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has been shown. However, to the best of our knowledge, this is the first report of a multiple IgE-mediated sensitization to different woods that caused occupational respiratory symptoms in the same worker.
A 40-year-old man who worked in carpentry for 12 years was referred for developing symptoms of nasal pruritus, blockage, sneezing, and runny nose while cutting and sanding wood. Symptoms started 10 years ago and were mild. The symptoms worsened in the last 2 years, with the patient developing episodes of dry cough, chest tightness, intense dyspnea, and wheezing 20 to 30 minutes after starting his working shift and needing treatment at the emergency department because of severe bronchospasm. The patient worked in a wood factory where, despite the ventilation, substantial amounts of wood dust were visible in the air. He related his symptoms with exposure to obeche (Triplochiton scleroxylon, also known as African maple or samba wood), iroko (Milicia excelsa), cerejeira (Amburana cearensis), oak (Quercus sp), and pine (Pinus sp) wood that were used regularly in his workplace. The physical examination of the patient was normal, and so were the hemogram and basic blood biochemistry. Total IgE was 275 IU/mL, and chest x-ray showed no abnormalities. Spirometry showed FEV1 87%, FEV1/FVC 85%, and mild obstruction of midlung volume flow (forced expiratory flow at 25%-75% of forced vital capacity 62%) with the negative bronchodilator test (<12% change in FEV1). Skin prick tests to a battery of common aeroallergens showed positive results to grass pollen and dust mites. To further investigate a possible sensitization to wood, in-house extracts were made with the 5 aforementioned woods provided by the patient as described
(see this article's Online Repository at www.jacionline.org). Skin prick tests with the extracts (1 mg/mL) were positive to obeche (8 mm), iroko (10 mm), cerejeira (3.5 mm), and pine (10 mm) and negative to oak. Specific IgE to these woods was measured by RAST as described,
showing positive results to obeche (38%) and pine tree (6.9%), a weak positive response to iroko (1.5%, positive cutoff point 0.5%), and negative results to cerejeira and oak. The same results were obtained by performing the ELISA test.
Skin prick testing, RAST, and ELISA assays were performed in 10 controls all of whom had negative responses. Written informed consent was obtained from all participants, and the ethical committee of our institution approved the study. Methacholine challenge was performed at baseline, yielding a PC20 value of 0.96 mg/mL (normal value > 16.0 mg/mL), and induced sputum before and after challenge. Specific inhalation challenges were performed by using the same extracts (1 mg/mL) in aqueous form with serial dilutions and using a DeVilbiss device (DeVilbiss, Somerset, Pa). Challenges were performed on different days for each wood and separated 4 weeks after a control day. Bronchial challenges were positive (≥20% fall in FEV1) to obeche (1:1000), iroko (1:100), cerejeira (1:10), and pine (1:100) extracts and negative to oak. All responses were immediate (30-45 minutes) except for obeche wood (90 minutes), which caused a significant proportional increase in sputum eosinophils (4.5% vs 16.9%) and a decrease in neutrophils (89.6% vs 59.5%) after challenge. Since a multiple sensitization was demonstrated, the possible cross-reactivity between woods was further analyzed by ELISA cross-inhibition assays (see this article's Online Repository at www.jacionline.org). As shown in Fig 1, A, pine showed a strong inhibition against obeche (85%), iroko showed a weak inhibition (below 50%), and cerejeira or oak extracts showed no inhibition. A similar pattern was observed when pine was used as solid phase (Fig 1, B), where obeche inhibits pine by almost 70%. Finally, when iroko was used as solid phase, we observed that once again pine acts as a potent inhibitor (90% inhibition), followed by a more modest although significant inhibition by obeche (64%) and cerejeira (62%). SDS-PAGE and Western blot assays with obeche and pine extracts (see Fig E1 in this article's Online Repository at www.jacionline.org) showed bands of approximately 14, 28, 38, and 58 kDa in the obeche extract but no apparent bands in the pine extract. As described in other woods such as red cedar or iroko,
a low-molecular-weight substance (abietic acid) could be the causative agent in asthma induced by pine. Our pine extract was then tested by using gas chromatography combined with mass spectrometry (see this article's Online Repository at www.jacionline.org), detecting abietic acid (23%) and dehydroabietic acid (51%) as principal low-molecular-weight compounds (Fig 2). An ELISA inhibition immunoassay did not show any significant inhibition of patient's IgE binding by using free abietic acid as a liquid phase inhibitor (up to 1000 μg/mL) and the pine extract as solid phase (30 μg/mL) (data not shown).
We report a case of a multiple sensitization to different woods demonstrated by in vivo and in vitro tests. Cross-reactivity among different woods was assessed by using ELISA cross-inhibition assays. Surprisingly, pine extract was a strong inhibitor of 2 tropical woods such as obeche (genus Sterculia, family Sterculiaceae) and iroko (genus Milicia, family Moreaceae), which are unrelated to pine (genus Pinus, family Pineaceae). Information about wood allergens is scarce with few exceptions,
and so the nature of the allergen(s) responsible for the multiple sensitizations in this patient is still unknown; although the presence of low-molecular-weight compounds such as abietic acid and dehydroabietic acid was demonstrated in the extract by using gas chromatography combined with mass spectrometry, no IgE-binding activity was detected. Further studies are necessary to characterize wood antigens and develop standardized extracts to improve the diagnosis of occupational asthma caused by wood dust.
Preparation of extracts
Wood dust was extracted with PBS buffer (0.1 mol/L sodium phosphate, pH 7.0, and 0.15 mol/L NaCl; 1× 1:5 [w/v], 1 hour, 4°C) and centrifuged (10,000×g, 30 minutes, 4°C). The supernatant was dialyzed (cutoff point, 3.5 kDa) against H2O and freeze-dried. The protein concentration was quantified according to the method of Bradford (Pierce Biotechnology, Inc, Rockford, Ill).
Elisa inhibition and cross-inhibition immunoassays
ELISA cross-inhibition studies were performed according to established methods.
The 96-well polystyrene plates (Costar) were coated with 30 μg/mL of each wood extract to which the patient showed positive specific IgE (obeche, pine, and iroko). Patient serum was preincubated with obeche, oak, pine, cerejeira, and iroko at final concentrations of 100, 10, 1, and 0.1 μg/mL at room temperature for 3 hours. Subsequently, the inhibitor mixtures (including serum with no inhibitor as positive control) were added to the obeche-, pine-, and iroko-coated plates and incubated at 37 °C for 1 hour. The assay was completed by incubating with rabbit anti-human IgE antibody (1:3000; DAKO A/S, Glostrup, Denmark). IgE binding was detected by using o-phenylenediamine tablets (OPD, DAKO), and the reaction was stopped by adding 2 mol/L of chloric acid. The absorbance (optical density [OD]) in each well was measured at 490 nm. For inhibition ELISA with pine and abietic acid, the plate was coated with 30 μg/mL of pine extract and patient serum was incubated with pine and abietic acid at final concentrations of 1000, 100, 10, 1, and 0.1 μg/mL.
The percentage of inhibition of IgE binding was calculated by using the following formula:
Western-blot assay with obeche and pine extracts
Obeche and pine wood dust extracts (30 μg) were separated on SDS-PAGE.
Proteins were electrotransferred to polyvinylidene difluoride membranes. Blocked membranes (Blocking solution, Sigma, St Louis, Mo) were incubated with patient serum (1:10 dilution). The IgE binding was revealed with anti-human IgE-peroxidase conjugate (Biosource, Camarillo, Calif; 1:3000 dilution) and chemiluminiscence (Amersham Supersignal West Pico Chemiluminutoescent Substrate, Rockford, Ill).
Gas chromatography–mass spectrometry analysis
Western Red cedar (Thuja plicata) and other wood dusts.
in: Bernstein I.L. Chan-Yeung M. Bernstein D. Asthma in the workplace. 3rd ed. Taylor & Francis Group Publishers,
New York (NY)2006: 505-524
This study was supported by Ministry of Health grant FIS/PI071175 , Andalusian Health Division grant PI-0182/2008 , and a grant from the Spanish Society of Allergy (SEAIC). The Ministry of Science and Innovation-DGI , Spain (grant BIO2009-07050 ), and the Thematic Network FIS-RIRAAF (grant no. RD07/0064 ) also supported the study.
Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.