A Rapid Sputum-based Lateral Flow Assay for Airway Eosinophilia using an RNA-cleaving DNAzyme Selected for Eosinophil Peroxidase.

The first protein-binding allosteric RNA-cleaving DNAzyme (RCD) obtained by direct in vitro selection against eosinophil peroxidase (EPX), a validated marker for airway eosinophilia, is described. The RCD has nanomolar affinity for EPX, shows high selectivity against related peroxidases and other eosinophil proteins, and is resistant to degradation by mammalian nucleases. An optimized RCD was used to develop both fluorescence and lateral flow assays, which were evaluated using 38 minimally processed patient sputum samples (23 non-eosinophilic, 15 eosinophilic), producing a clinical sensitivity of 100 % and specificity of 96 %. This RCD-based lateral flow assay should allow for rapid evaluation of airway eosinophilia as an aid for guiding asthma therapy.

Ex-vivo mucolytic and anti-inflammatory activity of BromAc in tracheal aspirates from COVID-19.

COVID-19 is a lethal disease caused by the pandemic SARS-CoV-2, which continues to be a public health threat. COVID-19 is principally a respiratory disease and is often associated with sputum retention and cytokine storm, for which there are limited therapeutic options. In this regard, we evaluated the use of BromAc®, a combination of Bromelain and Acetylcysteine (NAC). Both drugs present mucolytic effect and have been studied to treat COVID-19. Therefore, we sought to examine the mucolytic and anti-inflammatory effect of BromAc® in tracheal aspirate samples from critically ill COVID-19 patients requiring mechanical ventilation. METHOD: Tracheal aspirate samples from COVID-19 patients were collected following next of kin consent and mucolysis, rheometry and cytokine analysis using Luminex kit was performed. RESULTS: BromAc® displayed a robust mucolytic effect in a dose dependent manner on COVID-19 sputum ex vivo. BromAc® showed anti-inflammatory activity, reducing the action of cytokine storm, chemokines including MIP-1alpha, CXCL8, MIP-1b, MCP-1 and IP-10, and regulatory cytokines IL-5, IL-10, IL-13 IL-1Ra and total reduction for IL-9 compared to NAC alone and control. BromAc® acted on IL-6, demonstrating a reduction in G-CSF and VEGF-D at concentrations of 125 and 250 µg. CONCLUSION: These results indicate robust mucolytic and anti-inflammatory effect of BromAc® ex vivo in tracheal aspirates from critically ill COVID-19 patients, indicating its potential to be further assessed as pharmacological treatment for COVID-19.

lncRNA-NEAT1 Sponges miR-128 to Promote Inflammatory Reaction and Phenotypic Transformation of Airway Smooth Muscle Cells.

OBJECTIVE: Pediatric asthma is still a health threat to the children. Long noncoding RNA-NEAT1 (lncRNA-NEAT1) was reported to be positively correlated with the severity of asthma. We aimed to study the effects and mechanism of lncRNA-NEAT1on inflammatory reaction and phenotypic transformation of airway smooth muscle cells (ASMCs) in the bronchial asthma. METHOD: The degree of lncRNA-NEAT1 and miR-128 mRNA in children with bronchial asthma and healthy individuals was tested by qRT-PCR. After the inflammatory reaction and phenotypic transformation of PDGF-BB-induced ASMCs, the expression of lncRNA-NEAT1 or miR-128 in the AMSC was disturbed in the AMSC. Subsequently, the expression of lncRNA-NEAT1 and miR-128 was detected by the way of qRT-PCR, and western blot was applied to measure the expression of MMP-2, MMP-9, α-SMA, calponin, NF-κB, and so on in the cells. The content of TNF-α, IL-1ß, IL-6, and IL-8 in the cell culture supernatant was checked by ELISA. MTT, Transwell, and flow cytometry were used to detect cell proliferation, migration, and apoptosis. Further, the targeting relations between lncRNA-NEAT1 and miR-128 were evaluated by the dual-luciferase reporter assay. RESULT: In the sputum of children with bronchial asthma, lncRNA-NEAT1 was significantly upregulated while miR-128 was rapidly downregulated. Besides, lncRNA-NEAT1 and miR-128 were competitively combined and, for their expression, negatively correlated. CONCLUSION: lncRNA-NEAT1 sponges miR-128 to boost PDGF-BB-induced inflammatory reaction and phenotypic transformation of ASMCs to aggravate the occurrence and development of childhood bronchial asthma.

Are sputum eosinophils associated with a different phenotype in COPD patients? A retrospective study.

Sputum eosinophilia in Chronic Obstructive Pulmonary Disease (COPD) patients seems to be associated with a better response to inhaled corticosteroids (ICS). To verify if this feature could identify a specific subpopulation of COPD patients, we retrospectively compared functional and inflammatory parameters of 110 COPD patients according to the presence of sputum eosinophilia (>2%). Patient with eosinophilia were characterized by lower dyspnea score, lower functional impairment and lower ICS use, suggesting that airway eosinophilia may be associated to a lower COPD severity and some functional "asthma-like" characteristics, therefore explaining the better response to ICS in this subgroup of patients.

Cough hypersensitivity in patients with metabolic syndrome: a clinical finding and its possible mechanisms.

PURPOSE: To investigate the changes of cough sensitivity in patients with metabolic syndrome and its possible mechanisms. METHOD: A total of 29 metabolic syndrome (MetS) patients with OSAHS (group-1), 22 MetS patients without OSAHS (group-2), and 25 healthy controls (group-3) were included. All participants underwent a routine physical examination and completed the gastroesophageal reflux disease questionnaire (GerdQ), and the inflammatory mediator profile were determined. The cough threshold for capsaicin, induced sputum cell count and cell classification, and inflammatory mediators in induced sputum supernatants were compared. The correlation between capsaicin cough sensitivity and various indicators in the MetS population was analyzed. RESULTS: The minimum concentration of inhaled capsaicin needed to induce ≥ 5 coughs (C5) was significantly different among three groups (H = 14.393, P = 0.001) and lower for group-1 and group-2 than it for group-3 (P = 0.002, P = 0.005). The percentage of neutrophils in induced sputum and the concentrations of calcitonin gene-related peptide (CGRP), substance P (SP), and interleukin 8 (IL-8) in the sputum supernatant of group-1 and group-2 were significantly higher than those of group-3. Besides, the pepsin concentrations were significantly different among the 3 groups (F = 129.362, P < 0.001), which significantly was highest in group-1 (P < 0.001) and lowest in group-3 (P < 0.001). Triglycerides, AHI, pepsin concentration and BMI were risk factors of increased capsaicin cough sensitivity. CONCLUSION: Increased capsaicin cough sensitivity in MetS patients is closely related to sleep apnea and gastroesophageal reflux. For patients in MetS patients without OSAHS, gastroesophageal reflux is an important factor for increased capsaicin cough sensitivity. Airway inflammation, especially airway neurogenic inflammation, may also play a role in the pathogenesis of increased capsaicin cough sensitivity. Trial registration The protocol was registered in the Chinese Clinical Trials Register ( http://www.chictr.org.cn/ ) (ChiCTR1800014768). Written informed consent was obtained from all participants before enrollment.

Long-term Lung Cancer Risk Associated with Sputum Atypia: A 27-Year Follow-up Study of an Occupational Lung Screening Cohort in Yunnan, China.

BACKGROUND: Sputum cytologic atypia is associated with increased lung cancer risk. However, little is known about the long-term magnitude and temporal trend of this risk. METHODS: An extended follow-up was conducted in a prospective screening cohort among occupational tin miners in Yunnan, China. Sputum samples were collected prospectively at baseline and 7 annual screenings since enrollment. The associations between sputum cytologic results from baseline screening, the first 4 consecutive rounds of sputum screening, and lung cancer risk were analyzed by time-varying covariate Cox regression model. RESULTS: A moderate or worse cytologic result was associated with a significantly increased lung cancer risk. This relative hazard significantly decreased over time. Compared with negative screening results, the adjusted hazard ratios of baseline-moderate or worse atypia, at least one moderate or worse atypia in the first 4 consecutive screening rounds during the first 10 years of follow-up were 3.11 [95% confidence interval (CI): 2.37-4.07], 3.25 (95% CI: 2.33-4.54) respectively. This association was stronger for persistent atypia (adjusted hazard ratio = 17.55, 95% CI: 8.32-37.03); atypia identified in the recent screening rounds (adjusted HR = 4.14, 95% CI: 2.70-6.35), and those were old in age, had higher level of smoking, occupational radon, and arsenic exposure. In terms of histology, this increased risk was significant for squamous cell carcinoma and small cell lung cancer. CONCLUSIONS: Although decreasing over time, an increased lung cancer risk concerning moderate or worse sputum atypia can continue at least for 10 years. IMPACT: Sputum atypia might be helpful for identifying high-risk individuals for screening, surveillance, or chemoprevention of lung cancer.

Comprehensive Care Management in Conjunction with Sputum Cytometry-Guided Pharmacotherapy in a Post-Discharge Clinic for Patients with COPD.

Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are amongst the most common reasons for hospital admission, and recurrent episodes occur frequently. Comprehensive care management (CCM) strategies have modest effect in preventing re-admissions. The objectives of this study were to examine the utility of optimizing anti-inflammatory therapy guided by sputum cytometry in the post-hospitalization setting, and to assess the feasibility and effectiveness of a clinic combining CCM and sputum-guided therapy. This is an observational study examining patients who received open-label CCM and sputum cytometry-guided pharmacotherapy in a COPD post-discharge clinic. Referral was based on high risk for readmission after hospitalization for AECOPD. The primary outcome was the change in COPD-related healthcare utilization before and after Visit 1, and this was analyzed with a mixed-effects negative binomial model controlling for age, number of follow-up clinic visits, pack years, current smoking and FEV1. Of 138 patients referred to the clinic, 73% attended at least one visit. Mean FEV1 was 42.8 (19.3) % predicted. Of the patients attending clinic, 42.6% produced an adequate sputum sample, and 32.7% had an abnormal sputum. By individual, infectious bronchitis was the most common (25.7%), followed by eosinophilic bronchitis (13.9%). Comparing the 6-months prior to and after the first clinic visit, there was a lower incidence rate ratio after visit 1 for COPD-related healthcare utilization (0.26 (95%CI 0.22,0.33; p < 0.001)). A COPD post-discharge clinic combining sputum-guided treatment and CCM was feasible and associated with a nearly 75% reduction in the incidence of COPD-related healthcare utilization.

Raised sputum extracellular DNA confers lung function impairment and poor symptom control in an exacerbation-susceptible phenotype of neutrophilic asthma.

BACKGROUND: Extracellular DNA (e-DNA) and neutrophil extracellular traps (NETs) are linked to asthmatics airway inflammation. However, data demonstrating the characterization of airway inflammation associated with excessive e-DNA production and its impact on asthma outcomes are limited. OBJECTIVE: To characterize the airway inflammation associated with excessive e-DNA production and its association with asthma control, severe exacerbations and pulmonary function, particularly, air trapping and small airway dysfunction. METHODS: We measured e-DNA concentrations in induced sputum from 134 asthma patients and 28 healthy controls. We studied the correlation of e-DNA concentrations with sputum neutrophils, eosinophils and macrophages and the fractional exhaled nitric oxide (FeNO). Lung function was evaluated using spirometry, body plethysmography, impulse oscillometry and inert gas multiple breath washout. We stratified patients with asthma into low-DNA and high-DNA to compare lung function impairments and asthma outcomes. RESULTS: Patients with severe asthma had higher e-DNA concentration (54.2 ± 42.4 ng/µl) than patients with mild-moderate asthma (41.0 ± 44.1 ng/µl) or healthy controls (26.1 ± 16.5 ng/µl), (all p values < 0.05). E-DNA concentrations correlated directly with sputum neutrophils (R = 0.49, p < 0.0001) and negatively with sputum macrophages (R = - 0.36, p < 0.0001), but neither with sputum eosinophils (R = 0.10, p = 0.26), nor with FeNO (R = - 0.10, p = 0.22). We found that 29% of asthma patients (n = 39) had high e-DNA concentrations above the upper 95th percentile value in healthy controls (55.6 ng /µl). High-DNA was associated with broad lung function impairments including: airflow obstruction of the large (FEV1) and small airways (FEF50%, FEF25-75), increased air trapping (RV, RV/TLC), increased small airway resistance (R5-20, sReff), decreased lung elasticity (X5Hz) and increased ventilation heterogeneity (LCI), (all P values < 0.05). We also found that high e-DNA was associated with nearly three-fold greater risk of severe exacerbations (OR 2·93 [95% CI 1.2-7.5]; p = 0·012), worse asthma control test (p = 0.03), worse asthma control questionnaire scores (p = 0.01) and higher doses of inhaled corticosteroids (p = 0.026). CONCLUSION: Increased production of extracellular DNA in the airway characterizes a subset of neutrophilic asthma patients who have broad lung function impairments, poor symptom control and increased risk of severe exacerbations.

Eosinophilic Pleural Effusion in a Young Woman With Pleural Nodularity and Lytic Skeletal Lesions.

CASE PRESENTATION: A 21-year-old woman, a housewife with no known comorbidities, presented to the outpatient department with complaints of dry cough, left-sided pleuritic chest pain, modified Medical Research Council grade II breathlessness and backache. She had started developing these symptoms 1 month earlier. There was no history of fever, hemoptysis, or significant weight loss. She had no features suggestive of connective tissue disorder. There was no history of recurrent respiratory infections in the past. She was married for 1 year and had no children. Her sleep, bowel, and bladder habits were normal. No significant family history or medication history was noted.

The expression of IL17RA on sputum macrophages in asthma patients.

IL-17A and IL-25 (IL-17 cytokines family) play an important role in the development of asthma, and allergy. Both cytokines act by binding to heterodimeric receptors with IL17RA as a common subunit. This receptor is found on macrophages, and some other cell types. The aim of the study was to determine the expression of IL17RA on asthmatic and control macrophages from induced sputum (IS) with the regard to IL-17/IL-25 background and relation to clinical features of the disease. We found an elevated expression of IL17RA on sputum macrophages in asthma patients vs controls. A characteristic sputum profile of atopic asthmatic was as follows: high CD206 + IL17RA + macrophage percentage, elevated IL-25 level and low CD206 + IL17RA- macrophage percentage. Based on the above results, it seems that CD206 + sputum macrophages are the effector cells that express common subunit of the receptor for IL-17A and IL-25 in asthma. This may be related to the Th2-dependent environment in asthma and increased concentrations of IL-25 and IL-13 as well as eosinophils in the airways. To our knowledge, our study provides the first data on a possible link between immunological reaction orchestrating CD206 + expressing sputum macrophages and IL-25 via IL17RA pathway in the asthmatic airways.

Discontinuation of inhaled corticosteroids in patients with controlled asthma: The DISCO (Discontinuation of Inhaled Steroid in Controlled asthmatics Over 6 months) study.

BACKGROUND: Although inhaled corticosteroids (ICSs) are the recommended first-line therapy for asthma, determining whether to continue or discontinue ICS treatment in patients with mild asthma remains challenging for clinicians. Several studies have revealed that patients with mild-persistent asthma maintained a well-controlled state after ICS withdrawal. However, the long-term outcomes of ICS withdrawal have not yet been determined. OBJECTIVE: To determine the possible clinical outcomes of the discontinuation of ICS in patients with well-controlled mild asthma. METHODS: We investigated the clinical outcomes of discontinuing ICSs in patients with well-controlled mild asthma and compared the time to loss of control (LOC) between patients who stopped ICS treatment (ICS withdrawal group, IWG) and those who continued treatment for 3 years (continuous ICS group, CIG). RESULTS: A significant difference in the time to LOC was observed between the IWG and CIG (hazard ratio, 2.56; 95% confidence interval, 1.52-4.33; P < .001). Increasing fractional exhaled nitric oxide levels (P = 0.008) and sputum eosinophil counts (%) (P = 0.015) revealed a weak but significant association with LOC risk in the CIG. The sputum eosinophil counts (P = 0.039) and serum total immunoglobulin E levels (P = 0.014) were significantly higher in the LOC group than in the non-LOC group of the CIG. CONCLUSION: Our results suggest that the maintenance of ICS treatment may help keep patients' asthma under control. Furthermore, patients with LOC had significantly higher sputum eosinophil counts in the CIG than those in the non-LOC group. Therefore, continuous ICS use by patients with mild, well-controlled asthma could be associated with good clinical outcomes. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: KCT0002234.

Clinical evaluation of type 2 disease status in a real-world population of difficult to manage asthma using historic electronic healthcare records of blood eosinophil counts.

BACKGROUND: Blood eosinophil measurement is essential for the phenotypic characterization of patients with difficult asthma and in determining eligibility for anti-IL-5/IL-5Rα biological therapies. However, assessing such measures over limited time spans may not reveal the true underlying eosinophilic phenotype, as treatment, including daily oral corticosteroid therapy, suppresses eosinophilic inflammation and asthma is intrinsically variable. METHODS: We interrogated the electronic healthcare records of patients in the Wessex AsThma CoHort of difficult asthma (WATCH) study (UK). In 501 patients being evaluated in this tertiary care centre for difficult to control asthma, all requested full blood count test results in a 10-year retrospective period from the index WATCH assessment were investigated (n = 11,176). RESULTS: In 235 biological therapy-naïve participants who had 10 or more measures in this time period, 40.3% were eosinophilic (blood eosinophils ≥300 cells/µl) at WATCH enrolment whilst an additional 43.1%, though not eosinophilic at enrolment, demonstrated eosinophilia at least once in the preceding decade. Persistent eosinophilia was associated with worse post-bronchodilator airway obstruction and higher Fractional exhaled Nitric Oxide (FeNO). In contrast, the 16.6% of patients who never demonstrated eosinophilia at this blood eosinophil threshold showed preserved lung function and lower markers of Type 2 inflammation. CONCLUSIONS: This highlights the central role that type 2 inflammation, as indicated by blood eosinophilia, has in difficult asthma and suggests that longitudinal electronic healthcare record analysis can be an important tool in clinical asthma phenotyping, providing insight that may help understand disease progression and better guide more specific treatment approaches.

Duration of lead time in screening for lung cancer.

BACKGROUND: Screening for lung cancer has used chest radiography (CR), low dose computed tomography (LDCT) and sputum cytology (SC). Estimates of the lead time (LT), i.e., the time interval from detection of lung cancer by screening to the development of symptoms, have been derived from longitudinal studies of populations at risk, tumor doubling time (DT), the ratio between its prevalence at the first round of screening and its annual incidence during follow-up, and by probability modeling derived from the results of screening trials. OBJECTIVE: To review and update the estimates of LT of lung cancer. METHODS: A non-systematic search of the literature for estimates of LT and screening trials. Search of the reference sections of the retrieved papers for additional relevant studies. Calculation of LTs derived from these studies. RESULTS: LT since detection by CR was 0.8-1.1 years if derived from longitudinal studies; 0.6-2.1 years if derived from prevalence / incidence ratios; 0.2 years if derived from the average tumor DT; and 0.2-1.0 if derived from probability modeling. LT since detection by LDCT was 1.1-3.5 if derived from prevalence / incidence ratios; 3.9 if derived from DT; and 0.9 if derived from probability modeling. LT since detection of squamous cell cancer by SC in persons with normal CR was 1.3-1.5 if derived from prevalence/incidence ratios; and 2.1 years if derived from the DT of squamous cell cancer. CONCLUSIONS: Most estimates of the LT yield values of 0.2-1.5 years for detection by CR; of 0.9-3.5 years for detection by LDCT; and about 2 years or less for detection of squamous cell cancer by SC in persons with normal CR. The heterogeneity of the screening trials and methods of derivation may account for the variability of LT estimates.

Neutrophilic asthma features increased airway classical monocytes.

BACKGROUND: Monocytes and macrophages are critical innate immune cells of the airways. Despite their differing functions, few clinical studies discriminate between them and little is known about their regulation in asthma. OBJECTIVE: We aimed to distinguish and quantify macrophages, monocytes and monocyte subsets in induced sputum and blood and examine their relationship with inflammatory and clinical features of asthma. METHODS: We applied flow cytometry to distinguish macrophages, monocytes and subsets in sputum and blood (n = 53; 45 asthma, 8 non-asthma) and a second asthma sputum cohort (n = 26). Monocyte subsets were identified by surface CD14/CD16 (CD14++ CD16- classical, CD14+ CD16+ intermediate and CD14+ CD16++ non-classical monocytes). Surface CD206, a marker of monocyte tissue differentiation, was measured in sputum. Relationship to airway inflammatory phenotype (neutrophilic n = 9, eosinophilic n = 14, paucigranulocytic n = 22) and asthma severity (severe n = 12, non-severe n = 33) was assessed. RESULTS: Flow cytometry- and microscope-quantified sputum differential cell proportions were significantly correlated. Sputum macrophage number was reduced (p = .036), while classical monocyte proportion was increased in asthma vs non-asthma (p = .032). Sputum classical monocyte number was significantly higher in neutrophilic vs paucigranulocytic asthma (p = .013). CD206- monocyte proportion and number were increased in neutrophilic vs eosinophilic asthma (p < .001, p = .013). Increased sputum classical and CD206- monocyte numbers in neutrophilic asthma were confirmed in the second cohort. Blood monocytes did not vary with airway inflammatory phenotype, but blood classical monocyte proportion and number were increased in severe vs non-severe asthma (p = .022, p = .011). CONCLUSION AND CLINICAL RELEVANCE: Flow cytometry allowed distinction of sputum macrophages, monocytes and subsets, revealing compartment-specific dysregulation of monocytes in asthma. We observed an increase in classical and CD206- monocytes in sputum in neutrophilic asthma, suggesting co-recruitment of monocytes and neutrophils to the airways in asthma. Our data suggest further investigation of how airway monocyte dysregulation impacts on asthma-related disease activity is merited.

Disease burden of eosinophilic airway disease: Comparing severe asthma, COPD and asthma-COPD overlap.

BACKGROUND AND OBJECTIVE: There is less understanding of phenotypes and disease burden in asthma-COPD overlap (ACO) than either disease alone. Blood eosinophils may help identify the patients in the clinic with eosinophilic airway inflammation. The potential value of this approach requires an understanding of the illness burden associated with eosinophilic ACO, eosinophilic severe asthma and eosinophilic COPD, defined by blood eosinophils. METHODS: Participants from studies of multidimensional assessment in airway disease were pooled to identify patients with ACO (n = 106), severe asthma (n = 64) and COPD alone (n = 153). Patients were assessed cross-sectionally for demographic and clinical characteristics, including disease burden indicators such as health-related quality of life (HRQoL) and past-year exacerbation. Eosinophilic patients were identified using different thresholds of blood eosinophil count. RESULTS: Using a blood eosinophil count ≥0.3 × 109 /L, 41% had eosinophilic airway disease: 55% in ACO, 44% in severe asthma and 29% in COPD. Blood and sputum eosinophils were moderately correlated (rs = 0.51, n = 257, P < 0.001). Burden of disease was similar between eosinophilic and non-eosinophilic airway diseases, with poor HRQoL and high number of past-year exacerbations. Burden of disease was similar across eosinophilic severe asthma, COPD and ACO. Eosinophilic COPD tended to have poorer health status than eosinophilic ACO and severe asthma; however, in context of a high prevalence of eosinophilic ACO, cumulative population-level burden of eosinophilic disease was greater in ACO. CONCLUSION: Disease burden across eosinophilic ACO, eosinophilic severe asthma and eosinophilic COPD was high, particularly cumulative population-level burden in ACO. Factors beyond airway inflammation may drive disease burden in severe patients.

COPD patients with chronic bronchitis and higher sputum eosinophil counts show increased type-2 and PDE4 gene expression in sputum.

Chronic obstructive pulmonary disease (COPD) patients with higher eosinophil counts are associated with increased clinical response to phosphodiesterase-4-inhibitors (PDE4i). However, the underlying inflammatory mechanisms associated with this increased response is not yet elucidated. This post hoc analysis focused on sputum gene expression in patients with chronic bronchitis who underwent 32-day treatment with two doses of the inhaled PDE4i CHF6001 (tanimilast) or placebo on top of triple therapy. Biological characterization and treatment effects were assessed between patients with different sputum eosinophil levels (eosinophilhigh  ≥ 3%; eosinophillow  < 3%) at baseline (primary samples) or at the end of the treatment of the placebo arm (validation samples). Forty-one genes were differentially expressed in primary samples (p-adjusted for false discovery rate < 0.05); all up-regulated in eosinophilhigh patients and functionally enriched for type-2 and PDE4 inflammatory processes. Eleven out of nineteen genes having immune system biological processes annotations including IL5RA, ALOX15, IL1RL1, CLC, GATA1 and PDE4D were replicated using validation samples. The expression of a number of these inflammatory mediators was reduced by tanimilast treatment, with greater effects observed in eosinophilhigh patients. These findings suggest that type-2 and PDE4 overexpression in COPD patients with higher sputum eosinophil counts contribute to the differential clinical response to PDE4i observed in previous clinical trials.