title: Sputum RNA signature in allergic asthmatics following allergen bronchoprovocation test.
abstract: BACKGROUND
  Inhaled allergen challenge is a validated disease model of allergic asthma offering useful pharmacodynamic assessment of pharmacotherapeutic effects in a limited number of subjects.
  
  
  OBJECTIVE
  To evaluate whether an RNA signature can be identified from induced sputum following an inhaled allergen challenge, whether a RNA signature could be modulated by limited doses of inhaled fluticasone, and whether these gene expression profiles would correlate with the clinical endpoints measured in this study.
  
  
  METHODS
  Thirteen non-smoking, allergic subjects with mild-to-moderate asthma participated in a randomised, placebo-controlled, 2-period cross-over study following a single-blind placebo run-in period. Each period consisted of three consecutive days, separated by a wash-out period of at least 3 weeks. Subjects randomly received inhaled fluticasone ((FP) MDI; 500 mcg BID×5 doses in total) or placebo. On day 2, house dust mite extract was inhaled and airway response was measured by FEV1 at predefined time points until 7 h post-allergen. Sputum was induced by NaCl 4.5%, processed and analysed at 24 h pre-allergen and 7 and 24 h post-allergen. RNA was isolated from eligible sputum cell pellets (<80% squamous of 500 cells), amplified according to NuGEN technology, and profiled on Affymetrix arrays. Gene expression changes from baseline and fluticasone treatment effects were evaluated using a mixed effects ANCOVA model at 7 and at 24 h post-allergen challenge.
  
  
  RESULTS
  Inhaled allergen-induced statistically significant gene expression changes in sputum, which were effectively blunted by fluticasone (adjusted p<0.025). Forty-seven RNA signatures were selected from these responses for correlation analyses and further validation. This included Th2 mRNA levels for cytokines, chemokines, high-affinity IgE receptor FCER1A, histamine receptor HRH4, and enzymes and receptors in the arachidonic pathway. Individual messengers from the 47 RNA signatures correlated significantly with lung function and sputum eosinophil counts.
  
  
  CONCLUSIONS
  Our RNA extraction and profiling protocols allowed reproducible assessments of inflammatory signatures in sputum including quantification of drug effects on this response in allergic asthmatics. This approach offers novel possibilities for the development of pharmacodynamic (PD) biomarkers in asthma.
authors.affiliation: Centre for Human Drug Research, Leiden, The Netherlands; RZuiker@chdr.nl.
authors.forename: Rob G J A
authors.initials: RG
authors.lastname: Zuiker
authors.affiliation: Merck Research Laboratories, Rahway, New Jersey, USA.|Novartis, New York, NY, USA.
authors.forename: Catherine
authors.initials: C
authors.lastname: Tribouley
authors.affiliation: Centre for Human Drug Research, Leiden, The Netherlands.|Department of Respiratory Medicine and Allergology, Skane University Hospital, Lund, Sweden.|Department of Clinical & Pharmacology, University Medical Center Groningen, Groningen, The Netherlands.|Department of General Practice, University Medical Center Groningen, Groningen, The Netherlands.|QPS Netherlands, Groningen, The Netherlands.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Diamant
authors.affiliation: Centre for Human Drug Research, Leiden, The Netherlands.|Janssen Biologics B.V., Leiden, The Netherlands.
authors.forename: J Diderik
authors.initials: JD
authors.lastname: Boot
authors.affiliation: Centre for Human Drug Research, Leiden, The Netherlands.
authors.forename: Adam F
authors.initials: AF
authors.lastname: Cohen
authors.affiliation: Merck Research Laboratories, Rahway, New Jersey, USA.
authors.forename: K
authors.initials: K
authors.lastname: Van Dyck
authors.affiliation: Merck Research Laboratories, Rahway, New Jersey, USA.
authors.forename: I
authors.initials: I
authors.lastname: De Lepeleire
authors.affiliation: Merck Research Laboratories, Rahway, New Jersey, USA.
authors.forename: Veronica M
authors.initials: VM
authors.lastname: Rivas
authors.affiliation: Merck Research Laboratories, Rahway, New Jersey, USA.
authors.forename: Vladislav A
authors.initials: VA
authors.lastname: Malkov
authors.affiliation: Centre for Human Drug Research, Leiden, The Netherlands.
authors.forename: Jacobus
authors.initials: J
authors.lastname: Burggraaf
authors.affiliation: Merck Research Laboratories, Rahway, New Jersey, USA.|EMD Serono, Rockland, MA, USA.
authors.forename: Marcella K
authors.initials: MK
authors.lastname: Ruddy
keywords: DNA microarray; Th2 inflammation; allergen bronchial provocation test; asthma; fluticasone; sputum