Olive Pollen Extracts Research Articles

L’olivier, une richesse méditerranéenne au prix d’une allergie complexe

The olive tree (Olea europea L.) is a characteristic tree of Mediterranean basin countries, responsible for the most frequent pollinosis. This pollinosis is influenced by climate change, which can anticipate flowering dates, increase the quantity of pollen produced, and the pollen season length. The occurrence of olive pollinosis mainly depends on HLA class II, IL-13 and IL-4RA genes; and environment factors, such as socioeconomic and nutritional status, physical activity and exposure to different stimuli. Among deferent categories of molecular allergens involved in olive pollinosis, Ole e 1 is the main allergen, and the presence of homology between these allergens, and those of other species from the Oleaceae family and other different taxons (Poaceae), is responsible for cross-reactions. Polysensitization is a major challenge in the diagnostic and therapeutic approach and highlights the importance of using molecular allergens in the pollinosis diagnostic. Furthermore, allergy to the olive tree has nowadays benefited from progress in specific immunotherapy, thanks in particular to the development of a strategy based on allergenic peptides as an alternative to whole olive tree pollen extracts.

Detection of pan-allergens in commercial pollen extracts for allergen immunotherapy

BackgroundUp to 50% of patients with pollen allergy are sensitized to at least 1 of the 2 pollen pan-allergens profilin and polcalcin. These allergens could have clinical relevance but the content of profilin and polcalcin in commercial extracts for allergen immunotherapy (AIT) is unknown. ObjectiveTo detect these pan-allergens in commercial pollen extracts for AIT from various sources. MethodsImmunoglobulin E (IgE) reactivity to Phl p 7 and Bet v 2 of sera from 18 adults hypersensitive to profilin and/or polcalcin was investigated by enzyme-linked immunosorbent assay before and after absorption with grass, birch, ragweed, pellitory, and olive pollen extracts for AIT from different producers. Immunoblot inhibition experiments also were carried out using the same allergens. ResultsBirch, grass, ragweed, and olive pollen extracts for AIT contained large amounts of profilin, inducing 80% to 90% inhibition in most cases; Parietaria AIT extract appeared to contain little profilin. On immunoblot, grass and birch pollen extracts for sublingual AIT completely absorbed IgE specific for rBet v 2. Interestingly, only grass pollen extracts induced a significant inhibition of IgE binding to rPhl p 7 on enzyme-linked immunosorbent assay and immunoblot. A grass pollen allergoid lost most of its inhibitory potency, suggesting a much weakened affinity for specific IgE. ConclusionWith the exception of Parietaria, commercial extracts for AIT of most pollens are rich in profilin and, hence, potentially able to desensitize to this allergen; in contrast, only grass pollen extracts seem rich in polcalcin. These are the pollens to use in case of severe symptoms induced by pollen pan-allergens.

Therapeutic targets for olive pollen allergy defined by gene markers modulated by Ole e 1-derived peptides

Two regions of Ole e 1, the major olive-pollen allergen, have been characterized as T-cell epitopes, one as immunodominant region (aa91-130) and the other, as mainly recognized by non-allergic subjects (aa10-31). This report tries to characterize the specific relevance of these epitopes in the allergic response to olive pollen by analyzing the secreted cytokines and the gene expression profiles induced after specific stimulation of peripheral blood mononuclear cells (PBMCs). PBMCs from olive pollen-allergic and non-allergic control subjects were stimulated with olive-pollen extract and Ole e 1 dodecapeptides containing relevant T-cell epitopes. Levels of cytokines were measured in cellular supernatants and gene expression was determined by microarrays, on the RNAs extracted from PBMCs. One hundred eighty-nine differential genes (fold change >2 or <−2, P<0.05) were validated by qRT-PCR in a large population.It was not possible to define a pattern of response according the overall cytokine results but interesting differences were observed, mainly in the regulatory cytokines. Principal component (PCA) gene-expression analysis defined clusters that correlated with the experimental conditions in the group of allergic subjects. Gene expression and functional analyses revealed differential genes and pathways among the experimental conditions. A set of 51 genes (many essential to T-cell tolerance and homeostasis) correlated with the response to aa10-31 of Ole e 1. In conclusion, two peptides derived from Ole e 1 could regulate the immune response in allergic patients, by gene-expression modification of several regulation-related genes. These results open new research ways to the regulation of allergy by Oleaceae family members.

Chip‐based capillary electrophoresis profiling of olive pollen extracts used for allergy diagnosis and immunotherapy

Standardization of protein extracts for clinical purposes represents an important task in order to maintain adequate reactivity, presence of the relevant allergens, and safety among other factors. The main objective of this work was to explore the potential use of a chip-based automated CE system commercially available to analyze several of the most common forms of allergenic extracts from olive pollen used in allergy clinics. These include experimental extracts prepared from olive pollens, in-house reference extracts, extracts designed for skin prick test assays, and a panel of vaccine variants aimed to specific immunotherapy. As a major conclusion of the study, chip-based CE allowed in all cases to determine accurate protein profiles with different degrees of sensitivity, where several allergens (particularly the major olive pollen allergen Ole e 1) were easily recognized. Moreover, several purified allergens were also analyzed by this method, and proposed as specific standards for different purposes. In the present condition, the method can only provide the protein profile of the extracts with respect to a preestablished standard extract, but not allergen identification. However, these and other future developments and applications are discussed.

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

SummaryBackground: 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.

Evaluation of in Vivo and in Vitro Responses to Nole e1 in Systemic and Local Allergic Rhinitis

The aim of this study is evaluating in vivo and in vitro responses to purified allergen nOle e1 in local allergic rhinitis (LAR) compared to systemic allergic rhinitis (SAR). nOle e1 was purified from olive tree pollen by RP-HPLC chromatography. Subjects with positive skin prick test (SPT), specific IgE (sIgE) to olive >0.35 kU/L, and positive nasal challenge with commercial olive pollen extract were recruited (SAR subjects). Also, subjects with negative SPT, sIgE to olive <0.35 kU/L, and positive nasal challenge with commercial olive pollen extract were selected (LAR subjects). All subjects underwent SPT and nasal challenge with nOle e1 (0.5,1,5 and 10 mcg/ml) monitored by acoustic rhinometry. Basophil activation test (BAT) was performed at 0.1, 0.5,1 and 5 mcg/ml with nOle e1 and whole commercial extract (≥ 2 SI). Ethical committee approved the study. Six SAR subjects and 4 LAR subjects were selected for the study. SPT with nOle e1 was positive in 100% SAR subjects and all LAR subjects had negative skin tests. All SAR and LAR subjects had positive responses to nOle e1 nasal challenge. Of SAR subjects, 100% of them had positive BAT with whole commercial olive extract and 75% with nOle e1. LAR subjects showed a 75% positive BAT responses to whole commercial olive extract but no positive responses to nOle e1. Subjects with LAR showed positive responses to nOle e1 in vivo. In vitro responses to whole olive pollen extract were detected in a significant percentage of subjects with LAR.

Desensitization Effect of Preseasonal Seven-Injection Allergoid Immunotherapy with Olive Pollen on Basophil Activation: The Efficacy of Olive Pollen-Specific Preseasonal Allergoid Immunotherapy on Basophils

Background: It has previously been demonstrated that subcutaneous immunotherapy with allergoids positively affects clinical and immunological parameters even after 7 preseasonal injections. However, its effect on basophil activation remains unclear. We investigated the effect of preseasonal allergoid immunotherapy on basophils and concomitantly assessed its clinical and immunological efficacy in olive pollen-monosensitized patients. Methods: This study enrolled 437 consecutive patients with respiratory allergy and positive skin prick tests (SPTs); 212 (48.5%) patients were sensitized to olive pollen, and 33 (7.5%) patients were sensitized to olive pollen only. Of these patients, 23 received preseasonal immunotherapy with an olive pollen allergoid. The olive pollen-specific basophil activation, the titrated nasal provocation test, the nasal symptom score, and olive pollen-specific IgE, IgG1 and IgG4 levels were evaluated before immunotherapy and 8 months after the end of immunotherapy in the follow-up visit. Results: In comparison to baseline evaluation, 7 preseasonal injections of an allergoid resulted in a significant decrease in the percentage of basophils expressing CD63 (29 vs. 7%, respectively, p < 0.0001) and a significant increase in the titrated nasal provocative dose (1/10 vs. 1/1, respectively, p < 0.01). SPT induration diameters caused by an olive pollen extract decreased (12 mm at baseline vs. 5.5 mm at follow-up, p < 0.005), as did nasal symptom score (7 at baseline vs. 3 at follow-up, p < 0.01). Olive pollen-specific IgE (17.5 vs. 50 kU/l, p < 0.012), IgG1 (0.16 vs. 2.9 µg/ml, p < 0.0001) and IgG4 (0.07 vs. 1.92 µg/ml, p < 0.0001) levels significantly increased. Conclusions: Immunotherapy with 7 preseasonal injections of an olive pollen allergoid decreases olive pollen-specific basophil activation over 8 months, an effect observed in vitro and in vivo.

The Major Allergen of Olive Pollen Ole e 1 Is a Diagnostic Marker for Sensitization to Oleaceae

Background: Trees of the family Oleaceae are important allergen sources, with a strongly varying geographic distribution. For example, olive pollen is an important allergen source in Mediterranean countries, whereas ash pollen dominates in Northern and Central Europe and North America. The aim of this study was to compare the profiles of olive and ash pollen allergens and to study the degree of cross-reactivity using populations of allergic patients selectively exposed to olive or ash pollen. Methods: Olive and ash pollen extracts were analyzed by IgE immunoblotting using sera from Spanish patients highly exposed to olive pollen and Austrian patients without olive but ash pollen exposure. IgE cross-reactivity was studied by qualitative immunoblot inhibition assays and semiquantitative ELISA inhibitions using olive, ash, birch, mugwort, timothy grass pollen extracts and the major olive pollen allergen, Ole e 1. Results: Spanish and Austrian patients exhibited an almost identical IgE-binding profile to olive and ash pollen allergens, with major reactivity directed against Ole e 1, and its homologous ash counterpart, Fra e 1. IgE inhibition experiments demonstrated extensive cross-reactivity between olive and ash pollen allergens. However, whereas cross-reactions between profilins and calcium-binding allergens also occurred between unrelated plant species, cross-reactivity to Ole e 1 was confined to plants belonging to the Oleaceae. Conclusions: Ole e 1 is a marker allergen for the diagnosis of olive and ash pollen allergy.

Esche ist nicht Esche

Pollinosis during spring (March/April) is attributed mostly to birch (Betula sp.) Pollen (BP). Due to an increasing number of ash (Fraxinus sp.) pollen (AP) counted in Switzerland during the last decades, their clinical relevance was evaluated in a study from the Allergy Unit in Zurich, published 1994 in this journal. Patients suffering from the so-called spring pollinosis were sensitized not only to hazel, alder and birch pollen, but also frequently to AP. In 72% of the skin prick test-positive patients to AP, nasal provocation tests were also positive. Because of moderate cross-reactivity to BP and significant monovalent sensitization, it was concluded that AP should be included in the routine prick skin testing (SPT) of patients suffering from spring pollinosis, when AP are present in the environment. Therefore, AP should also be included in vaccines for specific immunotherapy (SIT) of patients with tree pollen allergies. The genus Fraxinus belongs to the family Oleaceae, closely related to jasmine (Jasminum), lilac (Syringa), privet (Ligustrum), forsythia (Forsythia) and olive (Olea). Using ash and olive pollen extracts for IgE inhibition studies, evidence was provided for the presence of cross-reactive allergens/epitopes in both pollen sources. This rises the possibility of symptoms of AP allergic patients from Northern Europe, when travelling in the Mediterranean area, where olive pollen (OP) are important allergens and vice versa for OP-sensitized patients. In clinical practice, the indication for SIT is often based on positive results of both, SPT and IgE determination, usually by the CAP method, as nasal provocation tests are too much time-consuming. Observations in the own private office about discrepancy between the results of SPT and CAP have shown that the CAP of Fraxinus americana (tl5), having been used until now in the clinical routine, is in the described cases, despite high cross-reactivity in former studies, less sensitive than a preparation from Fraxinus excelsior. Therefore, extracts from Fraxinus excelsior should be used for diagnosis (in vivo and in vitro) and therapy of AP-allergic patients in our countries.

Variability of Ole e 9 Allergen in Olive Pollen Extracts: Relevance of Minor Allergens in Immunotherapy Treatments

Background: Clustered severe adverse reactions to immunotherapy with olive pollen extracts have been occasionally reported in areas where olive trees are extensively grown. Allergic patients from these areas, in addition to the major olive pollen allergen Ole e 1, frequently recognize a recently described allergen, Ole e 9. Objective: We aimed to develop an immunoassay to measure Ole e 9 concentration and to study the variability of this allergen in olive pollen extracts. Methods: Monoclonal antibodies (mAb) to Ole e 9 were produced from mice immunized with the pure allergen. One of these mAbs was used to develop a sandwich ELISA with an anti-olive pollen extract rabbit serum as the tracer. Olive pollen batches from several suppliers were analyzed using this method. These batches were also analyzed for Ole e 1 content and biological activity. Results: A 10-fold variation between the extreme values was found for the biological activity of the batches analyzed. Ole e 1 concentration showed a 25-fold variation. Variability of Ole e 9 concentration was extremely high, up to 161 times. The ratio Ole e 1/Ole e 9 varied in a range from 0.6 to 390.4. Conclusion: The availability of a mAb-based ELISA for Ole e 9 made it possible for us to detect an important source of variability in olive pollen batches. This variability may be the cause of outbreaks of adverse reactions in the course of immunotherapy treatments, which have sometimes been observed among olive-allergic patients living in areas with very high levels of airborne olive pollen.

The role of major olive pollen allergens Ole e 1, Ole e 9, and Ole e 10 on mice sensitization

Olive pollen is an important cause of allergy in Mediterranean countries. To date, 10 allergens (Ole e 1 to Ole e 10) have been isolated and characterized. Animal models of olive pollen allergy are suitable tools for testing the efficacy and safety of new forms of immunotherapy. To characterize the immune response in mice sensitized with olive pollen extract and to compare it with that of allergic patients. BALB/c mice were sensitized by 4 intraperitoneal injections of olive pollen extract in aluminum hydroxide. The allergic state was proved by measuring serum specific IgG1 and total IgE antibody levels. The IgG1 responses to olive pollen allergens were assayed by immunoblotting and enzyme-linked immunosorbent assay. Competition experiments between human IgE and mouse IgG1 binding to olive pollen allergens were performed. Sensitization with olive pollen extract induced high levels of specific IgG1 and total IgE in all tested animals. Immunoblotting experiments showed that the mouse IgG1 binding pattern to pollen extract was complex and heterogeneous, as occurs with human IgE. High IgG1 antibody levels to the major olive pollen allergens described for humans were detected in serum samples from sensitized mice, whereas minor olive pollen allergens induced no significant IgG1 response. Coincubation of mouse serum samples with a cocktail of Ole e 1, Ole e 9, and Ole e 10 resulted in a significant decrease (60%) in IgG1 binding to olive pollen extract. Specific mouse IgG1 strongly inhibited human IgE binding to olive pollen allergens. This mouse model of olive pollen sensitization mimics immunologic features of human pollinosis and could be a useful tool for designing novel forms of immunotherapy for olive pollen allergy based on allergen cocktails.

Cloning and Expression of the Olea europaea Allergen Ole e 5, the Pollen Cu/Zn Superoxide Dismutase

Background: Recombinant DNA technology does provide pure, well-defined and reproducible products to be used for clinical purposes, by cloning and expressing the cDNA of allergens present in a specific extract. Ole e 5 is a pollen allergen of Olea europaea with an IgE-binding frequency of about 35%, which has been identified as a superoxide dismutase (SOD). The aim of this study was to clone the cDNA of Ole e 5, to express Ole e 5 in Escherichia coli and to characterize its immunoreactivity. Methods: cDNA of Ole e 5 was amplified by nested 3′-RACE PCR and cloned in pGEX vector 6P expression vector. After sequencing of some clones and homology analysis, the rOle e 5 was produced in an E. coli strain as a fusion protein with GST and purified. Then, the protein immunoreactivity was evaluated by patients’ IgE binding (ELISA, ELISA inhibition, and immunoblotting) and by rabbit anti-rOle e 5 binding (immunoblotting and immunoblotting inhibition). Results: The sequence analysis of Ole e 5 cDNA confirmed that Ole e 5 is a Cu/Zn SOD, with an identity from 90 to 80% with SOD from other species. rOle e 5 was recognized by IgE from 39% of olive pollen-allergic patients tested; moreover, this binding was inhibited by the olive pollen extract. An anti-rOle e 5 antiserum raised in rabbit strongly reacted with a natural component of about 16-kDa molecular weight present in the olive pollen extract; moreover, this binding was inhibited by the recombinant protein. Conclusions: Ole e 5 is the first Cu/Zn SOD identified as an allergen in a pollen source. Due to the widespread presence of this enzyme, rOle e 5 allergen, cloned and expressed in a complete form in E. coli, could represent a good tool to investigate the allergen cross-reactivity between O. europaea pollen and other allergenic sources, such as plant foods and other pollens.

Quality control of non-standardized allergenic extracts: Major allergen and IgE binding of birch, English plantain and olive pollen

Rationale The objective of these studies is to apply quality control techniques used to determine the potency of standardized allergenic extracts to assess the quality of non-standardized weight/volume, PNU, and alum adsorbed extracts. An outcome of this objective is the determination of appropriate methods to manufacture consistent well-characterized extracts. Methods Non-standardized birch, English plantain, and olive pollen extracts were tested. Multiple consecutive lots manufactured over 2-3 years were measured for major allergens Bet v 1, Pla l 1, and Ole e 1 using direct binding ELISAs obtained from ALK-Abelló, Madrid and validated in our laboratory. IgE binding ELISAs were performed using sera pools from at least 5 patients allergic to the allergen. Electrophoresis and immunoblotting were also performed. Results Most extracts had good consistency by either IgE binding or major allergen, although some lots show a significant deviation from the average. English plantain and birch had a few lots that were much lower in major allergen than the average. Aqueous extracted olive pollen extracts showed consistency as did glycerin extracted pollen, however a significant reduction in extractability of the major allergen with glycerin was observed. Some olive pollen raw material source lots showed significant differences in Ole e 1. IgE binding ability was well correlated with Bet v 1 in birch extracts. Major allergens were also measured in alum adsorbed extracts. Conclusions These studies demonstrate the benefit for testing potency in non-standardized extracts and will provide the means to produce consistent products in the interim before FDA standardized products become available.

Pollen from Different Olive Tree Cultivars Contains Varying Amounts of the Major Allergen Ole e 1

Background: Commercial olive pollen from uncertain cultivar origin is the common material used for clinical and biological studies. We aimed to assess the putative heterogeneity of olive cultivars with regard to the presence of the major pollen allergen Ole e 1 and to determine whether these differences have clinical relevance. Methods: The Ole e 1 content of several cultivars was determined by immunoblotting and ultrastructural immunocytochemistry and compared to that of a commercially available olive pollen extract designed for diagnosis. Reverse transcription-polymerase chain reaction analysis of Ole e 1 transcripts was also performed. Crude protein extracts were used to carry out skin prick tests (SPTs) on 30 allergic patients in order to evaluate the clinical importance of such differences. Results: Ole e 1 was present in all cultivars, although significant quantitative differences were detected. Ole e 1 transcripts positively correlated with the amount of the allergen. Significant variations in the average reactivity of allergic patients to SPTs were observed depending on the cultivar considered. Conclusions: The presence of the Ole e 1 allergen in all the cultivars suggests that this allergen may play an essential biological role. The expression of the allergen is controlled at the transcriptional level. The significant differences in the Ole e 1 content are likely responsible for the different average reactivity exhibited by patients to the cultivars studied, although the role of other allergens cannot be excluded. Our results suggest that the use of the commercial pollen mixtures currently available may lead to mistakes in allergy diagnosis and to limited success in immunotherapy. Therefore, further standardization is strongly recommended.

Rapid release of Ole e 1 from olive pollen using different solvents.

Solubility is an important characteristic of allergenic molecules. The aim of this study was to investigate the solubility of Ole e 1, a major allergen of Olea europaea, using different solvents. Olea europaea pollen was placed in a glass column and extracted using three different solvents: deionized water, phosphate buffer 0.01 M (PBS) and normal saline (NaCl 0.9%). Several fractions were collected after extraction with each solvent and pooled based on individual protein content. Each fraction corresponded to a different elution profile, as determined by linear regression analysis. After 130 min of extraction, the pollen that remained in the column was further extracted overnight. A control olive pollen extract was also prepared with each solvent. The antigenic and allergenic profiles of all the eluted and pooled fractions were analysed by SDS-PAGE and inmmunoblots. Protein and Ole e 1 content and the amount of protein needed to produce 50% inhibition were also calculated. Ten patients were skin prick tested with the fractions obtained with deionized water. Four elution profiles were obtained using deionized water as the extracting solution and three with the two other solvents. The three solvents produced different kinetics of allergen release. Ole e 1 was rapidly released when water was used, obtaining a total of 256 micro g of Ole e 1/ml after only 7 min of extraction (fraction EC1). Using PBS, or NaCl 0.9%, the release of Ole e 1 started after 4 and 9 min of extraction, respectively. The highest amount ofOle e 1 was eluted after 44 and 26 min, with a total concentration of 162 and 203 micro g of Ole e 1/ml, respectively. The presence of Ole e 1 in each phase was verified by SDS-PAGE and immunoblot analyses. The extracting solution seems to determine the antigenic profile of olive pollen extracts. Ole e 1 is rapidly released from the pollen grain after extraction in deionized water. The solubility seems to be affected by the use of other solvents. These techniques could be used to manipulate the Ole e 1 content in O. europaea extracts.

Monoclonal antibody-based method for measuring olive pollen major allergen Ole e 1

Olive tree pollen is an important cause of inhalant allergy in Mediterranean countries. The major allergen of this pollen, Ole e 1, has caused reactions in the sera of >80% of olive-sensitive patients. Accurate standardization of allergenic products for diagnosis and immunotherapy is essential to guarantee their quality, and measurement of the major allergen content is becoming an important aspect of standardization procedures. To develop a two-site enzyme-linked immunoadsorbent assay (ELISA) for the quantification of Ole e 1. BALB/c mice were immunized with purified natural Ole e 1. After fusion and screening by direct ELISA, one of the monoclonal antibodies (5A3) was selected as the capture antibody in an ELISA for Ole e 1 quantification. Bound allergens were detected by a combination of biotinylated Ole e 1-specific polyclonal rabbit antibody and peroxidase-conjugated streptavidin. This ELISA was subsequently evaluated and compared with other techniques. The developed ELISA was highly reproducible and sensitive, with a detection limit of 0.5 ng/mL and a practical range of 1 to 10 ng/mL. The Ole e 1 content ranged from 3 to 50% of the total protein among the nine Olea europaea pollen extracts studied. The assay also detected Ole e 1-like proteins in pollen from other Oleaceae. Correlation was good between the Ole e 1 content determined by ELISA and scanning densitometry and the immunoglobulin E-binding activity of the extracts. The described Ole e 1 ELISA is sensitive, reproducible, specific, and reliable, and therefore, can be helpful for standardization of olive pollen extracts intended for clinical use.

The allergenic relevance of profilin (Ole e 2) from Olea europaea pollen.

Many works have dealt with the study of the allergenic relevance of profilin from allergenic extracts, mainly derived from pollens and vegetable foods. Olive pollen extracts also contain a profilin allergen (Ole e 2). This protein has been characterized in detail, so the amino-acid sequence of three isoforms and the structural model of one of them are already known. The prevalence of Ole e 2 for olive allergenic patients has been evaluated by different in vivo and in vitro methods, and the results compared with those obtained for another pollen profilins.

Allergenic diversity of the olive pollen.

A great number of allergenic proteins have been detected in olive pollen extracts. To date, nine allergens have been isolated and characterized, which have been called Ole e 1 to Ole e 9. The most prevalent olive allergen is Ole e 1, which affects more than 70% of patients hypersensitive to olive pollen, but others, such as Ole e 2, Ole e 8, and Ole e 9, have been demonstrated to be major allergens, and Ole e 6 or Ole e 7 reach high values of clinical incidence. Many of these allergens, such as Ole e 2 (profilin) and Ole e 3 (polcalcin), are involved in cross-reactivities, which agrees with their adscription to panallergenic families. Among the many olive allergens of high molecular mass, only Ole e 9 (46 kDa) has been characterized. The allergen is a polymorphic and glycosylated beta-1,3-glucanase, which belongs to a pathogenesis-related (PR-2) protein family. In addition to the polypeptide epitopes, Ole e 1 also exhibits IgE-binding determinants in the carbohydrate, which are recognized by more than 60% of the sera from patients sensitive to the whole allergen, although the level of such glycan-specific IgE seems not to be clinically relevant in the overall content of the sera. Recent advances in the elucidation of the structure of the Ole e 1-oligosaccharide component allows us to explain the antigenicity of the molecule. Finally, the recombinant production of several allergens from olive pollen in both bacterial and eukaryotic cells has allowed us to resolve problems derived from the polymorphism and scarcity of the natural forms of these allergens. The biological equivalence between the natural and recombinant forms lets us initiate studies on the design of mixtures for clinical purposes, in which hypoallergenic derivatives of these allergens could play a definitive role.

Olive allergen-specific IgE responses in patients with Olea europaea pollinosis.

Olive tree (Olea europaea) pollen is an important cause of pollinosis in the countries of the Mediterranean area. This work aimed to study the IgE-binding frequency of Ole e 1, Ole e 2, Ole e 3, Ole e 6 and Ole e 7 from O. europaea pollen in a large population of olive pollen-allergic patients. We studied: 119 consecutive patients with seasonal rhinitis and/or asthma and a positive skin prick test to O. europaea pollen extract; 10 atopic patients without history of pollinosis and a negative skin prick test to O. europaea; and 10 healthy controls. Allergens were purified from O. europaea pollen extract by reverse phase HPLC and characterized by N-terminal amino acid sequencing, MALDI analysis, and specific IgE immunodetection. Skin prick tests and ELISA titration against above mentioned purified olive pollen allergens were performed in all pollinic patients and controls. One-hundred and seven (90.7%) patients had a positive skin response to Ole e 1; 88 (74.6%) reacted to Ole e 2; 57 (47.9%) reacted to both Ole e 6 and Ole e 7; and 43 (37.8%) reacted to Ole e 3. The allergenic activity determined by ELISA to Ole e 1 was found in 84%; to Ole e 2 in 61.3%; to Ole e 3 in 31.9%; to Ole e 6 in 39.4%; and to Ole e 7 in 41.2% of patients. All patients had positive skin responses to at least one of the allergens tested, However, a combination of Ole e 1 and Ole e 2 together with a minor allergen Ole e 6 or Ole e 7, disclosed the same diagnostic value that was obtained with the use of crude olive pollen extract. The nonatopic and atopic control subjects did not react to any purified allergens on the skin prick test. These results indicate that Ole e 1 and Ole e 2 are major allergens in patients with O. europaea pollinosis in our population. A combination of a few olive pollen allergens can substitute the crude extract for in vivo as well as in vitro diagnostic purposes.

Allergenic cross-reactivity of olive pollen.

Sera of patients allergic to olive (Olea europaea) pollen were used to analyze the IgE cross-reactivity between olive-pollen extract and other pollens obtained from phylogenetically unrelated species. We used IgE immunostaining of pollen extracts blotted to nitrocellulose membranes after SDS-PAGE and inhibition analysis of this binding. A high inhibition of the IgE binding on olive-pollen extract was exhibited by birch, mugwort, pine, and cypress pollens, suggesting that these extracts contain proteins which share common epitopes and thus can be recognized by olive-allergic sera. IgE binding to Gramineae pollen extracts was not inhibited by olive-pollen extract, indicating a primary sensitization of the patients to these species. From the inhibition assays, the presence of an allergen of 45 kDa in the olive pollen, which has no homologous counterparts in other allergenic species, has been inferred. Olive pollen contains allergens which cross-react with pollens from unrelated species, a fact that could simplify the diagnosis and treatment of pollinosis.