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1,3-beta-glucanases as candidates in latex-pollen-vegetable food cross-reactivity.

BACKGROUND: 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. OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Allergens↗

Phosphorylation of tobacco eukaryotic translation initiation factor 4A upon pollen tube germination.

Eukaryotic translation initiation factor eIF-4A is a member of the DEAD box family of RNA helicases and RNA-dependent ATPases. In tobacco, eIF-4A is encoded by a gene family with one isoform, eIF-4A8, being exclusively expressed in pollen. This pollen-specific isoform is a candidate for mediating translational control in the developing gametophyte. Here we show that eIF-4A is barely phosphorylated in mature pollen, but during pollen tube germination, two isoforms of eIF-4A become phosphorylated. Phosphoamino acid analysis indicated phosphorylation of threonine. In order to determine whether pollen-specific eIF-4A8 is among the phosphorylated isoforms, we raised transgenic tobacco plants overexpressing eIF-4A8 containing a histidine tag. Hereby, we could show that indeed eIF-4A8 is modified through phosphorylation. The biological relevance of the phosphorylation of eIF-4A is discussed.

Blotting, Western↗

Adaptation of the Pollen Tube Growth Assay to Cytotoxicity Testing of Cigarette Smoke.

We describe a recently developed in vitro assay for the assessment of the cytotoxicity of gaseous materials. The method is based on the pollen tube growth (PTG) test suited to determine the cytotoxicity of non-gaseous, water-soluble substances (Kristen and Kappler, 1995). Instead of the conventional PTG test, we used a semi-automatic, computer-controlled multi-channel device for the injection of gases and gas mixtures into a battery of incubation tubes containing germinating, suspension-cultured tobacco pollen as indicator of toxic effects. The efficiency of the new PTG gas test device was examined using the gas phase of cigarette smoke. The examination exhibited sufficient sensitivity of the method and high reproducibility of the toxicity data obtained. The assay can be adapted to various sources of exhaust gases.

Journal Article↗

Mechanistic models for wind dispersal.

The growing need for ecological forecasts of, for example, species migration, has increased interest in developing mechanistic models for wind dispersal of seeds, pollen and spores. Analytical models are only able to predict mean dispersal distances, whereas sophisticated trajectory simulation models are able to incorporate rare wind conditions causing long-distance dispersal and are therefore preferable. Despite the rapid development of mechanistic dispersal models, only a few studies have focused on comparing the performance of the models. To assess the level of model complexity needed, attention should be paid to model comparisons and the sensitivity of the predictions to model complexity. In addition to studying the movement of airborne particles, future modelling work should also focus on the processes of particle release and deposition.

Models, Biological↗

Possible induction of oral allergy syndrome during specific immunotherapy in patients sensitive to tree pollen.

BACKGROUND: Many studies have shown that specific immunotherapy may prevent the development of allergies to new allergens. Recently, however, there have been sporadic reports on the possibility of new allergies developing during immunotherapy. The aim of this study was to evaluate the possibility of the occurrence of food allergy and changes of sIgE concentration in patients sensitive to tree pollen during specific immunotherapy. MATERIAL/METHODS: We examined 12 patients subjected to desensitization treatment of pollinosis caused by allergy to the pollen of Betulaceae trees, in whom only Bet v 1 sIgE was found in serum prior to immunotherapy. The control group included 8 allergic patients who did not consent to this treatment. RESULTS: Bet v 2 sIgE was detected after 6 months in 3 patients (25%) and after 18 months in another 2 patients (41.7% total). In 2 cases, oral allergy syndrome (OAS) occurred in the course of therapy. These findings were accompanied by a concomitant decrease in nasal and ocular allergic signs during the birch pollen season in the overwhelming majority of patients (91.7%). In the control group, no serum occurrence of Bet v 2 sIgE was found during the 18-month observation period. Although no OAS signs appeared in any patient, the symptomatic intensity of pollinosis was not reduced. CONCLUSIONS: Due to the limitations of currently used vaccines, new allergies may be induced during immunotherapy. In sporadic cases these may be accompanied by the occurrence of specific clinical manifestations, usually mild.

Adult↗

Developmental regulation of the gene for chimeric calcium/calmodulin-dependent protein kinase in anthers.

Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) was cloned from developing anthers of lily (Lilium longiflorum Thumb. cv. Nellie White) and tobacco (Nicotiana tabacum L. cv. Xanthi). Previous biochemical characterization and structure/function studies had revealed that CCaMK has dual modes of regulation by Ca(2+) and Ca(2+)/calmodulin. The unique structural features of CCaMK include a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain. The existence of these three features in a single polypeptide distinguishes it from other kinases. Western analysis revealed that CCaMK is expressed in a stage-specific manner in developing anthers. Expression of CCaMK was first detected in pollen mother cells and continued to increase, reaching a peak around the tetrad stage of meiosis. Following microsporogenesis, CCaMK expression rapidly decreased and at later stages of microspore development, no expression was detected. A tobacco genomic clone of CCaMK was isolated and transgenic tobacco plants were produced carrying the CCaMK promoter fused to the beta-glucuronidase reporter gene. Both CCaMK mRNA and protein were detected in the pollen sac and their localizations were restricted to the pollen mother cells and tapetal cells. Consistent results showing a stage-specific expression pattern were obtained by beta-glucuronidase analysis, in-situ hybridization and immunolocalization. The stage- and tissue-specific appearance of CCaMK in anthers suggests that it could play a role in sensing transient changes in free Ca(2+) concentration in target cells, thereby controlling developmental events in the anther.

Amino Acid Sequence↗

Human monoclonal antibody-based quantification of group 2 grass pollen allergens.

BACKGROUND: Grasses belong to the most potent allergen sources worldwide. Group 2 grass pollen allergens are recognized by more than 100 million allergic patients. OBJECTIVE: The aim was to develop an assay for the specific detection and quantification of group 2 grass pollen allergens. METHODS: We have isolated a monoclonal human IgE Fab specific for group 2 grass pollen allergens by combinatorial cloning from lymphocytes of a grass pollen-allergic patient. This Fab was converted into a complete human IgG1 antibody and used together with rPh1 p 2 to develop a competitive ELISA for the specific measurement of group 2 allergens. ELISA plate-bound purified recombinant human Ph1 p 2-specific IgG1 is incubated with a constant amount of biotinylated rPh1 p 2 competing with increasing concentrations of group 2 allergens to be determined. Defined concentrations of purified rPhl p 2 are used to establish a standard curve. The concentration of unlabeled group 2 allergens can thus be deduced from the displacement of biotinylated rPh1 p 2, which can be detected with peroxidase-labeled streptavidin. RESULTS: The competition-ELISA measured rPh1 p 2 concentrations ranging from 10 ng/mL to 500 ng/mL and allowed to quantify group 2 allergens from 9 different grass families. The results were in good agreement with immunoblot data. CONCLUSIONS: The described assay can be used for standardization of diagnostic and therapeutic vaccines as well as for the quantification of group 2 allergens in environmental samples.

Allergens↗

Monoclonal antibody-based ELISA to quantify the major allergen of Artemisia vulgaris pollen, Art v 1.

BACKGROUND: Pollen of Artemisia vulgaris (mugwort) is a relevant cause of pollinosis in temperate and humid regions. Recently, the major allergen of this pollen, Art v 1, has been characterized. OBJECTIVE: To develop a monoclonal antibody (mAb)-based enzyme-linked immunosorbent assay (ELISA) to quantify Art v 1, and to assess the correlation of Art v 1 content with the biological activity of mugwort pollen extracts. METHODS: Art v 1-specific mAbs were obtained from a BALB/c mouse immunized with high-performance liquid chromatography (HPLC)-purified Art v 1. One of these antibodies (Av 3.7), which recognizes the N-terminal defensin-like domain of Art v 1, was used as the capture antibody in an ELISA method for allergen quantitation. An anti-A. vulgaris rabbit serum was used as the second antibody. Art v 1 was purified by immunoaffinity chromatography and used as the standard in the assay. RESULTS: The purity and identity of the affinity-purified Art v 1 was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometry, amino acid composition, and N-terminal amino acid sequencing. The prevalence of specific IgE against Art v 1, determined by radioallergosorbent test (RAST) in a population of 44 mugwort-allergic patients, was 79%. The Art v 1-ELISA developed displays a detection limit of 0.1 ng/ml, and a practical working range of 0.2-10 ng/ml. The concentration of Art v 1 was measured in 10 A. vulgaris pollen extracts, and a good correlation was observed between the Art v 1 content and the allergenic activity of the extracts. CONCLUSIONS: The results prove the usefulness of the Art v 1-ELISA for the standardization of A. vulgaris pollen extracts intended for clinical use.

Allergens↗

On the association between date of birth and pollen sensitization: is age an effect modifier?

An association between date of birth and development of allergy has been proposed by prior research. Yet, the presence of a dose-response relationship or any potential effect modification for this association has not been widely studied. The aims of our study were to investigate whether an association between birth during pollen season and symptomatic sensitization to pollens exists, whether this association is stronger for patients with high rather than low allergic reactivity to pollens, and whether this association is modified by the age of the patients. Among 3318 asthmatic and/or rhinitic outpatients, we selected 805 patients sensitized exclusively to pollens (78 with low reactivity [LR] and 727 with high reactivity [HR]) and 629 patients with negative skin-prick tests (SPT) (control group). The association between being born during pollen season (February-July) and each of the pollen reactivity levels was assessed by estimating the odds ratios (OR). HR pollinosis patients were more likely than SPT negative patients of being born in February-July (OR 1.38, 95% Confidence Intervals (CI) 1.11-1.71). The likelihood of having been born in pollen season significantly increased across the levels of reactivity to pollens (HR > LR > SPT negative). These findings were valid only among patients with an early onset of symptoms. Although the OR for being born in pollen season was 1.91 (95% CI 1.32-2.77) for HR pollinosis patients with onset of symptoms < or = 15 years, it was 1.13 (95% CI 0.87-1.48) for those with later onset of symptoms (test of homogeneity: p = 0.026). Our results suggest that the exposure to allergenic pollens in the first months of life increases the risk of developing clinically relevant sensitization to them, particularly in the first 15 years of life.

Adolescent↗

Predicting days of high allergenic risk during Betula pollination using weather types.

The aim of this study was to build up a picture of the influence of meteorological conditions on pollen and pollinosis, taking account of weather types, pollen concentrations in the air and pollinosis symptoms, with the aim of preventing allergic responses. The study took place in Burgundy from 1996 to 1998, during the pollination of the birch (Betula), which is the most important arborean allergen in this region. We used daily pollen data from four Hirst volumetric traps, identified weather types by Bénichou's classification, and obtained data on the occurrence of rhinitis, conjunctivitis, asthma and coughing from a sample of 100 patients. These data were analysed by multiple-component analysis. The results show that pollen dispersal is favoured by windy conditions, low relative humidity, precipitation below 2 mm and temperatures above 6 degrees C. Such weather also favours pollinosis, but other particular meteorological situations, even if they do not assist pollen dispersal, can act directly on the development of symptoms: a decrease of temperature (3 degrees C) led to the development of rhinitis and conjunctivitis, while strong winds were associated with many cases of conjunctivitis and asthma, owing to the irritant effect of cold or wind; asthma was favoured by temperature inversions with fog, probably because such weather corresponds to high levels of pollution, which act on bronchial hyperreactivity. Because the weather types favouring pollination and pollinosis are predicted by the meteorological office, this can constitute a tool for reducing the effect of high-risk allergenic days.

Asthma↗

Silencing gene expression of the ethylene-forming enzyme results in a reversible inhibition of ovule development in transgenic tobacco plants

To study the role of ethylene in plant reproduction, we constructed transgenic tobacco plants in which the expression of a pistil-specific gene coding for the ethylene-forming enzyme 1-aminocyclopropane-1-carboxylate oxidase was inhibited. Flowers from transgenic plants showed female sterility due to an arrest in ovule development. Megasporogenesis did not occur, and ovules did not reach maturity. When pollinated, pollen tubes were able to reach the ovary but did not penetrate into the immature ovule in transgenic plants. Flower treatment with an ethylene source resulted in a functional recovery of ovule development and restored guidance of the pollen tube tip into the ovule micropyle that resulted in seed set. The recovery was abolished if inhibitors of ethylene action were present. These results demonstrate that the plant hormone ethylene is required during the very early stages of female sporogenesis and ultimately to enable fertilization.

Journal Article↗

Onset of Graves' thyrotoxicosis after an attack of allergic rhinitis.

We previously reported that allergic rhinitis was an aggravating factor for Graves' disease and that thyrotoxicosis relapsed 2 months after an allergic attack. In this paper, we report a patient who showed onset of Graves' thyrotoxicosis after an attack of allergic rhinitis. The patient, a 30-year-old woman, was initially diagnosed with subclinical autoimmune thyroiditis. Interestingly, the patient showed weak activity of thyroid-stimulating antibody (TSAb), while TSH-binding inhibitory immunoglobulin (TBII) was negative and her thyroid function tests, including TSH, were completely normal. The patient developed severe allergic rhinitis in response to Japanese cedar pollen lasting from February until April in 1995 with an increase in serum antigen-specific immunoglobulin E and peripheral blood eosinophils. Two months later, she developed thyrotoxicosis in association with increase in TSAb and TBII. These findings suggest that allergic rhinitis not only aggravates Graves' disease but also induces the clinical onset of Graves' thyrotoxicosis.

Adult↗

MAP kinases in pollen.

The male gametophyte of flowering plants has a highly regulated developmental programme to ensure efficient fertilization of the ovule and the faithful transmission of genetic material to the offspring. Cell cycle control mechanisms dictate the formation of the vegetative and generative (sperm) cells, while an increase in transcriptional/translational activity and the accumulation of stored proteins and mRNA is followed by a quiescent state at maturation. A switch to a new developmental programme occurs after the pollen tube lands on the stigma with the formation of the pollen tube, growth through the style, and subsequent fertilization. Apart from the internal control mechanisms involved in this developmental programme, pollen grains must cope with physical changes during development within the anther (desiccation) and subsequently during germination on the stigma (rehydration). The metabolic and structural changes that occur throughout these processes should require signaling mechanisms to co-ordinate the appropriate response, and recent data demonstrate the presence in pollen of an array of molecules belonging to diverse signalling pathways, including mitogen-activated protein (MAP) kinases. The role of MAP kinases in pollen is discussed in the context of the various developmental and physical changes that occur throughout pollen maturation and germination.

MAP Kinase Signaling System↗

Heat requirement for the onset of the Olea europaea L. pollen season in several sites in Andalusia and the effect of the expected future climate change.

Olives are one of the largest crops in the Mediterranean region, especially in Andalusia, in southern Spain. A thermal model has been developed for forecasting the start of the olive tree pollen season at five localities in Andalusia: Cordoba, Priego, Jaen, Granada and Malaga using airborne pollen and meteorological data from 1982 to 2001. Threshold temperatures varied between 5 degrees C and 12.5 degrees C depending on bio-geographical characteristics. The external validity of the results was tested using the data for the year 2002 as an independent variable and it confirmed the model's accuracy with only a few days difference from predicted values. All the localities had increasingly earlier start dates during the study period. This could confirm that olive flower phenology can be considered as a sensitive indicator of the effects of climate fluctuations in the Mediterranean area. The theoretical impact of the predicted climatic warming on the olive's flowering phenology at the end of the century is also proposed by applying Regional Climate Model data. A general advance, from 1 to 3 weeks could be expected, although this advance will be more pronounced in mid-altitude inland areas.

Climate↗

Comparative pollen morphology and ultrastructure of the Callitrichaceae.

The Callitrichaceae are an aquatic family of dicots that include the single, geographically cosmopolitan genus Callitriche. Callitriche contains 40-50 terrestrial, amphibious, and obligately submersed species, and it is the only known genus in the plant kingdom with co-occurring aerial and hydrophilous pollination syndromes. Pollen morphology and ultrastructure were described for 13 Callitriche species using scanning electron and transmission electron microscopy. Representative taxa of each growth form were examined; these included three terrestrial species (C. deflexa, C. peploides, and C. nuttallii), nine amphibious species (C. brutia, C. cophocarpa, C. cophocarpa-stagnalis hybrid, C. cribrosa, C. hamulata, C. heterophylla var. heterophylla, C. lusitanica, C. marginata, and C. trochlearis), and one obligately submersed species (C. truncata). Of the amphibious taxa, C. heterophylla var. heterophylla and C. trochlearis had internal geitonogamy, a type of internal self-fertilization. Pollen from all taxa was spheroidal, small, intectate, and lacked well-defined apertures. Taxa primarily differed with respect to exine thickness, surface ornamentation, and the presence or absence of aperture-like regions. The pollen of terrestrial species, as well as that of C. marginata, had well-developed exines with thick sculptured and basal layers. In general, amphibious taxa produced pollen with distinct, but thinner, exines than that of terrestrial taxa. Pollen of the amphibious taxa with internal geitonogamy had a thicker basal layer than species without internal geitonogamy, whereas the overall exine was reduced in C. hamulata and absent in C. brutia and C. lusitanica. Pollen of the obligately submersed C. truncata also lacked an exine. These palynological data were correlated with growth habits and related pollination biologies, as well as with phylogenetic interpretations of Callitrichaceae. Exine reduction or loss has evolved at least twice in the family, and it is associated with aneuploid reduction in chromosome number.

Journal Article↗