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Allergen-specific immunotherapy in birch- and grass-pollen-allergic rhinitis. I. Efficacy estimated by a model reducing the bias of annual differences in pollen counts.

BACKGROUND: Evaluation of the efficacy of allergen-specific immunotherapy (IT) with pollen extracts is complicated by annual variation in pollen intensity. Our study aimed to evaluate the efficacy of birch and grass IT, taking into consideration these variations. METHODS: After 1 year of observation, 52 patients with rhinoconjunctivitis and allergy to birch as well as grass pollen were allocated to double-blinded clustered IT with aluminum-adsorbed extract produced from either birch (Betula verrucosa) pollen or grass (PIleum pratense) pollen. After 1 year of treatment, the patients continued IT with their original extract and also received the other extract. During the three consecutive pollen seasons, the rhinoconjunctivitis symptom score and the use of antihistamines, eye-drops, and oral prednisolone were recorded. Longitudinal data analysis was used to investigate the relation between different pollen counts and the magnitude of clinical efficacy. RESULTS: An effect of IT was found on symptom score, antihistamine intake, and eye-drop use for both birch and grass (P values <0.05). The mean reduction in symptom score/medication by IT ranged from 24% to 95%, depending on mean seasonal pollen counts. A minimum mean seasonal grass-pollen count of 20-30 pollen grains m3 was required for the efficacy of grass IT to emerge. CONCLUSIONS: A model was developed for evaluation of efficacy in longitudinal IT studies, taking the differences in annual pollen counts into consideration. The model showed a significant beneficial role of pollen IT in rhinoconjunctivitis patients allergic to birch and grass pollen.

Adolescent↗

Cell wall reactive proteins in the coat and wall of maize pollen: potential role in pollen tube growth on the stigma and through the style.

The surface of a pollen grain consists of an outermost coat and an underlying wall. In maize (Zea mays L.), the pollen coat contains two major proteins derived from the adjacent tapetum cells in the anthers. One of the proteins is a 35-kDa endoxylanase (Wu, S. S. H., Suen, D. F., Chang, H. C., and Huang, A. H. C. (2002) J. Biol. Chem. 277, 49055-49064). The other protein of 70 kDa was purified to homogeneity and shown to be a beta-glucanase. Its gene sequence and the developmental pattern of its mRNA differ from those of the known beta-glucanases that hydrolyze the callose wall of the microspore tetrad. Mature pollen placed in a liquid medium released about nine major proteins. These proteins were partially sequenced and identified via GenBank trade mark data bases, and some had not been previously reported to be in pollen. They appear to have wall-loosening, structural, and enzymatic functions. A novel pollen wall-bound protein of 17 kDa has a unique pattern of cysteine distribution in its sequence (six tandem repeats of CX3CX10-15) that could chelate cations and form signal-receiving finger motifs. These pollen-released proteins were synthesized in the pollen interior, and their mRNA increased during pollen maturation and germination. They were localized mainly in the pollen tube wall. The pollen shell was isolated and found to contain no detectable proteins. We suggest that the pollen-coat beta-glucanase and xylanase hydrolyze the stigma wall for pollen tube entry and that the pollen secrete proteins to loosen or become new wall constituents of the tube and to break the wall of the transmitting track for tube advance.

Allergens↗

Maize pollen coat xylanase facilitates pollen tube penetration into silk during sexual reproduction.

Cell wall hydrolases are well documented to be present on pollen, but their roles on the stigma during sexual reproduction have not been previously demonstrated. We explored the function of the tapetum-synthesized xylanase, ZmXYN1, on maize (Zea mays L.) pollen. Transgenic lines (xyl-less) containing little or no xylanase in the pollen coat were generated with use of an antisense construct of the xylanase gene-coding region driven by the XYN1 gene promoter. Xyl-less and wild-type plants had similar vegetative growth. Electron microscopy revealed no appreciable morphological difference in anther cells and pollen between xyl-less lines and the wild type, whereas immunofluorescence microscopy and biochemical analyses indicated an absence of xylanase on xyl-less pollen. Xyl-less pollen germinated as efficiently as wild-type pollen in vitro in a liquid medium but less so on gel media of increasing solidity or on silk, which is indicative of partial impaired water uptake. Once germinated in vitro or on silk, the xyl-less and wild-type pollen tubes elongated at comparable rates. Tubes of germinated xyl-less pollen on silk did not penetrate into the silk as efficiently as tubes of wild-type pollen, and this lower efficiency could be overcome by the addition of xylanase to the silk. For wild-type pollen, coat xylanase activity on oat spelled xylan in vitro and tube penetration into silk were inhibited by xylose but not glucose. The overall findings indicate that maize pollen coat xylanase facilitates pollen tube penetration into silk via enzymatic xylan hydrolysis.

Cloning, Molecular↗

Seasonal rise in interleukin-4 during pollen season is related to seasonal rise in specific IgE for pollens but not for mites.

Since IL-4 plays a key role in inducing and increasing the generation of not only primary polyclonal but also secondary specific IgE responses by B lymphocytes, a seasonal increase in IL-4 is likely to be involved in such seasonal rises in specific IgE in seasonal allergic rhinitis. The first aim of this study was to investigate the possible seasonal increase in serum IL-4 in patients with seasonal allergic rhinitis due to Japanese cedar pollens. If serum IL-4 increases in response to seasonal pollen exposure and is responsible for the seasonal increase in pollen-specific IgE in sera, this increase in IL-4 might theoretically affect specific IgE synthesis for other allergens. The second aim was to investigate the effect of natural pollen exposure on serum concentrations of house dust mite-specific IgE in patients who have seasonal allergic rhinitis and concurrent perennial allergic rhinitis due to house dust mites. This study included 55 adult patients with seasonal and perennial allergic rhinitis due to Japanese cedar pollens and Dermatophagoides farinae (D. farinae). Venous blood was collected twice from each patient, before and during the cedar pollen season 1996, to determine IL-4, cedar pollen-specific IgE and D. farinae-specific IgE in sera. Both IL-4 and pollen-specific IgE in sera were significantly increased during the pollen season, and the seasonal increase rate in pollen-specific IgE was significantly correlated with the seasonal increase rate in IL-4. By contrast, D. farinae-specific IgE was not changed during the pollen season in these patients. In conclusion, an elevation of IL-4 in sera during the pollen season may play an important part in the seasonal rise in pollen-specific IgE, and enhancement of specific IgE synthesis is likely to need not only an increase in IL-4 but also an increase in the number and/or capacity of specific IgE-secreting B cells.

Adolescent↗

Pollen dispersion, pollen viability and pistil receptivity in Leymus chinensis.

BACKGROUND AND AIMS: Leymus chinensis is an economically and ecologically important grass that is widely distributed across eastern areas of the Eurasian steppe. A major problem facing its propagation by man is its low sexual reproductivity. The causes of low fecundity are uncertain, largely because many aspects of the reproductive biology of this species remained unknown or incomplete. This study aims to address some of these issues. METHODS: Pollen dispersion, pollen viability, pollen longevity and pistil receptivity were studied in a representative, natural population of L. chinensis growing in Inner Mongolia. KEY RESULTS: Flowering of L. chinensis occurred at the end of June and lasted for 5 d. Pollination peaked between 1600 h and 1700 h, and about 56.1 % of the total pollen grains were released at this time. Pollen density was highest towards the middle of flowering spikes and lowest at the bottom over the 5 d measurement period. Pollen viability (62.4 %) assessed using TTC was more accurate than using IKI (85.6 %); 50 % of pollen arriving on stigmas germinated. Pollen remained viable for only 3 h and the pollen : ovule ratio was 79 333 : 1. Pistil receptivity lasted for only 3 h and, overall, 86.7 % of pistils were pollinated. Within the spike, the relative fecundity of different positions was middle > lower > upper throughout the period of pollination; daily variation of fecundity was similar to that of the pollen flow. The spikes that opened on the day of highest pollen density exhibited the highest fecundity (36.0 %). No seeds were produced by self-pollination. CONCLUSIONS: The data suggest that low pollen viability, short pollen longevity and short pistil receptivity all appear to contribute to the low seed production typical of this important forage crop.

Cell Survival↗

[Rice pollen asthma and pollinosis in childhood: seasonal asthma and allergic rhinoconjunctivitis during the period of rice pollen emission in the surrouding area of rice field].

Although in 1969, rice pollen was first reported as a cause of asthma, rice pollen allergy has not been studied after the first report and thus the allergic significance of rice pollen is not well recognized at present. We investigated the sensitization to various allergens and the residential areas in children with the symptoms of asthma or rhinoconjunctivitis during the first decade of August, and measured rice pollen-specific IgE antibodies. Eighty-eight children (57 boys and 31 girls, mean age 8.5+/-2.9 years) with bronchial asthma or allergic rhinitis/conjunctivitis were included in this study and divided into two groups: children with (n=21) or without (n=67) symptoms during this period. The positivity rate to orchard grass pollen and the rate of residence in the surrounding area of rice field were high (81%, P=0.008 and 86%, P<0.001, respectively) in children with allergic symptoms, as compared to the values (48% and 27%, respectively) in children without symptoms. As the rice pollen season in Nagano occurs in the first decade of August, we measured rice pollen-specific IgE antibodies in 8 patients with symptoms during this period; all of these patients showed positive IgE antibodies to rice pollen. The RAST-inhibition assay using orchard grass and rice pollen indicated cross-allergenecity between these two pollen and also the existence of rice pollen-specific allergens. These results suggest that rice pollen induces seasonal asthma and allergic rhinoconjunctivitis during the first decade of August, which is the rice pollen season, in the surrounding area of rice fields.

Adolescent↗

Pollen-pistil relationships and pollen size-number trade-off in species of the tribe Lycieae (Solanaceae).

Pollen volume may be involved in different associations with other floral traits. Particularly, the literature indicates that pollen volume can be implicated in a functional relationship with pistil length, and that it may be affected by a trade-off with pollen number because of the subdivision of limited resources. To assess these associations, pollen volume was subjected to correlation analyses with pollen number and pistil length in 20 Argentinean and Chilean taxa of the monophyletic tribe Lycieae. Depending on the mechanisms operating on pollen size and number, the variability of these traits may be different. Therefore, their coefficients of variation were compared. Pistil length and pollen volume showed a strong positive correlation. In contrast, pollen grain size and number were significantly correlated at neither inter- nor intraspecific levels. Results suggest that pollen size and pistil length may co-evolve. The central role of this interaction is discussed. The lack of a correlated variation in pollen size and number may be related to the similar constraints experienced by the species and/or because constraints are operating at the plant level and not at the species level. Lower variance in pollen size compared with pollen number denotes that pollen size may be the main trait subjected to natural selection.

Journal Article↗

Expression of green fluorescent protein in pollen of oilseed rape (Brassica napus L.) and its utility for assessing pollen movement in the field.

Transgene movement via pollen is an important component of gene flow from transgenic plants. Here, we present proof-of-concept studies that demonstrate the monitoring of short distant movement of pollen expressing a genetically encoded fluorescent tag in oilseed rape (Brassica napus L. cv. Westar). Transgenic oilseed rape plants were produced using Agrobacterium-mediated transformation method with the pBINDC1 construct containing a green fluorescent protein (GFP) variant, mGFP5-ER, under the control of the pollen-specific LAT59 promoter from tomato. Transgenic pollen was differentiated from non-transgenic pollen in vivo by a unique spectral signature, and was shown to be an effective tool to monitor pollen movement in the greenhouse and field. GFP-tagged pollen also served as a practical marker to determine the zygosity of plants. In a greenhouse pollen flow study, more pollen was captured at closer distances from the source plant plot with consistent wind generated by a fan. Under field conditions, GFP transgenic pollen grains were detected up to a distance of 15 m, the farthest distance from source plants assayed. GFP-tagged pollen was easily distinguishable from non-transgenic pollen using an epifluorescence microscope.

Brassica napus↗

A wheat genomic DNA fragment reduces pollen transmission of maize transgenes by reducing pollen viability.

A genomic DNA fragment from wheat carrying the Glu-1Dx5 gene has been shown to exhibit reduced pollen transmission in transgenic maize. To localize the region of the DNA fragment responsible for this reduced pollen transmission, we produced transgenic maize plants in which the wheat genomic DNA proximal to the 1Dx5 coding sequence was replaced with the maize 27 kDa gamma-zein promoter. Like the wheat promoter-driven Glu-1Dx5 transgene, this zein promoter-driven transgene functioned to produce 1Dx5 in maize endosperm. However, with the zein promoter-driven transgene, pollen transmission of the transgene loci was normal in most self- and cross-pollinations. We concluded that the wheat genomic DNA proximal to the wheat 1Dx5 coding sequence was required for reduced pollen transmission of the transgene in maize. In two of four transformation events of the wheat promoter-driven construct examined, pollen exhibited two morphological classes. In one class, pollen was normal in morphology and displayed average viability, and in the second, pollen was reduced in size and did not germinate on artificial media. DNA from the transgene was detectable in mature pollen from plants with reduced pollen transmission of transgene loci. To explain these observations, we hypothesize that elements within the transgene construct interfere with pollen development. We demonstrated that the wheat genomic DNA fragment can be used to control pollen transmission of an herbicide resistance transgene genetically linked to it. The wheat genomic DNA fragment may contain elements that are useful for controlling pollen transmission of transgene loci in commercial maize grain and seed production.

DNA↗

Degranulation of eosinophils from pollen-atopic patients with asthma is increased during pollen season.

The secretion of granule proteins from eosinophils and neutrophils was studied in isolated cells, obtained from 11 pollen-atopic patients with asthma, twice during and twice outside pollen season. Granulocytes were stimulated with serum-opsonized Sephadex particles, and the released amount of eosinophil cationic protein (ECP), eosinophil protein X (EPX), and myeloperoxidase (MPO) were measured by means of specific radioimmunoassay (RIA). Eosinophils from the pollen-atopic patients obtained during pollen season released significantly more (p less than 0.02) ECP and EPX than cells from the same patients obtained before pollen season. The released amount of ECP and EPX was correlated (r = 0.54; p less than 0.003) to the total pollen count. The release of MPO from neutrophils was only raised (p less than 0.01) at the end of the pollen season. Serum concentrations of ECP and EPX and blood eosinophil counts were significantly raised (p less than 0.002, p less than 0.001, and p less than 0.009, respectively) before pollen season and increased further at the end of the pollen season. There were no changes in lung function during pollen season and consequently no discernible relationships to eosinophil and neutrophil degranulation. We conclude that eosinophils and, to some extent, neutrophils from birch pollen-atopic subjects have an increased propensity to secrete their granule proteins during a pollen season. We suggest that these cells have been primed as a consequence of allergen exposure.

Adolescent↗

LAT52 protein is essential for tomato pollen development: pollen expressing antisense LAT52 RNA hydrates and germinates abnormally and cannot achieve fertilization.

The LAT52 gene of tomato is expressed in a pollen-specific manner. It is shown that LAT52 encodes a heat-stable, glycosylated protein that traverses the secretory pathway when expressed in a baculovirus expression system. The LAT52 protein shows some similarity with Kunitz trypsin inhibitors and with pollen proteins from maize, rice and olive, but the biological function of these pollen proteins is unknown. To test whether the LAT52 protein plays an important role during pollen development, tomato plants were transformed with an antisense LAT52 gene driven by the LAT52 promoter. Because the LAT52 gene is expressed gametophytically, only 50% of the pollen of the primary transformants would be expected to express the antisense construct. Selfprogeny of 19 of the primary transformants showed the predicted 3:1 segregation for a single locus insertion of the linked kanamycin-resistance gene. However, the self-progeny of the other 32 primary transformants showed a 1:1 segregation pattern and could not transmit the linked kanamycin-resistance gene through the male. A subset of these 1:1 segregation class plants was examined in detail. The pollen showed lower levels of LAT52 mRNA and LAT52 protein when compared with wild-type. In vitro, approximately 50% of the pollen grains appear to hydrate abnormally; this anomaly is not present when the same pollen grains are incubated in a medium with higher water potential. In vivo pollination experiments showed that the growth of around 50% of the pollen tubes is arrested in the style. The 3:1 segregation class plants showed no significant differences from untransformed control plants. Taken together, the results show a direct correlation between the reduced expression of LAT52 protein and abnormal pollen function, and suggest that the LAT52 protein plays a role in pollen hydration and/or pollen germination.

Amino Acid Sequence↗

An ankyrin repeat-containing protein, characterized as a ubiquitin ligase, is closely associated with membrane-enclosed organelles and required for pollen germination and pollen tube growth in lily.

Exhibiting rapid polarized growth, the pollen tube delivers the male gametes into the ovule for fertilization in higher plants. To get an overall picture of gene expression during pollen germination and pollen tube growth, we profiled the transcription patterns of 1,536 pollen cDNAs from lily (Lilium longiflorum) by microarray. Among those that exhibited significant differential expression, a cDNA named lily ankyrin repeat-containing protein (LlANK) was thoroughly studied. The full-length LlANK cDNA sequence predicts a protein containing five tandem ankyrin repeats and a RING zinc-finger domain. The LlANK protein possesses ubiquitin ligase activity in vitro. RNA blots demonstrated that LlANK transcript is present in mature pollen and its level, interestingly contrary to most pollen mRNAs, up-regulated significantly during pollen germination and pollen tube growth. When fused with green fluorescent protein and transiently expressed in pollen, LlANK was found dominantly associated with membrane-enclosed organelles as well as the generative cell. Overexpression of LlANK, however, led to abnormal growth of the pollen tube. On the other hand, transient silencing of LlANK impaired pollen germination and tube growth. Taken together, these results showed that LlANK is a ubiquitin ligase associated with membrane-enclosed organelles and required for polarized pollen tube growth.

Ankyrin Repeat↗

Seasonal appearance of grass pollen allergen in natural, pauci-micronic aerosol of various size fractions. Relationship with airborne grass pollen concentration.

In a study during the 1993 grass pollen season at Leiden, the relationship between atmospheric pollen allergen carried by five size fractions of pauci-micronic (few microns) particles and the grass pollen count was investigated. Sampling was carried out on dry days, and atmospheric pollen allergen in the particle fractions was assessed by a RAST-inhibition assay while grass pollen quantities were measured with a volumetric pollen trap. It appears that the atmospheric presence of grass pollen allergen in all size fractions is restricted mainly to the period of presence of grass pollen grains. Before and after the grass pollen season atmospheric grass pollen allergen quantities are generally very low. It is concluded that a routinely performed grass pollen count is a reliable measurement for the estimation of the amount of atmospheric grass pollen allergen, also in the pauci-micronic particle fraction.

Aerosols↗

Cross-reactivity to olive tree pollen and orchard grass pollen in patients with pollinosis.

We studied 92 patients with allergic rhinitis in Syodoshima, Japan, during the pollen season between April and June to evaluate the cross-reactivity to different antigens, including pollen from the olive tree (Olea europaea) and orchard grass (Dactylis glomerata). Olive tree pollen was present in the atmosphere for 23 days, from May 19 to June 12, 1994. Specific IgE antibodies for olive tree pollen antigen were present in 21 (26.9%) of the 78 patients with allergic rhinitis. Nine (24.3%) of the 37 patients with allergic rhinitis exhibited positive skin reactivity to an extract of olive tree pollen. Fifteen (88.2%) of the 17 patients who had IgE reactivity in their sera to olive tree pollen antigen demonstrated allergic reactions to an extract of olive tree pollen. Specific IgE antibodies for orchard grass pollen antigen were present in 43 (48.3%) of the 89 patients with allergic rhinitis and 20 (95.2%) of the 21 patients who had IgE reactivity in their sera to olive tree pollen antigen. The inhibition test using the CAP System revealed that the reactivity of the IgE antibody specific for olive tree pollen antigen was inhibited dose-dependently by an extract of orchard grass pollen. These findings show that there is a reaction in some patients with grass (Gramineae) pollinosis that might be induced by olive tree pollen.

Hemadsorption Inhibition Tests↗

[2001 survey of pollen in Wakayama City with a real-time pollen counter].

Self-care is gradually being recognized as important in the treatment of pollinosis, based up to now on data on airborne pollen. To determine the real-time numbers of airborne pollen would be more useful in self-care, however, so we studied the usefulness of the real-time pollen counter. Between Feb. 2, 2001, and Apr. 26, 2001, 4 types of airborne pollen i.e., Japanese cedar, Japanese cypress, black alder, and beech observed in Wakayama City were counted with a Durham pollen counter and a real-time pollen counter (Yamato Manufacturing Co. Ltd.). Correlation between the 2 pollen counters was r = 0.69 for Japanese cedar in March and r = 0.89 for Japanese cypress in April. A high correlation was observed between outcomes of the 2 pollen counters. The amount of pollen from black alder and beech was less than that from Japanese cedar and cypress. Unexpected peaks were observed not related to the pollen number is apparently due to snow. We have taken measure against snow, so we concluded that the real-time pollen counter was useful in counting the amount of airborne pollen over time.

Air Pollutants↗

[Identification of airborne pollen and airborne particles with pollen allergen (Cry j 1, Dac g) by aeroallergen immunoblotting technique].

After collection of airborne particles with a seven-day recording volumetric spore trap (Burkard model) using optically clear, pressure sensitive acrylic adhesive tapes, a dry PVDF membrane was pressed down firmly onto the adhesive tape, antigen-antibody reaction was performed in full contact with the tapes and PVDF membrane. Dark purple spots from airborne pollen allergens were examined under a light microscope to evaluate the form of pollens and particles wit h the antigenicity. It is clarified that identification of the form of pollens with the antigencity is possible not only pollens with the antigenicity but a pollen has already lost its shape, and it is also clarified that some airborne particular matters have the pollen antigencity. Airborne samples were collected during the Cryptomeria japonica or grass pollen season. Samples collected during the C. japonica pollen season were treated with anti-Cry j 1 monoclonal antibody, and those during the grass pollen season with anti-Dac g rabbit IgG. Samples collected during the C. japonica and the grass pollen peak season were treated with antibodies from sera of pollinosis patients. Spots originated not only from relevant pollen grains but also from parts of airborne particular matter have the pollen antigenicity and some spots could not observed for any kind of particles.

Air Pollutants↗

[A monoclonal antibody against ragweed pollen cross-reacting with yellow dock pollen].

Using monoclonal antibodies with different specificity against the major allergenic components of ragweed pollen, we analyzed their cross-reactivity with two tree pollens, two grass pollens and five other weed pollens which are common in Taiwan by the immunoblot method. It was found that besides reacting with AgE and AgK of the ragweed pollen, the monoclonal antibody 48-5 also reacted with antigens of yellow dock pollen with molecular weights of 40K, 38K, 24K, and 21K. In a preliminary study, sera of two patients containing IgE antibodies to ragweed pollen antigens also reacted to the 40K component of the yellow dock pollen. Furthermore, from the results of allergenic skin testings on 109 patients with bronchial asthma, we found that of 22 patients who had a positive reaction to a crude extract of ragweed pollen, 18(81.8%) also reacted to the crude extract of yellow dock pollen. In conclusion, our results suggest that there exists a common allergenic determinant between pollens of ragweed and yellow dock. It may play an important role in the expression of the sensitivity of patients to these two kinds of pollens.

Allergens↗