Nasal Provocation Challenges with Aedes aegypti Whole-Body Extract Induces Allergic Rhinitis.

BACKGROUND: Exposure to mosquitoes in the Tropics is perennial, and their somatic and saliva antigens have shown IgE binding capacity, although it is not clear whether this is due to cross-reactivity or primary sensitization. Inhalation of these allergens could trigger an allergic response. OBJECTIVE: The aim of the study was to evaluate the clinical relevance of sensitization to Aedes aegypti in a group of patients with allergic rhinitis. METHODS: A cross-sectional study with allergic rhinitis subjects and healthy controls sensitized to mosquito extract was performed. Sensitization to mosquito and house dust mites was evaluated using skin prick test (SPT) and antibody determination by ELISA. Nasal provocation test (NPT) with whole-body extract was used to determine clinical relevance. RESULTS: Allergic rhinitis patients were more sensitized to mosquito extract than controls with (+) SPT (66.6% vs. 7.6%). From these (+) SPT patients, 44.5% had (+) NPT, and just two (11%) presented mono-sensitization to mosquito. Antibody reactivity was similar between patients and controls; however, (+) NPT patients showed a tendency to had higher levels of IgE and IgG4. DISCUSSION: Mosquitoes are perennial in most tropical areas, and their body allergens could be associated with respiratory allergies.

Influence of environmental drivers on allergy to pollen grains in a case study in Spain (Madrid): meteorological factors, pollutants, and airborne concentration of aeroallergens.

The aim of this study was to compare airborne levels of Phl p 1 and Phl p 5, with Poaceae pollen concentrations inside and outside of the pollen season, and to evaluate their association with symptoms in grass allergic patients and the influence of climate and pollution. The Hirst and the Burkard Cyclone samplers were used for pollen and allergen quantification, respectively. The sampling period ran from 23 March 2009 to 27 July 2010. Twenty-three patients with seasonal allergic asthma and rhinitis used an electronic symptom card. The aerosol was extracted and quantified for Phl p 1 and Phl p 5 content. Descriptive statistics, non-parametric paired contrast of Wilcoxon, Spearman's correlations, and a categorical principal component analysis (CatPCA) were carried out. Significant variations in pollen, aeroallergen levels, pollen allergen potency, and symptoms score were observed in this study. Phl p 5 pollen allergen potency was higher at the beginning of the 2010 grass pollen season. Presence of Phl p 1 outside the pollen season with positive O3 correlation was clinically relevant. 45.5% of the variance was explained by two dimensions in the CatPCA analysis, showing the symptom relationships dissociated in two dimensions. In the first one, the more important relationship was with grass pollen grains concentration and Phl p 5 and to a lesser extent with Phl p 1 and levels of NO2 and O3, and in the second dimension, symptoms were associated with humidity and SO2. Clinically relevant out-season Phl p 1 was found with a positive O3 correlation. The effect of climate and pollution may have contributed to the higher seasonal allergic rhinitis symptom score recorded in 2009.

Prunus persica 9, a new occupational allergen from peach tree pollen involved in rhinitis and asthma.

OBJECTIVES: Several studies have described peach tree (PT) as an occupational allergen. The aim of this work was to assess the effect of Prunus persica 9 (Pru p 9), a recently identified allergen from PT pollen, in exposed workers. METHODS: The study included people who reported respiratory symptoms after handling PT in orchards during the flowering period (Blanca village, Murcia region, south-east Spain). After completing a detailed questionnaire, participants underwent skin prick test (SPT) and nasal provocation test (NPT). The IgE response was analysed by SDS-PAGE and immunoblotting assays. RESULTS: A total of 21 cases were included (mean age 45 years; 57% women). Most were polysensitised to common pollens, although one person was sensitised only to PT pollen. All cases had a positive SPT to this pollen, and 43% also to Pru p 9. All participants reported having rhinitis, and six participants reported having also asthma. Immunoblotting showed a heterogeneous IgE pattern for several proteins, with Pru p 9 recognised in nine cases. Most participants sensitised to PT pollen and Pru p 9 had positive NPTs, while those who were not sensitised to Pru p 9 tested negative. CONCLUSIONS: We demonstrate for the first time that Pru p 9, an allergen from PT pollen, can induce respiratory symptoms following occupational exposure. This must be considered a relevant allergen when people working with PT cultivars develop respiratory symptoms.

Novel vaccines targeting dendritic cells by coupling allergoids to nonoxidized mannan enhance allergen uptake and induce functional regulatory T cells through programmed death ligand 1.

BACKGROUND: Allergen immunotherapy (AIT) is the only curative treatment for allergy. AIT faces pitfalls related to efficacy, security, duration, and patient compliance. Novel vaccines overcoming such inconveniences are in demand. OBJECTIVES: We sought to study the immunologic mechanisms of action for novel vaccines targeting dendritic cells (DCs) generated by coupling glutaraldehyde-polymerized grass pollen allergoids to nonoxidized mannan (PM) compared with glutaraldehyde-polymerized allergoids (P) or native grass pollen extracts (N). METHODS: Skin prick tests and basophil activation tests with N, P, or PM were performed in patients with grass pollen allergy. IgE-blocking experiments, flow cytometry, confocal microscopy, cocultures, suppression assays, real-time quantitative PCR, ELISAs, and ELISpot assays were performed to assess allergen capture by human DCs and T-cell responses. BALB/c mice were immunized with PM, N, or P. Antibody levels, cytokine production by splenocytes, and splenic forkhead box P3 (FOXP3)(+) regulatory T (Treg) cells were quantified. Experiments with oxidized PM were also performed. RESULTS: PM displays in vivo hypoallergenicity, induces potent blocking antibodies, and is captured by human DCs much more efficiently than N or P by mechanisms depending on mannose receptor- and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-mediated internalization. PM endorses human DCs to generate functional FOXP3(+) Treg cells through programmed death ligand 1. Immunization of mice with PM induces a shift to nonallergic responses and increases the frequency of splenic FOXP3(+) Treg cells. Mild oxidation impairs these effects in human subjects and mice, demonstrating the essential role of preserving the carbohydrate structure of mannan. CONCLUSIONS: Allergoids conjugated to nonoxidized mannan represent suitable vaccines for AIT. Our findings might also be of the utmost relevance to development of therapeutic interventions in other immune tolerance-related diseases.

Structural studies of novel glycoconjugates from polymerized allergens (allergoids) and mannans as allergy vaccines.

Immunotherapy for treating IgE-mediated allergies requires high doses of the corresponding allergen. This may result in undesired side effects and, to avoid them, hypoallergenic allergens (allergoids) polymerized with glutaraldehyde are commonly used. Targeting allergoids to dendritic cells to enhance cell uptake may result in a more effective immunotherapy. Allergoids coupled to yeast mannan, as source of polymannoses, would be suitable for this purpose, since mannose-binding receptors are expressed on these cells. Conventional conjugation procedures of mannan to proteins use oxidized mannan to release reactive aldehydes able to bind to free amino groups in the protein; yet, allergoids lack these latter because their previous treatment with glutaraldehyde. The aim of this study was to obtain allergoids conjugated to mannan by an alternative approach based on just glutaraldehyde treatment, taking advantage of the mannoprotein bound to the polymannose backbone. Allergoid-mannan glycoconjugates were produced in a single step by treating with glutaraldehyde a defined mixture of allergens derived from Phleum pratense grass pollen and native mannan (non-oxidized) from Saccharomyces cerevisae. Analytical and structural studies, including 2D-DOSY and (1)H-(13)C HSQC nuclear magnetic resonance spectra, demonstrated the feasibility of such an approach. The glycoconjugates obtained were polymers of high molecular weight showing a higher stability than the native allergen or the conventional allergoid without mannan. The allergoid-mannan glycoconjugates were hypoallergenic as detected by the IgE reactivity with sera from grass allergic patients, even with lower reactivity than conventional allergoid without mannan. Thus, stable hypoallergenic allergoids conjugated to mannan suitable for using in immunotherapy can be achieved using glutaraldehyde. In contrast to mannan oxidation, the glutaraldehyde approach allows to preserve mannoses with their native geometry, which may be functionally important for its receptor-mediated recognition.

Recent advances in immunotherapy for allergic diseases.

Allergic diseases are a major health problem worldwide. The therapeutic approaches to treat allergic rhinitis (AR) and allergic asthma (AA) fall in three major categories. The first step is allergen avoidance, or reduction of exposure to the offending allergen(s). The second and most widely used therapeutic practice is the prescription of relevant medication to reduce symptoms. The third therapeutic element is specific allergy vaccination, also known as allergen specific immunotherapy. Allergen-specific immunotherapy (SIT) is the only etiologic treatment of allergic disorders that can alter the natural course of the disease. In this review, recent advances in immunotherapy and relevant patents are presented. General vaccine modifications could be applied for any type of allergen. New specific modifications in allergic vaccines have been developed for a variety of allergies such as house dust mites, horse, cat, parvalbumin and from birch, ragweed and parietaria pollen.

Olea europaea pollen counts and aeroallergen levels predict clinical symptoms in patients allergic to olive pollen.

BACKGROUND: Allergic symptoms are commonly related to atmospheric pollen counts in sensitized allergic individuals. However, concordance between symptoms, pollen counts, and aeroallergen concentrations is not always good. OBJECTIVES: To determine the correlation between olive pollen counts, aeroallergen levels, and clinical symptoms in patients with allergic asthma or rhinitis in Ciudad Real (Spain). METHODS: Two types of samplers were used to determine pollen exposure: a Burkard spore trap to collect pollen grains and a high-volume air sampler to collect airborne particles. A total of 366 air filters were collected. After extraction, they were analyzed by specific immunoglobulin E enzyme-linked immunosorbent assay inhibition using a serum pool containing high titers of olive-specific immunoglobulin E. Twenty olive-pollen monosensitized patients were asked to record their daily symptoms before, during, and after the olive pollen season. RESULTS: Olive pollen was detected between April 21 and June 30, 2004. Symptoms showed positive and significant correlations with pollen counts (r = 0.700, P < .001) and aeroallergen levels (r = 0.803, P < .001). Using a Poisson regression model, relative changes in aeroallergen concentrations and pollen counts were found to be similar and significant. Threshold levels for the induction of symptoms were 162 olive pollen grains/m(3) and 22.7 ng of olive pollen allergen/m(3) (equivalent to 0.9 ng/m(3) of Ole e 1). CONCLUSIONS: Olive aeroallergen concentrations and pollen counts are positively associated with symptoms of rhinitis and asthma in olive-allergic patients. Both data may be used in the clinical follow-up of these patients.

A modified protocol for RNA isolation from high polysaccharide containing Cupressus arizonica pollen. Applications for RT-PCR and phage display library construction.

RNA isolation is the first step in the study of gene expression and recombinant protein production. However, the isolation of high quantity and high-quality RNA from tissues containing large amounts of polysaccharides has proven to be a difficult process. Cupressus arizonica pollen, in addition to containing high polysaccharide levels, is a challenging starting material for RNA isolation due to the roughness of the pollen grain's walls. Here, we describe an improved technique for RNA isolation from C. arizonica pollen grains. The protocol includes a special disruption and homogenization process as well as a two-step modified RNA isolation technique which consists of an acid phenol extraction followed by a final cleanup using a commercial kit. Resulting RNA proved to be free of contaminants as determined by UV spectrophotometry. The quality of the RNA was analyzed on a bioanalyzer and showed visible 25S and 18S bands. This RNA was successfully used in downstream applications such as RT-PCR and phage display library construction.

Characterization of allergoids.

So far it has not been possible to measure the amount of major allergens in the complexes after chemical modification. Furthermore, the presence of minor allergens remained obscure, unless antibodies were successfully generated by animal immunization with allergoids and shown to be reactive with purified natural or recombinant allergens in immunological assays. Thus, we adapted and employed a set of physicochemical methods with the aim of elucidating the molecular size and allergen composition of allergoids. Using online-HPSEC light scattering and DLS, it was shown that two thirds of depigmented allergoid prepared from birch pollen extracts adopted a MW between 1000 and 2000 kDa. The question of reproducibility of the polymerization reaction was addressed by investigating four batches of P. pratense allergoid. Three out of the four batches contained 73 to 77% of polymerized molecules in the above-mentioned range of molecular sizes. One batch showed a significantly higher content of molecules with a MW exceeding 2 MDa. Analysis of allergen composition in B. alba allergoids revealed the presence of all relevant Bet v 1 isoforms and minor allergens except for Bet v 3 and Bet v 4, which was in good agreement with the allergens detected in the native extracts. It should be noted that Bet v 3 has not been detected at the protein level before. Similarly, good agreement in allergen composition between allergoid and native extract was also found for D. pteronyssinus. Presently, the European Directorate for Quality of Medicines and Healthcare (EDQM) is committed to the application of the 3R principles (i. e. replace, reduce, refine the use of animals) for the quality control of medicines wherever possible. This is reflected by the regular review of the monographs of the European Pharmacopoeia and the introduction of alternative tests. For instance, recently it was decided to replace the rabbit pyrogen test by an in vitro test. Furthermore, through the Biological Standardisation Programme the EDQM develops, validates, and establishes alternative test methods in the field of quality control of biologicals (personal communication with Karl-Heinz Buchheit, EDQM). Therefore, the approach presented here for the characterization of allergoids relying on physicochemical methods shall also serve the growing needs for alternative methods to animal testing.

Sensitization and allergy to Cannabis sativa leaves in a population of tomato (Lycopersicon esculentum)-sensitized patients.

BACKGROUND: Cases of allergy to Cannabis sativa have occasionally been reported, but both the allergenic profile and eventual cross-reactivity pattern remain unknown. OBJECTIVE: To analyze the allergenic profile of a population of patients from Spain sensitized to C. sativa and to characterize the C. sativa leaf extract. METHODS: A total of 32 subjects were enrolled in the study: group A, 10 individuals sensitized to tomato, reporting reactions by contact or inhalation to Cannabis; group B, 14 individuals sensitized to tomato, without reactions to Cannabis; group C, 8 individuals not sensitized to tomato and without reactions to Cannabis. Sensitivity to Cannabis, tomato and peach peel, Platanus hybrida and Artemisia vulgaris pollen extracts was measured by skin tests and specific IgE. Individual immunoblots and inhibition experiments with a pool of sera were conducted. RESULTS: All tomato-sensitized subjects (and 1 negative) had positive skin tests to C. sativa leaves and hashish. Specific IgE to C. sativa and peach peel was more common than to tomato. Immunoblot experiments showed 2 prominent bands of 10 and 14 kDa and 2 weakly recognized bands of 30 and 45 kDa. Tomato, peach and A. vulgaris extracts inhibited most of the bands present in C. sativa. P. hybrida inhibited only the high-molecular-weight bands. CONCLUSION: Sensitization to C. sativa with or without symptoms is frequent among patients in Spain sensitized to tomato. C. sativa leaves are a potential allergenic source and their allergens may cross-react with other allergenic sources from plants (fruit peels and pollen).

Comparison of the allergenicity and Ole e 1 content of 6 varieties of Olea europaea pollen collected during 5 consecutive years.

BACKGROUND: Numerous varieties of Olea europaea have been described in Mediterranean countries. OBJECTIVE: To investigate the immunochemical characteristics of 6 varieties of Olea europaea collected during 5 consecutive years. METHODS: The varieties Carrasquefio, Manzanillo, Acebuche (wild olive), Hojiblanco, Picual, and Nevado were analyzed. Pollen samples from each variety were collected for 5 consecutive years from the same cultivars by trained personnel. The antigenic and allergenic profiles of these extracts were evaluated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot using the serum of 29 O. europaea-allergic individuals. Ole e 1 was measured using enzyme-linked immunosorbent assay and purified Ole e 1 and rabbit polyclonal antibodies. Allergenic potency was determined using enzyme-linked immunosorbent assay inhibition and is expressed in histamine equivalent prick units per gram of raw material. RESULTS: Hojiblanco and Acebuche had the lowest mean +/- SD Ole e 1 content in the 5 years (0.045 +/- 0.029 and 0.059 +/-0.031 microg/microg of freeze-dried material, respectively). The variety with the highest mean +/- SD Ole e 1 content was Picual (0.19 +/-0.075 microg/microg). Hojiblanco had the lowest total biological potency throughout the study. A positive correlation was obtained between rainfall in the winter months and total allergenicity of the 6 varieties. CONCLUSIONS: The different varieties of O. europaea pollen demonstrated great differences in allergenic potency and Ole e 1 content. These differences were maintained throughout the study, suggesting that they are due to genetic differences intrinsic to the varieties, although certain climatic effects may also play a role.

Anaphylaxis to Cyphomandra betacea Sendth (tamarillo) in an obeche wood (Triplochiton scleroxylon)--allergic patient.

BACKGROUND: Anaphylaxis after the first exposure to a food allergen is uncommon unless a cross-reaction is present. OBJECTIVE: To investigate a possible relationship between the fruit Cyphomandra betacea Sendth (commonly known as tamarillo) and the wood of Triplochiton scleroxylon (obeche) in a patient with allergic occupational bronchial asthma due to obeche wood who began to experience anaphylaxis episodes after eating tamarillo. METHODS: A 33-year-old carpenter exposed to obeche wood in his occupation was initially seen with rhinitis and bronchial asthma. The causal relationship of these symptoms to obeche wood exposure was investigated by means of peak flow monitoring and bronchial inhalation testing. Furthermore, the patient had 2 acute episodes of anaphylaxis a few minutes after eating salad containing tamarillo. He had never tasted tamarillo before. The allergologic study included skin prick tests, serum specific IgE determinations, bronchial challenges, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting. RESULTS: Results of skin prick tests with common aeroallergens were negative. Strong skin prick test responses were obtained with obeche and tamarillo. Results of bronchial challenge testing with obeche extract were positive. In tamarillo extract, a 28-kDa band appeared as the most relevant IgE-binding antigen. A similar band of 28 kDa happens to be frequently detected in obeche-allergic patients. CONCLUSION: To our knowledge, this is the first reported case of anaphylaxis to tamarillo presented in a patient allergic to obeche, which raises the question of a new cross-reactivity antigen.

Characterization and improvement of apple extracts for the diagnosis of apple IgE-mediated allergy.

BACKGROUND: The production process of reliable fruit extracts is not well established. OBJECTIVES: To improve the overall quality of apple extracts by reducing protein loss during the manufacturing process and to evaluate the improved extracts using in vivo and in vitro experiments. METHODS: Two types of extracts were prepared from peels of Golden Delicious apples (Malus domesticus). Extract A was extracted, 1:2 wt/vol, for 30 minutes at 40 degrees C in 0.01 M phosphate-buffered saline, and extract B was extracted, 1:2 wt/vol, in phosphate-buffered saline with 20% polyvinylpolypyrrolidone and 2-mmol/L EDTA. Both extracts were filtered, dialyzed in 3.5-kDa dialysis membranes, and lyophilized. The antigenic and allergenic profiles were analyzed using immunoblot and enzyme-linked immunosorbent assay. Nine patients clinically sensitive to apples and 12 controls underwent skin testing with both extracts. RESULTS: Extracts A and B had dry weight yields of 0.71% and 1.86% and protein contents of 104.6 and 257 microg/mg of freeze-dried material, respectively. A steady and progressive loss of protein, greater in extract A than in extract B, was detected at different intervals during the manufacturing process of both extracts. Extract B produced larger wheal sizes than extract A (P = .008). Enzyme-linked immunosorbent assay inhibition results confirmed that extract B had a greater inhibition capacity than extract A. CONCLUSIONS: A progressive loss of protein content occurs during the manufacturing of apple extracts. Wheal sizes induced by extract B were significantly larger than those induced by extract A and prick-by-prick solutions. Extract B was also more potent in vitro than extract A.

Prevalence of skin reactions to aeroallergens in asthmatics of Puerto Rico

In Puerto Rico, although a high prevalence of asthma has been reported, the sensitization rates to aeroallergens in these patients is unknown. The purpose of this study using a case control design, was to determine and compare the rates of sensitization to common aeroallergens in an asthmatic population of 576 asthmatics and 144 healthy controls. A skin prick test was conducted using standardized extracts of Dermatophagoides farinae (Df) and D. pteronyssinus (Dp), house dust (HD), cat hair and epithelium (CT), dog hair and dander (DG), grass pollen mix (PG), tree pollen mix (PT), weed pollen mix (PW), Aspergillus mix (AM), mold mixes A (MA) and B (MB), Periplaneta americana (PA) and Alternaria-Hormodendrum mix (AH). In addition, an extract from the domestic mite Blomia tropicalis (Bt) was also used. A wheal > or = 3 mm2 was considered a positive reaction. In addition, a standardized questionnaire was administered and a preliminary domestic mite identification survey was conducted. The analysis of the data showed that 85.8 of the asthmatics had at least one positive reaction and 61.6 of them had positive skin reactions to atleast one mite species. Asthmatics reacted to domestic mites 6.19 times more than the control group (p < 0.0001) and was the largest significant difference found in this study for any allergen tested. Preliminary identification of the acarologic fauna in southern Puerto Rico demonstrated that Blomia tropicalis, Dermatophagoides pteronyssinus, D. farinae and E.maynei are the dominant domestic mite species found in homes of asthmatic individuals. These results demonstrate that domestic mites are a very important source of sensitizing aeroallergens for asthmatic patients in Puerto Rico. Based upon the mite survey, Blomia tropicalis plays an important role in allergic sensitization, in addition to Dermatophagoides pteronyssinus and D. farinae. The skin prevalence to pollens and to molds may not reflect the true prevalence of sensitization to these allergens. Pollen identification and counts, and a survey of microflora of Puerto Rico are needed in order to identify and validate important allergens that eventually could be incorporated into a more appropria).te panel for testing sensitization in susceptible individuals