O’Neil et al. Allergy, Asthma & Clinical Immunology 2010, 6:28 http://www.aacijournal.com/content/6/1/28 local OPN expression. The nasal biopsies used in the cur- rent study have previously been evaluated in other studies showing clear clinical effects of pollen exposure, as well as effects of a nasal glucocorticoid treatment on both symptoms of rhinitis, eosinophilia and expression of several cytokines [2,5]. Materials and methods Nasal biopsy samples were obtained as a part of a pre- viously published clinical study [2]. Briefly, grass pollen sensitised allergic rhinitis (AR) subjects were randomised to receive the intranasal glucocorticocoid, fluticasone propionate (FP) (200 μg/day) (n = 12; median age 30.5, range 18-40 years) or placebo (n = 16; median age 30, range 16-48 years) for the duration of the grass pollen season, starting approximately 2 weeks before the expected onset. The nasal biopsies were collected 1-2 months before the commencement of treatment and at the peak of the season. Ethics approval was obtained from the Ethics Review Committee of Clinical Research Studies at the University of Tartu, Estonia. Written informed consent was provided by all participants. As controls, nasal biopsies were taken from five healthy, non-allergic individuals prior to the pollen season [5]. Nasal biopsy sections were immersed in OCT com- pound in cryomoulds (Tissue-Tek, Sakura Finetek Europe, Zoeterwouede, Netherlands), snap frozen in liquid nitrogen and stored at -80°C prior to processing. Tissue sections of 5 μm thickness were prepared, wrapped in aluminium foil and stored at -80˚C. Thawed sections were fixed in 2% formaldehyde and treated with pre-heated PBS containing 0.0064% sodium azide, 0.18% glucose, 0.1% saponin, 1:3750 glucose oxidase, for 40 mins to inhibit endogenous peroxidise. Unspecific binding was blocked for 30 mins using 10% rabbit serum (DAKO Denmark A/S, Denmark). Sections were incubated with mouse anti-osteopontin monoclonal antibody (clone 190312. MAB1433) (R&D Systems, Inc. MN, USA) for 2 hrs, followed by incubation with a sec- ondary antibody (peroxidase conjugated rabbit F(ab’)2 anti-mouse IgG (Zymed Laboratories, CA, USA)). The positive staining was detected using the Liquid DAB Substrate-Chromogen System (DAKO), followed by counterstaining with Mayers Hematoxylin (Sigma- Aldrich, MO, USA) A matched isotype control, mouse IgG2B (clone 20116, MAB004) (Sigma), was used at the same concentration as the primary antibody. Represen- tative pictures were recorded prior to destaining the slides of hematoxylin with 1% HCl in 70% ethanol. The hematoxylin destained samples were assessed in a blinded fashion using a Leica DC 300F microscope (Leica Microsystems GmbH, Germany) at a magnifica- tion of ×200. The positively stained area of the entire tissue section was determined using quantitative imaging Page 2 of 4 software (Leica QWin) with the same threshold used for all sections. The data was expressed as a percentage of total tissue area. GraphPad 5 (GraphPad Software, Inc. CA, USA) was used for the non-parametric statistical analysis of the per- centage of positive staining in tissues. A Mann Whitney test was used to compare the controls with the patient groups and to compare the changes (Δ change) between the 2 patient groups over the season. The Wilcoxon signed rank test was used to compare the changes over the season. A P value of < 0.05 was considered statisti- cally significant. The data are presented in a scatter plot with mean and SEM of the treatment group. Results Immunohistochemistry revealed clear expression of intra- cellular OPN in epithelial cells (Figure 1A) and vascular, as well as submucosal gland (Figure 1B) endothelial cells. In most cells, the most prominent staining was observed in the nuclei. The most intense staining was observed in epithelial and endothelial cells, but also in the subepithelial layer to a lesser extent. Previous studies on these nasal biopsies have shown a clear increase in the number of EG2+ cells over the pollen season and no increase with FP treatment. To determine if the expression of OPN is related to the change in eosino- phil numbers previously seen in the AR nasal biopsies, the staining was compared to the EG2+ cell counts previously obtained [2]. The cellular location of the EG2+ staining was distinctly different to that of the OPN staining (data not shown). Additionally, no correlation between the EG2+ cell counts (epithelium or subepithelium) and OPN staining was observed (Spearman R 0.049-0.274). The mean OPN expression (% area) was similar in nasal biopsies from healthy controls (4.1 ± 0.8%) com- pared to pre-season patient samples (5.9 ± 0.4%; p = 0.0706). During the grass pollen season, no significant change in OPN expression was seen in AR patients trea- ted with placebo or FP (Figure 2). Comparison of the difference in OPN expression induced by the grass pol- len season revealed no difference between the placebo and FP treated patient groups (p = 0.82). Discussion There is an increased scientific interest in the putative role of OPN in several diseases, including asthma, allergy and rhinosinusitis [7,10,14,15]. In the current study, we have observed clear expression of OPN in the nasal mucosa of both healthy individuals and patients with AR. Natural exposure to pollen did not, however, change the expression of OPN in patients treated with placebo. Lastly, local treatment with a potent nasal glucocorticoid, did not affect the OPN expression in patients with AR during natural exposure to pollen.