Olive Pollen Research Articles

The reliability of geostatistic interpolation in olive field floral phenology

Floral phenology, as most of natural phenomena, shows, as an inherent feature, a high degree of spatial continuity. Geostatistics are a family of statistics that describe correlations through space/time and they can be used for both quantifying spatial correlation and interpolating no monitored sampling points. The combined use of Geographical Information Systems (GIS) and geostatistics can be an essential tool for spatial analysis in phenological and aerobiological studies. In the present work, Kriging interpolation by using linear geostatistic analysis has allow us to estimate phenological data of a wide olive crop area of the province of Cordoba (Andalusia, Southern Spain), on the basis of a limited number of␣phenology sampling points. The main a priori hypothesis was that 7 traditionally observed sampling points (with 10 olive trees in each site) uniformly distributed through the main olive crop areas could be enough for interpolating phenological information of the whole Cordoba olive area. Geostatistical results reject this hypothesis. The optimum/minimum number and location of sampling points was determined in 13 sites (including the original 7 sites and 6 new sites). The obtained phenological maps will improve␣olive pollen aerobiological information and forecasting in the area. The application of such new combined space analysis tools on floral phenology allows optimising human and economic resources on field phenology campaigns. Moreover, an appropriate use of GIS and geostatistic software to create phenological maps will be an essential complement in pollen aerobiological studies, given the increased interest in obtaining automatic aerobiological forecasting maps.

Role of Dau c 1 in three different patterns of carrot-induced asthma

Objective To assess the role of Dau c 1 in three patients with carrot induced asthma. Material and methods Patient 1 had asthma when handling raw carrots. Sensitization to pollens wasn’t detected. Patient 2 had rhinoconjunctivitis due to grass and olive pollen allergy. She had asthma when handling raw carrots. Patient 3 was diagnosed of rhinoconjunctivitis and asthma due to allergic sensitization to mites, several pollens and cat. She had asthma due to raw carrot ingestion and inhalation. IgE immunobot analysis and ELISA inhibition assay were used to investigate the allergens and specific antibodies. Results IgE Immunoblot Analysis: Dau c 1 from carrot extract and the recombinant rDau c 1 were recognized by IgE from patients 1 and 2. Band of Bet v 1 in birch pollen extract wasn’t recognized. Patient 3 didn’t recognize any of these allergens. Specific IgE to rDau c 1 was measured by ELISA. Specific IgE ELISA-inhibition with carrot as solid phase howed an intermediate inhibition (30 %) between carrot and rDau c 1 in patient 1; and a considerable inhibition (nearly 100 %) between carrot and rDau c 1 in patient 2. No inhibition was found in patient 3. Specific IgE ELISA inhibition between r Dau c 1 and r Bet v 1, employing r Dau c 1 as solid phase was made in patients 1 and 2. Bet v 1 showed less than 40 % of inhibition of r Dau c 1 in patient 1; and an intermediate inhibition (> 40 %) between r Bet v 1 and r Dau c 1 in patient 2. Conclusions Airborne carrot allergens are able to sensitize without the implication of a previous pollen allergy. Dau c 1 was the main allergen in patient 2. In patient 1, there was a band of 30 kd that looks like the predominant allergen. Patients 1 and 2 were sensitized directly from carrot allergens. In patient 3, Dau c 1 isn’t related to the carrot allergy. Allergy to carrot in patient 3 seems to be related to her allergy to different pollens; however, it wasn’t related to birch pollen. Mediterranean countries didn’t show the same patterns of food-related pollen allergy than Nordic countries.

Quality of life in allergic rhinitis and impact of high-dose sublingual immunotherapy: a real-life study

Allergic rhinitis (AR) may significantly impair quality of life (QoL), and therefore it is important to evaluate the effectiveness of the available treatments on such a parameter. We studied QoL by a real-life model in a large group of patients with rhinitis caused by the most common aeroallergens and assessed the effects of high-dose sublingual immunotherapy (SLIT). An overall number of 452 subjects (229 males, 223 females, mean age 25 ± 13 years) was included in the study. Of them, 208 were allergic to house dust mites, 196 to grass pollen, 104 to Parietaria pollen, 48 to olive pollen, 28 to ragweed pollen, and two to cypress pollen. QoL was measured before and after SLIT with the specific questionnaire Rhinoconjunctivitis Quality-of Life-Questionnaire (RQLQ) introduced and validated by Juniper. At the same time, patients evaluated their clinical status by visual analogue scale (VAS) from 0 (worst) to 10 (best). SLIT was performed by extracts from Stallergenes standardized in index of reactivity (IR) by a build-up phase in 11 days and a maintenance phase with 300 IR three times a week for the duration of the study. All side-effects were registered. The mean RQLQ was 2.93 ± 1.04 before SLIT and 1.50 ± 0.98 after SLIT (P < 0.001), with comparable improvement in the different allergen treatments. The respective mean VAS score was 4.47 ± 2.11 and 6.92 ± 1.64 (P < 0.001). SLIT was completely tolerated in 395 patients (87.4%), while in 57 (12.6%) it gave rise to slight reactions, mostly at the local level in the mouth or in the gastrointestinal tract. High dose SLIT, administered in a clinical model comparable with routine application, provides significant improvement of both clinical status and QoL in patients with AR caused by common aeroallergens.

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.

Profiling of Hydrophilic Proteins from Olea europaea Olive Pollen by MALDI TOF Mass Spectrometry

The antigenic profile of Olea europaea pollen from different Mediterranean cultivars was obtained by MALDI mass spectrometry. A simple procedure of chemical fractionation of the whole antigen extract was developed, whereby less complex, or pure, fractions of antigen candidate were obtained prior to mass spectrometric analysis. Some of the features of protein structure and distribution probably depend on cultivar adaptation to the environment. The profilings of pollen proteins thus obtained allow the distinction of the analyzed cultivars into three distinct groups: (i) those characterized by a low Ole e 1 content; (ii) those over-enriched in Ole e 1 and (iii) that containing Ole e 3 and Ole e 7 only. The latter consists of at least four isoforms differing by the degree of glycosilation. These results demonstrate that the proposed experimental procedure, can supply valuable information on the antigens' micro heterogeneity.

Forecasting airborne pollen concentration time series with neural and neuro-fuzzy models

Forecasting airborne pollen concentrations is one of the most studied topics in aerobiology, due to its crucial application to allergology. The most used tools for this problem are single lineal regressions and autoregressive models (ARIMA). Notwithstanding, few works have used more sophisticated tools based in Artificial Intelligence, as are neural or neuro-fuzzy models. In this work, we applied some of these models to forecast olive pollen concentrations in the atmosphere of Granada (Spain). We first studied the overall performance of the selected models, then considering the data segmented into intervals (low, medium and high concentration), to test how they behave on each interval. Experimental results show an advantage of the neuro-fuzzy models against classical statistical methods, although there is still room for improvement.1

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.

Prophylactic Intranasal Treatment with Fragments of 1,3-β-Glucanase Olive Pollen Allergen Prevents Airway Inflammation in a Murine Model of Type I Allergy

Background:Olive pollen is an important cause of allergy in Mediterranean countries. More than 50% of olive-pollen-allergic patients are sensitized against the 1,3-β-glucanase Ole e 9. To date, prophylactic and therapeutic treatments using purified recombinant allergens have not been studied in animal models of olive pollen allergy. Methods: BALB/c mice were immunized against Ole e 9 combining intraperitoneal injections of the allergen in Al(OH)₃ with airway allergen challenges. A prophylactic treatment was performed by intranasal administration of a mixture of the recombinant fragments of the allergen prior to Ole e 9 sensitization. Serum levels of specific IgE, IgG1, IgG2a and IgG2b were measured by ELISA, and total IgE levels by sandwich ELISA. Bronchoalveolar lavage and lungs from mice were collected to study airway inflammation by light microscopy. Results: BALB/c mice immunized against Ole e 9 developed a predominantly Th2-like immune response with allergen-specific immunoglobulin induction and airway inflammation accompanied by the infiltration of eosinophils, lymphocytes, and neutrophils in the lung. Prophylactic treatment by intranasal application of the recombinant fragments of Ole e 9 avoids airway inflammation induced by sensitization with this allergen although the levels of Ole e 9-specific antibodies remain unchanged. Conclusions: Prophylactic intranasal treatment with recombinant fragments of Ole e 9 prevents airway inflammation triggered by immunization to this allergen in a murine model of type I allergy.

Blackcurrant Allergy

RATIONALE: The blackcurrant is a temperate shrub that ussually grows by the edge of the pathways when not cultivated. It produces small edible berries, very dark purple/blue colour_almost black_hence the name. They belong to the Rosaceae family, Saxifragaceae order. Although the fruits of this family are a common cause of allergy in our enviroment (i.e. peaches, apple), allergy to blackcurrants has not been reported. METHODS: A 50-year-old woman, previously diagnosed of rhinitis due to arizonic, gramineae and olive pollen allergy, presented oral pruritus and pharyngeal occupation with dysphagia while eating either fresh fruit, jam or juice of peach, apricot and nectarine. The same symptoms appear after fresh fruit and jam of blackcurrant, in several times. She tolerates other fruits of the Rosaceae family (i.e. raspberry, strawberry, plum, apple and pear). RESULTS: Peach and raspberry prick tests were possitive. Skin test to blackcurrant resulted negative. Specific IgE determination (Pharmacia CAP System) to blackcurrant measured 5.7 kU/L, and 2.92 kU/L to peach. After SDS-PAGE-Immunoblot IgE reactive bands of >100-43 kd were detected in blackcurrant extract and >100 kd in raspberry extract. CONCLUSIONS: Cross-sensitivity among the different species of the Rosaceae family seems variable.We report a case of blackcurrant, peach, apricot and nectarine allergy due to immediate type hypersensitivity (IgE mediated) and subclinical sensitization to raspberry. In our knowledge, this might be the first case of blackcurrant allergy to be reported.

The Effect of Bactospeine and Three Insecticides on Olive Pollen Germination and Tube Growth

The Effect of Bactospeine and Three Insecticides on Olive Pollen Germination and Tube Growth

A recombinant functional variant of the olive pollen allergen Ole e 10 expressed in baculovirus system

Pollens have been reported as important sources of antigens causing type-I allergy and, among them, olive pollen has high clinical relevance in Mediterranean countries. The most recently described olive allergen, Ole e 10, is involved in cross-reactivity phenomena and related to asthma induction in allergic patients. These immunologic features make this allergen a good candidate to be included in diagnosis and therapy of protocols of allergic diseases. Since the availability of Ole e 10 from the olive pollen is limited, the allergen has been efficiently expressed in the baculovirus/insect cell system. The Ole e 10-cDNA inserted into the transfer vector pBacPAK8 allowed the expression of the recombinant protein in cultured Sf21 cells. Recombinant Ole e 10 (rOle e 10) was purified from the culture after dialysis and three chromatographic steps. Mass spectrometry, Edman degradation, IgE- and IgG-binding analyses were employed to characterize the recombinant allergen, which showed molecular and immunological equivalence with the natural protein. Affinity gel electrophoresis in presence of laminarin (1,3-β-glucan) revealed that rOle e 10 retains identical carbohydrate-binding capacity than the natural allergen. In conclusion, the recombinant expression of Ole e 10 in baculovirus/insect cell system produces a homogeneous and biologically active allergen that could be useful for clinical and scientific purposes.

Safety of sublingual-swallow immunotherapy in children aged 3 to 7 years

The minimum age to start specific immunotherapy with inhalant allergens in children has not been clearly established, and position papers discourage its use in children younger than 5 years. To assess the safety of high-dose sublingual-swallow immunotherapy (SLIT) in a group of children younger than 5 years. Sixty-five children (51 boys and 14 girls; age range, 38-80 months; mean +/- SD age, 60 +/- 10 years; median age, 60 months) were included in this observational study. They were treated with SLIT with a build-up phase of 11 days, culminating in a top dose of 300 IR (index of reactivity) and a maintenance phase of 300 IR 3 times a week. The allergens used were house dust mites in 42 patients, grass pollen in 11 patients, olive pollen in 5 patients, Parietaria pollen in 4 patients, and cypress pollen in 3 patients. All adverse reactions and changes in the treatment schedule were compared in 2 subgroups: children 38 to 60 months old and children 61. to 80 months old. The average cumulative dose of SLIT was 36,900 IR. Adverse reactions were observed in 11 children, none of them severe enough to require discontinuation of immunotherapy. Six reactions occurred in the 60 months or younger age group and 7 in the older than 60 months age group, with no differences between these 2 groups. High-dose immunotherapy in children younger than 5 years does not cause more adverse reactions than in children aged 5 to 7 years. There is no reason to forbear studies on safety and efficacy of these preparations in young children.

An olive pollen protein with allergenic activity, Ole e 10, defines a novel family of carbohydrate-binding modules and is potentially implicated in pollen germination

CBMs (carbohydrate-binding modules) are the most common non-catalytic modules associated with enzymes active in plant cell-wall hydrolysis. They have been frequently identified by amino acid sequence alignments, but only a few have been experimentally established to have a carbohydrate-binding activity. A small olive pollen protein, Ole e 10 (10 kDa), has been described as a major inducer of type I allergy in humans. In the present study, the ability of Ole e 10 to bind several polysaccharides has been analysed by affinity gel electrophoresis, which demonstrated that the protein bound 1,3-beta-glucans preferentially. Analytical ultracentrifugation studies confirmed binding to laminarin, at a protein/ligand ratio of 1:1. The interaction of Ole e 10 with laminarin induced a conformational change in the protein, as detected by CD and fluorescence analyses, and an increase of 3.6 degrees C in the thermal denaturation temperature of Ole e 10 in the presence of the glycan. These results, and the absence of alignment of the sequence of Ole e 10 with that of any classified CBM, indicate that this pollen protein defines a novel family of CBMs, which we propose to name CBM43. Immunolocalization of Ole e 10 in mature and germinating pollen by transmission electron microscopy and confocal laser scanning microscopy demonstrated the co-localization of Ole e 10 and callose (1,3-beta-glucan) in the growing pollen tube, suggesting a role for this protein in the metabolism of carbohydrates and in pollen tube wall re-formation during germination.

Analysis of IgE and IgG B-cell immunodominant regions of Ole e 1, the main allergen from olive pollen

Ole e 1 is a major allergen from olive pollen with an IgE-binding frequency around 80% among allergic population. Its diagnostic value has been demonstrated, and cross-reactive allergens have been found in ash, lilac and privet. We sought to determine IgE- and IgG-binding regions of Ole e 1. Ole e 1-specific polyclonal antiserum and sera from patients allergic to olive pollen were used to analyze IgG and IgE epitopes, respectively. Short overlapping synthetic peptides covering the complete sequence of Ole e 1 and point mutants of these peptides bound to membranes, as well as long recombinant peptides fused to GST were used in dot blot immunostaining and ELISA. Skin prick tests were performed on 14 allergic patients to assay the response in vivo to the recombinant fusion peptides. Residues at positions 8–11, 29, 32, 33, 55–59, 70, 107–110, 112, 120, 123, 141 of Ole e 1 sequence were found to be antigenically relevant in the IgG-binding. Although amino acids K137, L138, G139, Y141 and P142 were involved in the IgE-recognition of a pool of sera from allergic individuals, the response to the IgEs seemed to be preferentially conformational. IgE-binding capability of recombinant GST-fused peptide T114–M145 was demonstrated by in vivo (prick test) and in vitro (ELISA) experiments. Major IgG and IgE-binding regions of Ole e 1 have been identified being the C-terminal an immunodominant region. These data could help to design hypoallergenic forms of the allergen.

Expression of the major olive pollen allergen Ole e 10 in the yeast Pichia pastoris: Evidence of post-translational modifications

Olive pollen allergy is a clinical disorder that affects around 20% of the population in Mediterranean areas. The major olive pollen allergen, Ole e 10, is involved in cross-reactivity phenomena and asthma induction in allergic patients, and, besides its clinical interest, Ole e 10 is the first member of a new family of plant proteins. Ole e 10-specific cDNA has been cloned in the plasmid pPICZαA and expressed in the methylotrophic yeast Pichia pastoris. The recombinant protein has been purified in a two chromatographic-step procedure. N-Terminal sequencing, mass spectrometry, IgG, and IgE binding assays were employed to characterize the recombinant allergen. These analyses revealed that the product undergoes a proteolytic cleavage in the N-terminal end with the loss of the first six residues. Different strategies were used to solve this problem, such as changes in the fermentation conditions and the employment of protease-deficient yeast strains. Proteolytic cleavage was minimized and about 51% of rOle e 10 was obtained as a full-length protein. Moreover, a covalent modification was found in the N-terminal end of the full-length rOle e 10. Peptide mapping and mass spectrometry analyses pointed to the existence of a phosphorylation located in a serine residue of the N-terminal segment of rOle e 10 and it was confirmed after treatment of the sample with alkaline phosphatase. Finally, both full-length and truncated rOle e 10 retained most of the IgG- and IgE-binding capabilities of the natural protein isolated from the pollen.

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.

1,3‐β‐glucanases as candidates in latex–pollen–vegetable food cross‐reactivity

1,3-beta-glucanases (group 2 of pathogenesis-related proteins) are enzymes widely distributed among higher plants and have been recently proven to be significant allergens. The aim of this work was to study the potential implication of 1,3-beta-glucanases in cross-reactivities among latex, pollen and vegetable foods. The cDNA encoding the N-terminal domain (NtD) of Ole e 9, a major allergenic 1,3-beta-glucanase from olive pollen, was amplified by polymerase chain reaction and produced as a recombinant protein in Pichia pastoris (recombinant N-terminal domain, rNtD). Circular dichroism, ELISA, immunoblotting and immunoblotting inhibition experiments were carried out. Sera from olive pollen allergic patients and a rNtD-specific polyclonal antiserum were used. The NtD of Ole e 9 has been produced at high yield in the yeast P. pastoris and possesses 1,3-beta-glucanase activity. The expressed polypeptide conserves IgE and IgG immunodominant epitopes of the whole Ole e 9. A rNtD-specific polyclonal antiserum and sera from olive pollen allergic patients allowed detection of IgG and IgE reactive peptidic epitopes common to 1,3-beta-glucanase Ole e 9 in extracts from ash and birch pollen, tomato, potato, bell-pepper, banana and latex. rNtD and homologous glucanases are new molecules to be used in diagnostic protocols as they could help to identify allergic pollen patients who are at risk for developing allergic symptoms to fruits, vegetables and latex.

Preventive use of the co-administration of immunostimulatory DNA sequences (ISS-ODN) with olive pollen proteins in a mouse model of allergic asthma

RATIONALE: ISS-ODNs are potent immunomodulators and stimulators of innate immunity. We studied the effects of the co-administration of these sequences with olive pollen proteins (Olea) given prior to Olea sensitization in a mouse model of allergic asthma.

Intranasal tolerance induction in an established model of asthma limits eosinophils recruitment and IgE production via CD4-dependent mechanisms

Asthma Limits Eosinophils Recruitment and IgE Production Via CD4-Dependent Mechanisms C. Longaretti, C. Barbey, E. Pradervand, F. Spertini; Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois, Lausanne, SWITZERLAND. RATIONALE: We have recently shown that prophylactic induction of tolerance via the nasal route in ovalbumin (OVA) allergic mice was generating IL-10 producing regulatory CD4+ T cells. The aim of this study was to characterize, in an established model of asthma, the mechanisms leading to effective immune tolerance via the mucosal route. METHODS: OVA-sensitized asthmatic mice were intranasally treated with 1.5 mg OVA for 3 days. Mice were challenged by OVA aerosol 10 days later. Cells implicated in protection were characterized by transfer experiments. RESULTS: Upon challenge, intranasal OVA treatment (INT) drastically reduced inflammatory cell recruitment into bronchoalveolar lavage fluid (BALF), including eosinophils, inhibited OVA specific IgE rise and decreased bronchial hyperreactivity. T cell proliferation of ex vivo purified bronchial lymph node cells was inhibited as well as TH2 and TH1 cytokine production. Protection was effective for an extended period of time and was not associated with eotaxin reduction. Cell transfer experiment of CD4+ cells from INT OVA animals limited recruitment of inflammatory cells, including eosinophils, into BALF of recipient mice. CONCLUSIONS: INT with OVA was able to markedly limit asthmatic inflammation upon allergen challenge. CD4+ regulatory T cells appears to play a crucial role in this protection via mechanisms to be defined. Funding: Swiss National Science Foundation 1049 Preventive Use of the Co-administration of Immunostimulatory DNA Sequences (ISS-ODN) With Olive Pollen Proteins in a Mouse Model of Allergic Asthma L. Conejero1,2, Y. Higaki1,2, M. J. Fernandez-Bohorquez1, I. VarelaNieto2, M. L. Baeza1, J. M. Zubeldia1; 1Allergy Department, Hospital Gregorio Maranon, Madrid, SPAIN, 2Instituto Investigaciones Biomedicas Alberto Sols-CSIC, Madrid, SPAIN. RATIONALE: ISS-ODNs are potent immunomodulators and stimulators of innate immunity. We studied the effects of the co-administration of these sequences with olive pollen proteins (Olea) given prior to Olea sensitization in a mouse model of allergic asthma. METHODS: BALB/c mice were distributed in different groups and immunized by intradermal injections of ISS-ODN and Olea, ISS-ODN, Olea, or saline (SS) on days 0, 7 and 14. Mice were then sensitized by subcutaneous injections with Olea in alum twice. Blood samples were collected weekly to measure immunoglobulin levels. Mice were sacrificed and splenocytes were cultured with and without the allergen for 3 days. Supernatants were assayed to determine the level of different cytokines. Finally, bronchial reactivity to methacholine was determined by Whole Body Plethysmography. RESULTS: Concomitant administration of ISS-ODN with Olea before the mice were sensitized resulted in significant changes in the immunological and functional parameters: a) Production of Olea-specific IgE was greatly reduced (p= 0.05); b) Significant higher levels of specific IgG2a, IFNand IL-18 production were detected (p< 0.05); and c) Pulmonary function test revealed a greater reduction in the bronchial hypereactivity to methacholine (p< 0.05). CONCLUSIONS: The co-administration of ISS-ODN with Olea prior to sensitization prevents the development of an allergic phenotype in our mouse model of bronchial asthma. Funding: FIS 01/0598

Relationship between Flowering and Heat Units to Analyze Crop Efficiency of Olive Cultivars Located in Southern Italy

The relationship between heat units trends and reproductive development in olive (Olea europaea, L.) was studied over a 3-year period (1999-2001) in 15 areas in the southern Italian regions of Campania, Calabria, Puglia, and Sicily. Heat units were calculated using GDH and GDD formulas and the flowering phases in the olive groves were studied using volumetric pollen traps that aspirate fixed quantities of air. With this method, the olive pollen release and flowering trends were determined. The main objective of the study was to correlate the spring heat unit amounts and the phases of maximum pollen emission with the date of flowering. Moreover, the ranges of GDH and GDD in the different study areas were calculated to identify maps of olive pollen release.