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1.  The Value of Induced Sputum in the Diagnosis and Management of Children with Bronchial Asthma 
Clujul Medical  2014;87(3):171-176.
Induced sputum in children with bronchial asthma represents a non-invasive method of bronchial inflammation assessment. The main objective of our study was to analyze the cellularity of sputum in patients with bronchial asthma according to the level of disease control and the controlling therapy (with/without inhaled glucocorticoids). The second objective was to establish the correlation between sputum cellularity and other indirect parameters used to evidence bronchial inflammation (exhaled nitric oxide) and obstruction (forced expiratory volume in 1 second).
The study included children with bronchial asthma that were assessed clinically (physical exam, questionnaire on the control of bronchial asthma in children) and by medical tests (induced sputum, exhaled nitric oxide, spirometry).
In patients with partially controlled asthma and those with uncontrolled asthma, the eosinophils percentage in the sputum was higher than in patients with controlled asthma (19.8±26.4% respectively 9.2±20.5% versus 4.5±14.6%, p<0.001). Higher percentage of neutrophils in the sputum was found in the partially controlled and uncontrolled asthma than in the controlled asthma (43.9±20.1% respectively 51.6±38.3% versus 35±19.7%, p=0.009). We also evidenced a direct and statistically significant correlation between the exhaled nitric oxide and the neutrophils percentage in the sputum (r=0.67, p=0.0003). Also, an indirect, moderate to good correlation (r=−0.56, p=0.005) was evidenced between the values of the forced expiratory volume in 1 second and the high eosinophils percentage in the sputum.
In partially controlled and uncontrolled bronchial asthma the eosinophils and neutrophils count in the sputum is significantly higher than in patients with controlled asthma. There is an indirect correlation between the high eosinophils count in the sputum and the forced expiratory volume in 1 second, as well as a direct correlation between the neutrophils count and the exhaled nitric oxide, suggesting that induced sputum should be used in combination with other indirect parameters for the evidence of bronchial inflammation.
PMCID: PMC4508587  PMID: 26528019
induced sputum; child; bronchial asthma; eosinophils; inflammation
2.  Inhaled corticosteroid effects both eosinophilic and non-eosinophilic inflammation in asthmatic patients. 
Mediators of Inflammation  2004;13(4):285-291.
AIM: To determine induced sputum cell counts and interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha) and leukotriene B4 (LTB4) levels as markers of neutrophilic inflammation in moderate persistent asthma, and to evaluate the response to inhaled steroid therapy. METHODS: Forty-five moderate asthmatic patients and 10 non-smoker controls were included in this study. All patients received inhaled corticosteroid (800 microg of budesonide) for 12 weeks. Before and after treatment pulmonary function tests were performed, and symptom scores were determined. Blood was drawn for analysis of serum inflammatory markers, and sputum was induced. RESULTS: Induced sputum cell counts and inflammatory markers were significantly higher in patients with asthma than in the control group. The induced sputum eosinophil counts of 12 patients (26%) were found to be less than 5%, the non-eosinophilic group, and sputum neutrophil counts, IL-8 and TNF-alpha levels were significantly higher than the eosinophilic group (neutrophil, 50+/-14% versus 19+/-10%, p<0.01). In both groups, there was a significant decrease in sputum total cell counts and serum and sputum IL-8, TNF-alpha and LTB4 levels after the treatment. There was no change in sputum neutrophil counts. Although the sputum eosinophil count decreased only in the eosinophilic subjects, there was no significant difference in inflammatory markers between the groups. The symptom scores were significantly improved after treatment, while the improvement did not reach statistical significance on pulmonary function test parameters. CONCLUSION: Notably, in chronic asthma there is a subgroup of patients whose predominant inflammatory cells are not eosinophils. Sputum neutrophil counts and neutrophilic inflammatory markers are significantly higher in these patients. In the non-eosinophilic group, inhaled steroid caused an important decrease in inflammatory markers; however, there was no change in the sputum eosinophil and neutrophil counts.
PMCID: PMC1781566  PMID: 15545060
3.  Distribution of sputum cellular phenotype in a large asthma cohort: predicting factors for eosinophilic vs neutrophilic inflammation 
Phenotyping asthma according to airway inflammation allows identification of responders to targeted therapy. Induced sputum is technically demanding. We aimed to identify predictors of sputum inflammatory phenotypes according to easily available clinical characteristics.
This retrospective study was conducted in 508 asthmatics with successful sputum induction recruited from the University Asthma Clinic of Liege. Receiver-operating characteristic (ROC) curve and multiple logistic regression analysis were used to assess the relationship between sputum eosinophil or neutrophil count and a set of covariates. Equations predicting sputum eosinophils and neutrophils were then validated in an independent group of asthmatics.
Eosinophilic (≥3%) and neutrophilic (≥76%) airway inflammation were observed in 46% and 18% of patients respectively. Predictors of sputum eosinophilia ≥3% were high blood eosinophils, FENO and IgE level and low FEV1/FVC. The derived equation was validated with a Cohen’s kappa coefficient of 0.59 (p < 0.0001). ROC curves showed a cut-off value of 220/mm3 (AUC = 0.79, p < 0.0001) or 3% (AUC = 0.81, p < 0.0001) for blood eosinophils to identify sputum eosinophilia ≥3%. Independent predictors of sputum neutrophilia were advanced age and high FRC but not blood neutrophil count.
Eosinophilic and paucigranulocytic asthma are the dominant inflammatory phenotypes. Blood eosinophils provide a practical alternative to predict sputum eosinophilia but sputum neutrophil count is poorly related to blood neutrophils.
PMCID: PMC3657295  PMID: 23442497
Asthma; Induced sputum; Eosinophil; Neutrophil
4.  Association of Eosinophilic Inflammation with FKBP51 Expression in Sputum Cells in Asthma 
PLoS ONE  2013;8(6):e65284.
Airway eosinophilia is a predictor of steroid responsiveness in steroid-naïve asthma. However, the relationship between airway eosinophilia and the expression of FK506-binding protein 51 (FKBP51), a glucocorticoid receptor co-chaperone that plays a role in steroid insensitivity in asthma, remains unknown.
To evaluate the relationship between eosinophilic inflammation and FKBP51 expression in sputum cells in asthma.
The FKBP51 mRNA levels in sputum cells from steroid-naïve patients with asthma (n = 31) and stable asthmatic patients on inhaled corticosteroid (ICS) (n = 28) were cross-sectionally examined using real-time PCR. Associations between FKBP51 levels and clinical indices were analyzed.
In steroid-naïve patients, the FKBP51 levels were negatively correlated with eosinophil proportions in blood (r = −0.52) and sputum (r = −0.57), and exhaled nitric oxide levels (r = −0.42) (all p<0.05). No such associations were observed in patients on ICS. In steroid-naïve patients, improvement in forced expiratory volume in one second after ICS initiation was correlated with baseline eosinophil proportions in blood (r = 0.74) and sputum (r = 0.76) and negatively correlated with FKBP51 levels (r = −0.73) (all p<0.0001) (n = 20). Lastly, the FKBP51 levels were the lowest in steroid-naïve asthmatic patients, followed by mild to moderate persistent asthmatic patients on ICS, and the highest in severe persistent asthmatic patients on ICS (p<0.0001).
Lower FKBP51 expression in sputum cells may reflect eosinophilic inflammation and glucocorticoid responsiveness in steroid-naïve asthmatic patients.
PMCID: PMC3675168  PMID: 23762334
5.  Nitric oxide metabolites, eosinophils, and eosinophilic cationic protein in patients with asthma: sputum versus blood. 
Journal of Korean Medical Science  2003;18(4):489-493.
The monitoring of airway inflammation has assessed in bronchial asthma directly by sputum examination, and indirectly by measurements in peripheral blood. To investigate the diagnostic value of these two methods, we compared nitric oxide (NO) metabolites, eosinophils, and eosinophil cationic protein (ECP) in sputum and blood in patients with asthma and control subjects. Sputum and serum were obtained from fifteen patients with asthma, and then were examined before anti-asthma treatment, including steroid preparations. ECP was measured by fluoroimmunoassay. NO metabolites were assayed by using modified Griess reaction. Asthmatic patients, compared with control subjects, had significantly higher level of NO metabolites, higher proportion of eosinophils, and higher levels of ECP in sputum. Asthmatic patients, compared with control subjects, however, had significantly higher number of eosinophils, and were at higher levels of ECP in blood. FEV1, FEV1/FVC was negatively correlated with sputum eosinophils. The area under receiver operating characteristic (ROC) curve showed that eosinophils in sputum are significantly accurate markers than NO metabolites in sputum and blood. These findings suggest that the proportion of eosinophils in sputum have more accurate diagnostic marker of asthmatic airway inflammation than NO metabolites in sputum in differentiating asthmatic patients from control subjects.
PMCID: PMC3055074  PMID: 12923323
6.  Effect of inhaled steroids on airway hyperresponsiveness, sputum eosinophils, and exhaled nitric oxide levels in patients with asthma 
Thorax  1999;54(5):403-408.
BACKGROUND—Airway hyperresponsiveness, induced sputum eosinophils, and exhaled nitric oxide (NO) levels have all been proposed as non-invasive markers for monitoring airway inflammation in patients with asthma. The aim of this study was to compare the changes in each of these markers following treatment with inhaled glucocorticosteroids in a single study.
METHODS—In a randomised, double blind, placebo controlled, parallel study 25 patients with mild asthma (19-34 years, forced expiratory volume in one second (FEV1) >75% predicted, concentration of histamine provoking a fall in FEV1 of 20% or more (PC20) <4 mg/ml) inhaled fluticasone propionate (500 µg twice daily) for four weeks. PC20 to histamine, sputum eosinophil numbers, and exhaled NO levels were determined at weeks 0, 2, and 4, and two weeks after completing treatment. Sputum was induced by inhalation of hypertonic (4.5%) saline and eosinophil counts were expressed as percentage non-squamous cells. Exhaled NO levels (ppb) were measured by chemiluminescence.
RESULTS—In the steroid treated group there was a significant increase in PC20, decrease in sputum eosinophils, and decrease in exhaled NO levels compared with baseline at weeks 2 and 4 of treatment. Subsequently, each of these variables showed significant worsening during the two week washout period compared with week 4. These changes were significantly different from those in the placebo group, except for the changes in sputum eosinophils and exhaled NO levels during the washout period. There were no significant correlations between the changes in the three markers in either group at any time.
CONCLUSIONS—Treatment of asthmatic subjects with inhaled steroids for four weeks leads to improvements in airway hyperresponsiveness to histamine, eosinophil counts in induced sputum, and exhaled nitric oxide levels. The results suggest that these markers may provide different information when monitoring anti-inflammatory treatment in asthma.

PMCID: PMC1763792  PMID: 10212103
7.  Effectiveness of Electronic Reminders to Improve Medication Adherence in Tuberculosis Patients: A Cluster-Randomised Trial 
PLoS Medicine  2015;12(9):e1001876.
Mobile text messaging and medication monitors (medication monitor boxes) have the potential to improve adherence to tuberculosis (TB) treatment and reduce the need for directly observed treatment (DOT), but to our knowledge they have not been properly evaluated in TB patients. We assessed the effectiveness of text messaging and medication monitors to improve medication adherence in TB patients.
Methods and Findings
In a pragmatic cluster-randomised trial, 36 districts/counties (each with at least 300 active pulmonary TB patients registered in 2009) within the provinces of Heilongjiang, Jiangsu, Hunan, and Chongqing, China, were randomised using stratification and restriction to one of four case-management approaches in which patients received reminders via text messages, a medication monitor, combined, or neither (control). Patients in the intervention arms received reminders to take their drugs and reminders for monthly follow-up visits, and the managing doctor was recommended to switch patients with adherence problems to more intensive management or DOT. In all arms, patients took medications out of a medication monitor box, which recorded when the box was opened, but the box gave reminders only in the medication monitor and combined arms. Patients were followed up for 6 mo. The primary endpoint was the percentage of patient-months on TB treatment where at least 20% of doses were missed as measured by pill count and failure to open the medication monitor box. Secondary endpoints included additional adherence and standard treatment outcome measures. Interventions were not masked to study staff and patients. From 1 June 2011 to 7 March 2012, 4,292 new pulmonary TB patients were enrolled across the 36 clusters. A total of 119 patients (by arm: 33 control, 33 text messaging, 23 medication monitor, 30 combined) withdrew from the study in the first month because they were reassessed as not having TB by their managing doctor (61 patients) or were switched to a different treatment model because of hospitalisation or travel (58 patients), leaving 4,173 TB patients (by arm: 1,104 control, 1,008 text messaging, 997 medication monitor, 1,064 combined). The cluster geometric mean of the percentage of patient-months on TB treatment where at least 20% of doses were missed was 29.9% in the control arm; in comparison, this percentage was 27.3% in the text messaging arm (adjusted mean ratio [aMR] 0.94, 95% CI 0.71, 1.24), 17.0% in the medication monitor arm (aMR 0.58, 95% CI 0.42, 0.79), and 13.9% in the combined arm (aMR 0.49, 95% CI 0.27, 0.88). Patient loss to follow-up was lower in the text messaging arm than the control arm (aMR 0.42, 95% CI 0.18–0.98). Equipment malfunction or operation error was reported in all study arms. Analyses separating patients with and without medication monitor problems did not change the results. Initiation of intensive management was underutilised.
This study is the first to our knowledge to utilise a randomised trial design to demonstrate the effectiveness of a medication monitor to improve medication adherence in TB patients. Reminders from medication monitors improved medication adherence in TB patients, but text messaging reminders did not. In a setting such as China where universal use of DOT is not feasible, innovative approaches to support patients in adhering to TB treatment, such as this, are needed.
Trial Registration
Current Controlled Trials, ISRCTN46846388
In a cluster-randomized controlled trial, Katherine Fielding and colleagues examine the effectiveness of electronic reminders for improving tuberculosis medication adherence.
Editors' Summary
Tuberculosis—a contagious bacterial disease that usually infects the lungs—is a major global public health problem. Every year, about 9 million people develop tuberculosis and at least 1.3 million people die as a result. Mycobacterium tuberculosis, the organism that causes tuberculosis, is spread in airborne droplets when people with tuberculosis cough or sneeze. The symptoms of tuberculosis include cough, weight loss, and fever. Diagnostic tests for tuberculosis include sputum smear microscopy (microscopic analysis of mucus coughed up from the lungs), the growth of M. tuberculosis from sputum samples, and chest X-rays. Tuberculosis can be cured by taking antibiotics daily for several months (usually isoniazid, rifampicin, ethambutol, and pyrazinamide for two months followed by isoniazid and rifampicin for a further four months), but the emergence of multidrug-resistant M. tuberculosis is making tuberculosis increasingly hard to treat.
Why Was This Study Done?
Because tuberculosis treatment is long and unpleasant, patients often fail to take all their drugs. To improve medication adherence, the World Health Organization recommends that health care workers supervise patients while they take their medication (directly observed treatment, DOT). However, DOT can be hard to implement. In China, for example, where 11% of tuberculosis cases occur, DOT cannot be implemented in many parts of the country, and the national tuberculosis control policy permits self-administered treatment and treatment monitored by family members. It is estimated that over half of individuals with tuberculosis in China self-administer their treatment, but, in 2010, 20% of patients treated using nationally recommended case-management approaches were lost to follow-up or failed to take their medications regularly. In this pragmatic cluster-randomized trial, the researchers investigate whether reminders delivered by mobile phone or by medication monitor boxes (which provide audio reminders to patients and record when the box is opened) might improve tuberculosis medication adherence in China. A pragmatic trial asks whether an intervention works under real-life conditions; a cluster-randomized trial randomly assigns groups of people (here, people living in different counties/districts) to receive alternative interventions and compares outcomes in the differently treated “clusters.”
What Did the Researchers Do and Find?
The researchers assigned people newly diagnosed with tuberculosis in counties/districts to receive reminders about taking their antibiotics and about monthly follow-up visits via text messaging, a medication monitor, or both text messaging and a medication monitor (the intervention arms), or to receive standard nationally recommended care without electronic reminders (the control arm). All the trial participants (about 1,000 per arm) took their drugs out of a medication monitor box, but the box’s audio reminder function was switched off in the text messaging only and control arms. In the intervention arms, doctors were advised to switch participants with poor medication adherence (evaluated at follow-up visits) to either more intensive management or DOT, depending on the level of missed treatment doses. Compared to the control arm, the percentage of patient-months with at least 20% of the drug doses missed (called “poor adherence” and measured by pill counts and data from the medication monitor) was not significantly reduced in the text messaging arm, whereas poor adherence was significantly reduced by 42% and 51% in the medication monitor and the combined arms, respectively (a significant reduction is unlikely to have happened by chance). Notably, fewer patients were switched to intensive management or DOT than expected based on medication adherence evaluations.
What Do These Findings Mean?
These findings show that, in China, the use of an electronic medication monitor box to remind patients to take their anti-tuberculosis drugs improved medication adherence. Interestingly, text messaging alone, which has been shown to improve adherence to antiretroviral therapy among HIV-positive individuals, did not improve medication adherence among patients with tuberculosis, possibly because the messages were too frequent or too impersonal, although this intervention (but none of the others) did reduce patient loss to follow-up. Battery problems with the medication monitor may have resulted in over-estimation of poor adherence to treatment. Moreover, the researchers’ assumption that opening the medication monitor box is synonymous with taking the medication may have introduced some inaccuracies into these findings. Despite these limitations and the underuse of more intensive case management in patients with poor adherence, these findings suggest that using medication monitors to deliver electronic drug reminders to patients might improve medication adherence among patients with tuberculosis in China and in other settings.
Additional Information
This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at This study is further discussed in a PLOS Medicine Perspective by John Metcalfe, Max R. O’Donnell, and David R. BangsbergThe World Health Organization provides information (in several languages) on tuberculosis and on its Directly Observed Treatment Short Course (DOTS) strategy; the Global Tuberculosis Report 2014 provides information about tuberculosis around the worldThe Stop TB Partnership is working towards tuberculosis elimination and provides personal stories about tuberculosis (in English and Spanish); the Tuberculosis Vaccine Initiative (a not-for-profit organization) also provides personal stories about tuberculosisThe US Centers for Disease Control and Prevention provides information about tuberculosis and about treatment for tuberculosis (in English and Spanish)The US National Institute of Allergy and Infectious Diseases also has detailed information on all aspects of tuberculosisMedlinePlus has links to further information about tuberculosis (in English and Spanish)More information about this trial is available
PMCID: PMC4570796  PMID: 26372470
8.  A Large Subgroup of Mild-to-Moderate Asthma Is Persistently Noneosinophilic 
Rationale: Airway eosinophilia is typical of asthma, and many controller treatments target eosinophilic disease. Asthma is clinically heterogeneous, however, and a subgroup of people with asthma do not have airway eosinophilia. The size of this subgroup is uncertain because prior studies have not examined repeated measures of sputum cytology to determine when people with asthma have intermittent versus persistent sputum eosinophila and when they are persistently noneosinophilic.
Objectives: To determine the prevalence and clinical characteristics of the noneosinophilic asthma phenotype.
Methods: We analyzed sputum cytology data from 995 subjects with asthma enrolled in clinical trials in the Asthma Clinical Research Network where they had undergone sputum induction and measures of sputum cytology, often repeatedly, and assessment of responses to standardized asthma treatments.
Measurements and Main Results: In cross-sectional analyses, sputum eosinophilia (≥2% eosinophils) was found in only 36% of subjects with asthma not taking an inhaled corticosteroid (ICS) and 17% of ICS-treated subjects with asthma; an absence of eosinophilia was noted frequently, even in subjects with asthma whose disease was suboptimally controlled. In repeated measures analyses of people with asthma not taking an ICS, 22% of subjects had sputum eosinophilia on every occasion (persistent eosinophilia); 31% had eosinophilia on at least one occasion (intermittent eosinophilia); and 47% had no eosinophilia on every occasion (persistently noneosinophilic). Two weeks of combined antiinflammatory therapy caused significant improvements in airflow obstruction in eosinophilic asthma, but not in persistently noneosinophilic asthma. In contrast, bronchodilator responses to albuterol were similar in eosinophilic and noneosinophilic asthma.
Conclusions: Approximately half of patients with mild-to-moderate asthma have persistently noneosinophilic disease, a disease phenotype that responds poorly to currently available antiinflammatory therapy.
PMCID: PMC3326288  PMID: 22268133
asthma; eosinophil; noneosinophilic; obesity; neutrophil
9.  Use of induced sputum cell counts to investigate airway inflammation in asthma. 
Thorax  1992;47(1):25-29.
BACKGROUND: Airway inflammation is considered to be important in asthma but is relatively inaccessible to study. Less invasive methods of obtaining sputum from patients unable to produce it spontaneously should provide a useful investigational tool in asthma. METHODS: A method to induce sputum with inhaled hypertonic saline was modified for use in 17 asthmatic patients and 17 normal subjects who could not produce sputum spontaneously. The success rate and safety of the method, the reproducibility of cell counts, and differences in cell counts between the asthmatic and normal groups were examined. Hypertonic saline solution 3-5% was inhaled for up to 30 minutes after inhalation of salbutamol. Subjects were asked to expectorate sputum every five minutes. The quality of the sample was scored on the volume of plugs and the extent of salivary contamination. Plugs from the lower respiratory tract were selected for a total cell count and for differential cell counts of eosinophils and metachromatic cells (mast cells and basophils) in direct smears. RESULTS: Adequate samples from the lower respiratory tract were obtained in 76% of first attempts. The mean fall in the forced expiratory volume in one second (FEV1) during inhalation of saline was 5.3% and the maximum fall 20%. Eosinophil and metachromatic cell counts were reproducible (reliability coefficient 0.8 and 0.7 respectively). When compared with sputum from normal subjects sputum from asthmatic patients contained a significantly higher proportion of eosinophils (mean 18.5% (SE 3.8%) v 1.9% (0.6%)) and metachromatic cells (0.50% (0.18%) v 0.039% (0.014%)). In the asthmatic group the differential eosinophil count correlated with the baseline FEV1. CONCLUSION: Induced sputum is capable of detecting differences in cell counts between normal and asthmatic subjects and merits further development as a potential means of assessing airway inflammation in asthma.
PMCID: PMC463545  PMID: 1539140
10.  Eosinophil protein in airway macrophages: A novel biomarker of eosinophilic inflammation in patients with asthma 
Noneosinophilic asthma is common across asthma severities. However, in patients with moderate-to-severe disease, the absence of sputum eosinophilia cannot distinguish between asthmatic subjects with eosinophilic inflammation controlled by corticosteroids versus those in whom eosinophilic inflammation is not a component of the disease.
We sought to develop a method to quantify eosinophil proteins in airway macrophages as a novel biomarker of eosinophilic airway inflammation.
Eosinophil proteins in airway macrophages were assessed by means of flow cytometry, immunofluorescence, and cytoplasmic hue change after ingestion of apoptotic eosinophils. Airway macrophage median percentage of red-hued area in stained sputum cytospin preparations was assessed by means of image analysis from (1) subjects with mild-to-severe asthma, subjects with nonasthmatic eosinophilic bronchitis, and healthy control subjects; (2) subjects with eosinophilic severe asthma after treatment with prednisolone; and (3) subject with noneosinophilic asthma before corticosteroid withdrawal.
Eosinophil proteins were detected in airway macrophages, and cytoplasmic red hue increased after ingestion of apoptotic eosinophils. Airway macrophage percentage red-hued area was increased in subjects with moderate-to-severe asthma compared with that seen in subjects with mild asthma and healthy control subjects, was similar in those with or without a sputum eosinophilia, and was increased after corticosteroid therapy. In asthmatic subjects without sputum eosinophilia, the airway macrophage percentage red-hued area was increased in subjects who did versus those who did not have sputum eosinophilia after corticosteroid withdrawal.
Eosinophil proteins can be reliably measured in airway macrophages. In combination with sputum eosinophilia, the macrophage eosinophil protein content might further define the asthma phenotype and provide an additional tool to direct therapy.
PMCID: PMC3992372  PMID: 20639010
Asthma; macrophage; eosinophil; computer-assisted image analysis; induced sputum
11.  53 Association Between Eosinophil Apoptosis in Induced Sputum and Asthma Severity in Children 
There is increasing evidence that the disorder of eosinophil apoptosis contributes to the mechanism of prominent airway inflammation in asthma. However the relationship between dysregulation of eosinophil apoptosis and severity of childhood asthma is still unclear.
Investigate the relationship between eosinophil apoptosis in induced sputum and severity of asthma in children.
Eighty-six children aged 6 to 12 years with asthma and 32 age-matched healthy controls were observed. Diagnosis of asthma was made using a clinical questionnaire, physical examination and skin prick tests (SPTs). Lung function, and induced sputum analysis were measured in all patients. Total and antigen specific IgE levels were assessesed by ELISA. Eosonophils apoptosis was determined by staining nuclei with propidium iodide, and analyzed with a FACScan. Expression Apo-1/Fas antigen (CD95) in sputum eosinophils was assessed by immunohistochemical staining techniques.
Diagnosis of asthma was confirmed by positive SPT and increased total and specific IgE levels. Asthma severity (assessed by FEV1, peak expiratory flow (PEF) variability and daily symptom scores) complied with mild and moderate asthma. The percentage sputum eosinophils was expressively increased (threshold of <3%) in all asthmatic children (compared to control group) and directly correlated with peripheral blood eosinophilia, skin sensitization, increased level of total and specific IgE and clinical symptoms of asthma and all of these markers were more significant in children with moderate asthma (P < 0.05). Asthma children showed decreased eosinophils apoptosis (“apoptotic ratio”-AR) in induced sputum as compared to controls (P < 0.001), which directly correlated with predicted value of FEV1, PEF variability and inversely with symptoms score (P = 0.005), and was significantly lower in patients with moderate asthma than those in patients with mild (P = 0.001). More of that, these parameters also correlated with decreased expression of Apo-1/Fas antigen (CD95), especially in moderate asthmatic children (P < 0.05).
Our investigation: 1. Confirms that reduced eosinophil apoptosis in induced sputum associated with increased clinical severity of asthma in children. 2. Provides additional evidence that eosinophil apoptosis may be important in the resolution of eosinophilic airway inflammation in asthma, because of their prolonged survival that maintains inflammation.
PMCID: PMC3513001
12.  New drugs targeting Th2 lymphocytes in asthma 
Asthma represents a profound worldwide public health problem. The most effective anti-asthmatic drugs currently available include inhaled β2-agonists and glucocorticoids and control asthma in about 90-95% of patients. The current asthma therapies are not cures and symptoms return soon after treatment is stopped even after long term therapy. Although glucocorticoids are highly effective in controlling the inflammatory process in asthma, they appear to have little effect on the lower airway remodelling processes that appear to play a role in the pathophysiology of asthma at currently prescribed doses. The development of novel drugs may allow resolution of these changes. In addition, severe glucocorticoid-dependent and resistant asthma presents a great clinical burden and reducing the side-effects of glucocorticoids using novel steroid-sparing agents is needed. Furthermore, the mechanisms involved in the persistence of inflammation are poorly understood and the reasons why some patients have severe life threatening asthma and others have very mild disease are still unknown. Drug development for asthma has been directed at improving currently available drugs and findings new compounds that usually target the Th2-driven airway inflammatory response. Considering the apparently central role of T lymphocytes in the pathogenesis of asthma, drugs targeting disease-inducing Th2 cells are promising therapeutic strategies. However, although animal models of asthma suggest that this is feasible, the translation of these types of studies for the treatment of human asthma remains poor due to the limitations of the models currently used. The myriad of new compounds that are in development directed to modulate Th2 cells recruitment and/or activation will clarify in the near future the relative importance of these cells and their mediators in the complex interactions with the other pro-inflammatory/anti-inflammatory cells and mediators responsible of the different asthmatic phenotypes. Some of these new Th2-oriented strategies may in the future not only control symptoms and modify the natural course of asthma, but also potentially prevent or cure the disease.
PMCID: PMC2259400  PMID: 18315837
13.  Establishing the relationship of inhaler satisfaction, treatment adherence, and patient outcomes: a prospective, real-world, cross-sectional survey of US adult asthma patients and physicians 
Inhaled asthma medications are the mainstay of treatment for chronic asthma. However, nonadherence rates for long-term inhaler therapy among adults are estimated to exceed 50 %. Nonadherence is associated with unfavorable clinical outcomes and diminished quality of life. Research suggests that adherence is associated with patients’ satisfaction with their treatment regimen and other factors, such as concomitant allergic rhinitis and tobacco use.
This prospective, cross-sectional survey of physicians and their patients evaluated the relationship between patient satisfaction with attributes of inhaler devices, treatment adherence, and clinical outcomes. Primary care and specialist physicians completed a physician-reported patient record form for patients with a confirmed asthma diagnosis. Patients for whom a physician-reported form was completed were invited to complete a patient-reported form. Both surveys collected information about demographics, symptoms, exacerbation history, treatment, smoking status, comorbidities, type of inhaler device, and treatment adherence. Patients also indicated the degree to which they were satisfied with attributes of their currently prescribed inhaler device(s). Partial least squares path modeling quantified relationships between latent variables and clinical outcomes.
A total of 243 patients were included in our analysis and 41 % had poorly controlled asthma. More favorable clinical outcomes were significantly associated with greater patient satisfaction with drug delivery (P = 0.002), higher medication adherence (P = 0.049), no history of tobacco use (P < 0.001), and absence of comorbid allergic rhinitis (P = 0.005). Attributes associated with device satisfaction included patient perceptions of consistency in the amount of drug delivery to the lungs, ease of use, and feedback about the number of remaining doses.
Higher patient satisfaction with their asthma drug delivery inhaler device is a significant predictor of more favorable clinical outcomes while allergic rhinitis and smoking history were negatively associated with optimal control of asthma. These findings provide clinicians with opportunities to improve patients’ clinical outcomes by tailoring choice of inhaler device therapy and providing education about the correct way to use the device to ensure optimal outcomes. Patients will likely benefit from medical therapy to manage comorbid allergic rhinitis and smoking cessation interventions. Patients unable to stop smoking may require alternative medical therapies to improve their clinical outcomes.
PMCID: PMC4564954  PMID: 26417397
Adherence; Allergic rhinitis; Asthma; Inhaler device; Patient outcomes; Treatment satisfaction
14.  Clinical characteristics and long-term outcomes related to sputum eosinophilia in Korean asthmatics 
Asia Pacific Allergy  2011;1(1):16-24.
Bronchial asthma is usually associated with high sputum eosinophil levels. However, recent reports have suggested the importance of noneosinophilic asthma (NEA) as a distinct phenotype of asthma.
The aim of this study was to evaluate clinical significance of sputum eosinophilia and long-term treatment outcomes related to sputum eosinophilia in Korean asthmatics.
A total of 201 steroid-naive asthmatics who had undergone induced sputum analysis at baseline were selected from the Cohort for Reality and Evolution of Adult Asthma study population. Clinical evaluation, spirometry, a skin-prick test, a methacholine bronchial provocation test, and sputum eosinophil analysis were performed initially, and patients received the treatment recommended by the Global Initiative for Asthma. Lung function was evaluated every 6 months, and 53 patients completed 24 months of regular follow-up visits. Sputum eosinophilia was defined as a sputum eosinophil count of >3%.
Of the 201 steroid-naive asthmatics, 97 patients had NEA and 104 had eosinophilic asthma (EA). Only 52% of steroid-naive asthmatic subjects had elevated baseline sputum eosinophil levels. A higher percentage of sputum eosinophils was associated with a lower PC20 (r = -0.193; p = 0.009, Spearman correlation), but not with forced expiratory volume in one second (FEV1) (r = 0.045; p = 0.525). During the 24-month study period, the percentage change of FEV1 was significantly lower in the NEA group than in the EA group at 6, 12, 18, and 24 months (p < 0.05). The NEA group, unlike the EA group, showed no significant improvement in FEV1 at 6, 12, 18, or 24 months (p > 0.05).
A higher sputum eosinophil percentage was correlated with a higher airway hyperresponsiveness. Compared with EA patients, NEA patients had poor treatment outcomes in the 2-year follow-up of a Korean asthma cohort population.
PMCID: PMC3206232  PMID: 22053292
Asthma; Induced sputum; Eosinophil; Noneosinophilic asthma
15.  Effect of differing doses of inhaled budesonide on markers of airway inflammation in patients with mild asthma 
Thorax  1999;54(2):108-114.
BACKGROUND—It is desirable to prescribe the minimal effective dose of inhaled steroids to control asthma. To ensure that inflammation is suppressed whilst using the lowest possible dose, a sensitive and specific method for assessing airway inflammation is needed.
METHODS—The usefulness of exhaled nitric oxide (NO), sputum eosinophils, and methacholine airway responsiveness (PC20) for monitoring airway inflammatory changes following four weeks of treatment with an inhaled corticosteroid (budesonide via Turbohaler) were compared. Mild stable steroid naive asthmatic subjects were randomised into two double blind, placebo controlled studies. The first was a parallel group study involving three groups receiving either 100 µg/day budesonide (n = 8), 400 µg/day budesonide (n = 7), or a matched placebo (n = 6). The second was a crossover study involving 10 subjects randomised to receive 1600 µg budesonide or placebo. The groups were matched with respect to age, PC20, baseline FEV1 (% predicted), exhaled NO, and sputum eosinophilia.
RESULTS—There were significant improvements in FEV1 following 400 µg and 1600 µg budesonide (11.3% and 6.5%, respectively, p<0.05). This was accompanied by significant reductions in eosinophil numbers in induced sputum (0.7 and 0.9 fold, p<0.05). However, levels of exhaled NO were reduced following each budesonide dose while PC20 was improved only with 1600 µg budesonide. These results suggest that exhaled NO and PC20 may not reflect the control of airway inflammation as accurately as the number of eosinophils in sputum. There were dose dependent changes in exhaled NO, sputum eosinophils, and PC20 to inhaled budesonide but a plateau response of exhaled NO was found at a dose of 400 µg daily.
CONCLUSION—Monitoring the number of eosinophils in induced sputum may be the most accurate guide to establish the minimum dose of inhaled steroids needed to control inflammation. This, however, requires further studies involving a larger number of patients.

PMCID: PMC1745406  PMID: 10325913
16.  Inflammatory phenotypes underlying uncontrolled childhood asthma despite inhaled corticosteroid treatment: rationale and design of the PACMAN2 study 
BMC Pediatrics  2013;13:94.
The diagnosis of childhood asthma covers a broad spectrum of pathological mechanisms that can lead to similarly presenting clinical symptoms, but may nonetheless require different treatment approaches. Distinct underlying inflammatory patterns are thought to influence responsiveness to standard asthma medication.
The purpose of the PACMAN2 study is to identify inflammatory phenotypes that can discriminate uncontrolled childhood asthma from controlled childhood asthma by measures in peripheral blood and exhaled air. PACMAN2 is a nested, case–control follow-up study to the ongoing pharmacy-based “Pharmacogenetics of Asthma medication in Children: Medication with Anti-inflammatory effects” (PACMAN) study. The original PACMAN cohort consists of children aged 4–12 years with reported use of asthma medication. The PACMAN2 study will be conducted within the larger PACMAN cohort, and will focus on detailed phenotyping of a subset of the PACMAN children. The selected participants will be invited to a follow-up visit in a clinical setting at least six months after their baseline visit based on their adherence to usage of inhaled corticosteroids, their asthma symptoms in the past year, and their age (≥ 8 years). During the follow-up visit, current and long-term asthma symptoms, medication use, environmental factors, medication adherence and levels of exhaled nitric oxide will be reassessed. The following measures will also be examined: pulmonary function, exhaled volatile organic compounds, as well as inflammatory markers in peripheral blood and blood plasma. Comparative analysis and cluster-analyses will be used to identify markers that differentiate children with uncontrolled asthma despite their use of inhaled corticosteroids (ICS) (cases) from children whose asthma is controlled by the use of ICS (controls).
Asthmatic children with distinct inflammatory phenotypes may respond differently to anti-inflammatory therapy. Therefore, by identifying inflammatory phenotypes in children with the PACMAN2 study, we may greatly impact future personalised treatment strategies, uncover new leads for therapeutic targets and improve the design of future clinical studies in the assessment of the efficacy of novel therapeutics.
PMCID: PMC3691827  PMID: 23768206
Asthma; Child; Phenotypes; Inflammation; Proteomics; Volatile organic compounds; Corticosteroids
17.  Sputum eosinophilia can predict responsiveness to inhaled corticosteroid treatment in patients with overlap syndrome of COPD and asthma 
Chronic obstructive pulmonary disease (COPD) and asthma may overlap and converge in older people (overlap syndrome). It was hypothesized that patients with overlap syndrome may have different clinical characteristics such as sputum eosinophilia, and better responsiveness to treatment with inhaled corticosteroid (ICS).
Sixty-three patients with stable COPD (forced expiratory volume in 1 second [FEV1] ≤80%) underwent pulmonary function tests, including reversibility of airflow limitation, arterial blood gas analysis, analysis of inflammatory cells in induced sputum, and chest high-resolution computed tomography. The inclusion criteria for COPD patients with asthmatic symptoms included having asthmatic symptoms such as episodic breathlessness, wheezing, cough, and chest tightness worsening at night or in the early morning (COPD with asthma group). The clinical features of COPD patients with asthmatic symptoms were compared with those of COPD patients without asthmatic symptoms (COPD without asthma group).
The increases in FEV1 in response to treatment with ICS were significantly higher in the COPD with asthma group. The peripheral eosinophil counts and sputum eosinophil counts were significantly higher. The prevalence of patients with bronchial wall thickening on chest high-resolution computed tomography was significantly higher. A significant correlation was observed between the increases in FEV1 in response to treatment with ICS and sputum eosinophil counts, and between the increases in FEV1 in response to treatment with ICS and the grade of bronchial wall thickening. Receiver operating characteristic curve analysis revealed 82.4% sensitivity and 84.8% specificity of sputum eosinophil count for detecting COPD with asthma, using 2.5% as the cutoff value.
COPD patients with asthmatic symptoms had some clinical features. ICS should be considered earlier as a potential treatment in such patients. High sputum eosinophil counts and bronchial wall thickening on chest high-resolution computed tomography might therefore be a good predictor of response to ICS.
PMCID: PMC3346210  PMID: 22589579
COPD; asthma; HRCT; inhaled corticosteroid; pulmonary function
18.  Comparing the effects of two inhaled glucocorticoids on allergen-induced bronchoconstriction and markers of systemic effects, a randomised cross-over double-blind study 
Inhaled glucocorticoids are efficient in protecting against asthma exacerbations, but methods to compare their efficacy vs systemic effects have only been attempted in larger multi-centre studies. The aim of the current study was therefore to directly compare the effects of two separate inhaled glucocorticoids, mometasone and budesonide, to compare the effects on the early and late asthmatic responses to inhaled allergen in patients with mild allergic asthma, and sputum eosinophils, and to relate the clinical positive effects to any systemic effects observed.
Twelve patients with documented early and late asthmatic responses (EAR and LAR) to inhaled allergen at a screening visit were randomized in a double-blind fashion to treatment with mometasone (200 μg × 2 or 400 μg × 2), budesonide (400 μg × 2) or placebo in a double-blind crossover fashion for a period of seven days. Challenge with the total allergen dose causing both an EAR and LAR was given on the last day of treatment taken in the morning. Lung function was assessed using FEV1, and systemic glucocorticoid activity was quantified using 24 h urinary cortisol.
Mometasone and budesonide attenuate both EAR and LAR to allergen to a similar degree. No significant dose-related effects on the lung function parameters were observed. Both treatments reduced the relative amount of sputum eosinophils (%) after allergen. At the dose of 800 μg daily, mometasone reduced 24 h urinary cortisol by approximately 35%. Both drugs were well tolerated.
Mometasone and budesonide are equieffective in reducing early and late asthmatic responses induced by inhaled allergen challenge. Mometasone 800 μg given for seven days partially affects the HPA axis.
PMCID: PMC3339360  PMID: 22409907
allergen; asthma; budesonide; mometasone; inflammation
19.  Heterogeneity of bronchitis in airway diseases in tertiary care clinical practice 
Sputum cell counts have identified inflammatory subtypes of bronchitis in relatively small numbers of subjects with asthma, chronic obstructive pulmonary disease (COPD) and chronic cough in research studies. The prevalence of different subtypes of bronchitis in routine clinical practice, however, has not been reported.
To examine the heterogeneity of bronchitis and its relationship to the severity of airflow obstruction.
A retrospective cross-sectional survey based on a computerized database of spontaneous or induced sputum cell counts examined in a large university tertiary respiratory outpatient clinic.
The database contained 4232 consecutive sputum records from 2443 patients with chronic cough (39%), asthma (37%), asthma with COPD (9%), COPD (13%) and bronchiectasis (3%). Total and differential cell counts were obtained from 86% of successful sputum samples. Induced sputum provided more viable samples than spontaneous expectorate. Approximately one-third of patients with asthma and one-fifth of patients with COPD experience eosinophilic bronchitis. Asthmatic patients with moderate to severe airflow obstruction had a greater number of sputum eosinophils. There was a significantly higher number of total cell counts and percentage of neutrophils in the sputum of COPD patients with moderate and severe airflow obstruction than in those with mild airflow obstruction.
There is heterogeneity in the cellularity of sputum in various airway diseases. Patients with clinically stable airway diseases may have high sputum cell counts. During exacerbations, more patients may experience neutrophilic bronchitis. Severity of airflow obstruction is associated with eosinophilic bronchitis in patients with asthma, and neutrophilic bronchitis in patients with nonasthmatic COPD.
PMCID: PMC3328881  PMID: 21766077
Asthma; Bronchitis heterogeneity; Clinical practice; COPD; Sputum cell counts
20.  Detailed analysis of sputum and systemic inflammation in asthma phenotypes: are paucigranulocytic asthmatics really non-inflammatory? 
The technique of induced sputum has allowed to subdivide asthma patients into inflammatory phenotypes according to their level of granulocyte airway infiltration. There are very few studies which looked at detailed sputum and blood cell counts in a large cohort of asthmatics divided into inflammatory phenotypes. The purpose of this study was to analyze sputum cell counts, blood leukocytes and systemic inflammatory markers in these phenotypes, and investigate how those groups compared with healthy subjects.
We conducted a retrospective cross-sectional study on 833 asthmatics recruited from the University Asthma Clinic of Liege and compared them with 194 healthy subjects. Asthmatics were classified into inflammatory phenotypes.
The total non-squamous cell count per gram of sputum was greater in mixed granulocytic and neutrophilic phenotypes as compared to eosinophilic, paucigranulocytic asthma and healthy subjects (p < 0.005). Sputum eosinophils (in absolute values and percentages) were increased in all asthma phenotypes including paucigranulocytic asthma, compared to healthy subjects (p < 0.005). Eosinophilic asthma showed higher absolute sputum neutrophil and lymphocyte counts than healthy subjects (p < 0.005), while neutrophilic asthmatics had a particularly low number of sputum macrophages and epithelial cells. All asthma phenotypes showed an increased blood leukocyte count compared to healthy subjects (p < 0.005), with paucigranulocytic asthmatics having also increased absolute blood eosinophils compared to healthy subjects (p < 0.005). Neutrophilic asthma had raised CRP and fibrinogen while eosinophilic asthma only showed raised fibrinogen compared to healthy subjects (p < 0.005).
This study demonstrates that a significant eosinophilic inflammation is present across all categories of asthma, and that paucigranulocytic asthma may be seen as a low grade inflammatory disease.
Electronic supplementary material
The online version of this article (doi:10.1186/s12890-016-0208-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4820945  PMID: 27044366
Asthma; Phenotypes; Healthy subjects; Sputum cytology; Blood leukocyte count; CRP; Fibrinogen
21.  Efficacy of brief motivational interviewing to improve adherence to inhaled corticosteroids among adult asthmatics: results from a randomized controlled pilot feasibility trial 
Daily adherence to inhaled corticosteroid (ICS) regimens is one of the most important factors linked to achieving optimal asthma control. Motivational interviewing (MI) is a client-centered communication style that focuses on enhancing intrinsic motivation to engage in appropriate self-management behaviors. MI has been shown to improve a variety of health behaviors including medication adherence in other disorders, but its efficacy for the improvement of ICS adherence in asthmatics has yet to be examined. This pilot “proof of concept” trial assessed the feasibility of MI to improve daily ICS adherence and asthma control levels in adult asthmatics.
Fifty-four poorly controlled (Asthma Control Questionnaire [ACQ] score ≥1.5), highly nonadherent (filled <50% of ICS medication in the last year) adult asthmatics were recruited from the outpatient asthma clinic of a university-affiliated hospital. Participants underwent baseline assessments and were randomly assigned to MI (3×30 minutes sessions within a 6-week period, n=26) or a usual care (UC) control group (n=28). ICS adherence (% pharmacy refills) and asthma control (ACQ, Asthma Control Test [ACT]) were measured at 6 and 12 months postintervention. Mixed model repeated measure analyses for both intent-to-treat and per-protocol were used. Results were adjusted for a priori-defined covariates including baseline adherence. Patients in the MI group also reported their impressions of the intervention.
Six-month adjusted intent-to-treat analyses revealed a mean change in the percentage of ICS refills of 13% in the MI group vs 6% in the UC group (between group net improvement associated with MI =+6% [−12% to 24%]). Twelve-month analyses revealed a mean change in the percentage of ICS refills of 11% (MI group) vs 7% in the UC group (between group net improvement associated with MI =+3% [−11% to 18%]) representing an effect size (ES) of d=0.20 (medium). Six-month adjusted net improvement in ACQ and ACT scores between MI and control groups was −0.2 and +0.7, respectively, with improvements being even more pronounced at 12 months (ACQ =−0.5; ACT =+1.1; ES, d=0.12 and 0.18 [small], respectively). Interestingly, 6- and 12-month net improvements in asthma self-efficacy (which is specifically targeted by MI) improved by +0.4 and +0.4, respectively, with an ES of d=0.46 (marginally large). Patients in the MI group rated the intervention overwhelmingly positively in terms of usefulness, pertinence, feasibility, enjoyableness, and likelihood to change adherence behavior.
Results suggest that a brief (90 minutes) MI intervention that targets ICS adherence can produce clinically significant improvements in adherence behavior, asthma control levels, and asthma self-efficacy in poorly controlled nonadherent asthmatics at 6 months that are maintained for 1 year, and it is well accepted by patients. Future studies including larger sample sizes, modified intervention designs (eg, MI “booster” sessions), and assessments of the extent behavior changes translate into clinical improvements (eg, in asthma control and quality of life) are warranted.
PMCID: PMC4231985  PMID: 25422587
motivational interviewing; asthma; adherence; inhaled corticosteroids; asthma control
22.  Automated quantitation of circulating neutrophil and eosinophil activation in asthmatic patients 
Thorax  2000;55(6):471-477.
BACKGROUND—Asthma has been associated with eosinophil activation, measured in serum, sputum, bronchoalveolar lavage (BAL) fluid, and urine. A whole blood automated method was developed to assess eosinophil and neutrophil activity in terms of peroxidase content and cell morphology using the Bayer haematology analyser. The method was applied to an in vitro stimulation model when fMLP was added to whole blood and the samples were then analysed for changes in granularity and shape. In addition, cells stimulated with interleukin (IL)-8 were examined by electron microscopy.
METHODS—A cross sectional analysis was performed on venous blood from non-atopic, non-asthmatic normal subjects (n = 37), mild (n= 46) and symptomatic (n = 22) asthmatic patients on inhaled β2 agonist only, and more severe asthmatic patients (n = 17) on inhaled and oral corticosteroid therapy. Samples were analysed by the haematology analyser and peroxidase leucograms gated using the WinMDI software program.
RESULTS—There were significant differences in the amount of light scatter by the neutrophil populations in the symptomatic (p = 0.007) and severe asthmatic (p = 0.0001) groups compared with the control group. However, abnormalities in eosinophil populations were not observed. In vitro activation of whole blood with fMLP caused similar changes in neutrophil light scatter, suggesting that neutrophil activation is present in peripheral blood of symptomatic asthmatic patients. IL-8 caused a change in shape of the neutrophils seen using transmission electron microscopy.
CONCLUSIONS—Evidence of neutrophil activation can be seen in whole blood from patients with asthma using a novel automated method. This may potentially be applied to other inflammatory diseases.

PMCID: PMC1745769  PMID: 10817795
23.  Evaluation of correlation between airway and serum inflammatory markers in asthmatic patients 
Asthma is one of the most common chronic diseases all over the world, resulting from a state of persistent sub-acute inflammation of the airways. Beside local inflammation, systemic inflammation is also present, which can be shown by increased levels of C-reactive protein (CRP). One of the most important cells in this disease is eosinophil, and sputum eosinophilia is used for its diagnosis.
The purpose of the present study was to compare and evaluate the correlation between CRP level and sputum eosinophilia in asthmatic and control subjects.
Materials and Methods:
A total of 61 patients suffering from mild-to-moderate asthma participated in this study. They were divided into two groups based on whether they used inhaled steroid or not. Sputum was induced by ultrasonic nebulizer, and then samples of peripheral venous blood were collected to measure peripheral cell count and CRP by Enzyme-linked immunosorbent assay (ELISA). Thirty-seven healthy subjects were selected and their blood samples were taken. Thirty-seven healthy subjects were selected and their blood samples were taken.
Thirty asthmatic patients in user group (14 females/16 males) with the mean age of 39.4±9.37 years, 31 asthmatic patients in non-user group (13 females/18 males) with the mean age of 35.5±8.87 years, and 37 healthy controls (17 females/20 males) were included in our study. The mean serum concentration of CRP was 2.6 μg/mL, 3.32 μg/mL, and 1.16 μg/mL in user, non-user, and control groups, respectively. Compared to healthy controls, serum concentrations of high sensitivity-CRP (hs-CRP) significantly increased in the non-user group (P=0.0001), and user group as well. (P=0.016). The number of sputum eosinophils and peripheral blood eosinophils significantly increased in the non-users compared to the healthy controls (P=0.0001, P=0.003, respectively). In the non-user group, serum hs-CRP levels correlated negatively with FEV1 and positively with numbers of sputum eosinophils, which was not statistically significant. Atopy status, age, and sex did not affect hs-CRP levels in both asthmatic groups.
It was found that serum concentrations of hs-CRP significantly increased in asthmatic patients, and in the steroid-native group it partly correlated with FEV1 and numbers of sputum eosinophils. It suggests that serum hs-CRP can indirectly indicate the degree of airway inflammation.
PMCID: PMC3354488  PMID: 22628929
Asthma; C-reactive protein; eosinophilia; forced expiratory volume in one second; sputum
24.  Analyses of Asthma Severity Phenotypes and Inflammatory Proteins in Subjects Stratified by Sputum Granulocytes 
Patients with severe asthma have increased granulocytes in their sputum compared to patients with mild to moderate asthma.
We hypothesized that inflammatory granulocytes in sputum may identify specific asthma severity phenotypes and are associated with different patterns of inflammatory proteins in sputum supernatants.
This hypothesis was tested in 242 asthmatics enrolled in the Severe Asthma Research Program who provided sputum samples for cell count, differential cell determinations, cell lysates for Western blot, and supernatant analyses by inflammatory protein microarrays and ELISAs. ANOVA and multiple linear regression models tested mediator associations.
Stratified by sputum granulocytes, < or ≥2%eosinophils and < or ≥40%neutrophils, subjects with both increased eosinophils and neutrophils had the lowest lung function, increased symptoms and healthcare utilization. Subjects with elevated eosinophils with or without increased neutrophils had significantly increased FeNO, serum eosinophils and greater frequency of daily β-agonist use. Microarray data, stratified by granulocytes revealed 25–28 inflammatory proteins increased >2-fold in sputa with ≥40% neutrophils. Microarray analyses stratified by severity of asthma, identified 6–9 proteins increased >2-fold in sputa in subjects with severe asthma compared to nonsevere asthma. ELISA data, stratified by sputum granulocytes, showed significant increases in BDNF, IL-1β, and MIP-3α/CCL20 for those with ≥40%neutrophils; these mediators demonstrated positive associations with neutrophil counts.
Combined increased sputum eosinophils and neutrophils identified asthmatics with the lowest lung function and worse asthma control, increased symptoms and healthcare requirements. Inflammatory protein analyses of sputum supernatants found novel mediators increased in asthmatics, predominantly associated with increased sputum neutrophils.
PMCID: PMC2878277  PMID: 20398920
asthma phenotypes; protein microarrays; BDNF; CXCL13; TNFSF14; CCL20; CCL18
25.  Sputum IgE and Cytokines in Asthma: Relationship with Sputum Cellular Profile 
PLoS ONE  2013;8(3):e58388.
Local IgE production may play a role in asthma pathogenesis. The aim of the study was to assess sputum total IgE and cytokines in asthmatics according to sputum cellular phenotype.
We studied 122 subjects including 22 non atopic healthy subjects, 41 eosinophilic (sputum eosinophils ≥3%), 16 neutrophilic (sputum neutrophils >76%) and 43 pauci-granulocytic asthmatics (sputum eosinophils <3% and sputum neutrophils ≤76%) recruited from the asthma clinic at CHU Liege.
Sputum supernatant total IgE (tIgE) was measured by ImmunoCAP and sputum supernatant cytokines (IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IFN-γ and TNF-α) were measured with the Luminex xMAP Technology by using commercially available Fluorokine MAP kits.
After concentrating sputum samples, total IgE was detectable in the majority of subjects. Sputum IgE was raised in asthmatics when compared to healthy subjects. Overall, asthmatics did not significantly differ from healthy subjects with respect to cytokine levels. The eosinophilic asthma phenotype, however, was characterised by raised sputum tIgE, IL-5 and IL-13 compared to healthy subjects (p<0.001, p<0.001 and p<0.05 respectively) and pauci-granulocytic asthma (p<0.01, p<0.001 and p<0.05 respectively) and raised IL-5 compared to neutrophilic asthma (p<0.01). When patients were classified according to sputum IgE levels, it appeared that IL-5, IL-6, IL-17 and TNF-α sputum supernatant levels were raised in the “IgE high” asthmatics (IgE ≥0.1 kU/l) when compared to “IgE low” asthmatics (IgE<0.1 kU/l).
The eosinophilic asthma phenotype was associated with raised sputum IgE and a Th2 cytokine profile. Raised sputum IgE was associated with a heterogeneous cytokine overproduction.
PMCID: PMC3608646  PMID: 23555579

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