Environmental exposure and sensitization patterns in a Swiss alpine pediatric cohort

Background The level of environmental exposure throughout life may contribute to the prevalence of allergic sensitization and allergic disease. The alpine climate has been considered a healthy climate with little allergen exposure and pollution. We conducted a cross-sectional study to investigate local environmental exposure and concomitant prevalence of allergic sensitization among local school children born and raised in an alpine environment. Methods Clinical and demographic data were collected with a questionnaire. Allergen content was assessed in residential settled dust samples, lifetime exposure to pollen and air pollution was calculated using data from national pollen and air pollution monitoring stations, and the allergic sensitization profile was determined with component resolved diagnostics (ISAC®). Univariate and multivariate regression models were used to estimate the relation between exposure and sensitization. Results In a cohort of children born and raised in an alpine environment, sensitization to aeroallergens is quite common (38%), especially to grass (33%) and cat (16%). House dust mite allergen was detected in up to 38% of residential dust samples, but sensitization to HDM was low (2.5%). Pollutant levels were low, but an increasing trend was observed in the amount of ozone and PM10. Living close to a busy road was associated with increased odds OR (95% CI) for being sensitized to any allergen 2.7 (1.0–7.2), to outdoor allergens 2.8 (1.1–7.1) and being sensitized plus reporting symptoms of rhinoconjunctivitis 4.4 (1.3–14.8) and asthma 5.5 (1.4–21). Indoor living conditions, including the presence of visible mold, increased the odds of being sensitized to indoor allergens (1.9 (1.1–3.2) and being sensitized plus reporting symptoms of rhinoconjunctivitis 1.9 (1.0–3.6) and asthma 2.1 (1.0–4.1). Conclusion In a healthy alpine environment, pollution might still be an important factor contributing to allergic sensitization

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

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

Automatic and online pollen monitoring

BACKGROUND: Pollen are monitored in Europe by a network of about 400 pollen traps, all operated manually. To date, automated pollen monitoring has only been feasible in areas with limited variability in pollen species. There is a need for rapid reporting of airborne pollen as well as for alleviating the workload of manual operation. We report our experience with a fully automated, image recognition-based pollen monitoring system, BAA500. METHODS: The BAA500 sampled ambient air intermittently with a 3-stage virtual impactor at 60 m(3)/h in Munich, Germany. Pollen is deposited on a sticky surface that was regularly moved to a microscope equipped with a CCD camera. Images of the pollen were constructed and compared with a library of known samples. A Hirst-type pollen trap was operated simultaneously. RESULTS: Over 480,000 particles sampled with the BAA500 were both manually and automatically identified, of which about 46,000 were pollen. Of the automatically reported pollen, 93.3% were correctly recognized. However, compared with manual identification, 27.8% of the captured pollen were missing in the automatic report, with most reported as unknown pollen. Salix pollen grains were not identified satisfactorily. The daily pollen concentrations reported by a Hirst-type pollen trap and the BAA500 were highly correlated (r = 0.98). CONCLUSIONS: The BAA500 is a functional automated pollen counter. Its software can be upgraded, and so we expected its performance to improve upon training. Automated pollen counting has great potential for workload reduction and rapid online pollen reporting

Year-to-year variation in release of Bet v 1 allergen from birch pollen: evidence for geographical differences between West and South Germany

The release of the aeroallergen Bet v 1 from pollen is a major determinant in the etiology of allergic airway disease due to birch pollen. OBJECTIVE: We determined the release of the major birch pollen allergen Bet v 1 from pollen of birch trees growing in 2 different geographic regions in Germany for 2 consecutive years. METHODS: Catkins were collected during pollination in 2002 and 2003 from 82 healthy trees in South (Munich) and West Germany (North Rhine-Westphalia). The release of Bet v 1 from pollen samples was determined by a Bet v 1-specific ELISA. RESULTS: Pollen from South Germany released about 3 times more Bet v 1 than those from West Germany in both 2002 and 2003 (p = 0.034 and p = 0.007, respectively). This was independent of the number of pollen during the pollen flight season. In 2003, the release of Bet v 1 from pollen was more than 5 times higher than in 2002 in both regions (South Germany 6.1 times, p < 0.001; West Germany 5.4 times, p = 0.003). CONCLUSIONS: Despite large individual differences, there seem to be regional and year-to-year variations in Bet v 1 release from birch pollen. Therefore, the combination of pollen count and release of Bet v 1 from this pollen must be assessed to estimate Bet v 1 exposure reliably. 2007 S. Karger AG, Base

Predicting the main pollen season of Broussonetia Papyrifera (paper mulberry) tree

Paper mulberry pollen, declared a pest in several countries including Pakistan, can trigger severe allergies and cause asthma attacks. We aimed to develop an algorithm that could accurately predict high pollen days to underpin an alert system that would allow patients to take timely precautionary measures. We developed and validated two prediction models that take historical Nov 15, 2023 2/18 pollen and weather data as their input to predict the start date and peak date of the pollen season in Islamabad, the capital city of Pakistan. The first model is based on linear regression and the second one is based on phenological modelling. We tested our models on an original and comprehensive dataset from Islamabad. The mean absolute errors (MAEs) for the start day are 2.3 and 3.7 days for the linear and phenological models, respectively, while for the peak day, the MAEs are 3.3 and 4.0 days, respectively. These encouraging results could be used in a website or app to notify patients and healthcare providers to start preparing for the paper mulberry pollen season. Timely action could reduce the burden of symptoms, mitigate the risk of acute attacks and potentially prevent deaths due to acute pollen-induced allergy

High environmental ozone levels lead to enhanced allergenicity of Birch pollen

BACKGROUND: Evidence is compelling for a positive correlation between climate change, urbanisation and prevalence of allergic sensitisation and diseases. The reason for this association is not clear to date. Some data point to a pro-allergenic effect of anthropogenic factors on susceptible individuals. OBJECTIVES: To evaluate the impact of urbanisation and climate change on pollen allergenicity. METHODS: Catkins were sampled from birch trees from different sites across the greater area of Munich, pollen were isolated and an urbanisation index, NO(2) and ozone exposure were determined. To estimate pollen allergenicity, allergen content and pollen-associated lipid mediators were measured in aqueous pollen extracts. Immune stimulatory and modulatory capacity of pollen was assessed by neutrophil migration assays and the potential of pollen to inhibit dendritic cell interleukin-12 response. In vivo allergenicity was assessed by skin prick tests. RESULTS: The study revealed ozone as a prominent environmental factor influencing the allergenicity of birch pollen. Enhanced allergenicity, as assessed in skin prick tests, was mirrored by enhanced allergen content. Beyond that, ozone induced changes in lipid composition and chemotactic and immune modulatory potential of the pollen. Higher ozone-exposed pollen was characterised by less immune modulatory but higher immune stimulatory potential. CONCLUSION: It is likely that future climate change along with increasing urbanisation will lead to rising ozone concentrations in the next decades. Our study indicates that ozone is a crucial factor leading to clinically relevant enhanced allergenicity of birch pollen. Thus, with increasing temperatures and increasing ozone levels, also symptoms of pollen allergic patients may increase further

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

COEXPRESSION OF CYTOCHROME P4502A6 AND HUMAN NADPH-P450 OXIDOREDUCTASE IN THE BACULOVIRUS SYSTEM

ABSTRACT: Heterologous expression using baculovirus vectors has become a popular method for the production of catalytically active cytochrome P450s (CYPs). We have systematically optimized the multiplicity of infection (MOI) for a coinfection approach for the coexpression of CYP2A6 (viral vector designated v2A6) and NADPH-P450 oxidoreductase (OR; viral vector designated vOR) using Sf9 insect cells. A 3000-fold range of MOI was examined in stationary culture and stirred suspension culture. Surprisingly, our results indicate that the best CYP2A6 catalytic activity (850-1300 pmol/ min/mg total lysate protein as measured by coumarin 7-hydroxylase activity) was obtained only when using a low MOI of v2A6 (1.5-3 ؋ 10 ؊2 ) and a vOR of 10-to 20-fold less. This activity was ϳ7-to 11-fold higher than the best activity obtained when infecting cells with v2A6 alone. At this level of coinfection, the P450 content ranged from 180 to 250 pmol/mg total lysate protein, and the NADPH cytochrome c reductase activity ranged from 350 to 520 nmol/min/mg total lysate protein. Increasing the MOI of both viruses to 50-fold higher resulted in lower overall activity with the optimum (250 pmol/min/mg total lysate protein) being seen earlier postinfection (60 vs. 72 hr). Increasing the MOI of vOR to levels comparable with those of v2A6, decreased coumarin 7-hydroxylase activity 14-fold. These results suggest that the best CYP2A6 catalytic activity depends on properly posttranslationally modified proteins accumulating in a right ratio as a result of primary, secondary, and possibly tertiary infection of both viruses. These results also suggest that high OR expression results in degradation of P450. CYPs 1 are a multienzyme, membrane-bound system that metabolizes many drugs and other xenobiotics (1). The catalytic activity of CYP enzymes requires the presence of NADPH-CYP OR. In addition, cytochrome b 5 stimulates catalytic activity for some CYP forms (2). Several efficient systems for the heterologous expression of mammalian CYP enzymes have been developed, including bacterial, yeast, and mammalian and baculovirus/insect cell-based systems (reviewed in ref. 2). Of the systems available, the baculovirus system has distinct advantages for the production of high levels of active, native CYP enzyme. Although efficient mammalian CYP expression is possible in bacterial systems, modification of the amino acid sequence is usually required for high level expression (2). Three distinct approaches have been taken for production of catalytically active CYP enzymes using baculovirus system. Expression of the CYP enzyme alone and then reconstitution with OR using purified P450s or total cell lysate (3-5), coexpression of the CYP and OR using a single virus (6), and coinfection with independent viruses containing CYP or OR (7). Each approach has unique advantages and disadvantages. Purification/reconstitution provides flexibility in controlling the CYP to OR ratio, but is time-and labor-intensive because of the need for column purifications. The single virus approach is more time-and labor-efficient (simple cell lysate can be used), but, because of the small number of promoters characterized for baculovirus expression, the ability to control the CYP to OR ratio is limited The human CYP2A6 is the only enzyme known to date responsible for coumarin 7-hydroxylase activity in human liver (9, 10). CYP2A6 also metabolically activates certain carcinogens and promutagens (for review, see refs. 1, 9, and 10). It has the highest activity for activation of the mutagen N-nitrosodiethylamine among all human P450s studied (9, 11). It also activates tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal, n-nitrosonornicotine (12, 13), and the mycotoxin aflatoxin B1 In this study, we describe a systematic characterization of a coinfection approach using CYP2A6 and human OR. To obtain an overall phenomena for coexpression of P450 and OR proteins in the baculovirus system, we examined a wide range of MOI for both v2A6 and vOR, with different ratios for coinfection to determine the best ratio for optimal coumarin 7-hydroxylase catalytic activity in insect cell lysate. Materials and Methods Rabbit anti-rat 2A1 (16) and rabbit anti-rat OR (17) antibodies were described in the previous studies. Recombinant baculoviruses v2A6 and vOR This study was supported in part by SBIR Contract N43-ES-31001 from the National Institute of Environmental Health Sciences. 1 Abbreviations used are: CYP, cytochrome P450; OR, NADPH-P450 oxidoreductase; P450, cytochrome P450; MOI, multiplicity of infection (the number of virus used per cell); v2A6, a recombinant baculovirus containing the human CYP2A6 cDNA; vOR, a recombinant baculovirus containing the human OR cDNA; Sf9, Spodoptera frugiperda; pfu, plaque-forming units (measurement of infectivity of a virus); AcMNPV, Autographa californica nuclear polyhedrosis virus. Send reprint requests to: Dr. Liping Chen