Isolation, Characterization and IgE Binding of Two 2S Albumins of Pomegranate Seeds.

Literature reports suggest that the presence of proteins in pomegranate seeds is responsible for sensitization and IgE-mediated allergic reactions. The objective of this study was the analysis of a pomegranate seed extract and the isolation and characterization of proteins contained in high amounts. The extract characterization showed a protein profile with main bands at about 18 kDa and below 10 kDa upon SDS-PAGE, and molecules were recognized by specific IgEs upon immunoblotting. Then, two new 2S albumins, a monomeric and a heterodimeric one, were isolated by using classical biochemical methods. They were identified via direct protein sequencing and mass spectrometry, and their primary structure was analyzed and compared with homologous allergenic proteins via bioinformatics. In an Italian population of 703 suspected allergic patients, analyzed by using the FABER® test, the frequency of sensitization to the monomeric and heterodimeric 2S albumins was 1.7% and 0.28%, respectively. This study reports for the first time the isolation and characterization of two 2S albumins from pomegranate seeds. The clinical relevance of these molecules needs further investigation, for instance in populations having different exposures and allergy profiles.

Plant and Arthropod IgE-Binding Papain-like Cysteine Proteases: Multiple Contributions to Allergenicity.

Papain-like cysteine proteases are widespread and can be detected in all domains of life. They share structural and enzymatic properties with the group's namesake member, papain. They show a broad range of protein substrates and are involved in several biological processes. These proteases are widely exploited for food, pharmaceutical, chemical and cosmetic biotechnological applications. However, some of them are known to cause allergic reactions. In this context, the objective of this review is to report an overview of some general properties of papain-like cysteine proteases and to highlight their contributions to allergy reactions observed in humans. For instance, the literature shows that their proteolytic activity can cause an increase in tissue permeability, which favours the crossing of allergens through the skin, intestinal and respiratory barriers. The observation that allergy to PLCPs is mostly detected for inhaled proteins is in line with the reports describing mite homologs, such as Der p 1 and Der f 1, as major allergens showing a frequent correlation between sensitisation and clinical allergic reactions. In contrast, the plant food homologs are often digested in the gastrointestinal tract. Therefore, they only rarely can cause allergic reactions in humans. Accordingly, they are reported mainly as a cause of occupational diseases.

Comparative Analysis of the Immune Response and the Clinical Allergic Reaction to Papain-like Cysteine Proteases from Fig, Kiwifruit, Papaya, Pineapple and Mites in an Italian Population.

Several plant papain-like cysteine proteases are exploited by the food, cosmetic, pharmaceutical and textile industries. However, some of these enzymes can cause allergic reactions. In this context, we investigated the frequency of sensitization and allergic reactions to some fruit and/or latex cysteine proteases, which are used as additives by the food industry to improve and modify the quality of their products. The FABER test was used to analyse the patients' sensitization towards five plants and, for comparison, two homologous mite cysteine proteases. In an Italian population of 341 allergic patients, 133 (39%) had IgE specific for at least one of the seven cysteine proteases under investigation. Most of the patients were IgE positive for Der p 1 and/or Der f 1 (96.38%) reported a clinical history suggestive of respiratory allergy to mites, whereas none of the subjects sensitized to the homologs from papaya, pineapple and fig reported allergy symptoms following ingestion of these foods. Only one patient referred symptoms from ingesting kiwifruit. Therefore, the obtained results showed that sensitization to the fruit enzymes was only rarely concomitant with allergic reactions. These observations, together with the literature reports, suggest that the allergy to plant papain-like cysteine proteases might mainly be an occupational disease.

Detection of Allergenic Proteins in Foodstuffs: Advantages of the Innovative Multiplex Allergen Microarray-Based Immunoassay Compared to Conventional Methods.

Several factors can affect the allergen content and profile of a specific food, including processing procedures often leading to a decrease in allergenicity, although no change, or even an increase, have also been reported. Evaluation of the effectiveness of a processing procedure requires the availability of reliable methodologies to assess the variation in molecules able to induce allergic reactions in the analyzed food. Conventional and innovative strategies and methodologies can be exploited to identify allergenic proteins in foodstuffs. However, depending on the specific purposes, different methods can be used. In this review, we have critically reviewed the advantages of an innovative method, the multiplex allergen microarray-based immunoassay, in the detection of allergens in foodstuffs. In particular, we have analyzed some studies reporting the exploitation of an IgE-binding inhibition assay on multiplex allergen biochips, which has not yet been reviewed in the available literature. Unlike the others, this methodology enables the identification of many allergenic proteins, some of which are still unknown, which are recognized by IgE from allergic patients, with a single test. The examined literature suggests that the inhibition test associated with the multiplex allergen immunoassay is a promising methodology exploitable for the detection of IgE-binding proteins in food samples.

Pattern of sensitization to Juniperus oxycedrus 4EF-hand polcalcin, Jun o 4, compared with the 2EF-hand grass homolog Phl p 7 in a general Italian population of subjects suffering from pollinosis.

Cupressaceae pollen causes allergic reactions worldwide with long-lasting symptomatic periods. Currently, no cypress polcalcin is available for diagnostic purposes. With the aim to investigate the pattern of sensitization to a cypress polcalcin, a synthetic gene of Jun o 4, the Juniperus oxycedrus 4EF-hand polcalcin, was cloned and expressed in Escherichia coli. Its features were investigated in comparison with the grass 2EF-hand Phl p 7. Rhinitis was the symptom most frequently reported in a cohort of Italian patients sensitized to rJun o 4 and/or rPhl p 7. The detection of many pollen allergic patients sensitized to the cypress polcalcin, but negative to Phl p 7, indicates that Phl p 7 cannot be further considered a marker of sensitization towards all the polcalcins. A 4EF-hand cypress polcalcin claims the inclusion in allergy diagnostic tests. In addition, the sensitivity of polcalcins to gastrointestinal digestion is reported and discussed for the first time.

When the Frequencies of Sensitization and Elicitation of Allergic Reaction Do Not Correlate-The Case of Apple Gibberellin-Regulated Protein Tested in an Italian Population.

Background: The literature reports describing allergic symptoms against apples in the patients sensitized to the gibberellin-regulated proteins (GRPs) suggested the presence of an allergenic GRP in this fruit. Objective: This study aimed to assess the presence of a GRP protein in apples and investigate its allergenicity. Methods: The protein was isolated and identified by the classical biochemical methods. The bioinformatics tools were used for similar searches and molecular modeling. The immunological features were investigated using the multiplex FABER test. Clinical data were collected by the allergy specialists. Results: A GRP was detected in the apple peel and pulp and it was named applemaclein. This protein displays 94% of sequence identity with peamaclein, Pru p 7, representing the prototype of this allergen family. The applemaclein molecular model shows a very irregular surface with grooves/clefts that may potentially accommodate small molecular ligands. In a population of 4,721 patients in Italy, 187 (4.0%) were sensitized to any allergenic GPR. Of those, 115 (61.5%), 61 (32.6%), 30 (16.0%), and 99 (52.9%) had immunoglobulin E (IgE) to apple, peach, pomegranate, and cypress GRP, respectively. However, in a cohort of the patients in Italy, most individuals IgE positive to the apple GRP did not report allergic reactions against this fruit. Conclusion: Compared with the peach Pru p 7, applemaclein shows some different structural features and higher sensitization frequency, which is often not associated with allergic reactions against apple. Further studies are needed to understand a possible correlation between the applemaclein structural properties, the interaction with still unknown molecules, and immunological behavior.

Formulations for Allergen Immunotherapy in Human and Veterinary Patients: New Candidates on the Horizon.

Allergen immunotherapy is currently the only causal treatment for allergic diseases in human beings and animals. It aims to re-direct the immune system into a tolerogenic or desensitized state. Requirements include clinical efficacy, safety, and schedules optimizing patient or owner compliance. To achieve these goals, specific allergens can be formulated with adjuvants that prolong tissue deposition and support uptake by antigen presenting cells, and/or provide a beneficial immunomodulatory action. Here, we depict adjuvant formulations being investigated for human and veterinary allergen immunotherapy.

Molecular approach to a patient's tailored diagnosis of the oral allergy syndrome.

Oral allergy syndrome (OAS) is one of the most common IgE-mediated allergic reactions. It is characterized by a number of symptoms induced by the exposure of the oral and pharyngeal mucosa to allergenic proteins belonging to class 1 or to class 2 food allergens. OAS occurring when patients sensitized to pollens are exposed to some fresh plant foods has been called pollen food allergy syndrome (PFAS). In the wake of PFAS, several different associations of allergenic sources have been progressively proposed and called syndromes. Molecular allergology has shown that these associations are based on IgE co-recognition taking place between homologous allergens present in different allergenic sources. In addition, the molecular approach reveals that some allergens involved in OAS are also responsible for systemic reactions, as in the case of some food Bet v 1-related proteins, lipid transfer proteins and gibberellin regulated proteins. Therefore, in the presence of a convincing history of OAS, it becomes crucial to perform a patient's tailored molecule-based diagnosis in order to identify the individual IgE sensitization profile. This information allows the prediction of possible cross-reactions with homologous molecules contained in other sources. In addition, it allows the assessment of the risk of developing more severe symptoms on the basis of the features of the allergenic proteins to which the patient is sensitized. In this context, we aimed to provide an overview of the features of relevant plant allergenic molecules and their involvement in the clinical onset of OAS. The value of a personalized molecule-based approach to OAS diagnosis is also analyzed and discussed.

Boiling down the cysteine-stabilized LTP fold - loss of structural and immunological integrity of allergenic Art v 3 and Pru p 3 as a consequence of irreversible lanthionine formation.

BACKGROUND: Non-specific lipid transfer proteins (LTPs) are important allergens in fruits, pollen, vegetables, nuts and latex. Due to their compact structure, LTPs are highly resistant to heat treatment. Here, Art v 3 from mugwort pollen and Pru p 3 from peach were used as model allergens to in-depth investigate structural and immunological properties upon thermal treatment at different buffer conditions. METHODS: Recombinant Art v 3 and Pru p 3 were purified from E. coli and incubated at 95 °C up to 120 min using sodium phosphate buffer pH 3.4 or 7.3. Physicochemical properties of allergens were analyzed in circular dichroism spectroscopy, Fourier transform infrared spectroscopy, dynamic light scattering, size exclusion chromatography, and mass spectrometry. The crystal structure of Art v 3.0201 was determined to 1.9 Šresolution. IgG and IgE binding was investigated in ELISA using murine and LTP allergic patients' sera. RESULTS: Highly pure and homogenous recombinant allergens were obtained from bacterial production. The crystal structure of Art v 3.0201 revealed an antiparallel four helix bundle with a C-terminal extension mediating an asymmetric, transient dimer interface and differently sized cavities. Both allergens showed high thermal stability at acidic conditions. In contrast, extensive heat treatment in neutral buffer induced irreversible structural changes due to lanthionine-based cysteine rearrangement. This fostered loss of the typical α-helical structure, increased molecular size and abrogation of IgG and IgE binding epitopes. Pru p 3 lost its structural integrity at shorter heat stress duration than Art v 3, which did however only partially affect the molecule's IgE binding epitopes. CONCLUSION: During thermal treatment, susceptibility to structural changes of the LTP-fold is highly dependent on the surrounding environment but also on intrinsic features of individual LTPs. This is a crucial fact to consider when processing LTP-containing food or food products as this will directly influence their allergenic potential.

Isolation of cypress gibberellin-regulated protein: Analysis of its structural features and IgE binding competition with homologous allergens.

The presence in cypress pollen of an important allergen, belonging to the gibberellin-regulated protein (GRP) family, has been suggested for many years. However, it has never been isolated and sometimes the homologous peach allergen, Pru p 7, has been used as a surrogate to perform immunological investigations. The aim of this study has been the isolation and molecular characterization of the GRP contained in the Cupressus sempervirens pollen. This protein, named Cypmaclein, has been purified from the natural source using conventional biochemical methods consisting in different chromatographic separations. Cypmaclein has been identified by direct protein sequencing of the N-terminal region and of internal fragments of the molecule. In SDS-PAGE, its apparent molecular mass is slightly higher than that of Pru p 7. Nevertheless, the mass spectrometry experiments reveal that the exact molecular mass of Cypmaclein (6821.88 Da) is very close to that of Pru p 7 (6909.90 Da). Two regions of Cypmaclein have been sequenced providing 50% of its primary structure. A high overall sequence identity of Cypmaclein with all the analyzed GRP has been observed, although in the N-terminal region the high identity is limited to the homolog of Cryptomeria japonica. In circular dichroism experiments Cypmaclein produced a spectrum overlapping that of Pru p 7. However, the comparative analysis of Cypmaclein, Pru p 7 and Pun g 7 IgE reactivity revealed a behavior that was not completely overlapping, thus suggesting that the IgE epitopes are only partially shared. In single point highest inhibition achievable assays performed with the FABER test, Cypmaclein efficiently competed with the allergenic peach and pomegranate GRP in the binding of specific IgE of patients sensitized to Pru p 7. In conclusion, the natural cypress pollen GRP has been isolated for the first time, its structural features have been investigated and its cross-reactivity with Pru p 7 and Pun g 7 has been demonstrated. This protein is now available for further investigations aimed at understanding its clinical relevance in the allergy to cypress pollen. In addition, the prevalence of sensitization directly to Cypmaclein, and not limited to the homologs, can be defined.

ENEA, a peach and apricot IgE-binding protein cross-reacting with the latex major allergen Hev b 5.

Peach and apricot can cause allergic reactions with symptoms ranging from mild to very severe, including anaphylaxis. Sometimes subjects allergic to fruits of the Prunus genus have been reported to be also allergic to rubber latex products. The objective of this study is the characterization of a newly identified peach and apricot protein showing similarities with the allergens Hev b 5 from rubber latex and Man e 5 from manioc. This protein has been named ENEA on the basis of the single letter amino acid code of the first four N-terminal residues of the isolated molecule. It has been found in very variable amounts in different peach cultivars and batches. ENEA was isolated from peach pulp extracts by chromatographic separations and identified by direct protein sequencing. At that time, the full length sequence was available only for the homologous protein of the taxonomically closely related apricot, which was produced as a recombinant molecule in Escherichia coli. The following availability of the full length sequence of peach ENEA revealed a very high identity (97%) with the apricot homolog. Similarly to Hev b 5 and to Man e 5, the structural characterization indicated that ENEA is an intrinsically disordered protein. The immunological properties, investigated by dot blotting, the ABA system and the FABER test, showed that ENEA is recognized by specific IgE of allergic patients. In a selected population of 31 patients reporting allergic reactions to peach fruit and/or IgE positive to Hev b 5, 28 and 27 subjects resulted co-sensitized to rENEA and Hev b 5 in the ABA and ISAC test, respectively. In a random population of 3305 suspected allergic patients, analyzed with the FABER test, 17 of them were sensitized to rENEA and 10 of them were also positive to Hev b 5. In addition, both the natural molecule from peach and the recombinant protein of apricot partially inhibited the IgE binding to Hev b 5. In conclusion, a new peach and apricot IgE-binding protein, cross-reacting with the major latex allergen Hev b 5, has been identified. Its variable concentration in the fruit might explain some occasionally occurring allergic reactions. The apricot molecule has recently been registered by the WHO/IUIS Allergen Nomenclature Sub-Committee with the allergen name Pru ar 5. The recombinant form of apricot ENEA, now available, will contribute to allergy diagnosis.

Pomegranate chitinase III: Identification of a new allergen and analysis of sensitization patterns to chitinases.

Allergy to pomegranate is often associated with severe symptoms. Two allergens have previously been described: 9k-LTP Pun g 1 and pommaclein Pun g 7. This study describes the isolation of a chitinase III, identified by direct protein sequencing and mass spectrometry. It is a 29-kDa protein showing 69% sequence identity with the latex hevamine and IgE binding in dot blotting, immunoblotting and FABER®test. Chitinase-specific IgE were detected in 69 of 357 patients sensitized to one or more pomegranate allergenic preparations present on the FABER®test. Using this test, 19.2% of the patients sensitized to kiwifruit chitinase IV were also sensitized to pomegranate chitinase III, rather than to latex chitinase I (7.2%) with which it shares the N-terminal hevein-like domain. In conclusion, a new allergen has been identified, contributing to improving food allergy diagnosis. This study reveals the important role of chitinases III and IV as allergy sensitizers and prompts further investigations.

Pomegranate Cultivars: Identification of the New IgE-Binding Protein Pommaclein and Analysis of Antioxidant Variability.

The consumption of pomegranate is increasing as it is considered a health-promoting food. Nevertheless, it can trigger allergic reactions, sometimes severe. The LTP Pun g 1 is the only pomegranate allergen so far reported. Based on preliminary clinical observations, the main aim of this study was the investigation of still unknown allergens contained in this fruit. Pommaclein, a homologue of peamaclein, the peach allergen Pru p 7, was isolated, identified by protein sequencing, and characterized as an IgE-binding protein by different test systems. RP-HPLC protein profiles revealed significant variations of LTP and pommaclein content in the red pulp of selected cultivars and accessions. Conversely, the mesocarp appeared free of proteins and much richer in antioxidants. In conclusion, a new allergen has been identified, and it could contribute to improving allergy diagnosis. The study highlights that pomegranate mesocarp could represent a rich and safe source of nutraceuticals also for allergic subjects.

Structural features, IgE binding and preliminary clinical findings of the 7kDa Lipid Transfer Protein from tomato seeds.

Allergic reactions caused by 9kDa Lipid Transfer Proteins (9k-LTP), such as Pru p 3, have been widely investigated, whereas a possible contribution of components of 7kDa LTP (7k-LTP) sub-family in triggering allergic symptoms has been overlooked so far. With the aim to investigate the contribution of 7k-LTPs to the food allergies, we have identified, isolated and characterised a tomato seed 7k-LTP (Sola l 7k-LTP). The protein was purified by chromatographic separations, identified by direct protein sequencing and mass spectrometry and a molecular model was built. Functional evaluation of the allergen has been performed by skin testing. Sola l 7k-LTP consists of 68 amino acids producing a molecular mass of 7045Da and displays 41% sequence identity with Pru p 3, the allergenic 9k-LTP from peach. IgE antibodies specifically recognising Sola l 7k-LTP were found within the population claiming tomato ingestion-related symptoms, but also in subjects tolerant on tomato exposure. A few subjects were mono-sensitised to Sola l 7k-LTP, which is biologically active as shown by the positive skin test. In line with the immunological results, the molecular model shows structural similarities between the IgE binding regions of the two sub-families. Therefore, Sola l 7k-LTP shares some structural and immunological features with Pru p 3, but it also displays individual features that could be responsible for mono-specific IgE binding. In conclusion, Sola l 7k-LTP is a new identified allergenic LTP, the description of which may contribute to the improvement of allergy diagnosis and to the formulation of a safe and personalised diet. In addition, to avoid current confusing classifications, a new nomenclature policy for LTP sub-families is proposed in this paper. We now suggest that 7-kDa LTP (so far named LTP2) be renamed 7k-LTP and 9-kDa LTP (so far named LTP1) be renamed 9k-LTP.

Structure, stability, and IgE binding of the peach allergen Peamaclein (Pru p 7).

Knowledge of the structural properties of allergenic proteins is a necessary prerequisite to better understand the molecular bases of their action, and also to design targeted structural/functional modifications. Peamaclein is a recently identified 7 kDa peach allergen that has been associated with severe allergic reactions in sensitive subjects. This protein represents the first component of a new allergen family, which has no 3D structure available yet. Here, we report the first experimental data on the 3D-structure of Peamaclein. Almost 75% of the backbone resonances, including two helical stretches in the N-terminal region, and four out of six cysteine pairs have been assigned by 2D-NMR using a natural protein sample. Simulated gastrointestinal digestion experiments have highlighted that Peamaclein is even more resistant to digestion than the peach major allergen Pru p 3. Only the heat-denatured protein becomes sensitive to intestinal proteases. Similar to Pru p 3, Peamaclein keeps its native 3D-structure up to 90°C, but it becomes unfolded at temperatures of 100-120°C. Heat denaturation affects the immunological properties of both peach allergens, which lose at least partially their IgE-binding epitopes. In conclusion, the data collected in this study provide a first set of information on the molecular properties of Peamaclein. Future studies could lead to the possible use of the denatured form of this protein as a vaccine, and of the inclusion of cooked peach in the diet of subjects allergic to Peamaclein.

Biological therapy in a pediatric Crohn disease population at a referral center.

OBJECTIVE: The antitumor necrosis factor α (TNFα) antibodies infliximab and adalimumab are effective in inducing and maintaining remission in pediatric patients with Crohn disease (CD). The aim of the study was to evaluate the long-term efficacy and safety of biological therapy in pediatric patients with CD followed at a referral center. METHODS: This work is a retrospective observational study enrolling patients with CD treated with infliximab or adalimumab beyond the induction protocol. The patients' data were collected from the unit's IBD database (maximum follow-up evaluation after 36 months of treatment). The efficacy was evaluated by the Pediatric Crohn Disease Activity Index score and by analysis of the cumulative probability of continuing therapy; the safety was assessed in terms of adverse events. RESULTS: We enrolled 78 patients; the mean therapy duration was 27.2 ± 16.7 months, and the mean age at enrollment was 15 ± 3.1 years. The Kaplan-Meier analysis showed a cumulative probability of continuing therapy of 81%, 54%, and 33% at 1, 2, and 3 years, respectively, from the introduction of therapy. No association between the patients' baseline characteristics and the long-term outcome was found. The evaluation of the concomitant therapy with immunomodulators and anti-TNFα therapy versus anti-TNFα alone did not show a different outcome. No serious adverse events were recorded. CONCLUSIONS: The study indicates that biological therapy is effective and safe in pediatric patients with CD in a longer follow-up period. The response to treatment was not influenced by the patients' baseline characteristics or by the immunomodulator association.

Tolerability of a fully maturated cheese in cow's milk allergic children: biochemical, immunochemical, and clinical aspects.

BACKGROUND: From patients' reports and our preliminary observations, a fully maturated cheese (Parmigiano-Reggiano; PR) seems to be well tolerated by a subset of cow's milk (CM) allergic patients. OBJECTIVE AND METHODS: To biochemically and immunologically characterize PR samples at different maturation stage and to verify PR tolerability in CM allergic children. Seventy patients, with suspected CM allergy, were enrolled. IgE to CM, α-lactalbumin (ALA), ß-lactoglobulin (BLG) and caseins (CAS) were tested using ImmunoCAP, ISAC103 and skin prick test. Patients underwent a double-blind, placebo-controlled food challenge with CM, and an open food challenge with 36 months-maturated PR. Extracts obtained from PR samples were biochemically analyzed in order to determine protein and peptide contents. Pepsin and trypsin-chymotrypsin-pepsin simulated digestions were applied to PR extracts. Each PR extract was investigated by IgE Single Point Highest Inhibition Achievable assay (SPHIAa). The efficiency analysis was carried out using CM and PR oral challenges as gold standards. RESULTS: The IgE binding to milk allergens was 100% inhibited by almost all PR preparations; the only difference was for CAS, mainly α(S1)-CAS. Sixteen patients sensitized to CM tolerated both CM and PR; 29 patients tolerated PR only; 21 patients, reacted to both CM and PR, whereas 4 patients reactive to CM refused to ingest PR. ROC analysis showed that the absence of IgE to BLG measured by ISAC could be a good marker of PR tolerance. The SPHIAa using digested PR preparations showed a marked effect on IgE binding to CAS and almost none on ALA and BLG. CONCLUSIONS: 58% of patients clinically reactive to CM tolerated fully maturated PR. The preliminary digestion of CAS induced by PR maturation process, facilitating a further loss of allergenic reactivity during gut digestion, might explain the tolerance. This hypothesis seems to work when no IgE sensitization to ISAC BLG is detected.

Varying allergen composition and content affects the in vivo allergenic activity of commercial Dermatophagoides pteronyssinus extracts.

BACKGROUND: Diagnosis and immunotherapy of house-dust mite (HDM) allergy is still based on natural allergen extracts. The aim of this study was to analyze commercially available Dermatophagoides pteronyssinus extracts from different manufacturers regarding allergen composition and content and whether variations may affect their allergenic activity. METHODS: Antibodies specific for several D. pteronyssinus allergens (Der p 1, 2, 5, 7, 10 and 21) were used to analyze extracts from 10 different manufacturers by immunoblotting. Sandwich ELISAs were used to quantify Der p 1 and Der p 2 in the extracts. Mite-allergic patients (n = 45) were skin-tested with the extracts and tested for immunoglobulin E (IgE) reactivity to a panel of 10 mite allergens (Der p 1, 2, 4, 5, 7, 8, 10, 14, 20 and 21) by dot blot. RESULTS: Only Der p 1 and Der p 2 were detected in all extracts but their concentrations and ratios showed high variability (Der p 1: 6.0-40.8 µg ml(-1); Der p 2: 1.7-45.0 µg ml(-1)). At least 1 out of 4 allergens (i.e. Der p 5, 7, 10 and 21) was not detected in 8 of the studied extracts. Mite-allergic subjects showed different IgE reactivity profiles to the individual mite allergens, the extracts showed different allergenic activity in skin-prick tests and false-negative results. CONCLUSIONS: Commercially available D. pteronyssinus extracts lack important allergens, show great variability regarding allergen composition and content and some gave false-negative diagnostic test results in certain patients.