Allergenicity at component level of sub-pollen particles from different sources obtained by osmolar shock: A molecular approach to thunderstorm-related asthma outbreaks.

BACKGROUND: The so-called "thunderstorm asthma" (TA) is an uncommon but dramatic outbreak of asthma attacks occurring during a thunderstorm in the pollen and moulds season. Mechanisms which make the pollen able to enter the deeper airways and provoke severe asthma symptoms are still unclear. OBJECTIVE: To test the hypothesis that sub-pollen particles (SPPs) originated from the rupture by an osmotic shock of pollen associated with TA contain allergens. METHODS: After hydration, SPPs released from pollen grains of grass, pellitory, olive, cypress, ragweed and birch were isolated and determined by microscopy. Allergens were determined by in vitro ELISA inhibition tests indirectly using the sera from 10 polyreactive patients. An inhibition <50% was considered as negative, 50%-75% moderate and > 75% complete. RESULTS: The inhibition experiments showed that the SPPs from birch and cypress were unable to inhibit serum IgE reactivity to Bet v 1 and Cup a 1, respectively. Ragweed SPPs inhibited ragweed pollen extract and Amb a 1 by 75.8 ± 0.11% and 81.2 ± 0.15%, respectively. Olive and pellitory SPPs retained almost the whole IgE-binding capability in all cases tested. Grass SPPs inhibited 32 ± 0.06% of Lolium perenne Lol p 1 and 65% of Phleum pratense extracts, but results were highly variable for individual allergens (97.5%-0.03% for Phl p 2, 45.3 ± 0.12% for Phl p 5, 24.7 ± 0.22% for Phl p 6, and 38.3 ± 0.2% for Phl p 1). CONCLUSIONS: Inhibition experiments confirm the hypothesis that SSPs obtained after the osmotic shock of pollen involved in TA, namely grass, pellitory and olive tree pollen, contain allergens and therefore they can induce severe asthma attacks during thunderstorms.

Tree pollen exposure is associated with reduced lung function in children.

BACKGROUND: Allergic disease is a recognized global epidemic and a significant cause of ill health and poor quality of life. The prevalence of pollen allergy is high throughout the world, and pollen exposure itself plays a role in emergency department presentations and hospitalizations for asthma. Lung function and airway inflammation are important measures of asthma activity and control. OBJECTIVE: To examine associations between exposure to multiple pollen types and lung function and markers of airway inflammation at 8 and 14 years of age, and to explore potential modification by residential greenness. METHODS: A cohort of high-risk children living in Sydney, Australia had spirometry and fractional exhaled nitric oxide (FeNO) measured at 8 and 14 years of age. Ambient pollen concentration on the day of lung function measurement and up to three days prior was used as the exposure measure. Residential greenness was derived from satellite imagery. We modelled the association between six pollen types and lung function and FeNO. We also assessed modifying effects of residential greenness. RESULTS: Casuarina, cypress and Pinus pollen in the air the day before measurement and 3 days prior respectively, were associated with reduced lung function in 8-year-olds. The pollen exposures were associated with decreases in FEV1 and FVC; however, the FEV1 /FVC ratio was not affected. Effect modification by greenness was not observed due to loss of power. CONCLUSIONS & CLINICAL RELEVANCE: Airborne tree pollen of cypress, Casuarina and Pinus and not grass in some regions may be detrimental to childhood lung function.

Isolation of cypress gibberellin-regulated protein: Analysis of its structural features and IgE binding competition with homologous allergens.

The presence in cypress pollen of an important allergen, belonging to the gibberellin-regulated protein (GRP) family, has been suggested for many years. However, it has never been isolated and sometimes the homologous peach allergen, Pru p 7, has been used as a surrogate to perform immunological investigations. The aim of this study has been the isolation and molecular characterization of the GRP contained in the Cupressus sempervirens pollen. This protein, named Cypmaclein, has been purified from the natural source using conventional biochemical methods consisting in different chromatographic separations. Cypmaclein has been identified by direct protein sequencing of the N-terminal region and of internal fragments of the molecule. In SDS-PAGE, its apparent molecular mass is slightly higher than that of Pru p 7. Nevertheless, the mass spectrometry experiments reveal that the exact molecular mass of Cypmaclein (6821.88 Da) is very close to that of Pru p 7 (6909.90 Da). Two regions of Cypmaclein have been sequenced providing 50% of its primary structure. A high overall sequence identity of Cypmaclein with all the analyzed GRP has been observed, although in the N-terminal region the high identity is limited to the homolog of Cryptomeria japonica. In circular dichroism experiments Cypmaclein produced a spectrum overlapping that of Pru p 7. However, the comparative analysis of Cypmaclein, Pru p 7 and Pun g 7 IgE reactivity revealed a behavior that was not completely overlapping, thus suggesting that the IgE epitopes are only partially shared. In single point highest inhibition achievable assays performed with the FABER test, Cypmaclein efficiently competed with the allergenic peach and pomegranate GRP in the binding of specific IgE of patients sensitized to Pru p 7. In conclusion, the natural cypress pollen GRP has been isolated for the first time, its structural features have been investigated and its cross-reactivity with Pru p 7 and Pun g 7 has been demonstrated. This protein is now available for further investigations aimed at understanding its clinical relevance in the allergy to cypress pollen. In addition, the prevalence of sensitization directly to Cypmaclein, and not limited to the homologs, can be defined.

Exposure to Cypress Pollens and Subsequent Symptoms: A Panel Study.

OBJECTIVE: The objective of this panel study was to document the relationship between exposure to cypress pollen and allergic symptoms. METHODS: The study group included 47 patients with allergy to cypress pollen who completed a daily diary and a weekly evaluation of quality of life (QoL) during the cypress pollen season. Different patients were included in three consecutive pollen seasons: 2014-2015, 2015-2016, and 2016-2017. Daily cypress pollen counts were obtained from the National Aerobiological Network. Air pollution and meteorological data were recorded on a daily basis. The pollen-symptoms relationship was quantified by calculating odds ratios for an increase of 1 log of grains of pollen/m3, taking into account potential confounding factors. The QoL score was expressed with a beta coefficient that increased with 1 grain of pollen/m3. RESULTS: There was a marked increase in rhinitis and ocular symptoms during the pollen season, with a plateau effect at high levels of exposure, but no relationship with bronchial symptoms. The QoL score had a linear and significant relationship with the cypress pollen count. We did not detect any threshold level. CONCLUSION: This panel study demonstrated a significant association between exposure to cypress pollens and allergic symptoms, with a plateau effect for high exposures.

Pru p 7 sensitization is a predominant cause of severe, cypress pollen-associated peach allergy.

BACKGROUND: Peach is a common elicitor of food allergic reactions. Peach-induced immediate reactions may occur as benign pollen-food syndromes, usually due to birch pollen-related PR-10 cross-reactivity in temperate climates, and as potentially severe primary food allergies, predominantly related to nsLTP Pru p 3 in Mediterranean regions. The newly described peach allergen Pru p 7 has gained recent attention as a potential peach allergy severity marker. Sensitization to Pru p 7 and its allergenic homologues of the gibberellin-regulated protein family occurs in areas with high Cupressaceae tree pollen exposure. OBJECTIVE: We sought to investigate the distribution, clinical characteristics and molecular associations of Pru p 7 sensitization among subjects with suspected peach allergy in different regions of France. METHODS: Subjects with suspected peach allergy (n = 316) were included. Diagnostic work-up was performed according to current guidelines, including open food challenge when required. IgE antibody measurements and competition experiments were performed using the ImmunoCAP assay platform. RESULTS: Sensitization to Pru p 7 was present in 171 (54%) of all subjects in the study and in 123 of 198 (62%) diagnosed as peach allergic, more than half of whom were sensitized to no other peach allergen. Frequency and magnitude of Pru p 7 sensitization were associated with the presence of peach allergy, the clinical severity of peach-induced allergic reactions and the level of cypress pollen exposure. Cypress pollen extract completely outcompeted IgE binding to Pru p 7. Pru p 7 was extremely potent in basophil activation tests. CONCLUSION AND CLINICAL RELEVANCE: A subtype of Cupressaceae pollinosis, characterized by Pru p 7 sensitization, can be an underlying cause of severe peach allergy.

Analysis of factors associated with cedar pollen sensitization and development of pollinosis in a young Japanese adult population.

BACKGROUND: Genetic and environmental factors are proposed to be involved in cedar pollen allergy sensitization and onset. The impact of these factors will provide key information for the prevention of cedar pollen sensitization and allergy onset, which we investigated in this cross-sectional study. METHODS: Subjects were 382 young adult volunteers who completed a self-administered questionnaire on self-reported subjective symptoms of pollinosis, physician-diagnosed pollinosis, and background factors. We also measured their serum IgE antibody titers specific for cedar, cypress, and mites. Factors associated with subjective symptoms, physician diagnosis, and the three specific antigens were determined using both univariate and multivariate analyses. RESULTS: Sensitization to cedar, cypress, and mites, defined as specific IgE levels of class 1 or above, was found in 78.8%, 64.4%, and 56.0% of subjects, respectively. The prevalence of cedar pollinosis was 41.2% based on subjective symptoms and 22.2% based on physician diagnosis. Factors associated with increased cedar pollen sensitization were mite sensitization, comorbid allergic rhinitis, and family history of cedar pollinosis. Risk-reducing factors for cedar pollen sensitization were keeping a cat, number of common colds, and hours of sleep. Risk-increasing factors for both subjective pollinosis symptoms and physician-diagnosed pollinosis were comorbid allergic rhinitis and family history of cedar pollinosis. CONCLUSIONS: Sensitization to cedar pollen in this population was extremely high. Both common and distinct factors were associated with sensitization to pollen and with the development of pollinosis. The distinct factors were associated with sensitization to cedar and cypress antigens.

Cypress pollen allergy is responsible for two distinct phenotypes of allergic rhinitis different from other pollinosis.

Summary: Different phenotypes of allergic rhinitis have been identified based on the seasonality of the allergen involved. Within pollinosis, importance has to be paid to the responsible pollen species. Guidelines for clinical management are mostly based on studies performed in patients with grass pollen allergy. Only few data is available on tree pollen allergy and more specifically on cypress pollen allergy. We focused on the clinical and biological features of cypress pollen allergy to determine whether it is associated with a specific phenotype of allergic rhinitis or not. Our results suggest that cypress pollen can be responsible for two distinct phenotypes of rhinitis, both different from other pollinosis. In the most common phenotype, cypress pollen was not responsible for bronchial hyperresponsiveness or systemic inflammation. Close attention has to be paid to the allergen involved in allergic rhinitis. Different phenotypes leading to different pharmacological strategies may apply.

Identification of a polygalacturonase (Cup s 2) as the major CCD-bearing allergen in Cupressus sempervirens pollen.

As IgE glyco-epitopes, also referred to as cross-reactive carbohydrate determinants (CCDs), can share significant structural homologies between different plants, they are prone to extensive cross-reactivity among allergen pollen extracts. Here, cypress pollen allergens, especially a polygalacturonase (PG), were further characterized using double one-dimensional electrophoresis (D1-DE). The presence of specific IgE directed against CCDs was investigated by bromelain IgE inhibition and concanavalin A binding assays using sera of cypress pollen-sensitized patients. Our results showed that IgE reactivity to CCDs in Cupressus sempervirens pollen extracts is mainly related to bromelain-type epitopes of a newly identified cypress PG. This glycoprotein has been further characterized through an immunoproteomic approach and officially indexed as Cup s 2 by the WHO/IUIS allergen nomenclature. Cup s 2 could thus be associated with the increased prevalence of IgE reactivity to cypress pollen extracts because of CCD interference.

Allergic sensitization prevalence in a children and adolescent population of northeastern Greece region.

OBJECTIVES: To evaluate the prevalence of allergic sensitization in a childhood and adolescent population, to explore age- and gender-specific variations and finally to discover co-sensitivities among allergens. METHODS: A two-stage cross-sectional survey among school-aged children. The two stages of the study involved enrollment of schools and then skin prick testing (SPT) within schools. A total of 675 school children were included in the study. Of those, 231 were diagnosed with allergic rhinitis (AR), according to the medical history as provided by parental-completed questionnaires and positive SPT results. The antigen panel consisted of common allergens and more specifically house dust mites-HDM (Dermatophagoides farinae and Dermatophagoides pteronyssinus), grass mix, trees (olive, cypress and pine), weeds (Parietaria spp.), cat and dog epithelium and moulds (Alternaria spp., Cladosporium spp.). The SPT sensitivity was graded according to SPT-USA Standards. RESULTS: The overall prevalence rate of AR was 34.22%. In total, 93 school children (40.3%) were mono- and 138 (59.7%) were poly-sensitized. Overall, the most prevalent sensitizations in decreasing order were to HDM (59.74%), to grasses (48.9%), to Alternaria (34.6%) and to olive (14.71%). There were no age- and sex-specific differences, except for Alternaria mould that showed a significant prevalence among primary school-aged children and predominance in the female gender, by contrast to grass pollen allergy that was predominant to males. A 32% of SPT-positive individuals were not aware of their allergy, with no statistically significant differences between ages. Co-sensitivities were detected for grass pollens and pine and olive trees, for Alternaria and Cladosporium moulds, for cypress and pine trees, and finally for dog and cat danders. CONCLUSIONS: Given data among school-aged children should be a baseline from which to monitor disease trends and is considered important for the optimal management of AR patients.

Characteristics of laryngeal symptoms induced in patients with allergic rhinitis in an environmental challenge chamber.

BACKGROUND: People with allergic rhinitis often have laryngeal symptoms (LSs) in addition to nasal symptoms during the pollen-scattering season. OBJECTIVE: To clarify the characteristics of the LSs induced by pollen exposure using an environmental challenge chamber. METHODS: Cypress pollen exposure using an environmental challenge chamber for 25 participants with cypress pollen-induced allergic rhinitis was performed for 3 hours for 2 consecutive days in 3 study courses: namely, pollen exposure under normal nasal breathing and pollen or sham pollen exposure with nasal blockage, which eliminated any allergic reactions in the nasal mucosa. The nasal and LSs scores and the levels of serum inflammatory mediators, including eosinophil cationic protein (ECP), were monitored. Laryngeal examinations and physiologic lung tests were also conducted. RESULTS: Various LSs were reported, and these LSs were significantly elevated during pollen exposure and even under sham exposure with artificial nasal blockage. The pollen exposure with artificial nasal blockage exaggerated the LSs in 32% of the participants and also increased the serum ECP levels. The serum ECP levels did not change after sham exposure. The findings of both laryngeal examinations and lung tests failed to reveal any significant changes. CONCLUSION: Nasal obstruction could induce significant LSs even without pollen exposure. LSs were enhanced by pollen exposure and allergic reactions in the larynx could thus be involved in this enhancement. TRIAL REGISTRATION: clinicaltrials.gov Identifier: UMIN000015667.

Effect of desert dust exposure on allergic symptoms: A natural experiment in Japan.

BACKGROUND: Desert dust originating from arid and semiarid areas is transported to widespread regions, including Japan. Desert dust particles exert adjuvant effects in animals. OBJECTIVE: To examine whether desert dust enhances allergic symptoms in real-life settings and to explore its effect modifiers. METHODS: We conducted an observational study of 3,327 pregnant women during spring and fall in October 2011 to May 2013 in 3 regions in Japan as an adjunct study of the Japan Environment & Children's Study. We acquired participants' daily symptom scores by sending a questionnaire to their mobile phones on high desert-dust days (>0.07/km) and on some randomly selected other days (control days) for each participant. RESULTS: Pregnant women had an increased risk of allergic symptoms on high desert-dust days (adjusted odds ratio [OR], 1.10; 95% CI, 1.04-1.18). The increased OR was mostly driven by those who showed positive IgE to Japanese cedar pollen when pollen simultaneously dispersed (adjusted OR, 1.25; 95% CI, 1.13-1.38), whereas no clear risk increase was observed in the absence of pollen or for participants with negative IgE to Japanese cedar pollen. The risk elevation was observed from low levels of desert dust in a dose-dependent manner even on control days. CONCLUSION: Ambient desert dust level was associated with an increased risk of allergic symptoms in pollen-sensitized pregnant women when pollen was present in the air. The risk increase was dose dependent and was observed from low levels of desert dust. These results support a hypothesis that ambient desert dust particles exert adjuvant effects in human in real-life settings. TRIAL REGISTRATION: clinicaltrials.gov Identifier: UMIN000010826.

Cross-reactivity between major IgE core epitopes on Cry j 2 allergen of Japanese cedar pollen and relevant sequences on Cha o 2 allergen of Japanese cypress pollen.

BACKGROUND: Cry j 2 and Cha o 2 are major allergens in Japanese cedar (Cryptomeria japonica; CJ) and Japanese cypress (Chamaecyparis obtusa; CO) pollen, respectively. Here, we assessed the epitopes related to the cross-reactivity between Cry j 2 and Cha o 2 using in vitro analyses. METHODS: Peptides were synthesized based on Cry j 2 sequential epitopes and relevant Cha o 2 amino acid sequences. Four representative monoclonal antibodies (mAbs) against Cry j 2 were used according to their epitope recognitions. Serum samples were collected from 31 patients with CJ pollinosis. To investigate cross-reactivity between Cry j 2 and Cha o 2, ELISA and inhibition ELISA were performed with mAbs and sera from patients with CJ pollinosis. RESULTS: Two of four mAbs had reactivity to both Cry j 2 and Cha o 2. Of these two mAbs, one mAb (T27) recognized the amino acid sequence (169)KVVNGRTV(176) on Cha o 2. This is related to the core epitope (169)KWVNGREI(176) on Cry j 2, which is an important IgE epitope. In addition, we found that these correlative sequences and purified allergens showed cross-reactivity between Cry j 2 and Cha o 2 in IgE of CJ patients. CONCLUSIONS: We demonstrated the importance of (169)KVVNGRTV(176) in Cha o 2 for cross-reactivity with the Cry j 2 epitope (169)KWVNGREI(176), which plays an important role in allergenicity in CJ pollinosis. Our results are useful for the development of safer and more efficient therapeutic strategies for the treatment of CJ and CO pollen allergies.

Different allergenicity of pollen extracts of three Mediterranean cypress species accounted for cytological observations.

BACKGROUND: Cypresses play an important role in the urban landscape of the Mediterranean region, releasing a huge amount of allergenic airborne pollen which causes a specific pollinosis in exposed people. OBJECTIVE: The aim of this work is to evaluate, in vivo and in vitro, the potential allergenicity of Cupressus macrocarpa pollen, and to compare it with the allergenicity observed for C. arizonica and C. sempervirens. METHODS: Pollen extracts of the three species were prepared, to determine their protein profile through SDS PAGE analysis and to evaluate their allergenic potential through EAST inhibition assays and SPT. Pollen grain composition was evaluated using a cytochemical approach with optical microscopy. RESULTS: SDS PAGE, EAST inhibition and SPT indicated the higher allergenic potential of C. arizonica compared to C. sempervirens and C. macrocarpa. No significant differences in allergenic potential were found between the latter two species. Cytochemical observations reveal higher ß-glucans and protein content in the intine of C. arizonica during hydration. CONCLUSION: The higher protein content found in C. arizonica pollen grains extract may be due to higher enzyme activity leading to the movement of ß-glucans and pectins from the intine to the partially developed pollen cell wall during hydration. This could explain the higher potential allergenicity of C.arizonica in respect to C. macrocarpa and C. sempervirens.

Strong Induction of Minor Terpenes in Italian Cypress, Cupressus sempervirens, in Response to Infection by the Fungus Seiridium cardinale.

Seiridium cardinale, the main fungal pathogen responsible for cypress bark canker, is the largest threat to cypresses worldwide. The terpene response of canker-resistant clones of Italian cypress, Cupressus sempervirens, to two differently aggressive isolates of S. cardinale was studied. Phloem terpene concentrations, foliar terpene concentrations, as well as foliar terpene emission rates were analyzed 1, 10, 30, and 90 days after artificial inoculation with fungal isolates. The phloem surrounding the inoculation point exhibited de novo production of four oxygenated monoterpenes and two unidentified terpenes. The concentrations of several constitutive mono- and diterpenes increased strongly (especially α-thujene, sabinene, terpinolene, terpinen-4-ol, oxygenated monoterpenes, manool, and two unidentified diterpenes) as the infection progressed. The proportion of minor terpenes in the infected cypresses increased markedly from the first day after inoculation (from 10% in the control to 30-50% in the infected treatments). Foliar concentrations showed no clear trend, but emission rates peaked at day 10 in infected trees, with higher δ-3-carene (15-fold) and total monoterpene (10-fold) emissions than the control. No substantial differences were found among cypresses infected by the two fungal isolates. These results suggest that cypresses activate several direct and indirect chemical defense mechanisms after infection by S. cardinale.

Cypress pollen: an unexpected major sensitizing agent in different regions of Italy.

OBJECTIVES: In this multicenter survey, we assessed the impact of sensitization to cypress in atopic patients in Italy and determined whether cypress pollen concentration changed over time. METHODS: Allergists were required to collect the results of 100-200 consecutive skin prick tests (SPTs) performed during 2012. Seasonal symptoms were also recorded, as were airborne cypress pollen concentrations (data from the Italian Aerobiology Association) in 1998-2000 and 2010-2012. RESULTS: We examined 2258 atopic outpatients (56% females; age, 2-84 years) sensitized to at least 1 of the aeroallergens tested (Dermatophagoides species, grass, pellitory, olive, cypress, birch, Alternaria tenuis, and dog and cat dander). We found that 62.9%, 16.1%, and 32.7% of patients living in central, northern, and southern Italy, respectively, were sensitized to cypress (P < .0001). The cypress pollen concentration peak was delayed from February to March in 1998-2000 and 2010-2012 in all 3 regions, with a shift in pollination towards spring. Patients who were monosensitized to cypress reported mainly rhinitis (90.7%-97.6%) and conjunctivitis (38.1%-100%). In polysensitized patients, the prevalence of rhinitis, conjunctivitis, and asthma increased progressively (P < .0001) from southern to northern Italy. The same trend was observed for the prevalence of reported winter symptoms typical of cypress allergy (28%-65%). CONCLUSIONS: Today, cypress pollen is the most frequent sensitizing aeroallergen (assessed by SPT) in several areas of central Italy. Variations in the timing of the cypress pollination period may have favored this increased sensitization. Rhinitis and conjunctivitis are the predominant symptoms. The clinical impact of this allergy was poor in southern Italy and increased in central areas before reaching its peak in northern regions.

Development of a rice-based peptide vaccine for Japanese cedar and cypress pollen allergies.

Peptide immunotherapy using dominant T-cell epitopes is a safe treatment alternative to conventional subcutaneous injection of natural crude allergen extract, which is sometimes accompanied by anaphylactic shock. For Japanese cedar pollinosis (JCP), hybrid peptides composed of six to seven major T-cell epitopes (7Crp peptide) from the causative allergens Cry j 1 and Cry j 2 have been developed on the basis of different human leukemia antigen class II restrictions, because of the diversity of patients' genetic backgrounds. However, other dominant T-cell epitopes that are produced in some patients are not covered by these peptides. To develop a more universal peptide vaccine for JCP, we generated transgenic rice seeds containing seven new T-cell epitopes (Crp3) in addition to the T-cell epitopes used in the 7Crp peptide. Next, we co-expressed unique T-cell epitopes (6Chao) from the Japanese cypress pollen allergens Cha o 1 and Cha o 2 in transgenic rice seeds, with 7Crp and Crp3. These transgenic rice seeds, containing many highly homologous T-cell epitopes derived from cedar and cypress allergens, are expected to be applicable to a wide range of patients suffering from these pollen allergies.

Determination of allergenic load and pollen count of Cupressus arizonica pollen by flow cytometry using Cup a1 polyclonal antibody.

BACKGROUND: There is an increase in the incidence of pollen related allergy, thus information on pollen schedules would be a great asset for physicians to improve the clinical care of patients. Like cypress pollen sensitization shows a high prevalence among the causes of allergic rhinitis, and therefore it is of interest to use it like a model of study, distinguishing cypress pollen, pollen count, and allergenic load level. In this work, we use a flow cytometry based technique to obtain both Cupressus arizonica pollen count and allergenic load, using specific rabbit polyclonal antibody Cup a1 and its comparison with optical microscopy technique measurement. METHODS: Airborne samples were collected from Burkard Spore-Trap and Burkard Cyclone Cupressus arizonica pollen was studied using specific rabbit polyclonal antibody Cup a1, labeled with AlexaFluor(®) 488 or 750 and analysed by Flow Cytometry in both an EPICS XL and Cyan ADP cytometers (Beckman Coulter(®) ). Optical microscopy study was realized with a Leica optical microscope. Bland and Altman was used to determine agreement between both techniques measured. RESULTS: We can identify three different populations based on rabbit polyclonal antibody Cup a1 staining. The main region (44.5%) had 97.3% recognition, a second region (25%) with 28% and a third region (30.5%) with 68% respectively. Immunofluorescence and confocal microscopy showed that main region corresponds to whole pollen grains, the second region are pollen without exine and the third region is constituted by smaller particles with allergenic properties. Pollen schedule shows a higher correlation measured by optical microscopy and flow cytometry in the pollen count with a P-value: 0.0008 E(-2) and 0.0002 with regard to smaller particles, so the Bland and Altman measurement showed a good correlation between them, P-value: 0.0003. CONCLUSION: Determination of pollen count and allergenic load by flow cytometry represents an important tool in the determination of airborne respiratory allergens. We showed that not only whole pollen but also smaller particles could induce allergic sensitization. This is the first study where flow cytometry is used for calculating pollen counts and allergenic load.