Ratko Djukanovic

Effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyper-responsìveness, and the late asthmatic response

BACKGROUND Interleukin-5 (IL-5) is essential for the formation of eosinophils, which are thought to have a major role in the pathogenesis of asthma and other allergic diseases. We aimed to assess the effects of monoclonal antibody to IL-5 on blood and sputum eosinophils, airway hyper-responsiveness, and the late asthmatic reaction to inhaled allergen in patients with mild asthma. METHODS We did a double-blind randomised placebo-controlled trial, in which a single intravenous infusion of humanised (IgG-K) monoclonal antibody to IL-5 (SB-240563) was given at doses of 2.5 mg/kg (n=8) or 10.0 mg/kg (n=8). The effects of treatment on responses to inhaled allergen challenge, sputum eosinophils, and airway hyper-responsiveness to histamine were measured at weeks 1 and 4 with monitoring of blood eosinophil counts for up to 16 weeks. FINDINGS Monoclonal antibody against IL-5 lowered the mean blood eosinophil count at day 29 from 0.25x10(9)/L (95% CI 0.16-0.34) in the placebo group to 0.04x10(9)/L (0.00-0.07) in the 10 mg/kg group (p<0.0001), and prevented the blood eosinophilia that follows allergen challenge. After inhaled allergen challenge, 9 days after treatment, the percentage sputum eosinophils were 12.2% in the placebo group and lowered to 0.9% (-1.2 to 3.0; p=0.0076) in the 10 mg/kg group, and this effect persisted at day 30 after the dose. There was no significant effect of monoclonal antibody to IL-5 on the late asthmatic response or on airway hyper-responsiveness to histamine. INTERPRETATION A single dose of monoclonal antibody to IL-5 decreased blood eosinophils for up to 16 weeks and sputum eosinophils at 4 weeks, which has considerable therapeutic potential for asthma and allergy. However, our findings question the role of eosinophils in mediating the late asthmatic response and causing airway hyper-responsiveness.

International ERS/ATS guidelines on definition, evaluation and treatment of severe asthma

Severe or therapy-resistant asthma is increasingly recognised as a major unmet need. A Task Force, supported by the European Respiratory Society and American Thoracic Society, reviewed the definition and provided recommendations and guidelines on the evaluation and treatment of severe asthma in children and adults. A literature review was performed, followed by discussion by an expert committee according to the GRADE (Grading of Recommendations, Assessment, Development and Evaluation) approach for development of specific clinical recommendations. When the diagnosis of asthma is confirmed and comorbidities addressed, severe asthma is defined as asthma that requires treatment with high dose inhaled corticosteroids plus a second controller and/or systemic corticosteroids to prevent it from becoming “uncontrolled” or that remains “uncontrolled” despite this therapy. Severe asthma is a heterogeneous condition consisting of phenotypes such as eosinophilic asthma. Specific recommendations on the use of sputum eosinophil count and exhaled nitric oxide to guide therapy, as well as treatment with anti-IgE antibody, methotrexate, macrolide antibiotics, antifungal agents and bronchial thermoplasty are provided. Coordinated research efforts for improved phenotyping will provide safe and effective biomarker-driven approaches to severe asthma therapy. ERS/ATS guidelines revise the definition of severe asthma, discuss phenotypes and provide guidance on patient management http://ow.ly/roufI

The ENFUMOSA cross-sectional European multicentre study of the clinical phenotype of chronic severe asthma

Since severe asthma is a poorly understood, major health problem, 12 clinical specialist centres in nine European countries formed a European Network For Understanding Mechanisms Of Severe Asthma (ENFUMOSA). In a cross-sectional observational study, a total of 163 subjects with severe asthma were compared with 158 subjects whose asthma was controlled by low doses of inhaled corticosteroids (median dose of beclomethasone equivalents 666 µg). Despite being treated with higher doses of inhaled corticosteroids (median dose 1773 µg) and for a third of the severe asthmatics also being treated with regular, oral-steroid therapy (median daily dose 19 mg), the subjects with severe asthma met the inclusion criteria. The criteria required subjects to have undergone at least one asthma exacerbation in the past year requiring oral steroid treatment. Females dominated the severe asthma group (female/male ratio 4.4:1 versus 1.6:1 in the controlled asthmatics), and compared with controlled asthmatics, they had a predominantly neutrophilic inflammation (sputum neutrophils, 36 versus 28%) and evidence of ongoing mediator release but less atopy. From these findings and other physiological and clinical data reported in this paper, it is suggested that severe asthma might be a different form of asthma rather than an increase in asthma symptoms. The findings prompt for longitudinal studies and interventions to define the mechanisms in severe asthma.

Defective epithelial barrier function in asthma

BACKGROUND Asthma is a complex disease involving gene and environment interactions. Although atopy is a strong predisposing risk factor for asthma, local tissue susceptibilities are required for disease expression. The bronchial epithelium forms the interface with the external environment and is pivotally involved in controlling tissue homeostasis through provision of a physical barrier controlled by tight junction (TJ) complexes. OBJECTIVES To explain the link between environment exposures and airway vulnerability, we hypothesized that epithelial TJs are abnormal in asthma, leading to increased susceptibility to environmental agents. METHODS Localization of TJs in bronchial biopsies and differentiated epithelial cultures was assessed by electron microscopy or immunostaining. Baseline permeability and the effect of cigarette smoke and growth factor were assessed by measurement of transepithelial electrical resistance and passage of fluorescently labeled dextrans. RESULTS By using immunostaining, we found that bronchial biopsies from asthmatic subjects displayed patchy disruption of TJs. In differentiated bronchial epithelial cultures, TJ formation and transepithelial electrical resistance were significantly lower (P < .05) in cultures from asthmatic donors (n = 43) than from normal controls (n = 40) and inversely correlated with macromolecular permeability. Cultures from asthmatic donors were also more sensitive to disruption by cigarette smoke extract. Epidermal growth factor enhanced basal TJ formation in cultures from asthmatic subjects (P < .01) and protected against cigarette smoke-induced barrier disruption (P < .01). CONCLUSIONS Our results show that the bronchial epithelial barrier in asthma is compromised. This defect may facilitate the passage of allergens and other agents into the airway tissue, leading to immune activation and may thus contribute to the end organ expression of asthma.

Eridothelin immunoreactivity of airway epithelium in asthmatic patients

There is extensive pharmacological and physiological evidence that endothelin-1 influences airway calibre. In mammals, endothelin receptor occur on airway smooth muscle, local storage and release of the peptide have been demonstrated, and inhalation of endothelin-1 induces bronchoconstriction. To investigate the relation between endothelins and asthma the expression of this peptide in endobronchial biopsy specimens was examined immunohistochemically with an antiserum against endothelin-1. Biopsy specimens from 17 asthmatic patients and 11 atopic and non-atopic healthy controls revealed striking differences, with endothelin expression being evident in airways epithelium and vascular endothelium in 11 of the 17 asthmatic patients but in only 1 of 11 controls. These results suggest that endothelins may play a part in the exaggerated bronchomotor tone of asthma.

Standardised methodology of sputum induction and processing

Research into the pathogenetic mechanisms and clinical monitoring of inammatory diseases of any system is complete only if it involves the study of both the underlying pathological features and the physio- logical consequences that result from what are always complex inammatory processes. Respiratory dis- eases, including those of the airways, are no exception in this respect. It is, therefore, not surprising that the development over the last 25 years of techniques enabling the study of inammatory processes in the airways has revolutionised understanding of the commonest pulmonary diseases, asthma and chronic obstructive pulmonary disease (COPD). Airways inammation is now an established feature and a central consideration of any treatment strategy for both of these conditions. Most of the initial observations made in asthma, documenting the involvement of eosinophils, mast cells, T-cells and more recently structural cells, ® broblasts, endothelial cells and epithelial cells, were made in studies using ® breoptic bronchoscopy in

Relationship between peripheral airway dysfunction, airway obstruction, and neutrophilic inflammation in COPD

Background: Considerable research has been conducted into the nature of airway inflammation in chronic obstructive pulmonary disease (COPD) but the relationship between proximal airways inflammation and both dynamic collapse of the peripheral airways and HRCT determined emphysema severity remains unknown. A number of research tools have been combined to study smokers with a range of COPD severities classified according to the GOLD criteria. Methods: Sixty five subjects (11 healthy smokers, 44 smokers with stage 0–IV COPD, and 10 healthy non-smokers) were assessed using lung function testing and HRCT scanning to quantify emphysema and peripheral airway dysfunction and sputum induction to measure airway inflammation. Results: Expiratory HRCT measurements and the expiratory/inspiratory mean lung density ratio (both indicators of peripheral airway dysfunction) correlated more closely in smokers with the severity of airflow obstruction (r = −0.64, p<0.001) than did inspiratory HRCT measurements (which reflect emphysema severity; r = −0.45, p<0.01). Raised sputum neutrophil counts also correlated strongly in smokers with HRCT indicators of peripheral airway dysfunction (r = 0.55, p<0.001) but did not correlate with HRCT indicators of the severity of emphysema. Conclusions: This study suggests that peripheral airway dysfunction, assessed by expiratory HRCT measurements, is a determinant of COPD severity. Airway neutrophilia, a central feature of COPD, is closely associated with the severity of peripheral airway dysfunction in COPD but is not related to the overall severity of emphysema as measured by HRCT.

Inflammatory cells in the airways in COPD

Airway inflammation is central to the pathogenesis of both airway remodelling and parenchymal destruction in chronic obstructive pulmonary disease (COPD). Neutrophils, macrophages, and CD8+ T lymphocytes have been implicated in a number of studies, but a detailed profile of disease-phenotype specific inflammation has yet to emerge. The heterogeneity of the disease has hindered data interpretation while extrapolation of the results of relatively non-invasive studies to the actual pathology found in the distal lung is difficult. Moreover, prominent studies have had frequently conflicting results. Further investigations are needed to marry the different clinical phenotypes of COPD to their respective inflammatory profiles in the airways and thus improve our understanding of the pathogenesis of the disease as a whole.

Anti-immunoglobulin E treatment with omalizumab in allergic diseases: an update on anti-inflammatory activity and clinical efficacy.

Omalizumab is a humanized monoclonal anti‐IgE antibody developed for the treatment of allergic disease, with established efficacy in patients with moderate‐to‐severe allergic asthma and in patients with intermittent (seasonal) and persistent (perennial) allergic rhinitis (AR). Omalizumab is known to result in a marked reduction in serum levels of free IgE and down‐regulation of IgE receptors on circulating basophils. Recent work has shed further light on its mechanism of action, showing significant and profound reductions in tissue (nasal and bronchial) eosinophils and in bronchial IgE+ cells (mast cells), as well as T cells and B cells. Omalizumab treatment was also shown to be associated with down‐regulation of IgE receptors on circulating (precursor) dendritic cells, suggesting that blocking IgE may inhibit more chronic aspects of allergic inflammation involving T cell activation. Further work with omalizumab demonstrated it to have important benefits in patients with poorly controlled asthma despite high‐dose inhaled corticosteroid therapy, and analysis of clinical data suggests that the patients who are the best ‘responders’ to anti‐IgE treatment are those with asthma at the more severe end of the spectrum. Notably, systemic anti‐IgE therapy with omalizumab has been shown to improve symptoms, quality of life and disease control (asthma exacerbations) in patients with concomitant asthma and persistent AR. These impressive clinical data and the studies elucidating the anti‐inflammatory profile of omalizumab also serve to emphasize the fundamental importance of IgE in the pathogenesis of allergic diseases.

Methods of sputum processing for cell counts, immunocytochemistry and in situ hybridisation

Since the first attempts to use standardised methods for sampling induced airways sputum, two methods for processing the expectorate have evolved. The first involves selecting all viscid or denser portions from the expectorated sample with the aid of an inverted microscope 1, 2. This method has been extensively evaluated and reported in detail 2–4. The second approach involves processing the entire expectorate, comprising sputum plus variable amounts of saliva 5. Recent modifications to this method include collecting saliva and sputum separately in order to reduce salivary contamination 6–8. Both methods have advantages and disadvantages. The advantages of using selected sputum are: squamous cell contamination is 20% of all recovered cells 4. There is conflicting data as to whether or not differential cell counts (DCCs) differ between the two methods. One study reported a higher percentage of eosinophils in sputum processed by the selection method compared to the entire expectorate 9 but this has not been confirmed in other studies 2, 6, 10. Although, both the selected …