Estudo para o desenvolvimento de métodos para quantificação de Phl p5 em amostras de ar exterior colhidas na cidade de Évora

Estudo para o desenvolvimento de métodos para quantificação de Phl p5 em amostras de ar exterior colhidas na cidade de Évora R Ribeiro1, CM Antunes1,4 & R Brandão2,3 1Departamento de Química, 2Instituto de Ciências Agrárias e Ambientais Mediterrânicas (ICAAM); 3Departamento Biologia, Universidade de Évora, Largo dos Colegiais 2, 7000 Évora UE; 4Centro de Neurociências e Biologia Celular (CNC), Universidade de Coimbra, 3004-517 Coimbra; [email protected] As doenças alérgico-respiratórias decorrentes da exposição a pólenes na atmosfera constituem na actualidade um problema de saúde pública, afectando cerca de 40% da população europeia. Em Portugal, os pólenes das gramíneas são uma das principais fontes de aeroalergénios atmosféricos, sendo o Phl p5 o principal alergénio da família Phleum pratense. e responsável por 80 a 90% das rinites e asma brônquica que atingem das crianças e adolescentes e 40 a 50% dos adultos já asmáticos. Assim, a realização de estudos para o desenvolvimento de metodologias que permitam prever o potencial alergénico da atmosfera e assim contribuir para desenvolver estratégias de prevenção da patologia alérgica são muito relevantes. Neste trabalho pretende-se desenvolver um teste imunoquímico para detecção e quantificação do alergénio Phl p5. Numa plataforma meteorológica situada no centro da cidade de Évora (Colégio Luís António Verney, Fase III), foram monitorizados em simultâneo e diariamente o conteúdo polínico atmosférico de Poaceae e o aeroalergénio Phl p5. Para a contagem dos primeiros, utilizou-se um colector volumétrico do tipo “Hirst” (Burkard Seven Day Recording Volumetric Spore Trap) e os segundos foram monitorizados mediante a utilização de um colector de impacto de médio volume (CHEMVOL) com filtros de poli-uretano, entre os dias 30 de Abril e 8 de Julho de 2009. Após extracção, a quantificação dos aeroalergénios efectuou-se mediante uma técnica de ELISA tipo “sandwich”, utilizando anticorpos específicos. Durante a época polínica de 2009 observaram-se níveis elevados de pólenes de gramíneas (entre 2 de Maio e 1 de Junho de 2009). Observou-se ainda que o conteúdo de alergénio Phl p5 estava directamente correlacionado com as contagens polínicas de pólenes de Poaceae. Estes resultados sugerem que a quantificação directa dos aeroalergénios pode contribuir, em conjunto com as contagens polínicas, para definir com melhor precisão do risco alérgico. Este estudo prosseguirá na época polínica de 2010 com vista à certificação do método. Agradecimentos: Este trabalho realiza-se no âmbito do projecto europeu HIALINE (“grant agreement” 20081107 II.5.1)

Estudo para o desenvolvimento de métodos para quantificação de Ole e1 em amostras de ar exterior colhidas na cidade de Évora

Estudo para o desenvolvimento de métodos para quantificação de Ole e1 em amostras de ar exterior colhidas na cidade de Évora. R Ferro1*, CM Antunes1,4 & R Brandão2,3 1Departamento de Química, 2Instituto de Ciências Agrárias e Ambientais Mediterrânicas (ICAAM); 3Departamento Biologia, Universidade de Évora, Largo dos Colegiais 2, 7000 Évora UE; 4Centro de Neurociências e Biologia Celular (CNC), Universidade de Coimbra, 3004-517 Coimbra; [email protected] As doenças alérgico-respiratórias decorrentes da exposição a pólenes na atmosfera representam um dos principais problemas clínicos da actualidade, afectando cerca de 40% da população europeia. Em Portugal, a polinização da oliveira é seguramente uma das principais fontes de aeroalergénios atmosféricos e consequentemente uma das maiores causas de alergia respiratória, sendo a Ole e1 a glicoproteína mais abundante do grão de pólen e o alergénio que mais significativamente contribui para o grau de alergenicidade deste pólen. Assim, o desenvolvimento de metodologias que permitam prever o potencial alergénico da atmosfera são da maior relevância já que poderão contribuir para desenvolver estratégias de prevenção da patologia alérgica. Neste trabalho pretende-se desenvolver e optimizar testes imunoquímicos para detecção e quantificação de Ole e1. Numa plataforma meteorológica situada no centro da cidade de Évora (Colégio Luís António Verney - Fase III), foram monitorizados em simultâneo e diariamente o conteúdo polínico atmosférico da Olea europaea e do aeroalergeno Ole e1. Para a contagem do primeiro, utilizou-se um colector volumétrico do tipo “Hirst” (Burkard Seven Day Recording Volumetric Spore Trap) e o segundo foi monitorizado mediante a utilização de um colector de impacto de médio volume (CHEMVOL) com filtros de poli-uretano, entre os dias 30 de Abril e 8 de Julho de 2009. Após extracção, a quantificação do aeroalergénio efectuou-se mediante uma técnica de ELISA tipo “sandwich”, utilizando um anticorpo específico. Durante a época polínica de 2009, observaram-se níveis elevados de pólen de oliveira entre 2 de Maio e 30 de Maio. Observou-se ainda que o conteúdo do alergénio Ole e1 estava directamente correlacionado com as contagens polínicas de Oleaceae. Estes resultados sugerem que a quantificação directa dos aeroalergénios pode contribuir, em conjunto com as contagens polínicas, para definir com melhor precisão o risco alérgico. Este estudo continuará na época polínica de 2010 com vista à certificação do método. Agradecimentos: Este trabalho realiza-se no âmbito do projecto europeu HIALINE (“grant agreement” 20081107 II.5.1)

Evidence from cross-border mergers and acquisitions

Funding agencies: European Research Council, Fundacao para a Ciencia e Tecnologia (grant nr.: PTDC/IIM-FIN/2977/2014) and Mayo Center for Asset ManagementWe test the hypothesis that foreign direct investment promotes corporate governance spillovers in the host country. Using firm-level data from 64 countries during the period 2005-2014, we find that cross-border M&A activity is associated with subsequent improvements in the governance of nontarget firms when the acquirer country has stronger investor protection than the target country. The effect is more pronounced when the target industry is more competitive. Cross-border M&As are also associated with increases in investment and valuation of nontarget firms. Alternative explanations, such as access to global financial markets and cultural similarities, do not appear to explain our findings.authorsversionpublishe

Comparison between Airborne Pollen and Aeroallergen Quantification with the ChemVol Impact Sampler. Olive pollen vs Ole e 1

Comparison between Airborne Pollen and Aeroallergen Quantification with the ChemVol Impact Sampler. Olive pollen vs Ole e 1. Torres M.C.1, C. Antunes2, M.J. Velasco1, R. Ferro2, H. García-Mozo1, R. Ribeiro2, R.Brandao3, Galán, C1 and the HIALINE team4 1Department of Botany, Ecology and Plant Physiology, University of Córdoba 2Department of Chemistry, University of Évora, Portugal 3Department of Biology, University of Évora, Portugal 4J.T.M. Buters, Germany, M. Thibaudon, France, M. Smith, Great Britain, C. Galan, Spain, R. Brandao and C. Antunes, Portugal, G. Reese, Germany, R. Albertini, Italy, L. Grewling, Poland, A. Rantio-Lehtimäki, Finland, S. Jäger and U. Berger, Austria, I. Sauliene, Lithuania, L. Cecchi, Italy Nowadays, pollinosis is affecting a large percentage of population in the countries with a western life style. The existence of allergenic activity in the atmosphere is not only associated to pollen grains and fungal spores, but also to submicronic and paucimicronic biological particles. The origin of these allergens can be due to the rupture of pollen transported in the atmosphere or to the presence of allergens from other parts of the plant making amorphous material with an allergen load. Olive pollen is recognized as one of the main causes of allergic disease in the Mediterranean area. In this study we have tried to compare olive pollen count in the air and Ole e 1 as major allergen of this species, at two different localities in South of Europe: Evora (Portugal) and Córdoba (Spain). At each location both samplers were placed side-by-side. Pollen grains have been sampled using a volumetric Hirst type spore trap. Chemvol high-volume cascade impactor equipped with stages PM>10µm, 10 µm>PM>2.5µm were used for detecting aeroallergens. Ole e 1 major allergen was determined using allergen specific ELISA´s. Similar behaviour between pollen and the total allergenic load was observed during the pollen season. Nevertheless, at some occasions, during the previous and later period of the pollen season, airborne allergenic load was detected in South Spain, due to the contributions from other Oleaceae species. For this reason the use of these two different methodologies allow a better understanding of the allergenic load in the atmosphere. This work was supported in part by the European Agency for Health and Consumers EAHC, Luxembourg, under the grant agreement 20081107

Comparison between Poaceae Airborne Pollen Counts and Phl p5 Aeroallergen Quantification in South Europe

The European project HIALINE: Comparison between Poaceae Airborne Pollen Counts and Phl p5 Aeroallergen Quantification in South Europe C. Antunes1,2, R. Ferro2, R. Ribeiro2, Torres M.C.4, M.J. Velasco4H. García-Mozo4, Galán, C4 , R.Brandao1,3, M.Thibaudon5, R. Albertini6 Ugolotti. M.6, Usberti I.6, Dall’Aglio P.6 and the HIALINE team7 1DInstitute of Mediterranean Agricultural and Environmental Sciences –ICAAM, University of Évora, Portugal 2Department of Chemistry, University of Évora, Portugal 3Department of Biology, University of Évora, Portugal 4Department of Botany, Ecology and Plant Physiology, University of Córdoba 5Réseau National de l Surveillance Aerobiologique, Saint-Genis-l’Argentière, France 6Department of Clinical Medicine, Nephrology and Health Sciences, University of Parma, Italy 7J.T.M. Buters, Germany, M. Thibaudon, France, M. Smith, Great Britain, C. Galan, Spain, R. Brandao and C.M. Antunes, Portugal, G. Reese, Germany, R. Albertini, Italy, L. Grewling, Poland, A. Rantio-Lehtimäki, Finland, S. Jäger and U. Berger, Austria, I. Sauliene, Lithuania, L. Cecchi, Italy Introduction: Nowadays, pollinosis is affecting a large percentage of population in countries with a western life style. The existence of allergenic activity in the atmosphere is not only associated to pollen grains and fungal spores, but also to submicronic and paucimicronic biological particles. The origin of these allergens can be due to the rupture of pollen transported in the atmosphere or to the presence of allergens from other parts of the plant making amorphous material with an allergen load. Poaceae pollen is recognized as one of the main causes of allergic disease in all Europe. In this study we have tried to compare Poaceae pollen counts in the air and Phl p 5, one of the major allergens of this family, through the use of a high-volume cascade impactor (Chemvol). This study was done in the frame of the European project HIALINE and it compares the results obtained in 2009 by 4 different partners participating in this project: in Córdoba (Spain), Évora (Portugal), Lyon (France) and Parma (Italy). Methodology: Pollen grains were sampled using a 7-day volumetric Hirst type spore trap. Chemvol high-volume cascade impactor equipped with stages PM>10µm, 10 µm>PM>2.5µm was used for detecting aeroallergens. In each stage polyurethane filters were use as an impacting substrate. Phl p 5 allergen was determined using an allergen specific ELISA. Antibodies for analysis were delivered by Allergopharma Joachim Ganzer KG, the industrial partner in this project. At each location both samplers were placed side-by-side. Results: Most of the allergen was collected in the PM>10µm fraction. Similar profiles between airborne pollen and the total allergenic load was observed during the pollen season. A good correlation was obtained between pollen count and allergen content of the air and a value of 2.5 pg/pollen grain of Poaceae was estimated. Discussion: This is the first year of this project. Nevertheless, results suggest that the allergenic load in outdoor air might be mainly due to pollen bursts. It supports the hypothesis that monitoring the allergens itself in ambient air might be an improvement in allergen exposure assessment. This work was supported in part by the European Agency for Health and Consumers EAHC, Luxembourg, under the grant agreement 2008110

The European project HIALINE: Comparison between Poaceae Airborne Pollen Counts and Phl p5 Aeroallergen Quantification in SW Europe

The European project HIALINE: Comparison between Poaceae Airborne Pollen Counts and Phl p5 Aeroallergen Quantification in SW Europe C. Antunes1*, R. Ferro1, R. Ribeiro1, Torres M.C.3, M.J. Velasco3H. García-Mozo3, Galán, C3 , R.Brandao2, M.Thibaudon4 and the HIALINE team5 1Department of Chemistry, University of Évora, Portugal 2Department of Biology, University of Évora, Portugal 3Department of Botany, Ecology and Plant Physiology, University of Córdoba 4Réseau National de l Surveillance Aerobiologique, Saint-Genis-l’Argentière, France 5J.Buters, Germanny, M. Thibaudon, France, M. Smith, Great Britain, C. Galan, Spain, R. Brandao and C.M. Antunes, Portugal, R. Albertini, Italy, L. Grewling, Poland, A. Rantio-Lehtimäki, Finland, S. Jäger and U. Berger, Austria, I. Sauliene, Lithuania, L. Cecchi, Italy * Presenting Author: Antunes, C. Department of Chemistry, University of Évora, Portugal. Phone +351 266745319 ; email: [email protected] Introduction: Nowadays, pollinosis is affecting in a large percentage of population in the developed countries. The existence of allergenic activity in the atmosphere is not only associated to pollen grains and fungal spores, but also to submicronic and paucimicronic biological particles. The origin of these allergens can be due to the rupture of pollen transported in the atmosphere or to the presence of allergens from other parts of the plant making amorphous material with allergenic load. Poaceae pollen is recognized as one of the main causes of allergic disease in all Europe. In this study we have tried to compare Poaceae pollen counts in the air and Phl p 5, one of the major allergens of this family, through the use of a high-volume cascade impactor (Chemvol). This study was done in the frame of the European project HIALINE and it compares the results obtained in 2009 by 3 different partners participating in this project: in Córdoba (Spain), Évora (Portugal) and Lyon (France). Methodology: Pollen grains have been taken out using a 7-day volumetric Hirst type spore trap. Chemvol high-volume cascade impactor equipped with stages PM>10µm, 10 µm>PM>2.5µm were used for detecting aeroallergens. In each stage polyurethane filters were applied. Phl p5 allergen was determined using allergen specific ELISA´s. Antibodies for analysis are delivered by Allergopharma Joachim Ganzer KG, the industrial partner in this project. Both samplers were placed side-by-side. Results: Most of the allergen was collected in the PM>10µm fraction. Similar profiles between airborne pollen and the total allergenic load was observed during the pollen season. A good correlation was obtained and a value of 2.5 pg/pollen grain of Poaceae was estimated. Discussion: These results suggest that the allergenic load in outdoor air might be mainly due to pollen bursts. It supports the hypothesis that monitoring the allergens itself in ambient air might be an improvement in allergen exposure assessment. This work was supported in part by the European Agency for Health and Consumers EAHC, Luxembourg, under the grant agreement 2008110

Differential Ole e 1 Release from Olea Airborne Pollen in the Southwest Iberian Peninsula. Results from the HIALINE Study

Background: Ole e 1 is the major allergen of olive pollen (Olea europaea L.), the second largest cause of pollinosis in some areas from the Mediterranean Region. Although it has been assumed that airborne pollen is a representative parameter for allergen exposure, variability of allergen content and/or release from pollen has been demonstrated for other taxa. The aim of this study was to: i) estimate the correlation between daily airborne olive pollen and Ole e 1 in ambient air; ii) evaluate the annual and geographical variation of pollen and allergenic loads in southwest Iberian Peninsula; iii) evaluate the contribution of meteorological parameters to ambient Ole e 1 loads variations. Methods: Airborne Ole e 1 and olive pollen were assessed simultaneously in Cordoba, Spain and Evora, Portugal. Aeroallergens were collected in 2009-2011 using prewashed polyurethane foam as impacting substrate (Rupprecht & Patashnick ChemVol®2400 high-volume cascade impactor, Albany, NY, USA). Flow was adjusted to 800 L/min with a rotameter controlled high-volume pump (Digitel DHM-60, Ludesch, Austria). After extraction, Ole e 1 was quantified by ELISA. Airborne Olea pollen was monitored with a Burkard Hirst type Seven-Day Recording Volumetric SporeTrap®. Both samplers were placed side-by-side with the air input at the same level. Results: The aeroallergen and airborne pollen profiles overlapped during pollen seasons, however, deviations between pollen counts and allergen load were found. Annual pollen index of Olea was 3-4 folds higher in Spain (29,956, 26,274 and 42,223 in Spain versus 12,524, 7,144 and 10,499 in Portugal). A 4-9 fold difference in aeroallergen load was observed (14,375, 18,913 and 20,989 in Portugal and 108,720, 80,972 and 171,248 in Spain). Annual Ole e 1/pollen was 3.1-4.0 in Spain, 0.8-2.6 in Portugal and was positively correlated with precipitation prior to pollen season. Conclusions: These results have shown that Ole e 1 is mostly associated with olive pollen grains but aeroallergen load was not always directly proportional to airborne pollen counts. This suggests that Ole e 1 quantification is a better marker for olive allergen exposure. In conclusion, aeroallergen monitoring may contribute to a better understanding of the Ole e 1 exposure from airborne pollen. Acknowledgments: This study is integrated in the European project HIALINE (Executive Agency for Health and Consumers, grant agreement No 2008 11 07). 1st&2nd author equally contributed to the work

Release of Bet v 1 from birch pollen from 5 European countries. Results from the HIALINE study

Exposure to allergens is pivotal in determining sensitization and allergic symptoms in individuals. Pollen grain counts in ambient air have traditionally been assessed to estimate airborne allergen exposure. However, the exact allergen content of ambient air is unknown. We therefore monitored atmospheric concentrations of birch pollen grains and the matched major birch pollen allergen Bet v 1 simultaneously across Europe within the EU-funded project HIALINE (Health Impacts of Airborne Allergen Information Network). Pollen count was assessed with Hirst type pollen traps at 10 l min 1 at sites in France, United Kingdom, Germany, Italy and Finland. Allergen concentrations in ambient air were sampled at 800 l min 1 with a Chemvol high-volume cascade impactor equipped with stages PM > 10 mm, 10 mm > PM > 2.5 mm, and in Germany also 2.5 mm > PM > 0.12 mm. The major birch pollen allergen Bet v 1 was determined with an allergen specific ELISA. Bet v 1 isoform patterns were analyzed by 2D-SDS-PAGE blots and mass spectrometric identification. Basophil activation was tested in an Fc 3R1-humanized rat basophil cell line passively sensitized with serum of a birch pollen symptomatic patient. Compared to 10 previous years, 2009 was a representative birch pollen season for all stations. About 90% of the allergen was found in the PM > 10 mm fraction at all stations. Bet v 1 isoforms pattern did not vary substantially neither during ripening of pollen nor between different geographical locations. The average European allergen release from birch pollen was 3.2 pg Bet v 1/pollen and did not vary much between the European countries. However, in all countries a >10-fold difference in daily allergen release per pollen was measured which could be explained by long-range transport of pollen with a deviating allergen release. Basophil activation by ambient air extracts correlated better with airborne allergen than with pollen concentration. Although Bet v 1 is a mixture of different isoforms, its fingerprint is constant across Europe. Bet v 1 was also exclusively linked to pollen. Pollen from different days varied >10-fold in allergen release. Thus exposure to allergen is inaccurately monitored by only monitoring birch pollen grains. Indeed, a humanized basophil activation test correlated much better with allergen concentrations in ambient air than with pollen count. Monitoring the allergens themselves together with pollen in ambient air might be an improvement in allergen exposure assessmen

THE HIALINE PROJECT: ALLERGEN RELEASE FROM POLLEN ACROSS 10 EUROPEAN COUNTRIES

Exposure to allergens is one of severa1 factors determining sensitization and allergic symptoms in individuals. Exposure to aeroallergens from pollen is assessed by counting allergenic pollen in ambient air. However, proof is lacking that pollen count is representative for allergen exposure. We therefore monitored simultaneously birch, grass and olive pollen counts and their corresponding major pollen allergens Bet v 1, Phl p 5 and Ole e 1 across Europe. Already at one location in Europe in Munich, Germany, it has been found that the same amount of pollen from different years, different trees and even different days released up to lO-fold different amounts of Bet v 1. Thus exposure to allergen is poorly monitored by only monitoring pollen countl-2. Monitoring the allergen itself in ambient air might be an improvement in allergen exposure assessment. The objective of the HIALINE-project is to evaluate if these effects found in Munich, Germany are also measurable over a bigger geographic area like Europe, and at the same time implement an outdoor allergen early warning network, in addition to the pollen forecasts. Climatic factors that influence allergen exposure will be extracted and will be used to calculate the effect of climate change on local airborne allergen exposure. The major allergens from the top 3 airborne allergens in Europe (grasses, birch and olive) are sampled with a cascade impactor, extracted and analyzed by allergen specific ELISA 's. Pollen counts are measured by standard pollen traps and correlated with the weather data. Allergen forecast will be calculated by incorporating the SILAM chemical transport model and compared with the observations of HIALINE aiming at a comprehensive parameterization of the allergen release and transport. Expected outcomes are the implementation of a network of European outdoor allergen measurements to better predict allergic symptoms. Also the climatic factors that govern allergen exposure in outdoor air will be established. These can be used to calculate the effect of climate change on the health effects of airborne allergens The research leading to these results has received funding from the Executive Agency for Health and Consumers under grant agreement No 2008 11 07

Detecção e Quantificação de Aeroalergenos Polínicos de Gramíneas e Olea spp. em Amostras de Ar Exterior e sua Correlação com as Contagens Polínicas

Detecção e Quantificação de Aeroalergenos Polínicos de Gramíneas e Olea spp. em Amostras de Ar Exterior e sua Correlação com as Contagens Polínicas C.M Antunes1,5 R Ferro1*, R Ribeiro1*, E Caeiro4, L. Lopes8, C. Nunes6 R Brandão2,3 & M. Morais-Almeida7 e o HIALINE working group9 1Dep. De Química, Universidade de Évora, Portugal; 2Mediterranean Inst. Crop and Environment Sciences, Univ.Evora, Portugal; 3Depto. Biologia, Universidade de Évora, Portugal; 4Soc.Portuguesa Alergol.Imunologia Clínica , Portugal 5Center for NeuroSciences and Cell Biology-University of Coimbra, Portugal; 6Centro de Imunoalergologia do Algarve, Portugal 7Hospital CUF Descobertas 8Hospital Stª Luzia, Elvas, Portugal 9 M. Thibaudon, France, M. Smith, United Kingdom, C. Galan, Spain R. Albertini, Italy, L. Grewling, Poland, G. Reese, Germany, A. Rantio-Lehtimäki, Finland, S. Jäger and U. Berger, Austria, M. Sofiev, Finland, I. Sauliene, Lithuania, L. Cecchi, Italy Introducão: As Doenças Alérgicas Respiratórias desencadeadas após uma exposição ao pólen atmosférico, como a asma e a rinite alérgica, representam actualmente um grave problema de saúde pública. Em Portugal, o pólen de Poaceae (gramíneas) e de Olea europaea constitui uma das principais fontes de aeroalergenos polínicos atmosféricos e, como tal, uma das principais causas da alergia respiratória. Por essa razão, é importante o desenvolvimento de novas estratégias para a respectiva prevenção e tratamento. O objectivo deste estudo, desenvolvido no âmbito do projecto europeu Hialine, consistiu em avaliar a correlação entre o doseamento, por imunoensaios, de 2 importantes aeroalergenos – Phl p5 e Ole e1 – e as contagens polínicas do pólen de gramíneas e Olea europaea.. Metodologia: Numa plataforma meteorologica situada no centro da cidade de Évora (sul de Portugal), foram recolhidas amostras do ar ambiente a 800L/min com um colector Chemvol high-volume cascade impactor, equipado com filtros para os níveis PM>10µm e 10 µm>PM>2.5µm. As amostras dos filtros em polyurethane foram extraídas com 0.1M NH4HCO3, pH 8.1, suplementado com 0.1% BSA. Os aeroalergenos Phleum p 5 e Ole e 1 foram determinados por ELISA´s específicas. O polen atmosférico de Poaceae e Olea europaea foi monitorizado em simultâneo com um Burkard Seven Day Recording Volumetric Spore Trap* , entre 30 de Abril e 8 de Julho de 2009. Ambos os colectores estiveram colocados lado a lado, com o nível de entrada de ar à mesma altura. Resultados: Durante a estação polínica de 2009, foram registados elevados valores de gramíneas entre os dias 2 de Maio e 1 de Junho. Foi observado também que o conteúdo das amostras de ar dos aeroalergenos Phl p5 ou Ole e1 estavam directamente correlacionados com as contagens polínicas de Poaceae e Oleaceae, respectivamente. Conclusões: Estes resultados sugerem que a quantificação directa de aeroalergenos pode contribuir, juntamente com as contagens polínicas atmosféricas, para definir o risco alergénico com maior precisão. Agradecimentos: Este estudo está integrado no projecto europeu HIALINE (Executive Agency for Health and Consumers under grant agreement No 2008 11 07