FcαRI Dynamics Are Regulated by GSK-3 and PKCζ During Cytokine Mediated Inside-Out Signaling.

IgA binding to FcαRI (CD89) is rapidly enhanced by cytokine induced inside-out signaling. Dephosphorylation of serine 263 in the intracellular tail of FcαRI by PP2A and PI3K activation are instrumental in this process. To further investigate these signaling pathways, we targeted downstream kinases of PI3K. Our experiments revealed that PI3K activates PKCζ, which subsequently inhibits GSK-3, a constitutively active kinase in resting cells and found here to be associated with FcαRI. We propose that GSK-3 maintains FcαRI in an inactive state at homeostatic conditions. Upon cytokine stimulation, GSK-3 is inactivated through a PI3K-PKCζ pathway, preventing the maintenance of phosphorylated inactive FcαRI. The concomitantly activated PP2A is then able to dephosphorylate and activate FcαRI. Moreover, FRAP and FLIP studies showed that FcαRI activation coincides with an increased mobile fraction of the receptor. This can enhance FcαRI valency and contribute to stronger avidity for IgA immune complexes. This tightly regulated inside-out signaling pathway allows leukocytes to respond rapidly and efficiently to their environment and could be exploited to enhance the efficacy of future IgA therapeutics.

Patterns of amino acid metabolism by proliferating human mesenchymal stem cells.

The nutritional requirements of stem cells have not been determined; in particular, the amino acid metabolism of stem cells is largely unknown. In this study, we investigated the amino acid metabolism of human mesenchymal stem cells (hMSCs), with focus on two questions: Which amino acids are consumed and/or secreted by hMSCs and at what rates? To answer these questions, hMSCs were cultured on tissue culture plastic and in a bioreactor, and their amino acid profile was analyzed. The results showed that the kinetics of hMSCs growth and amino acid metabolism were significantly higher for hMSCs in tissue culture plastic than in the bioreactor. Despite differences in culture conditions, 8 essential and 6 nonessential amino acids were consumed by hMSCs in both tissue culture plastic and bioreactor cultures. Glutamine was the most consumed amino acid with significantly higher rates than for any other amino acid. The metabolism of nonessential amino acids by hMSCs deviated significantly from that of other cell lines. The secretion of alanine, glycine, glutamate, and ornithine by hMSCs showed that there is a strong overflow metabolism that can be due to the high concentrations of amino acids provided in the medium. In addition, the data showed that there is a metabolic pattern for proliferating hMSCs, which can contribute to the design of medium without animal serum for stem cells. Further, this study shows how to implement amino acid rates and metabolic principles in three-dimensional stem cell biology.

Effects of glucocorticoids on the neutrophil count: a cohort study among hospitalized patients.

BACKGROUND: Systemic glucocorticoids are often used in clinical practice for a large variety of indications. Clinical observations have shown that patients using glucocorticoids often have higher neutrophil counts. Debate remains whether this observed neutrophilia is associated with glucocorticoid use or that other factors, like disease and severity of disease, should be considered. The objective of this study was to investigate the effect of systemic glucocorticoids on the absolute neutrophil count in hospitalized patients. METHODS: A cohort study was conducted using data from the Utrecht Patient Oriented Database which comprises clinical data of patients of the University Medical Center Utrecht. We identified all adult patients, hospitalized in 2005 with at least two blood samples for hematological testing during admission and compared in-hospital glucocorticoid use with non-use. RESULTS: A total of 809 glucocorticoid users and 2658 non-users were included in the study with comparable neutrophil counts at admission (8.2.10(9)/l for glucocorticoid users and 8.0.10(9)/l for non-users). Overall analysis showed a slight association between glucocorticoid use and an increase in neutrophil count (RR 1.3; 95% CI 1.1-1.5). However, within diagnostic subgroups there was no increase in neutrophil count in glucocorticoid users. Furthermore, among all no dose response relationship, no effect of time between the two samples, and no effect of anti-inflammatory/sodium retaining potency was found. CONCLUSION: Observed neutrophilia in users of systemic glucocorticoids is probably associated with underlying disease, rather than glucocorticoid use itself.

Identification of exacerbations in obstructive lung disease through biomarkers.

Inflammation has been identified as an important factor for disease exacerbation in obstructive lung disease. In this study, we used neutrophil and eosinophil counts as biomarkers for exacerbation in obstructive lung disease. We conducted a case-control study within a cohort of patients frequenting an outpatient clinic of Respiratory Medicine using data from the Utrecht Patient Oriented Database (UPOD). Cases were patients with a hospital admission for obstructive lung disease in 2005. For each case, one control patient was sampled from the same study base. We identified 143 cases (118 patients with chronic obstructive pulmonary disease and 25 asthma patients) and 143 controls. Admission was associated with both neutrophilia (adjusted odds ratio (OR) 4.3; 95% confidence interval (CI) 2.2-8.5), and eosinophilia (adjusted OR 2.6; 95% CI 1.1-6.2). The association with eosinophilia was only seen in asthma patients. In conclusion, neutrophil and eosinophil counts seem to be useful biomarkers for identifying exacerbations in pharmacoepidemiological studies on obstructive lung disease.

Pharmacogenetics of anti-inflammatory treatment in children with asthma: rationale and design of the PACMAN cohort.

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.

Hematocytometry analysis as discriminative marker for asthma phenotypes.

BACKGROUND: There is an increasing demand for easy to measure biomarkers in clinical practice. We created the relational database Utrecht Patient Oriented Database (UPOD) to develop tools for identifying new biomarkers for disease. In this study, we used UPOD to identify better biomarkers for discriminating different asthma phenotypes. METHODS: We performed a prospective study at the University Medical Center (UMC) Utrecht using blood from patients with asthma and a healthy reference group. Since asthma is an inflammatory disease, absolute leukocyte counts and leukocyte differential parameters were analyzed using raw data files and a logistic regression model. RESULTS: We compared 17 difficult-to-treat asthma (DTA) cases, 13 non-difficult-to-treat asthma cases, and 19 healthy volunteers. Absolute leukocyte counts and differential parameters for leukocytes were able to discriminate asthma patients from healthy volunteers. However, among patients with asthma, difficult-to-treat cases could be more accurately defined with a neutrophil morphology change (OR 8.0; 95% CI 1.5-42.0), compared to the absolute neutrophil count (OR 4.0; 95% CI 0.8-21.0). CONCLUSIONS: In this asthma patient population, we were able to define asthma phenotypes more precisely using neutrophil morphology parameters, compared to absolute counts. Using UPOD with differential parameters, it is possible to conduct larger scale biomarker studies, combining clinical, laboratory medicine, and epidemiological techniques.

What drives prescribing of asthma medication to children? A multilevel population-based study.

PURPOSE: Diagnosing asthma in children with asthmatic symptoms remains a challenge, particularly in preschool children. This challenge creates an opportunity for variability in prescribing. The aim of our study was to investigate how and to what degree patient, family, and physician characteristics influence prescribing of asthma medication in children. METHODS: We undertook a multilevel population-based study using the second Dutch national survey of general practice (DNSGP-2), 2001. Participants were 46,371 children aged 1 to 17 years belonging to 25,537 families registered with 109 general practitioners. Using a multilevel multivariate logistic regression analysis with 3 levels, our main outcome measure was the prescribing of asthma medication, defined as at least 1 prescription for beta(2)-adrenergic agonists, inhaled corticosteroids, cromones, or montelukast during the 1-year study period. RESULTS: We identified characteristics significantly associated with prescribing asthma medication on all 3 levels (child, family, and physician). The variance in prescribing among physicians was significantly higher with children who were younger than 6 years than with children aged 6 years and older (95% CI, 3.5%-25.2% vs 2.4%-13.4%). Several diagnoses other than asthma and asthmatic complaints were strongly associated with prescribing asthma medication, including bronchitis/bronchiolitis (OR = 9.04; 95% CI, 7.57-10.8) and cough (OR = 6.51; 95% CI, 5.68-7.47). CONCLUSIONS: Our study shows a much higher variance in prescribing patterns among general practitioners for children younger than 6 years compared with older children, which could be a direct result of the diagnostic complexities found in young children with asthmatic symptoms. Thus diagnostic gaps may lead to more physician-driven prescribing irrespective of the clinical context.

Inside-out regulation of Fc alpha RI (CD89) depends on PP2A.

To achieve a correct cellular immune response toward pathogens, interaction between FcR and their ligands must be regulated. The Fc receptor for IgA, FcalphaRI, is pivotal for the inflammatory responses against IgA-opsonized pathogens. Cytokine-induced inside-out signaling through the intracellular FcalphaRI tail is important for FcalphaRI-IgA binding. However, the underlying molecular mechanism governing this process is not well understood. In this study, we report that PP2A can act as a molecular switch in FcalphaRI activation. PP2A binds to the intracellular tail of FcalphaRI and, upon cytokine stimulation, PP2A becomes activated. Subsequently, FcalphaRI is dephosphorylated on intracellular Serine 263, which we could link to receptor activation. PP2A inhibition, in contrast, decreased FcalphaRI ligand binding capacity in transfected cells but also in eosinophils and monocytes. Interestingly, PP2A activity was found crucial for IgA-mediated binding and phagocytosis of Neisseria meningitidis. The present findings demonstrate PP2A involvement as a molecular mechanism for FcalphaRI ligand binding regulation, a key step in initiating an immune response.

Patients' understanding of the reasons for starting and discontinuing inhaled corticosteroids.

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 beta(2)-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.

Persistence of asthma medication use in preschool children.

OBJECTIVE: In young children with asthmatic symptoms diagnostic difficulties lead to use of trials of asthma medication as a diagnostic tool. Our aim is to quantify the persistent use of asthma medication, initiated in the first year of life and identify determinants of this persistent use. PATIENTS AND METHODS: We identified 165 children within the PIAMA (Prevention and Incidence of Asthma and Mite Allergy) birth cohort who used asthma medication before the age of one. Persistent use was investigated during three years after the first prescription. A Cox regression analysis was performed to identify factors associated with persistent use. RESULTS: A total of 58.8% of children continued using asthma medication after the first prescription and 10.3% continued during three years. Children with doctor-diagnosed asthma (Hazard ratio of discontinuation (HR)=0.64, 95% CI: 0.45-0.91) or prescribed inhaled corticosteroids in the first year of life (HR of discontinuation=0.59, 95% CI: 0.40-0.86) were 1.6-1.7 times more likely to continue using asthma medication. CONCLUSIONS: Persistence of asthma medication, prescribed in the first year of life is very low and is positively associated with doctor-diagnosed asthma and use of inhaled corticosteroids. Characterizing persistent users of asthma medication is important to understand prescribing of asthma medication in this age group.

Use of beta2 agonists and risk of acute myocardial infarction in patients with hypertension.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: * Use of beta(2) agonists has been associated with tachycardia, an abnormal ECG and atrial fibrillation. * Previous observational studies of the association between use of beta(2) agonists and the risk of acute myocardial infarction (MI) have demonstrated conflicting results. * Instead of a causal effect, the positive association between beta(2) agonist use and MI may be explained by latent ischaemic heart disease, which has symptoms that appear similar to respiratory complaints in chronic obstructive pulmonary disease. WHAT THIS STUDY ADDS: * The majority of beta(2) agonist users in our study population did not have an increased risk of nonfatal acute MI. * Only patients with ischaemic heart disease and who had recently started beta(2) agonists had an increased risk of acute MI. * It is likely that this increased risk was related to latent cardiovascular disease rather than direct effects of beta(2) agonists. AIM: Observational retrospective studies of the association between use of beta(2) agonists and the risk of acute myocardial infarction (MI) have demonstrated conflicting results, particularly among first-time users. The aim of this study was to examine the association between beta(2) agonist use and first nonfatal acute MI. METHODS: We conducted a case-control study (2476 cases) nested in a cohort of antihypertensive drug users in the Dutch PHARMO RLS database. PHARMO RLS consists of drug dispensing linked to the national hospitalizations register. Each case of nonfatal acute MI was matched with up to 12 control patients by gender, age and region. Drug and disease history and the severity of the underlying respiratory disease were adjusted for. RESULTS: Risk of acute MI was increased in current beta(2) agonist users [crude odds ratio (OR) 1.36, 95% confidence interval (CI) 1.15, 1.61]. However, this excess risk was reduced after adjustment for severity of asthma and chronic obstructive pulmonary disease (adjusted OR 1.18, 95% CI 0.93, 1.49). The risk was highest in patients with ischaemic heart disease and low cumulative dose of beta(2) agonists (adjusted OR 2.47, 95% CI 1.60, 3.82). CONCLUSION: Most users of beta(2) agonists did not have an increased risk of acute MI. Only patients with ischaemic heart disease with low cumulative exposure to beta(2) agonists had an increased risk of acute MI. It is likely that this increased risk was related to latent cardiovascular disease rather than to the direct effects of beta(2) agonists.

Prescription of respiratory medication without an asthma diagnosis in children: a population based study.

BACKGROUND: In pre-school children a diagnosis of asthma is not easily made and only a minority of wheezing children will develop persistent atopic asthma. According to the general consensus a diagnosis of asthma becomes more certain with increasing age. Therefore the congruence between asthma medication use and doctor-diagnosed asthma is expected to increase with age. The aim of this study is to evaluate the relationship between prescribing of asthma medication and doctor-diagnosed asthma in children age 0-17. METHODS: We studied all 74,580 children below 18 years of age, belonging to 95 GP practices within the second Dutch national survey of general practice (DNSGP-2), in which GPs registered all physician-patient contacts during the year 2001. Status on prescribing of asthma medication (at least one prescription for beta2-agonists, inhaled corticosteroids, cromones or montelukast) and doctor-diagnosed asthma (coded according to the International Classification of Primary Care) was determined. RESULTS: In total 7.5% of children received asthma medication and 4.1% had a diagnosis of asthma. Only 49% of all children receiving asthma medication was diagnosed as an asthmatic. Subgroup analyses on age, gender and therapy groups showed that the Positive Predictive Value (PPV) differs significantly between therapy groups only. The likelihood of having doctor-diagnosed asthma increased when a child received combination therapy of short acting beta2-agonists and inhaled corticosteroids (PPV = 0.64) and with the number of prescriptions (3 prescriptions or more, PPV = 0.66). Both prescribing of asthma medication and doctor-diagnosed asthma declined with age but the congruence between the two measures did not increase with age. CONCLUSION: In this study, less than half of all children receiving asthma medication had a registered diagnosis of asthma. Detailed subgroup analyses show that a diagnosis of asthma was present in at most 66%, even in groups of children treated intensively with asthma medication. Although age strongly influences the chance of being treated, remarkably, the congruence between prescribing of asthma medication and doctor-diagnosed asthma does not increase with age.

Use of inhaled corticosteroids and the risk of non-fatal acute myocardial infarction.

BACKGROUND: Use of inhaled corticosteroids may reduce the risk of acute myocardial infarction (MI) through reductions in systemic inflammation and C-reactive protein. OBJECTIVES: To examine the association between the use of inhaled corticosteroids and the risk of non-fatal acute MI. METHODS: In the Dutch PHARMO record linkage system database, we conducted a case-control study (2476 MI cases), nested in a cohort of antihypertensive drug users. The use of inhaled corticosteroids 100 days before the index date was compared with never use. We adjusted the analyses for the severity of the underlying respiratory disease and general drug and disease history. RESULTS: We found that the use of inhaled corticosteroids was not associated with a decreased risk of non-fatal MI in antihypertensive drug users after adjustment for the underlying respiratory disease severity, adjusted odds ratio (OR) 1.24, 95% confidence interval (CI) 0.97-1.57. A higher daily dose (adjusted OR 1.82, 95% CI 0.80-4.13) and longer duration of use (adjusted OR 1.28, 95% CI 0.90-1.81) were not associated with a decreased risk of non-fatal MI. An inhaled corticosteroid dispensing in the 30 days before the index date was not protective but resulted in a 1.7-fold increased risk of non-fatal MI. CONCLUSION: Our results do not support the hypothesis that inhaled corticosteroids protect against the risk of non-fatal MI by a reduction of systemic inflammation.

Beliefs about medicines predict refill adherence to inhaled corticosteroids.

OBJECTIVE: Despite the importance of the chronic use of inhaled corticosteroids (ICS) in maintaining asthma control, reported adherence varies between 40% and 60%. The Beliefs about Medicines Questionnaire (BMQ) has been shown to correlate with self-reported adherence. The aim of this study is to investigate whether beliefs about ICS (necessity and concerns), as measured by the BMQ, relate to adherence objectively measured by prescription-refill records. METHODS: In a cross-sectional study of patients aged 18-45 years who filled at least two ICS prescriptions in 11 community pharmacies in The Netherlands, perceptions of ICS were assessed using the BMQ. Additionally, self-reported adherence was assessed using the Medication Adherence Report Scale. ICS prescription-refill adherence rates for a 12-month period prior to the survey were obtained from automated pharmacy dispensing records. Four attitudinal groups were defined using the necessity and concerns constructs. Statistical tests were used to examine associations between ICS adherence (assessed by subjective self-report and objective pharmacy records), specific beliefs about and attitudes towards ICS, and more general beliefs about pharmaceuticals. RESULTS: Questionnaires were returned by 238 patients (51.1%). Both self-reported adherence (r=.38) and adherence by pharmacy records (rho=0.32) correlated with ICS necessity beliefs and concerns. Patients defined as skeptical, indifferent, ambivalent, or accepting, on the basis of these constructs, differed with respect to both their attitudes towards medicines in general and their adherence to medication. CONCLUSIONS: Patients' beliefs about ICS correlate not only with adherence by self-report but also with a more objective measure of medication adherence calculated by pharmacy dispensing records. The necessity-concerns constructs offer a potentially useful framework to help clinicians elicit key treatment beliefs influencing adherence to ICS.

Expression of activated Fc gamma RII discriminates between multiple granulocyte-priming phenotypes in peripheral blood of allergic asthmatic subjects.

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 alpha-chain of Mac-1 (alpha m)/CD11b, L-selectin/CD62L, and an activation epitope present on Fc gamma RII/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 Fc gamma RII was found in patients experiencing a late asthmatic response and not in patients with a single early asthmatic response. In contrast, expression of alpha m/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 alpha m 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.

Distinguishing patterns in the dynamics of long-term medication use by Markov analysis: beyond persistence.

BACKGROUND: In order to accurately distinguish gaps of varying length in drug treatment for chronic conditions from discontinuation without resuming therapy, short-term observation does not suffice. Thus, the use of inhalation corticosteroids (ICS) in the long-term, during a ten-year period is investigated. To describe medication use as a continuum, taking into account the timeliness and consistency of refilling, a Markov model is proposed. METHODS: Patients, that filled at least one prescription in 1993, were selected from the PHARMO medical record linkage system (RLS) containing >95% prescription dispensings per patient originating from community pharmacy records of 6 medium-sized cities in the Netherlands.The probabilities of continuous use, the refilling of at least one ICS prescription in each year of follow-up, and medication free periods were assessed by Markov analysis. Stratified analysis according to new use was performed. RESULTS: The transition probabilities of the refilling of at least one ICS prescription in the subsequent year of follow-up, were assessed for each year of follow-up and for the total study period.The change of transition probabilities in time was evaluated, e.g. the probability of continuing ICS use of starters in the first two years (51%) of follow-up increased to more than 70% in the following years. The probabilities of different patterns of medication use were assessed: continuous use (7.7%), cumulative medication gaps (1-8 years 69.1%) and discontinuing (23.2%) during ten-year follow-up for new users. New users had lower probability of continuous use (7.7%) and more variability in ICS refill patterns than previous users (56%). CONCLUSION: In addition to well-established methods in epidemiology to ascertain compliance and persistence, a Markov model could be useful to further specify the variety of possible patterns of medication use within the continuum of adherence. This Markov model describes variation in behaviour and patterns of ICS use and could also be useful to investigate continuous use of other drugs applied in chronic diseases.

Use of beta-2 agonists and risk of hip/femur fracture: a population-based case-control study.

INTRODUCTION: Administration of beta-2 agonists decreased bone mineral density in rats. But the association between bronchodilators and fracture risk has not been studied in humans. OBJECTIVES: To examine the association between use of beta-2 agonists and risk of hip/femur fracture. METHODS: We conducted a population-based case-control study (6763 cases) in the Dutch PHARMO database. Current beta-2 agonist use was compared to never use. We adjusted for severity of the underlying respiratory disease and disease and drug history. RESULTS: A hospitalisation for asthma/COPD in the year before index date increased risk of hip/femur fracture: crude OR 2.17 (95% CI, 1.41-3.34). Patients using higher doses of beta-2 agonists had increased risk of hip/femur fracture: crude OR 1.94 (95% CI, 1.41-2.66) for daily dosages of >or=1600 microg albuterol equivalent. The excess fracture risk reduced after adjustment for disease severity (1.46; 95% CI, 1.02-2.08) and after exclusion of oral glucocorticoid users (1.31; 95% CI, 0.80-2.15). Risk of hip/femur fracture was similar between users of beta-2 agonists, inhaled glucocorticoids and anticholinergics. CONCLUSION: We found increases in the risk of hip/femur fracture in patients using higher doses of beta-2 agonists. However, the excess risk of hip/femur fracture substantially reduced after exclusion of oral glucocorticoid users and after adjustment for the underlying disease. Risk of hip/femur fracture was similar between users of beta-2 agonists, inhaled glucocorticoids and anticholinergics. The severity of the underlying disease, rather than the use of beta-2 agonists, may play an important role in the aetiology of hip/femur fractures in patients using beta-2 agonists.

Fracture risk with intermittent high-dose oral glucocorticoid therapy.

OBJECTIVE: To evaluate the risk of fracture in patients receiving intermittent therapy with high-dose oral glucocorticoids (GCs). METHODS: The study group comprised 191,752 patients from the UK General Practice Database who were 40 years of age and older and received therapy with GCs. The followup time period was divided into the categories of "current" and "no exposure." The daily dose and cumulative dose for each time period were determined. Relative risks were estimated using Cox proportional hazards models, adjusted for age, sex, body mass index, smoking, disease history, and drug history. Fractures of the radius/ulna, humerus, rib, femur/hip, pelvis, or vertebrae were included in the evaluation. RESULTS: Patients who intermittently received high-dose GCs (daily dose > or =15 mg) and had no or little previous exposure to GCs (cumulative exposure < or =1 gm) had a small increased risk of osteoporotic (but not hip/femur) fracture; this risk increased substantially with increasing cumulative exposure. Among patients who received a daily dose > or =30 mg and whose cumulative exposure was >5 gm, the relative risk (RR) of osteoporotic fracture was 3.63 (95% confidence interval [95% CI] 2.54-5.20), the RR of fracture of the hip/femur was 3.13 (95% CI 1.49-6.59), and the RR of vertebral fracture was 14.42 (95% CI 8.29-25.08). CONCLUSION: Intermittent use of high-dose oral GCs (daily dose > or =15 mg and cumulative exposure < or =1 gm) may result in a small increased risk of osteoporotic fracture. Conversely, patients who receive several courses of high-dose GCs (daily dose > or =15 mg and cumulative exposure >1 gm) have a substantially increased risk of fracture.

Differential regulation of TNFalpha and GM-CSF induced activation of P38 MAPK in neutrophils and eosinophils.

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.

Rapid selective priming of FcalphaR on eosinophils by corticosteroids.

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 FcalphaR (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 FcalphaR, eosinophil FcgammaRII (CD32) was not affected by Dex. Further characterization of the Dex-induced inside-out regulation of FcalphaR 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 FcalphaR. This work demonstrates that corticosteroids selectively activate the FcalphaR on eosinophils by activation of p38 MAPK.