title: Kinetics of TH2 biomarkers in sputum of asthmatics following inhaled allergen.
abstract: BACKGROUND
  Allergen-induced late airway response offers important pharmacodynamic targets, including T helper 2 (TH2) biomarkers. However, detection of inflammatory markers has been limited in dithiothreitol-processed sputum.
  
  
  OBJECTIVE
  To test whether allergen-induced TH2 inflammatory markers can be reproducibly quantified by sensitive detection techniques in ultracentrifuged sputum and the effect of fluticasone (FP) on these endpoints.
  
  
  METHODS
  Thirteen allergic asthmatics with dual allergen-induced airway responses, documented during a single-blind placebo run-in period, participated in a double-blind, two-period crossover study. Each period consisted of three consecutive days, separated by ≥3 weeks. Following randomization, subjects inhaled FP (500 µg bid, five doses total) or placebo. On Day 2 in each study period, allergen challenge was performed and airway response measured by forced expiratory volume in 1 sec (FEV1) until 7 h post-challenge. Sputum was induced 24 h pre-allergen and 7 and 24 h post-allergen. Sputum samples were split into two portions: TH2 biomarkers were quantified by Meso Scale multiplex platform following ultracentrifugation, and cell differentials were counted on Giemsa-May-Grünwald-stained cytospins. Allergen-induced changes in inflammatory endpoints were compared between FP and placebo using a mixed model ANCOVA.
  
  
  RESULTS
  Inhaled allergen induced dual airway responses in all subjects during both placebo periods with reproducible late asthmatic response (LAR) and increased sputum inflammatory biomarkers (IL-2, IL-4, IL-13, and eotaxin-1) and eosinophil counts. FP effectively blunted both the LAR and the inflammatory biomarkers.
  
  
  CONCLUSIONS
  Combining novel, sensitive quantification methods with ultracentrifugation allows reproducible quantification of sputum biomarkers following allergen challenge, reversed by FP. This approach allows non-invasive identification of pharmacodynamic targets for anti-asthma therapies.
authors.affiliation: Centre for Human Drug Research, Leiden, The Netherlands.
authors.forename: Rob G J A
authors.initials: RG
authors.lastname: Zuiker
authors.affiliation: EMD Serono, Rockland, MA, USA.
authors.forename: Marcella K
authors.initials: MK
authors.lastname: Ruddy
authors.affiliation: VU Medical Center, Amsterdam, The Netherlands.
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Morelli
authors.affiliation: Janssen Pharmaceutical Companies of Johnson & Johnson, Spring House, PA, USA.
authors.forename: Robin
authors.initials: R
authors.lastname: Mogg
authors.affiliation: Merck Research Laboratories, Rahway, NJ, USA.
authors.forename: Veronica M
authors.initials: VM
authors.lastname: Rivas
authors.affiliation: Merck Research Laboratories, Brussels, Belgium.
authors.forename: Kristien
authors.initials: K
authors.lastname: van Dyck
authors.affiliation: Merck Research Laboratories, Brussels, Belgium.
authors.forename: Inge
authors.initials: I
authors.lastname: De Lepeleire
authors.affiliation: Merck Research Laboratories, Rahway, NJ, USA.
authors.forename: Michael R L
authors.initials: MR
authors.lastname: Tanen
authors.affiliation: HAL Allergy 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: Ingrid M C
authors.initials: IM
authors.lastname: Kamerling
authors.affiliation: Skane University Hospital, Department of Respiratory Medicine and Allergology, Institute for Clinical Science, Skane University, Lund, Sweden ; Departments of Clinical Pharmacy & Pharmacology and General Practice, University Medical Center Groningen, Groningen, The Netherlands ; QPS Netherlands, Groningen, The Netherlands.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Diamant
keywords: TH2 inflammation; allergen bronchial provocation test; asthma; fluticasone; sputum