Eng Kok Ong

A Human Monoclonal IgE Antibody Defines a Highly Allergenic Fragment of the Major Timothy Grass Pollen Allergen, Phl p 5: Molecular, Immunological, and Structural Characterization of the Epitope-Containing Domain

Almost 90% of grass pollen-allergic patients are sensitized against group 5 grass pollen allergens. We isolated a monoclonal human IgE Fab out of a combinatorial library prepared from lymphocytes of a grass pollen-allergic patient and studied its interaction with group 5 allergens. The IgE Fab cross-reacted with group 5A isoallergens from several grass and corn species. By allergen gene fragmentation we mapped the binding site of the IgE Fab to a 11.2-kDa N-terminal fragment of the major timothy grass pollen allergen Phl p 5A. The IgE Fab-defined Phl p 5A fragment was expressed in Escherichia coli and purified to homogeneity. Circular dichroism analysis revealed that the rPhl p 5A domain, as well as complete rPhl p 5A, assumed a folded conformation consisting predominantly of an α helical secondary structure, and exhibited a remarkable refolding capacity. It reacted with serum IgE from 76% of grass pollen-allergic patients and revealed an extremely high allergenic activity in basophil histamine release as well as skin test experiments. Thus, the rPhl p 5A domain represents an important allergen domain containing several IgE epitopes in a configuration optimal for efficient effector cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation and, perhaps later, for the development of generally applicable allergen-specific therapy strategies.

Incidence ofCladosporium, alternaria and total fungal spores in the atmosphere of melbourne (Australia) over three years

A Burkard spore trap was used to monitor daily fungal spore counts in the atmosphere of Melbourne (Australia) between October 1991 and December 1994. Annual total spore counts varied widely (range 345 770 in 1994 to 1 106 037 in 1992), of which approximately half were identified asCladosporium sp. and only about 1%Alternaria sp. Highest daily total spore counts were recorded late-summer through to mid-winter, probably corresponding to senescence of annual grasses and leaf fall. Spore counts were negatively correlated with rainfall (P<0.05), significantly correlated with average temperature (P<0.001), and showed a highly significant linear relationship with cumulative temperature throughout the year.

Immunological relationships among group I and group V allergens from grass pollen

Specific IgE antibodies have been affinity-purified from recombinant grass pollen allergens, and used to identify isoforms of the two major allergens of rye-grass pollen, Lol p I and Lol p V and cross-reactive allergens in other grasses. Lol p I-specific IgE (affinity-purified from the recombinant protein expressed by clone 13R which encodes amino acids 96-240 of Lol p I) identified four isoforms of the allergen. The same probe recognized cross-reactive epitopes in pollen proteins from 14 out of 16 grasses. The allergens identified by Lol p V-specific IgE (affinity-purified from the recombinant protein expressed by clones 12R or 19R which encode the full Lol p V protein) varied more in their physicochemical characteristics than the Group I isoforms. At least eight isoforms of Lol p V were identified by the Lol p V-specific IgE. The same probe recognized cross-reactive epitopes in pollen protein from 13 out of 16 grasses. Group I proteins were identified in grasses from two sub-families of the Poaceae, while the Group V allergens were only identified in pollen of grasses from one sub-family, the Pooideae.

Seasonal distribution of pollen in the atmosphere of melbourne: an airborne pollen calendar

Environmental monitoring of pollen grains in the atmosphere of Melbourne has been achieved using Burkard volumetric traps. Twenty-two families of flowering plants and confiers were identified in the pollen counts. About 62% of these pollen grains belonged to trees, 20% to grasses and 9% to herbs and weedy plants. During spring and summer, the atmosphere contained about 70% of the total annual pollen count. Tree pollen, predominantly elm and cypress, occurred abundantly in late winter and spring, with grass pollen predominantly in spring and early summer. These three types of pollen grains occurred in significant amounts, together accounting for more than 60% of the total annual catch. A seasonal incidence chart (pollen calendar) for Melbourne based on 2 years observation has been constructed. This pollen calendar is useful in identifying sources of allergies against particular seasonal airborne pollen types. Comparison of the time of occurrence of a particular pollen type using the pollen calendar and the time of allergic symptoms, can lead to accurate diagnosis and preventive measures being taken. This study has confirmed that grass pollen is the major source of allergenic pollen in the external environment triggering hay fever and allergic asthma in spring and early summer in Melbourne, Australia.Environmental monitoring of pollen grains in the atmosphere of Melbourne has been achieved using Burkard volumetric traps. Twenty-two families of flowering plants and confiers were identified in the pollen counts. About 62% of these pollen grains belonged to trees, 20% to grasses and 9% to herbs and weedy plants. During spring and summer, the atmosphere contained about 70% of the total annual pollen count. Tree pollen, predominantly elm and cypress, occurred abundantly in late winter and spring, with grass pollen predominantly in spring and early summer. These three types of pollen grains occurred in significant amounts, together accounting for more than 60% of the total annual catch. A seasonal incidence chart (pollen calendar) for Melbourne based on 2 years observation has been constructed. This pollen calendar is useful in identifying sources of allergies against particular seasonal airborne pollen types. Comparison of the time of occurrence of a particular pollen type using the pollen calendar and the time of allergic symptoms, can lead to accurate diagnosis and preventive measures being taken. This study has confirmed that grass pollen is the major source of allergenic pollen in the external environment triggering hay fever and allergic asthma in spring and early summer in Melbourne, Australia.

Mapping of the antigenic and allergenic epitopes of Lol p VB using gene fragmentation

The recombinant proteins of Lol p VA and Lol p VB expressed in E. coli reacted with IgE antibodies from sera of allergic patients and mAbs FMC A7 and PpV1. Cross-absorption analyses using these recombinant proteins showed that Lol p VA and Lol p VB possess both similar and unique IgE binding determinants. Gene fragmentation was utilized to localize the antigenic and allergenic determinants of Lol p VB. When full-length cDNA of Lol p VB was digested into three fragments and expressed as the fusions from the glutathione transferase of pGEX vectors, fragments Met1-Val196 and Asp197-Val339 bound IgE while fragment Met1-Pro96 did not. The data suggest that there are at least two IgE binding determinants in Lol p VB. In addition, only fragment Met1-Val196 reacted with mAb PpV1. The localization of these determinants was further resolved using random fragment expression libraries. The mAb PpV1 determinant was near the N-terminal region of Lol p VB molecule. The IgE binding determinants were distributed in the central region: region I (amino acids 111-195) and II (199-254). These IgE binding determinants are conserved in Lol p VA.

Forecasting the onset of the grass pollen season in Melbourne (Australia)

In Melbourne, a southern hemisphere city with a cool temperate climate, the grass pollen season has been monitored using a Burkard spore trap for 12 years (11 pollen seasons, which extend from October through January). The onset of the grass pollen season (OGPS) has been defined in various ways using both arbitrary cumulative scores (Sum 75, Sum 100) and percentages (10% Pollen Fly). OGPS, based on the forecast model of pollen season devised by Lejoly-Gabriel (Acta Geogr. Lovan., 13 (1978) 1–260) has been most widely used in efforts to forecast the beginning of the pollen season. OGPS occurred in Melbourne between 20 October to 24 November (average 6 November), a difference of 35 days. Duration of the pollen season ranged from 46 to 81 days, with a mean of 55 days, one of the longest reported. The relationships between onset and various weather parameters for July have enabled us to modify a model, using linear regression analysis, to predict onset. The prediction model is based on a negative correlation between date of onset and the sum of rainfall for July (a winter month). The error of prediction (Ep) is 24% and predicted day of OGPS was precisely predicted on 2 occasions, and on others with a range of accuracy of 3 to 14 days.

Grass pollen in the atmosphere of Melbourne: Seasonal distribution over nine years

Abstract Both the seasonal and diurnal incidence of grass pollen in the atmosphere of Melbourne have been established using data from seven pollen seasons (1975–1980 and 1991–1993). Grass pollen represented an average of 26% (ranging from 22 to 33.1%) of the total pollen count. November and December are the most important months with an average of 81% of the total grass pollen. There is marked variation in the total seasonal and daily grass pollen counts. The total seasonal grass pollen count varied from 2319 to 6798 grains, while the daily grass pollen count fluctuated from 0 to 549 grains m-3 of air. In this study, we arbitrarily assigned days during the grass pollen season with > 100 grains m-3 of air as extremely high, > 50 grains m-3 of air as high, 21–50 grains m-3 of air as moderate and < 20 grains m-3 of air as low. The number of days with extremely high counts varied from 3 to 26. The number of days with high counts varied from 7 to 21, with the highest number of days occurring in seasons with mo...

Aeroallergens of plant origin: Molecular basis and aerobiological significance

This review presents an update on the sources and molecular basis of aeroallergens of plants, derived from pollen, seeds, leaf and stem detritus and their protein molecules. These aeroallergens are a natural component of the atmosphere, either because of their natural function or human activity. Pollen is a source of allergens within the 10–200 μm size range, and while most allergenic pollen types account for only 20–30% of total annual pollen catch, during their flowering season, they are usually the dominant type. Tree pollen commences the season in winter, with birch pollen counts in Scandinavia being the highest daily pollen counts yet reported and a major allergen, a 14-kDa protein, which is similar to pathogenesis-related proteins. Grass pollen follows in spring, and is unique as its two immunodominant allergens, a 35-kDa glycoprotein and 28–32-kDa protein, are in different cellular sites: the cytosol and surface of pollen grains; and in intracellular starch granules. The allergens at the pollen surface are not inhalable and can interact only with the eyes, nasal and oral cavities. Starch granules are released to the atmospheric aerosol when grains rupture in rainwater. These are a major source of allergen-containing micronic particles, which are important because they are inhalable. At the same time, allergen molecules are present in the aerosol, and these can bind to soot particles, and so be respired deep into the airways. The major Japanese cedar pollen allergen has been detected both within the pollen and in orbicules; particles less than 1 μm that line the anther cavity and can be released into the air when dehiscence occurs. Ragweed is the major cause of late summer hayfever in eastern North America, where its pollen accounts for up to 41% of the annual pollen catch. It is a major source of aeroallergens in both respirable and non-respirable size ranges. As a result of human activity, dusts derived from seeds and cereal grains during transport, storage and milling provide a source of micronic particles, containing potent allergens that can trigger allergic disease.

Immunodetection methods for grass pollen allergens on western blots

A comparison is made of eight different methods to detect allergenic proteins in Western blots of rye-grass pollen extracts. Horseradish peroxidase-based enhanced chemiluminescence (ECL) provides a sensitive method for the detection of allergenic proteins. The method has been modified to use more dilute solutions of ECL substrate to reduce the background, can be applied to a standard nitrocellulose membrane, and used with Kodak X-ray film. The assays can be performed rapidly, replacing use of radiolabelled probes. Increased resolution is obtained. This makes the method suitable for detection of cDNA clones on plaque lifts, and for rapid and specific purification of proteins following immunodetection on nitrocellulose membranes.

Recombinant expression and epitope mapping of grass pollen allergens.

We have studied the expression of recombinant forms of Group 1 allergens from rye-grass and Bermuda grass pollens. Recombinant Lol p 1 expressed in bacteria bound serum IgE from allergic patients. Based on analysis of fragments of the Lol p 1 cDNA clone, the major IgE-reactive epitope has been mapped to the C-terminus. However, although SDS-denatured natural Cyn d 1 (from Bermuda grass) bound IgE, the full or partial recombinant proteins expressed in bacteria did not bind IgE. We have since expressed Cyn d 1 in the yeast Pichia pastoris and restored IgE binding. cDNA clones encoding two isoforms of Lol p 5, Lol p 5A and Lol p 5B, have been expressed in bacteria and resulting polypeptides show IgE-binding. Random fragments of these clones have been generated and when expressed as partial recombinant proteins in bacteria, allowed us to identify the major IgE-binding epitopes. The allergenic epitopes were localised towards the C-terminal half of the molecule. Although both isoforms shared similar IgE-reactive epitopes, Lol p 5B did not recognise the Lol p 5A-specific monoclonal antibody A7. At sequence level, there appear to be several amino acid differences between the antigenic epitopes of these two isoallergens. These results aid in the design of diagnostics and in grass pollen immunotherapy.