Andrew J. Tan

Topical corticosteroid inhibits interleukin‐4, ‐5 and ‐13 in nasal secretions following allergen challenge

Background Cytokines and chemokines produced by allergen‐reactive T‐helper type 2 (Th2) cells may be pivotal to the pathophysiology of allergic disorders.

A novel method for assessing unchallenged levels of mediators in nasal epithelial lining fluid

To the Editor: There is a need to develop noninvasive methods to sample epithelial lining fluid (ELF) from the respiratory system. The nasal mucosa is easily accessible, and it has long been recognized that there is a strong functional and immunologic relationship between the nose and bronchi. It is possible to obtain samples of ELF by means of nasal lavage, which has been used to measure inflammatory protein secretion after nasal allergen challenge. However, nasal allergen challenge exaggerates natural exposures, and the unknown dilution factor from nasal lavage is a significant confounder and might dilute the mediators to less than the detection limit of the assay. Therefore there is a need to measure mediators in the undisturbed ELF. We propose a method for sampling nasal ELF using a synthetic absorptive matrix (SAM), separating the fluid by means of centrifugation, and analyzing the sample with a multiplex quantitative protein assay for a panel of inflammatory mediators. The advantage of this technique is that it analyzes undiluted ELF at the tissue interface without need for stimulation of the mucosa. We conducted a case-control study of children with symptomatic allergic rhinitis and healthy control subjects from the Copenhagen Prospective Study on Asthma in Childhood birth cohort to assess whether unchallenged levels of nasal mediators could be detected in ELF collected with SAM and whether such levels associated with symptoms of allergic rhinitis. The study was conducted August to September 2008 and was approved by the Copenhagen Ethics Committee. Case status was determined based on allergic rhinitis with troublesome symptoms in the previous 24 hours, and control

Rapid effect of inhaled ciclesonide in asthma: a randomized, placebo-controlled study.

BACKGROUND Ciclesonide is a novel inhaled corticosteroid for the treatment of asthma, and it is important to measure the onset of effect of this therapy on airway hyperresponsiveness (AHR), exhaled nitric oxide (NO), and levels of eosinophils in induced sputum. METHODS In a randomized, double-blind, crossover study, 21 patients with mild asthma inhaled ciclesonide 320 microg (ex-actuator) qd, ciclesonide 640 microg (ex-actuator) bid, and placebo for 7 days. Exhaled NO and AHR to adenosine monophosphate (AMP), measured as the provocative concentration of AMP producing a 20% reduction in FEV1 (PC20FEV1), were assessed after inhalation on days 1, 3 and 7. Eosinophil levels in induced sputum were also measured. RESULTS Ciclesonide 320 microg qd and 640 microg bid produced significantly greater improvements in PC20FEV1 compared with placebo on day 1 (within 2.5 h), and on days 3 and 7 (all p < 0.0001). On day 3, both ciclesonide doses significantly reduced exhaled NO levels by - 17.7 parts per billion (p < 0.0001) and - 15.4 parts per billion (p < 0.003) vs placebo, respectively. Significant reductions were maintained during the study with both ciclesonide doses (p < 0.01). A nonsignificant trend towards a decrease in eosinophil cell numbers was observed after 7 days of ciclesonide treatment, especially in patients receiving the higher dose. CONCLUSIONS A single dose of ciclesonide decreased AHR to AMP and exhaled NO within 3 h, while FEV, improved at 3 days and 7 days.

The nasal mucosal late allergic reaction to grass pollen involves type 2 inflammation (IL-5 and IL-13), the inflammasome (IL-1β), and complement

Non-invasive mucosal sampling (nasosorption and nasal curettage) was used following nasal allergen challenge with grass pollen in subjects with allergic rhinitis, in order to define the molecular basis of the late allergic reaction (LAR). It was found that the nasal LAR to grass pollen involves parallel changes in pathways of type 2 inflammation (IL-4, IL-5 and IL-13), inflammasome-related (IL-1β), and complement and circadian-associated genes. A grass pollen nasal spray was given to subjects with hay fever followed by serial sampling, in which cytokines and chemokines were measured in absorbed nasal mucosal lining fluid, and global gene expression (transcriptomics) assessed in nasal mucosal curettage samples. Twelve of 19 subjects responded with elevations in interleukin (IL)-5, IL-13, IL-1β and MIP-1β/CCL4 protein levels in the late phase. In addition, in these individuals whole-genome expression profiling showed upregulation of type 2 inflammation involving eosinophils and IL-4, IL-5 and IL-13; neutrophil recruitment with IL-1α and IL-1β; the alternative pathway of complement (factor P and C5aR); and prominent effects on circadian-associated transcription regulators. Baseline IL-33 mRNA strongly correlated with these late-phase responses, whereas a single oral dose of prednisone dose-dependently reversed most nasal allergen challenge-induced cytokine and transcript responses. This study shows that the LAR to grass pollen involves a range of inflammatory pathways and suggests potential new biomarkers and therapeutic targets. Furthermore, the marked variation in mucosal inflammatory events between different patients suggests that in the future precision mucosal sampling may enable rational specific therapy.

Nasal Lipopolysaccharide Challenge and Cytokine Measurement Reflects Innate Mucosal Immune Responsiveness

Background Practical methods of monitoring innate immune mucosal responsiveness are lacking. Lipopolysaccharide (LPS) is a component of the cell wall of Gram negative bacteria and a potent activator of Toll-like receptor (TLR)-4. To measure LPS responsiveness of the nasal mucosa, we administered LPS as a nasal spray and quantified chemokine and cytokine levels in mucosal lining fluid (MLF). Methods We performed a 5-way cross-over, single blind, placebo-controlled study in 15 healthy non-atopic subjects (n = 14 per protocol). Doses of ultrapure LPS (1, 10, 30 or 100μg/100μl) or placebo were administered by a single nasal spray to each nostril. Using the recently developed method of nasosorption with synthetic adsorptive matrices (SAM), a series of samples were taken. A panel of seven cytokines/chemokines were measured by multiplex immunoassay in MLF. mRNA for intercellular cell adhesion molecule-1 (ICAM-1) was quantified from nasal epithelial curettage samples taken before and after challenge. Results Topical nasal LPS was well tolerated, causing no symptoms and no visible changes to the nasal mucosa. LPS induced dose-related increases in MLF levels of IL-1β, IL-6, CXCL8 (IL-8) and CCL3 (MIP-1α) (AUC at 0.5 to 10h, compared to placebo, p<0.05 at 30 and 100μg LPS). At 100μg LPS, IL-10, IFN-α and TNF-α were also increased (p<0.05). Dose-related changes in mucosal ICAM-1 mRNA were also seen after challenge, and neutrophils appeared to peak in MLF at 8h. However, 2 subjects with high baseline cytokine levels showed prominent cytokine and chemokine responses to relatively low LPS doses (10μg and 30μg LPS). Conclusions Topical nasal LPS causes dose-dependent increases in cytokines, chemokines, mRNA and cells. However, responsiveness can show unpredictable variations, possibly because baseline innate tone is affected by environmental factors. We believe that this new technique will have wide application in the study of the innate immune responses of the respiratory mucosa. Key Messages Ultrapure LPS was used as innate immune stimulus in a human nasal challenge model, with serial sampling of nasal mucosal lining fluid (MLF) by nasosorption using a synthetic absorptive matrix (SAM), and nasal curettage of mucosal cells. A dose response could be demonstrated in terms of levels of IL-1β, IL-6, CXCL8 and CCL3 in MLF, as well as ICAM-1 mRNA in nasal curettage specimens, and levels of neutrophils in nasal lavage. Depending on higher baseline levels of inflammation, there were occasional magnified innate inflammatory responses to LPS. Trial Registration Clinical Trials.gov NCT02284074

Nasal testing for novel anti-inflammatory agents

The nose is much more accessible than the airways to assess the effects of anti-inflammatory therapy. Hence, it is possible to obtain repeated samples of nasal exudates and mucosa cells before and after nasal allergen challenge (NAC) in a relatively non-invasive way by techniques such as nasal lavage, filter paper, and nasal brushing and scraping. A comprehensive review of the extensive clinical research experience with these nasal methodologies has recently been published by Howarth et al. [1]. Nasal samples can then be analysed by novel semiautomated analytical methods to assess chemokines and cytokines, inflammatory mediators, mRNA, and transcription factors. Inhaled allergen challenge commonly calls a profound decrease in forced expiratory volume in 1 s, while the nasal symptoms that follow NAC are generally mild. Furthermore, it is much easier to recruit potential subjects with allergic rhinitis (AR) because of grass pollen outside the hayfever season, rather than subjects with a dual early and late reaction to an inhaled allergen. Following NAC it is possible to measure symptoms, use acoustic rhinomanometry, and measure levels of cells and mediators to evaluate new drugs for AR and asthma [2–4]. It has long been recognized that there is a strong functional and immunological relationship between the nose and bronchi [5, 6], especially in terms of infiltrating leucocytes and inflammatory mediators when comparing AR and allergic asthma [7]. The upper and lower airways have related respiratory epithelium and similar responses to allergen challenge. Indeed, AR and asthma commonly coexist [8], as allergy is a systemic disorder that can affect various organs within the unified immune system [9, 10]. This is in line with the WHO Initiative on Allergic Rhinitis and its Impact on Asthma (ARIA) stressing the concept of a single airway disease [11]. However, the nasal model involves a different vasculature to that in the airways, while the bronchi have added airway smooth muscle. At a pathological level, the extent of nasal remodelling in AR seems to be much less than that in the bronchi of asthmatic patients [12, 13]. There is strong evidence that allergen-reactive type 2 T helper (Th2) cells play an important role in the induction and maintenance of the allergic inflammatory cascade [14]. Cytokines and chemokines produced by Th2 cells (IL-4, IL-5, IL-9, and IL-13) may be pivotal to the pathophysiology of allergic disorders involving production of IgE, recruitment and activation of mast cells and eosinophils, mucus hypersecretion, subepithelial fibrosis, and tissue remodelling. Several studies have demonstrated significant expression of various cytokines and chemokines in inflammatory cells at sites of nasal allergic inflammation [15–19]. Excessive production of IL-5 and IL-13 may be critical to the allergic response [14, 20]. Maintenance treatment with topical steroids exerts a range of anti-inflammatory nasal effects on production of eotaxin [21], RANTES, MIP-1a, IL-8, IL-1b [17], TNF-a [22], and IL-5 [23]. Topical allergen challenge increases the levels of mucosal mRNA of IL-5 and IL-13 [19, 23] but nasal cytokines and chemokines may be produced at low concentration in nasal secretions, and may be undetectable when using conventional ELISAs. A single dose of topical corticosteroid has been shown to reduce levels of granulocyte macrophage-colony stimulating factor and IL-5 detected by absorption with filter paper following nasal challenge with grass pollen in AR [23, 24]. In order to sample nasal exudates for allergic inflammatory mediators, the classical methods of nasal lavage are those described by Naclerio et al. [25], the nasal pool method of Greiff et al. [26], and the use of a Foley’s catheter by Grünberg et al. [27]. Lavage is performed with saline at volumes between 1 and 10mL. The repeatability and validity of different nasal lavage methods have been compared [28]. An important demonstration of the utility of this methodology for assessing therapy was the demonstration that pretreatment with topical corticosteroids causes inhibition of release of histamine, kinins, and symptoms after NAC [29]. Peptidyl leukotrienes are also released [30] and there is a later increase in histamine during the late nasal reaction [31]. More recently, increases in IL-5 and eotaxin have been detected in nasal lavage fluid in the early and late reactions following NAC [21, 32]. In this edition of Clinical and Experimental Allergy, Rami Salib, Laurie Lau, and Peter Howarth demonstrate elegantly that nasal lavage levels of eotaxin-1 (CCL11) are elevated in symptomatic AR compared with controls [33]. Tommy Sim and colleagues have developed the use of filter paper strips, which are placed on the turbinates to absorb nasal secretions [34]. The nasal filter paper method has the advantage of directly sampling nasal secretions that are less diluted and can therefore pick up protein signals, which are below the detection limits of nasal lavage. The matrix or filter paper method has been used to measure chemokines and cytokines after NAC [17, 24, 35, 36]. However, it should be noted that nasal lavage probably represents an extracellular signal, while nasal sampling by absorption into filter paper strips probably represents both an intracellular and extracellular signal, as cells that adhere to the surface of the filter paper may lyse and release their intracellular contents. Cells samples may be obtained from the nasal mucosal using small nylon dental flossing brushes which are gently rotated over the epithelium; then the attached cells are dislodged in balanced salt solution [37, 38]. It has been demonstrated that nasal brushing can be used as an Clin Exp Allergy 2005; 35:981–985 doi:10.1111/j.1365-2222.2005.02311.x

Chapter 49 – Anticholinergic Bronchodilators

Publisher Summary This chapter considers the pharmacology, biochemistry, and potential anti-inflammatory actions of anticholinergic bronchodilators, including possible anti-inflammatory actions, as well as clinical trial evidence for the safety and efficacy of these therapies. Important outcome measures for LAMAs involve considerations of effects on symptoms, lung function, dynamic hyperinflation and exercise responses, quality of life, health economics, exacerbations, survival, and the natural history of chronic obstructive pulmonary disease (COPD). Anticholinergics are muscarinic receptor antagonists, inhibiting cholinergic reflex bronchoconstriction, and reducing vagal cholinergic tone, which is the main reversible component in COPD. Normal airways have a small degree of vagal cholinergic tone, but because the airways are patent this has no perceptible effect and does not reduce airflow. Anticholinergics may reduce mucus hypersecretion. Anticholinergics have no apparent effect on pulmonary vessels, and therefore do not cause a fall in Pao2, as may sometimes be seen with β2-agonists and theophylline. The existence of several subtypes of muscarinic receptor in human airways has suggested that more selective muscarinic antagonists may have advantages over nonselective agents, such as ipratropium bromide and oxitropium bromide. Interestingly, anticholinergic may benefit asthmatics with COPD-like inflammation in asthma, viral hyperreactivity, and emergency asthma.

New drugs for COPD based on advances in pathophysiology

There is a pressing need to develop new treatments for chronic obstructive pulmonary disease (COPD), as no currently available drug has been shown to reduce the relentless progression of this disease. Furthermore, recognition of the global importance and rising prevalence of COPD and the absence of effective therapies has now led to a concerted effort to develop new drugs for this disease [1, 2]. However, there have been disappointingly few therapeutic advances in the drug therapy of COPD, in contrast to the enormous advances made in asthma management that reflect a much better understanding of the underlying disease [3, 4].

Evaluation of New Drugs for Asthma and COPD: Endpoints, Biomarkers and Clinical Trial Designs

The incidence of both asthma and chronic obstructive pulmonary disease (COPD) is increasing throughout the world, and acts as a major incentive for the development of new and improved drug therapy. For the large range of bronchodilator and anti-inflammatory agents in current clinical development, reliable decision-making is imperative in phase II, before entering large-scale phase III clinical studies. With anti-inflammatory therapies for asthma, many studies have been performed utilising the inhaled allergen challenge as a proof of concept study, effects on airway hyper-reactivity (AHR) can be assessed, and it is also possible to directly study limited numbers of symptomatic asthma patients. Additional clinical trial designs in asthma include studies to assess bronchodilation, bronchoprotection against a variety of inhaled constrictor agents, exercise tolerance, add-on and titration studies with inhaled and oral corticosteroids, and prevention and treatment of exacerbations. In contrast, it is a major issue for the development of new anti-inflammatory drugs for COPD that large-scale phase II studies are generally required in this disease in order to detect clinical efficacy. In COPD, clinical trial designs range from studies on lung function, symptoms and exercise performance, inflammatory biomarkers, natural history of chronic stable disease, prevention and treatment of exacerbations, and effects on cachexia and muscle function. Compared with asthma, inclusion criteria, monitoring parameters, comparator therapies and trial design are less well established for COPD. The large variety of potential clinical endpoints includes lung function, symptoms, walking tests, hyperinflation, health-related quality of life (HR-QOL), airway reactivity, and frequency and severity of exacerbations. In addition, surrogate biomarkers may be assessed in blood, exhaled breath, induced sputum, bronchial mucosal biopsy and bronchoalveolar lavage (BAL), and advanced radiographic imaging employed. Of particular utility is ex vivo whole blood stimulation to enable pharmacokinetic/pharmacodynamic modelling in establishing an optimal dosage regimen relatively early in human clinical studies. There have been considerable recent advances in the development of non-invasive biomarkers and novel clinical trial designs, as well as clarification of regulatory requirements, that will facilitate the development of new therapies for patients with asthma and COPD.