Allergenicity to worldwide invasive grass Cortaderia selloana as environmental risk to public health

Setting

This study was conducted in Cantabria, a region of the North coast of Spain.

Design and patients

A cross-sectional study with prospective data collection was performed at the Allergy Services of the Marqués de Valdecilla University Hospital in Santander and the Sierrallana Hospital in Torrelavega (Cantabria, Spain).

98 patients diagnosed of rhinoconjunctivitis, asthma or both, caused by sensitization to grass pollen, were included in a sequential way from October 2015 to March 2016.

Written informed consent was obtained from all patients before entering the study. The study met the principles of the 1975 Helsinki declaration and was reviewed and approved by the local Research Committee of Cantabria (CEIC reference number 2015.207).

A serum sample was obtained from each patient and stored at – 20 °C until used.

Pollen extract preparation

All methods were performed in accordance with the relevant guidelines and regulations.

Cortaderia selloana (CS) pollen was obtained commercially (Iber-Polen, Jaén, Spain) and then extracted at a 1:10 (w/v) ratio in PBS pH 6.5 with magnetic stirring for 90 min. at 5 °C. The soluble fraction was separated by centrifugation. After dialysis against PBS, the extract was filtered through 0, 22 µm filters. Protein content was determined by Bradford method (BioRad, Hercules, CA, USA). Two different batches were obtained (07 and 09) with consistent results.

Part of the extract was adjusted to 0.25 mg protein/ml and formulated in PBS with 50% glycerol, phenol 0.51% (SPT buffer). The remaining extract was stored in aliquots at − 20 °C.

Phleum pratense (Phl) pollen extract was made as described for CS. The origin of the pollen in this case was ALK Source Materials, Post Falls, Idaho, USA.

The protein profiles of the CS or the Phl extracts were determined by polyacrylamide electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) under reducing conditions (Invitrogen-Novex tricine gels 10–20% acrylamide, Fisher Scientific, SL, Madrid Spain).

Skin prick test

Patients were skin prick tested (SPT) with a commercial extract (ALK-Abelló, S.A. Madrid, Spain) of Phl and the CS extract. Histamine dihydrochloride solution (10 mg/ml) and SPT buffer were used as positive and negative control (no reaction), respectively.

The SPT wheal areas were measured by planimetry. A cut-off area of 7 mm² (about 3 mm average diameter) or higher was considered a positive test result (histamine).

The CS extract was tested in 10 control subjects, that were not sensitised to grass pollen, with negative result (no reaction).

IgE assays

Serum samples were tested for IgE antibodies against Phleum pratense (Phl) pollen extract and the allergens Phl p 1, Phl p 5, Phl p 7 (polcalcin) and Phl p 12 (profilin) (ImmunoCap FEIA, Thermo Fisher Scientific, Barcelona, Spain).

In addition, specific IgE against Phl and CS pollen extracts was determined by RAST (Radio Allergo Sorbent Test). Paper discs were activated with CNBr and sensitised with the pollen extracts as described by Ceska et al.²¹. Phl and CS discs were incubated overnight with 50 µL of the patient’s serum and after washing (0.1% Tween-20 in PBS), with approximately 100,000 cpm of the iodine 125–labeled anti-IgE mAb HE-2 for 3 h as described²². Finally, the discs were washed, and their radioactivity was determined in a gamma counter. sIgE values in kilounits per litre were determined by interpolating in a standard curve built up with Lolium perenne—sensitised discs and 4 dilutions of a serum pool from patients with grass allergy, which was previously calibrated in arbitrary kU/l.

A cut-off value of 0.35 kU/l was considered positive for both ImmunoCap and RAST. There was a very significant correlation between the sIgE against Phl determined by both methods (r Spearman = 0.8874, p < 0.0001).

RAST inhibition assay

Paper discs were sensitised as above in the IgE assays section and then incubated with 50 µL of a serum pool from all patients combined. 50 µL of (inhibitory) CS extract solution (in serial dilutions) were added onto the paper discs and incubated overnight at room temperature. All other incubations were performed as indicated above in the IgE assays section. The % of inhibition was determined for each extract dilution by radioactive counts (cpm) and calculated by means of the following equation:

Cpmx corresponds to the mean radioactivity of the discs incubated with inhibitor at a given X dilution. cpm100% corresponds to the blank control samples of the assay (no serum pool added). cpm0% corresponds to the signal obtained with no inhibitor extract added.

Source: Ecology - nature.com