Allergens of non-biting midges (Diptera: Chironomidae): a systematic survey of chironomid haemoglobins

Various genera of non-biting midges (Diptera: Chironomidae) possess haemoglobins in larvae and adults. For certain species, these haemoglobins have been implicated in human allergic disease in several countries. The present study confirms and extends observations that haemoglobin is present in many species of Chironomidae, establishes that it is retained from the larval to the adult stage and shows that haemoglobin persists both in live and in dead dry flies. Previous suggestions that Chironomidae should be seen as important environmental and occupational allergens are clearly substantiated.     

The polyprotein allergens of nematodes (NPAs) - structure at last, but still mysterious

We are engaged in structural and functional studies of several types of lipid binding protein that are only found in nematodes. Amongst these are the nematode polyprotein allergens (NPAs) and we now report the solution structure of ABA-1A (As-NPA-A1), the most repeated unit within the NPA array of Ascaris suum, which is almost identical in amino acid sequence to that of Ascaris lumbricoides. The protein forms a slightly flattened, compact, globular fold consisting of a long central helix that participates in two flanking helical bundles. Two pockets lined with apolar amino acid sidechains are apparent, one in the carboxy-terminal region of the protein, and another smaller one in the amino-terminal region. The former appears to be the main site of fatty acid binding, and the latter may have different, though possibly overlapping, ligand binding propensities. The structure of the binding sites indicates that lipid ligands are anchored within them with their hydrophobic tails oriented towards the core of the protein and their polar headgroups bound to charged sidechains at the mouth of the pockets. The three-dimensional architectures of the amino- and carboxy-terminal halves of ABA-1A are closely similar, thereby strengthening the long-suspected idea that the repeated units of NPAs themselves originate from an ancient duplication event.     

Lipid transfer proteins in Parietaria judaica L. pollen grains: immunocytochemical localization and function

Parietariajudaica L. (Urticaceae) pollen is considered one of the most common causes of allergic respiratory symptoms in the Mediterranean area. The localization of lipid transfer proteins (LTPs) in P. judaica mature and hydrated-activated pollen grains was investigated applying a combination of transmission electron microscopy (TEM) with immunocytochemical methods. Our results show that the content of LTPs in P. judaica pollen grains changes during the process of hydration. The localization of judaica LTPs in the cytoplasm and in the lipid bodies associated with vacuoles demonstrated that LTPs represent primarily intracellular proteins. On the other hand, exposure of the pollen grains to germination medium induced the release of LTPs from the pollen grain. Thus, LTPs are cytoplasmic proteins that are secreted to become available for pollen-stigma interactions and probably induce the IgE antibody responses in allergic patients.     

Expression of tropomyosin from Blattella germanica as a recombinant non-fusion protein in Pichia pastoris and comparison of its IgE reactivity with its native counterpart

Tropomyosins derived from invertebrates are well-known pan allergens. However, the allergenicities of recombinant tropomyosins are variable. Here, we undertook to compare the IgE-binding reactivities of native and recombinant German cockroach tropomyosins. Native tropomyosin was purified by ammonium sulfate fractionation, hydroxyapatite column chromatography, and electroelution, and recombinant tropomyosin was expressed in Pichia pastoris. The allergenicities of the native and recombinant tropomyosins were compared by ELISA inhibition analysis. Native German cockroach tropomyosin showed 18% IgE-binding reactivity to German cockroach sensitized sera. Recombinant tropomyosin was produced without fusion protein and its N-terminus was blocked like that of the native counterpart. The IgE-binding reactivity of the recombinant was found to be comparable to that of native tropomyosin over the concentration range 1-1000 ng/ml by ELISA inhibition testing. Recombinant German cockroach tropomyosin expressed in Pichia pastoris showed better allergenicity than that expressed in Escherichia coli. Other factors in addition to the structural differences of native and recombinant proteins may also influence the IgE reactivities of tropomyosins.     

Predicting the grass pollen count from meteorological data with regard to estimating the severity of hayfever symptoms in Melbourne (Australia)

In Melbourne, Australia, grass pollen is the predominant cause of hayfever in late spring and summer. The grass pollen season has been monitored in Melbourne, using a Burkard spore trap, for 13 years (1975–1981, 1985 and 1991–1997). Total counts for grass pollen were highly variable from one season to the next (approximately 1000 to >8000 grains/m³). The daily grass pollen counts also showed a high variability (0 to approximately 400 grains/m³). In this study, the grass pollen counts of the 13 years (12 grass pollen seasons, extending from October to January) have been compared with meteorological data in order to identify the conditions that can determine the daily amounts of grass pollen in the air. It was found that the seasonal total of grass pollen was directly correlated with the rainfall sum of the preceding 12 months (1 September–31 August): seasonal total of grass pollen (counts/m³)=18.161 × rainfall sum of the preceding 12 months (mm) −8541.5 (r _s=0.74,P<0.005,n=12). The daily amounts of grass pollen in the air were positively correlated with the corresponding daily average ambient temperatures (P<0.001). The daily amount of grass pollen which was to be expected with a certain daily average temperature was linked to the seasonal total of grass pollen: in years with high total grass pollen counts, a lower daily average temperature was required for a high daily pollen count than in years with low total grass pollen counts. As the concentration of airborne grass pollen determines the severity of hayfever in sensitive patients, an estimation of daily grass pollen counts can provide an indication of potential pollinosis symptoms. We compared daily grass pollen counts with the reported symptomatic responses of hayfever sufferers in November 1985 and found that hayfever symptoms were significantly correlated to the grass pollen counts (P<0.001 for nasal,P<0.005 for eye symptoms). Thus, a combination of meteorological information (i.e. rainfall and temperature) allows for an estimation of the potential daily pollinosis symptoms during the grass pollen season. Here we propose a symptom estimation chart, allowing a quick prediction of eye and nasal symptoms that are likely to occur as a result of variations in meteorological conditions, thus enabling both physicians and patients to take appropriate avoidance measures or therapy.     

Electrophoretic profiles and amino acid composition of rice endosperm proteins of a mutant with enhanced lysine and total protein after backcrosses for germplasm improvements

 Seed storage proteins from in vitro-derived rice mutants improved by several backcrosses to ‘Calrose 76’ and BC₂ and BC₃ were characterized for changes in five different solubility classes. Albumins, rsealb (water-soluble globulins), true salt-soluble globulins, prolamins and glutelins were SDS-PAGE separated in a single dimension, and some two-dimensionally, to identify protein modifications. The genetic transmission of the enhanced-lysine mutants in backcrosses and the linkage of lysine with grain chalkiness were confirmed. Advanced lines had altered globulin profiles similar to those of unimproved lines. Chalky/ enhanced-lysine phenotypes had similar prolamin and glutelin profiles in the mutant and controls at the same protein level. Mutants had increased levels of globulins at 50 kDa and 33 kDa but had substantially less protein at 25 kDa than the controls. High protein in the mutant contributed to an increase in prolamins and the major storage proteins in both the globulins and glutelins. A significant decrease in low-molecular-weight, 15- to 18-kDa albumins was associated with the chalky/enhanced-lysine mutant phenotype. Two proteins in the 15- to 18-kDa group were amino acid sequenced, and database comparisons identified these proteins as allergens. Advanced lines downregulated for allergens and with enhanced-lysine/protein but with normal fertility and seed weight should be useful in breeding programs for nutritional quality. Received: 14 October 1996 / Accepted: 8 November 1996     

Localization and release of allergens from tapetum and pollen grains ofBetula pendula

Summary Although intact pollen grains are assumed to be the primary carrier of pollen allergens, specific immunoreactive components have been found in other aerosol fractions, e.g., starch grains and remains of tapetal cells Cryo-scanning-electron-microscopy results demonstrate the presence of a clear network of strands connecting the tapetum with the microspores. The distribution of protein in tapetal orbicules, pollen wall, and pollen cytoplasm was tested by histochemical stains for light microscopy and transmission electron microscopy. The protein is mainly localized at the apertures and starch grains in the cytoplasm of pollen and in the core and on the surface of tapetal orbicules. Monoclonal antibodies Bv-10, BIP3, and BIP4 have been used to locate the cellular sites of pollen and tapetal allergens inBetula pendula (syn.B. verrucosa). The application of rapid-freeze fixation prevented relocation of allergens from their native sites. The allergens are predominantly found in the starch grains and to lesser extent in the exine. We also tested interactions between mature birch pollen and human fluids: saliva, nostrils fluid, and eyes solution. The aim was to mimic more closely the in vivo situation during allergenic response. In all cases we observed several pollen grains that were burst and had released their cytoplasmic contents. In the nose the allergens are released from the pollen within minutes. In rhinitis, nasal pH is increased from the normal pH 6.0 to 8.0. When we used nasal fluid at pH 8.0, the number of ruptured pollen grains increased. The mechanism that might induce formation of small allergen-bearing particles from living plant cells is discussed.     

Bioinformatics applied to allergy: allergen databases, from collecting sequence information to data integration. The Allergome platform as a model

Allergens are proteins or glycoproteins that are recognized by IgE produced by the immune system of allergic individuals. Until now around 1,500 allergenic structures have been identified and this number seems not have reached a plateau after 3-4 decades of research and the advent of molecular biology. Several allergen databases are available on Internet. Different aims and philosophies lead to different products. Here we report about main feature of web sites dedicated to allergens and we describe in more details our current work on the Allergome platform. The web server Allergome (www.allergome.org) represent a free independent open resource whose goal is to provide an exhaustive repository of data related to all the IgE-binding compounds. The main purpose of Allergome is to collect a list of allergenic sources and molecules by using the widest selection criteria and sources. A further development of the Allergome platform has been represented by the Real Time Monitoring of IgE sensitization module (ReTiME) that allows uploading of raw data from both in vivo and in vitro testing, thus representing the first attempt to have IT applied to allergy data mining. More recently, a new module (RefArray) representing a tool for literature mining has been released.     

Expression of lymphocyte cell surface markers in workers exposed to different respiratory hazards: biomarkers of occupational respiratory disease

This study was designed to evaluate the potential of flow cytometry to measure biomarkers of airways inflammation in the peripheral blood of two cohorts of workers reporting work related respiratory symptoms, who were exposed to different respiratory hazards. Nine bakers exposed to wheat flour and 10 glass bottle manufacturers exposed to a range of irritant chemicals were selected for study. Phenotypic and inducible cell surface markers were measured by flow cytometry. Results were compared with a control population of 58 volunteers reporting no respiratory problems. The bakers showed a significant increase above control values for cell types associated with inflammation; in particular CD3 CD4 cells p 0.005 and CD4 CD25 cells p 0.01 . In contrast, the workers reporting work related respiratory symptoms who were exposed to a range of irritant chemicals showed a different pattern of cell surface lymphocyte markers, with a significant decrease in the total T cell population p 0.05 . Comparison of results from a subset of smoking controls with the population of bakers who were all heavy smokers confirmed that the increase in CD3 CD4 cells and CD4 CD25 cells could not be ascribed to the effects of smoking alone. We have shown activation of helper T cells in the peripheral blood of bakers reporting work related respiratory symptoms consistent with the changes observed in mild to severe asthmatics. However, workers with similar symptoms who were exposed to irritant chemicals did not show this pattern of phenotypic or inducible cell surface markers, reflecting an absence of airways inflammation in these individuals. Our results suggest that flow cytometry may be of use as an objective test for detecting workers with airways inflammation to allow the identification of workers at risk of developing occupational asthma.     

An approach to the knowledge of pollen and allergen diversity through lipid transfer protein localisation in taxonomically distant pollen grains

The aim of the present study is to localise non‐specific lipid transfer proteins (nsLTPs), obtained from Rosaceae (Prunus persica (L.) Batsch) in pollen grains of taxonomically distant plants, such as Iridaceae (Aristea latifolia G.J. Lewis), Platanaceae (Platanus acerifolia (Aiton) Willd.) and Urticaceae (Parietaria judaica L.), in order to compare pollen and nsLTP diversity. A combination of transmission electron microscopy, immunocytochemical techniques, and rabbit specific antiserum against peach nsLTPs (Pru p 3) were used. Abundant labelling to Pru p 3‐like proteins was observed in the cytoplasm, walls and pollenkitt of A. latifolia pollen grains. The presence of nsLTPs associated with the pollenkitt proves that it takes part in the defence mechanism of pollen grains. The labelling was less intense in the cytoplasm and walls of P. acerifolia. Immuno‐stained gold particles were associated with the vacuoles, lipid inclusions, endoplasmic reticulum and Golgi complex. No significant labelling was found in the P. judaica pollen grains incubated with anti‐Pru p 3 polyclonal antibodies. These results indicate important variations in the nsLTPs of different pollen species. Consequently, no taxonomic relationship between pollen grains and nsLTPs could be established.