RESUMO
The cross-linking of effector cell-bound IgE antibodies by allergens induces the release of inflammatory mediators which are responsible for the symptoms of allergy. We demonstrate that a recombinant hybrid molecule consisting of the major birch (Bet v 1) and grass (Phl p 5) pollen allergen exhibited reduced allergenic activity as compared to equimolar mixes of the isolated allergens in basophil activation experiments. The reduced allergenic activity of the hybrid was not due to reduced IgE reactivity as demonstrated by IgE binding experiments using sera from allergic patients. Physicochemical characterization of the hybrid by size exclusion chromatography, dynamic light scattering, negative-stain electron microscopy and circular dichroism showed that the hybrid occurred as folded aggregate whereas the isolated allergens were folded monomeric proteins. IgG antibodies raised in rabbits against epitopes of Bet v 1 and Phl p 5 showed reduced reactivity with the hybrid compared to the monomeric allergens. Our results thus demonstrate that aggregation can induce changes in the conformation of allergens and lead to the reduction of allergenic activity. This is a new mechanism for reducing the allergenic activity of allergens which may be important for modifying allergens to exhibit reduced side effects when used for allergen-specific immunotherapy.
Assuntos
Alérgenos/imunologia , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Proteínas Recombinantes/imunologia , Animais , Reações Cruzadas/imunologia , Dessensibilização Imunológica/métodos , Epitopos/imunologia , Humanos , Proteínas de Plantas/imunologia , Pólen/imunologia , Coelhos , Ratos , Rinite Alérgica Sazonal/imunologiaRESUMO
BACKGROUND: The calcium-binding 2EF-hand protein Phl p 7 from timothy grass pollen is a highly cross-reactive pollen pan-allergen that can induce severe clinical symptoms in allergic patients. Recently, a human monoclonal Phl p 7-specific IgG4 antibody (mAb102.1F10) was isolated from a patient who had received grass pollen-specific immunotherapy (SIT). METHODS: We studied epitope specificity, cross-reactivity, affinity and cross-protection of mAb102.1F10 towards homologous calcium-binding pollen allergens. Sequence comparisons and molecular modelling studies were performed with ClustalW and SPADE, respectively. Surface plasmon resonance measurements were made with purified recombinant allergens. Binding and cross-reactivity of patients' IgE and mAb102.1F10 to calcium-binding allergens and peptides thereof were studied with quantitative RAST-based methods, in ELISA, basophil activation and IgE-facilitated allergen presentation experiments. RESULTS: Allergens from timothy grass (Phl p 7), alder (Aln g 4), birch (Bet v 4), turnip rape (Bra r 1), lamb's quarter (Che a 3) and olive (Ole e 3, Ole e 8) showed high sequence similarity and cross-reacted with allergic patients' IgE. mAb102.1F10 bound the C-terminal portion of Phl p 7 in a calcium-dependent manner. It cross-reacted with high affinity with Ole e 3, whereas binding and affinity to the other allergens were low. mAb102.1F10 showed limited cross-inhibition of patients' IgE binding and basophil activation. Sequence comparison and surface exposure calculations identified three amino acids likely to be responsible for limited cross-reactivity. CONCLUSIONS: Our results demonstrate that a small number of amino acid differences among cross-reactive allergens can reduce the affinity of binding by a SIT-induced IgG and thus limit cross-protection.
Assuntos
Alérgenos/imunologia , Reações Cruzadas/imunologia , Epitopos/imunologia , Imunoglobulina G/imunologia , Imunoterapia , Pólen/imunologia , Alérgenos/química , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos/imunologia , Antígenos de Plantas , Cálcio/metabolismo , Epitopos/química , Humanos , Imunoglobulina E/imunologia , Modelos Moleculares , Peptídeos/imunologia , Ligação Proteica/imunologia , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologiaRESUMO
BACKGROUND: We have identified a monoclonal anti-human immunoglobulin E (IgE) antibody, which recognizes FcepsilonRI-bound IgE and prevents binding of IgE to FcepsilonRI. In this study, we assessed the binding kinetics and affinity of monoclonal antibody 12 (mAb12) for IgE and investigated whether mAb12 can be used for depletion of IgE and isolation of IgE-bearing cells from peripheral blood. METHODS: Binding kinetics and affinity for IgE were studied using Biacore surface plasmon resonance technique experiments. IgE antibodies were depleted from serum using sepharose-coupled mAb12 and IgE-bearing cells were enriched from heparinized blood samples with mAb12. The extent and biological relevance of IgE depletion were studied by quantitative IgE measurements and basophil histamine release experiments. Specific binding of mAb12 to IgE-bearing cells (basophils, mast cells, IgE-secreting plasma cells) was demonstrated by FACS. RESULTS: Monoclonal antibody 12 shows rapid association (k(a) = 5.46e5/Ms) with IgE, almost no dissociation (k(d) = 8.8e-5/s) and an affinity for IgE (K(D) = 1.61e-10 M), which is as high as that of FcepsilonRI. Immobilized mAb12 could be used to deplete IgE antibodies and isolate IgE-bearing cells from peripheral blood in a single-step procedure. CONCLUSIONS: Monoclonal antibody 12 is a high affinity anti-human IgE antibody, which efficiently removes IgE and IgE-bearing cells from peripheral blood and may thus be used for extracorporeal depletion of IgE and IgE-bearing cells.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Imunoglobulina E/imunologia , Receptores de IgE/imunologia , Rinite Alérgica Sazonal/imunologia , Adulto , Alérgenos/imunologia , Anticorpos Monoclonais/metabolismo , Basófilos/imunologia , Basófilos/metabolismo , Feminino , Liberação de Histamina , Humanos , Imunoglobulina E/sangue , Masculino , Mastócitos/imunologia , Mastócitos/metabolismo , Pessoa de Meia-Idade , Plasmócitos/imunologia , Plasmócitos/metabolismo , Pólen/imunologia , Receptores de IgE/metabolismoRESUMO
Transfusion-related morbidity is an emerging challenge in chronically transfused patients with low-risk myelodysplastic syndromes (MDS). In these patients, transfusion-induced iron overload may represent a leading medical problem. However, although iron-chelating drugs are available, little is known about optimal diagnostic tools, predisposing factors, and the optimal management of these patients. In the current article, we provide recommendations for the diagnosis, prevention and treatment of iron overload in MDS and propose treatment response criteria. Consensus criteria and resulting recommendations were discussed and formulated by members of the MDS platform of the Austrian Society of Haematology and Oncology in a series of meetings and conferences in 2006 and 2007. These recommendations should facilitate and assist in recognition of iron overload, selection of patients, timing of treatment, drug selection and the measurement of treatment responses.
Assuntos
Terapia por Quelação/métodos , Transfusão de Eritrócitos/efeitos adversos , Quelantes de Ferro/uso terapêutico , Sobrecarga de Ferro/terapia , Síndromes Mielodisplásicas/terapia , Ferritinas/sangue , Guias como Assunto , Humanos , Sobrecarga de Ferro/fisiopatologia , Sobrecarga de Ferro/prevenção & controle , Síndromes Mielodisplásicas/complicaçõesRESUMO
We recently identified the ectoenzyme CD203c as a novel basophil activation antigen that is upregulated in response to FcepsilonRI cross-linkage. We investigated the effects of various interleukins (ILs) on expression of CD203c on blood basophils using an antibody against CD203c and flow cytometry. Of all cytokines tested, only IL-3 was found to upregulate expression of CD203c on basophils above baseline levels. The effects of IL-3 were dose- and time-dependent (EC(50): 0.1-1 ng/ml) without differences observed between healthy and allergic donors. Whereas anti-IgE induced maximum upregulation of CD203c within 15 minutes, the IL-3-induced upregulation showed a maximum after 180 minutes. IgE-receptor cross-linking resulted in enhanced expression of both CD63 and CD203c, whereas IL-3 enhanced the levels of CD203c without promoting expression of CD63. The IL-3-induced upregulation of CD203c was also observed in highly enriched basophils and was counteracted by a blocking antibody against the alpha chain of the IL-3 receptor (CD123). The IL-3-induced upregulation of CD203c was also found to depend on the presence of calcium. To analyze signaling pathways involved in IL-3-induced upregulation of CD203c, pharmacologic inhibitors were applied. The PI3-kinase inhibitors, wortmannin and LY294002 counteracted the IL-3-induced expression of CD203c, whereas MEK- and PKC inhibitors showed no effects. In conclusion, IL-3 upregulates expression of CD203c on basophils through a specific receptor and via a PI3-kinase-dependent signaling-pathway. Compared to FcepsilonRI-mediated cell activation, IL-3-induced upregulation of CD203c is a late(r) event and is not accompanied by upregulation of CD63.
Assuntos
Basófilos/imunologia , Basófilos/metabolismo , Betula/imunologia , Interleucina-3/fisiologia , Diester Fosfórico Hidrolases/genética , Pólen/imunologia , Pirofosfatases/genética , Rinite Alérgica Sazonal/imunologia , Antígenos CD/biossíntese , Antígenos CD/genética , Células Cultivadas , Regulação da Expressão Gênica/fisiologia , Humanos , Diester Fosfórico Hidrolases/biossíntese , Glicoproteínas da Membrana de Plaquetas/biossíntese , Glicoproteínas da Membrana de Plaquetas/genética , Pirofosfatases/biossíntese , Tetraspanina 30RESUMO
BACKGROUND: Almost 100 million allergic patients are sensitized to the major birch pollen allergen, Bet v 1, a 17 kDa protein containing most of the IgE epitopes present in pollens of trees belonging to the Fagales order and plant-derived food. OBJECTIVE: Our aim was to develop an approach for the rational design of B cell epitope-derived, non-allergenic peptide allergy vaccines. METHODS: According to the three-dimensional (3-D) structure of birch pollen allergen, Bet v 1, six peptides comprising 25-32 preferably solvent-exposed amino acids were synthesized. RESULTS: Because of lack of secondary structure, the peptides showed no allergenic activity in allergic patients. In a mouse model of birch pollen allergy, peptide vaccination induced Bet v 1-specific IgG and prevented IgE-mediated allergic sensitization to Bet v 1. The protective role of peptide-induced blocking antibodies is demonstrated by inhibition of allergic patients IgE binding to the allergen and by blocking of allergen-induced basophil degranulation. CONCLUSION: Our results indicate the mechanistic importance of blocking antibodies for allergy vaccination and present a B cell epitope-based approach for the rational design of safe peptide allergy vaccines whenever the structure of the disease-eliciting allergen is known.
Assuntos
Alérgenos/imunologia , Betula/imunologia , Hipersensibilidade Imediata/prevenção & controle , Pólen/imunologia , Vacinas/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Plantas , Linfócitos B/imunologia , Basófilos/imunologia , Dessensibilização Imunológica/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/imunologia , Liberação de Histamina/imunologia , Humanos , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Camundongos , Peptídeos/imunologia , Estrutura Terciária de Proteína , Coelhos , Ratos , Testes Cutâneos/métodosRESUMO
IgE-mediated allergy affects >25% of the population in industrialized countries. Repeated contact with the disease-eliciting allergens induces rises of allergen-specific IgE Abs and progression of the disease to more severe manifestations. Our study uses a type of vaccine that is based on genetically modified allergen derivatives to treat allergic patients. We developed hypoallergenic derivatives of the major birch pollen allergen, Bet v 1, by genetic engineering and vaccinated birch pollen-allergic patients (n = 124) in a double-blind, placebo-controlled study. Active treatment induced protective IgG Abs that inhibited allergen-induced release of inflammatory mediators. We also observed a reduction of cutaneous sensitivity as well as an improvement of symptoms in actively treated patients. Most important, rises of allergen-specific IgE induced by seasonal birch pollen exposure were significantly reduced in vaccinated patients. Vaccination with genetically engineered allergen derivatives is a therapy for allergy that not only ameliorates allergic reactions but also reduces the IgE production underlying the disease.
Assuntos
Alérgenos/genética , Rinite Alérgica Sazonal/terapia , Vacinas/genética , Vacinas/uso terapêutico , Betula/genética , Betula/imunologia , Reações Cruzadas , Método Duplo-Cego , Alimentos , Humanos , Hipersensibilidade Imediata/prevenção & controle , Imunoglobulina E/biossíntese , Imunoglobulina G/biossíntese , Memória Imunológica , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Pólen/genética , Pólen/imunologia , Engenharia de Proteínas , Rinite Alérgica Sazonal/imunologia , Rinite Alérgica Sazonal/prevenção & controle , Estações do Ano , Vacinas Sintéticas/genética , Vacinas Sintéticas/uso terapêuticoRESUMO
BACKGROUND: Allergen-specific immunotherapy represents a causal form of treatment for IgE-mediated allergies. The allergen extract-based analyses of immunotherapy-induced effects yielded highly controversial results regarding a beneficial role of therapy-induced IgG antibodies. OBJECTIVE: We analysed allergen-specific IgE, IgG subclass, and IgM responses in patients treated with a grass pollen allergy vaccine adjuvanted with monophosphoryl lipid A (MPL), a Th1-inducing agent, and in a placebo group using recombinant timothy grass pollen allergen molecules (rPhl p 1, rPhl p 2, rPhl p 5). RESULTS: The strong induction of allergen-specific IgG1 and IgG4 antibodies observed only in the actively treated group was associated with significant clinical improvement. Therapy-induced allergen-specific IgM and IgG2 responses were also noted in several actively treated patients. An inhibition of allergen-dependent basophil histamine release was only obtained with sera containing therapy-induced allergen-specific IgG, but not with sera obtained before therapy or from placebo-treated patients. Moreover, patients with therapy-induced allergen-specific IgG antibodies showed a reduced induction of allergen-specific IgE responses during seasonal grass pollen exposure. CONCLUSION: Successful immunotherapy with the MPL-adjuvanted grass pollen allergy vaccine is associated with the production of allergen-specific IgG antibodies. These blocking antibodies may have protective effects by inhibiting immediate-type reactions and systemic increases of IgE responses caused by seasonal allergen exposure.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Alérgenos/imunologia , Imunoglobulina E/biossíntese , Imunoterapia/métodos , Lipídeo A/análogos & derivados , Lipídeo A/uso terapêutico , Basófilos/imunologia , Método Duplo-Cego , Ensaio de Imunoadsorção Enzimática/métodos , Liberação de Histamina/imunologia , Humanos , Hipersensibilidade Imediata/imunologia , Hipersensibilidade Imediata/prevenção & controle , Imunoglobulina E/imunologia , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Imunoglobulina M/biossíntese , Imunoglobulina M/imunologia , Lipídeo A/imunologia , Phleum/imunologia , Pólen/imunologia , Estações do Ano , Vacinas/uso terapêuticoRESUMO
BACKGROUND: Allergy to grass pollen is typically associated with serum IgE antibodies to group 1 and/or group 5 allergens, and additionally often to one or several less prominent allergens. Most of the grass pollen allergens identified to date have been characterized in detail by molecular, biochemical and immunological methods, timothy grass being one of the most thoroughly studied species. However, a 20-kDa allergen frequently recognized by IgE antibodies from grass pollen allergics has so far escaped cloning and molecular characterization. OBJECTIVE: To clone and characterize the 20 kDa timothy grass pollen allergen Phl p 11. METHODS: Phl p 11 cDNA was cloned by PCR techniques, utilizing N-terminal amino acid sequence obtained from the natural allergen. Phl p 11 was expressed as a soluble fusion protein in Escherichia coli, purified to homogeneity and used for serological analysis and to study Phl p 11 specific induction of histamine release from basophils and skin reactivity in sensitized and control subjects. RESULTS: Phl p 11 cDNA defined an acidic polypeptide of 15.8 kDa with homology to pollen proteins from a variety of plant species and to soybean trypsin inhibitor. The sequence contained one potential site for N-linked glycosylation. Serological analysis revealed that recombinant Phl p 11 shared epitopes for human IgE antibodies with the natural protein and bound serum IgE from 32% of grass pollen-sensitized subjects (n = 184). Purified recombinant Phl p 11 elicited skin reactions and dose-dependent histamine release from basophils of sensitized subjects, but not in non-allergic controls. CONCLUSION: As the first representative of group 11 grass pollen allergens, Phl p 11 has been cloned and produced as a recombinant protein showing allergenic activity. One-third of grass pollen-sensitized subjects showed specific IgE reactivity to recombinant Phl p 11, corresponding in magnitude to a significant proportion of specific IgE to grass pollen extract.
Assuntos
Proteínas de Ciclo Celular/genética , Phleum/imunologia , Pólen , Sequência de Aminoácidos , Reações Antígeno-Anticorpo , Sequência de Bases , Proteínas de Ciclo Celular/imunologia , Eletroforese em Gel de Poliacrilamida , Escherichia coli/imunologia , Granulócitos/imunologia , Liberação de Histamina , Humanos , Hipersensibilidade Imediata/imunologia , Immunoblotting , Imunoglobulina E/imunologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes/imunologia , Análise de Sequência de DNA , Testes CutâneosRESUMO
Worldwide more than 200 million individuals are allergic to group 1 grass pollen allergens. We have used the major timothy grass pollen allergen Phl p 1, which cross-reacts with most grass-, corn-, and monocot-derived group 1 allergens to develop a generally applicable strategy for the production of hypoallergenic allergy vaccines. On the basis of the experimentally determined B cell epitopes of Phl p 1, we have synthesized five synthetic peptides. These peptides are derived from the major Phl p 1 IgE epitopes and were between 28-32 amino acids long. We demonstrate by nuclear magnetic resonance that the peptides exhibit no secondary and tertiary structure and accordingly failed to bind IgE antibodies from grass pollen allergic patients. The five peptides, as well as an equimolar mixture thereof, lacked allergenic activity as demonstrated by basophil histamine release and skin test experiments in grass pollen allergic patients. When used as immunogens in mice and rabbits, the peptides induced protective IgG antibodies, which recognized the complete Phl p 1 wild-type allergen and group 1 allergens from other grass species. Moreover, peptide-induced antibodies inhibited the binding of grass pollen allergic patients IgE antibodies to the wild-type allergen. We thus demonstrate that synthetic hypoallergenic peptides derived from B cell epitopes of major allergens represent safe vaccine candidates for the treatment of IgE- mediated allergies.
Assuntos
Alérgenos/imunologia , Epitopos de Linfócito B/imunologia , Hipersensibilidade/prevenção & controle , Peptídeos/imunologia , Proteínas de Plantas/imunologia , Poaceae/imunologia , Pólen/imunologia , Alérgenos/química , Anafilaxia , Animais , Epitopos de Linfócito B/química , Humanos , Imunoglobulina E/imunologia , Camundongos , Peptídeos/síntese química , Peptídeos/química , Proteínas de Plantas/síntese química , Proteínas de Plantas/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Coelhos , VacinaçãoRESUMO
Almost 90% of grass pollen-allergic patients are sensitized against group 5 grass pollen allergens. We isolated a monoclonal human IgE Fab out of a combinatorial library prepared from lymphocytes of a grass pollen-allergic patient and studied its interaction with group 5 allergens. The IgE Fab cross-reacted with group 5A isoallergens from several grass and corn species. By allergen gene fragmentation we mapped the binding site of the IgE Fab to a 11.2-kDa N-terminal fragment of the major timothy grass pollen allergen Phl p 5A. The IgE Fab-defined Phl p 5A fragment was expressed in Escherichia coli and purified to homogeneity. Circular dichroism analysis revealed that the rPhl p 5A domain, as well as complete rPhl p 5A, assumed a folded conformation consisting predominantly of an alpha helical secondary structure, and exhibited a remarkable refolding capacity. It reacted with serum IgE from 76% of grass pollen-allergic patients and revealed an extremely high allergenic activity in basophil histamine release as well as skin test experiments. Thus, the rPhl p 5A domain represents an important allergen domain containing several IgE epitopes in a configuration optimal for efficient effector cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation and, perhaps later, for the development of generally applicable allergen-specific therapy strategies.
Assuntos
Alérgenos/química , Anticorpos Monoclonais/química , Epitopos/química , Imunoglobulina E/química , Fragmentos Fab das Imunoglobulinas/química , Proteínas de Plantas/química , Poaceae/imunologia , Pólen/química , Alérgenos/imunologia , Alérgenos/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos/genética , Basófilos/metabolismo , Sítios de Ligação de Anticorpos/genética , Sítios de Ligação de Anticorpos/imunologia , Dicroísmo Circular , Reações Cruzadas , Mapeamento de Epitopos , Epitopos/imunologia , Epitopos/metabolismo , Liberação de Histamina/imunologia , Humanos , Hipersensibilidade Imediata/imunologia , Imunoglobulina E/genética , Imunoglobulina E/metabolismo , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/isolamento & purificação , Mapeamento de Peptídeos , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Proteínas de Plantas/isolamento & purificação , Poaceae/química , Pólen/imunologia , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Zea mays/química , Zea mays/imunologiaRESUMO
Due to the wide distribution and heavy pollen production of grasses, approximately 50% of allergic patients are sensitized against grass pollen allergens. cDNAs coding for two isoforms and four fragments of a major timothy grass (Phleum pratense) pollen allergen, Phl p 6, were isolated by IgE immunoscreening from a pollen expression cDNA library. Recombinant Phl p 6 (rPhl p 6), an acidic protein of 11.8 kDa, was purified to homogeneity as assessed by mass spectrometry and exhibited almost exclusive alpha-helical secondary structure as determined by circular dichroism spectroscopy. Phl p 6 reacted with serum IgE from 75% of grass pollen-allergic patients (n = 171). IgE binding experiments with rPhl p 6 fragments indicated that the N terminus of the allergen is required for IgE recognition. Purified rPhl p 6 elicited dose-dependent basophil histamine release and immediate type skin reactions in patients allergic to grass pollen. A rabbit antiserum raised against purified rPhl p 6 identified it as a pollen-specific protein that, by immunogold electron microscopy, was localized on the polysaccharide-containing wall-precursor bodies (P-particles). The association of Phl p 6 with P-particles may facilitate its intrusion into the deeper airways and thus be responsible for the high prevalence of IgE recognition of Phl p 6. Recombinant native-like Phl p 6 can be used for in vitro as well as in vivo diagnoses of grass pollen allergy, whereas N-terminal deletion mutants with reduced IgE binding capacity may represent candidates for immunotherapy of grass pollen allergy with a low risk of anaphylactic side effects.
Assuntos
Poluentes Atmosféricos/química , Alérgenos/imunologia , Alérgenos/isolamento & purificação , Proteínas de Plantas/imunologia , Proteínas de Plantas/isolamento & purificação , Pólen/química , Pólen/imunologia , Poluentes Atmosféricos/imunologia , Alérgenos/genética , Alérgenos/ultraestrutura , Sequência de Aminoácidos , Sítios de Ligação de Anticorpos , DNA Complementar/química , DNA Complementar/isolamento & purificação , Relação Dose-Resposta Imunológica , Epitopos/imunologia , Liberação de Histamina , Humanos , Hipersensibilidade Imediata/imunologia , Imunoglobulina E/metabolismo , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/ultraestrutura , Poaceae , Pólen/ultraestrutura , Conformação Proteica , Dobramento de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Group 1 grass pollen allergens are recognized by IgE antibodies of almost 40% of allergic individuals and therefore belong to the most important elicitors of Type I allergy worldwide. We have previously isolated the cDNA coding for the group 1 allergen from timothy grass, Phl p 1, and demonstrated that recombinant Phl p 1 contains most of the B cell as well as T cell epitopes of group 1 allergens from a variety of grass and corn species. Here we determine continuous B cell epitopes of Phl p 1 by gene fragmentation. IgE antibodies of grass pollen allergic patients identified five continuous epitope-containing areas that on an average bound 40% of Phl p 1-specific IgE antibodies and were stably recognized in the course of disease. In contrast to untreated patients, patients undergoing grass pollen immunotherapy started to mount IgG(4) antibodies to the recombinant IgE-defined fragments in the course of immunotherapy. The protective role of these IgG(4) antibodies is demonstrated by observations that 1) increases in rPhl p 1 fragment-specific IgG(4) were in parallel with decreases in Phl p 1-specific IgE, and 2) preincubation of rPhl p 1 with patients sera containing rPhl p 1 fragment-specific IgG(4) blocked histamine release from basophils of an untreated grass pollen allergic patient. We propose to use recombinant Phl p 1 fragments for active immunotherapy in order to induce protective IgG responses against IgE epitopes in grass pollen allergic patients. This concept may be applied for the development of allergy vaccines whenever the primary sequence or structure of an allergen is available.
Assuntos
Alérgenos/imunologia , Linfócitos B/imunologia , Hipersensibilidade/imunologia , Proteínas de Plantas/imunologia , Pólen , Receptores de Antígenos de Linfócitos B/imunologia , Sequência de Aminoácidos , Sequência de Bases , Mapeamento de Epitopos , Epitopos/genética , Epitopos/imunologia , Humanos , Dados de Sequência Molecular , Proteínas de Plantas/genética , Receptores de Antígenos de Linfócitos B/genéticaRESUMO
Whether the modulation of antibody responses can contribute to the improvement of clinical symptoms in patients receiving allergen immunotherapy represents a controversial issue. We have used purified [seven recombinant (r) and one natural] timothy grass pollen allergens as well as recombinant B cell epitope-containing fragments of the major timothy grass pollen allergen, Phl p 1, to investigate humoral immune responses in eight allergic patients receiving grass pollen-specific immunotherapy. We found that the administration of aluminium hydroxide-adsorbed grass pollen extract induced complex changes in allergen/epitope-specific antibody responses: increases in IgG subclass (IgG1, IgG2, IgG4) responses against allergens recognized before the therapy were observed. All eight patients started to mount IgE and IgG4 responses to continuous Phl p 1 epitopes not recognized before the therapy and a de novo induction of IgE antibodies against new allergens was found in one patient. Evidence for a protective role of IgG antibodies specific for continuous Phl p 1 epitopes was provided by the demonstration that preincubation of rPhl p 1 with human serum containing therapy-induced Phl p 1-specific IgG inhibited rPhl p 1-induced histamine release from basophils of a grass pollen-allergic patient. Our finding that immunotherapy induced antibody responses against previously not recognized B cell epitopes indicates the vaccination character of this treatment. The fact that patients started to mount de novo IgE as well as protective IgG responses against epitopes may explain the unpredictability of specific immunotherapy performed with allergen extracts and emphasizes the need for novel forms of component-resolved immunotherapy.
Assuntos
Alérgenos/imunologia , Linfócitos B/imunologia , Dessensibilização Imunológica , Imunoglobulina E/biossíntese , Imunoglobulina G/biossíntese , Rinite Alérgica Sazonal/imunologia , Rinite Alérgica Sazonal/terapia , Adulto , Basófilos/imunologia , Epitopos , Liberação de Histamina , Humanos , Imunoglobulina G/classificação , Técnicas In Vitro , Proteínas de Plantas/imunologia , Pólen/imunologiaRESUMO
Type I allergy, an immunodisorder that affects almost 20% of the population worldwide, is based on the immunoglobulin E (IgE) recognition of per se innocuous antigens (allergens). Pollen from wind-pollinated plants belong to the most potent allergen sources. We report the isolation of a cDNA coding for a 8.6 kDa two EF-hand calcium binding allergen, Phl p 7, from a timothy grass (Phleum pratense) pollen expression cDNA library, using serum IgE from a grass pollen allergic patient. Sequence analysis identified Phl p 7 as a member of a recently discovered subfamily of pollen-specific calcium binding proteins. Recombinant Phl p 7 was expressed in Escherichia coli and purified to homogeneity as determined by mass spectroscopy. Approximately 10% of pollen allergic patients displayed IgE reactivity to rPhl p 7 and Phl p 7-homologous allergens present in pollens of monocotyledonic and dicotyledonic plants. Circular dichroism analysis of the calcium-bound and apo-rPhl p 7 indicated that differences in IgE recognition may be due to calcium-induced changes in the protein conformation. The fact that patients mount IgE antibodies against different protein conformations is interpreted as a footprint of a preferential sensitization against either form. The biological activity of rPhl p 7 was demonstrated by its ability to induce basophil histamine release and immediate type skin reactions in sensitized individuals. In conclusion, IgE binding to Phl p 7 represents an example for the conformation-dependent IgE recognition of an allergen. Recombinant Phl p 7 may be used for diagnosis and perhaps treatment of a group of patients who suffer from allergy to pollens of many unrelated plant species.
Assuntos
Alérgenos/imunologia , Alérgenos/metabolismo , Cálcio/metabolismo , Imunoglobulina E/metabolismo , Pólen/imunologia , Pólen/metabolismo , Alérgenos/genética , Sequência de Aminoácidos , Animais , Apoproteínas/genética , Apoproteínas/imunologia , Apoproteínas/metabolismo , Sequência de Bases , Reações Cruzadas , Primers do DNA/genética , DNA Complementar/genética , DNA Complementar/isolamento & purificação , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Escherichia coli/genética , Liberação de Histamina , Humanos , Hipersensibilidade Imediata/etiologia , Técnicas In Vitro , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Proteínas de Plantas/metabolismo , Poaceae/genética , Poaceae/imunologia , Pólen/genética , Conformação Proteica , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosAssuntos
Alérgenos/uso terapêutico , Dessensibilização Imunológica , Hipersensibilidade/tratamento farmacológico , Proteínas de Plantas/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Alérgenos/genética , Alérgenos/imunologia , Antígenos de Plantas , Engenharia Genética , Humanos , Hipersensibilidade/imunologia , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Pólen , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
BACKGROUND: The interaction of immune complexes consisting of allergens and allergen-specific IgE with the high-affinity Fcepsilon receptor represents the key event in the induction of symptoms in type I allergic individuals. Immediate-type symptoms result from the release of biological mediators due to allergen-induced cross-linking of FcepsilonRI receptors on mast cells and basophils, whereas FcepsilonRI-mediated presentation of allergen-IgE complexes may contribute to late-phase symptoms through enhanced T cell activation. The interaction of allergens/allergen-specific IgE/FcepsilonRI represents, therefore, an important target for therapeutic intervention strategies in type I allergy. METHODS AND RESULTS: A molecular model of the allergen-IgE-FcepsilonRI interaction was established. It consists of recombinant purified Bet v 1, the major birch pollen allergen, a chimeric Bet v 1 specific monoclonal IgE antibody, and the baculovirus-expressed purified human alpha chain of FcepsilonRI. The chimeric Bet v 1-specific IgE antibody consists of the light chain and the heavy chain variable region of a mouse monoclonal Bet v 1 specific antibody, Bip 1, and the constant region of human IgE. The interaction of rBet v 1, chimeric Bip 1, and human alpha chain was investigated by overlay experiments. Nitrocellulose-immobilized recombinant alpha chains was incubated with chimeric Bip 1 and, for control purposes, with mouse-derived Bip 1. Bound chimeric Bip 1 was detected with 125I-labeled rBet v 1. The specific interaction of rBetv 1, chimeric Bip 1, and recombinant human alpha chain is demonstrated. We thus establish a molecular model of the allergen/IgE/alpha chain interaction. The usefulness of the described in vitro system is exemplified by the identification of a mouse monoclonal antihuman IgE antibody which blocked the IgE-alpha chain interaction. CONCLUSIONS: The module system consisting of rBet v 1, chimeric Bip 1, and recombinant alpha chain may be used for the identification of competitors of the allergic effector reaction by means of high throughput screening of compounds or by combinatorial chemistry.
Assuntos
Alérgenos , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Proteínas de Plantas/imunologia , Receptores de IgE/imunologia , Animais , Reações Antígeno-Anticorpo , Antígenos de Plantas , Humanos , Camundongos , Proteínas de Plantas/genética , Pólen , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
Serum IgE was used to isolate a cDNA coding for a 9.4-kDa two EF-hand calcium-binding allergen, Aln g 4, from a lambda gt11 expression cDNA library constructed from alder (Alnus glutinosa) pollen. rAln g 4 was overexpressed in Escherichia coli and purified to homogeneity. It reacted with serum IgE from 18% of pollen-allergic patients (n = 122); shared IgE epitopes with homologous allergens present in tree, grass, and weed pollens; and thus belongs to a family of highly cross-reactive pollen allergens. Exposure of two E. coli-expressed rAln g 4 fragments comprising amino acids 1-41 and 42-85 to patients' IgE Abs, as well as to a rabbit antiserum raised against purified rAln g 4, indicated that most of the B cell epitopes reside in the N-terminal portion of the protein. IgE recognition of Aln g 4 was strongly modulated by the presence or absence of calcium. Circular dichroism analysis of rAln g 4 revealed that the protein consisted mostly of alpha helical secondary structure and possessed a remarkable thermal stability and refolding capacity, a property that was greatly reduced after calcium depletion. Circular dichroism analysis of the calcium-bound and apo form of rAln g 4 indicated that calcium-induced modulation of IgE binding could be due to changes in the protein conformation. Purified rAln g 4 elicited dose-dependent basophil histamine release and immediate type skin reactions in sensitized patients. It may hence be useful for allergy diagnosis and for specific immunotherapy.
Assuntos
Alérgenos/imunologia , Cálcio/metabolismo , Sequências Hélice-Alça-Hélice , Imunoglobulina E/imunologia , Proteínas de Plantas/imunologia , Pólen/imunologia , Alérgenos/química , Alérgenos/genética , Sequência de Aminoácidos , Animais , Reações Antígeno-Anticorpo/efeitos dos fármacos , Antígenos de Plantas , Apoproteínas/química , Sequência de Bases , Cálcio/farmacologia , Dicroísmo Circular , Reações Cruzadas , DNA Complementar/genética , Dermatite Alérgica de Contato/imunologia , Dermatite Alérgica de Contato/terapia , Dessensibilização Imunológica , Escherichia coli , Liberação de Histamina , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/uso terapêutico , Conformação Proteica , Estrutura Secundária de Proteína , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Rinite Alérgica Sazonal/imunologia , Rinite Alérgica Sazonal/terapia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade , ÁrvoresRESUMO
OBJECTIVE: Stem cell factor (SCF), the ligand for the SCF receptor (c-kit) expressed on precursors and mature mast cells (MC), is a major agonist for human MC (e.g., SCF induces MC development, chemotaxis, activation, proliferation of MC precursors, mediates MC adhesion, and changes MC releasability). We investigated expression of SCF and c-kit in synovial membrane with particular reference to the mechanism of local MC hyperplasia and inflammation in arthritis. METHODS: We conducted single and double labeling immunohistochemistry (ABC, APAAP, indirect immunofluorescence techniques) with antibodies to SCF, c-kit, MC tryptase, Ki-67 antigen (marker for proliferating cells), and CD68 (monocyte/macrophage marker). Synovial specimens analyzed were from 31 patients: traumatic arthritis (TrA, n=9), osteoarthritis (OA, n=12), and rheumatoid arthritis (RA, n=10). Control experiments were performed on human lung, skin, and buccal mucosa tissues, on the HMC-1 mast cell line, and isolated lung MC. Morphometry was performed by computerized image analysis. RESULTS: Synovial c-kit expression was found to be restricted to MC, whereas SCF is detected in synovial lining cells, stromal fibroblasts, monocyte/macrophages, endothelial cells, and in vascular basement membranes. SCF staining was localized to MC as well, but it was not possible to specify whether this represents SCF produced by or bound (via c-kit) to MC. In inflamed synovial membranes/areas, SCF was found to be redistributed into the extracellular matrix. Redistribution of SCF was accompanied by degranulation and/or accumulation of c-kit+ MC, the hyperplasia of which correlated positively with histologic inflammation/inflammatory cell densities, but did not appear to involve MC proliferation in situ. These findings appeared to be common for all the conditions (TrA, OA, RA) studied. CONCLUSION: In addition to the demonstration/characterization of SCF and c-kit protein expression in human synovium, results of this study suggest the hypothesis that, in arthritis, local mobilization of SCF may play a role in the development of synovial MC hyperplasia without inducing in situ proliferation of MC, and that the synovial SCF/MC c-kit system may contribute to the local nonspecific inflammatory response/arthritic flares in TrA, OA, and RA.
Assuntos
Artrite/metabolismo , Proteínas Proto-Oncogênicas c-kit/biossíntese , Fator de Células-Tronco/biossíntese , Membrana Sinovial/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular , Feminino , Humanos , Hiperplasia , Imuno-Histoquímica , Inflamação , Antígeno Ki-67/análise , Pulmão/citologia , Pulmão/metabolismo , Masculino , Mastócitos/patologia , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-kit/análise , Fator de Células-Tronco/análise , Membrana Sinovial/citologia , Membrana Sinovial/patologiaRESUMO
Recently we demonstrated that a high percentage of atopic dermatitis (AD) patients displayed specific immunoglobulin E reactivity to human proteins. Here we show that IgE autoreactivity is found predominantly in AD patients with severe skin manifestations and reveal the molecular nature of four IgE autoantigens. An expression cDNA library constructed from a human epithelial cell line (A 431) was screened with serum IgE from two AD patients. DNA sequence analysis of three IgE-reactive clones identified the alpha-chain of the nascent polypeptide-associated complex, cytokeratin type II, and the BCL7B oncogen as atopy-related IgE autoantigens (ara). The fourth cDNA coded for an IgE autoantigen containing a typical calcium binding motif that occurred in histogenetically different cells and tissues (keratinocytes, muscle, brain). Recombinant Escherichia coli-expressed IgE autoantigens bound IgE from AD but not from patients with other immunologically mediated disorders (graft vs. host disease, systemic lupus erythematosus) and elicited immediate type skin reactions in AD patients. In serum samples collected from an AD patient over a period of 5 years, IgE anti-ara NAC antibody levels peaked during disease exacerbation. Our finding that ara BCL7B was detected in serum bound to IgE antibodies suggests that intracellular IgE autoantigens can become released after tissue damage and may occur as IgE immune complexes. Via binding to antigen presenting cells as well as to effector cells, IgE autoantigen immune complexes may contribute to exacerbation and/or perpetuation of severe atopic diseases even in the absence of exogenous allergens.