Crude Pollen Research Articles

Functional characterization of complete and immunodominant epitopes of a novel pollen allergen from Parthenium hysterophorus.

Background. Parthenium hysterophorus pollen induces chronic clinical conditions such as allergic rhinitis and bronchial asthma. Among the plethora of proteins in the pollens, only few were reported to induce allergy. Currently sensitization to P. hysterophorus pollen allergen is diagnosed by skin prick test (SPT) using the entire pollen extract instead of using the specific allergen. Methods. In P. hysterophorus sensitized patients, SPT was done using the crude pollen extract, 40 kDa allergenic pollen protein and two commercially synthesized allergen epitopes (17 and 24) of P. hysterophorus. Dot-blot of allergen epitopes was done using P. hysterophorus sensitized sera. Crude pollen extract (1, 1.25, 2.5, 5 and 10 µg/mL), 40 kDa allergenic protein (3 µg/mL), and allergen epitopes (3µg/mL) were used to perform Basophil Activation Test (BAT). Results. Crude pollen extract at 2.5, 5, 10 μg/mL and 40 kDa allergenic protein at 3μg/mL concentrations induced wheal and flare reaction by around 15 minutes, whereas commercially synthesized allergen epitopes at 3μg/mL induced wheal and flare reactions in less than 10 minutes. Allergen epitopes (3 µg/mL) revealed strong reactivity with sensitized patient's IgE in dot-blot analysis. Basophil activation Test using crude pollen extract (2.5, 5, 10 µg/mL), 40 kDa allergenic protein (3 µg/mL), and allergenic epitopes (3µg/mL) indicated significant basophil activation (as measured by CD63 expression) in sensitized patients. Conclusions. The 40 kDa allergenic protein and its allergenic epitopes (17 and 24) induced phenotypic and cellular immune responses in P. hysterophorus sensitized individuals. The tested allergenic epitopes (17 and 24) induced faster wheal and flare reactions in comparison with the crude extract and the 40 kDa allergenic protein. The novel 40kDa allergenic protein and its allergen epitopes identified here may be useful for the development of component-resolved diagnosis (CRD) while also serving as a potential therapeutic lead for desensitization treatment for P. hysterophorus pollen induced allergy.

Characterization of Pollen Grain and Pollen Extract from Common Allergenic Plants in the Philippines

Allergic rhinitis and asthma are prevalent in tropical countries like the Philippines. Pollen grains from anemophilous plant species may trigger these respiratory allergies. Pollen extracts from common allergenic plants are currently produced locally in the Philippines and are utilized for the diagnosis and immunotherapy of patients with respiratory allergies. Further improvement of their quality is necessary to avoid false negative or false positive diagnoses, as well as to increase the effectiveness of immunotherapy and avoid adverse reactions. This study aimed to characterize the morphology of the collected pollen grains and assess the pollen purity, protein concentration, and electrophoretic profile of the allergenic pollen extracts. Pollen identification and percent purity of the pollen collections were assessed via microscopy and hemocytometer. Lowry assay was performed to identify the total protein concentration of the pollen extracts before subjecting them to SDS-PAGE and silver staining. The pollen samples from varying plant families have distinct features that will aid in their proper identification. Collected aggregates of pollen samples with pollen purity of < 90%, like T. procumbens, A. saman, L. leucocephala, and M. indica, manifest darker colors compared to their actual color. On the other hand, the color of collected pollen samples with pollen purity ≥ 90% are commonly yellow or off-white, which is their natural color. The total protein concentration of the crude pollen extracts ranges from 2.87–9.63 mg/mL. The pollen extracts revealed protein bands at molecular weights ranging from 10–250 kD on SDS-PAGE. Proper characterization of a particular pollen may help in assessing the purity of the collected pollen. Higher pollen purity may contribute to the assurance that the positive diagnosis was due to the target pollen of a plant and not due to contaminations from other components. The result of this study may be considered for the improvement of the crude pollen allergen extract production for clinical use. It is also recommended to assess the effect of the environmental condition where the pollen was collected in terms of the actual proteins present after allergen extraction.

Assessing cross-reactivity in allergen immunotherapy.

Knowledge of patterns of pollen cross-reactivity is crucial for formulation of immunotherapy vaccines. As phylogenetic relationships have become better clarified through the use of tools such as gene sequencing, it is apparent that cross-reactivity reflects taxonomy in the vast majority of cases. Contradictory observations of unexpected cross-reactivity between distantly related plants require explanation. There are many proteins, presumably performing vital functions, tightly preserved throughout the evolutionary tree from plants to animals. Examples are profilins, lipid transfer proteins, and pathogenesis-related proteins. These may function as panallergens. The small differences that exist between these ubiquitous proteins may explain why these are frequently minor allergens, not reacting in the majority of allergic sera. This article summarizes cross-reactivity from older studies using crude pollen extracts as well as newer research of purified or recombinant allergenic proteins. The patterns of cross-allergenicity that emerge should be helpful in guiding therapeutic decisions.

Plant Stress Scenarios Differentially Affect Expression and IgE Reactivity of Grass Group-1 Allergen (β-Expansin) in Maize and Rice Pollen.

Grass pollen is among the most common outdoor aeroallergens eliciting pollen allergies throughout the world. Grass group-1 allergen or β-expansin is recognized as a major pollen allergen, particularly in the grass family Poaceae. Expression of β-expansin has been shown to be dynamic and can be influenced by environmental stresses. This study evaluated the relative expression of β-expansin and IgE-binding ability of crude pollen extract protein of rice and maize under three different stress conditions: flood, salt, and drought. After 1 week of treatments, anthers containing pollen were collected followed by RNA extraction and cDNA synthesis. To evaluate relative expression, qRT-PCR was performed using specific primers for β-expansin and reference genes. Physiological characteristics of treated and untreated maize and rice: plant height; fresh weight of anthers; number of inflorescences, anthers, and pollen grains were also recorded. To assess IgE-binding ability of proteins in rice pollen extracts, soluble crude proteins were extracted and IgE immunoblot and ELISA were performed using serum samples from grass-allergic subjects and healthy control donors. Results showed that plant height, fresh weight of anthers, number of inflorescences, anthers, and pollen grains of both maize and rice decreased significantly under drought stress conditions, but not in other conditions. Expression of β-expansin in pollen of rice showed an apparent increase in all stress treatments relative to control samples. In contrast, a significant decrease of β-expansin expression was detected in maize pollen under all stress-treated conditions. IgE-reactive protein bands from rice pollen extract proteins were ~30 kDa, as expected of the grass-group 1 protein. The intensity of IgE-reactive protein bands and the level of IgE to rice pollen proteins showed significant differences among stress conditions. In conclusion, environmental stresses—flood, salt, and drought, can elicit a change of β-expansin expression and IgE reactivity to grass group-1 pollen allergens. Changes in expression level of this gene likely reflected its importance during stress. However, the response is highly dependent on different schemes employed by each plant species.

Phoenix dactylifera L. date tree pollen fertility effects on female rats in relation to its UPLC-MS profile via a biochemometric approach

Date palm (Phoenix dactylifera L.) is among the most ancient cultivated crops, of special value owing to its fruits high nutritive and economic benefits. Asides, date palm pollen is a high energy material that has been used traditionally used for fertility enhancement. In this study, effects of date palm pollen crude extract and its fractions viz., petroleum ether, methylene chloride, ethyl acetate and n-butanol on the female reproductive system were evaluated for the first time in relation to its metabolite fingerprint.Fertility activity was evaluated in immature female rats by assessing their FSH-, LH- and estrogen- activities. To pinpoint active hormonal agents in crude pollen extract and fractions, UPLC- MS analysis was employed for metabolites profiling, and in correlation to extract/fraction bioassays using multivariate OPLS analysis. Results revealed that both polar n-butanol and non-polar petroleum ether fractions exhibited the strongest activities; with a significant increase in FSH (25.7 mIU/ml in n-butanol group), estradiol (414.7 pg/ml in petroleum ether group) and progesterone levels (122.4 pg/ml in n-butanol group). Correlation between UPLC-MS and fraction bioassays was attempted using multivariate OPLS analysis to reveal for bioactive hits in these fractions. This study provides the first report on the fertility effect of date palm pollen in female rats and in relation to its metabolite fingerprint.

Ultrasound-Assisted Extraction of Polyphenols From Crude Pollen.

The aim of this study was to evaluate the extraction efficiency of polyphenols from crude pollen by an ultrasonic process. Prior to the polyphenols extraction, the crude pollen was defatted. The extraction from defatted pollen was carried out by varying four extraction parameters: ultrasonic amplitude (20%, 60% and 100%), solid/liquid ratio (10 g/L, 20 g/L and 30 g/L), temperature (35, 50 and 65 °C) and time (10, 20 and 30 min). The extracts were analyzed in terms of extraction yield (%), total phenolic content (TPC) and total flavones content (TFC). The extracted oil was analyzed in terms of fatty acids composition; myristic acid (159.1 µg × g−1) and cis-14-pentadecenoic acid (106.6 µg·g−1) were found in the highest amount in the pollen oil. The optimum conditions of extraction were determined and were, as follows: 100% amplitude of ultrasonic treatment, 30 g/L solid/liquid ratio, 40.85 °C and 14.30 min, which led to the extraction of 366.1 mg GAE/L of TPC and 592.2 mg QE/g of TFC, and also to an extraction yield of 1.92%.

Quality assessment of pollen typhae by high-performance liquid chromatography fingerprint, hierarchical cluster analysis, and principal component analysis

Aim: This study aims to establish the quality assessment methods of Pollen Typhae. Materials and Methods: High performance liquid chromatography (HPLC) fingerprint analysis, hierarchical cluster analysis (HCA), and principal component analysis (PCA) were used for quality evaluation of Pollen Typhae from different origins together with microscopic identification. Then, the quantity of 43 crude Pollen Typhae samples in the market was collected and analyzed. Results: In true and false test, four False Pollen Typhae samples, 13 Net Pollen Typhae (NPT) samples, and 26 Grass Pollen Typhae (GPT) samples were identified by microscopic identification. In quality test, the amounts and percentages of Qualified Pollen Typhae, Unqualified Pollen Typhae were 24 (55.81%) and 19 (44.19%), respectively with typhaneoside and isorhamnetin-3-O-neohesperidoside determined by HPLC according to China Pharmacopeia. We analyzed 43 samples from 20 regions and established their fingerprints, then selected 31 peaks as characteristic peaks and calculated their relative peak areas. To express the HPLC fingerprints quantitatively, peak 16, 18, 22, 23, and 26 were verified as typhaneoside, isorhamnetin-3-O-neoheptanoside, rutin, quercetin, and isorhamnetin. The similarity of correlation coefficients in chromatogram was 0.954 ± 0.007 and 0.922 ± 0.004 for NPT and GPT, respectively, while 0.67 ± 0.008 for 43 samples. The analysis of HCA and PCA can distinguish true or false, qualified or unqualified of Pollen Typhae. Conclusion: HPLC fingerprint combined with HCA and PCA provides a very efficient and comprehensive method for quality evaluation of Pollen Typhae. Abbreviations used: HCA: Hierarchical cluster analysis; PCA: Principal component analysis; FPT: False Pollen Typhae; NPT: Net Pollen Typhae; GPT: Grass Pollen Typhae; QPT: Qualified Pollen Typhae; UPT: Unqualified Pollen Typhae; RSDs: The relative standard deviations; CASE: Computer Aided Similarity Evaluation; TCM: Traditional Chinese medicine.

Multi-Residue Analysis of Pesticide Residues in Crude Pollens by UPLC-MS/MS.

A multi-residue method for the determination of 54 pesticide residues in pollens has been developed and validated. The proposed method was applied to the analysis of 48 crude pollen samples collected from eight provinces of China. The recovery of analytes ranged from 60% to 136% with relative standard deviations (RSDs) below 30%. Of the 54 targeted compounds, 19 pesticides were detected. The major detection rates of each compound were 77.1% for carbendazim, 58.3% for fenpropathrin, 56.3% for chlorpyrifos, 50.0% for fluvalinate, 31.3% for chlorbenzuron, and 29.2% for triadimefon in crude pollen samples. The maximum values of each pesticide were 4516 ng/g for carbendazim, 162.8 ng/g for fenpropathrin, 176.6 ng/g for chlorpyrifos, 316.2 ng/g for fluvalinate, 437.2 ng/g for chlorbenzuron, 79.00 ng/g for triadimefon, and so on. This study provides basis for the research on the risks to honeybee health.

Effects of sublingual immunotherapy on a murine model of Japanese cedar pollinosis (P4198)

Abstract Japanese cedar (Cryptomeria japonica) pollinosis (JCP) is one of the most common allergic diseases in Japan. Sublingual immunotherapy (SLIT) has been performed as a safer and more beneficial treatment to induce allergen-specific immune tolerance. However, immunological mechanisms and therapeutic biomarkers for SLIT remain unclear. In this study, we have established a new murine model of JCP that resembles human JCP with crude pollen grains and without the use of adjuvant and examined the effects of SLIT. SLIT was performed by the graded doses of cedar pollen extract painting onto the ventral surface of tongue for 28 days. [Results] The frequency of sneezing and the duration of rubbing for 10 min after the final challenge, serum IgE titer, pollen-specific IgG1, and the ratio of eosinophils in BALF and nasal mucosae were significantly elevated in the pollinosis mice. SLIT efficiently reduced the above manifestations. SLIT significantly decreased pollen-specific IL-4, IL-5, IL-13 and IL-10 production by splenic T cells and DC activation status such as MHC class II, CD86, and B7-H1 expression in the regional lymph nodes. Histological examination revealed that repeated pollen extract painting onto the sublingual mucosae replaced sublingual resident DCs with newly recruited CD11b+ DCs. These results suggest that our new pollinosis model is beneficial to evaluate and develop a new immunotherapy and therapeutic biomarkers.

Prophylaxis of Intranasally Induced Pollen Allergy in a BALB/C Mouse Model Using a Potential Prebiotic β-1, 4 Mannobiose

Dietary supplementation with unique prebiotic nondigestible carbohydrates has been shown to suppress allergy. In the present study, the prophylactic efficacy of a disaccharide β-1, 4 mannobiose (MNB) in a BALB/C mouse model of intranasally-induced pollen allergy was characterized. Balb/c mice were pretreated with MNB orally and sensitized with pollen extract intraperitoneally and intranasally and challenged with histamine and crude pollen extract. Outcomes were measured as clinical signs, antibody isotypes, cytokine gene and protein expression patterns. The MNB-treated mice had lower sneezing frequency as compared to the positive control mice (P < 0.05). The low dose MNB-treated mice had less histamine (P < 0.05). However, the Cry j1 and Cry j 2-specific IgE, IgG, IgG1 and IgG2a antibody activity did not differ between groups (P > 0.05). The MNB-treated mice had increased IFN-γ (P < 0.05), and decreased IL-4 (P < 0.05). Mice in the high dose group had increased IL-10 (P < 0.05). However, TGF-β and IL-17 concentration did not differ between groups (P > 0.05). Both total and Cry j1 and Cry j 2-specific IgA were increased in the high dose group. Real-time RT-PCR analysis indicated that IL-4 and IL-17 mRNA expression were lower in MNB-treated mice (P < 0.05). This work provides insights into using MNB as a potential prebiotic immunomodulator via decreased clinical signs, improved type1/type 2 balance, and IgA production, thus validating the potential use of MNB as a prophylactic prebiotic candidate to attenuate allergic response.

Evaluation of Molecular Basis of Cross Reactivity between Rye and Bermuda Grass Pollen Allergens

Allergenic cross reactivity between the members of the Pooids (Lolium perenne, Phleum pratense, and Poa pratensis) and Chloridoids (Cynodon dactylon and Paspalum notatum) is well established. Studies using crude extracts in the past have demonstrated limited cross reactivity between the Pooids and the Chloridoids suggesting separate diagnosis and therapy. However, little is known regarding the molecular basis for the limited cross reactivity observed between the 2 groups of grasses. The present study was undertaken to gain insights into the molecular basis of cross allergenicity between the major allergens from rye and Bermuda grass pollens. Immunoblot inhibition tests were carried out to determine the specificity of the proteins involved in cross reactivity. Crude pollen extract and bacterially expressed and purified recombinant Lol p 1and Lol p 5 from rye grass were subjected to cross inhibition experiments with crude and purified recombinant Cyn d 1 from Bermuda grass using sera from patients allergic to rye grass pollen. The immunoblot inhibition studies revealed a high degree of cross inhibition between the group 1 allergens. In contrast, a complete lack of inhibition was observed between Bermuda grass group 1 allergen rCyn d 1, and rye grass group 5 allergen rLol p 5. Crude rye grass extract strongly inhibited IgE reactivity to Bermuda grass, whereas crude Bermuda grass pollen extract showed a weaker inhibition. Our data suggests that a possible explanation for the limited cross reactivity between the Pooids and Chloridoids may, in part, be due to the absence of group 5 allergen from Chloridoid grasses. This approach of using purified proteins may be applied to better characterize the cross allergenicity patterns between different grass pollen allergens.

Structural and immunological properties of arabinogalactan polysaccharides from pollen of timothy grass ( Phleum pratense L.)

Extracts from pollen of timothy grass ( Phleum pratense L.) contain up to 20% arabinogalactan proteins (AGPs). Separation of the AGP polysaccharide moieties by tryptic digestion, size exclusion chromatography (GPC), and reverse phase HPLC yielded arabinogalactan fractions AG-1 and AG-2 with molecular weights of ∼15,000 and ∼60,000 Da, respectively. The backbones of both polysaccharides are composed of (1→6)-linked β- d-galactopyranosides with β- d-GlcUA p or 4- O-Me-β- d-GlcUA p at their terminal ends as revealed by chemical analysis, FT-IR, MALDI-MS, and NMR spectroscopy. AG-1 contains a small number of β- l-Ara f side chains while AG-2 possesses a variety of (1→3)-linked units, which consist of β- l-Ara f-(1→, α- l-Ara f-(1→3)-β- l-Ara f-(1→, and α- l-Ara f-(1→5)-β- l-Ara f-(1→ as well as a small number of longer arabinogalactan side chains. In contrast to crude pollen extracts, the immunological properties of the arabinogalactan mixture reveal an IgG4 reactivity instead of IgE reactivity. Structural properties of timothy pollen arabinogalactan might thus influence the immune response.

Patterns of pollen cross-allergenicity

Knowledge of patterns of pollen cross-reactivity is crucial for diagnostics and especially for formulation of immunotherapy vaccines in times of diminishing availability of pollen extract constituents. As phylogenetic relationships have become better clarified, it becomes apparent that cross-reactivity does reflect taxonomy in the very great majority of cases. Contradictory observations of unexpected cross-reactivity between unrelated plants, sometimes remarkably distant ones, require explanation. There are many proteins, presumably performing vital functions, that are tightly preserved throughout the evolutionary tree from plants to animals, such as profilins, lipid transfer proteins, and pathogenesis-related proteins. These might function as panallergens. The small differences that exist between these ubiquitous proteins explain why these are frequently minor allergens not reacting in the majority of allergic sera. This review summarizes cross-reactivity studies with both crude pollen extracts and purified or recombinant allergenic proteins. The patterns of cross-allergenicity that emerge should be helpful in guiding both diagnostic and therapeutic decisions. (J Allergy Clin Immunol 2003;112:229-39.)

Successive glycosyltransfer activity and enzymatic characterization of pectic polygalacturonate 4-alpha-galacturonosyltransferase solubilized from pollen tubes of Petunia axillaris using pyridylaminated oligogalacturonates as substrates.

Polygalacturonate 4-alpha-galacturonosyltransferase (pectin synthase) was solubilized from pollen tubes of Petunia axillaris and characterized. To accomplish this, an assay method using fluorogenic pyridylaminated-oligogalacturonic acids (PA-OGAs) as acceptor substrates was developed. When the pollen tube enzyme was solubilized with 0.5% (v/v) Triton X-100 and was incubated with PA-OGA and UDP-galacturonic acid (UDP-GalUA), successive transfer activity of more than 10 GalUAs from UDP-GalUA to the nonreducing end of PA-OGA was observed by diethylaminoethyl high-performance liquid chromatography. This activity was time- and enzyme concentration-dependent. The optimum enzyme activity was observed at pH 7.0 and 30 degrees C. Among the PA-OGAs investigated, those with a degree of polymerization of more than 10 were preferred as substrates. The crude pollen tube enzyme had an apparent K(m) value of 13 microM for the PA-OGA with a degree of polymerization 11 and 170 microM for UDP-GalUA. The characteristics of the P. axillaris pollen tube enzyme and the usefulness of fluorogenic PA-OGAs for the assay of this enzyme are discussed.

WHAT GOVERNS PROTEIN CONTENT OF POLLEN: POLLINATOR PREFERENCES, POLLEN–PISTIL INTERACTIONS, OR PHYLOGENY?

Pollen ranges from 2.5% to 61% protein content. Most pollen proteins are likely to be enzymes that function during pollen tube growth and subsequent fertilization, but the vast range of protein quantity may not reflect only pollen–pistil interactions. Because numerous vertebrate and invertebrate floral visitors consume pollen for protein, protein content may influence floral host choice. Additionally, many floral visitors pollinate their host plants. If protein content influences pollinator visitation, then pollinators are hypothesized to select for increased protein content of host plants. We analyzed or gleaned from the literature crude pollen protein concentrations of 377 plant species from 93 plant families. Using this database, we compared pollen protein concentration with (1) pollination mode, (2) pollen collection by bees, and (3) distance from stigma to ovule, after accounting for phylogeny through paired phylogenetic comparisons and a nested ANOVA including taxonomic rank. We found that pollen pr...

Successful Technique for the Selective Production of Monoclonal Antibodies Against a Major Allergenic Component in Timothy Pollen Extract

Monoclonal antibodies (MAbs) were selectively raised against a major allergenic component of 38 kD in timothy grass pollen (Phleum pratense). We used a special prefractionating technique to isolate the 38 kD allergen, because immunizations with crude pollen extract had resulted in a wide variety of antibodies against other determinants, and cross-reactions between different-sized proteins occurred. Pollen extract was separated by Western blotting. Strips of the nitrocellulose membrane containing the allergen were excised, dispersed into allergen bearing particles, and used for immunization of BALB/c mice. Five MAbs were obtained that reacted with the 38 kD allergen. Four of these antibodies (BF 1, DC 9, EB 4, GE 2) exclusively detected the 38 kD protein, while one MAb (EB 6) additionally bound to a 32 kD allergen. Epitope mapping with the 3 IgG 1 antibodies (BF 1, EB 6, DC 9) was performed by ELISA inhibition tests using the purified 38 kD component. The antibodies did not interfere with each other. This confirms that they bind to different sites of the 38 kD molecule. Only a weak inhibitory effect of the MAbs on the binding of patient's IgE was determined, we suppose that the MAbs do not bind to IgE reactive epitopes. For standardization and further characterization of the 38 kD protein by peptide mapping the MAbs will be useful tools.

Measurement of serum IgE antibodies against Japanese cedar pollen (Cryptomeria japonica) in Japanese monkeys (Macaca fuscata) with pollinosis

IgE antibodies against allergens of Japanese cedar (Cryptomeria japonica, CJ) pollen in the serum of seven Japanese monkeys (Macaca fuscata) with pollinosis were measured by fluorometric indirect enzyme-linked immunosorbent assay (ELISA). All of the monkeys were found to have specific IgE to the crude pollen antigen. The specific IgE levels were well correlated with those determined by the Pharmacia CAP system. IgE antibodies were then assayed with two kinds of purified allergens (Cry j I and Cry j II) by the ELISA. We found that five monkeys had specific IgE to both allergens, although the other two had IgE only to Cry j I or Cry j II; there is different immune responsiveness to the two major allergens in the monkeys.

Pollen allergens from differentDactylis glomeratavarieties harvested between 1986 and 1988

Abstract Pollen was collected from different cultivated varieties of Dactylis glomerata in 1986, 1987 and 1988 and compared with an undifferentiated stock of Dactylis glomerata pollen harvested and stored dry at +4°C since 1981. The allergen content of the crude pollen extracts was established on the basis of the IgE antibodies from the sera of three different patients allergic to grass pollen, using the nitrocellulose immunoprint technique following isoelectric focusing (IEF) and separation in agarose gels. Coomassie blue and silver staining patterns were also compared. They showed some marked differences in the isoelectric points of the constituants of these extracts. Using this major allergen recognition by patient sera as a selection criteria we were able to delimit 8 Dactylis varieties with low or undetectable Dac g IV allergen amounts and 7 varieties which contained this allergen. Two other allergens used as markers enabled us to suggest a kind of taxonomy, based upon the allergen presence, of these 15 varieties. The effect of the storage temperature was studied for 8 different varieties kept at +4°C, -20°C and -40°C. The allergen recognition and silver staining patterns after IEF separation of the crude pollen extracts revealed a selective persistance of some constituants and the disappearance of others at +4°C. The effect of freeze-drying was also analysed in the same way. Finally the pollen constituants of one Dactylis glomerata variety, harvested in 1986 and 1987 and kept at different temperatures were compared using the same techniques.

Experimental Cedar Pollinosis in Rhesus Monkeys

In order to develop an experimental model for human cedar pollinosis, 4 rhesus monkeys (Macaca mulatta) were subcutaneously immunized with crude cedar pollen antigen, which contained 1.0 microgram or 10 micrograms protein with aluminium hydroxide gel, six times every month. All 4 animals showed immediate-type cutaneous reactivity. Serum IgE antibody responses against the pollen antigen, as quantitated by the Prausnitz-Küstner reaction, were seen in 3 of 4 monkeys. Radioallergosorbent test scores increased in 2 monkeys and remained at higher levels throughout the study. Histamine release from leukocytes was positive in all animals. In conjunctival provocation tests, they showed clinical signs such as eye-scratching after the antigen was dropped into their eyes. This reaction is similar to that observed in humans and suggests this animal model might be useful in studying human cedar pollinosis.

Selection of patients for biological standardization as exemplified by standardization of mugwort, goosefoot and English plantain pollen allergen extracts/preparations.

The biological activity of a partly purified, biochemically/immunochemically characterized mugwort pollen allergen preparation and crude pollen extracts of mugwort, goosefoot and English plantain was determined by means of skin prick test (SPT). The patient inclusion criteria with mugwort were a well-defined positive clinical history and a positive SPT. Symptoms related to goosefoot/English plantain pollens are difficult to define, as these weeds flower during the grass pollen season. Thus patients tested with these allergens did not fulfill the most important inclusion criterion for so-called biological standardization. To elicit a wheal of the same size as that produced by histamine 1 mg/ml required 100 to 10,000 times more material from these weeds, than from mugwort and other pollen allergen extracts investigated earlier. One thousand Biological Units/ml (BU/ml) corresponded to 8.3 micrograms dry weight (dw/ml) of the crude and 1.8 micrograms dw/ml of the purified mugwort pollen allergen preparation. Only 7/22 goosefoot-and English plantain-tested patients were positive at conjunctival or nasal challenge. All three weeds showed a similar composition with 5-10 allergens by CIE/CRIE analysis and 10-13 by immunoblotting analysis. One dominating allergen (approx. 15,000 d), could be identified for each weed species by protein gel blot after separation by SDS g-PAGE. There was no other explanation for the difference in biological activity than the criteria of selection. If there is no obvious clinical history, which is the main patient inclusion criterion in biological standardization, then additional criteria should be used.