a major clinical problem that is linked to activation of innate immune cells. Levels of IFN-γ and LPS are often increased in these patients. Cooperative signaling between IFN-γ/LPS induces macrophage-dependent steroid-resistant airway hyperresponsiveness (AHR) in mouse models. MicroRNAs (miRs) are small noncoding RNAs that regulate the function of innate immune cells by controlling mRNA stability and translation. Their role in regulating glucocorticoid responsiveness and AHR remains unexplored. OBJECTIVE: IFN-γ and LPS synergistically increase the expression of miR-9 in macrophages and lung tissue, suggesting a role in the mechanisms of steroid resistance. Here we demonstrate the role of miR-9 in IFN-γ/LPS-induced inhibition of dexamethasone (DEX) signaling in macrophages and in induction of steroid-resistant AHR. METHODS: MiRNA-9 expression was assessed by means of quantitative RT-PCR. Putative miR-9 targets were determined in silico and confirmed in luciferase reporter assays. miR-9 function was inhibited with sequence-specific antagomirs. The efficacy of DEX was assessed by quantifying glucocorticoid receptor (GR) cellular localization, protein phosphatase 2A (PP2A) activity, and AHR. RESULTS: Exposure of pulmonary macrophages to IFN-γ/LPS synergistically induced miR-9 expression; reduced levels of its target transcript, protein phosphatase 2 regulatory subunit B (B56) δ isoform; attenuated PP2A activity; and inhibited DEX-induced GR nuclear translocation. Inhibition of miR-9 increased both PP2A activity and GR nuclear translocation in macrophages and restored steroid sensitivity in multiple models of steroid-resistant AHR. Pharmacologic activation of PP2A restored DEX efficacy and inhibited AHR. MiR-9 expression was increased in sputum of patients with neutrophilic but not those with eosinophilic asthma. CONCLUSION: MiR-9 regulates GR signaling and steroid-resistant AHR. Targeting miR-9 function might be a novel approach for the treatment of steroid-resistant asthma.

Salmeterol attenuates chemotactic responses in rhinovirus-induced exacerbation of allergic airways disease by modulating protein phosphatase 2A.

BACKGROUND: ß-Agonists are used for relief and control of asthma symptoms by reversing bronchoconstriction. They might also have anti-inflammatory properties, but the underpinning mechanisms remain poorly understood. Recently, a direct interaction between formoterol and protein phosphatase 2A (PP2A) has been described in vitro. OBJECTIVE: We sought to elucidate the molecular mechanisms by which ß-agonists exert anti-inflammatory effects in allergen-driven and rhinovirus 1B-exacerbated allergic airways disease (AAD). METHODS: Mice were sensitized and then challenged with house dust mite to induce AAD while receiving treatment with salmeterol, formoterol, or salbutamol. Mice were also infected with rhinovirus 1B to exacerbate lung inflammation and therapeutically administered salmeterol, dexamethasone, or the PP2A-activating drug (S)-2-amino-4-(4-[heptyloxy]phenyl)-2-methylbutan-1-ol (AAL[S]). RESULTS: Systemic or intranasal administration of salmeterol protected against the development of allergen- and rhinovirus-induced airway hyperreactivity and decreased eosinophil recruitment to the lungs as effectively as dexamethasone. Formoterol and salbutamol also showed anti-inflammatory properties. Salmeterol, but not dexamethasone, increased PP2A activity, which reduced CCL11, CCL20, and CXCL2 expression and reduced levels of phosphorylated extracellular signal-regulated kinase 1 and active nuclear factor κB subunits in the lungs. The anti-inflammatory effect of salmeterol was blocked by targeting the catalytic subunit of PP2A with small RNA interference. Conversely, increasing PP2A activity with AAL(S) abolished rhinovirus-induced airway hyperreactivity, eosinophil influx, and CCL11, CCL20, and CXCL2 expression. Salmeterol also directly activated immunoprecipitated PP2A in vitro isolated from human airway epithelial cells. CONCLUSIONS: Salmeterol exerts anti-inflammatory effects by increasing PP2A activity in AAD and rhinovirus-induced lung inflammation, which might potentially account for some of its clinical benefits.

Inhibiting AKT phosphorylation employing non-cytotoxic anthraquinones ameliorates TH2 mediated allergic airways disease and rhinovirus exacerbation.

BACKGROUND: Severe asthma is associated with T helper (TH) 2 and 17 cell activation, airway neutrophilia and phosphoinositide-3-kinase (PI3K) activation. Asthma exacerbations are commonly caused by rhinovirus (RV) and also associated with PI3K-driven inflammation. Anthraquinone derivatives have been shown to reduce PI3K-mediated AKT phosphorylation in-vitro. OBJECTIVE: To determine the anti-inflammatory potential of anthraquinones in-vivo. METHODS: BALB/c mice were sensitized and challenged with crude house dust mite extract to induce allergic airways disease and treated with mitoxantrone and a novel non-cytotoxic anthraquinone derivative. Allergic mice were also infected with RV1B to induce an exacerbation. RESULTS: Anthraquinone treatment reduced AKT phosphorylation, hypoxia-inducible factor-1α and vascular endothelial growth factor expression, and ameliorated allergen- and RV-induced airways hyprereactivity, neutrophilic and eosinophilic inflammation, cytokine/chemokine expression, mucus hypersecretion, and expression of TH2 proteins in the airways. Anthraquinones also boosted type 1 interferon responses and limited RV replication in the lung. CONCLUSION: Non-cytotoxic anthraquinone derivatives may be of therapeutic benefit for the treatment of severe and RV-induced asthma by blocking pro-inflammatory pathways regulated by PI3K/AKT.

The E3 ubiquitin ligase midline 1 promotes allergen and rhinovirus-induced asthma by inhibiting protein phosphatase 2A activity.

Allergic airway inflammation is associated with activation of innate immune pathways by allergens. Acute exacerbations of asthma are commonly associated with rhinovirus infection. Here we show that, after exposure to house dust mite (HDM) or rhinovirus infection, the E3 ubiquitin ligase midline 1 (MID1) is upregulated in mouse bronchial epithelium. HDM regulates MID1 expression in a Toll-like receptor 4 (TLR4)- and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-dependent manner. MID1 decreases protein phosphatase 2A (PP2A) activity through association with its catalytic subunit PP2Ac. siRNA-mediated knockdown of MID1 or pharmacological activation of PP2A using a nonphosphorylatable FTY720 analog in mice exposed to HDM reduces airway hyperreactivity and inflammation, including the expression of interleukin-25 (IL-25), IL-33 and CCL20, IL-5 and IL-13 release, nuclear factor (NF)κB activity, p38 mitogen-activated protein kinase (MAPK) phosphorylation, accumulation of eosinophils, T lymphocytes and myeloid dendritic cells, and the number of mucus-producing cells. MID1 inhibition also limited rhinovirus-induced exacerbation of allergic airway disease. We found that MID1 was upregulated in primary human bronchial epithelial cells upon HDM or rhinovirus exposure, and this correlated with TRAIL and CCL20 expression. Together, these findings identify a key role of MID1 in allergic airway inflammation and links innate immune pathway activation to the development and exacerbation of asthma.