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.

Isolation of cDNA encoding a newly identified major allergenic protein of rye-grass pollen : intracellular targeting to the amyloplast

We have identified a major allergenic protein from rye-grass pollen, tentatively designated Lol pIb of 31kDa and with pI 9.0. A cDNA clone encoding Lol pIb has been isolated, sequenced, and characterized. Lol pIb is located mainly in the starch granules. This is a distinct allergen from Lol pI, which is located in the cytosol. Lol pIb is synthesized in pollen as a pre-allergen with a transit peptide targeting the allergen to amyloplasts. Epitope mapping of the fusion protein localized the IgE binding determinant in the C-terminal domain.

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.

Development of plastids in pollen and tapetum of rye-grass, Lolium perenne L

Proplastids of both tapetal cells and microsporocytes were present early in anther development. Tapetal proplastids differentiated—probably into elaioplasts—at late microspore stage. The tapetal cytoplasm was completely resorbed by early tricellular pollen stage. Microspore proplastids differentiated into amyloplasts at early bicellular stage, and were present in both vegetative and generative cells. In the generative cell, the amyloplasts were ephemeral and apparently degenerated within autophagic vacuoles. Plastids were absent from sperm cells. Vegetative cell amyloplasts increased in number apparently by fission such that one amyloplast produced one amyloplast and one proplastid per division. Mature pollen grains were estimated to contain between 550 and 820 amyloplasts with only one starch granule per plastid.

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.

Book review: Allan Bedford The unofficial Lego builder’s guide

This is a review of : The unofficial Lego builder's guide, by Allan Bedford, No Starch Press, San Francisco, USA, 2005. ISBN 9781593270544.

Clarification and concentration of sugar cane juice through ultra, nano and reverse osmosis membranes

The performance of ultrafiltration (UF) membranes with molecular weight cut off (MWCO) of 1000 and 3500 Da in clarifying sugar cane juice was investigated, as well as the performance of a nanofiltration (NF) membrane with MWCO of 200 Da and a reverse osmosis (RO) membrane in concentrating sugar cane juice. For both cases the sugar cane juice had been limed and partially clarified. The UF membranes were found to be effective at clarifying the sugar cane juice in terms of purity rise and reduction in turbidity, colour, starch and protein. A purity rise of approximately 6 was achieved by both UF membranes at trans-membrane pressures (TMP) from 15 to 25 bar. However, Brix reduction in the permeate was between 14.5 and 41.85% and 12.11 and 26.52% for 1000 Da and 3500 Da membranes respectively. For the 200 Da and RO membranes the Brix in the concentrate was increased from 7.65 to 12.3 after 3 hours of operation for the 200 Da membrane at a TMP of 10 bar, whilst the Brix in the concentrate was increased from 15.65 to 27.6 after 3 hours of operation for the RO membrane at a TMP of 35 bar. Overall, UF membranes were found to be unsuitable for clarification of sugar cane juice since significant amount of Brix is reduced in the permeate, whilst RO membranes were found to be effective for concentration of sugar cane juice.

Grain excretion by goats fed whole or processed cereals with various roughages

Despite cereal grains being grown on 5 continents where goats are kept, there is little information on the excretion of whole cereal grains when fed to goats. We determined the effects of various dietary treatments on whole grain and starch loss in the faeces of Angora goats. In Experiment 1 there were 4 replicates of the factorial design: (a) 2 grain types (barley, oats); (b) whole grain or processing (milled barley or rolled oats); (c) 2 roughage qualities (Persian clover hay, barley straw); and (d) 2 feeding levels (level 1, 150 g/d of grain, 250 g/d of roughage; level 2, 250 g/d of grain, ad libitum roughage). In Experiment 2, which immediately followed Experiment 1, and aimed to detect carry over effects of previous feeding of barley straw and grain processing, feed levels were either 650 g/d grain or 400 g/d grain with 550 g/d Persian clover hay. Data were analysed by ANOVA. In Experiment 1, processing had no effect on digestible dry matter intake. The number of whole grains lost per 100 g of fresh faeces and whole grains loss as the % of grain dry matter intake were affected by an interaction between processing and roughage quality. Whole grain fed with Persian clover hay had greater grain loss than all other diets. Whole grain loss was greater with whole grain than with processed grain. Level of feeding had no effects on grain loss. In Experiment 2, more whole grains were lost in fresh faeces when fed with Persian clover hay than when fed without hay, an effect of previous feeding with barley straw reduced whole grain excretion, and more barley grains were lost than oat grain. Faecal starch was affected, with higher levels when whole barley grain was fed, particularly with Persian clover hay, or when previously fed barley straw at a high level. Feeding grain at 650 g/d did not increase grain or starch excretion. Whole grains represented a small loss of grain dry matter intake in faeces, averaging 0.8% with a maximum recorded of 2.6%. Faecal concentration of the whole grains may be altered by grain size and the digestibility of the roughage component of the diet. In this study an additional cost of 3% for processing grains would not have provided economic benefits.

Carbon nanotube based elastomer composites-an approach towards multifunctional materials

The current study focuses on giving a basic understanding of tubular graphene sheets or carbon nanotubes (CNTs) and points towards their role in fabricating elastomer composites. Since the properties and the performance of CNT reinforced elastomer composites predominantly depend on the rate of dispersion of fillers in the matrix, the physical and chemical interaction of polymer chains with the nanotubes, crosslinking chemistry of rubbers and the orientation of the tubes within the matrix, here, a thorough study of these topics is carried out. For this, various techniques of composite manufacturing such as pulverization, heterocoagulation, freeze drying, etc. are discussed by emphasizing the dispersion and alignment of CNTs in elastomers. The importance of the functionalization technique as well as the confinement effect of nanotubes in elastomer media is derived. In a word, this article is aimed exclusively at addressing the prevailing problems related to the CNT dispersion in various rubber matrices, the solutions to produce advanced high-performance elastomeric composites and various fields of applications of such composites, especially electronics. Special attention has also been given to the non-linear viscoelasticity effects of elastomers such as the Payne effect, Mullin's effect and hysteresis in regulating the composite properties. Moreover, the current challenges and opportunities for efficiently translating the extraordinary electrical properties of CNTs to rubbery matrices are also dealt with.

Maternal overnutrition programs changes in the expression of skeletal muscle genes that are associated with insulin resistance and defects of oxidative phosphorylation in adult male rat offspring.

Children of obese mothers have increased risk of metabolic syndrome as adults. Here we report the effects of a high-fat diet in the absence of maternal obesity at conception on skeletal muscle metabolic and transcriptional profiles of adult male offspring. Female Sprague Dawley rats were fed a diet rich in saturated fat and sucrose [high-fat diet (HFD): 23.5% total fat, 9.83% saturated fat, 20% sucrose wt:wt] or a normal control diet [(CD) 7% total fat, 0.5% saturated fat, 10% sucrose wt:wt] for the 3 wk prior to mating and throughout pregnancy and lactation. Maternal weights were not different at conception; however, HFD-fed dams were 22% heavier than controls during pregnancy. On a normal diet, the male offspring of HFD-fed dams were not heavier than controls but demonstrated features of insulin resistance, including elevated plasma insulin concentration [40.1 ± 2.5 (CD) vs 56.2 ± 6.1 (HFD) mU/L; P = 0.023]. Next-generation mRNA sequencing was used to identify differentially expressed genes in the offspring soleus muscle, and gene set enrichment analysis (GSEA) was used to detect coordinated changes that are characteristic of a biological function. GSEA identified 15 upregulated pathways, including cytokine signaling (P < 0.005), starch and sucrose metabolism (P < 0.017), inflammatory response (P < 0.024), and cytokine-cytokine receptor interaction (P < 0.037). A further 8 pathways were downregulated, including oxidative phosphorylation (P < 0.004), mitochondrial matrix (P < 0.006), and electron transport/uncoupling (P < 0.022). Phosphorylation of the insulin signaling protein kinase B was reduced [2.86 ± 0.63 (CD) vs 1.02 ± 0.27 (HFD); P = 0.027] and mitochondrial complexes I, II, and V protein were downregulated by 50-68% (P < 0.005). On a normal diet, the male offspring of HFD-fed dams did not become obese adults but developed insulin resistance, with transcriptional evidence of muscle cytokine activation, inflammation, and mitochondrial dysfunction. These data indicate that maternal overnutrition, even in the absence of prepregnancy obesity, can promote metabolic dysregulation and predispose offspring to type 2 diabetes.

Strain-responsive polyurethane/PEDOT:PSS elastomeric composite fibers with high electrical conductivity

It is a challenge to retain the high stretchability of an elastomer when used in polymer composites. Likewise, the high conductivity of organic conductors is typically compromised when used as filler in composite systems. Here, it is possible to achieve elastomeric fiber composites with high electrical conductivity at relatively low loading of the conductor and, more importantly, to attain mechanical properties that are useful in strain-sensing applications. The preparation of homogenous composite formulations from polyurethane (PU) and poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) that are also processable by fiber wet-spinning techniques are systematically evaluated. With increasing PEDOT:PSS loading in the fiber composites, the Young's modulus increases exponentially and the yield stress increases linearly. A model describing the effects of the reversible and irreversible deformations as a result of the re-arrangement of PEDOT:PSS filler networks within PU and how this relates to the electromechanical properties of the fibers during the tensile and cyclic stretching is presented. Conducting elastomeric fibers based on a composite of polyurethane (PU) and PEDOT:PSS, produced by a wet-spinning method, have high electrical conductivity and stretchability. These fibers can sense large strains by changes in resistance. The PU/PEDOT:PSS fiber is optimized to achieve the best strain sensing. PU/PEDOT:PSS fibers can be produced on a large scale and integrated into conventional textiles by weaving or knitting. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Achieving outstanding mechanical performance in reinforced elastomeric composite fibers using large sheets of graphene oxide

A simple fiber spinning method used to fabricate elastomeric composite fibers with outstanding mechanical performance is demonstrated. By taking advantage of the large size of as-prepared graphene oxide sheets (in the order of tens of micrometers) and their liquid crystalline behavior, elastomeric composite fibers with outstanding low strain properties have been fabricated without compromising their high strain properties. For example, the modulus and yield stress of the parent elastomer improved by 80- and 40-fold, respectively, while maintaining the high extensibility of ∼400% strain inherent to the parent elastomer. This outstanding mechanical performance was shown to be dependent upon the GO sheet size. Insights into how both the GO sheet size dimension and dispersion parameters influence the mechanical behavior at various applied strains are discussed.

Does dynamic vulcanization induce phase separation?

Immiscible and miscible blends of poly(vinylidene fluoride) (PVDF) and acrylic rubber (ACM) were subjected to dynamic vulcanization to investigate the effect of crosslinking on phase separation. As a result of different processability, mixing torque behavior of miscible and immiscible blends was significantly different from one another. Scanning electron microscopy (SEM) was used to investigate the morphology of the system. After dynamic vulcanization, submicron ACM droplets were observed in the samples near the binodal curve of the system under mixing conditions. Small angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC) analysis were used to investigate the effect of dynamic vulcanization on the lamellar structure of the system. It was shown that for samples near the boundary of phase separation, increasing the crosslink density led to a decrease in the lamellar long period (L) as a sign of increment of crosslink density induced phase decomposition. Effects of shear rate on the final morphology of the system were investigated by changing the mixing temperature and by comparing the results of dynamic vulcanization at one phase and two phase regions.

Ductile thermoset polymers via controlling network flexibility

We report the design and synthesis of a polymer structure from a cross-linkable epoxy-ionic liquid system which behaves like a hard and brittle epoxy thermoset, perfectly ductile thermoplastic and an elastomer, all depending on controllable network compositions.