Estudo do perfil em alergénios do pólen de Platanus hybrida e da sua repercussão na doença alérgica respiratória sazonal na população de Évora

O plátano (Platanus hybrida), uma árvore ornamental e de sombra, produz pólen em grande quantidade, sendo responsável pela exposição elevada a pólen no início da primavera. Desconhece-se, porém, o seu perfil alergénico. Este trabalho teve como objetivo caracterizar o perfil em alergénios do pólen de plátano em Évora, Alentejo. Obtiveram-se extratos de pólen que foram liofilizados e conservados a -80ºC. O perfil em alergénios foi avaliado por western blot utilizando soros humanos (obtidos mediante consentimento informado de doentes do Hospital do Espírito Santo de Évora – HESE). Identificaram-se diversas bandas imunorreativas de pólen de P. hybrida, sugerindo a presença de alergénios. Evidenciou-se também a existência de reatividade cruzada com pólen de gramíneas. Estes resultados sugerem que o pólen de plátano, dada a sua grande abundância na cidade de Évora, poderá contribuir para o agravamento a sintomatologia da população que sofre de polinose, em particular no início da primavera; ABSTRACT: Study on the pollen allergen profile from Platanus hybrida and its contribution to pollinosis in the population of Évora Plane trees, hence pollen, are highly abundant in the city of Évora, nonetheless allergen profile has not yet been evaluated. The aim of this work was to characterize the allergen profile of pollen from Platanus hybrida, one of the most representative species in Évora showing pollination prior to the main pollen season in Alentejo. Pollen extracts were lyophilized and stored at -80ºC until analysis. Protein content was determined by the Bradford method. SDS-PAGE followed by western blot, using allergic patient sera (obtained from the Hospital do Espírito Santo de Évora – HESE), were performed. Western blot have shown several immunoreactive bands. Cross-reactivity of P. hybrida with Q. rotundifolia and D. glomerata was found. These results evidenced allergens found in P. hybrida pollen. Moreover, cross–reactivity between P. hybrida and highly allergenic species such as D. glomerata was found which probably contributes for the aggravation of pollinosis in the early spring

Cupressaceae Pollen in the City of Évora, South of Portugal: Disruption of the Pollen during Air Transport Facilitates Allergen Exposure

Research Highlights: Daily airborne Cupressaceae pollen disruption ranged from 20 to 90%; relative humidity (RH), rainfall and atmospheric pressure (AtP) were the major meteorological determinants of this phenomenon. Background and Objectives: Cupressaceae family includes several species that are widely used as ornamental plants pollinating in late winter-early spring and might be responsible for allergic outbreaks. Cupressaceae pollen disruption may favour allergen dissemination, potentiating its allergenicity. The aim of this work was to characterize the Cupressaceae pollen aerobiology in Évora, South of Portugal, in 2017 and 2018, particularly the pollen disruption, and to identify the meteorological parameters contributing to this phenomenon. Materials and Methods: Pollen was collected using a Hirst type 7-day pollen trap and was identified following the standard methodology. Temperature, RH, rainfall, global solar radiation (Global Srad), AtP, wind speed and direction were obtained from a weather station installed side-by-side to the Hirst platform. Back trajectories (12-h) of air masses arriving at Évora were calculated using the HYSPLIT model. Results: Cupressaceae pollen index was higher in 2017 compared to 2018 (>5994 and 3175 pollen/m3, respectively) and 36 ± 19% (2017) and 64 ± 17% (2018) of the pollen was disrupted. Higher levels of disrupted pollen coincided with RH > 60% and rainfall. Temperature, Global Srad and AtP correlated negatively with pollen disruption. Wind speed and wind direction did not significantly correlate with pollen disruption. Intra-diurnal pollen pattern peaked between 9:00 am–2:00 pm, suggesting local origin, confirmed by the back trajectory analysis. Intra-diurnal pollen disruption profile followed hourly pollen pattern and it negatively correlated with AtP, temperature and Global Srad but was uncorrelated with RH. Conclusions: The results suggest that RH, rainfall and AtP are the main factors affecting airborne Cupressaceae pollen integrity and in conjunction with daily pollen concentration may be used to predict the risk of allergy outbreaks to this pollen type

Q. rotundifolia and P. hybrida pollen extracts induced basophil degranulation: study using a cell line expressing human FcERI.

Nowadays skin prick tests remain the favourite methodology in allergy diagnosis to aeroallergens. These tests, however, cause discomfort to the patient. Several biochemical methods are available but have limited diagnostic power as biological response, hence allergic reaction, is not predicted by these tests. A biological assay allowing the evaluation of allergic response to allergens is lacking. The aim of this work was to investigate the value of a rat basophil cell line expressing human high affinity IgE receptor (FcERI) for the evaluation of specific response to allergens. FACS analysis was used to determine FcERI expression (APC-A). Pollen extracts from different species were prepared with ammonium bicarbonate buffer, lyophilized and stored at -80ºC until use. Batches of RBL-h21 cells were sensitized with selected pools of human sera (purified IgE was used as control) and were stimulated with pollen extracts or Anti-IgE antibody. Histamine release (% degranulation) was determined a ß-hesoaminidase (tracer) assay. Batches of RBL-h21 cells sensitized (human sera) to grasses (D. glomerata), olive, oak or plane exhibited selective and dose-dependent degranulation upon selective stimulation with pollen extracts in the range of 1-200μg/mL. Maximal degranulation (>25%) was observed for 50, 120 and 62μg/mL for grass, oak and plane, respectively, while the lowest concentration with observed effect was 12μg/mL for any of the extracts. The results show that RBL-h21 cells sensitized with human sera exhibit specific and dose-dependent degranulation upon stimulation with pollen extract containing allergens suggesting this bioassay may constitute a useful tool to evaluate allergic responses both for research and/or diagnostics

Differential Quercus spp pollen-particulate matter interaction is dependent on geographical areas

Particulate matter (PM) and pollen interaction, either airborne or at the respiratory mucosa needs further clarification, as allergic reaction intensification can be related to the PM physical characteristics and toxicity. This study aimed to investigate the physical-chemical properties of PM that can adhere to the pollen wall during its transport or inhalation, using Quercus spp. as a model, in three Portuguese cities with different geographical locations, meteorological influence and urbanization levels. Possible sources were evaluated through air masses trajectory analysis using the HYSPLIT model and correlation with meteorological factors. The sampling was performed using a 7-days Hirst-type volumetric sampler, and the pollen grains were observed using a Field Emission Electron Probe Microanalyser for PM analysis. A secondary electron image of each pollen grain was taken, to determine the adhered particles characteristics and energy dispersive x-ray spectroscopy (EDS) spectra were obtained for individual particles. A total of 484 pollen grains was observed, with 7683 particles counted and 1914 EDS spectra analyzed. The particle's equivalent diameter ranged from 0.3-16 μm, with most having a diameter < 3 μm. For the three cities, there were significant differences in the number of particles per pollen and the % area occupied by the particles. Particles adhered were mainly Si-rich, but variations in other dominant groups were observed. For Évora and Guarda, Ca-rich, SO-rich were second and third more representative, while Porto were Organic and Cl-rich. Metals&Oxides were found in all cities with the highest number in Porto. P-rich particles were only found in Évora. Sea salt particles were observed in Évora, coincide with air mass trajectories possible carrying them from the Mediterranean Sea. In conclusion, the PM physical characteristics are similar between the studied cities, however, the dominant chemical composition is different, certainly impacting the exposome influence and pollen-allergy intensification towards the same pollen type and concentration

Air pollutants NO2- and O3-induced Dactylis glomerata L. pollen oxidative defences and enhanced its allergenic potential.

Air pollutants impact airborne pollen biochemistry. Oxidative damage to lipids, proteins and nucleic acids or protein nitration are among ozone (O3) and nitrogen dioxide (NO2) described deleterious effects possibly causing pollen physiology damage and enhanced allergenic activity, contributing to aggravate pollen driven respiratory allergy in urban areas. The goal of this research was to evaluate the effects of O3, NO2, alone and combined, on Dactylis glomerata pollen reactive oxygen species scavenging enzymes, on pollen germination and their potential contribution to the allergenicity. D. glomerata pollen was in vitro exposed to pollutants. Protein extracts were prepared and superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) activities were evaluated. Western blot with pooled sera or with IgG against group 5 allergens and profilin was performed. Pollen germination capacity was increased by NO2 and was unaffected by O3 or O3 + NO2 but showed longer pollen tubes in the latter. Exposure to O3 did not affect SOD activity but induced a twofold increase in catalase activity. SOD activity was twofold higher in pollen exposed to NO2. Exposure to O3 + NO2 induced a twofold and fivefold increase of SOD and catalase activities, respectively. Pollen GPx was unaffected by the pollutants. IgE-recognition of proteins in the molecular weight range of 42–57 kDa were amplified by NO2 and O3 + NO2 and O3 amplified proteins with molecular weight of 13 (profilin), 29 (Group 5), and 31 kDa. Taken together, these results show that pollen oxidative defences are activated by common air pollutants affecting both its germination capacity and its allergenic activity

XXI SPB CONGRESS BOOK

The University of Évora welcomes YOU at the XXI SPB National Congress of Biochemistry 2020 in 14-16 October 2021 either in person or online! Under challenging conditions, due to the COVID-19 pandemic, we have managed to organize the National Congress of Biochemistry in a hybrid format, where at least 2/3 of the participants will come to Évora in person. With the pandemic under control and we hope to carry out the Congress both successfully as well as safely. This is the main meeting point for Portuguese Biochemistry Academy, fostering the discussion and dissemination of high-quality research in Biochemistry, both fundamental and applied, taking place in Portugal. The Scientific Program covers a wide range of issues, from Health and Disease to Environment and Drugs development, where Biochemistry is either fundamental or instrumental in the study of complex and transdisciplinary problems in the society. The Congress is a moment of Science and Innovation in several Biochemistry domains, sharing experiences and fostering healthy confraternization. Despite the difficult context, over 160 confirmed registrations and >120 abstracts were submitted, involving the whole Portuguese Biochemical community. Moreover, this year for the first time the Congress has gone eco-friendly, with ePosters only where short poster presentations are encouraged. We hope the congress meets your expectations! The Organizing Committee, on behalf of the Portuguese Biochemical Society, looks forward meeting you, at Colégio do Espírito Santo, University of Évora!Sociedade Portuguesa de Bioquímica - SPB; Biochem; Instituto de Ciências da Terra - ICT; Fundação Eugénio de Almeida; Câmara Municipal de Évora; Universidade de Évora; Labor Spirit; Terrius; Nitrifresco; Evora Hotel

Efeito de poluentes atmosféricos e caracterização da matéria particulada adsorvida a grãos de polén aerossolizados

O pólen é um bioaerossol atmosférico capaz de induzir reação alérgica respiratória em indivíduos suscetíveis. O objetivo deste trabalho foi estudar a disseminação, distribuição, transporte e alterações bioquímicas de alguns tipos polínicos alergizantes resultantes da interação com os parâmetros meteorológicos e com os poluentes atmosféricos. Observou-se que os parâmetros meteorológicos influenciam a emissão, distribuição e transporte do pólen, afetando a época polínica e a integridade do pólen. Observou-se ainda que a composição da matéria particulada adsorvida ao pólen é variável entre regiões de acordo com as características topográficas e bioclimáticas. Confirmou-se que os poluentes atmosféricos provocam alterações químicas e bioquímicas (imunorreconhecimento e/ou conteúdo em alergénios) no pólen, com significativas implicações alergológicas. Estes resultados contribuem para o conhecimento dos fatores que afetam a exposição a alergénios polínicos e, consequentemente, constituem uma ferramenta para o desenvolvimento futuro de sistemas de alerta que contribuam para melhorar o controlo da doença alérgica respiratória; Abstract: Effect of air pollutants and characteristic of particulate matter adsorbed to pollen aerosols Pollen is an atmospheric bioaerosol capable of inducing respiratory allergic reaction in susceptible individuals. The objective of this work was to study the dissemination, distribution, transport, and biochemical changes of some allergenic pollen types, related to the interaction with meteorological parameters and air pollutants. It was observed that meteorological parameters influence emission, distribution, and transport, thus affecting the pollen season and integrity. It was also observed that the distribution of pollen-adsorbed particulate matter composition varied between regions according to topography and bioclimate characteristics. It was confirmed that atmospheric pollutants evoked pollen chemical and biochemical changes (allergen immune-recognition patterns and/or content), with significant allergological implications. This work highly contributes to the knowledge of the factors affecting human exposure to pollen allergens and, consequently, constitute a tool for the future development of effective alert systems contributing to improve respiratory allergic disease control

Atmospheric pollutants NO2 and O3 enhance allergenic potential of Dactylis glomerata pollen.

Background : Airborne pollen during its transport become in contact with a variety of atmospheric chemicals, including the common air pollutants O 3 and NO 2 . Nevertheless, the effect of these pollutants, alone or in combination, is not fully elucidated. With a strong oxidiz-ing potential, ozone may affect pollen redox balance and contribute to change the ratio of ROS scavenging enzymes and in the presence of NO 2 , may also contribute to the nitration of proteins, including allergens, affecting pollen allergenicity. Traffic- related pollution may as such contribute to an increased prevalence of pollen driven aller-gic diseases in urban areas. The goal of this research was to study the effects of O 3 , NO 2 and the mixture of both pollutants on IgE- reactivity patterns to Dactylis glomerata pollen, as well as on the levels of Dac g 5 and profilin by immunoblot. Method : D. glomerata pollen was treated in an environmental cham-ber during 6 hours. Protein extracts from unexposed (Control) or exposed to air pollutants (O 3 , NO 2 or O 3 +NO 2 ) pollen samples were prepared in phosphate buffered saline (PBS) and frozen until analy-sis. SDS- page separated proteins were blotted and used to obtain IgE- reactivity patterns with pooled sera (from four D. glomerata EAST- positive sera) and with antibodies against grasses group 5 al-lergens and profilin. Results : IgE- reactivity have shown bands with MW of 15, 29, 33, 37, 46, 52, 57, 60, 69 and 142 kDa (intensity >4%). Bands with 15, 57 and 60 kDa were intensified by NO 2 exposure compared to control. Pollen exposure to O 3 induced an amplification of IgE- reactivity in 40% of the detected bands (29, 33, 60, 66, 69, 74 and 80 kDa). Pollen exposed to O 3 +NO 2 showed augmented IgE- reactivity in bands with 14, 15, 18, 57, 66 and 80 kDa. Some of the bands identified to be affected by the pollen exposure to air pollutants of MW 29, 33 and 60 kDa, have correspondence to known allergens, respectively, Dac g 5, Dac g 1, Dac g 4. Additionally, immunoblot performed with IgG anti- Dac g 5 and anti- profilin confirmed overexpressed bands with, respectively, ~30 kDa and ~13 kDa, in O 3 treated pollen. Conclusion : D. glomerata pollen exposure to O 3 and NO 2 showed enhanced IgE recognition of several proteins, some of which are de-scribed allergens. These results suggest that common air pollutants contribute to an increased pollen allergenicity and directly correlat-ing with the higher incidence of respiratory allergic diseases in urban areas

Allergy medications sales: An usefulsurrogate marker for pollinosis health impacts in Alentejo

ntroduction:The burden of pollen allergies has been increasingworldwide. Due to their environmental-related aetiology, manage-ment of the disease in a changing environment has proven challeng-ing. Several strategies have been used aiming the monitoring of thedisease health impacts with limited outcomes.Objectives:The aim of this work was to investigate the associationbetween the sale of antiallergic drugs with air allergenic loads andwhether it constitutes a marker for health impacts of pollinosis inAlentejo, Portugal.Results:Olive and Poaceae pollen and their major allergens Ole e 1and Phl p 5 were obtained within the HIALINE project (2010-2011).The pollen was monitored using a 7-day Hirst type sampler and theindexes were determined by the standard methodology. Allergenload was monitored using a high-volume cascade impactor andspecific allergens were quantified by specific ELISA. The medicationsales data (2010-2011) were provided by CEFAR (Centro de Estudose Avaliac~ao em Sa ude, Associac~ao Nacional de Farm acias), and weredivided into two groups: group 1 (G1: medication used throughoutthe year—histamine antagonists) and group 2 (G2: medication usedin SOS—corticosteroids,b2 agonists and a LTR antagonist). Spearman’s test was used in the correlation studies. Sales of anti-allergic medication are higher between March and June (<2-fold) forboth groups compared to the rest of the year, corresponding to theSpring time in Alentejo region. The sales peaked in May, coincidingwith pollen and allergen season peak.Sales of G1 showed association with both Olea (0.735) and Poaceae(0.729) pollen and Ole e 1 (0.776) and Phl p 5 (0.505) allergens.Sales of G2 also correlated both with pollen (0.619 for Olea and0.593 for Poaceae) and allergens (0.658 and 0.437 for Ole e 1 andPhl p 5, respectively). Correlation was stronger when the total pollenor allergen load was considered for G1 (0.744 and 0.627, respec-tively) and G2 (0.675 and 0.743, respectively).Conclusions:In summary, it was observed that allergy drug salespositively correlated with airborne pollen and allergen loads; A stron-ger correlation was observed for G1, particularly when the totalallergenic load is considered. These results suggest that the sales ofG1 medication might be used as a surrogate marker of healthimpacts of pollinosis during the pollen season of highly allergenicspecies in Alentejo region, Portugal

Study of the allergenic profile of pollen from Platanus hybrida in the city of Evora, Portugal

Background and Aim: Although grasses and olive are the most relevant allergenic species in the Alentejo region, aggravation of allergic symptoms in the early spring, unrelated with those species pollen seasons, has been reported, particularly in urban environment. Plane trees, hence pollen, are highly abundant in the city of Évora, nonetheless allergen pollen profile has not yet been evaluated. The aim of this work was to characterize the allergen profile of pollen from Platanus hybrida, one of the most representative species in Evora showing pollination prior to the main pollen season in Alentejo. Methods: Pollen from Platanus hybrida and Dactylis glomerata was extracted with ammonium bicarbonate buffer, lyophilized and stored at -80ºC until analysis. Protein content was determined by the Bradford method. SDS-PAGE followed by western blot, using allergic patient sera (obtained from the Hospital do Espírito Santo de Évora – HESE), were performed to evaluate the allergen profile of the pollen. Sensitization and cross-reactivity was assessed by solid phase immunoblot. Results: Half of the patient exhibited sensitization to pollen extracts of P. Hybrida. Western blot have shown several immunoreactive bands in the Mr 10-90 kDa range. Immunoreactive bands were also observed in the protein profile according to the pI in the pI range 4.0-6.1. Cross-reactivity of P. hybrida with D. glomerata was found. Although several bands are common to D. glomerata, a band with ~50kDa was observed in P. hybrida but not in D. glometata. Conclusion: These results evidenced allergens found in P. hybrida pollen. Moreover, cross–reactivity between P. hybrida and highly allergenic species such as D. glomerata was found which probably contributes for aggravation of pollinosis in the early spring. Acknowledgments: This work was supported by FEDER through the “Programa Operacional Fatores de Competitividade – COMPETE” (Strategic projects of ICAAM and ICT 2013-2015)