Inducible gene expression of IκB-kinase ε is dependent on nuclear factor-κB in human pulmonary epithelial cells.

While IκB-kinase-ε (IKKε) induces immunomodulatory genes following viral stimuli, its up-regulation by inflammatory cytokines remains under-explored. Since airway epithelial cells respond to airborne insults and potentiate inflammation, IKKε expression was characterized in pulmonary epithelial cell lines (A549, BEAS-2B) and primary human bronchial epithelial cells grown as submersion or differentiated air-liquid interface cultures. IKKε expression was up-regulated by the pro-inflammatory cytokines, interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNFα). Thus, mechanistic interrogations in A549 cells were used to demonstrate the NF-κB dependence of cytokine-induced IKKε. Furthermore, chromatin immunoprecipitation in A549 and BEAS-2B cells revealed robust recruitment of the NF-κB subunit, p65, to one 5' and two intronic regions within the IKKε locus (IKBKE). In addition, IL-1ß and TNFα induced strong RNA polymerase 2 recruitment to the 5' region, the first intron, and the transcription start site. Stable transfection of the p65-binding regions into A549 cells revealed IL-1ß- and TNFα-inducible reporter activity that required NF-κB, but was not repressed by glucocorticoid. While critical NF-κB motifs were identified in the 5' and downstream intronic regions, the first intronic region did not contain functional NF-κB motifs. Thus, IL-1ß- and TNFα-induced IKKε expression involves three NF-κB-binding regions, containing multiple functional NF-κB motifs, and potentially other mechanisms of p65 binding through non-classical NF-κB binding motifs. By enhancing IKKε expression, IL-1ß may prime, or potentiate, responses to alternative stimuli, as modelled by IKKε phosphorylation induced by phorbol 12-myristate 13-acetate. However, since IKKε expression was only partially repressed by glucocorticoid, IKKε-dependent responses could contribute to glucocorticoid-resistant disease.

Benralizumab for allergic asthma: a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Benralizumab induces rapid and near-complete depletion of eosinophils from blood and lung tissue. We investigated whether benralizumab could attenuate the allergen-induced late asthmatic response (LAR) in participants with allergic asthma. METHODS: Participants with allergic asthma who demonstrated increased sputum eosinophils and LAR at screening were randomised to benralizumab 30 mg or matched placebo given every 4 weeks for 8 weeks (3 doses). Allergen challenges were performed at weeks 9 and 12 when blood, sputum, bone marrow and bronchial tissue eosinophils and LAR were assessed. RESULTS: 46 participants (mean age 30.9 years) were randomised to benralizumab (n=23) or placebo (n=23). Eosinophils were significantly reduced in the benralizumab group compared with placebo in blood at 4 weeks and sputum and bone marrow at 9 weeks after treatment initiation. At 7 h after an allergen challenge at week 9, sputum eosinophilia was significantly attenuated in the benralizumab group compared to placebo (least squares mean difference -5.81%, 95% CI -10.69- -0.94%; p=0.021); however, the LAR was not significantly different (least squares mean difference 2.54%, 95% CI 3.05-8.12%; p=0.363). Adverse events were reported for seven (30.4%) and 14 (60.9%) participants in the benralizumab and placebo groups, respectively. CONCLUSION: Benralizumab administration over 8 weeks resulted in a significant attenuation of blood, bone marrow and sputum eosinophilia in participants with mild allergic asthma; however, there was no change in the LAR, suggesting that eosinophils alone are not a key component of allergen-induced bronchoconstriction.

Synergy between Interleukin-1ß, Interferon-γ, and Glucocorticoids to Induce TLR2 Expression Involves NF-κB, STAT1, and the Glucocorticoid Receptor.

Glucocorticoids act via the glucocorticoid receptor (GR; NR3C1) to downregulate inflammatory gene expression and are effective treatments for mild to moderate asthma. However, in severe asthma and virus-induced exacerbations, glucocorticoid therapies are less efficacious, possibly due to reduced repressive ability and/or the increased expression of proinflammatory genes. In human A549 epithelial and primary human bronchial epithelial cells, toll-like receptor (TLR)-2 mRNA and protein were supra-additively induced by interleukin-1ß (IL-1ß) plus dexamethasone (IL-1ß+Dex), interferon-γ (IFN-γ) plus dexamethasone (IFN-γ+Dex), and IL-1ß plus IFN-γ plus dexamethasone (IL-1ß+IFN-γ+Dex). Indeed, ∼34- to 2100-fold increases were apparent at 24 hours for IL-1ß+IFN-γ+Dex, and this was greater than for any single or dual treatment. Using the A549 cell model, TLR2 induction by IL-1ß+IFN-γ+Dex was antagonized by Org34517, a competitive GR antagonist. Further, when combined with IL-1ß, IFN-γ, or IL-1ß+IFN-γ, the enhancements by dexamethasone on TLR2 expression required GR. Likewise, inhibitor of κB kinase 2 inhibitors reduced IL-1ß+IFN-γ+Dex-induced TLR2 expression, and TLR2 expression induced by IL-1ß+Dex, with or without IFN-γ, required the nuclear factor (NF)-κB subunit, p65. Similarly, signal transducer and activator of transcription (STAT)-1 phosphorylation and γ-interferon-activated sequence-dependent transcription were induced by IFN-γ These, along with IL-1ß+IFN-γ+Dex-induced TLR2 expression, were inhibited by Janus kinase (JAK) inhibitors. As IL-1ß+IFN-γ+Dex-induced TLR2 expression also required STAT1, this study reveals cooperation between JAK-STAT1, NF-κB, and GR to upregulate TLR2 expression. Since TLR2 agonism elicits inflammatory responses, we propose that synergies involving TLR2 may occur within the cytokine milieu present in the immunopathology of glucocorticoid-resistant disease, and this could promote glucocorticoid resistance. SIGNIFICANCE STATEMENT: This study highlights that in human pulmonary epithelial cells, glucocorticoids, when combined with the inflammatory cytokines interleukin-1ß (IL-1ß) and interferon-γ (IFN-γ), can synergistically induce the expression of inflammatory genes, such as TLR2. This effect involved positive combinatorial interactions between NF-κB/p65, glucocorticoid receptor, and JAK-STAT1 signaling to synergistically upregulate TLR2 expression. Thus, synergies involving glucocorticoid enhancement of TLR2 expression may occur in the immunopathology of glucocorticoid-resistant inflammatory diseases, including severe asthma.

Interplay between nuclear factor-κB, p38 MAPK, and glucocorticoid receptor signaling synergistically induces functional TLR2 in lung epithelial cells.

While glucocorticoids act via the glucocorticoid receptor (GR; NR3C1) to reduce the expression of many inflammatory genes, repression is not an invariable outcome. Here, we explore synergy occurring between synthetic glucocorticoids (dexamethasone and budesonide) and proinflammatory cytokines (IL1B and TNF) on the expression of the toll-like receptor 2 (TLR2). This effect is observed in epithelial cell lines and both undifferentiated and differentiated primary human bronchial epithelial cells (pHBECs). In A549 cells, IL1B-plus-glucocorticoid-induced TLR2 expression required nuclear factor (NF)-κB and GR. Likewise, in A549 cells, BEAS-2B cells, and pHBECs, chromatin immunoprecipitation identified GR- and NF-κB/p65-binding regions ∼32 kb (R1) and ∼7.3 kb (R2) upstream of the TLR2 gene. Treatment of BEAS-2B cells with TNF or/and dexamethasone followed by global run-on sequencing confirmed transcriptional activity at these regions. Furthermore, cloning R1 or R2 into luciferase reporters revealed transcriptional activation by budesonide or IL1B, respectively, while R1+R2 juxtaposition enabled synergistic activation by IL1B and budesonide. In addition, small-molecule inhibitors and siRNA knockdown showed p38α MAPK to negatively regulate both IL1B-induced TLR2 expression and R1+R2 reporter activity. Finally, agonism of IL1B-plus-dexamethasone-induced TLR2 in A549 cells and pHBECs stimulated NF-κB- and interferon regulatory factor-dependent reporter activity and chemokine release. We conclude that glucocorticoid-plus-cytokine-driven synergy at TLR2 involves GR and NF-κB acting via specific enhancer regions, which combined with the inhibition of p38α MAPK promotes TLR2 expression. Subsequent inflammatory effects that occur following TLR2 agonism may be pertinent in severe neutrophilic asthma or chronic obstructive pulmonary disease, where glucocorticoid-based therapies are less efficacious.

Role of Brain Imaging in the Prediction of Intracerebral Hemorrhage Following Endovascular Therapy for Acute Stroke.

Currently most acute ischemic stroke patients presenting with a large vessel occlusion are treated with endovascular therapy (EVT), which results in high rates of successful recanalization. Despite this success, more than half of EVT-treated patients are significantly disabled 3 months later partly due to the occurrence of post-EVT intracerebral hemorrhage. Predicting post-EVT intracerebral hemorrhage is important for individualizing treatment strategies in clinical practice (eg, safe initiation of early antithrombotic therapies), as well as in selecting the optimal candidates for clinical trials that aim to reduce this deleterious outcome. Emerging data suggest that brain and vascular imaging biomarkers may be particularly relevant since they provide insights into the ongoing acute stroke pathophysiology. In this review/perspective, we summarize the accumulating literature on the role of cerebrovascular imaging biomarkers in predicting post-EVT-associated intracerebral hemorrhage. We focus on imaging acquired before EVT, during the EVT procedure, and in the early post-EVT time frames when new therapeutic therapies could be tested. Accounting for the complex pathophysiology of post-EVT-associated intracerebral hemorrhage, this review may provide some guidance for future prospective observational or therapeutic studies.

Inhaled anti-TSLP antibody fragment, ecleralimab, blocks responses to allergen in mild asthma.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) is a key upstream regulator driving allergic inflammatory responses. We evaluated the efficacy and safety of ecleralimab, a potent inhaled neutralising antibody fragment against human TSLP, using allergen inhalation challenge (AIC) in subjects with mild atopic asthma. METHODS: This was a 12-week, randomised, double-blind, placebo-controlled, parallel-design, multicentre allergen bronchoprovocation study conducted at 10 centres across Canada and Germany. Subjects aged 18-60 years with stable mild atopic asthma were randomised (1:1) to receive 4 mg once-daily inhaled ecleralimab or placebo. Primary end-points were the allergen-induced change in forced expiratory volume in 1 s (FEV1) during the late asthmatic response (LAR) measured by area under the curve (AUC3-7h) and maximum percentage decrease (LAR%) on day 84, and the safety of ecleralimab. Allergen-induced early asthmatic response (EAR), sputum eosinophils and fractional exhaled nitric oxide (F ENO) were secondary and exploratory end-points. RESULTS: 28 subjects were randomised to ecleralimab (n=15) or placebo (n=13). On day 84, ecleralimab significantly attenuated LAR AUC3-7h by 64% (p=0.008), LAR% by 48% (p=0.029), and allergen-induced sputum eosinophils by 64% at 7 h (p=0.011) and by 52% at 24 h (p=0.047) post-challenge. Ecleralimab also numerically reduced EAR AUC0-2h (p=0.097) and EAR% (p=0.105). F ENO levels were significantly reduced from baseline throughout the study (p<0.05), except at 24 h post-allergen (day 43 and day 85). Overall, ecleralimab was safe and well tolerated. CONCLUSION: Ecleralimab significantly attenuated allergen-induced bronchoconstriction and airway inflammation, and was safe in subjects with mild atopic asthma.

Amide proton transfer imaging in stroke.

Amide proton transfer (APT) imaging, a variant of chemical exchange saturation transfer MRI, has shown promise in detecting ischemic tissue acidosis following impaired aerobic metabolism in animal models and in human stroke patients due to the sensitivity of the amide proton exchange rate to changes in pH within the physiological range. Recent studies have demonstrated the possibility of using APT-MRI to detect acidosis of the ischemic penumbra, enabling the assessment of stroke severity and risk of progression, monitoring of treatment progress, and prognostication of clinical outcome. This paper reviews current APT imaging methods actively used in ischemic stroke research and explores the clinical aspects of ischemic stroke and future applications for these methods.

Sounding the alarmins-The role of alarmin cytokines in asthma.

The alarmin cytokines thymic stromal lymphopoietin (TSLP), interleukin (IL)-33, and IL-25 are epithelial cell-derived mediators that contribute to the pathobiology and pathophysiology of asthma. Released from airway epithelial cells exposed to environmental triggers, the alarmins drive airway inflammation through the release of predominantly T2 cytokines from multiple effector cells. The upstream positioning of the alarmins is an attractive pharmacological target to block multiple T2 pathways important in asthma. Blocking the function of TSLP inhibits allergen-induced responses including bronchoconstriction, airway hyperresponsiveness, and inflammation, and subsequent clinical trials of an anti-TSLP monoclonal antibody, tezepelumab, in asthma patients demonstrated improvements in lung function, airway responsiveness, inflammation, and importantly, a reduction in the rate of exacerbations. Notably, these improvements were observed in patients with T2-high and with T2-low asthma. Clinical trials blocking IL-33 and its receptor ST2 have also shown improvements in lung function and exacerbation rates; however, the impact of blocking the IL-33/ST2 axis in T2-high versus T2-low asthma is unclear. To date, there is no evidence that IL-25 blockade is beneficial in asthma. Despite the considerable overlap in the cellular functions of IL-25, IL-33, and TSLP, they appear to have distinct roles in the immunopathology of asthma.

Estimating Perfusion Deficits in Acute Stroke Patients Without Perfusion Imaging.

BACKGROUND: Perfusion weighted imaging (PWI) is critical for determining whether stroke patients presenting in an extended time window are candidates for mechanical thrombectomy. However, PWI is not always available. Fluid-attenuated inversion recovery hyperintense vessels (FHVs) are seen in patients with a PWI lesion. We investigated whether a scale measuring the extent FHV could serve as a surrogate for PWI to determine eligibility for thrombectomy. METHODS: The National Institutes of Health (NIH) FHV score was developed to quantify the burden of FHV and applied to magnetic resonance imaging scans of stroke patients with fluid-attenuated inversion recovery and perfusion imaging. The NIH-FHV was combined with the diffusion weighted image volume to estimate the diffusion-perfusion mismatch ratio. Linear regression was used to compare PWI volumes and mismatch ratios with estimates from the NIH-FHV score. Receiver operating characteristic analysis was used to test the ability of the NIH-FHV score to identify a significant mismatch. RESULTS: There were 101 patients included in the analysis, of whom 78% had a perfusion deficit detected on PWI with a mean lesion volume of 47 (±59) mL. The NIH-FHV score was strongly associated with the PWI lesion volume (P<0.001; R2=0.32; ß-coefficient, 0.57). When combined with diffusion weighted image lesion volume, receiver operating characteristic analysis testing the ability to detect a mismatch ratio ≥1.8 using the NIH-FHV score resulted in an area under the curve of 0.94. CONCLUSIONS: The NIH-FHV score provides an estimate of the PWI lesion volume and, when combined with diffusion weighted imaging, may be helpful when trying to determine whether there is a clinically relevant diffusion-perfusion mismatch in situations where perfusion imaging is not available. Further studies are needed to validate this approach.

Simultaneous Hemodynamic and Structural Imaging of Ischemic Stroke With Magnetic Resonance Fingerprinting Arterial Spin Labeling.

BACKGROUND: Perfusion and structural imaging play an important role in ischemic stroke. Magnetic resonance fingerprinting (MRF) arterial spin labeling (ASL) is a novel noninvasive method of ASL perfusion that allows simultaneous estimation of cerebral blood flow (CBF), bolus arrival time (BAT), and tissue T1 map in a single scan of <4 minutes. Here, we evaluated the utility of MRF-ASL in patients with ischemic stroke in terms of detecting hemodynamic and structural damage and predicting neurological deficits and disability. METHODS: A total of 34 patients were scanned on 3T magnetic resonance imaging. MRF-ASL, standard single-delay pseudo-continuous ASL, T2-weighted, and diffusion magnetic resonance imaging were performed. Regions of interest of lesion and contralateral normal tissues were manually delineated. CBF (with 2 different compartmental models), BAT, and tissue T1 parameters were quantified. Cross-sectional linear regression analyses were performed to examine the relationship between MRF-ASL parameters and National Institutes of Health Stroke Scale (NIHSS) and modified Rankin Scale. Receiver operating characteristic analyses were performed to determine the utility of MRF-ASL in the classification of stroke lesion voxels. RESULTS: MRF-ASL derived parameters revealed a significant difference between stroke lesion and contralateral normal regions of interest, in that lesion regions manifested a lower CBF1-compartment (P<0.001), lower CBF2-compartment (P<0.001), longer BAT (P=0.002), and longer T1 (P<0.001) compared with normal regions of interest. NIHSS scores at acute stage revealed a strong association with lesion-normal differences in CBF1-compartment,diff (ß=-0.11, P=0.008), CBF2-compartment,diff (ß=-0.16, P=0.003), and T1,diff (ß=0.008, P=0.001). MRF-ASL parameters were also predictive of NIHSS score and modified Rankin Scale scale measured at a later stage, although the degree of the associations was weaker. These associations tended to be even stronger when the MRF-ASL data were acquired at the acute/subacute stage. Compared with standard pseudo-continuous ASL, the multiparametric capability of MRF-ASL yielded higher area under curve values in the receiver operating characteristic analyses of stroke voxel classifications. CONCLUSIONS: MRF-ASL may provide a new approach for quantitative hemodynamic and structural imaging in ischemic stroke.

Microvascular Dysfunction in Blood-Brain Barrier Disruption and Hypoperfusion Within the Infarct Posttreatment Are Associated With Cerebral Edema.

BACKGROUND: Factors contributing to cerebral edema in the post-hyperacute period of ischemic stroke (first 24-72 hours) are poorly understood. Blood-brain barrier (BBB) disruption and postischemic hyperperfusion reflect microvascular dysfunction and are associated with hemorrhagic transformation. We investigated the relationships between BBB integrity, cerebral blood flow, and space-occupying cerebral edema in patients who received acute reperfusion therapy. METHODS: We performed a pooled analysis of patients treated for anterior circulation large vessel occlusion in the EXTEND-IA TNK and EXTEND-IA TNK part 2 trials who had MRI with dynamic susceptibility contrast-enhanced perfusion-weighted imaging 24 hours after treatment. We investigated the associations between BBB disruption and cerebral blood flow within the infarct with cerebral edema assessed using 2 metrics: first midline shift (MLS) trichotomized as an ordinal scale of negligible (<1 mm), mild (≥1 to <5 mm), or severe (≥5 mm), and second relative hemispheric volume (rHV), defined as the ratio of the 3-dimensional volume of the ischemic hemisphere relative to the contralateral hemisphere. RESULTS: Of 238 patients analyzed, 133 (55.9%) had negligible, 93 (39.1%) mild, and 12 (5.0%) severe MLS at 24 hours. The associated median rHV was 1.01 (IQR, 1.00-1.028), 1.03 (IQR, 1.01-1.077), and 1.15 (IQR, 1.08-1.22), respectively. MLS and rHV were associated with poor functional outcome at 90 days (P<0.002). Increased BBB permeability was independently associated with more edema after adjusting for age, occlusion location, reperfusion, parenchymal hematoma, and thrombolytic agent used (MLS cOR, 1.12 [95% CI, 1.03-1.20], P=0.005; rHV ß, 0.39 [95% CI, 0.24-0.55], P<0.0001), as was reduced cerebral blood flow (MLS cOR, 0.25 [95% CI, 0.10-0.58], P=0.001; rHV ß, -2.95 [95% CI, -4.61 to -11.29], P=0.0006). In subgroup analysis of patients with successful reperfusion (extended Treatment in Cerebral Ischemia 2b-3, n=200), reduced cerebral blood flow remained significantly associated with edema (MLS cOR, 0.37 [95% CI, 0.14-0.98], P=0.045; rHV ß, -2.59 [95% CI, -4.32 to -0.86], P=0.004). CONCLUSIONS: BBB disruption and persistent hypoperfusion in the infarct after reperfusion treatment is associated with space-occupying cerebral edema. Further studies evaluating microvascular dysfunction during the post-hyperacute period as biomarkers of poststroke edema and potential therapeutic targets are warranted.

Reduced Severity of Tissue Injury Within the Infarct May Partially Mediate the Benefit of Reperfusion in Ischemic Stroke.

BACKGROUND: Emerging data suggest tissue within the infarct lesion is not homogenously damaged following ischemic stroke but has a gradient of injury. Using blood-brain-barrier (BBB) disruption as a marker of tissue injury, we tested whether therapeutic reperfusion improves clinical outcome by reducing the severity of tissue injury within the infarct in patients with ischemic stroke. METHODS: In a pooled analysis of patients treated for anterior circulation large vessel occlusion in the EXTEND-IA TNK (Tenecteplase Versus Alteplase Before Endovascular Therapy for Ischemic Stroke) and EXTEND-IA part-2 (Determining the Optimal Dose of Tenecteplase Before Endovascular Therapy for Ischaemic Stroke) trials, post-treatment BBB permeability at 24 hours was calculated based on the extent of T1-brightening by extravascular gadolinium on T2* perfusion-weighted imaging and measured within the diffusion-weighted-imaging lesion. First, to determine the clinical significance of BBB disruption as a marker of severity of tissue injury, we examined the association between post-treatment BBB permeability and functional outcome. Second, we performed an exploratory (reperfusion, BBB permeability, functional outcome) mediation analysis to estimate the proportion of the reperfusion-outcome relationship that is mediated by change in BBB permeability. RESULTS: In the 238 patients analyzed, an increased BBB permeability measured within the infarct at 24 hours was associated with a reduced likelihood of favorable outcome (90-day modified Rankin Scale score of ≤2) after adjusting for age, baseline National Institutes of Health Stroke Scale, premorbid modified Rankin Scale, infarct topography, laterality, thrombolytic agent, sex, parenchymal hematoma, and follow-up infarct volume (adjusted odds ratio, 0.86 [95% CI, 0.75-0.98]; P=0.023). Mediation analysis suggested reducing the severity of tissue injury (as estimated by BBB permeability) accounts for 18.2% of the association between reperfusion and favorable outcome, as indicated by a reduction in the regression coefficient of reperfusion after addition of BBB permeability as a covariate. CONCLUSIONS: In patients with ischemic stroke, reduced severity of tissue injury within the infarct, as determined by assessing the integrity of the BBB, is independently associated with improved functional outcome. In addition to reducing diffusion-weighted imaging-defined infarct volume, reperfusion may also improve clinical outcome by reducing tissue injury severity within the infarct.

Transcriptome-Level Interactions between Budesonide and Formoterol Provide Insight into the Mechanism of Action of Inhaled Corticosteroid/Long-Acting ß 2-Adrenoceptor Agonist Combination Therapy in Asthma.

In 2019, the Global Initiative for Asthma treatment guidelines were updated to recommend that inhaled corticosteroid (ICS)/long-acting ß 2-adrenoceptor agonist (LABA) combination therapy should be a first-in-line treatment option for asthma. Although clinically superior to ICS, mechanisms underlying the efficacy of this combination therapy remain unclear. We hypothesized the existence of transcriptomic interactions, an effect that was tested in BEAS-2B and primary human bronchial epithelial cells (pHBECs) using formoterol and budesonide as representative LABA and ICS, respectively. In BEAS-2B cells, formoterol produced 267 (212 induced; 55 repressed) gene expression changes (≥2/≤0.5-fold) that were dominated by rapidly (1 to 2 hours) upregulated transcripts. Conversely, budesonide induced 370 and repressed 413 mRNAs, which occurred predominantly at 6-18 hours and was preceded by transcripts enriched in transcriptional regulators. Significantly, genes regulated by both formoterol and budesonide were over-represented in the genome; moreover, budesonide plus formoterol induced and repressed 609 and 577 mRNAs, respectively, of which ∼one-third failed the cutoff criterion for either treatment alone. Although induction of many mRNAs by budesonide plus formoterol was supra-additive, the dominant (and potentially beneficial) effect of budesonide on formoterol-induced transcripts, including those encoding many proinflammatory proteins, was repression. Gene ontology analysis of the budesonide-modulated transcriptome returned enriched terms for transcription, apoptosis, proliferation, differentiation, development, and migration. This "functional" ICS signature was augmented in the presence of formoterol. Thus, LABAs modulate glucocorticoid action, and comparable transcriptome-wide interactions in pHBECs imply that such effects may be extrapolated to individuals with asthma taking combination therapy. Although repression of formoterol-induced proinflammatory mRNAs should be beneficial, the pathophysiological consequences of other interactions require investigation. SIGNIFICANCE STATEMENT: In human bronchial epithelial cells, formoterol, a long-acting ß 2-adrenoceptor agonist (LABA), enhanced the expression of inflammatory genes, and many of these changes were reduced by the glucocorticoid budesonide. Conversely, the ability of formoterol to enhance both gene induction and repression by budesonide provides mechanistic insight as to how adding a LABA to an inhaled corticosteroid may improve clinical outcomes in asthma.

Fluid-Attenuated Inversion Recovery Hyperintense Ischemic Stroke Predicts Less Favorable 90-Day Outcome after Intravenous Thrombolysis.

INTRODUCTION: The absence of an ischemic lesion on MRI fluid-attenuated inversion recovery (FLAIR) is helpful in predicting stroke onset within 4.5 h. However, some ischemic strokes become visible on FLAIR within 4.5 h. We hypothesized that the early lesion visibility on FLAIR may predict stroke outcome 90 days after intravenous (IV) thrombolysis, independent of time. MATERIALS AND METHODS: We analyzed data from acute ischemic stroke patients presenting over the last 10 years who were screened with MRI and treated with IV thrombolysis within 4.5 h from onset. Three independent readers assessed whether ischemic lesions seen on diffusion-weighted imaging were also FLAIR positive based on visual inspection. Multivariable regression analyses were used to obtain an adjusted odds ratio of favorable clinical and radiological outcomes based on FLAIR positivity. RESULTS: Of 297 ischemic stroke patients, 25% had lesion visibility on initial FLAIR. The interrater agreement for the FLAIR positivity assessment was 84% (κ = 0.604, 95% CI: 0.557-0.652). Patients with FLAIR-positive lesions had more right hemispheric strokes (57 vs. 41%, p = 0.045), were imaged later (129 vs. 104 min, p = 0.036), and had less frequent favorable 90-day functional outcome (49 vs. 63%, p = 0.028), less frequent early neurologic improvement (30 vs. 58%, p = 0.001), and more frequent contrast extravasation to the cerebrospinal fluid space (44 vs. 26%, p = 0.008). CONCLUSIONS: Early development of stroke lesion on FLAIR within 4.5 h of onset is associated with reduced likelihood of favorable 90-day outcome after IV thrombolysis.

Rhinovirus and Bacteria Synergistically Induce IL-17C Release from Human Airway Epithelial Cells To Promote Neutrophil Recruitment.

Virus-bacteria coinfections are associated with more severe exacerbations and increased risk of hospital readmission in patients with chronic obstructive pulmonary disease (COPD). The airway epithelium responds to such infections by releasing proinflammatory and antimicrobial cytokines, including IL-17C. However, the regulation and role of IL-17C is not well understood. In this study, we examine the mechanisms regulating IL-17C production and its potential role in COPD exacerbations. Human bronchial epithelial cells (HBE) obtained from normal, nontransplanted lungs or from brushings of nonsmokers, healthy smokers, or COPD patients were exposed to bacteria and/or human rhinovirus (HRV). RNA and protein were collected for analysis, and signaling pathways were assessed with pharmacological agonists, inhibitors, or small interfering RNAs. HBE were also stimulated with IL-17C to assess function. HRV-bacterial coinfections synergistically induced IL-17C expression. This induction was dependent on HRV replication and required NF-κB-mediated signaling. Synergy was lost in the presence of an inhibitor of the p38 MAP kinase pathway. HBE exposed to IL-17C show increased gene expression of CXCL1, CXCL2, NFKBIZ, and TFRC, and release CXCL1 protein, a neutrophil chemoattractant. Knockdown of IL-17C significantly reduced induction of CXCL1 in response to HRV-bacterial coinfection as well as neutrophil chemotaxis. HBE from healthy smokers release less IL-17C than cells from nonsmokers, but cells from COPD patients release significantly more IL-17C compared with either nonsmokers or healthy smokers. These data suggest that IL-17C may contribute to microbial-induced COPD exacerbations by promoting neutrophil recruitment.

Blood-brain barrier integrity of stroke patients presenting in an extended time window.

BACKGROUND: Current guidelines limit thrombolytic treatment of stroke to those patients who present within 4.5 h to minimize the risk of hemorrhagic complications. Risk of hemorrhage increases with increasing blood-brain barrier (BBB) disruption. This study aimed to determine, in a cohort of patients presenting outside of an IV-tPA treatment window, whether disruption of the BBB is time dependent, and what proportion of patients could be safely treated. METHODS: We analyzed untreated stroke patients, seen between 2011 and 2015, who had MRI studies in the time window of 4 to 24 h from symptoms onset. Permeability of the BBB was measured within the ischemic tissue using an application of dynamic susceptibility contrast imaging. Patients were dichotomized into two groups based on a 20% threshold of BBB disruption and compared using logistic regression. RESULTS: Of the 222 patients included in the final analysis, over half, 129 (58%), had preserved BBB integrity below the 20% threshold. There was no relationship between time imaged after symptom onset and the amount of BBB disruption (p = 0.138) across the population; BBB disruption varied widely. CONCLUSIONS: Estimating BBB integrity may help to expand the treatment window for stroke patients by identifying those individuals for whom thrombolytic therapy can be considered.

Assessment of the long-term safety of mepolizumab and durability of clinical response in patients with severe eosinophilic asthma.

BACKGROUND: Mepolizumab has demonstrated favorable safety and efficacy profiles in placebo-controlled trials of 12 months' duration or less; however, long-term data are lacking. OBJECTIVE: We sought to evaluate the long-term safety and efficacy of mepolizumab in patients with severe eosinophilic asthma (SEA). METHODS: COLUMBA (Open-label Long Term Extension Safety Study of Mepolizumab in Asthmatic Subjects, NCT01691859) was an open-label extension study in patients with SEA previously enrolled in DREAM (Dose Ranging Efficacy And Safety With Mepolizumab in Severe Asthma, NCT01000506). Patients received 100 mg of subcutaneous mepolizumab every 4 weeks plus standard of care until a protocol-defined stopping criterion was met. Safety end points included frequency of adverse events (AEs), serious AEs, and AEs of special interest. Efficacy end points included annualized exacerbation rates, changes from baseline in Asthma Control Questionnaire 5 scores, and blood eosinophil counts. Immunogenicity was also assessed. RESULTS: Overall, 347 patients were enrolled for an average of 3.5 years (maximum, 4.5 years; total exposure, 1201 patient-years). On-treatment AEs were reported in 94% of patients (exposure-adjusted rate, 3688 events/1000 patient-years). The most frequently reported on-treatment AEs were respiratory tract infection, headache, bronchitis, and asthma worsening. Seventy-nine (23%) patients experienced 1 or more on-treatment serious AEs; there were 6 deaths, none of which were assessed as related to mepolizumab. For patients with 156 weeks or greater enrollment, the exacerbation rate was 0.74 events/y (weeks 0-156), a 56% reduction from the off-treatment period between DREAM and COLUMBA. For all patients, at the first postbaseline assessment, the mean Asthma Control Questionnaire 5 score was reduced by 0.47 points, and blood eosinophil counts were reduced by 78%, with similar improvements maintained throughout the study. The immunogenicity profile (8% anti-drug antibodies) was consistent with previous studies. CONCLUSION: These data support the long-term safety and efficacy of mepolizumab in patients with SEA.

Routine use of FLAIR-negative MRI in the treatment of unknown onset stroke.

BACKGROUND: Treatment of FLAIR-negative stroke in patients presenting in an unknown time window has been shown to be safe and effective. However, implementation can be challenging due to the need for hyper-acute MRI screening. The purpose of this study was to review the routine application of this practice outside of a clinical trial. METHODS: Patients presenting from 3/1/16 to 8/22/18 in a time window <4.5 h from symptom discovery but >4.5 h from last known normal were included if they had a hyper-acute MRI performed. Quantitative assessment based on the MR WITNESS trial and qualitative assessment based on the WAKE-UP trial were used to grade the FLAIR images. The MR WITNESS trial used a quantitative assessment of FLAIR change where the fractional increase in signal change had to be <1.15, whereas the WAKE-UP trial used a visual assessment requiring the absence of marked FLAIR signal changes. RESULTS: During the study period, 136 stroke patients presented and were imaged in the specified time window. Of these, 17 (12.5%) received IV tPA. Three patients had hemorrhage on 24-h MRI follow up; none had an increase in NIHSS ≥4. Of the 119 patients who were screened but not treated, 18 (15%) were eligible based on FLAIR quantitative assessment and 55 (46%) were eligible based on qualitative assessment. In all cases where patients were not treated, there was an identifiable exclusion based on trial criteria. During the study period, IV tPA utilization was increased by 5.6% due to screening and treating patients with unknown onset stroke. CONCLUSIONS: Screening stroke patients in an unknown time window with MRI is practical in a real-world setting and increases IV tPA utilization.

Fat grafting and platelet-rich plasma for the treatment of diabetic foot ulcers: A feasibility-randomised controlled trial.

Chronic, nonhealing diabetic foot ulcers (DFU) are increasing in prevalence and are often unresponsive to conventional therapy. Adipose tissue, containing adipose-derived stem cells, and platelet rich plasma (PRP) are regenerative therapies rich in growth factors which may provide a solution to chronic wound healing. This study aimed to assess the feasibility of conducting a definitive randomised controlled trial (RCT) to investigate the efficacy of these therapies for the treatment of DFU. This was a single centre, feasibility, three-arm, parallel group RCT. Eligible DFU patients were randomised on a 1:1:1 basis to three intervention arms: control (podiatry); fat grafting; fat grafting with PRP. The intervention was delivered once and patients were followed-up for 12 weeks. The primary objective was to assess measures of trial feasibility. Clinical outcomes and health-related quality of life (HRQoL) were also evaluated. Three hundred and thirty four patients were screened and 32 patients (9.6%) were deemed eligible with 18 enrolled in the trial (6 per arm) over 17 months. All participants completed the trial with no withdrawals or crossover. Participant engagement was high with most HRQoL questionnaires returned and only 4.8% follow-up appointments missed. There were five adverse events (AEs) related to the trial with no serious AEs. Five (28%) of the wounds healed. There was no difference between any of the groups in terms of clinical outcomes. This feasibility study demonstrated that a multi-centre RCT is safe and feasible with excellent patient engagement. We have highlighted crucial information regarding methodology and recruitment, which will guide future trial design. Registration number: NCT03085550 clinicaltrials.gov. Registered 01/03/2017.

Rapid Apparent Diffusion Coefficient Evolution After Early Revascularization.

Background and Purpose- In this era of endovascular therapy (EVT) with early, complete recanalization and reperfusion, we have observed an even more rapid apparent diffusion coefficient (ADC) normalization within the acute ischemic lesion compared with the natural history or IV-tPA-treated patient. In this study, we aimed to evaluate the effect of revascularization on ADC evolution within the core lesion in the first 24 hours in acute ischemic stroke patients. Methods- This retrospective study included anterior circulation acute ischemic stroke patients treated with EVT with or without intravenous tPA (IVT) from 2015 to 2017 compared with a consecutive cohort of IVT-only patients treated before 2015. Diffusion-weighted imaging and ADC maps were used to quantify baseline core lesions. Median ADC value change and core reversal were determined at 24 hours. Diffusion-weighted imaging lesion growth was measured at 24 hours and 5 days. Good clinical outcome was defined as modified Rankin Scale score of 0 to 2 at 90 days. Results- Twenty-five patients (50%) received IVT while the other 25 patients received EVT (50%) with or without IVT. Between these patient groups, there were no differences in age, sex, baseline National Institutes of Health Stroke Scale, interhospital transfer, or IVT rates. Thirty-two patients (64%) revascularized with 69% receiving EVT. There was a significant increase in median ADC value of the core lesion at 24 hours in patients who revascularized compared with further ADC reduction in nonrevascularization patients. Revascularization patients had a significantly higher rate of good clinical outcome at 90 days, 63% versus 9% (P=0.003). Core reversal at 24 hours was significantly higher in revascularization patients, 69% versus 22% (P=0.002). Conclusions- ADC evolution in acute ischemic stroke patients with early, complete revascularization, now more commonly seen with EVT, is strikingly different from our historical understanding. The early ADC normalization we have observed in this setting may include a component of secondary injury and serve as a potential imaging biomarker for the development of future adjunctive therapies. Clinical Trial Registration- URL: https://www.clinicaltrials.gov. Unique identifier: NCT00009243.