Localisation of allergens in ryegrass pollen and in airborne micronic particles

In Melbourne, Australia, grass pollen allergens, especially from ryegrass, are a major cause of allergic hayfever and asthma. This review outlines recent developments in our understanding of how grass pollen allergens find their way into the atmosphere and how they are transported in particulate form. Much of this work has relied on antibody technology in immunological and immunocytochemical investigations. The localisation of allergens in situ has proved difficult due to their water-soluble character. Recently, allergens have been localised in developing ryegrass pollen by dryfixation, rapid-freeze and freeze-substitution techniques. This involved anthers being substituted in a mixture of aldehydes, organic solvents, and 2,2-dimethoxypropane. Incubation in dimethylsulfoxide prior to embedding in LR Gold resin provided good infiltration with freeze-substituted material. Immunogold-labelled sections show that the major allergens, Lol p 1 and Lol p 5, are synthesised in the pollen cytoplasm from the early bicellular stage, soon after the first starch granules are formed. From the early tricellular stage, Lol p 5 moves into the starch granules where it remains until maturity. Lol p 1 is localised in the cytoplasm of mature pollen grains. The incidence of airborne grass pollen, as measured in pollen traps, correlates with hayfever symptoms. Forecasting models which rely on rainfall and temperature data have been produced for the grass pollen (daily and seasonal) counts in Melbourne. Research over the past six years has shed light on the causes of grass-pollen-induced asthma. Micronic particles in the atmosphere may be starch granules originating from pollen grains osmotically ruptured by rainwater. Ultrastructural and immunological characterisation of micronic particles collected from outdoor air filters confirm the presence of airborne starch granules. These are loaded with grass pollen allergens, occur in the atmosphere especially after rainfall, and correlate significantly with instances of allergic asthma. Diesel particles might also play a role in the transmission of grass pollen allergens and thus become an extra asthma trigger. A variation in the mode of release of micronic particles occurs in other species, such as birch, where such particles are derived from burst birch pollen tubes. These particles are positive for Bet v 1 and are starch granules which are released into the atmosphere after light rain as a result of pollen germination on, e.g., leaves. After subsequent rupture of pollen tubes their contents are released when conditions become drier.

Concentrations of major grass group 5 allergens in pollen grains and atmospheric particles : implications for hay fever and allergic asthma sufferers sensitized to grass pollen allergens

Background

Grass pollen allergens are the most important cause of hay fever and allergic asthma during summer in cool temperate climates. Pollen counts provide a guide to hay fever sufferers. However, grass pollen, because of its size, has a low probability of entering the lower airways to trigger asthma. Yet, grass pollen allergens are known to be associated with atmospheric respirable particles.
Objective

We aimed (1) to determine the concentration of group 5 major allergens in (a) pollen grains of clinically important grass species and (b) atmospheric particles (respirable and nonrespirable) and (2) to compare the atmospheric allergen load with clinical data to assess different risk factors for asthma and hay fever.
Methods

We have performed a continuous 24 h sampling of atmospheric particles greater and lower than 7.2 μm in diameter during the grass pollen season of 1996 and 1997 (17 October 1996–16 January 1997) by means of a high volume cascade impactor at a height of about 15 m above ground in Melbourne. Using Western analysis, we assessed the reactivity of major timothy grass allergen Phl p 5 specific monoclonal antibody (MoAb) against selected pollen extracts. A MoAb-based ELISA was then employed to quantify Phl p 5 and cross-reactive allergens in pollen extracts and atmospheric particles larger and smaller than 7.2 μm.
Results

Phl p 5-specific MoAb detected group 5 allergens in tested grass pollen extracts, indicating that the ELISA employed here determines total group 5 allergen concentrations. On average, 0.05 ng of group 5 allergens were detectable per grass pollen grain. Atmospheric group 5 allergen concentrations in particles > 7.2 μm were significantly correlated with grass pollen counts (rs = 0.842, P < 0.001). On dry days, 37% of the total group 5 allergen load, whereas upon rainfall, 57% of the total load was detected in respirable particles. After rainfall, the number of starch granule equivalents increased up to 10-fold; starch granule equivalent is defined as a hypothetical potential number of airborne starch granules based on known pollen count data. This indicates that rainfall tended to wash out large particles and contributed to an increase in respirable particles containing group 5 allergens by bursting of pollen grains. Four day running means of group 5 allergens in respirable particles and of asthma attendances (delayed by 2 days) were shown to be significantly correlated (P < 0.001).
Conclusion

Here we present, for the first time, an estimation of the total group 5 allergen content in respirable and nonrespirable particles in the atmosphere of Melbourne. These results highlight the different environmental risk factors for hay fever and allergic asthma in patients, as on days of rainfall following high grass pollen count, the risk for asthma sufferers is far greater than on days of high pollen count with no associated rainfall. Moreover, rainfall may also contribute to the release of allergens from fungal spores and, along with the release of free allergen molecules from pollen grains, may be able to interact with other particles such as pollutants (i.e. diesel exhaust carbon particles) to trigger allergic asthma.

Source of Bet v 1 loaded inhalable particles from birch revealed

Allergenic proteins present in pollen grains, when inhaled, interact with the airways to cause an attack of asthma in susceptible humans. In one system, grass pollen grains rupture osmotically in rainfall, releasing allergen-containing inhalable particles into the atmosphere. In contrast, birch tree pollen grains do not rupture under these conditions, yet the major allergen, Bet v 1, has been detected in the atmosphere in inhalable particles of unknown origin. It is possible that Bet v 1 may diffuse from intact settled pollen grains and the allergenic material may again become airborne, interacting with settled fine particles from other sources prior to resuspension. This study investigates the mechanism for the release of birch pollen allergen-containing inhalable particles from pollen grains. We propose the hypothesis that (1) airborne birch pollen grains settle on nearby leaf surfaces; (2) then, following light rainfall, the grains germinate and, (3) later, pollen tubes burst, releasing inhalable particles carrying Bet v 1 into the atmospheric aerosol.   We used microscopic analyses of pollen behaviour following anther opening, a Burkard volumetric trap for pollen counts and a high volume air sampler with a two-stage cascade impactor for quantitative immunochemical analyses of Bet v 1. On dry days of high birch pollen count (48 grains/m3, 1.5 ng/m3 of Bet v 1), we found that the surfaces of birch leaves became coated with pollen. This ”pollen rain” is a source of secondary emission of allergens into the atmosphere. We observed that following light rainfall (<1 mm per day), about 80% of the birch pollen grains germinated, producing pollen tubes, especially in the sticky surface secretions of leaf glands. These pollen tubes may grow up to 300 μm in length prior to rupturing, each releasing about 400 starch granules coated with allergen molecules that may, after drying, be dispersed into the aerosol. On these days following light rainfall, the highest atmospheric levels of Bet v 1 (1.18 ng/m3) are associated with inhalable particles. Following heavy rainfall, both pollen and inhalable particles are washed from the atmosphere. Immunoprinting studies show that Bet v 1 is associated with starch granules rather than the smaller orbicules. Bet v 1 is present in the atmosphere in large particles, i.e. in particular pollen grains and in inhalable particles, i.e. in particular starch granules.

A new approach to the investigation of allergenic respirable particles using a modified Anderson Impactor

Pollen allergens have been shown to occur in fine particles much smaller than pollen grains. As such allergenic particles have a high probability of entering the lower human airways, it is important to further investigate their origin and their biological and immunological properties. Accordingly, we have modified the particle collection surface of an Andersen Impactor using adhesive coated microscope slides to enable identification by high resolution microscopic analysis and immunodetection of allergenic particulates.

Major grass pollen allergen Lol p 1 binds to diesel exhaust particles : implications for asthma and air pollution

Background Grass pollen allergens are known to be present in the atmosphere in a range of particle sizes from whole pollen grains (approx. 20 to 55 μim in diameter) to smaller size fractions < 2.5 μ (fine particles, PM2.5). These latter particles are within the respirable range and include allergen-containing starch granules released from within the grains into the atmosphere when grass pollen ruptures in rainfall and are associated with epidemics of thunderstorm asthma during the grass pollen season. The question arises whether grass pollen allergens can interact with other sources of fine particles, particularly those present during episodes of air pollution.

Objective We propose the hypothesis that free grass pollen allergen molecules, derived from dead or burst grains and dispersed in microdroplets of water in aerosols, can bind to fine particles in polluted air.

Methods We used diesel exhaust carbon particles (DECP) derived from the exhaust of a stationary diesel engine, natural highly purified Lol p 1, immunogold labelling with specific monoclonal antibodies and a high voltage transmission electron -microscopic imaging technique

Results DECP are visualized as small carbon spheres, each 30–60 nm in diameter, forming fractal aggregates about 1–2μ in diameter. Here we test our hypothesis and show by in vitro experiments that the major grass pollen allergen, Lol p I. binds to one defined class of fine particles, DECP.

Conclusion DECP are in the respirable size range, can bind to the major grass pollen allergen Lol p I under in vitro conditions and represent a possible mechanism by which allergens can become concentrated in polluted air and thus trigger attacks of asthma.

Thinning of a vertical free draining aqueous film incorporating colloidal particles

The drainage under gravity of a vertical foam film formed on a wire frame has been investigated. Dual-wavelength optical interferometry was used so that unambiguous fringe order assignments could be made, enabling absolute film thicknesses to be calculated with confidence. Films were stabilized by nonionic polypropylene glycol surfactant. Halfmicrometer silica particles with varying degrees of hydrophobicity were added to the film-forming liquid to investigate their effect on film drainage rate and stability. Hydrophilic particles had little or no effect, while hydrophobic particles slowed the drainage of the film and caused a minor increase in film lifetime, from ∼10 to ∼30 s. In both the hydrophilic and hydrophobic cases the films ruptured when they reached a thickness of ∼2 particle diameters. Particles of intermediate hydrophobicity had the most significant effect, increasing film lifetime by an order of magnitude over that for hydrophilic particles. The intermediate particles allowed films to thin down to a thickness less than the particle diameter, indicating that particles bridge across the entire film. This did not occur with more hydrophobic particles even though they were embedded in each of the two film surfaces. These results correlate well with previous literature on particle-laden foams. The film thickness and drainage measurements allow drainage mechanisms for the different particles to be identified, thus providing a mechanistic explanation for the observation by several previous authors that foams formed in the presence of particles, for example during mineral processing, have the greatest stability when the particles are of intermediate hydrophobicity.

On the impact of second phase particles on twinning in magnesium alloys

Deformation twinning is an important deformation mode in magnesium alloys. Despite this, little is known on the extent to which the stress for twinning can be altered by a dispersion of second phase particles. The current paper presents a series of findings on the role of differently shaped particles on both the stress required for twinning and the characteristics of the twins that form. It is shown that plate shaped particles are, as one might expect, an effective strengthener to {10-12} twinning. When precipitate plates form on the basal planes, the relative hardening of basal slip is minor in comparison to that seen for twinning. This provides opportunity for the alloy designer to control the apparent critical resolved shear stresses (CRSS) for the different deformation modes. Possible sources for the hardening of twins are discussed.