Effect of Caspase Inhibitors on CSE-Induced Apoptosis.

<p>PBEC obtained from 4 non-asthmatic and 4 asthmatic donors were treated for 24h with 20%CSE alone or in combination with Ac-DEVD-CHO (C3i) (a) or Z-LEHD-FMK (C9i) (b). Changes in EA were evaluated with AxV staining. The results are displayed as a box plot showing median and 5–95% confidence intervals of fold changes from CSE only in both groups.</p

Characteristics of subjects with asthma.

<p>¹Inhaled corticosteroid (ICS) dose is given as μg of beclomethasone (BDP) used per day, expressed as the mean (sd).</p><p>²Values for FEV₁ as a percentage of the predicted FEV₁ are given as a mean and standard deviation (sd).</p><p>Characteristics of subjects with asthma.</p

Ability of GSH, but not AA, to protect against CSE-induced cell death.

<p>PBEC obtained from non-asthmatic or asthmatic donors were treated for 24h with 20% CSE alone or in the presence of GSH (1mM) or AA (250nM). Changes in cell viability (a) and EA (b) were then evaluated with AxV staining. The results are displayed as a box plot showing median, interquartile range and 5–95% confidence intervals of fold changes from CSE alone using PBECs from 10 non-asthmatic (○) and 10 asthmatic (Δ) donors. <b>*</b> = p<0.05 according to Wilcoxon Signed Rank tests comparing GSH+CSE with CSE only. <b>∞</b> = p<0.05 according to a Mann Whitney U test comparing CSE+GSH treatment between PBECs from non-asthmatic and asthmatic donors.</p

Ability of GSH and AA to protect against H₂O₂-induced cell death.

<p>PBEC obtained from non-asthmatic or asthmatic donors were treated for 24h with 400μM H₂O₂ alone or in the presence of GSH (1mM) or AA (250nM). Changes in cell viability (a) and EA (b) were then evaluated with AxV staining. The results are displayed as a box plot showing median, interquartile range and 5–95% confidence intervals of fold changes from H₂O₂ alone for PBECs from 10 non-asthmatic (○) and 10 asthmatic (Δ) donors. * = p<0.02 according to Wilcoxon Signed Rank tests comparing antioxidants with H₂O₂ only. There was no statistical difference comparing antioxidants treatment between PBECs from non-asthmatic or asthmatic donors according to a Mann Whitney U test.</p

Effect of CSE on PBEC Viability and Apoptosis.

<p>PBEC obtained from non-asthmatic and asthmatic donors were treated for 24h with 20% CSE. Cell viability (a) and early apoptosis (b) were then evaluated with AnnexinV staining. The results are displayed as a box plot showing median, interquartile range and 5–95% confidence intervals using PBECs from 10 non-asthmatic (○) and 10 asthmatic (Δ) volunteers. * represents significance (p<0.05) according to Wilcoxon Signed Rank tests comparing treatment with untreated control. ∞ represents significance (p = 0.003) according to a Mann Whitney U test comparing CSE treatment between PBECs from non-asthmatic and asthmatic donors.</p

Involvement of AIF in CSE-induced Apoptosis.

<p>PBECs were left untreated (A), or were treated for 24h with 20% CSE alone (B), or with a combination of CSE and GSH (1mM) (C) or AA (250nM) (D). Cells were then washed, fixed and stained with a primary antibody directed towards AIF, followed by a secondary FITC-conjugated antibody. The figure shows that in basal conditions, PBEC expressed AIF in their cytoplasm especially inside their mitochondria (A, insert); CSE treatment caused translocation of AIF from the mitochondria to inside the nuclei (B, green arrows and insert); GSH pre-treatment caused a significant decrease in the number of nuclear-AIF positive cells whereas AA was not so effective. Bar = 30μm. Data are representative of experiments performed with PBECs from 2 donors.</p

DataSheet_1_Remission outcomes in severe eosinophilic asthma with mepolizumab therapy: Analysis of the REDES study.docx

IntroductionClinical remission as a multicomponent treatment goal in severe asthma is being explored in clinical practice. This post hoc analysis used data from the REDES study to assess the proportion of patients with severe eosinophilic asthma achieving our multicomponent definitions of clinical remission after 1 year of mepolizumab treatment.MethodsThe real-world, retrospective observational REDES study enrolled patients with severe eosinophilic asthma who were newly prescribed mepolizumab and with ≥12 months of medical records pre-enrolment. Multicomponent clinical remission was defined as: oral corticosteroid (OCS)-free; exacerbation-free; asthma control test (ACT) score ≥20; and with or without post-bronchodilator forced expiratory volume in 1 second ≥80%. Baseline characteristics were also assessed in those who did/did not achieve clinical remission.Results37% and 30% of patients with severe eosinophilic asthma met our proposed three- and four-component on-treatment clinical remission definitions; an increase from 2% and 3% at baseline. Most frequently achieved individual components of clinical remission were: OCS-free; ACT score ≥20. For patients fulfilling the multicomponent clinical remission definitions, at baseline we observed higher blood eosinophil counts, better ACT scores and lung function, lower maintenance OCS use, and a slightly lower rate of prior exacerbations versus those who did not.DiscussionClinical remission is a realistic target in clinical practice for a subset of patients with severe eosinophilic asthma receiving mepolizumab. Further studies are required to elucidate whether features linked to the underlying endotype can help predict treatment outcomes, increase rates of clinical remission, and potentially modify disease progression.</p

Image_1_Remission outcomes in severe eosinophilic asthma with mepolizumab therapy: Analysis of the REDES study.tif

IntroductionClinical remission as a multicomponent treatment goal in severe asthma is being explored in clinical practice. This post hoc analysis used data from the REDES study to assess the proportion of patients with severe eosinophilic asthma achieving our multicomponent definitions of clinical remission after 1 year of mepolizumab treatment.MethodsThe real-world, retrospective observational REDES study enrolled patients with severe eosinophilic asthma who were newly prescribed mepolizumab and with ≥12 months of medical records pre-enrolment. Multicomponent clinical remission was defined as: oral corticosteroid (OCS)-free; exacerbation-free; asthma control test (ACT) score ≥20; and with or without post-bronchodilator forced expiratory volume in 1 second ≥80%. Baseline characteristics were also assessed in those who did/did not achieve clinical remission.Results37% and 30% of patients with severe eosinophilic asthma met our proposed three- and four-component on-treatment clinical remission definitions; an increase from 2% and 3% at baseline. Most frequently achieved individual components of clinical remission were: OCS-free; ACT score ≥20. For patients fulfilling the multicomponent clinical remission definitions, at baseline we observed higher blood eosinophil counts, better ACT scores and lung function, lower maintenance OCS use, and a slightly lower rate of prior exacerbations versus those who did not.DiscussionClinical remission is a realistic target in clinical practice for a subset of patients with severe eosinophilic asthma receiving mepolizumab. Further studies are required to elucidate whether features linked to the underlying endotype can help predict treatment outcomes, increase rates of clinical remission, and potentially modify disease progression.</p

Exacerbation Profile and Risk Factors in a T2-Low Severe Asthma Population.

Although present in a minority of severe asthmatics, very little is known about the mechanisms underlying T2-low asthma, making it a significant unmet need in asthma research. METHODS: Exacerbation assessment was a pre-specified secondary analysis of data from a RCT comparing the use of biomarkers & symptoms to adjust steroid treatment in a T2-low severe asthma-enriched cohort. Participants were phenotyped as T2LOW(fractional exhaled nitric oxide [FeNO] ≤20 ppb & blood eosinophil count [PBE] ≤150 cells/µL) or T2HIGH ( FeNO>20 or PBE>150) at study enrolment & at each exacerbation. We report comparison of exacerbators & non-exacerbators, physiological changes at exacerbation in T2LOW & T2HIGH ,& stability of inflammatory phenotypes. RESULTS: 60.8% (183/301) ≥1 self-reported exacerbations (total of 390). Exacerbators were more likely to be female, have a higher BMI & more exacerbations requiring oral corticosteroid (OCS) & unscheduled primary care attendances for exacerbations. At enrolment, 23.6% (71/301) were T2LOW, & 76.4% (230/301) T2HIGH. The T2LOW group had more asthma primary care attendances, were more likely to have a previous admission to HDU/ICU & to be receiving maintenance OCS. At exacerbation the T2LOW events were indistinguishable from T2HIGH exacerbations in terms of lung function & symptom increase, with no increase in T2 biomarkers from stable to exacerbation state in the T2LOW exacerbations. CONCLUSION: Asthma exacerbations demonstrating a T2LOW phenotype were physiologically & symptomatically similar to T2HIGHexacerbations. The clinically significant T2LOW exacerbations highlights the unmet & pressing need to further understand the mechanisms at play in non-T2 asthma.</p

Blood transcriptomic signature in type-2 biomarker low severe asthma and asthma control.

BackgroundPatients with Type-2 (T2) cytokine-low severe asthma often have persistent symptoms despite suppression of T2-inflammation with corticosteroids (CS).ObjectivesTo analyze whole blood transcriptome from 738 samples in T2-biomarker high/low severe asthma patients to relate transcriptomic signatures to T2-biomarkers and asthma symptom scores.MethodsBulk RNAseq data were generated for blood samples (baseline, Week24, Week48) from 301 participants recruited to a randomized clinical trial of CS optimization in severe asthma. Unsupervised clustering, differential gene expression analysis, and pathway analysis were performed. Patients were grouped by T2-biomarker status and symptoms. Associations between clinical characteristics and differentially expressed genes (DEGs) associated with biomarker and symptom levels were investigated.ResultsUnsupervised clustering identified two clusters; Cluster 2 patients were blood eosinophil low/symptom high and more likely to be receiving oral CS (OCS). Differential gene expression analysis of these clusters, with and without stratification for OCS, identified 2,960 and 4,162 DEGs respectively. 627/2,960 genes remained after adjusting for OCS by subtracting OCS signature genes. Pathway analysis identified dolichyl-diphosphooligosaccharide biosynthesis and assembly of RNA polymerase I complex as significantly enriched pathways. No stable DEGs were associated with high symptoms in T2-biomarker low patients, but numerous associated with elevated T2-biomarkers, including 15 that were up-regulated at all time-points irrespective of symptom level.ConclusionsOCS have a considerable effect on whole blood transcriptome. DEG analysis demonstrates a clear T2-biomarker transcriptomic signature, but no signature was found in association with T2-biomarker low patients, including those with a high symptom burden