Allergenicity and structural properties of new Cor a 1 isoallergens from hazel identified in different plant tissues.

The hazel allergen Cor a 1 is a PR-10 protein, closely related to the major birch pollen allergen Bet v 1. Hazel allergies are caused by cross-reactive IgE antibodies originally directed against Bet v 1. Despite the importance of PR-10 proteins in allergy development, their function and localization in the plant remain largely elusive. Therefore, the presence of Cor a 1 mRNA and proteins was investigated in different tissues, i.e., the female flower, immature and mature nuts, catkins, and pollen. Four yet unknown Cor a 1 isoallergens, i.e., Cor a 1.0501-1.0801, and one new Cor a 1.03 variant were discovered and characterized. Depending on the isoallergen, the occurrence and level of mRNA expression varied in different tissues, suggesting different functions. Interestingly, Cor a 1.04 previously thought to be only present in nuts, was also detected in catkins and pollen. The corresponding Cor a 1 genes were expressed in Escherichia coli. The purified proteins were analysed by CD and NMR spectroscopy. Immunoblots and ELISAs to determine their allergenic potential showed that the new proteins reacted positively with sera from patients allergic to birch, hazel and elder pollen and were recognized as novel isoallergens/variants by the WHO/IUIS Allergen Nomenclature Sub-Committee.

Imaging Flow Cytometry as a Quick and Effective Identification Technique of Pollen Grains from Betulaceae, Oleaceae, Urticaceae and Asteraceae.

Despite the continuous and intensive development of laboratory techniques, a light microscope is still the most common tool used in pollen grains differentiation. However, microscopy is time-consuming and needs well-educated and experienced researchers. Other currently used techniques can be categorised as images and non-images analysis, but each has certain limitations. We propose a new approach to differentiate pollen grains using the Imaging Flow Cytometry (IFC) technique. It allows for high-throughput fluorescence data recording, which, in contrast to the standard FC, also enables real-time control of the results thanks to the possibility of digital image recording of cells flowing through the measuring capillary. The developed method allows us to determine the characteristics of the pollen grains population based on the obtained fluorescence data, using various combinations of parameters available in the IDEAS software, which can be analysed on different fluorescence channels. On this basis, we distinguished pollen grains both between and within different genera belonging to the Betulaceae, Oleaceae, Urticaceae and Asteraceae families. Thereby, we prove that the proposed methodology is sufficient for accurate, fast, and cost-effective identification and potentially can be used in the routine analysis of allergenic pollen grains.

Isolation and quantification of diarylheptanoids from European hornbeam (Carpinus betulus L.) and HPLC-ESI-MS/MS characterization of its antioxidative phenolics.

Detailed polyphenol profiling of European hornbeam (Carpinus betulus L.) bark, leaf, male and female catkin extracts was performed by high-performance liquid chromatography-diode array detection coupled to tandem mass spectrometry (HPLC-DAD-MS/MS). A total of 194 compounds were characterized and tentatively identified. Gallo- and ellagitannins dominated in the methanol extracts, while flavonol glycosides and methoxylated flavones prevailed in the ethyl acetate samples. In the quest for diarylheptanoids, twelve compounds were isolated by the combination of subsequent reversed-phase flash chromatographic and high-performance liquid chromatographic methods. The structural elucidation of the isolated components was performed by ultrahigh-performance liquid chromatography-Orbitrap mass spectrometry (UHPLC-Orbitrap-MS) as well as 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. Six known cyclic diarylheptanoids, together with a new compound were described in Carpinus betulus for the first time. The occurrence of a linear diarylheptanoid and a lignan has also been unprecedented in the genus Carpinus. Moreover, three known flavonol glycosides were isolated. Based on the identification of characteristic fragment ions, a new mass spectrometric fragmentation pathway for meta,meta-cyclophane-type diarylheptanoids was proposed. Quantities of the four major cyclic diarylheptanoids in European hornbeam were determined by a validated UHPLC-DAD method for the first time. The antioxidant properties of the extracts and the isolated compounds were assessed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Contribution of the individual constituents to the total radical scavenging activity of the samples was evaluated by an off-line DPPH-HPLC-DAD method. This allowed the identification of gallo- and ellagitannin derivatives as the constituents being primarily responsible for the antioxidant capacity of the extracts.

Inter- and intra-tree variability of carbon and oxygen stable isotope ratios of modern pollen from nine European tree species.

Stable carbon and oxygen isotope ratios of raw pollen sampled from nine abundant tree species growing in natural habitats of central and northern Europe were investigated to understand the intra- and inter-specific variability of pollen-isotope values. All species yielded specific δ13Cpollen and δ18Opollen values and patterns, which can be ascribed to their physiology and habitat preferences. Broad-leaved trees flowering early in the year before leaf proliferation (Alnus glutinosa and Corylus avellana) exhibited on average 2.6‰ lower δ13Cpollen and 3.1‰ lower δ18Opollen values than broad-leaved and coniferous trees flowering during mid and late spring (Acer pseudoplatanus, Betula pendula, Carpinus betulus, Fagus sylvatica, Picea abies, Pinus sylvestris and Quercus robur). Mean species-specific δ13Cpollen values did not change markedly over time, whereas δ18Opollen values of two consecutive years were often statistically distinct. An intra-annual analysis of B. pendula and P. sylvestris pollen revealed increasing δ18Opollen values during the final weeks of pollen development. However, the δ13Cpollen values remained consistent throughout the pollen-maturation process. Detailed intra-individual analysis yielded circumferential and height-dependent variations within carbon and oxygen pollen-isotopes and the sampling position on a tree accounted for differences of up to 3.5‰ for δ13Cpollen and 2.1‰ for δ18Opollen. A comparison of isotope ranges from different geographic settings revealed gradients between maritime and continental as well as between high and low altitudinal study sites. The results of stepwise regression analysis demonstrated, that carbon and oxygen pollen-isotopes also reflect local non-climate environmental conditions. A detailed understanding of isotope patterns and ranges in modern pollen is necessary to enhance the accuracy of palaeoclimate investigations on δ13C and δ18O of fossil pollen. Furthermore, pollen-isotope values are species-specific and the analysis of species growing during different phenophases may be valuable for palaeoweather reconstructions of different seasons.

Production of Ellagitannin Hexahydroxydiphenoyl Ester by Spontaneous Reduction of Dehydrohexa-hydroxydiphenoyl Ester.

Amariin is an ellagitannin with two dehydrohexahydroxydiphenoyl (DHHDP) moieties connecting glucose 2,4- and 3,6-hydroxy groups. This tannin is predominant in the young leaves of Triadica sebifera and Carpinus japonica. However, as the leaves grow, the 3,6-DHHDP is converted to its reduced form, the hexahydroxydiphenoyl (HHDP) group, to generate geraniin, a predominant ellagitannin of the matured leaves. The purified amariin is unstable in aqueous solution, and the 3,6-(R)-DHHDP is spontaneously degraded to give HHDP, whereas 2,4-(R)-DHHDP is stable. The driving force of the selective reduction of the 3,6-DHHDP of amariin is shown to be the conformational change of glucose from O,3B to 1C4. Heating geraniin with pyridine affords 2,4-(R)-DHHDP reduction products. Furthermore, the acid hydrolysis of geraniin yields two equivalents of ellagic acid. Although the reaction mechanism is still ambiguous, these results propose an alternative biosynthetic route of the ellagitannin HHDP groups.

Impact of Tree Pollen Distribution on Allergic Diseases in Serbia: Evidence of Implementation of Allergen Immunotherapy to Betula verrucosa.

Background and objectives: The relationship between air pollen quantity and the sensitization of allergic patients is crucial for both the diagnosis and treatment of allergic diseases. Weather conditions influence the distribution of allergenic pollen and increases in pollen concentration may negatively affect the health of allergic patients. The aim of this study was to analyze the implementation of allergen immunotherapy with regard to air pollen concentration. Material and Methods: Here we examined the relationship between Betula air pollen concentration and the usage of Betula verrucosa allergen immunotherapy in Serbia. Examination covered the period from 2015 to 2018. Measurement of airborne pollen concentration was performed with Lanzoni volumetric pollen traps. The evidence of the usage of sublingual allergen immunotherapy (SLIT) was gathered from patients with documented sensitization to specific pollen. Results: During this period tree pollens were represented with 58% ± 21% of all measured air pollen species, while Betula pollen represented 15% ± 8% of all tree pollens. Betula pollination peaked in April. Allergen immunotherapy to Betula verrucosa in Serbia is entirely conducted as sublingual immunotherapy and represents 47.1% ± 1.4% of issued tree pollen SLIT. The use of pollen SLIT increased by 68% from 2015 to 2018, with an even greater increase in usage recorded for Betula SLIT-80%. Conclusions: This analysis shows a clear causative relationship between pollination and the type/prevalence of applied allergen immunotherapy. Information about the flowering seasons of allergenic plants is very important for people who suffer from allergy, for clinical allergologists, as well as for governing authorities. The presented data is of practical importance to the proper timing of immunotherapy initiation and of importance for urban landscaping. The obtained data can be the starting point for the instatement of a thorough epidemiological study and the inclusion of Serbia on the pollen map of Europe.

Cross-reactivity between the Betulaceae family and fallout in the real atmospheric aeroallergen load.

Betulaceae family is a dominant tree pollen type in the atmosphere at Northwest Spain, being a major cause of allergenic rhinitis or asthma symptoms. Alnus pollen cause symptoms in the 9-20% of the total hay fever sufferers mean while the 41.89% of patients present a positive skin-prick-test for Betula allergens. Aln g1 and Bet v1 aeroallergens belong to PR-10 protein family and are associated to cross-reactivity processes. Airborne pollen and aeroallergens from Alnus and Betula were sampled during their Main Pollen Season from 2016 to 2019. Pollen sampling was conducted by means of a Hirst volumetric trap and aeroallergens were sampled using a Multi Vial Cyclone Sampler. Alnus flowering took place from January to February, with an average duration of 44 days. Betula bloom occurred from April to May with an average pollen season of 33 days. The major innovation of our study was the first detection of Aln g1 allergens in the atmosphere by using Bet v1 antibodies. This fact verified the cross-reactivity between the main allergens of Betula and Alnus pollen. Along the study period, an average of 18 days/year and 14 days/year with high potential risk of allergy due to Alnus pollen and allergen respectively, was registered. For Betula an average of 16 days/year with high potential risk of allergy due to pollen and 22 days for allergen was registered. The main consequence of the successive bloom of both trees would be the so-called "priming effect". Urban population sensitized to Betula pollen could suffer allergic symptoms during winter (as a consequence of Alnus), and in spring with the manifestation of higher symptoms under low birch pollen grain levels in the atmosphere. The traditional information to prevent allergies, such as the airborne pollen concentrations, should be combined with the data of aeroallergen to identify the real allergenic load in the atmosphere.

Anti-acne vulgaris effect including skin barrier improvement and 5α-reductase inhibition by tellimagrandin I from Carpinus tschonoskii.

BACKGROUND: Carpinus tschonoskii (CT) has been previously studied for various activities in the improvement of skin diseases. In the present study, we examined the in vitro anti-acne vulgaris (AV) effect of CT leaves (CTL) and tellimagrandin I (TI), one of the main ellagitannins from CT, including skin barrier improvement and 5α-reductase inhibitory activity. METHODS: To test the anti-AV activities of CTL and TI, firstly, anti-oxidative and anti-inflammatory activities including DPPH radical scavenging activity, nitric oxide (NO) inhibitory activity, and cytokines [interleukin (IL)-6 and IL-8] were tested. Skin barrier improvement experiments were tested using developing cornified envelope (CE) formation, and filaggrin mRNA expression level was determined by RT-PCR. The 5α-reductase inhibitory activity was determined by measuring the testosterone levels in rat liver microsomes. RESULTS: CTL and TI showed potent anti-oxidative activity and anti-inflammatory activities. Especially, the cytokine production inhibitory activities of TI were found to be similar to the positive control, epigallocatechin gallate (EGCG). CTL and TI enhanced the CE formation and filaggrin mRNA expression levels and showed potent activities compared to that in the positive control, 1.5 mM Ca2+. In additionally, CTL and TI showed 5α-reductase inhibitory activities in a dose-dependent manner. CONCLUSION: The results showed that CTL and TI inhibit AV endogenous factors such as 5α-reductase and inflammatory cytokines and affect exogenous factors such as developing skin barrier function (CE and filaggrin levels). Therefore, CTL and TI may be plant-derived agent, promising in the treatment of acne vulgaris.

Impact of the combination of sourdough fermentation and hop extract addition on baking properties, antioxidant capacity and phenolics bioaccessibility of rice bran-enhanced bread.

The effect of hop sourdough fermentation combined with rice bran (RB) addition was investigated for improving technological, bioactive and sensory properties of wheat bread by optimizing the quantities of RB, yeast, sourdough and hop extract contents based on response surface methodology. The bread produced according to the optimized formula was compared with control bread, sourdough or RB-enhanced bread with regard to quality attributes, bioaccessible phenolics and antioxidant activity. Sourdough fermentation combined with RB flour increased volume of loaves and consumer acceptance with no significant variations in crumb moisture content and crumb firmness during storage. RB darkened crumb while no effects on colour were observed for sourdough bread. Sourdough technology allowed increasing phenolic content of RB bread with no significant differences in its antioxidant activity. The bioaccessible ferulic and sinapic acids, the main phenolics released in vitro digestion of optimized bread were lower compared to those of control bread.

Taxanes content and cytotoxicity of hazel cells extract after elicitation with silver nanoparticles.

The toxicity of silver nanoparticles (AgNPs) has been attributed to the generation of Ag+ ions as well as production of ROS. The latter can elicit defensive response of plant cells in different ways e.g., enhancement of secondary metabolite productions. In the present study this hypothesis was evaluated by assessment of taxanes production by suspension-cultured hazel (Corylus avellana L.) cells after treatment with AgNPs. The cells were treated with different concentrations of AgNPs (0, 2.5, 5, and 10 ppm), in their logarithmic growth phase (d7) and were harvested after 1 weak. The growth of cells and their membrane integrity decreased but extracellular electro conductivity and total dissolved solids increase by AgNPs (probably due to loosening of cell membrane). Treatment of hazel cells with AgNPs (in particular of 5 ppm) rapidly and remarkably increased the yields of two major taxanes, i.e., Taxol and baccatin III; so that 24 h of the treatment their contents reached to 378% and 163% of the control, respectively. Increase of Taxanes was accompanied by the increase of total soluble phenols. The extracts of AgNPs-treated cells were able to inhibit the growth of cancerous HeLa cells and reduce their viability to 60% of the control. The results suggest the elicitation of suspension-cultured hazel cells with AgNPs as a procedure for rapid enhancement of anticancer taxanes biosynthesis by the cells.

Seasonal variation of pheophorbide a and flavonoid in different organs of two Carpinus species and its correlation with immunosuppressive activity.

The genus Carpinus of Betulaceae is the most widely distributed in the European landscape. This study reports a comparative study based on the pheophorbide a and flavonoid content from the two main species of the genus Carpinus, Carpinus betulus and Carpinus turczaninowii, respectively, in Nanjing, China. The pheophorbide a and flavonoid content depends on the organ, species, and season. HPLC analysis showed that the pheophorbide a and flavonoid levels were the highest in May and June, respectively, from the leaves of C. betulus 'Fastigiata.' In contrast, the content of pheophorbide a and flavonoid in the stems of C. betulus 'Fastigiata' or in other species was low. The immunosuppressive effects of the ethyl acetate extracts and methanol extracts from the two Carpinus species were also evaluated. The ethyl acetate extracts of C. betulus 'Fastigiata' in May and the methanol extracts of C. betulus 'Fastigiata' in June showed better immunosuppressive activity than in other seasons, which coincided with the content of pheophorbide a and flavonoid, respectively. Our findings indicated that C. betulus 'Fastigiata' can serve as a medicinal plant against inflammation because of its pheophorbide a and flavonoid content.

Effects of atmospheric pollutants (CO, O3, SO2) on the allergenicity of Betula pendula, Ostrya carpinifolia, and Carpinus betulus pollen.

Pollen of Betula pendula, Ostrya carpinifolia, and Carpinus betulus was exposed in vitro to relatively low levels of the air pollutants, namely carbon monoxide, ozone, and sulfur dioxide. The allergenicity of the exposed pollen was compared with that of non-exposed pollen samples to assess if air pollution exposition affects the allergenicity potential of pollen. The immunodetection assays indicated higher IgE recognition by all sera of allergic patients to the pollen protein extracts in all exposed samples in comparison to the non-exposed samples. These results show that the pollen exposition to low pollutants' levels induces increased allergic reaction to sensitized individuals.

Effect of air pollutant NO2 on Betula pendula, Ostrya carpinifolia and Carpinus betulus pollen fertility and human allergenicity.

Pollen of Betula pendula, Ostrya carpinifolia and Carpinus betulus was exposed in vitro to two levels of NO2 (about 0.034 and 0.067 ppm) - both below current atmospheric hour-limit value acceptable for human health protection in Europe (0.11 ppm for NO2). Experiments were performed under artificial solar light with temperature and relative humidity continuously monitored. The viability, germination and total soluble proteins of all the pollen samples exposed to NO2 decreased significantly when compared with the non-exposed. The polypeptide profiles of all the pollen samples showed bands between 15 and 70 kDa and the exposure to NO2 did not produce any detectable changes in these profiles. However, the immunodetection assays indicated higher IgE recognition by patient sera sensitized to the pollen extracts from all exposed samples in comparison to the non-exposed samples. The common reactive bands to the three pollen samples correspond to 58 and 17 kDa proteins.

In vitro exposure of Ostrya carpinifolia and Carpinus betulus pollen to atmospheric levels of CO, O3 and SO 2.

Ostrya spp. and Carpinus spp. pollen was in vitro exposed to three atmospheric pollutants: CO, O3 and SO2. Two levels of each pollutant were used, and the first level corresponds to a concentration about the atmospheric hour-limit value acceptable for human health protection in Europe and the second level to about the triple of the first level. Experiments were done under artificial solar light with temperature and relative humidity controlled. The viability of the exposed pollen samples showed a significant decrease. Also, the germination percentage showed a significant decrease in both exposed pollens, and the effect was most pronounced for SO2, followed by O3 and CO. A general decreasing trend in the total soluble protein content of the exposed pollen samples when compared with the control was observed, but it was only statistically significant for the Ostrya spp pollen. The results showed marked effects were observed on the Ostrya spp. and Carpinus spp. pollen when exposed to air pollutant levels that can be considered safe for human health protection.

Tree pollen spectra and pollen allergy risk in the Osijek-Baranja County.

The forests of north-eastern Croatia, as well as various plants and trees in the parks and streets of the Osijek-Baranja County, produce large amounts of pollen during the pollen season, which can cause allergy symptoms in pollen sensitive individuals. The aim of this study was to determine the most frequent types of pollen in this area and estimate possible health risks, especially the risk of allergy. In 2009 and 2010, the staff of the Health Ecology Department of the Osijek Public Health Institute monitored tree pollen concentrations in four cities from the Osijek - Baranja County (Osijek, Nasice, Dakovo and Beli Manastir) using a Burkard volumetric instrument. The results were affected by weather conditions. Windy and sunny days facilitated the transfer of pollen, whereas during rainy days, the concentration of pollen grains decreased. High pollen concentrations of Cupressaceae/Taxaceae, Betulaceae, Salicaceae and Aceraceae could be the cause for symptoms of pollen allergy. In 2009, conifers, birch and poplar pollen were dominant at all monitoring stations with 5000 pollen grains (PG), 3188 PG and 3113 PG respectively. The highest number of pollen grains was recorded at measuring site Osijek. The variations in airborne pollen concentration between pollen seasons were recorded at all monitoring stations. The most obvious variations were recorded at measuring site Osijek. The usual pollination period lasts two to three months, which means that most pollen grains remain present from February to early June. However, the Cupressaceae / Taxaceae pollination periods last the longest and their pollen grains remain present until the end of summer. The risk of allergy was determined at four monitored measuring stations and the obtained data confirmed that the largest number of days with a high health risk was at the Dakovo measuring station for a species of birch. The research information aims to help allergologists and individuals allergic to plant pollen develop preventive measures and proper treatment therapies.

Inhibitory effects of Carpinus tschonoskii leaves extract on CpG-stimulated pro-inflammatory cytokine production in murine bone marrow-derived macrophages and dendritic cells.

This report describes the anti-inflammatory effects of MeOH extract from leaves of Carpinus tschonoskii (CE) on primary bone marrow-derived macrophage (BMDMs) and dendritic cells (BMDCs). Primary BMDMs and BMDCs were used for pro-inflammatory cytokine production and Western blot analysis. Human embryonic kidney cell line 293 T (HEK293 T) was used to access NF-κB activity. In all cases, CpG DNA was used to stimulate the cells. The CE (0-150 µg/ml) was treated to BMDMs, BMDCs, and HEK293T cells. CE pre-treatment in CpG-stimulated BMDMs and BMDCs showed a dose-dependent inhibitory effect on pro-inflammatory cytokine (e.g., IL-12 p40, IL-6, and TNF-α) production as compared to non-treated controls. The CE pre-treatment had no significant inhibition on mitogen-activated protein kinases (MAPKs) phosphorylation but strongly inhibited IκBα degradation. In NF-κB reporter gene assay, the CE pre-treatment inhibited NF-κB-dependent luciferase activity in a dose-dependent manner. Taken together, these data suggest that CE has significant inhibitory effect on pro-inflammatory cytokine production and warrant further studies concerning potentials of CE for medicinal uses.

Potential anticancer activity of young Carpinus betulus leaves.

As part of our continuing research for anticancer compounds from the Walloon Region forest, EtOAc extract from Carpinus betulus leaves was phytochemically studied, leading to the bioguided isolation of pheophorbide a, which is responsible of anticancer properties of C. betulus young leaves. This compound was identified using nuclear magnetic resonance and mass spectrophotometric data and comparison with a commercial standard. Evaluation of the growth inhibitory activities of pheophorbide a using MTT colorimetric assay and phase-contrast microscopy in various human cancer cell lines confirmed the photoactivable properties of this compound. Our research showed, for the first time, the presence of pheophorbide a, a chlorophyll derived compound, which we quantified in high quantities in young leaves of C. betulus. This is in contrast with the literature which generally describes pheophorbide a as a catabolic product of chlorophyll, then preferentially present in old leaves.

Thirty-year survey on airborne pollen concentrations in Genoa, Italy: relationship with sensitizations, meteorological data, and air pollution.

BACKGROUND: Pollen allergy represents a relevant health issue. Betulaceae sensitization significantly increased in Genoa, Italy, in the last decades. This study investigated possible relationships among pollen count, meteorological changes, air pollution, and sensitizations in this city during a 30-year period. METHODS: Betulaceae, Urticaceae, Gramineae, and Oleaceae pollen counts were measured from 1981 to 2010 in Genoa. Sensitization to these pollens was also considered in large populations of allergic patients. Meteorological parameters and pollutants were also measured in the same area. RESULTS: Betulaceae sensitization increased over time. All pollen species significantly increased over this time. Pollen season advanced for Betulaceae and Urticaceae. Only Urticaceae season significantly increased. Temperature increased while rainfall decreased over the time. Pollutants significantly decreased. There were some relationships between pollen changes and climatic and air pollution parameters. CONCLUSION: This 30-year study conducted in an urbanized area provided evidence that Betulaceae sensitization significantly increased, pollen load significantly augmented, and climate and air pollution changed with a possible influence on pollen release.

Effects of traffic air pollution and hornbeam pollen on adult asthma hospitalizations in Zagreb.

BACKGROUND: Environmental factors play an important role in asthma morbidity, although the contribution of individual pollutants or pollens in exacerbating asthma is not completely elucidated. Despite the evidence of importance of the hornbeam pollen in inducing allergic sensitization, its role in provoking asthma exacerbation has not been evaluated. The aim of the present study was to investigate effects of traffic pollutants on adult asthma hospitalization adjusting for pollens including hornbeam. METHODS: During a 3-year period, health and environmental data were collected and analyzed. Daily asthma hospitalizations were regressed on pollutants and potential confounding variables using an autoregressive Poisson model. RESULTS: The risk of asthma hospitalization was associated significantly with the 95th to 99th percentile increase in levels of nitrogen dioxide (RR = 1.22; 95% CI: 1.05-1.40), carbon monoxide (RR = 1.25; 95% CI: 1.01-1.55) and hornbeam pollen (RR = 1.21; 95% CI: 1.11-1.30). The effect of nitrogen dioxide was delayed by 5 days. No statistically significant increase in the risk of asthma hospitalization was found for PM(10) particles. A comparison among the standardized regression coefficients and their respective p values indicates that the most important risk factor for asthma hospitalization is associated with hornbeam pollen levels. No statistically significant interactions between pollutants and pollens were detected. CONCLUSIONS: The current results suggest that traffic-related air pollution is associated with increased risk of adult asthma hospitalization. Nonetheless, the most significant risk for asthma hospitalization is associated with hornbeam pollen levels in the city of Zagreb.