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BACKGROUND: Due to high IgE recognition frequency and high allergenic activity, Der p 5 and Der p 21 are clinically important house dust mite (HDM) allergens. The objective of this study was to characterize the immunodominant IgE epitopes of Der p 5 and Der p 21 responsible for their high allergenic activity. METHODS: A panel of 12 overlapping peptides spanning the Der p 5 and Der p 21 sequence were synthesized to search for sequential IgE epitopes by direct testing for allergic patients' IgE reactivity. Peptide-specific antibodies raised in rabbits were used in inhibition studies for localizing conformational IgE epitopes which were visualized on the surfaces of the allergen structures by molecular modelling. IgE cross-reactivity between the allergens was investigated by IgE inhibition studies. RESULTS: Immunodominant IgE epitopes defined by allergic patients' IgE on Der p 5 and Der p 21 were primarily of the conformational, discontinuous type including N- and C-terminal portions of the protein. They could be located on each allergen on one area with similar localization, but despite similar structure of the allergens, no relevant IgE cross-reactivity could be detected. CONCLUSION: Our study shows that Der p 5 and Der p 21 contain a major conformational IgE epitope-containing area located on similar portions of their structure, but they lack relevant IgE cross-reactivity. These data are important for the development of modern allergy vaccines based on defined molecules for allergen-specific immunotherapy of HDM allergy.
Assuntos
Alérgenos/imunologia , Antígenos de Dermatophagoides/química , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/química , Proteínas de Artrópodes/imunologia , Reações Cruzadas/imunologia , Epitopos/química , Imunoglobulina E/imunologia , Pyroglyphidae/imunologia , Animais , Proteínas de Artrópodes/síntese química , Descoberta de Drogas , Mapeamento de Epitopos , Epitopos/imunologia , Humanos , Imunização , Imunoglobulina E/sangue , Imunoglobulina G/imunologia , Modelos Moleculares , Conformação Molecular , Conformação Proteica em alfa-Hélice , Dobramento de Proteína , Coelhos , Vacinas SintéticasRESUMO
BACKGROUND: Recombinant hypoallergenic allergen derivatives have been used in clinical immunotherapy studies, and clinical efficacy seems to be related to the induction of blocking IgG antibodies recognizing the wild-type allergens. However, so far no treatment-induced IgG antibodies have been characterized. OBJECTIVE: To clone, express, and characterize IgG antibodies induced by vaccination with two hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1 in a nonallergic subject. METHODS: A phage-displayed combinatorial single-chain fragment (ScFv) library was constructed from blood of the immunized subject and screened for Bet v 1-reactive antibody fragments. ScFvs were tested for specificity and cross-reactivity to native Bet v 1 and related pollen and food allergens, and epitope mapping was performed. Germline ancestor genes of the antibody were analyzed with the ImMunoGeneTics (IMGT) database. The affinity to Bet v 1 and cross-reactive allergens was determined by surface plasmon resonance measurements. The ability to inhibit patients' IgE binding to ELISA plate-bound allergens and allergen-induced basophil activation was assessed. RESULTS: A combinatorial ScFv library was obtained from the vaccinated donor after three injections with the Bet v 1 fragments. Despite being almost in germline configuration, ScFv (clone H3-1) reacted with high affinity to native Bet v 1 and homologous allergens, inhibited allergic patients' polyclonal IgE binding to Bet v 1, and partially suppressed allergen-induced basophil activation. CONCLUSION: Immunization with unfolded hypoallergenic allergen derivatives induces high-affinity antibodies even in nonallergic subjects which recognize the folded wild-type allergens and inhibit polyclonal IgE binding of allergic patients.
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Especificidade de Anticorpos/imunologia , Antígenos de Plantas/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/isolamento & purificação , Alérgenos/imunologia , Sequência de Aminoácidos , Sequência de Bases , Basófilos/imunologia , Basófilos/metabolismo , Reações Cruzadas/imunologia , Epitopos/imunologia , Biblioteca Gênica , Humanos , Imunização , Imunoglobulina E/imunologia , Imunoglobulina G/química , Imunoglobulina G/genética , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Ressonância de Plasmônio de SuperfícieRESUMO
BACKGROUND: Companion animals are also affected by IgE-mediated allergies, but the eliciting molecules are largely unknown. We aimed at refining an allergen microarray to explore sensitization in horses and compare it to the human IgE reactivity profiles. METHODS: Custom-designed allergen microarray was produced on the basis of the ImmunoCAP ISAC technology containing 131 allergens. Sera from 51 horses derived from Europe or Japan were tested for specific IgE reactivity. The included horse patients were diagnosed for eczema due to insect bite hypersensitivity, chronic coughing, recurrent airway obstruction and urticaria or were clinically asymptomatic. RESULTS: Horses showed individual IgE-binding patterns irrespective of their health status, indicating sensitization. In contrast to European and Japanese human sensitization patterns, frequently recognized allergens were Aln g 1 from alder and Cyn d 1 from Bermuda grass, likely due to specific respiratory exposure around paddocks and near the ground. The most prevalent allergen for 72.5% of the tested horses (37/51) was the 2S-albumin Fag e 2 from buckwheat, which recently gained importance not only in human but also in horse diet. CONCLUSION: In line with the One Health concept, covering human health, animal health and environmental health, allergen microarrays provide novel information on the allergen sensitization patterns of the companion animals around us, which may form a basis for allergen-specific preventive and therapeutic concepts.
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Alérgenos/imunologia , Antígenos de Plantas/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , Fagopyrum/efeitos adversos , Animais , Mapeamento de Epitopos/métodos , Epitopos/genética , Feminino , Cavalos , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , MasculinoRESUMO
BACKGROUND: IgE reactivity to antigens from Gram-positive and Gram-negative bacteria is common in patients suffering from respiratory and skin manifestations of allergy, but the routes and mechanisms of sensitization are not fully understood. The analysis of the genome, transcriptome and microbiome of house dust mites (HDM) has shown that Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) species are abundant bacteria within the HDM microbiome. Therefore, our aim was to investigate whether HDM are carriers of bacterial antigens leading to IgE sensitization in patients suffering from atopic dermatitis. METHODS: Plasma samples from patients with AD (n = 179) were analysed for IgE reactivity to a comprehensive panel of microarrayed HDM allergen molecules and to S. aureus and E. coli by IgE immunoblotting. Antibodies specific for S. aureus and E. coli antigens were tested for reactivity to nitrocellulose-blotted extract from purified HDM bodies, and the IgE-reactive antigens were detected by IgE immunoblot inhibition experiments. IgE antibodies directed to bacterial antigens in HDM were quantified by IgE ImmunoCAP™ inhibition experiments. RESULTS: IgE reactivity to bacterial antigens was significantly more frequent in patients with AD sensitized to HDM than in AD patients without HDM sensitization. S. aureus and E. coli antigens were detected in immune-blotted HDM extract, and the presence of IgE-reactive antigens in HDM was demonstrated by qualitative and quantitative IgE inhibition experiments. CONCLUSION: House dust mites (HDM) may serve as carriers of bacteria responsible for the induction of IgE sensitization to microbial antigens.
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Alérgenos/imunologia , Antígenos de Bactérias/imunologia , Imunização , Imunoglobulina E/imunologia , Pyroglyphidae/imunologia , Animais , Anticorpos Antibacterianos , Dermatite Atópica/imunologia , Dermatite Atópica/metabolismo , Epitopos/imunologia , Escherichia coli/imunologia , Feminino , Humanos , Hipersensibilidade/epidemiologia , Hipersensibilidade/imunologia , Imunoglobulina G/imunologia , Masculino , Prevalência , Coelhos , Índice de Gravidade de Doença , Staphylococcus aureus/imunologia , Suécia/epidemiologiaRESUMO
BACKGROUND: IgG to galactose-α-1,3-galactose (α-gal) are highly abundant natural antibodies (Ab) in humans. α-Gal-specific IgE Ab cause a special form of meat allergy characterized by severe systemic reactions 3-7 h after consumption of red meat. We investigated 20 patients who experienced such reactions and characterized their α-gal-specific IgE and IgG responses in more detail. METHODS: α-Gal-specific IgE was determined by ImmunoCAP. IgE reactivity to meat extract and bovine gamma globulin (BGG) was assessed by immunoblotting and ELISA, respectively. In some experiments, sera were pre-incubated with α-gal or protein G to deplete IgG Ab. α-Gal-specific IgG1-4 Ab in individuals with and without meat allergy were assessed by ELISA. RESULTS: In immunoblots, BGG was the most frequently recognized meat protein. Binding of IgE and IgG to BGG was confirmed by ELISA and completely abolished after pre-incubation with α-gal. Neither the depletion of autologous α-gal-specific IgG Ab nor the addition of α-gal-specific IgG Ab from nonallergic individuals changed the IgE recognition of BGG of meat-allergic patients. Meat-allergic patients showed significantly higher α-gal-specific IgG1 and IgG3 Ab than nonallergic individuals, whereas the latter showed significantly higher levels of α-gal-specific IgG4 Ab. CONCLUSION: Patients with delayed meat allergy display IgE and IgG Ab that selectively recognize the α-gal epitope on BGG. Their enhanced α-gal-specific IgE levels are accompanied by high levels of α-gal-specific IgG1 devoid of IgE-blocking activity. This subclass distribution is atypical for food allergies and distinct from natural α-gal IgG responses in nonallergic individuals.
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Alérgenos/imunologia , Anticorpos/imunologia , Hipersensibilidade Alimentar/imunologia , Galactose/imunologia , Hipersensibilidade Tardia/imunologia , Carne Vermelha/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Especificidade de Anticorpos/imunologia , Feminino , Humanos , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
House dust mites, cats and dogs are amongst the most frequent sources of indoor allergens in Europe. The fact that the allergens of house dust mites cause allergic disease through inhalation of house dust was discovered in 1964. The diagnosis of mite allergy is regularly complicated by its often nonspecific symptoms, which frequently develop insidiously and by no means always include attacks of paroxysmal sneezing and itching. Antibody-based immunological detection methods can be used to measure exposure to mite allergens. The structure and function of more than 20 allergens from Dermatophagoides pteronyssinus and D. farina are known. Other relevant indoor allergens come from mammals kept in households. Here again, allergens have been described and diagnostic as well as exposure-measurement tools are available. It is important to remember indoor pests and other "unwelcome lodgers" as a possible cause in the case of unexplained symptoms experienced indoors. This short overview summarizes the current key points on the subject of "mites and other indoor allergens". The present article provides an overview of several articles published in a special issue of the German journal Allergologie [February 2015; 38(2)] on the subject of "Mites and other indoor allergens".
RESUMO
BACKGROUND: Wheat is one of the most common food allergen sources for children and adults. The aim of this study was to characterize new wheat allergens using an IgE discovery approach and to investigate their IgE epitopes. METHODS: A cDNA expression library representing the wheat transcriptome was constructed in phage lambda gt11 and screened with IgE antibodies from wheat food allergic patients. IgE-reactive cDNA clones coding for portions of high molecular weight (HMW) glutenin subunits were identified by sequence analysis of positive clones. IgE epitopes were characterized using recombinant fragments from the HMW Bx7 and synthetic peptides thereof for testing of allergic patients' sera and in basophil degranulation assays. RESULTS: We found that the major IgE-reactive areas of HMW glutenins are located in the repetitive regions of the protein and could show that two independent IgE-reactive fragments from HMW Bx7 contained repetitive IgE epitopes. CONCLUSIONS: Our results demonstrate that IgE antibodies from wheat food allergic patients can recognize repetitive epitopes in one of the important wheat food allergens. Recombinant HMW Bx7 may be included into the panel of allergens for component-resolved diagnosis of wheat food allergy.
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Alérgenos/imunologia , Epitopos de Linfócito B/imunologia , Glutens/química , Glutens/imunologia , Hipersensibilidade a Trigo/imunologia , Sequência de Aminoácidos , Teste de Degranulação de Basófilos , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina E/imunologia , Dados de Sequência Molecular , Proteínas Recombinantes/imunologiaRESUMO
BACKGROUND: Vaccines consisting of allergen-derived peptides lacking IgE reactivity and allergen-specific T cell epitopes bound to allergen-unrelated carrier molecules have been suggested as candidates for allergen-specific immunotherapy. OBJECTIVE: To study whether prophylactic and therapeutic vaccination with carrier-bound peptides from the major birch pollen allergen Bet v 1 lacking allergen-specific T cell epitopes has influence on Bet v 1-specific T cell responses. METHODS: Three Bet v 1-derived peptides, devoid of Bet v 1-specific T cell epitopes, were coupled to KLH and adsorbed to aluminium hydroxide to obtain a Bet v 1-specific allergy vaccine. Groups of BALB/c mice were immunized with the peptide vaccine before or after sensitization to Bet v 1. Bet v 1- and peptide-specific antibody responses were analysed by ELISA. T cell and cytokine responses to Bet v 1, KLH, and the peptides were studied in proliferation assays. The effects of peptide-specific and allergen-specific antibodies on T cell responses and allergic lung inflammation were studied using specific antibodies. RESULTS: Prophylactic and therapeutic vaccination with carrier-bound Bet v 1 peptides induced a Bet v 1-specific IgG antibody response without priming/boosting of Bet v 1-specific T cells. Prophylactic and therapeutic vaccination of mice with the peptide vaccine induced Bet v 1-specific antibodies which suppressed Bet v 1-specific T cell responses and allergic lung inflammation. CONCLUSION AND CLINICAL RELEVANCE: Vaccination with carrier-bound allergen-derived peptides lacking allergen-specific T cell epitopes induces allergen-specific IgG antibodies which suppress allergen-specific T cell responses and allergic lung inflammation.
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Anticorpos Neutralizantes/imunologia , Antígenos de Plantas/farmacologia , Betula , Epitopos de Linfócito T/farmacologia , Peptídeos/farmacologia , Rinite Alérgica Sazonal , Vacinação , Vacinas/farmacocinética , Adjuvantes Imunológicos/farmacologia , Hidróxido de Alumínio/farmacologia , Animais , Antígenos de Plantas/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/imunologia , Rinite Alérgica Sazonal/imunologia , Rinite Alérgica Sazonal/prevenção & controle , Vacinas/imunologiaRESUMO
BACKGROUND: It has been shown that birch pollen immunotherapy can induce IgG antibodies which enhance IgE binding to Bet v 1. We aimed to develop a serological assay to predict the development of antibodies which enhance IgE binding to Bet v 1 during immunotherapy. METHODS: In 18 patients treated by Bet v 1-fragment-specific immunotherapy, the effects of IgG antibodies specific for the fragments on the binding of IgE antibodies to Bet v 1 were measured by ELISA. Blocking and possible enhancing effects on IgE binding were compared with skin sensitivity to Bet v 1 after treatment. RESULTS: We found that fragment-specific IgG enhanced IgE binding to Bet v 1 in two patients who also showed an increase of skin sensitivity to Bet v 1. CONCLUSION: Our results indicate that it may be possible to develop serological tests which predict the induction of unfavourable IgG antibodies enhancing the binding of IgE to Bet v 1 during immunotherapy.
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Alérgenos/imunologia , Antígenos de Plantas/imunologia , Betula/efeitos adversos , Dessensibilização Imunológica , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Adulto , Especificidade de Anticorpos/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Rinite Alérgica Sazonal/diagnóstico , Rinite Alérgica Sazonal/imunologia , Rinite Alérgica Sazonal/terapia , Adulto JovemRESUMO
BACKGROUND: The production of allergen-specific IgE antibodies is a hallmark of IgE-mediated allergy but the contribution of blood cells to allergen-specific IgE production in allergic patients has not been studied in detail. OBJECTIVE: Aim of this study was the characterization of IgE-producing cells in the blood of allergic patients and the determination of the amount of IgE antibodies which are produced by these cells in relation to total amounts of circulating specific IgE. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from allergic patients and cell populations were purified or depleted using magnetically labelled antibodies directed against specific cell surface markers (CD19, CD20, CD22, CD27, CD38, CD126, CD138, CD203c). Allergen-specific IgE was measured in serum samples and cell culture supernatants by quantitative ImmunoCAP measurements and by ELISA using purified recombinant allergens. IgE transcripts were detected using RT-PCR with primers specific for human IgE. RESULTS: We found that allergen-specific IgE levels in PBMC supernatants correlated strongly with specific serum IgE but represented less than 1% of circulating IgE. Depletion of basophils resulted in substantial reduction of allergen-specific IgE levels in PBMC culture supernatants suggesting that an important source of allergen-specific IgE in PBMC supernatants could be IgE derived from the surface of basophils. Newly synthesized IgE was derived from CD138+ plasma cells, but not from B and B memory cells, and accounted for only approximately 0.2% of circulating IgE in blood. CONCLUSION AND CLINICAL RELEVANCE: Our finding that the majority of allergen-specific IgE in the peripheral blood is not derived from IgE-secreting cells in the blood suggests local IgE production in tissues as a major source for allergen-specific IgE and possible target for therapeutic intervention.
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Alérgenos/imunologia , Especificidade de Anticorpos , Linfócitos B/imunologia , Hipersensibilidade Imediata/imunologia , Imunoglobulina E/biossíntese , Plasmócitos/imunologia , Células Cultivadas , Citometria de Fluxo , Humanos , Imunoglobulina E/sangue , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Sindecana-1/metabolismoRESUMO
BACKGROUND: Wheat (Triticum aestivum) is an important allergen source responsible for various clinical manifestations of allergy (i.e. food allergy, pollen allergy, respiratory allergy to flour-Baker's asthma). OBJECTIVE: The objective of this study was the molecular and immunological characterization of new recombinant wheat allergens and to evaluate their usefulness for the diagnosis of allergy to wheat. METHODS: A T. aestivum cDNA library was constructed and screened with serum IgE from patients suffering from wheat allergy to identify cDNAs coding for new wheat allergens. The allergen-encoding cDNAs were expressed in Escherichia coli and purified to homogeneity. IgE reactivity of recombinant proteins was analysed in RAST-based, non-denaturing dot blot experiments and by ELISA with sera from wheat allergic patients and their allergenic activity was assessed in basophil degranulation experiments. RESULTS: We report the molecular characterization, recombinant expression and purification of five wheat allergens, a thioredoxin h isoform, glutathione transferase, 1-Cys-peroxiredoxin, profilin and dehydrin. Homologous proteins were identified by sequence comparisons in various plants. 1-Cys-peroxiredoxin appeared to be the most relevant of the newly identified wheat allergens according to prevalence of IgE recognition and results from basophil degranulation experiments. It showed IgE cross-reactivity with seed proteins from barley, rye, rice, maize, soy, oat and spelt. 1-Cys-peroxiredoxin, glutathione transferase and dehydrin were mainly recognized by patients with baker's asthma but not wheat-induced food allergy. CONCLUSION AND CLINICAL RELEVANCE: The characterized recombinant wheat allergens may be useful for the development of serological tests which allow the discrimination of different clinical manifestations of wheat allergy.
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Alérgenos/química , Alérgenos/isolamento & purificação , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Hipersensibilidade a Trigo/imunologia , Adulto , Alérgenos/imunologia , Sequência de Aminoácidos , Asma/imunologia , Degranulação Celular/imunologia , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Immunoblotting , Imunoglobulina E , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas de Plantas/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
BACKGROUND: More than 90% of house dust mite-allergic patients are sensitized to the major Dermatophagoides pteronyssinus allergen, Der p 2. The aim of this study was to develop and characterize an allergy vaccine based on carrier-bound Der p 2 peptides, which should allow reducing IgE- and T-cell-mediated side-effects during specific immunotherapy (SIT). METHODS: Five Der p 2 peptides (P1-P5) were synthesized and analyzed regarding IgE reactivity and allergenic activity. Lymphoproliferative and cytokine responses induced with Der p 2 and Der p 2 peptides were determined in peripheral blood mononuclear cells from mite-allergic patients. Der p 2-specific IgG antibodies induced with carrier-bound Der p 2 peptides in mice and rabbits were tested for their capacity to inhibit IgE binding and basophil activation in allergic patients. RESULTS: Of five overlapping peptides (P1-P5) covering the Der p 2 sequence, two peptides (P2 and P4) were identified, which showed no relevant IgE reactivity, allergenic activity, and induced lower Der p 2-specific T-cell activation than Der p 2. However, when coupled to a carrier, P2 and P4 induced Der p 2-specific IgG antibodies in animals, which inhibited allergic patients' IgE binding to the allergen and allergen-induced basophil activation similar as antibodies induced with Der p 2. CONCLUSIONS: Carrier-bound Der p 2 peptides should allow avoiding IgE-mediated side-effects, and because of their low potential to activate allergen-specific T cells, they may reduce late-phase side-effects during SIT. Further, these peptides may be also useful for prophylactic vaccination.
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Alérgenos/imunologia , Anticorpos Bloqueadores/imunologia , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/imunologia , Basófilos/imunologia , Hipersensibilidade/imunologia , Imunoglobulina G/imunologia , Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos/imunologia , Antígenos de Dermatophagoides/química , Proteínas de Artrópodes/química , Basófilos/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Hemocianinas/química , Humanos , Imunoglobulina E/imunologia , Imunoglobulina E/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peptídeos/química , Diester Fosfórico Hidrolases/metabolismo , Ligação Proteica/imunologia , Pyroglyphidae/imunologia , Pirofosfatases/antagonistas & inibidores , Pirofosfatases/metabolismo , Coelhos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Vacinas Conjugadas/imunologiaRESUMO
BACKGROUND: Tropomyosins represent clinically relevant seafood allergens but the role of mite tropomyosin, Der p 10, in house dust mite (HDM) allergy has not been studied in detail. OBJECTIVE: To express and purify a recombinant Der p 10 with equivalent IgE reactivity as natural Der p 10 and to evaluate its IgE reactivity and allergenic activity in HDM-allergic patients. METHODS: rDer p 10 was expressed in Escherichia coli, purified and characterized by mass spectrometry and circular dichroism. It was tested for IgE reactivity in 1322 HDM-allergic patients. Detailed IgE-reactivity profiles to six HDM allergens (Der p 1, 2, 5, 7, 10, 21) were established for subgroups of Der p 10-positive and -negative patients. The allergenic activity of rDer p 10 was evaluated in basophil degranulation experiments. RESULTS: rDer p 10 is an α-helical protein sharing IgE epitopes with nDer p 10. It is recognized by 15.2% of HDM-allergic patients. Der p 10-negative patients were primarily sensitized to Der p 1 and/or Der p 2, whereas Der p 10-positive patients reacted to several other HDM allergens besides the major allergens (Der p 1, Der p 2) or showed a rather selective Der p 10 reactivity. The allergenic activity of Der p 10 was generally low but patients could be identified who suffered from clinically relevant HDM allergy due to Der p 10 sensitization. CONCLUSION AND CLINICAL RELEVANCE: Der p 10 may be a diagnostic marker for HDM-allergic patients with additional sensitization to allergens other than Der p 1 and Der p 2. Such patients may require attention when allergen-specific immunotherapy is considered.
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Antígenos de Dermatophagoides/genética , Proteínas de Artrópodes/genética , Hipersensibilidade Imediata/diagnóstico , Tropomiosina/genética , Adolescente , Adulto , Idoso , Animais , Antígenos de Dermatophagoides/química , Proteínas de Artrópodes/química , Criança , Dicroísmo Circular , Dermatophagoides pteronyssinus/imunologia , Dermatophagoides pteronyssinus/metabolismo , Dessensibilização Imunológica/métodos , Poeira/imunologia , Feminino , Humanos , Hipersensibilidade Imediata/imunologia , Imunoglobulina E/sangue , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Tropomiosina/química , Adulto JovemRESUMO
BACKGROUND: Allergen-specific subcutaneous immunotherapy (SCIT) is an antigen-specific therapy of IgE-mediated allergies. In the present study, we analyze the epitope specificities of antibody responses induced by SCIT with allergen extracts from pollen of trees belonging to the order Fagales (birch, alder, hazel) adsorbed onto aluminum hydroxide. METHODS: The IgE, IgG1-4 and IgA responses to defined recombinant allergens (birch pollen: Bet v 1; alder pollen: Aln g 1; hazel pollen: Cor a 1; apple: Mal d 1) as well as to Bet v 1-derived recombinant fragments and synthetic peptides were analyzed in sera from patients who had undergone SCIT for different periods of time. RESULTS: Long-term SCIT (>1 year; cumulative dose >1,000,000 SQ units) induced more pronounced IgG1, IgG2 and IgG4 responses to Bet v 1 and Bet v 1-related allergens according to the degree of sequence homology (Bet v 1>Aln g 1>Cor a 1>Mal d 1) than short-term SCIT (<1 year; cumulative dose <1,000,000 SQ units). In contrast to patients treated for <1 year, patients treated for >1 year mounted distinct IgG1, IgG2 and IgG4 responses against sequential Bet v 1 epitopes. No relevant allergen-specific IgA or IgG3 responses were induced by short- or long-term SCIT. Using a competitive ELISA assay, it could be shown that serum IgG from patients undergoing long-term SCIT inhibited IgE reactivity to Bet v 1 better than IgG from patients undergoing short-term SCIT. CONCLUSION: SCIT with allergen extracts adsorbed onto aluminum hydroxide induces IgG responses against new epitopes that block IgE binding and cross-react with structurally related allergens depending, among other factors, on duration of treatment and cumulative injected dose.
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Alérgenos/administração & dosagem , Proteínas Recombinantes/administração & dosagem , Rinite Alérgica Sazonal/terapia , Adjuvantes Imunológicos/farmacologia , Adolescente , Adulto , Alérgenos/imunologia , Hidróxido de Alumínio/farmacologia , Dessensibilização Imunológica , Mapeamento de Epitopos , Feminino , Humanos , Imunoglobulina A/sangue , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Injeções Subcutâneas , Masculino , Pessoa de Meia-Idade , Pólen/imunologia , Proteínas Recombinantes/imunologia , Rinite Alérgica Sazonal/sangue , Rinite Alérgica Sazonal/imunologia , Adulto JovemRESUMO
BACKGROUND: The association between cigarette smoke exposure and allergic airway disease is a matter for debate. We sought to investigate in an in vitro system whether active smoking reduces the integrity and barrier function of the respiratory epithelium and thus facilitates allergen penetration. METHODS: We cultured the human bronchial epithelial cell line 16HBE14o- in a transwell culture system as a surrogate for the intact respiratory epithelium. The cell monolayer was exposed to standardized cigarette smoke extract (CSE). The extent and effects of trans-epithelial allergen penetration were measured using 125I-labelled purified major respiratory allergens (rBet v 1, rPhl p 5 and rDer p 2) and histamine release experiments. RESULTS: Exposure of cells to concentrations of CSE similar to those found in smokers induced the development of para-cellular gaps and a decrease in trans-epithelial resistance. CSE exposure induced a more than threefold increase in allergen penetration. Increased subepithelial allergen concentrations provoked a substantial augmentation of histamine release from sensitized basophils. CONCLUSIONS: Our results indicate that cigarette smoke is a potent factor capable of reducing the barrier function of the respiratory epithelium for allergens and may contribute to increased allergic inflammation, exacerbation of allergic disease and boosting of IgE memory.
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Alérgenos/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Nicotiana/efeitos adversos , Mucosa Respiratória/metabolismo , Alérgenos/imunologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/imunologia , Citocinas , Citometria de Fluxo , Liberação de Histamina/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Mucosa Respiratória/imunologia , FumaçaRESUMO
BACKGROUND: House dust mites (HDM) Dermatophagoides pteronyssinus are a frequent indoor allergen source. Our aim was to determine the frequencies of IgE reactivity to purified HDM allergen molecules in mite allergic patients from different parts of Europe in order to establish an allergen panel for diagnosis of HDM allergy. MATERIALS AND METHODS: Populations of D. pteronyssinus-allergic patients from Austria (n = 56), France (n = 55), Italy (n = 67) and Sweden (n = 65) and storage mite allergic patients from Sweden (n = 31) were analysed for IgE reactivity to eight purified natural (n) and recombinant (r) D. pteronyssinus allergens (nDer p 1, rDer p 2, nDer p 4, rDer p 5, rDer p 7, rDer p 8, rDer p 10 and rDer p 14) in RAST-based dot blot assays. RESULTS: Using a combination of Der p 1 and Der p 2, at least 97% of the D. pteronyssinus-allergic patients could be diagnosed in each of the HDM allergic populations. However, more than 50% of the patients also reacted with other allergens and significant variabilities regarding the frequencies of IgE reactivity to individual allergen molecules were found. Patients with a predominant storage mite allergy showed none or only very weak IgE reactivity to purified D. pteronyssinus allergens. CONCLUSIONS: Purified Der p 1 and Der p 2 are sufficient for the diagnosis of > or = 97% of D. pteronyssinus allergic patients in Europe, but other allergens may also play an important role for the diagnosis and treatment of HDM allergy.
Assuntos
Alérgenos/imunologia , Antígenos de Dermatophagoides/imunologia , Dermatophagoides pteronyssinus/imunologia , Hipersensibilidade Imediata/imunologia , Imunoglobulina E/metabolismo , Animais , Europa (Continente)/epidemiologia , Humanos , Hipersensibilidade Imediata/epidemiologiaRESUMO
BACKGROUND: The house dust mite (HDM) Dermatophagoides pteronyssinus is a major allergen source eliciting allergic asthma. The aim of the study was to identify new important HDM allergens associated with allergic asthma. METHODS: A cDNA coding for a new mite allergen, designated Der p 21, was isolated using immunoglobulin E (IgE) antibodies from patients with allergic asthma out of a D. pteronyssinus expression cDNA library and expressed in Escherichia coli. RESULTS: Circular dichroism analysis of the purified allergen showed that rDer p 21 (14 726 Da) is one of the few mite allergens with an alpha-helical secondary structure. The protein exhibited high thermal stability and refolding capacity, and, as determined by small angle X-ray scattering, formed a dimer consisting of two flat triangles. rDer p 21 bound high levels of patients' IgE antibodies and showed high allergenic activity in basophil activation experiments. Rabbit anti-Der p 21 IgG antibodies inhibited mite-allergic patients' IgE binding and allowed the ultrastructural localization of the allergen in the midgut (epithelium, lumen and faeces) of D. pteronyssinus by immunogold electron microscopy. Der p 21 revealed sequence homology with group 5 mite allergens, but IgE and IgG reactivity data and cross-inhibition studies identified it as a new mite allergen. CONCLUSIONS: Der p 21 is a new important mite allergen which is liberated into the environment via faecal particles and hence may be associated with allergic asthma.
Assuntos
Alérgenos/química , Alérgenos/imunologia , Antígenos de Dermatophagoides/química , Antígenos de Dermatophagoides/imunologia , Asma/imunologia , Dermatophagoides pteronyssinus/imunologia , Alérgenos/genética , Alérgenos/isolamento & purificação , Sequência de Aminoácidos , Animais , Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/isolamento & purificação , Sequência de Bases , Basófilos/imunologia , Dicroísmo Circular , DNA Complementar , Dermatophagoides pteronyssinus/ultraestrutura , Poeira/imunologia , Células Epiteliais/imunologia , Células Epiteliais/ultraestrutura , Humanos , Imunoglobulina E/imunologia , Intestinos/imunologia , Intestinos/ultraestrutura , Microscopia Imunoeletrônica , Dados de Sequência MolecularRESUMO
IgE-dependent activation of basophils is associated with upregulation of several surface molecules. We recently identified the surface enzyme aminopeptidase N (CD13) as a novel activation antigen on human basophils. In the present study, we asked whether CD13 can be employed as a novel marker of allergen-induced activation of basophils in allergic individuals. Patients allergic to major allergens from grass pollen (Phl p 1, Phl p 5), birch pollen (Bet v 1), or house dust mites (Der p 2), were examined. Blood basophils were exposed to various concentrations of recombinant allergens for 15 minutes, and examined for expression of CD13 by multicolor flow cytometry. The allergen-induced upregulation of CD13 was compared with allergen-dependent increases in expression of CD63 and CD203c. Exposure to recombinant allergens resulted in an increase in expression of CD13 on basophils in all sensitized individuals, whereas no increase in CD13 was seen in healthy controls. The effects of the recombinant allergens on CD13-expression were dose- and time-dependent, were not observed in the absence of extracellular calcium, and were counteracted by preincubation of basophils with the PI3-kinase-targeting drugs staurosporin and LY294002. There was a good correlation between allergen-induced upregulation of CD13, CD63, and CD203c on basophils. In aggregate, our data show that recombinant allergens promote expression of CD13 on basophils in sensitized individuals. The functional significance and diagnostic implications of this observation remain to be determined.
Assuntos
Alérgenos/imunologia , Basófilos/imunologia , Antígenos CD13/sangue , Hipersensibilidade/imunologia , Adolescente , Adulto , Antígenos CD/sangue , Basófilos/enzimologia , Basófilos/fisiologia , Cálcio/fisiologia , Feminino , Humanos , Interleucina-3/farmacologia , Masculino , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinases/fisiologia , Diester Fosfórico Hidrolases/sangue , Glicoproteínas da Membrana de Plaquetas , Pirofosfatases/sangue , Proteínas Recombinantes/imunologia , Tetraspanina 30RESUMO
Type I allergy represents an important health problem that affects more than 25% of the population in industrialized countries. Specific immunotherapy is one of the few causative treatment approaches for type I allergy and is currently performed with crude allergen extracts, which consist of a mixture of allergenic and nonallergenic components, are difficult to standardize and cannot be applied according to the patient's reactivity profile. With the introduction of molecular biological techniques into allergy research, a large panel of individual recombinant allergens has become available. Recombinant allergens can be used for improved diagnosis of allergy to determine the patient's sensitization profile, which is a prerequisite to select the allergens for patient-tailored immunotherapy. They allow the elucidation of the properties of allergens and of the mechanisms of allergy as well as of the mechanisms of immunotherapy. Moreover, recombinant allergens allow the development of hypoallergenic allergen derivatives with reduced allergenic activity and retained immunogenicity. First immunotherapy trials with hypoallergenic allergen derivatives have shown that this treatment might improve immunotherapy in the near future. This review summarizes the results, which were obtained with recombinant allergens and hypoallergenic allergen derivatives. The experiences from the in vitro and in vivo evaluation of the hypoallergenic derivatives and from clinical studies as well as the contribution of hypoallergenic derivatives to develop new treatment strategies and possibly prophylactic vaccination strategies are discussed.
Assuntos
Alérgenos , Hipersensibilidade/diagnóstico , Hipersensibilidade/terapia , Imunoterapia/métodos , Proteínas Recombinantes , Alérgenos/genética , Antialérgicos/uso terapêutico , Ensaios Clínicos como Assunto , Terapia Combinada , Feminino , Previsões , Humanos , Hipersensibilidade/genética , Masculino , Prognóstico , Proteínas Recombinantes/genética , Medição de Risco , Testes Cutâneos/métodos , Resultado do TratamentoRESUMO
BACKGROUND: House dust mites (HDMs) represent significant indoor allergen sources for patients with atopic dermatitis (AD). Subcutaneous allergen-specific immunotherapy (SCIT) has been shown to be successful in patients with allergic rhinitis and mild asthma and might represent an attractive therapeutic option for the long-term treatment of HDM sensitizations in AD patients. However, only a few studies have been conducted on the effectiveness of HDM SCIT in AD, resulting in controversial clinical results. Data on immunological changes induced by SCIT in AD patients are rare. OBJECTIVES: We performed an open pilot study to assess clinical changes and objective laboratory parameters and evaluate the benefit of HDM SCIT in 25 AD patients with IgE-mediated sensitization against HDM. METHODS: The severity of AD was evaluated by the severity scoring of atopic dermatitis system (SCORAD). Specific IgE and IgG4 against HDM and serum levels of TARC/CCL17, MDC/CCL22, IL-16, IL-4, IFN-gamma, IL-10 and TGF-beta1 were measured during SCIT. RESULTS: Subjective and objective SCORAD improved significantly within only 4 weeks of treatment. The level of the tolerogenic cytokine IL-10 increased, whereas CCL17 and IL-16 decreased in the sera of the patients during SCIT. Allergen specific IgE decreased, while IgG4 increased during SCIT. CONCLUSION: In this open-label pilot study, SCIT with an HDM extract in patients with AD led to a significant improvement of AD mirrored by a reduction of SCORAD as well as serological and immunological changes, which might serve as valuable parameters to estimate the therapeutic effect of SCIT.