VH gene usage in immunoglobulin E responses of seasonal rhinitis patients allergic to grass pollen is oligoclonal and antigen driven

Background: IgE is the pivotal-specific effector molecule of allergic reactions yet it remains unclear whether the elevated production of IgE in atopic individuals is due to superantigen activation of B cell populations, increased antibody class switching to IgE or oligoclonal allergen-driven IgE responses. Objectives: To increase our understanding of the mechanisms driving IgE responses in allergic disease we examined immunoglobulin variable regions of IgE heavy chain transcripts from three patients with seasonal rhinitis due to grass pollen allergy. Methods: Variable domain of heavy chain-epsilon constant domain 1 cDNAs were amplified from peripheral blood using a two-step semi-nested PCR, cloned and sequenced. Results: The VH gene family usage in subject A was broadly based, but there were two clusters of sequences using genes VH 3-9 and 3-11 with unusually low levels of somatic mutations, 0-3%. Subject B repeatedly used VH 1-69 and subject C repeatedly used VH 1-02, 1-46 and 5a genes. Most clones were highly mutated being only 86-95% homologous to their germline VH gene counterparts and somatic mutations were more abundant at the complementarity determining rather than framework regions. Multiple sequence alignment revealed both repeated use of particular VH genes as well as clonal relatedness among clusters of IgE transcripts. Conclusion: In contrast to previous studies we observed no preferred VH gene common to IgE transcripts of the three subjects allergic to grass pollen. Moreover, most of the VH gene characteristics of the IgE transcripts were consistent with oligoclonal antigen-driven IgE responses.

A rapid and efficient two-step gel electrophoresis method for the purification of major rye grass pollen allergens

Purified proteins are mandatory for molecular, immunological and cellular studies. However, purification of proteins from complex mixtures requires specialised chromatography methods (i.e., gel filtration, ion exchange, etc.) using fast protein liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC) systems. Such systems are expensive and certain proteins require two or more different steps for sufficient purity and generally result in low recovery. The aim of this study was to develop a rapid, inexpensive and efficient gel-electrophoresis-based protein purification method using basic and readily available laboratory equipment. We have used crude rye grass pollen extract to purify the major allergens Lol p 1 and Lol p 5 as the model protein candidates. Total proteins were resolved on large primary gel and Coomassie Brilliant Blue (CBB)-stained Lol p 1/5 allergens were excised and purified on a secondary "mini"-gel. Purified proteins were extracted from unstained separating gels and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses. Silver-stained SDS-PAGE gels resolved pure proteins (i.e., 875 μg of Lol p 1 recovered from a 8 mg crude starting material) while immunoblot analysis confirmed immunological reactivity of the purified proteins. Such a purification method is rapid, inexpensive, and efficient in generating proteins of sufficient purity for use in monoclonal antibody (mAb) production, protein sequencing and general molecular, immunological, and cellular studies.

Allergenic Cross-Reactivity between Parthenium and Ragweed Pollen Allergens

Cross-reactivity of allergens from the pollen of the Compositae weeds, Parthenium hysterophorus (American feverfew) and Ambrosia (ragweed), in 2 groups of patients with different geographic distributions was studied. Parthenium-sensitive Indian patients, who were never exposed to ragweed, elicited positive skin reactions with ragweed pollen extracts. A significant correlation in the RAST scores of Parthenium and ragweed-specific IgE was observed with the sera of Parthenium and ragweed-sensitive Indian and US patients, respectively. RAST inhibition experiments demonstrated that the binding of IgE antibodies in the sera of ragweed-sensitive patients to short (Wl) and giant (W3) ragweed allergen discs could be inhibited by up to 94% by Parthenium pollen extracts. Similar inhibition (up to 82%) was obtained when the sera of Parthenium rhinitis patients were incubated with ragweed allergen extracts. A dose-dependent proliferation of lymphocytes from a Parthenium-sensitive rhinitis patient with elevated levels of both Parthenium and ragweed-specific IgE was observed when incubated with Parthenium and ragweed pollen extracts. A 1.6-fold higher proliferation, however, was observed with Parthenium pollen extract at a concentration of 100 µg/ml. These results suggest that shared epitopes present on Parthenium and ragweed pollen allergens are recognized by both Indian and US patients sensitized by exposure to Parthenium and ragweed pollen, respectively. The high degree of cross-reactivity between Parthenium and ragweed pollen allergens suggests that individuals sensitized to Parthenium may develop type-I hypersensitivity reactions to ragweed and vice versa when they travel to regions infested with the weed to which they had not been previously exposed.

Standardization of pollen allergens of Parthenium hysterophoms and selection of an in-house reference extract

A standardized in-house reference extract from the pollen of Parthenium hysterophorus, which is responsible for the high incidence of allergic rhinitis in India, was generated and examined by skin test, radio-allergosorbent test inhibition and isoelectric focusing. Parthenium reference allergen discs and positive reference serum were also generated. These reference reagents could not only be used for the quantitation of Parthenium-specific IgE in the sera of rhinitis patients but also for the evaluation of allergenic activity (relative potency and lot-to-lot variation) of different batches of Parthenium pollen.

Identification of the main allergen sensitizers in an Iran asthmatic population by molecular diagnosis

Background: There has been a significant growth in the prevalence of allergy, mainly associated to IgE-mediated disorders such as asthma and rhinitis. The identification of atopy in asthmatic patients through the measurement of specific IgE can help to identify risk factors that cause asthmatic symptoms in patients. The development and use of individualized allergen-based tests by the Component Resolved Diagnosis has been a crucial advance in the accurate diagnosis and control of allergic patients. The objective of this work was to assess the usefulness of molecular diagnosis to identify environmental allergens as possible factors influencing the development and manifestation of asthma in a group of asthmatic patients from Iran. Methods: Studied population: 202 adult asthmatic patients treated at the Loghman Hakim Hospital and Pasteur Institute of Teheran (Iran) from 2011 to 2012. Specific IgE determined by the ImmunoCAP system were used to both evaluate the patients' atopic condition and the molecules involved in the allergic sensitization. SDS-PAGE IgE-immunoblotting associated with mass spectrometry was carried out to study the cockroach IgE-binding sensitizing proteins. Results: Forty-five percent of all patients could be considered atopic individuals. Eighty-two percent of atopic patients were sensitized to pollen allergens. The Salsola kali (Sal k 1) and the Phleum pratense (rPhl p 1 and/or rPhl p 5) major allergens were the most common sensitizers among pollens (71% and 18%, respectively). Thirty-five percent of the atopic population was sensitized to cockroach. Four different allergens, including a previously unknown alpha-amylase, were identified in the cockroach extract. No significant associations could be demonstrated between the severity of asthma and the specific IgE levels in the atopic population. Statistical analysis identified the Sal k 1 as the main protein allergen influencing the development and expression of asthma in the studied population. Conclusions: Pollen and cockroach were the most relevant allergen sources in the asthmatic population. The Salsola kali major allergen was the main cause for sensitization in the atopic patients suffering asthma. Using the Component Resolved Diagnosis, it was possible to identify a new Blattella germanica cockroach allergen (Blattella alpha amylase 53 kDa) that could sensitize a relevant percentage of this population.

Regional Importance of Alnus Pollen as an Aeroallergen: A Comparative Study of Alnus Pollen Counts from Worcester (UK) and Poznań (Poland)

Daily average Alnus pollen counts (1996-2005) from Worcester (UK) and Poznań (Poland) were examined with the aim of assessing the regional importance of Alnus pollen as an aeroallergen. The average number of Alnus pollen grains recorded annually at Poznań was more than 2.5 times that of Worcester. Furthermore, daily average Alnus pollen counts exceeded the thresholds of 100, 500 and 1,000 grains/m3 more times at Poznań than Worcester. Skin prick test results (1996-2005) and allergen-specific IgE(asIgE) measurements using the CAP (Pharmacia) system (2002-2005), were supplied by the Allergic Diseases Diagnostic Centre in Poznań. The annual number of positive skin prick tests to Alnus pollen allergens was significantly related (p<0.05) to seasonal variations in the magnitude of the Alnus pollen catch recorded at Poznań (r=0.70). The symptoms of patients with positive skin prick tests to Alnus pollen allergens were: 51% pollinosis, 43% atopic dermatitis, 4% asthma, 1% chronic urticaria and 1% eczema. On a scale of 0-6, 20.5% of patients examined for serum asIgE in relation to Alnus pollen allergens had asIgE measurements in classes 5 and 6. Alnus pollen is generally considered to be mildly allergenic. However, the amount of Alnus pollen released into the atmosphere in places such as Poznań may increase its impact on the population and make it one of the more important aeroallergens present.

Mesoscale Atmospheric Transport of Ragweed Pollen Allergens from Infected to Uninfected Areas

Allergenic ragweed (Ambrosia spp.) pollen grains, after being released from anthers, can be dispersed by air masses far from their source. However, the action of air temperature,humidity and solar radiation on pollen grains in the atmosphere could impact on the ability of long distance transported (LDT) pollen to maintain allergenic potency. Here, we report that the major allergen of Ambrosia artemisiifolia pollen (Amb a 1) collected in ambient air during episodes of LDT still have immunoreactive properties. The amount of Amb a 1 found in LDT ragweed pollen grains was not constant and varied between episodes. In addition to allergens in pollen sized particles, we detected reactive Amb a 1 in subpollen sized respirable particles. These findings suggest that ragweed pollen grains have the potential to cause allergic reactions, not only in the heavily infested areas but, due to LDT episodes, also in the regions unaffected by ragweed populations.

Cellular localization of water soluble, allergenic proteins in rye-grass (Lolium perenne) pollen using monoclonal and specific IgE antibodies with immunogold probes

A postembedding method has been developed for localizing water soluble allergens in rye-grass pollen. This uses dry fixation in glutaraldehyde vapour, followed by 2,2-dimethoxypropane, prior to a 100% ethanol series leading into embedment in LR Gold. This has allowed the attachment of specific monoclonal antibodies to the allergen, which are themselves probed with specific immunogold labels to the antibodies. Wall and cytoplasmic sites have been identified, representing an improvement of fixation and localization of allergens over previous studies employing polyclonal, broad spectrum antibodies.

Rye-grass allergens are labelled in mature pollen grains in the exine (tectum, nexine and central chamber), and in the electron opaque areas of the cytoplasm, especially mitochondria. The allergens are absent from the intine, polysaccharide (P) particles, amyloplasts, Golgi bodies and endoplasmic reticulum. IgE antibodies derived from humans allergic to rye-grass pollen, bind to similar sites in the cytoplasm but only to the outer surface of the pollen grain wall. This method now provides a valuable tool for further developmental studies on the pollen grains, in order to establish the site/s of synthesis of the allergens.

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.

Immunologic significance of respirable atmospheric starch granules containing major birch allergen Bet v 1

Background: Birch-pollen allergens are an important cause of early spring hay fever and allergic asthma. Recently, we reported a mechanism for the release of respirable allergenic particles from birch pollen containing the major allergen Bet v 1. In this study, we aimed to assess the immunologic significance of the released Bet v 1-containing starch granules in the environment.

Methods: A two-site monoclonal antibody-based assay (ELISA) was employed to quantitate Bet v 1 in high-volume air sampler filter extracts, and immunogold-labelling was used on sections of these extracts to localize Bet v 1. Immunoblot analyses were performed with pooled sera from patients sensitive to birch pollen.

Results: Atmospheric starch granules contained Bet v 1, and the concentration increased upon light rainfall. Sera from patients allergic to birch allergens recognized extracts from isolated starch granules.

Conclusions: The clinical implications of these findings are that starch granules released from birch pollen are potentially able to trigger allergic asthmatic reactions to Bet v 1, since the allergen occurs in respirable particles. Thus, clinicians can advise asthma patients to remain indoors on days of light rainfall during the birch-pollen season to avoid high levels of allergen exposure.

Influence of climate changes on cross allergies: possible involvement of pollen transglutaminase

Allergies are a complex of symptoms derived from altered IgE-mediated reactions of the immune system towards substances known as allergens. Allergic sensibilization can be of food or respiratory origin and, in particular, apple and hazelnut allergens have been identified in pollens or fruits. Allergic cross-reactivity can occur in a patient reacting to similar allergens from different origins, justifying the research in both systems as in Europe a greater number of people suffers from apple fruit allergy, but little evidence exists about pollen. Apple fruit allergies are due to four different classes of allergens (Mal d 1, 2, 3, 4), whose allergenicity is related both to genotype and tissue specificity; therefore I have investigated their presence also in pollen at different time of germination to clarify the apple pollen allergenic potential. I have observed that the same four classes of allergens found in fruit are expressed at different levels also in pollen, and their presence might support that the apple pollen can be considered allergenic as the fruit, deducing that apple allergy could also be indirectly caused by sensitization to pollen. Climate changes resulting from increases in temperature and air pollution influence pollen allergenicity, responsible for the dramatic raise in respiratory allergies (hay fever, bronchial asthma, conjunctivitis). Although the link between climate change and pollen allergenicity is proven, the underlying mechanism is little understood. Transglutaminases (TGases), a class of enzymes able to post-translationally modify proteins, are activated under stress and involved in some inflammatory responses, enhancing the activity of pro-inflammatory phospholipase A2, suggesting a role in allergies. Recently, a calcium-dependent TGase activity has been identified in the pollen cell wall, raising the possibility that pollen TGase may have a role in the modification of pollen allergens reported above, thus stabilizing them against proteases. This enzyme can be involved also in the transamidation of proteins present in the human mucosa interacting with surface pollen or, finally, the enzyme itself can represent an allergen, as suggested by studies on celiac desease. I have hypothesized that this pollen enzyme can be affected by climate changes and be involved in exhacerbating allergy response. The data presented in this thesis represent a scientific basis for future development of studies devoted to verify the hypothesis set out here. First, I have demonstrated the presence of an extracellular TGase on the surface of the grain observed either at the apical or the proximal parts of the pollen-tube by laser confocal microscopy (Iorio et al., 2008), that plays an essential role in apple pollen-tube growth, as suggested by the arrest of tube elongation by TGase inhibitors, such as EGTA or R281. Its involvement in pollen tube growth is mainly confirmed by the data of activity and gene expression, because TGase showed a peak between 15 min and 30 min of germination, when this process is well established, and an optimal pH around 6.5, which is close to that recorded for the germination medium. Moreover, data show that pollen TGase can be a glycoprotein as the glycosylation profile is linked both with the activation of the enzyme and with its localization at the pollen cell wall during germination, because from the data presented seems that the active form of TGase involved in pollen tube growth and pollen-stylar interaction is more exposed and more weakly bound to the cell wall. Interestingly, TGase interacts with fibronectin (FN), a putative SAMs or psECM component, inducing possibly intracellular signal transduction during the interaction between pollen-stylar occuring in the germination process, since a protein immunorecognised by anti-FN antibody is also present in pollen, in particular at the level of pollen grain cell wall in a punctuate pattern, but also along the shank of the pollen tube wall, in a similar pattern that recalls the signal obtained with the antibody anti TGase. FN represents a good substrate for the enzyme activity, better than DMC usually used as standard substrate for animal TGase. Thus, this pollen enzyme, necessary for its germination, is exposed on the pollen surface and consequently can easily interact with mucosal proteins, as it has been found germinated pollen in studies conducted on human mucus (Forlani, personal communication). I have obtained data that TGase activity increases in a very remarkable way when pollen is exposed to stressful conditions, such as climate changes and environmental pollution. I have used two different species of pollen, an aero allergenic (hazelnut, Corylus avellana) pollen, whose allergenicity is well documented, and an enthomophylus (apple, Malus domestica) pollen, which is not yet well characterized, to compare data on their mechanism of action in response to stressors. The two pollens have been exposed to climate changes (different temperatures, relative humidity (rH), acid rain at pH 5.6 and copper pollution (3.10 µg/l)) and showed an increase in pollen surface TGase activity that is not accompanied to an induced expression of TGase immunoreactive protein with AtPNG1p. Probably, climate change induce an alteration or damage to pollen cell wall that carries the pollen grains to release their content in the medium including TGase enzyme, that can be free to carry out its function as confirmed by the immunolocalisation and by the in situ TGase activity assay data; morphological examination indicated pollen damage, viability significantly reduced and in acid rain conditions an early germination of apple pollen, thus possibly enhancing the TGase exposure on pollen surface. Several pollen proteins were post-translationally modified, as well as mammalian sPLA2 especially with Corylus pollen, which results in its activation, potentially altering pollen allergenicity and inflammation. Pollen TGase activity mimicked the behaviour of gpl TGase and AtPNG1p in the stimulation of sPLA2, even if the regulatory mechanism seems different to gpl TGase, because pollen TGase favours an intermolecular cross-linking between various molecules of sPLA2, giving rise to high-molecular protein networks normally more stable. In general, pollens exhibited a significant endogenous phospholipase activity and it has been observed differences according to the allergenic (Corylus) or not-well characterized allergenic (Malus) attitude of the pollen. However, even if with a different intensity level in activation, pollen enzyme share the ability to activate the sPLA2, thus suggesting an important regulatory role for the activation of a key enzyme of the inflammatory response, among which my interest was addressed to pollen allergy. In conclusion, from all the data presented, mainly presence of allergens, presence of an extracellular TGase, increasing in its activity following exposure to environmental pollution and PLA2 activation, I can conclude that also Malus pollen can behave as potentially allergenic. The mechanisms described here that could affect the allergenicity of pollen, maybe could be the same occurring in fruit, paving the way for future studies in the identification of hyper- and hypo- allergenic cultivars, in preventing environmental stressor effects and, possibly, in the production of transgenic plants.

Continuous apple consumption induces oral tolerance in birch-pollen-associated apple allergy

Patients with birch pollen allergy (major allergen: Bet v 1) have often an associated oral allergy syndrome (OAS) to apple, which contains the cross-reactive allergen Mal d 1. As successful birch pollen immunotherapy does not consistently improve apple related OAS symptoms, we evaluated whether regular apple consumption has an effect on OAS and immune parameters of Mal d 1 or Bet v 1 allergy.

Surfactant protein D modulates allergen particle uptake and inflammatory response in a human epithelial airway model

Background Allergen-containing subpollen particles (SPP) are released from whole plant pollen upon contact with water or even high humidity. Because of their size SPP can preferentially reach the lower airways where they come into contact with surfactant protein (SP)-D. The aim of the present study was to investigate the influence of SP-D in a complex three-dimensional human epithelial airway model, which simulates the most important barrier functions of the epithelial airway. The uptake of SPP as well as the secretion of pro-inflammatory cytokines was investigated. Methods SPP were isolated from timothy grass and subsequently fluorescently labeled. A human epithelial airway model was built by using human Type II-pneumocyte like cells (A549 cells), human monocyte derived macrophages as well as human monocyte derived dendritic cells. The epithelial cell model was incubated with SPP in the presence and absence of surfactant protein D. Particle uptake was evaluated by confocal microscopy and advanced computer-controlled analysis. Finally, human primary CD4+ T-Cells were added to the epithelial airway model and soluble mediators were measured by enzyme linked immunosorbent assay or bead array. Results SPP were taken up by epithelial cells, macrophages, and dendritic cells. This uptake coincided with secretion of pro-inflammatory cytokines and chemokines. SP-D modulated the uptake of SPP in a cell type specific way (e.g. increased number of macrophages and epithelial cells, which participated in allergen particle uptake) and led to a decreased secretion of pro-inflammatory cytokines. Conclusion These results display a possible mechanism of how SP-D can modulate the inflammatory response to inhaled allergen.

Graph based study of allergen cross-reactivity of plant lipid transfer proteins (LTPs) using microarray in a multicenter study.

The study of cross-reactivity in allergy is key to both understanding. the allergic response of many patients and providing them with a rational treatment In the present study, protein microarrays and a co-sensitization graph approach were used in conjunction with an allergen microarray immunoassay. This enabled us to include a wide number of proteins and a large number of patients, and to study sensitization profiles among members of the LTP family. Fourteen LTPs from the most frequent plant food-induced allergies in the geographical area studied were printed into a microarray specifically designed for this research. 212 patients with fruit allergy and 117 food-tolerant pollen allergic subjects were recruited from seven regions of Spain with different pollen profiles, and their sera were tested with allergen microarray. This approach has proven itself to be a good tool to study cross-reactivity between members of LTP family, and could become a useful strategy to analyze other families of allergens.