Bart Hilvering

Clinical utility of asthma biomarkers: from bench to bedside

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.

Evidence for the efficacy and safety of anti-interleukin-5 treatment in the management of refractory eosinophilic asthma

Two recent phase III trials in patients with severe eosinophilic asthma have shown that anti-interleukin 5 (IL-5) therapy with mepolizumab reduces the frequency of asthma attacks, improves symptoms and allows patients to reduce oral glucocorticoid use without loss of control of asthma. An earlier large 616 patient Dose Ranging Efficacy And safety with Mepolizumab in severe asthma (DREAM) study had shown that the only variables associated with treatment efficacy were a prior history of asthma attacks and the peripheral blood eosinophil count. The link between blood eosinophil counts and treatment efficacy is biologically obvious given that IL-5 has a pivotal role in eosinophil production, proliferation and chemotaxis. It is also clinically relevant as the blood eosinophil count is routinely measured and thus readily available in patients with asthma. Recognition of the link between airway or blood eosinophilia and treatment response was also important in the clinical testing of the alternative IL-5 blocker, such as reslizumab, which is currently being evaluated in a phase III randomized controlled trial (RCT) after having shown to improve lung function, improve symptom score and reduce sputum eosinophilia in a smaller phase IIb study. In addition, benralizumab, an IL-5α receptor blocker, has shown good effects in a phase IIb RCT with patients with severe asthma that had sputum eosinophilia and more recently in a phase IIa trial with patients with eosinophilic chronic obstructive pulmonary disease. Therefore anti-IL-5 treatment seems generally effective in eosinophilic asthma, either assessed by blood or airway eosinophilia. This factor together with the impressive clinical efficacy and good safety profile make anti-IL-5 (mepolizumab, reslizumab) and benralizumab (anti-IL-5 receptor α) very promising drugs for the treatment of patients with severe eosinophilic asthma, a subgroup that is in desperate need of better treatments.

Similar activation state of neutrophils in sputum of asthma patients irrespective of sputum eosinophilia

Inflammatory phenotypes of asthma are associated with differences in disease characteristics. It is unknown whether these inflammatory phenotypes are reflected by the activation status of neutrophils in blood and sputum. We obtained peripheral blood and induced sputum from 21 asthma patients and stratified our samples based on sputum eosinophilia resulting in two groups (>3% eosinophils: n = 13, <3%: n = 8). Eosinophils and neutrophils from blood and sputum were analysed for expression of activation and degranulation markers by flow cytometry. Data were analysed by both classical, non‐parametric statistics and a multi‐dimensional approach, using principal component analysis (PCA). Patients with sputum eosinophilia were characterized by increased asthma control questionnaire (ACQ) scores and blood eosinophil counts. Both sputum neutrophils and eosinophils displayed an activated and degranulated phenotype compared to cells obtained from blood. Specifically, degranulation of all granule types was detected in sputum cells, combined with an increased expression of the activation markers (activated) Mac‐1 (CD11b), programmed death ligand 1 (PD‐L1) (CD274) and a decreased expression of CD62L. CD69 expression was only increased on sputum eosinophils. Surface marker expression of neutrophils was similar in the presence or absence of eosinophilia, either by single or multi‐dimensional analysis. Sputum neutrophils were highly activated and degranulated irrespective of sputum eosinophilia. Therefore, we conclude that differences in granulocyte activation in sputum and/or blood are not associated with clinical differences in the two groups of asthma patients. The finding of PD‐L1 expression on sputum granulocytes suggests an immunomodulatory role of these cells in the tissue.

What goes up must come down: biomarkers and novel biologicals in severe asthma

Asthma is a heterogeneous airway disease characterized by typical symptoms in combination with variable airway obstruction. Most patients with asthma have well controlled symptoms and a low risk of asthma attacks with inhaled corticosteroid (ICS) treatment. However, a clinically important subgroup (~ 10%) remains symptomatic and/or at risk of asthma attacks despite maximum inhaled therapy. Patients with severe asthma are responsible for a significant proportion of healthcare costs attributable to asthma and have a large unmet need for better treatments. An important advance in recent years has been the recognition that severe asthma is heterogeneous with respect to clinical problems and the pattern of lower airway inflammation. Identification of eosinophilic inflammation in the airways has become an important priority as novel biologicals that target Th2 cytokines, such as anti‐IL5, anti‐IL‐13 and combined anti‐IL‐4/13 are showing considerable promise as treatments for this subgroup. It has also become clear that anti‐IgE (Omalizumab), the first monoclonal antibody registered for treatment of severe asthma, is only active in patients with active eosinophilic airway inflammation. The future will be identification of potentially responsive patients on the basis of raised biomarkers and, as suggested by the title of this review, targeted treatment with specific cytokine blockade that has a direct effect on the biomarkers. In this review, we outline an approach to the clinical assessment of patients potentially suitable for biological treatment and describe in detail the likely clinical impact of established and new biological treatments.

Diagnosing eosinophilic asthma using a multivariate prediction model based on blood granulocyte responsiveness

The identification of inflammatory asthma phenotypes, using sputum analysis, has proven its value in diagnosis and disease monitoring. However due to technical limitations of sputum analysis, there is a strong need for fast and noninvasive diagnostics. This study included the activation state of eosinophils and neutrophils in peripheral blood to phenotype and monitor asthma.

Emerging Biologics in Severe Asthma

Asthma is a heterogeneous disease that can be classified into different clinical endotypes, depending on the type of airway inflammation, clinical severity, and response to treatment. This article focuses on the eosinophilic endotype of asthma, which is defined by the central role that eosinophils play in the pathophysiology of the condition. It is characterized by persistently elevated sputum and/or blood eosinophils and by a significant response to treatments that suppress eosinophilia. Eosinophil activity in the airway may be more important than their numbers and this needs to be investigated. Transcriplomic or Metabolomic signatures may also be useful to identify this endotype.

Colloidal stability of (functionalised) fullerenes in the presence of dissolved organic carbon and electrolytes

Carbon-based nanoparticles such as fullerenes have been widely applied in personal care products, drug delivery systems, and solar cells. The properties of nanoparticles have been increasingly studied because of their applications and their potential risks to the environment and human health. Many studies have focused on the environmental fate and properties of C60. However, there is limited information available on the environmental properties of functionalised fullerenes. This study focuses on the colloidal stability of two fullerenes (C60 and [6,6]-diphenyl-C62-bis(butyric acid methyl ester)) in water in the presence of dissolved organic carbon (DOC) and different electrolytes (NaCl and CaCl2). Suspended fullerene concentrations were determined with high resolution Orbitrap mass spectrometry. The size was determined by multi-angle light scattering, dynamic light scattering and for the first time by flow cytometry. The suspended concentrations of the fullerenes were stabilised by low concentrations of DOC (2 mg C per L) in the presence of NaCl. However, sedimentation of DOC occurred at low concentrations of CaCl2 (>~2 mM) which caused removal of (functionalised) fullerenes. The results show that (functionalised) fullerenes can be rapidly removed in natural aqueous systems in the presence of low concentrations of DOC and multivalent inorganic electrolytes.

IL-33–Dependent Th2 Response after Rhinovirus Infection in Asthma: More Information Needed

We read with great interest the article by Jackson and colleagues regarding IL-33–dependent type 2 inflammation during rhinovirus-induced asthma exacerbations (1). Their rhinovirus 16 (RV16) challenge model is a promising approach to investigate the nature of virally induced exacerbations in asthma. However, the strong statements they make about the role of IL-33 and Th2 cytokine production in the in vivo model are not justified by the data presented. IL-33 and Th2 cytokine nasal levels were measured before RV16 inoculation (baseline) and at 2, 3, 4, 5, 7, 10, and 42 days after inoculation. However, in the article, only baseline, “infection,” and Day 42 are presented (their Figure 2A). Looking more closely at the results, it becomes clear the infection is the peak value of IL-33 selected for each patient, regardless of the time point. When a single baseline measurement is compared with the peak value of multiple measures, an increase is to be expected as a result of “regression to the peak.” It would be easier to accept the authors’ conclusions if we were shown the full time course of IL-33 and Th2 cytokine release after inoculation. The only data presented that allow us to draw a confident conclusion are the bronchial lining fluid cytokine levels, measured before and at a single time point after inoculation (Table E4 in their online supplement). We note that there is no evidence of virally induced IL-33 production in the bronchial lining fluid at Day 4, a time when the viral load was decreasing and the immune response against RV16 was likely to be at maximum. The small differences in bronchial lining fluid Th2 cytokine levels before and after inoculation might have been significant within groups, but it is not clear whether the between-group comparisons compare the change in cytokine levels. This comparison is required to conclude that this is an asthmaspecific effect. We therefore do not accept that Jackson and colleagues have shown convincingly that IL-33 and Th2 cytokine production is induced by RV16 infection in asthma. These issues are also relevant to the earlier published data on the IL-25 response after RV16 infection in the same patients (2). The authors’ conclusion that virus-induced asthma exacerbations involve a type 2 response is challenging and, if correct, clinically very important. However, before accepting this conclusion, we would like to see the data presented and analyzed in a more transparent and appropriate way. n

Novel data analysis method for multicolour flow cytometry links variability of multiple markers on single cells to a clinical phenotype

Multicolour Flow Cytometry (MFC) produces multidimensional analytical data on the quantitative expression of multiple markers on single cells. This data contains invaluable biomedical information on (1) the marker expressions per cell, (2) the variation in such expression across cells, (3) the variability of cell marker expression across samples that (4) may vary systematically between cells collected from donors and patients. Current conventional and even advanced data analysis methods for MFC data explore only a subset of these levels. The Discriminant Analysis of MultiAspect CYtometry (DAMACY) we present here provides a comprehensive view on health and disease responses by integrating all four levels. We validate DAMACY by using three distinct datasets: in vivo response of neutrophils evoked by systemic endotoxin challenge, the clonal response of leukocytes in bone marrow of acute myeloid leukaemia (AML) patients, and the complex immune response in blood of asthmatics. DAMACY provided good accuracy 91–100% in the discrimination between health and disease, on par with literature values. Additionally, the method provides figures that give insight into the marker expression and cell variability for more in-depth interpretation, that can benefit both physicians and biomedical researchers to better diagnose and monitor diseases that are reflected by changes in blood leukocytes.

Similar activation of sputum granulocytes in eosinophilic and non-eosinophilic asthma

Method We obtained peripheral blood and induced sputum from 21 asthma patients and defined two patient groups based on sputum eosinophilia (cut-off 3%). Eosinophils and neutrophils from blood and sputum were analysed by flow cytometry for expression of activation and degranulation markers. Data were analysed by both classical, non-parametric statistics and a more advanced multi-dimensional approach, using principal component analysis (PCA).