Rye Pollen Research Articles

Constant relative humidity chambers using phosphoric acid for controlled desiccation of small recalcitrant biological samples

For successful storage and cryopreservation of recalcitrant (short-lived or desiccation-sensitive) plant seeds, excised seed embryos or pollen, precise control of water content and/or slow drying rates are often crucial. To realize such controlled desiccation, we developed simple and safe constant relative humidity (RH) chambers using a single compound, phosphoric acid (PA). It consisted of a bowl of PA-water solution placed in a hermetically sealed box. With this apparatus, RH ranges of 4-88% were obtained from 100-10% PA solutions, respectively. Similar RH-PA concentration profiles were obtained at 24 and 7°C. Constant RH was ensured at a constant temperature as temperature fluctuations resulted in mirror-image RH oscillations within the chamber. When the temperature was greatly changed within 5-30°C ranges, the chamber regained the same equilibrium RH in 4-8 h. Using this system, typical recalcitrant winter rye pollen was successfully stored under RH50% at 7°C for 10 days. Alginate beads, irrespective of sucrose concentrations (0.7-1.2 M), were desiccated to equilibrium water contents of 11.5-32% (fresh weight basis) under constant RH of 27-78% at 24°C. This system is simple, economical and safer than conventional RH chamber systems using saturated solutions or sulfuric acid. It allows constant RH available at various temperatures and is useful for controlled desiccation of small recalcitrant genetic or in vitro resources for dry-cold or cryogenic storage.

Adsorption of rye pollen and egg-white allergens on aerosil

The surface of aerosils – substances representing highly disperse nontoxic silica gels – is characterized by a very large area and a specific morphology. These factors account for the high adsorption capacity of aerosils with respect to proteins, enzymes, exoand endotoxins, and microorganisms. The absence of developed pores ensures a high rate of adsorption on the aerosil surface, which makes these media promising sorbents and carriers for immobilization of a wide spectrum of biologically active substances [1]. Sorption methods are used in the course of specific immunotherapy for the immobilization of allergens. Adsorbents used for such purposes include both inorganic (calcium phosphate [2, 3], aluminum hydroxide and phosphate [4]) and organic (L-tyrosine [5]) media; it was also suggested to use activated charcoal as a metric for diagnostic purposes [3]. The aim of this work was to study the process of immobilization of some pollen and food-born allergens on aerosil. We have studied the properties of A-380 aerosil, which had never been used before as a medium for allergen immobilization. This sorbent, obtained from the Institute of Surface Chemistry (National Academy of Sciences of Ukraine, Kiev), is a highly disperse nonporous silica gel with a specific surface of 380 m g, an average particle size of 7 nm, and a surface concentration of silicon atoms of 5.09 mmole g. The main adsorption centers on the aerosil surface are silanol groups (SiOH2 ). The experiments were performed with commercial preparations of rye pollen allergen (RPA) and egg-white allergen (EWA) in the form of aqueous-salt solutions obtained from the “Immunolog” Company (Vinnitsy), which were standardized with respect to protein nitrogen (PNU cm) and stabilized with 0.4% of phenol. RPA and EWA are representatives of the wide classes of pollen and food-born allergens, which are used both for diagnostic purposes and for specific immunotherapy [6, 7] in the complex treatment of allergic disorders. These agents are widespread and characterized by high allergen activity. As is known, the main allergen components in both rye pollen (molecular weight, 38 – 40,000) and in egg-white albumin are glycoproteins (lysozyme, ovalbumin, avomucoid). We have studied the dependence of the allergen adsorption on the protein carrier ratio and the time. The content of

Contact Allergy in Agricultural Workers

Agricultural workers (n = 121) referred to the Nofer Institute of Occupational Medicine for suspected occupational disease were subjected to dermatological and allergological examinations. All were patch and prick tested with standard occupational and environmental allergen sets. Contact dermatitis was diagnosed in 60 (49.6%) patients. In the women, the dominant allergy was that to nickel, benzalkonium, palladium, cobalt, thimerosal, fragrances and balsam of Peru, while the men were most frequently sensitive to chromates, cobalt, 4-phenylenediamine, fragrances, captan, formaldehyde, 4-aminoazobenzene, wool alcohols and cinnamic alcohol. Phenylmercuric chloride caused allergic reactions in 6 women and 2 men, while 6 workers were sensitive to neomycin. Allergy to glutaraldehyde was diagnosed in 3 workers, to lysol in 4, to chlorhexidine in 1, to chloramine in 1. Three patients reacted to thiurams, 3 to diphenylguanidine, 3 to mercaptobenzothiazole. Of the 9 workers sensitive to 4-phenylenediamine, only 2 men reacted additionally to N-isopropyl-N-4-phenylenediamine. Ziram (zinc dimethyldithiocarbamate) and copper sulfate caused allergy in 1 agricultural worker each. Immediate allergy was diagnosed in 43 (35.5%) patients. The dominant allergens in that category included straw dust, hay dust, wheat threshing, rye pollen and cow epithelium. The final diagnosis was based on the clinical picture and the results of the patch and prick tests. Allergic contact dermatitis was diagnosed in 27 (22.3%), atopic dermatitis in 27, irritant contact dermatitis in 15 (12.4%) and urticaria in 9 (7.5%) agricultural workers. Other dermal diseases were diagnosed in single patients. The clinical examinations showed no dermal lesions in 31 (25.6%) patients.

The effect of short-term immunotherapy with molecular standardized grass and rye allergens on eosinophil cationic protein and tryptase in nasal secretions

Background: Activation of mast cells and eosinophils under pollen exposure can be inhibited by specific immunotherapy. Objective: The effect of short-term immunotherapy with 7 preseasonal injections of molecular standardized allergens from grass and rye pollen on eosinophil cationic protein (ECP) and tryptase levels in nasal secretions has been compared with symptomatic drug treatment in an open, randomized study with 48 patients. Methods: Nasal reactivity and mediator levels in nasal secretions were measured at baseline, before season, in season, and after season. Results: Symptom scores in the immunotherapy group were 134.5 (95% CI, 65 to 336) versus 386.0 (95% CI, 185 to 563), significantly lower as in the drug-treated group. ECP and tryptase levels increased significantly during natural allergen exposition. The seasonal levels in the immunotherapy group were significantly lower than in the drug-treated group with 272.1 ng/mL (252.0 to 293.9 ng/mL; immunotherapy) versus 470.4 ng/mL (SEM, 435.6 to 508.0 ng/mL; drugs) for ECP and with 8.73 ng/mL (SEM, 8.20 to 9.29 ng/mL) versus 17.47 ng/mL (16.42 to 18.60 ng/mL) for tryptase (all, P < .001). The ECP level induced by nasal provocation was 105.6 ng/mL (99.0 to 112.6 ng/mL) versus 180.4 ng/mL (169.2 to 192.4 ng/mL), significantly lower ( P < .001) in the immunotherapy group, as was the tryptase level with 12.12 ng/mL (11.53 to 12.75 ng/mL) versus 8.19 ng/mL (7.79 to 8.62 ng/mL; P < .001) at the after-season visit. Conclusion: Short-term immunotherapy is able to reduce tryptase and ECP in nasal secretions more effectively than drug treatment in patients with allergic rhinitis. (J Allergy Clin Immunol 1999;103:47-53.)

Identification of Allergens in Oilseed Rape (Brassica napus) Pollen

Background: Pollen from oilseed rape (OSR), Brassica napus, an increasingly cultivated oilplant from the Brassicaceae, has been recognized as a potential cause of allergic sensitization. Allergens have been hardly investigated. Methods: We characterized IgE binding proteins in OSR pollen by immunoblot, immunoblot inhibition and specific monoclonal antibodies using sera from 89 patients sensitized to OSR. Results: Two low–molecular–weight allergens of 6/8 kD and 14 kD as well as a high molecular–weight cluster (27–69 kD) comprising six cross–reactive peptides could be identified. The three allergens were recognized by 50, 34 and 80% of patients, respectively. Immunoblot IgE binding to OSR could be totally inhibited by rye pollen and moderately by birch pollen (6/8 and 14 kD) while mugwort had little effect. An anti–profilin–specific monoclonal antibody bound specifically to a 14–kD protein in OSR. Binding to the 6/8–kD rape allergen could be effectively inhibited by rAln g 2, a calcium–binding protein from alder. Periodate treatment led to a significant reduction in IgE binding to the 27 to 69–kD OSR allergens indicating that carbohydrate determinants are involved in IgE binding. OSR proteins were capable to quench IgE binding to timothy grass pollen proteins of ≥60 kD suggesting that grass pollen group 4 allergens cross–react with the 27 to 69–kD cluster in OSR. Conclusions: The data demonstrate that OSR pollen is allergenic and indicate that the identified allergens represent cross–reacting homologues of well–known pollen allergens, i.e. calcium–binding proteins, profilins, and high–molecular–weight glycoproteins. Via cross–reactivity, exposure to OSR pollen may be a prolonging and aggravating factor in underlying birch and grass pollen allergy.

Short-term immunotherapy: A prospective, randomized, double-blind, placebo-controlled multicenter study of molecular standardized grass and rye allergens in patients with grass pollen-induced allergic rhinitis

Background: Short-term immunotherapy (STI) can be beneficial for patients who are noncompliant with long-term specific immunotherapy. Objective: The efficacy and tolerance of STI with seven preseasonal injections of molecular standardized allergens from grass and rye pollen has been investigated in a double-blind, placebo-controlled multicenter study with 87 patients at 12 German University hospitals. Methods: Symptoms of the eyes, nose, and bronchi and use of symptomatic drugs were documented daily in diaries by patients with allergic rhinitis to grass and/or rye pollen and without bronchial asthma. Patients were monitored by skin prick test titration and measurement of levels of specific IgE and IgG4. Results: The median nasal score for the 10 weeks with the strongest symptoms during the grass pollen season was significantly lower ( p = 0.014) with 35.0 for STI ( n = 41) versus 69.0 for placebo ( n = 40); the overall symptom score was 54.0 for STI versus 97.5 for placebo ( p = 0.020). Only STI-treated patients exposed to less than 40 pollen grains per cubic meter per week showed a significantly lower nasal symptom score of 39.0 versus 75.0 for placebo ( p = 0.006); these patients also had fewer nasal symptoms and less use of topical nasal drugs ( p < 0.001). The threshold dose in skin prick tests was significantly higher, being 9.06 histamine equivalent for skin prick test (HEP) for STI-treated patients who received the maximum dose ( n = 22) versus 4.33 HEP for placebo ( p = 0.005). Specific IgE levels were significantly higher, being 55.9 SU/ml for STI versus 39.2 SU/ml for placebo after seven injections ( p = 0.006) and level of specific IgG4 was 5.36% for STI versus 1.28% for placebo ( p < 0.001). No severe systemic reactions were observed. Conclusion: STI with seven preseasonal injections with molecular standardized allergens is effective and well tolerated. J Allergy Clin Immunol 1997;100:23-9.

Prevalence of IgE-mediated allergy to latex in hospital nursing staff.

IgE-mediated hypersensitivity to latex proteins has become a significant clinical problem over the last decade. Nursing and medical staff are at risk because of their occupational exposure to latex. To determine the prevalence of type I hypersensitivity to latex allergens in the nursing staff of an Australian hospital. A questionnaire which asked about symptoms associated with the use of latex gloves was completed by 140 nurses working in the Alfred Hospital (72 in general medical wards, 68 in intensive care units). Skin prick tests with eluates of five different types of latex glove as well as common aeroallergens (rye pollen and house dust mite) and banana extract were performed. Thirty-one nurses (22%) were skin prick test positive to at least one of the five latex glove eluates. All of these nurses were atopic, having positive skin prick tests to rye pollen or house dust mite. Symptoms of local dryness, itch and erythema associated with glove use were reported by more than half the study group, but not more frequently by those who were skin prick test positive to latex. Urticaria associated with glove use was reported more frequently by those with positive latex skin prick tests (13% vs 4%, p = 0.05). Eighty-seven per cent of the nurses who were latex skin test positive were also positive to banana extract. IgE-mediated hypersensitivity to latex is common in nurses working in an Australian hospital. Glove associated symptoms were frequently reported, but in most cases the symptoms were more typical of irritant or contact dermatitis rather than type I hypersensitivity reactions. However, the extent of subclinical sensitisation to latex found in this study suggests that symptomatic latex allergy is likely to emerge as an increasing problem for nursing staff in this country.

An in vitro assay for incompatibility between Triticum aestivum L. and Secale cereale L.

summaryA liquid medium was developed for in vitro germination and growth of rye (Secale cereale L.) pollen. Addition of style extracts from ‘Otane’ wheat which were incompatible with rye selectively inhibited pollen tube growth, compared with compatible ‘Chinese Spring’ wheat. Dialysed extracts were most suitable for incompatibility assays. Rye pollen was also selectively inhibited by extracts from self–compatible styles. Intergeneric incompatibility in wheat appears to be controlled by inhibitory style proteins.

Allergens from rye pollen (Secale cereale). I. Study of protein release by rye pollen during a 19-hour extraction process. Allergen identification.

We have studied the proteins and allergens released by rye pollen in the course of a 19-h pollen incubation process. Nearly 40% of the total extracted proteins were collected during the first 5 min, and most of them had a molecular weight less than 28 kDa. Between 5 and 30 min, 15% of the proteins from total extract were released, showing in the SDS-PAGE analysis an increase in which components moved close to 30 kDa standard. From 30 min to 19 h several extracts were collected. Electrophoretical profile of components from these extracts reveals that bands moving below 28 kDa were practically absent and those of 28 and 23 kDa became very intense. At the end of the process there was a rise of 67 kDa proteins. Dot-immunobinding and immunoblotting techniques reveal that allergens leave the rye pollen, for the most part, after 5 min incubation and are proteins with 28 kDa, 33 kDa, 48 kDa and 67 kDa molecular weights.

Allergens from rye pollen (Secale cereale). II. Characterization and partial purification.

Rye pollen was incubated for 30 min and proteins extracted at this time were collected as extract A (EA). The same pollen grains were resuspended in buffer and incubated for 18.5 h. Proteins extracted in this period were designated extract B (EB). Both extracts were subfractionated by DEAE ion-exchange chromatography and allergen presence in peaks detected by the dot-immunobinding technique. The results reveal that unretained proteins (peaks 1 and 2) and proteins eluted at 0.2 M NaCl from extract B contain the highest proportion of allergens. SDS-PAGE of chromatographic peaks showed that peak 2 from extract B contains a highly purified 28 kDa band. On the skin of allergic patients this band gave a stronger positive prick test than for the crude extract.

Hybridization barriers between wheat and rye: In vitro pollen assays and electrophoretic survey

Five- to seven-day-old ovaries from Triticum aestivum vars Hope (non-crossable), Chinese Spring (CS, crossable) and the chromosomal substitution line Chinese Spring/Hope 5B (CS/H5B) were used to test the hypothesis that an ovarian substance inhibits rye (Secale cereale var Wrens) pollen tube growth. In vitro assays on a semi-solid medium demonstrated that a soluble, dialyzed lysate from Hope or CS/H5B ovaries inhibited rye pollen tube elongation significantly more than a similar lysate from CS ovaries (26%, 13% and 5% respectively compared to controls). When only the 20 longest tubes from each sample were included in the analysis the relationship was maintained (33%, 22% and 15% for Hope, CS/H5B and CS respectively). Heating the Hope lysate at 100° C for 10 min reduced the inhibition from 26% to 14% for all pollen tubes measured (n = 51 per sample) and from 33% to 20% when only the 20 longest pollen tubes per sample were analyzed. Isoelectric focusing followed by sodium dodecyl sulfate polyacrylamide electrophoresis (IEF/SDS-PAGE) and non-equilibrium pH gradient electrophoresis (NEPHGE) revealed several differences in the polypeptide profiles of soluble lysates from vars Hope, CS and CS/H5B. While two striking differences were observed, a 50 kilodalton (kD) polypeptide with an isoelectric point (pI) of approximately 8.5 and 100 kD polypeptide (pI∼4) from CS lysates which were considerably reduced in lysates of Hope and CS/H5B, none were obviously associated with the Hope 5B chromosome and therefore cross-incompatibility.

Airborne pollen survey of the warsaw area

Abstract Airborne and allergenic pollen types were identified in the area of Warsaw in the years 1983–1987. Over the same period of time, skin tests reactions were analysed in 400 hay fever patients. The aim of our study was to determine whether the most abundant pollen types at a given time did evoke hay fever symptoms in our allergy patients as confirmed by positive skin responses. In February, March and April 10.8% of patients showed allergy to deciduous tree pollen, while in May allergy to conifer pollen was confirmed in 2%. The most numerous group of patients demonstrating pollinosis due to grass and rye pollen attended the Clinic in June and July. In the autumn (September-October) symptoms due to weed pollens predominated in our patients.

Monoclonal Antibodies to Proteins from Cocksfoot Grass &lt;i&gt;(Dactylis glomerata)&lt;/i&gt; Pollen: Isolation and N-Terminal Sequence of a Major Allergen

Monoclonal antibodies (Mabs) raised to an aqueous extract of cocksfoot grass (Dactylis glomerata) pollen have been characterised. Mab 1B9 was demonstrated by SDS-PAGE and Western blotting to recognise a major allergen of an approximate molecular weight of 28 kD in this extract, termed DG3, and a component with a molecular weight of between 35 and 40 kD in an extract of Secale cereale (cultivated rye) pollen. The 28 kD component of cocksfoot grass pollen isolated by affinity chromatography using Mab 1B9 was recognised by IgE antibodies in 80% (8 of 10) atopic sera, but only weakly by 25% (1 of 4) non-atopic sera tested. N-terminal sequencing of DG3 purified by affinity chromatography, 2 D electrophoresis and electroblotting to polyvinylidenedifluoride revealed significant homology with a group-V allergen (Phl p V) from timothy grass (Phleum pratense).

Early seed development in wheat*rye crosses following pollination with irradiated rye pollen.

Early seed development was studied in wheat×rye crosses, in which irradiated (0-50 Gy) rye pollen was used for pollination. The number of cells and nuclei in the embryos and endosperms were scored daily for 6 and 3 days after pollination (DAP), respectively. Embryo and endosperm growth rates in the 2 Gy treatment were similar to the control (no irradiation). In other treatments, pollen irradiation greatly reduced the growth rates in both the tissues. The 2 Gy and control ovules had 7 to 10 times more embryo cells and 2 to 3 times more endosperm nuclei than the other treatments at 6 and 3 DAP, respectively. Several abnormalities were observed in the developing ovules of pistils pollinated with irradiated pollen. Degeneration of the endosperm began 3 DAP in the 5 Gy and higher radiation treatments but later in the 2 Gy treatment. Embryo abortion also occurred at a later stage in the 2 Gy treatment than in the higher radiation treatments. These studies indicate that gamma radiation doses beyond 2 Gy are quite deleterious to the developing ovule and in most cases the embryo sac succumbs to higher radiation treatments.

In vitro synthesis of IgE by human peripheral blood leucocytes: VI. Seasonal variations in synthesis of rye pollen (Lolium perenne) specific IgE.

Fractionation by Percoll density centrifugation of peripheral blood leucocyte cells, from atopic subjects with seasonal hay fever, unmasked IgE-B cell populations whose individual capacities to synthesize IgE in vitro were obscured in cultures of unfractionated B cells. B cell cultures from all six subjects in the study released rye pollen-specific IgE during the 6 days of culture, but actual synthesis was significant only in October, the pollen season. Synthesis in October occurred most frequently in cultures of mature, low density B cells, which generally responded to the addition of autologous T cells with enhanced synthesis (T-help). T-help was also found for high density B cells in the mid-winter (July) cultures. Total IgE synthesis in vitro demonstrated a less seasonal relationship, although it tended to be maximal for low density B cell cultures in October and for high density B cells in May. All B cell cultures contained preformed total and rye-specific IgE antibody which persisted throughout the pre- and post-pollen seasons, particularly in the low density B cell fractions, even in the absence of de novo synthesis. Moreover, the intracellular levels of rye pollen-specific IgE antibody were often higher in the winter than in the peak of the pollen season. The relevance of this preformed IgE remains to be established.

Analysis of Rye Pollen &lt;i&gt;(Secale cereale)&lt;/i&gt; Allergens Using Patients’ IgE, Immunoprint, Western Blot and Monoclonal Antibodies

Rye pollen (Secale cereale) was investigated by Immunoprint and Western Blot analysis. Using Immunoprint, more than 30 proteins could be shown to be allergens in terms of their IgE binding, and 9 were determined as major allergens. In Western Blot 17 allergens were discovered, 3 of them major allergens. 4 monoclonal antibodies raised against a pollen extract of 6 grasses showed cross-reactivity to rye pollen, thus indicating common epitopes. 1 of 3 monoclonal antibodies produced against an extract of rye pollen was only reacting to this pollen. Looking at the overall binding pattern of the 7 monoclonal antibodies, they showed partial identity to patients' IgE in Immunoprint and Western Blot. The possibility of defining epitopes using preparative isoelectric focusing and monoclonal antibodies is discussed.

Monoclonal antibodies to the major lolium perenne (rye grass) pollen allergen Lol p I (Rye I)

Thirteen monoclonal antibodies (MAbs) were produced against Lol p I (Rye I), the major Lolium perenne (rye grass) pollen allergen. Spleen cells from A/J and SJL mice immunized with highly purified Lol p I ( Lol I) were allowed to fuse with cells from the non-secreting Sp2/0-Ag14 myeloma cell line. Each MAb was analyzed for antigenic specificity by radioimmunoassay (RIA) using 125I- LolI. The epitope specificities of seven of the MAbs were examined by competitive binding against a labelled standard MAb for the Lol I antigen (Ag). The dissociation constant, K d, of one MAb (No. 3.2) that was studied most extensively was determined by double Ab RIA to be 3.5 × 10 −6 L/M. This MAb recognized the related 27,000–30,000 Group I glycoproteins found in the pollens of nine other species of grass pollens tested, including weak binding to Bermuda grass Group I ( Cyn d I), which by conventional analysis using polyclonal anti- Lol I serum shows no detectable binding. Monoclonal antibody No. 3.2 was coupled covalently to Sepharose 4B and used to prepare highly purified Lol I from a partially purified rye pollen extract. Finally, an RIA was developed which permitted the analysis of the Group I components in rye grass and nine other grass pollen species. The latter assay is likely to prove useful in the standardization of grass pollen extracts according to their Group I contents.

IS “HAY-FEVER” A CORRECT DESCRIPTION OF DISEASES CAUSED BY ALLERGIC REACTIONS TO POLLEN

“Hay fever” is the comprehensive term for allergic pollen reactions. In allergen challenge tests with extracts of rye pollen (Secale cereale) either by inhalation or oral ingestion it could be shown with the thrombopenic index by Storck that pollen allergy is not only a reaction of the affected organ e.g. the eyes, the nose or the bronchi but a systemic immunological reaction of the total organism. Therefore, it is concluded that the term “hay fever” better demonstrates these general reactions than the term “pollinosis”. This should be included in the considerations of further research on allergic diseases.

Rationale for specific immunotherapy of grass pollen allergy with extracts of rye pollen. Skin test reactivity and immunochemical relationship between pollen allergens from rye and other common grasses.

In immunotherapy of grass pollen allergy, an extract of rye (Secale cereale) is often included. The aim of this study was to investigate by skin prick test (SPT) and immunochemical methods whether rye pollen contains specific allergens justifying the use of this extract separately. Twenty grass pollen allergic patients were skin prick tested with a dialysed freeze-dried raw extract of rye pollen (Sc), timothy extract (Soluprick SQ, 1 HEP) and two other rye extracts (Soluprick). Sera from the patients were RAST-tested using Sc and timothy (Pp). CRIE was performed using Sc and rabbit-anti grass (aNG) antibodies. The antigenic relations between rye and common grasses were investigated by CLIE using Sc and aNG as references, and by RAST inhibition. The ability of aNG to absorb the allergen activity of Sc was also tested. Significant correlations were found between timothy and rye when compared by means of SPT and RAST. The immunochemical analyses did not reveal any rye antigens containing rye epitopes only. However, the possibility of rye antigens with several epitopes, of which at least one is specific for rye, could not be excluded. Clinical symptoms supposedly elicited by rye alone can be explained quantitatively by the strongly time-limited and concentrated natural exposition. Diagnosis and treatment can, however, be performed with extracts of common grasses.

Crossed radioimmunoelectrophoretic analysis of cultivated rye (Secale cereale) pollen allergens.

Cultivated rye pollen extracts were characterized by means of crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE). 32 antigens were identified in CIE and among these 16 were radiostained in CRIE analysis of a rye pollen sensitive patient panel. Crossed line immunoelectrophoresis revealed that some of the rye pollen antigens were immunological partially identical with antigens of wheat flour and rye flour.