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Aim: International collaboration is needed to enable large-scale pharmacogenomics studies in childhood asthma. Here, we describe the design of the Pharmacogenomics in Childhood Asthma (PiCA) consortium. Materials & methods: Investigators of each study participating in PiCA provided data on the study characteristics by answering an online questionnaire. Results: A total of 21 studies, including 14,227 children/young persons (58% male), from 12 different countries are currently enrolled in the PiCA consortium. Fifty six percent of the patients are Caucasians. In total, 7619 were inhaled corticosteroid users. Among patients from 13 studies with available data on asthma exacerbations, a third reported exacerbations despite inhaled corticosteroid use. In the future pharmacogenomics studies within the consortium, the pharmacogenomics analyses will be performed separately in each center and the results will be meta-analyzed. Conclusion: PiCA is a valuable platform to perform pharmacogenetics studies within a multiethnic pediatric asthma population.

Objectives: To study topical corticosteroid use in Dutch asthmatic children using pharmacy dispensing data and to assess whether Dutch physicians prescribe topical corticosteroids in this population according to clinical guidelines. Methods: Medication histories of children using asthma medication were extracted from the pharmacy dispensing system in 100 Dutch community pharmacies. The incidence rate and the potency of topical corticosteroid prescriptions per age were assessed. The topical corticosteroid incidence rates of the different age groups were compared using the Pearson chi-square test. Generalized linear models were used to study the prescription behavior of general practitioners and atopic dermatitis-related specialists regarding different classes of topical corticosteroids. Results: Thirty percent of the infants received a topical corticosteroid prescription, compared with 15%-18% of the children aged 4 and older. Similarly, the mean number of topical corticosteroid prescriptions in infants was 2.2 per year, compared with 1.6-1.9 in children aged 4 and older. In concordance with the clinical guidelines, we observed that atopic dermatitis-related specialists more often prescribed first prescriptions of potent and very potent topical corticosteroids than general practitioners (relative risk = 2.55, 95% confidence interval = 1.79-3.63). Statistically significant differences (P

Asthma medicines (e.g. inhaled corticosteroids (ICS) and inhaled β-agonists) are the most commonly chronically used medication in children.(1) In the Netherlands, asthma is treated according to a stepwise approach, which is mainly derived from the British Thoracic Society (BTS) guidelines.(2, 3) The guidelines advice to start treatment at the most appropriate step according to clinical severity. Step up of asthma treatment is advised if a child does not reach asthma control in the current step, and step down is advised if a child is well controlled for a period of 3 months.(2, 4) Little is known about how well the stepwise approach in the guidelines is followed in clinical practice. This article is protected by copyright. All rights reserved.

Pieters LE, Vijverberg SJH, Raaijmakers JAM, Van der Ent CK, Maitland-van der Zee AH. Astmadiagnose bij kinderen. Huisarts Wet 2014;57(9):446-51. Het onderzoeken van de overeenstemming tussen door de ouder en door de huisarts gerapporteerde astmadiagnose bij kinderen die astmamedicatie gebruiken. We hebben 996 kinderen (4-12 jaar) uit het PACMAN-cohort onderzocht. Alle kinderen hadden recentelijk astmamedicatie gebruikt. Tijdens een onderzoeksbezoek in de apotheek vulden ouders en kinderen een vragenlijst in over astmadiagnose, klachten, medicatiegebruik en therapietrouw. Huisartsen hebben we schriftelijk benaderd met de vraag of het kind astma had. We onderzochten de invloed van verschillende patientkarakteristieken met multivariate logistische regressieanalyse. Bij 63,5% van de kinderen rapporteerden zowel de huisarts als de ouders een astmadiagnose. Er was meer overeenstemming tussen de ouders en de huisarts in de aan- of afwezigheid van een astmadiagnose als het kind ouder was (OR 1,14; p = 0,008), de astmaklachten minder onder controle waren (OR 1,82; p = 0,02) en het kind inhalatiecorticosteroiden (OR 2,93; p = 0,001) of kortwerkende beta-2-agonisten (OR 2,04; p = 0,03) gebruikte. Afkomst en opleidingsniveau van de ouders hingen niet samen met de mate van overeenstemming. Therapietrouw aan onderhoudsmedicatie was het hoogst (63,1%) bij de groep kinderen van wie zowel de ouders als de huisartsen hadden aangegeven dat het kind een astmadiagnose had. De overeenstemming tussen de ouders en de huisarts in het aangeven van een astmadiagnose bij kinderen die astmamedicatie gebruiken is relatief hoog, maar er is nog steeds ruimte voor verbetering.

Background: Current evidence suggests that asthma patients with the ADRB2 Arg16 genotype have a poorer response to long-acting β2-agonists (LABA), but the results remain inconsistent. Aim: This study assessed the association between Arg16 variants and treatment outcome in children treated with inhaled corticosteroids (ICS) and LABA. Materials & methods: ADRB2 Arg16 was genotyped in 597 children (4–12 years of age) participating in the PACMAN cohort study. A questionnaire was used to assess asthma control, frequency of asthma-related emergency department visits and use of oral corticosteroids in the past year. Results: Arg/Arg carriers with a reported use of ICS and LABA had an increased risk of oral corticosteroid use (odds ratio: 14.9; 95% CI: 1.59–140.1) and emergency department visits in the past year (odds ratio: 11.9; 95% CI: 1.22–115.8) compared to Gly/Gly carriers. This effect was not observed in Arg/Arg genotype carriers reporting ICS use only. Conclusion: Children who are homozygous for ADRB2 Arg16 have an increased risk of exacerbations when treated with combined LABA and ICS. Original submitted 4 September 2013; Revision submitted 27 September 2013

Background: Genetic variation may partly explain heterogeneity in response to asthma treatment . Recently, the P harmacogenomics i n C hildhood A sthma (PiCA) consortium was initiated to perform large-scale pharmacogenomics studies. Aims: To describe the characteristics of the asthmatic children currently included in PiCA and to assess treatment response definitions within this growing consortium. Methods: An online survey including questions about data collection, patient characteristics and outcome phenotypes was send to the 20 studies included in the PiCA consortium. Results: In total, 14,634 children/young adults (up to 25 years) from 11 different countries are enrolled in the PiCA consortium. Preliminary analysis of patients showed that 60% of the patients are male. PiCA is ethnically diverse: 47% Caucasians, 25% Latinos, 14% Asians, 9% African-Americans, 2% have mixed/other-ethnicity and 3% is unknown. PiCA currently includes data of 2,056 severe asthmatics. Furthermore, PiCA has data of 8,907 short acting β 2 -adrenergic agonist (SABA) users, 7,908 ICS users, 2,054 leukotriene modifier (LTM) users and 1,543 users of long acting β 2 -adrenergic agonists (LABA). Ninety-five percent of the studies have information regarding asthma exacerbations and lung function measurements and 80% have data on asthma symptoms. Discussion: The children within the PiCA consortium are a reflection of the global heterogeneous pediatric asthma population. Different outcome measures reflect different dimensions of asthma. By classifying three commonly used outcome measures using data that are available within the PiCA consortium, we will be able to study distinct response phenotypes in relation to genetics and clinical factors.

To cite this article: Vijverberg SJH, Koster ES, Koenderman L, Arets HGM, van der Ent CK, Postma DS, Koppelman GH, Raaijmakers JAM, Maitland-van der Zee A-H. Exhaled NO is a poor marker of asthma control in children with a reported use of asthma medication: a pharmacy-based study. Pediatr Allergy Immunol 2012: 23: 529536. Abstract Background: A high fraction of nitric oxide in exhaled breath (FeNO) has been suggested to be a marker of ongoing airway inflammation and poorly controlled disease in asthma. The usefulness of FeNO to monitor asthma control is still debated today. Aim: To assess the validity of FeNO as a marker of asthma control in children with reported use of asthma medication. Methods: Fraction of nitric oxide in exhaled breath was measured in 601 children (aged 412 yr) with reported use of asthma medication in the past 6 months and in 63 healthy non-asthmatic children (aged 512). Asthma control was assessed by the Asthma Control Questionnaire (ACQ). A receiver-operator characteristics (ROC) curve was generated to assess the accuracy of FeNO as a marker for asthma control. Logistic regression analysis was used to study whether clinical, healthcare, medication, and environmental factors are associated with high FeNO levels (>25 ppb). Results: Fraction of nitric oxide in exhaled breath had a poor accuracy to discriminate well-controlled from not well-controlled asthma [area under the ROC curve: 0.56 (95% CI: 0.520.61, p = 0.008)]. In addition, high FeNO (>25 ppb) was associated with lower medication adherence rates (OR: 0.4; 95% CI 0.30.6), fewer antibiotic courses in the past year (OR: 0.6; 95% CI: 0.40.9), fewer leukotriene antagonists use in the past year (OR: 0.4; 95% CI: 0.20.9), and fewer visits to a (pulmonary) pediatrician (OR: 0.6; 95% CI: 0.40.9). Children living in a non-urban environment had more often high FeNO levels (OR: 1.7; 95% CI: 1.12.6). Conclusion: High FeNO is a poor marker of asthma control in children with reported use of asthma medication. Various other factors, including medication adherence and medication use, are associated with increased FeNO levels.

Objective To evaluate, among new users of inhaled corticosteroids that did not persist treatment, knowledge of inhaled corticosteroids' actions and whether they were instructed on the use of their inhaler. Setting Fifteen community pharmacies in The Netherlands. Methods Patients were interviewed by telephone. Their general practitioners provided diagnostic information and automated dispensing records were retrieved. Main outcome measures Knowledge of patients about the actions of inhaled corticosteroids. Results 230 (80.1%) of 287 patients were willing to participate. The majority (79.1%) of 230 patients was not aware of the anti-inflammatory actions of inhaled corticosteroids. Most patients were instructed on the use of their inhaler, predominantly by their physician (53%) or pharmacy (35.2%). Conclusions Although most patients reported inhaler instruction by at least one health care provider, the majority was unaware of inhaled corticosteroids' actions. Physicians and pharmacists should reconsider the instructions they provide especially to patients who should continuously use inhaled corticosteroids.

BACKGROUND: The T2206C FCER2 variant was found previously to be associated with IgE levels, exacerbation rates and decreased FCER2 expression in children on inhaled corticosteroids (ICS) participating in a clinical trial. This finding has not been replicated. We sought to replicate the association between the FCER2 gene and exacerbations in children with asthma. In addition, we tested the hypothesis that the T2206C variant may be associated with other markers of steroid resistance such as asthma symptom scores and asthma medication use. METHODS: The influence of the T2206C variant on asthma exacerbations (emergency department visits or hospitalization), symptoms scores and medication use was explored using data from two populations of asthmatic children using ICS: Pharmacogenetics of Asthma medication in Children: Medication with ANti-inflammatory effects study (n = 386) and BREATHE study (n = 939). RESULTS: The T2206C variant was associated with increased risk of asthma-related hospital visits in both cohorts (OR: 1.91, 95% CI: 1.08-3.40), and meta-analysis with previously published results was highly significant (OR: 2.38, 95% CI: 1.47-3.85, P = 0.0004). The FCER2 variant was also associated with increased risk of uncontrolled asthma measured by Asthma Control Questionnaire (OR: 2.64, 95% CI: 1.00-6.98) and was associated with increased daily steroid dose (OR: 2.46, 95% CI: 1.38-4.39). CONCLUSION: The association between the FCER2 T2206C variant and asthma-related hospitalizations in steroid-treated asthma appears robust and may also be associated with other indicators of lack of ICS efficacy such as asthma symptoms and a requirement for higher daily doses of ICS. Our results suggest that the FCER2 T2206C variant might be a useful pharmacogenetic predictor of steroid refractory patients.

Aims Poor adherence with inhaled corticosteroids (ICSs) has been reported frequently and may be associated with uncontrolled asthma. A better understanding of factors influencing adherence may help to achieve higher adherence rates for a larger part of the population, which will eventually lead to better asthma control. The aim of this study was to investigate factors associated with adherence in paediatric ICS users. Methods We included 527 children using ICSs who participated in the Pharmacogenetics of Asthma Medication in Children: Medication with Anti-inflammatory Effects (PACMAN) cohort study. The outcome, a parent-reported adherence, was assessed by using the Medication Adherence Report Scale. Four categories of determinants were studied: child characteristics, family characteristics, medication use (parental beliefs towards medication; using Beliefs about Medicines Questionnaire) and environmental factors. Results Good adherence was observed in 302 children (57%). Increased fractional exhaled nitric oxide values (indication for airway inflammation) were associated with a lower chance of good adherence (OR = 0.25, 95%CI = 0.15–0.41). Parental necessity beliefs about medication were associated with higher adherence (OR = 2.32, 95%CI = 1.59–3.39). Dutch origin was also associated with higher adherence rates (OR = 2.11, 95%CI = 1.09–4.07). Furthermore, younger age (

Seasonal variation in asthma has been widely recognized. The aim of this study was to describe seasonal patterns of asthma symptoms and asthma medication use in a cohort of pediatric asthma medication users and to study determinants of seasonal childhood asthma.For this study, 602 children participating in the Pharmacogenetics of Asthma medication in Children: Medication with Anti-inflammatory effects) cohort were included. Parents were asked about their child's respiratory symptoms and quick-reliever medication use over the past year. Logistic regression analysis was used to study determinants of seasonality in asthma control (the level of disease control based on symptoms, limitations in activities, and rescue medication use).There was a decline in asthma symptoms and asthma medication use during the summer period and a peak occurred from autumn to spring. The prevalence of wheeze ranged from 32% in summer to 56% in autumn. The prevalence of respiratory symptoms and medication use was significantly lower during summer (p.0001). Oral steroids and antibiotics use and strong parental necessity beliefs were associated with uncontrolled asthma, regardless of seasonality. Allergic rhinitis was associated with an increased risk of uncontrolled asthma during spring [odds ratio (OR): 1.9; 95% confidence interval (CI): 1.3-2.8] and summer (OR: 1.9; 95% CI: 1.2-3.0). Eczema was associated with a higher risk of uncontrolled asthma during autumn (OR: 1.5; 95% CI: 1.0-2.2) and winter (OR: 1.3; 95% CI: 1.0-1.9).We showed seasonal patterns in asthma symptoms and medication use. Associations were shown between allergic rhinitis and asthma control during spring/summer and eczema was associated with uncontrolled asthma during autumn/winter. Seasonality in asthma morbidity and health-care use is most likely associated with atopic constitution and viral infections, which are common during fall, winter, and spring.

Background: Several studies have shown that predictors of asthma treatment outcomes differ depending on the definition of the outcome chosen. This provides evidence that different outcomes studied may reflect distinct aspects of asthma control. To assess predictors of asthma control, we need firm outcome phenotypes. The aim of this study was to investigate the association between measurements of current and long-term asthma control. Methods: We included 527 children using inhaled corticosteroids participating in the Pharmacogenetics of Asthma medication in Children: Medication with ANti-inflammatory effects cohort. Current asthma control (previous week) was defined using the Asthma Control Questionnaire. Long-term asthma control was based on Global Initiative for Asthma guidelines. Not well-controlled asthma in a season was defined as =3 of the following items present in a season: (i) day-time or (ii) night-time symptoms, (iii) limitations in activities, and (iv) rescue medication use. Asthma control during (i) the previous season and (ii) the year preceding the pharmacy visit was used as long-term asthma control definitions. Current and long-term asthma control were compared to investigate agreement. Results: Long-term uncontrolled asthma rates were highest in autumn and winter (50%) and lowest in summer (32%) (p

Summary Asthma is one of the most common chronic diseases worldwide. There is a large interindividual variability in response to asthma treatment. Most patients respond well to standard therapy; however, a small proportion of the patients remain symptomatic despite treatment with high dosages of corticosteroids. Uncontrolled asthma leads to a decreased quality of life. Therefore, it is important to identify individuals who will respond poorly to standard asthma medication, especially to standard maintenance therapy with inhaled corticosteroids, at an early stage. Response to anti-inflammatory therapy is generally monitored by the assessment of clinical symptoms, which only partially correlates with underlying airway inflammation. The identification of specific inflammatory biomarkers might help to guide treatment or predict a corticosteroid response more accurately. Some inflammatory biomarkers are already finding their way into clinical practice (e.g. fraction of nitric oxide in exhaled breath), whereas others are predominantly used as a research tool (e.g. profiles of volatile organic compounds). Currently, there is no inflammatory biomarker used in routine clinical practice to predict a corticosteroid response. More knowledge on the underlying biological mechanism(s) of heterogeneous therapeutic responses could help to identify novel biomarkers. This review will focus on inflammatory patterns and genetic variations that may underlie differences in treatment response in patients with asthma, and will provide an overview of inflammatory biomarkers that could potentially serve as response predictors.

Background: Despite existing effective treatment options, asthma is uncontrolled in a considerable proportion of patients. The aim of this study was to identify determinants of uncontrolled asthma at age 8 in children participating in the PIAMA birth cohort study. Methods: One hundred seventy children using inhaled corticosteroids in the previous 12 months at age 8 were included. Uncontrolled asthma was defined as: >= 3 items present in the past month: (1) day-time or (2) night-time asthma symptoms, (3) limitations in activities, (4) rescue medication use, (5) FEV(1)

PURPOSE: This study was conducted to assess the validity of parental reported use of inhaled corticosteroids (ICS) in children. METHODS: ICS users were identified within the Prevention and Incidence of Asthma and Mite Allergy (PIAMA) birth cohort study and the PIAMA pharmacy sub-cohort which is nested within the PIAMA study. Complete medication histories were available for the first 8 years of life for children within the PIAMA pharmacy sub-cohort. Parental reported ICS use was measured by using data from questionnaires. ICS use in the pharmacy records was determined by using the Anatomical Therapeutic Chemical (ATC) codes. The proportion of overall agreement and kappa statistics with their corresponding 95% confidence intervals were calculated to quantify agreement between self-reported medication use and pharmacy prescription data. RESULTS: At all ages overall agreement was very high (>97%) and Cohen's kappa's ranged from 0.80 to 0.88 which also reflects excellent agreement between parental reported use of ICS and pharmacy prescription data. CONCLUSIONS: Our finding suggests that parental report of medication use is a reliable source of data to asses ICS use in children. The questionnaire-based medication data collected within the PIAMA study can be used to study asthma medication use in a large group of children.

The detailed knowledge of the human genome has not fulfilled its promise as yet. It seems fair to say that we are far from treating existing diseases by therapeutic interventions developed on the basis of genetic knowledge. However, pharmacogenetics has shown to be useful in improving our understanding of pharmacotherapy. Industry is starting to embed this knowledge in the design of innovative drugs and there are three important areas of interest: safety, efficacy and target identification. Application of pharmacogenetics e.g. in patient selection are leading to the direction of more personalised medicine. The future will bring more of such applications. However, current knowledge also leads to a more integrated approach of pharmacogenetics as part of systems biology, providing an even more complete image of reality surrounding disease and therapy, including for example environmental factors and behaviour. In addition, collaborative efforts with academic partners are very much welcomed by the pharmaceutical industry and are expected to have a synergistic effect on progression in this field.

Aims: To investigate the effects of genetic variation on treatment response to asthma medication in children and to identify (profiles of) SNPs that characterize response phenotypes. Material & methods: The Pharmacogenetics of Asthma medication in Children: Medication with Anti-inflammatory effects (PACMAN) study was initiated in April 2009 as an observational retrospective pharmacy-based study, including at least 1000 children with asthma medication (aged 4–12 years). Data on respiratory symptoms and medication use behavior will be collected using a questionnaire; complete medication histories will be extracted from the pharmacy information system; additional health information will be requested from the general practitioner; quality of inhalation technique and lung function measurements will be performed and saliva samples for DNA extraction and genotyping will be collected. Two groups of patients will be defined based on questionnaire data and lung function measurements: responders and nonresponders to anti-inflammatory asthma treatment. These two groups will be compared with respect to genetic variation. Corrections will be made for potential confounding factors. Results: The main study end point is treatment response, including asthma control, medication use and exhaled nitric oxide as a measure of airway inflammation. Whilst our focus is on genetic factors, this study allows us to also investigate other treatment response determinants, such as inhalation technique and therapy adherence. Conclusion: Results from the PACMAN study could eventually lead to a more individualized therapy approach. PACMAN will focus on pharmacogenetics of asthma medication in children, while knowledge will be gained of relevant interest to the treatment of the asthma population at large.

Background Allergic asthma is associated with chronic airway and systemic immune responses. Systemic responses include priming of peripheral blood eosinophils, which is enhanced after allergen challenge. In a subpopulation of asthmatic subjects, neutrophils are associated with bronchial inflammation. Objective We sought to monitor systemic granulocyte priming in allergic asthmatic subjects as a consequence of chronic and acute inflammatory signals initiated by allergen challenge. Methods Blood was taken at baseline and 6 to 24 hours after allergen challenge in asthmatic subjects with and without late asthmatic responses. Systemic granulocyte priming was studied by using expression of cellular markers, such as α-chain of Mac-1 (αm)/CD11b, L-selectin/CD62L, and an activation epitope present on FcγRII/CD32 recognized by monoclonal phage antibody A17. Results Eosinophils of asthmatic subjects have a primed phenotype identified by cell-surface markers. Neutrophils of these patients were subtly primed, which was only identified after activation with N-formyl-methionyl-leucyl-phenylalanine. After allergen challenge, an acute increase in eosinophil priming characterized by enhanced expression of activated FcγRII was found in patients experiencing a late asthmatic response and not in patients with a single early asthmatic response. In contrast, expression of αm/CD11b and L-selectin on granulocytes was not different between control and asthmatic subjects and was not affected by allergen challenge. Interestingly, expression of both adhesion molecules was positively correlated, and αm expression on eosinophils and neutrophils correlated positively with bronchial hyperresponsiveness. Conclusion Different phases, phenotypes, or both of allergic asthma are associated with distinct priming profiles of inflammatory cells in peripheral blood. Clinical implications Insight in differences of systemic innate responses will lead to better definition of asthma subtypes and to better designs of new therapeutic options.

P38 MAPK is a central mediator in cytokine signalling in human leukocytes. P38 MAPK is activated by phosphorylation of a conserved Thr180-X-Tyr182 motif by dual phosphorylation via the upstream kinases MKK3 and MKK6. Alternatively, P38 MAPK can be activated via autophosphorylation when associated with TAB1. In this study P38 MAPK phosphorylation and activation (measured via phosphorylation of P38 MAPK downstream target MK2) were investigated upon engagement of the GM-CSF- and TNFalpha-receptors expressed on both eosinophils and neutrophils. The MKK3/MKK6 pathway mediated neutrophil P38 MAPK activation after stimulation with TNFalpha (100U/ml) or GM-CSF (10(-10)M). Under these conditions the activation but not phosphorylation of P38 MAPK could be inhibited by SB203580 (10(-5)M or 10(-6)M). In eosinophils SB203580 (10(-6)M) inhibited both the phosphorylation and activation of P38 MAPK after stimulation with several doses of TNFalpha (10-10000 U/ml) or GM-CSF (10(-11) to 10(-9)M), indicating that P38 MAPK activation is mediated via autophosphorylation in eosinophils. This hypothesis was supported by the finding that in marked contrast to neutrophils, MKK3/MKK6 did not show enhanced phosphorylation in eosinophils after cytokine stimulation, but were constitutively phosphorylated. Therefore, the involvement of TAB1 was investigated with regard to this cytokine-induced autophosphorylation. Co-immunoprecipitation experiments showed that TAB1 was constitutively associated with P38 MAPK in eosinophils and neutrophils and that cytokine-induced autophosphorylated P38 MAPK was co-precipitated with TAB1. These findings are consistent with the hypothesis that cytokine-induced autophosphorylation of P38 MAPK in primary granulocytes depends on the interaction with TAB1.

There is increasing discussion in the Netherlands about the introduction of a threshold value for the costs per extra year of life when reimbursing costs of new drugs. The Medicines Committee ('Commissie Geneesmiddelen'), a division of the Netherlands National Healthcare Institute ('Zorginstituut Nederland'), advises on reimbursement of costs of new drugs. This advice is based upon the determination of therapeutic value of the drug and the results of economic evaluations. Mathematical models that predict future costs and effectiveness are often used in economic evaluations; these models can vary greatly in transparency and quality due to author assumptions. Standardisation of cost-effectiveness models is one solution to overcome the unwanted variation in quality. Discussions about the introduction of a threshold value can only be meaningful if all involved are adequately informed, and by high quality in cost-effectiveness research and, particularly, economic evaluations. Collaboration and discussion between medical specialists, patients or patient organisations, health economists and policy makers, both in development of methods and in standardisation, are essential to improve the quality of decision making.

Background:GLCCI1 rs37972 has previously been associated with decreased lung function improvement upon treatment with inhaled corticosteroids (ICS) in asthmatics. Aim: To assess whether variation in rs37972 is associated with altered ICS efficacy in north European asthmatic children and young adults with a reported use of ICS. Patients & methods: rs37972 was genotyped in three cohort studies of asthmatic children with a reported use of ICS. As an indicator for asthma exacerbations, asthma-related hospital visits and oral corticosteroid use were studied. Asthma control was assessed using a questionnaire. Results: rs37972 T allele was not significantly associated with an increased risk of oral corticosteroid use (summary odds ratio: 1.20; 95% CI: 0.99–1.45), an increased risk of asthma-related hospital visits (summary odds ratio: 1.07; 95% CI: 0.89–1.29), uncontrolled symptoms (summary odds ratio: 1.01; 95% CI: 0.75–1.36) or higher ICS dosages (summary β: 0.01, 95% CI: -0.06–0.08). Conclusion: Variation in GLCCI1 rs37972 genotype does not seem to affect ICS efficacy in north European asthmatic children. Original submitted 26 November 2013; Revision submitted 13 February 2014

The small guanosine triphosphate (GTPase) p21rac is highly expressed in human neutrophils where it is thought to play a role in cytoskeletal reorganization and superoxide production. Using the p21rac binding domain of PAK (PAK-RBD) as an activation-specific probe, we have investigated agonist-stimulated activation of p21rac. Stimulation of neutrophils with the chemoattractants fMet-Leu-Phe (fMLP) or platelet-activating factor (PAF) induced an extremely rapid and transient p21rac activation, being optimal within 5 seconds. This activation correlates with the rapid changes of intracellular free Ca2+ ([Ca2+]i) stimulated by fMLP; however, changes in [Ca2+]i were neither sufficient nor required for p21rac activation. Furthermore, fMLP-induced p21rac activation was not inhibited by broad tyrosine kinase inhibitors or specific inhibitors of ERK, p38 mitogen activated protein kinase, Src, or phosphatidylinositol 3-kinases. Surprisingly, the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor- did not cause p21rac activation or modulate fMLP-induced p21rac activation. AlF−, a potent activator of heterotrimeric G-protein -subunits, however, was found to activate p21rac. Stimulation of neutrophils with phorbol myristate acetate (PMA) strongly activated the respiratory burst, but did not induce p21rac activation, suggesting that superoxide production per se can occur independently of p21rac activation. These data suggest that in human granulocytes, G-protein coupled receptors, but not cytokine receptors, activate p21rac via a rapid, novel exchange-mechanism independently of changes in [Ca2+]i, tyrosine phosphorylation, or PI3K.

Interleukin-5 (IL-5), IL-3, and granulocyte macrophage colony-stimulating factor (GM-CSF) are hematopoietic cytokines which signal through a common beta subunit (betac) of a heterodimeric receptor. Among the intracellular signaling pathways activated via betac is the JAK/STAT pathway. We show that different STAT5 isoforms are activated by IL-5 and GM-CSF in eosinophils, neutrophils, and differentiated eosinophilic HL-60 cells. Whereas IL-5 activated the wild-type STAT5A and STAT5B proteins in HL60-eos cells, a carboxyl-terminally truncated 80-kDa STAT5 isoform was activated in mature eosinophils and neutrophils. Surprisingly, while both isoforms bind strongly to an element from the beta-casein promoter, only p80 STAT5 binds to the ICAM1-IRE. Consequently, a carboxyl-terminal truncated STAT5 is capable of blocking STAT3-mediated transcription of an IREtkCAT reporter construct. The cell type-specific expression of these functionally distinct STAT5 isoforms might contribute to the pleiotropic effects of IL-5 and GM-CSF on different target cells.

In contrast to the general model of cytokine-induced JAK/STAT signaling, tyrosine phosphorylation of the IL-5R ß chain seems to be dispensable for STAT activation in cells overexpressing exogenous STAT proteins. In this study we expressed IL-5 receptor mutants in 293 cells and studied IL-5- induced endogenous STAT-dependent transcription. Our results indicate that: (a) tyrosine phosphorylation of the IL-5R ß chain is required for endogenous STAT5 activation, (b) multiple tyrosine residues are phosphorylated upon IL-5 stimulation, including Tyr^(577) , Tyr^(612) , Tyr^(695) , and Tyr^(750) , and (c) Tyr^(612) , Tyr^(695) , and Tyr^(750) are all capable of inducing activation of STAT5, demonstrating a high level of functional redundancy within the IL-5R ß chain.

With approximately 300 million patients worldwide, asthma is a major global health problem. The disease is the most common chronic disease among children [101]. Candidate gene approaches and, to a lesser extent, whole-genome association studies have identified several genetic loci that are associated with poor treatment response [1–3]. However, replication of studies in distinct (pediatric) study populations is uncommon and reported effect estimates are generally moderate to low, questioning the value of pharmacogenomics for clinical asthma practice. Randomized controlled trials (RCTs) involving pharmacogenomics (as an intervention to guide treatment or ad hoc pharmacogenomics analysis), which are still the gold standard for evaluating the effectiveness of clinical interventions, are very rare in asthmatic children.

Asthma is a chronic disease characterized by airway inflammation, bronchial hyperresponsiveness, and recurrent episodes of reversible airway obstruction. The disease is very heterogeneous in onset, course, and response to treatment, and seems to encompass a broad collection of heterogeneous disease subtypes with different underlying pathophysiological mechanisms. There is a strong need for easily interpreted clinical biomarkers to assess the nature and severity of the disease. Currently available biomarkers for clinical practice - for example markers in bronchial lavage, bronchial biopsies, sputum, or fraction of exhaled nitric oxide (FeNO) - are limited due to invasiveness or lack of specificity. The assessment of markers in peripheral blood might be a good alternative to study airway inflammation more specifically, compared to FeNO, and in a less invasive manner, compared to bronchoalveolar lavage, biopsies, or sputum induction. In addition, promising novel biomarkers are discovered in the field of breath metabolomics (eg, volatile organic compounds) and (pharmaco)genomics. Biomarker research in asthma is increasingly shifting from the assessment of the value of single biomarkers to multidimensional approaches in which the clinical value of a combination of various markers is studied. This could eventually lead to the development of a clinically applicable algorithm composed of various markers and clinical features to phenotype asthma and improve diagnosis and asthma management.

Background 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. Methods/design 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). Discussion 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.

A high fraction of nitric oxide in exhaled breath (FeNO) has been suggested to be a marker of ongoing airway inflammation and poorly controlled disease in asthma. The usefulness of FeNO to monitor asthma control is still debated today.To assess the validity of FeNO as a marker of asthma control in children with reported use of asthma medication.Fraction of nitric oxide in exhaled breath was measured in 601 children (aged 4-12 yr) with reported use of asthma medication in the past 6 months and in 63 healthy non-asthmatic children (aged 5-12). Asthma control was assessed by the Asthma Control Questionnaire (ACQ). A receiver-operator characteristics (ROC) curve was generated to assess the accuracy of FeNO as a marker for asthma control. Logistic regression analysis was used to study whether clinical, healthcare, medication, and environmental factors are associated with high FeNO levels (25 ppb).Fraction of nitric oxide in exhaled breath had a poor accuracy to discriminate well-controlled from not well-controlled asthma [area under the ROC curve: 0.56 (95% CI: 0.52-0.61, p = 0.008)]. In addition, high FeNO (25 ppb) was associated with lower medication adherence rates (OR: 0.4; 95% CI 0.3-0.6), fewer antibiotic courses in the past year (OR: 0.6; 95% CI: 0.4-0.9), fewer leukotriene antagonists use in the past year (OR: 0.4; 95% CI: 0.2-0.9), and fewer visits to a (pulmonary) pediatrician (OR: 0.6; 95% CI: 0.4-0.9). Children living in a non-urban environment had more often high FeNO levels (OR: 1.7; 95% CI: 1.1-2.6).High FeNO is a poor marker of asthma control in children with reported use of asthma medication. Various other factors, including medication adherence and medication use, are associated with increased FeNO levels.

Asthma is a chronic lung disorder characterized by recurrent episodes of wheezing, shortness of breath and coughing [101]. It is a common dis­ ease in children and its prevalence in Europe is approximately 10% [102]. It is generally accepted that asthma is not simply caused by one under­ lying factor but that it can be caused by different disease mechanisms [1]. This heterogeneity can be determined by sputum ana lysis [1], disease control [103], and also sensitivity to treatment [104]. Until now, none of these domains have been able to fully explain the heterogeneity in phenotypes that lead to the same symptoms that are so characteristic for the disease. The treatment of asthma can be summarized into two categories: symptom­relieving therapy via broncho dilatation, mainly using inhaled b­agonists on an as­needed basis, and in chronic asthma, continuous anti­inflammatory therapy with inhaled corticosteroids (ICS). For the majority of children with asthma these therapeutic approaches enable them to lead a normal life. However, 10–15% of children have serious asthma symptoms despite optimal treatment with ICS and are often referred to as having “difficult­to­treat asthma” [2]. This means an important reduction in the quality of life in this group of children. When looking into this in more detail, there appears to be a significant interindividual variability in ICS responsiveness; however, the intraindividual response is highly consistent [3]. This strongly suggests the influence of a genetic component, which may be responsible for the heterogeneity of response. Several pharmacogenetic studies have been performed, and several candidate genes have been identif ied (e.g., CRHR1, TBX21 and NR3C1) [4]. However, by far the majority of these findings still need more replication and, until recently, none of these findings appear to have come close to clinical relevance. Recently

We report that responses of normal human eosinophils toward the chemokines RANTES and interleukin-8 (IL-8) are modulated and upregulated by priming with IL-5. In a modified Boyden chamber assay, we studied migratory responses toward the members of the chemokine family RANTES, monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1 alpha (MIP-1 alpha) (C-C subfamily), and IL-8, platelet factor-4 (PF-4), and neutrophil-activating peptide-2 (NAP-2) (C-x-C subfamily). These chemokines were also studied in terms of actin polymerization and ([Ca2+]i)-mobilizing properties, intracellular signals that are thought to play a role during migratory responses. We found that eosinophils showed significant migratory responses toward RANTES and IL-8 at concentrations of 10(-9) to 10(-7) mol/L only after priming with IL-5 (10 pmol/L). At these concentrations, PF-4, NAP-2, MCP-1, and MIP-1 alpha induced no significant migratory responses after priming. Unprimed eosinophils only showed a significant migratory response toward RANTES (10(-6) mol/L). Changes in [Ca2+]i were found after addition of RANTES, MIP-1 alpha, and NAP-2 (10 nmol/L) to unprimed eosinophils. RANTES (10(-9) to 10(-7) mol/L) significantly induced actin polymerization both in primed and unprimed eosinophils, whereas IL-8 (10(-9) to 10(-8) mol/L) and MIP-1 alpha (10(-8) mol/L) only induced actin polymerization after priming with IL-5. NAP-2, PF-4, and MCP-1 did not affect actin polymerization. These findings are further evidence for the hypothesis that cytokines like IL-5 and locally secreted chemokines like RANTES and IL-8 are both at the basis of specific eosinophil influx into the allergic inflammatory locus.

Several studies have shown that predictors of asthma treatment outcomes differ depending on the definition of the outcome chosen. This provides evidence that different outcomes studied may reflect distinct aspects of asthma control. To assess predictors of asthma control, we need firm outcome phenotypes. The aim of this study was to investigate the association between measurements of current and long-term asthma control.We included 527 children using inhaled corticosteroids participating in the Pharmacogenetics of Asthma medication in Children: Medication with ANti-inflammatory effects cohort. Current asthma control (previous week) was defined using the Asthma Control Questionnaire. Long-term asthma control was based on Global Initiative for Asthma guidelines. Not well-controlled asthma in a season was defined as ≥ 3 of the following items present in a season: (i) day-time or (ii) night-time symptoms, (iii) limitations in activities, and (iv) rescue medication use. Asthma control during (i) the previous season and (ii) the year preceding the pharmacy visit was used as long-term asthma control definitions. Current and long-term asthma control were compared to investigate agreement.Long-term uncontrolled asthma rates were highest in autumn and winter (50%) and lowest in summer (32%) (p0.05). Overall agreement between current and long-term asthma control was limited (66% for previous season and 68% for previous year).Congruence between current and long-term asthma control was limited. Furthermore, we showed significant seasonal differences. It is therefore important to calculate asthma control over a longer period of time, instead of using current asthma control as indicator.

Despite existing effective treatment options, asthma is uncontrolled in a considerable proportion of patients. The aim of this study was to identify determinants of uncontrolled asthma at age 8 in children participating in the PIAMA birth cohort study.One hundred seventy children using inhaled corticosteroids in the previous 12 months at age 8 were included. Uncontrolled asthma was defined as: ≥3 items present in the past month: (1) day-time or (2) night-time asthma symptoms, (3) limitations in activities, (4) rescue medication use, (5) FEV(1)0% predicted and (6) unscheduled physician visits because of asthma. Binomial regression was performed to study five groups of determinants representing asthma control: child and parental characteristics, environmental factors, therapy adherence and parental perception towards medication use (Beliefs about Medicines Questionnaire).Seventy seven children (45%) had uncontrolled asthma. Low maternal education (RR 1.6, 95% CI: 1.0-2.4) was associated with uncontrolled asthma. Parental necessity beliefs about medication use to maintain present and future health and parental concerns about potential adverse consequences of medication were also associated with uncontrolled asthma (RR 1.6, 95% CI: 1.1-2.2; and 1.6, 95% CI: 1.0-2.5, respectively).Environmental factors and therapy adherence were not associated with asthma control. In our cohort, uncontrolled asthma is associated with low maternal education and with strong parental beliefs about medication necessity and higher concern about potential side effects of medication.

Asthma is a chronic airway disease with a relatively high prevalence. Although the etiology of asthma is still not fully clear, there are effective treatments available. The treatment strategy for asthma consists of two steps: maintenance therapy with anti-inflammatory agents (inhaled corticosteroids and/or leukotriene antagonists) to reduce airway inflammation and retain proper lung function and secondly, the use of beta-agonists for quick symptomatic relief. Furthermore, oral steroids are used to treat acute exacerbations. There are large differences in response to drug therapy. This may be due to many factors, such as severity and type of disease, compliance, co-morbidity, co-medication (drug-drug interactions), environmental exposures and age. However, calculations of repeatability of treatment response suggest that part of this variance in response to pharmacotherapy could be due to genetic factors. Pharmacogenetics may explain the inter-individual variability in drug response due to genetic variation. Pharmacogenetics is a relatively new emerging research field that provides the opportunity to discover associations between genetic variation and response to a variety of drugs. This review will discuss the pharmacogenetics of antiinflammatory agents (corticosteroids and leukotriene antagonists) used in the treatment of asthma.

Response to pharmacotherapy can be highly variable amongst individuals. Pharmacogenomics may explain the interindividual variability in drug response due to genetic variation. However, besides genetics, many other factors can play a role in the response to pharmacotherapy, including disease severity, co-morbidity, environmental factors, therapy adherence and co-medication use. Better understanding of these factors and inter-relationships should bring about a much more effective approach to disease management. Systems biology that studies organisms as integrated and interacting networks of genes, proteins and biochemical reactions can contribute to this. Organisms are no longer studied part by part, but in a more integral manner. Integration of the genetic data with intermediate and end point phenotypic characterization may prove essential to define the inherent nature of drug effects. Therefore, in the future, a multidisciplinary systems-based approach will be necessary to deal with the bulk of the biological data that is available and, ultimately, to reach the goal of personalized prescribing.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Early discontinuation of treatment in new users of inhaled corticosteroids (ICS) has been widely discussed, but patients' reasons for this have not been investigated in depth. WHAT THIS STUDY ADDS • In contrast to what might have been expected, the majority of patients mentioned a wide range of symptoms or conditions as the reason for the start of ICS therapy. • There is no clear indication for prescribing ICS in these patients. • Most of these conditions are self limiting and may be expected to be of short duration, consequently a decrease in symptoms was the main and justifiable reason for discontinuing ICS. • However, a non-negligible proportion of patients has significant residual symptoms and seems to be in need of continuous ICS use. AIM Although early discontinuation of treatment in new users of inhaled corticosteroids (ICS) has been widely discussed, the reasons for stopping have not been investigated in depth. We aimed to describe reasons for discontinuation from a patient's perspective in relation to their experience of symptoms at the time of the investigation. METHODS A cross-sectional study among new users that discontinued ICS use in the Netherlands was performed. Patients were interviewed by telephone, aiming to identify the symptoms for which they were prescribed ICS, the reasons for discontinuing treatment and the respiratory symptoms patients still experienced at the time of the survey. In addition, automated dispensing records of all patients were retrieved. RESULTS From 287 eligible patients, 230 (80.1%) were interviewed. Only 22 patients (9.6%) mentioned asthma as the reason for a first ICS prescription. A decrease in symptoms was the main reason for discontinuation (45%). Thirty patients (13%) reported clinically significant residual symptoms. These patients reported more seasonal variation of symptoms and were more often prescribed short-acting β2-agonists. CONCLUSIONS The majority of patients mentioned a wide range of symptoms and conditions, other than asthma or chronic obstructive pulmonary disease, as the reason for the start of ICS therapy. Most of these conditions may be expected to be of short duration. Not surprisingly a decrease in symptoms was the main, and justifiable, reason for discontinuing ICS. However, a non-negligible proportion of patients reported residual symptoms that suggest the need of continued ICS use. Physicians and pharmacists could cooperate in identifying those patients for which ICS are really indicated and motivate them to continue the use of ICS.

Method VOCs were measured in thirty-three asthmatic children (age: 11.8 yrs, SD 2.3 yrs) who participated in the PACMAN2 study. All children were current users of ICS. Current asthma control was assessed using the Asthma Control Questionnaire. Long-term asthma control was based on reported symptoms in the four seasons preceding baseline and follow-up visit. A single vital capacity volume of exhaled air was collected upon 5 minutes of normal breathing through a three-way non-re-breathing valve with a VOC filter in a Tedlar bag. The VOCs in the breath sample were subsequently captured on Tenax GR Tubes and analyzed offline on a validated panel of four eNoses with different sensor technologies (Owlstone Lonestar, Sensigent Cyranose 320, Comon Invent eNose and Tor Vergata TEN). Breathprints were analysed per eNose using principal component analyses (PCA). ROC curves and cross-validated accuracy values of significant (t-test) principal components were used to assess the accuracy of the devices to discriminate between controlled and uncontrolled patients. Additionally, the fraction of exhaled nitric oxide (FeNO) was measured with a NIOX Mino and an expiration time of 6 seconds.

Preactivation or priming of eosinophils by (proinflammatory) cytokines is important in the pathogenesis of allergic diseases. Several priming-dependent eosinophil responses, such as migration and adhesion, are reduced by treatment with corticosteroids. Many inhibitory effects of corticosteroids are mediated by the glucocorticoid receptor via genomic mechanisms, which are evident only after prolonged interaction (>30 min). However, also faster actions of corticosteroids have been identified, which occur in a rapid, nongenomic manner. In this study, fast effects of corticosteroids were investigated on the function of eosinophil opsonin receptors. Short term corticosteroid treatment of eosinophils for maximal 30 min with dexamethasone (Dex) did not influence eosinophil cell surface CD11b/CD18 expression, adhesion, and/or chemokinesis. In marked contrast, incubation with Dex resulted in a rapid increase in binding of IgA-coated beads to human eosinophils, showing that Dex can up-regulate the activation of FcαR (CD89). This priming response by Dex was dose dependent and optimal between 10−8 and 10−6 M and was mediated via the glucocorticoid receptor as its selective antagonist RU38486 (10−6 M) blocked the priming effect. In contrast to FcαR, eosinophil FcγRII (CD32) was not affected by Dex. Further characterization of the Dex-induced inside-out regulation of FcαR revealed p38 MAPK as the central mediator. Dex dose dependently enhanced p38 MAPK phosphorylation and activation in situ as measured by phosphorylation of its downstream target mitogen-activated protein kinase-activated protein kinase 2. The dose responses of the Dex-induced activation of these kinases were similar as seen for the priming of FcαR. This work demonstrates that corticosteroids selectively activate the FcαR on eosinophils by activation of p38 MAPK.

Cytokine receptors consist of multiple subunits, which are often shared between different receptors, resulting in the functional redundancy sometimes observed between cytokines. The interleukin 5 (IL-5) receptor consists of an IL-5-specific alpha-subunit (IL-5Ralpha) and a signal-transducing beta-subunit (betac) shared with the IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors. In this study, we sought to find a role for the cytoplasmic domain of IL-5Ralpha. We show that syntenin, a protein containing PSD-95/Discs large/zO-1 (PDZ) domains, associates with the cytoplasmic tail of the IL-5Ralpha. Syntenin was found to directly associate with the transcription factor Sox4. Association of syntenin with IL-5Ralpha was required for IL-5-mediated activation of Sox4. These studies identify a mechanism of transcriptional activation by cytokine-specific receptor subunits.

Signal transducers and activators of transcription (STATs) are a family of transcription factors that were originally identified as mediators of cytokine-induced gene expression. We and others have recently shown that STAT5 also plays a major role in cellular transformation by the Bcr-Abl oncogene. Here we show that the antiapoptotic bcl-xL gene product and the cell cycle regulator cyclin D1 are targets of STAT5 in Bcr-Abl-transformed cells. In the CML cell line K562 and in BaF3 cells ectopically expressing Bcr-Abl, both the cyclin D1 and bcl-x promoters are highly active. The activity of these promoters can be strongly repressed by cotransfection of a dominant negative (DN) mutant of STAT5. Moreover, the cyclin D1 and bcl-x promoters contain STAT binding sites to which STAT5 constitutively binds in Bcr-Abl transformed cells. These results suggest that STAT5 contributes to transformation by Bcr-Abl by induction of cyclin D1 and bcl-xL expression.

Background: Human polymorphic nuclear granulocytes (PMNs) such as neutrophils and eosinophils play a critical role in mediating inflammatory responses to microbial and parasitic infections. Exposure of these leukocytes to cytokines leads to an amplification of granulocyte effector functions by a mechanism termed "priming." Although many studies have investigated the effects of granulocyte priming, little is known concerning the molecular mechanisms that lead to this phenomenon. Objective: The purpose of this study was to identify potential markers for granulocyte priming and thus also to gain further insight into the pathogenesis of inflammatory responses. Methods: We used a modified differential display technique, random arbitrary primed–PCR to identify genes regulated during the priming of human polymorphic nuclear granulocytes by GM-CSF in vitro. Genes identified were validated by Northern blot analysis of in vitro and in vivo primed leukocytes. Results: Several genes were identified and their expression characterized in vitro. One of these genes, 5-lipoxygenase–activating protein, was also found to be up-regulated in leukocytes isolated after allergen challenge of allergic asthmatic patients. Conclusion: The use of differential display technology is a rapid and effective means of identifying genes whose expression is regulated by priming in vitro and in vivo. Further analysis will lead to a better understanding of the priming phenotype and may provide further insight into the pathologic mechanisms of inflammatory processes. (J Allergy Clin Immunol 2000;105:760-8.)

Granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5 belong to a family of cytokines that regulate proliferation, differentiation and function of haematopoietic cells. Their receptor consists of a ligand specific alpha-chain and a signal transducing beta-chain (betac). While, the role of phosphotyrosine residues in the betac as mediators of downstream signalling cascades has been established, little is known about non-phosphotyrosine mediated events. To identify proteins interacting with betac, we screened a yeast two-hybrid library with the intracellular domain of betac. We found that RACK1, a molecule associating with activated PKC, PLCgamma and Src kinases, associated with the membrane proximal region of betac in both yeast two-hybrid, immunoprecipitation and GST-pull-down assays. The association of RACK1 was constitutive, demonstrating no alteration upon cellular stimulation. Furthermore, upon stimulation of cells with IL-5 or PMA, a complex of betac and PKCbeta was found. Together, these findings suggest a novel role for RACK1 as a possible adapter molecule associating with the intracellular domain of cytokine receptors.

Interleukin-5 (IL-5) and granulocyte macrophage-colony stimulating factor (GM-CSF) are important cytokines for the proliferation, differentiation, and activation of myeloid lineages. The JAK/STAT pathway is one of the signaling pathways implicated in mediating biological responses induced by these cytokines. Previous studies have demonstrated that these cytokines predominantly activate an 80 kDa STAT5 isoform in mature granulocytes. To better understand the role of STAT proteins during growth and differentiation of granulocytes, we evaluated differentiation of human CD34+ hematopoietic stem cells ex vivo toward eosinophils and neutrophils. Bandshift experiments showed that in an early stage of both differentiation pathways (14 days), the 94 kDa STAT5B protein was activated by both IL-5 (eosinophil lineage) and GM-CSF (neutrophil lineage). However, during maturation of both lineages (days 21 and 28), increased expression of a functionally distinct 80 kDa STAT5 isoform was observed, resulting in heterodimer DNA-binding complexes containing both the 94 and 80 kDa STAT5 proteins. The finding that functionally distinct isoforms of STAT5 are activated during the early and late differentiation stages of granulocytes suggests that they might be involved in regulating different biological functions in these cells.

Interleukin-5 (IL-5) plays a crucial role in the proliferation, differentiation and activation of eosinophils. The IL-5 receptor is composed of an IL-5-specific alpha subunit, which is expressed by eosinophils and basophils, and a beta c-subunit shared with the receptors for IL-3 and GM-CSF. We identified an AP-1 element which is important for IL-5R alpha promoter activity in eosinophilic HL60 cells. The AP-1 site and the previously identified EOS1 site cooperate, since single mutation of either of the sites decreased promoter activity. We show that the AP-1 site of the IL-5R alpha promoter binds multiple proteins, including cJun, CREB, and CREM.

Background: Human eosinophils are strongly modulated by the eosinophilotrophic cytokines IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF). A clear intracellular effect of these cytokines is the induction of tyrosine phosphorylation of multiple cellular substrates. However, the relevance of tyrosine phosphorylation for eosinophil functioning has not been established. Objective: In this study we have investigated dose-response and time curves of IL-5-, IL-3-, and GM-CSF–induced tyrosine phosphorylation in eosinophils. Moreover, we have evaluated the importance of IL-5-induced tyrosine phosphorylation for priming of human eosinophils. Methods: Cytokine-induced tyrosine phosphorylation was monitored on western blot with an antiphosphotyrosine antibody (4G10). To probe the relevance of tyrosine phosphorylation for priming, eosinophils were primed with IL-5 in the presence of the tyrosine kinase inhibitor herbimycin A. Platelet activating factor (PAF) was used as a control priming agent. Subsequently, the eosinophils were incubated with serum-treated zymosan (STZ) to activate the respiratory burst. Binding of STZ was determined by FACS analysis. Results: IL-5-, IL-3-, and GM-CSF–induced tyrosine phosphorylation was found at concentrations that primed eosinophil effector mechanism (median effective dose values: ≅5.10 −11 mol/L, ≅5.10 −10 mol/L, and ≅5.10 −12 mol/L for IL-5, IL-3, and GM-CSF, respectively). Cytokine-induced tyrosine phosphorylation was transient with an optimum value at 15 minutes. IL-5 priming of STZ–induced activation of the respiratory burst was blocked by herbimycin A, whereas PAF still primed this response. In fact, herbimycin A inhibited IL-5 priming of STZ binding to human eosinophils. On the other hand, PAF priming of STZ binding was not affected by herbimycin A. Both IL-5-induced and PAF–induced tyrosine phosphorylation were inhibited by herbimycin A. Conclusion: These data demonstrate for the first time that IL-5 priming of opsonized particle-induced responses is mediated by tyrosine kinase activity in human eosinophils. (J Allergy Clin Immunol 1998;101:103-9.)

Interleukin-5 (IL-5) is one of the major regulators of eosinophilic granulocytes in vivo. IL-5 exerts its pleiotropic effects by binding to the IL-5 receptor, which is composed of an IL-5-specific alpha chain and a common betac chain shared with the receptors for IL-3 and granulocyte-macrophage colony-stimulating factor. Previous studies have shown that binding of IL-5 to its receptor triggers the activation of multiple signaling cascades, including the Ras/mitogen-activated protein kinase, the phosphatidyl -3'-kinase, and the Janus kinase/signal transducer and activator of transcription pathways. Here we describe that IL-5 activates the serine/threonine protein kinase Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway. We show that IL-5 activates TPA response element (TRE)-dependent transcription in transfection experiments. TRE activation by IL-5 is mediated by a region of the betac (577-581) that is also responsible for activation of JNK/SAPK and for activation of dyad symmetry element (DSE)-dependent transcription. Dominant-negative SAPK or ERK kinase-1 was used to demonstrate that JNK/SAPK activation is necessary for induction of DSE- and TRE-dependent transcription by IL-5, whereas extracellular signal-regulated kinase 2 was not essential for TRE- and DSE-dependent transcription. By contrast, IL-5-induced activation of the tyrosine kinase Janus kinase 2 seems to be a prerequisite for TRE- and DSE-dependent transcription. Taken together, we show for the first time that IL-5 activates kinases of the JNK/SAPK family, and that this activation is linked to IL-5-induced TRE- and DSE-dependent transcription.