Selection and characterization of DNA aptamers for highly selective recognition of the major allergen of olive pollen Ole e 1.

In this study, single-stranded DNA aptamers with binding affinity to Ole e 1, the major allergen of olive pollen, were selected using systematic evolution of ligands by exponential enrichment (SELEX) method. Binding of the aptamers was firstly established by enzyme-linked oligonucleotide assay (ELONA) and aptaprecipitation assays. Additionally, aptamer-modified monolithic capillary chromatography was used in order to evaluate the recognition of this allergenic protein against other non-target proteins. The results indicated that AptOle1#6 was the aptamer that provided the highest affinity for Ole e 1. The selected aptamer showed good selective recognition of this protein, being not able to retain other non-target proteins (HSA, cyt c, and other pollen protein such as Ole e 9). The feasibility of the affinity monolithic column was demonstrated by selective recognition of Ole e 1 in an allergy skin test. The stability and reproducibility of this monolithic column was suitable, with relative standard deviations (RSDs) in retention times and peak area values of 7.8 and 9.3%, respectively (column-to-column reproducibility). This is the first study that describes the design of an efficient DNA aptamer for this relevant allergen.

Peptide Allergen Immunotherapy: A New Perspective in Olive-Pollen Allergy.

Allergic diseases are highly prevalent disorders, mainly in industrialized countries where they constitute a high global health problem. Allergy is defined as an immune response "shifted toward a type 2 inflammation" induced by the interaction between the antigen (allergen) and IgE antibodies bound to mast cells and basophils that induce the release of inflammatory mediators that cause the clinical symptoms. Currently, allergen-specific immunotherapy (AIT) is the only treatment able to change the course of these diseases, modifying the type 2 inflammatory response by an allergenic tolerance, where the implication of T regulatory (Treg) cells is considered essential. The pollen of the olive tree is one of the most prevalent causes of respiratory allergic diseases in Mediterranean countries, inducing mainly nasal and conjunctival symptoms, although, in areas with a high antigenic load, olive-tree pollen may cause asthma exacerbation. Classically, olive-pollen allergy treatment has been based on specific immunotherapy using whole-olive pollen extracts. Despite extracts standardization, the effectiveness of this strategy varies widely, therefore there is a need for more effective AIT approaches. One of the most attractive is the use of synthetic peptides representing the B- or T-cell epitopes of the main allergens. This review summarizes experimental evidence of several T-cell epitopes derived from the Ole e 1 sequence to modulate the response to olive pollen in vitro, associated with several possible mechanisms that these peptides could be inducing, showing their usefulness as a safe preventive tool for these complex diseases.

Assessment of heterogeneity of two cultivars of Olea europaea based on the study of their Ole e 1 protein content.

Olive pollen is one of the main causes of allergic disease in the Mediterranean area. Ten different proteins with allergenic activity have been described in olive pollen, with major allergen Ole e 1. Olea europaea L. may cause allergenic effects of different severity depending on the Ole e 1 content of cultivars. In this paper, we aimed to assess the heterogeneity of two olive cultivars concerning concentrations of the major allergen Ole e 1 during a period of 2 years. Pollens from two most common olive cultivars, known as "Gemlik" and "Celebi," were analyzed on regular basis. Ole e 1 amounts were measured by double-sandwich enzyme-linked immunosorbent assay (ELISA). The results were expressed as µg of Ole e 1 per µg of total freeze-dried extract. Comparisons of Ole e 1 levels were made both between individual trees and between cultivars. It was analyzed the influence of some meteorological parameters on pollen counts/allergenic content on a local scale, for 2 years. Pollen sampling was carried out continuously for 2 years, using a Hirst-type volumetric trap. "Gemlik" had the higher value (mean ± standard deviation) of Ole e 1 content (2.44 ±0.70 and 1.87 ±1.03 µg/µg, respectively) when compared to "Celebi" (2.16 ±0.86 and 0.20 ±0.30 µg/µg, respectively) in the years 2013 and 2015. In our research, daily variations were observed in pollen samples of two olive cultivars and even different trees of the same cultivar. Furthermore, during certain sampling days, discrepancies between airborne pollen counts and Ole e 1 concentrations were detected for both cultivars. It was found that meteorological changes, especially temperature and precipitation fluctuations, could affect airborne pollen and Ole e 1 allergen levels in the atmosphere. Therefore, pollen samples of different O. europaea cultivars demonstrated great differences in Ole e 1 content. We believe that these findings were a result of alternate bearing behavior modulated by meteorological factors.

Secondary Outcomes of the Ole e 1 Proteins Involved in Pollen Tube Development: Impact on Allergies.

Ole e 1 protein is involved in olive fertilization mechanisms controlling pollen tube development. Similarly to the process by which pollen grains hydrated and form a pollen tube upon arrival at the female gametophyte, when pollen grains fall on the nasal mucosa the expression of Ole e 1 protein induce allergic reaction in sensitive individuals. The research was conducted in Ourense (North-western Spain), during the 2009-2018 period. Ole e 1 protein was collected using a Cyclone Sampler and processed with the ELISA methodology. Airborne Olea pollen were monitored using a Hirst type volumetric sampler. Allergy risk episodes identified by pollen concentrations were detected in five of the 10 studied years, all with moderate risk. Actual risk episodes of allergy increased when the combination of pollen and Ole e 1 concentrations were considered. Moderate risk episodes were detected during 9 years and high-risk episodes during 3 years. In addition, some years of low annual pollen concentrations recorded high total amounts of Ole e 1. During the years with lower pollen production, the tree increases the synthesis of Ole e 1 to ensure proper pollen tube elongation in order to complete a successful fertilization. This fact could justify higher sensitization rates in years in which a lower pollen production is expected. The present method contributes to the determination of the real exposure to Ole e 1 allergen evaluating the role of this protein as an aeroallergen for sensitized population. The allergen content in the atmosphere should be considered to enhance the prevention of pollinosis clinical symptomatology and the reduction of medicine consumption.

Immunochemical and Physical Quantitation of Grass and Olive Pollen Allergens: Correlation With Asthma Admissions in Cáceres, Spain.

BACKGROUND AND OBJECTIVES: The association between pollen counts and allergen levels in the air is controversial. Objectives: The aims of the study were to quantify total and major allergen levels of Phleum pratense and Olea europaea and to analyze their correlation with grass and olive pollen counts and the number of asthma attacks attended at Complejo Hospitalario Universitario, Cáceres, Spain. MATERIAL AND METHODS: A volumetric air sampler and a Burkard spore trap were used for pollen and aeroallergen collection during April- June 2011. Filters were extracted, and major allergens were quantified using enzyme-linked immunosorbent assay. RESULTS: May was the main grass pollination period, with a maximum peak of 1362 grains/m3 (May 13). The main pollination period for olive was April 30-May 20, with a maximum peak of 851 grains/m3 (May 11). A moderate correlation was observed between asthma exacerbations and grass pollen counts or Phleum total allergen levels; this became stronger when a 3-day offset was introduced. A significant association was observed between asthma exacerbations and total olive allergen or olive pollen grain levels when a 1-day offset was introduced. The maximum correlation (moderate-high) was observed 4 days and 6 days away from the maximum olive pollen peak and the maximum Ole e 1 peak level, respectively. CONCLUSIONS: This study reveals a significant correlation between grass and olive pollination and an increase in the number of visits to the emergency room for asthma attacks. The aerobiological pattern of allergen levels in the air is similar to that of pollen counts during the grass and olive pollination periods.

Endolysosomal Degradation of Allergenic Ole e 1-Like Proteins: Analysis of Proteolytic Cleavage Sites Revealing T Cell Epitope-Containing Peptides.

Knowledge of the susceptibility of proteins to endolysosomal proteases provides valuable information on immunogenicity. Though Ole e 1-like proteins are considered relevant allergens, little is known about their immunogenic properties and T cell epitopes. Thus, six representative molecules, i.e., Ole e 1, Fra e 1, Sal k 5, Che a 1, Phl p 11 and Pla l 1, were investigated. Endolysosomal degradation and peptide generation were simulated using microsomal fractions of JAWS II dendritic cells. Kinetics and peptide patterns were evaluated by gel electrophoresis and mass spectrometry. In silico MHC (major histocompatibility complex) class II binding prediction was performed with ProPred. Cleavage sites were assigned to the primary and secondary structure, and in silico docking experiments between the protease cathepsin S and Ole e 1 were performed. Different kinetics during endolysosomal degradation were observed while similar peptide profiles especially at the C-termini were detected. Typically, the identified peptide clusters comprised the previously-reported T cell epitopes of Ole e 1, consistent with an in silico analysis of the T cell epitopes. The results emphasize the importance of the fold on allergen processing, as also reflected by conserved cleavage sites located within the large flexible loop. In silico docking and mass spectrometry results suggest that one of the first Ole e 1 cleavages might occur at positions 107-108. Our results provided kinetic and structural information on endolysosomal processing of Ole e 1-like proteins.

Polymorphism and structure of style-specific arabinogalactan proteins as determinants of pollen tube growth in Nicotiana.

BACKGROUND: Pollen tube growth and fertilization are key processes in angiosperm sexual reproduction. The transmitting tract (TT) of Nicotiana tabacum controls pollen tube growth in part by secreting pistil extensin-like protein III (PELPIII), transmitting-tract-specific (TTS) protein and 120 kDa glycoprotein (120 K) into the stylar extracellular matrix. The three arabinogalactan proteins (AGP) are referred to as stylar AGPs and are the focus of this research. The transmitting tract regulates pollen tube growth, promoting fertilization or rejecting pollen tubes. RESULTS: The N-terminal domain (NTD) of the stylar AGPs is proline rich and polymorphic among Nicotiana spp. The NTD was predicted to be mainly an intrinsically disordered region (IDR), making it a candidate for protein-protein interactions. The NTD is also the location for the majority of the predicted O-glycosylation sites that were variable among Nicotiana spp. The C-terminal domain (CTD) contains an Ole e 1-like domain, that was predicted to form beta-sheets that are similar in position and length among Nicotiana spp. and among stylar AGPs. The TTS protein had the greatest amino acid and predicted O-glycosylation conservation among Nicotiana spp. relative to the PELPIII and 120 K. The PELPIII, TTS and 120 K genes undergo negative selection, with dn/ds ratios of 0.59, 0.29 and 0.38 respectively. The dn/ds ratio for individual species ranged from 0.4 to 0.9 and from 0.1 to 0.8, for PELPIII and TTS genes, respectively. These data indicate that PELPIII and TTS genes are under different selective pressures. A newly discovered AGP gene, Nicotiana tabacum Proline Rich Protein (NtPRP), was found with a similar intron-exon configuration and protein structure resembling other stylar AGPs, particularly TTS. CONCLUSIONS: Further studies of the NtPRP gene are necessary to elucidate its biological role. Due to its high similarity to the TTS gene, NtPRP may be involved in pollen tube guidance and growth. In contrast to TTS, both PELPIII and 120 K genes are more diverse indicating a possible role in speciation or mating preference of Nicotiana spp. We hypothesize that the stylar AGPs and NtPRP share a common origin from a single gene that duplicated and diversified into four distinct genes involved in pollen-style interactions.

Correlation between airborne Olea europaea pollen concentrations and levels of the major allergen Ole e 1 in Córdoba, Spain, 2012-2014.

Olea europaea L. pollen is the second-largest cause of pollinosis in the southern Iberian Peninsula. Airborne-pollen monitoring networks provide essential data on pollen dynamics over a given study area. Recent research, however, has shown that airborne pollen levels alone do not always provide a clear indicator of actual exposure to aeroallergens. This study sought to evaluate correlations between airborne concentrations of olive pollen and Ole e 1 allergen levels in Córdoba (southern Spain), in order to determine whether atmospheric pollen concentrations alone are sufficient to chart changes in hay fever symptoms. The influence of major weather-related variables on local airborne pollen and allergen levels was also examined. Monitoring was carried out from 2012 to 2014. Pollen sampling was performed using a Hirst-type sampler, following the protocol recommended by the Spanish Aerobiology Network. A multi-vial cyclone sampler was used to collect aeroallergens, and allergenic particles were quantified by ELISA assay. Significant positive correlations were found between daily airborne allergen levels and atmospheric pollen concentrations, although there were occasions when allergen was detected before and after the pollen season and in the absence of airborne pollen. The correlation between the two was irregular, and pollen potency displayed year-on-year variations and did not necessarily match pollen-season-intensity.

Oleaceae cross-reactions as potential pollinosis cause in urban areas.

It is worth noting the allergological problems induced by a not accurate design of the ornamental vegetation in the parks and streets of the cities. Usually, in the Oleaceae family, only the olive pollen is considered an important aeroallergen but other species of the family could be an important source of airborne pollen allergens. Pollen from Fraxinus, Olea and Ligustrum and its main aeroallergens were sampled in the atmosphere of an urban area in North-Western Spain during 2011. The allergen bioaerosol content was quantified by using specific 2-site ELISA and Ole e 1 antibodies. The Fra e 1 and Lig v 1 allergens were detected by means Ole e 1 antibodies. This fact demonstrates the cross-reactivity between the main allergens of Fraxinus, Olea and Ligustrum, plants widely species used as ornamental in the cities. Therefore, the urban allergenic people sensitized to Olea pollen could present allergenic reactions during the winter (due to ash pollen allergens), the spring (caused by olive pollen allergens) and the early summer (triggered by the privet flowering). As a consequence, sensitivity to the pollen of one species may favour development of sensitivity to all three species as consequence of the priming effect. The combination of pollen count and the allergen quantification must be assessed in the epidemiologic study of allergic respiratory diseases.

Immunologic responses to the major allergen of Olea europaea in local and systemic allergic rhinitis subjects.

OBJECTIVE: To evaluate the in vivo and in vitro responses to nOle e 1 in allergic rhinitis (AR) and local allergic rhinitis (LAR) patients sensitized to olive tree pollen (OL) confirmed by nasal allergen provocation test (NAPT). METHODS: Twelve subjects with AR, 12 with LAR and 12 subjects as control group (CG) were selected. Skin testing and NAPT with nOle e 1 were performed. Eosinophilic cationic protein (ECP) and tryptase were measured in nasal lavages before and after NAPT. Serum IgE to OL allergens was measured by ELISA. Basophil activation tests (BAT) with OL and nOle e 1 and dendritic cell maturation/proliferation studies were carried out. RESULTS: All AR (12/12) and 10/12 (83%) of LAR had a +NAPT to nOle e 1. ECP levels in nasal lavages were significantly increased after NAPT in both AR and LAR compared with CG at 15 min (P < 0.05). Serum IgE was positive only in AR. All AR had +BAT responses to OL and 10/12 to nOle e 1 (83%); 8/12 LAR (66.6%) had a +BAT to OL and 4/12 (33%) to nOle e 1, with only one subject of the CG with a +BAT to both OL and nOle e 1 (8%). Dendritic cell proliferation to nOle e 1 was increased in AR compared to LAR and CG (P = 0.019 and P = 0.001, respectively). CONCLUSION: Both AR and LAR had a similar in vivo response to nOle e 1 with release of inflammatory mediators. Specific basophil activation with OL and nOle e 1 was observed in LAR confirming previous data obtained with dust mites.

Ash pollen allergy: reliable detection of sensitization on the basis of IgE to Ole e 1.

Background: Alongside hazel, alder and birch pollen allergies, ash pollen allergy is a relevant cause of hay fever during spring in the European region. For some considerable time, ash pollen allergy was not routinely investigated and its clinical relevance may well have been underestimated, particularly since ash and birch tree pollination times are largely the same. Ash pollen extracts are not yet well standardized and diagnosis is therefore sometimes unreliable. Olive pollen, on the other hand, is strongly cross-reactive with ash pollen and is apparently better standardized. Therefore, the main allergen of olive pollen, Ole e 1, has been postulated as a reliable alternative for the detection of ash pollen sensitization. Methods: To determine to what extent specific IgE against Ole e 1 in patients with ash pollen allergy is relevant, we included 183 subjects with ash pollen allergy displaying typical symptoms in March/April and positive skin prick test specific IgE against Ole e 1 (t224) and ash pollen (t25) and various birch allergens (Bet v 1, Bet v 2/v 4) in a retrospective study. Results: A significant correlation was seen between specific IgE against Ole e 1 and ash pollen, but also to a slightly lesser extent between IgE against Ole e 1 and skin prick test with ash pollen, the latter being even higher than IgE and skin prick test both with ash pollen. No relevant correlation was found with birch pollen allergens, demonstrating the very limited cross-reactivity between ash and birch pollen. Conclusion: It appears appropriate to determine specific IgE against Ole e 1 instead of IgE against ash pollen to detect persons with ash pollen allergy. Our findings may also support the idea of using possibly better standardized or more widely available olive pollen extracts instead of ash pollen extract for allergen-specific immunotherapy.