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1.
J Food Sci ; 84(8): 2357-2363, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31364176

RESUMO

Peanut allergic consumers rely on food package labels to avoid foods containing peanut. The inadvertent presence of peanut in foods due to cross-contact can be fatal if ingestion of such food leads to an allergic reaction. Analytical methods are available to detect undeclared peanut in foods. However, depending on the type of food matrix and food processing parameters, method performance can be adversely affected due to reduction in the extraction efficiency of peanut proteins. Temperature and probe sonication were used as a preincubation treatment for peanut flour slurries to assess their effect on the total peanut protein solubility from raw, light-roasted, and dark-roasted peanut flours. The effect of these treatments on the immunoreactivity of peanut allergens (Ara h 1, Ara h 2, Ara h 3, and Ara h 6) was determined by an indirect enzyme-linked immunosorbent assay using antibodies raised against these individual peanut proteins. Preincubation at 50 °C did not significantly improve the peanut protein solubility, whereas an increase in protein solubility was observed when light- and dark-roasted peanut flour slurries were preincubated at 90 °C or sonicated. The immunoreactivity of peanut allergens varied depending on the degree of peanut flour roasting and type of preincubation treatment. Overall, the immunoreactivity of peanut allergens from most peanut flour slurries was unaffected when preincubated at 50 °C for up to 60 min or sonicated with a probe for up to 5 min, whereas preincubation at 90 °C resulted in a time-dependent reduction in immunoreactivity of peanut allergens. Sonication treatment may improve peanut protein extraction without markedly affecting their immunoreactivity. PRACTICAL APPLICATION: Extraction of peanut proteins is vital for developed analytical methods to estimate peanut allergens in foods. The manuscript describes the effect of two different temperatures (50 and 90 °C) and probe-type sonication on peanut protein solubility. The findings suggest sonication can improve peanut protein solubility without markedly affecting their immunoreactivity.


Assuntos
Arachis/imunologia , Antígenos de Plantas/análise , Antígenos de Plantas/imunologia , Antígenos de Plantas/isolamento & purificação , Arachis/química , Ensaio de Imunoadsorção Enzimática , Farinha/análise , Manipulação de Alimentos , Humanos , Proteínas de Plantas/análise , Proteínas de Plantas/imunologia , Proteínas de Plantas/isolamento & purificação , Temperatura
2.
Allergol. immunopatol ; 47(4): 357-364, jul.-ago. 2019. tab, graf
Artigo em Inglês | IBECS | ID: ibc-186507

RESUMO

Introduction: Amaranthus retroflexus (Redroot Pigweed) is one of the main sources of allergenic pollens in temperate areas. Polcalcin is a well-known panallergen involved in cross-reactivity between different plants. The aim of this study was the molecular cloning and expression of polcalcin, as well as evaluating its IgE-reactivity with A. retroflexus sensitive patients' sera. Methods: Allergenic extract was prepared from A. retroflexus pollen and the IgE-reactivity profile was determined by ELISA and immunoblotting using sera from twenty A. retroflexus sensitive patients. Polcalcin-coding sequence was amplified by conventional PCR method and the product was inserted into pET-21b(+) vector. The recombinant protein was expressed in E. coli BL21 and purified by metal affinity chromatography. The IgE-binding capability of the recombinant protein was analyzed by ELISA and immunoblotting assays, and compared with crude extract. Results: Of 20 skin prick test positive patients, 17 patients were positive in IgE-specific ELISA. Western blotting confirmed that approximately 53% of ELISA positive patients reacted with 10kDa protein in crude extract. The A. retroflexus polcalcin gene, encoding to 80 amino acid residues was cloned and expressed as a soluble protein and designated as Ama r 3. The recombinant polcalcin showed rather identical IgE-reactivity in ELISA and western blotting with 10 kDa protein in crude extract. These results were confirmed by inhibition methods, too. Conclusion: The recombinant form of A. retroflexus polcalcin (Ama r 3) could be easily produced in E. coli in a soluble form and shows rather similar IgE-reactivity with its natural counterpart


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Assuntos
Humanos , Masculino , Feminino , Adolescente , Adulto Jovem , Adulto , Alérgenos/imunologia , Amaranthus/imunologia , Antígenos de Plantas/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Pólen/imunologia , Rinite Alérgica Sazonal/imunologia , Testes Cutâneos , Alérgenos/isolamento & purificação , Antígenos de Plantas/isolamento & purificação , Proteínas de Ligação ao Cálcio/isolamento & purificação , Clonagem Molecular , Reações Cruzadas , Escherichia coli/genética , Expressão Gênica , Imunoglobulina E/metabolismo , Extratos Vegetais , Proteínas Recombinantes/isolamento & purificação
3.
Allergol Immunopathol (Madr) ; 47(4): 357-364, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30770138

RESUMO

INTRODUCTION: Amaranthus retroflexus (Redroot Pigweed) is one of the main sources of allergenic pollens in temperate areas. Polcalcin is a well-known panallergen involved in cross-reactivity between different plants. The aim of this study was the molecular cloning and expression of polcalcin, as well as evaluating its IgE-reactivity with A. retroflexus sensitive patients' sera. METHODS: Allergenic extract was prepared from A. retroflexus pollen and the IgE-reactivity profile was determined by ELISA and immunoblotting using sera from twenty A. retroflexus sensitive patients. Polcalcin-coding sequence was amplified by conventional PCR method and the product was inserted into pET-21b(+) vector. The recombinant protein was expressed in E. coli BL21 and purified by metal affinity chromatography. The IgE-binding capability of the recombinant protein was analyzed by ELISA and immunoblotting assays, and compared with crude extract. RESULTS: Of 20 skin prick test positive patients, 17 patients were positive in IgE-specific ELISA. Western blotting confirmed that approximately 53% of ELISA positive patients reacted with 10kDa protein in crude extract. The A. retroflexus polcalcin gene, encoding to 80 amino acid residues was cloned and expressed as a soluble protein and designated as Ama r 3. The recombinant polcalcin showed rather identical IgE-reactivity in ELISA and western blotting with 10kDa protein in crude extract. These results were confirmed by inhibition methods, too. CONCLUSION: The recombinant form of A. retroflexus polcalcin (Ama r 3) could be easily produced in E. coli in a soluble form and shows rather similar IgE-reactivity with its natural counterpart.


Assuntos
Alérgenos/imunologia , Amaranthus/imunologia , Antígenos de Plantas/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Pólen/imunologia , Rinite Alérgica Sazonal/imunologia , Adolescente , Adulto , Alérgenos/isolamento & purificação , Antígenos de Plantas/isolamento & purificação , Proteínas de Ligação ao Cálcio/isolamento & purificação , Clonagem Molecular , Reações Cruzadas , Escherichia coli/genética , Feminino , Expressão Gênica , Humanos , Imunoglobulina E/metabolismo , Masculino , Extratos Vegetais , Proteínas Recombinantes/isolamento & purificação , Testes Cutâneos , Adulto Jovem
4.
Food Res Int ; 116: 1059-1065, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30716889

RESUMO

In this work, we explored the "deep" seed peanut proteome by using both two dimensional electrophoresis (2-DE)-based analysis run under reducing and non-reducing condition (protein-centric) and LC-MS/MS gel-free proteomic (peptide-centric). The former approach allowed to identify high molecular weight disulfide-linked Ara h 1 and Ara h 3 heteroligomers and Ara h 1 homoligomers linked through covalent bonds other than disulfides. The occurrence of these protein complexes revealed natural interactions between Ara(s) subunits with a possible involvement in the allergenic potential of peanut. The second approach, also referred to as shot-gun proteomics, allowed the identification of 149 gene products, including low-abundance proteins escaped the 2-DE detection. Interestingly, we identified 60 proteins never catalogued previously. The complementary exploitation of two proteomic approaches enabled the access to new relevant information about the complexity of the peanut proteome, with special emphasis to the complement of allergens (allergome).


Assuntos
Antígenos de Plantas/isolamento & purificação , Arachis/química , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel Bidimensional , Nozes/química , Hipersensibilidade a Amendoim/imunologia , Proteínas de Plantas/isolamento & purificação , Proteômica/métodos , Espectrometria de Massas em Tandem , Antígenos de Plantas/imunologia , Arachis/imunologia , Proteínas de Membrana/isolamento & purificação , Nozes/imunologia , Proteínas de Plantas/imunologia , Proteoma
5.
Molecules ; 23(12)2018 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-30544764

RESUMO

The soy protein isolates (SPI) extracted from different extruded full-fat soybean flakes (FFSF), and their conformational and functional properties were characterized. Overall, the free thiol (SH) content of SPI increased when the extrusion temperature was below 80 °C and decreased at higher temperatures. Soy glycinin (11S) showed higher stability than ß-conglycinin (7S) during extrusion. Results also indicated that the increase in some hydrophobic groups was due to the movement of hydrophobic groups from the interior to the surface of the SPI molecules at extrusion temperatures from 60 to 80 °C. However, the aggregation of SPI molecules occurred at extrusion temperatures of 90 and 100 °C, with decreasing levels of hydrophobic groups. The extrusion temperature negatively affected the emulsifying activity index (EAI); on the other side, it positively affected the emulsifying stability index (ESI), compared to unextruded SPI.


Assuntos
Proteínas de Soja/química , Proteínas de Soja/isolamento & purificação , Soja/metabolismo , Antígenos de Plantas/química , Antígenos de Plantas/isolamento & purificação , Antígenos de Plantas/metabolismo , Temperatura Baixa , Globulinas/química , Globulinas/isolamento & purificação , Globulinas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Peso Molecular , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/isolamento & purificação , Proteínas de Armazenamento de Sementes/metabolismo , Proteínas de Soja/metabolismo , Compostos de Sulfidrila/análise
6.
J Agric Food Chem ; 66(41): 10855-10863, 2018 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-30284821

RESUMO

The 2S albumin Ara h 6 is one of the most important peanut allergens. A post-translationally cleaved Ara h 6 (pAra h 6) was purified from Virginia type peanuts, and the cleavage site was mapped using high-resolution mass spectrometry. Compared to intact Ara h 6, pAra h 6 lacks a 5-amino acid stretch, resembling amino acids 43-47 (UniProt accession number Q647G9) in the nonstructured loop. Consequently, pAra h 6 consists of two chains: an N-terminal chain of approximately 5 kDa and a C-terminal chain of approximately 9 kDa, held together by disulfide bonds. Intermediate post-translationally cleaved products, in which this stretch is cleaved yet still attached to one of the subunits, are also present. The secondary structure and immunoglobulin E (IgE) binding of pAra h 6 resembles that of intact Ara h 6, indicating that the loss of the nonstructured loop is not critical for maintaining the protein structure. Commercially available monoclonal and polyclonal immunoglobulin G (IgG) antibodies directed to Ara h 6 react with both intact Ara h 6 and pAra h 6, suggesting that the involved epitopes are not located in the area that is post-translationally cleaved. No differences between intact Ara h 6 and pAra h 6 in terms of IgE binding were found, suggesting that the area that is post-translationally cleaved is not involved in IgE epitopes either. For all main cultivars Runner, Virginia, Valencia, and Spanish, intact Ara h 6 and pAra h 6 occur in peanut at similar levels, indicating that pAra h 6 is a consistent and important contributor to the allergenic potency of peanut.


Assuntos
Albuminas 2S de Plantas/química , Albuminas 2S de Plantas/isolamento & purificação , Antígenos de Plantas/química , Antígenos de Plantas/isolamento & purificação , Arachis/química , Albuminas 2S de Plantas/imunologia , Sequência de Aminoácidos , Aminoácidos/química , Antígenos de Plantas/imunologia , Epitopos/imunologia , Glicoproteínas/química , Glicoproteínas/imunologia , Glicoproteínas/isolamento & purificação , Humanos , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Hipersensibilidade a Amendoim/metabolismo , Hipersensibilidade a Amendoim/prevenção & controle , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Proteínas de Plantas/isolamento & purificação , Estrutura Secundária de Proteína
7.
Sci Rep ; 8(1): 10512, 2018 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-30002383

RESUMO

Fag s 1 is a member of the Pathogen Related protein family 10 (PR-10) and can elicit cross-reaction with IgE antibodies produced against the birch pollen allergen Bet v 1. The Nuclear Magnetic Resonance (NMR) structure of Fag s 1 is presented along with its dynamic properties. It shares 66% identity with Bet v 1 and exhibits the expected three α-helices and seven ß-sheets arranged as a semi-beta barrel and exposing the residues mapped as the Bet v 1 IgE epitope. The structural dynamics of Fag s 1 were monitored on the fast and intermediate timescales, using relaxation rates. The complex dynamics of Fag s 1 are closely related to the internal cavity, and they modulate IgE and ligand binding.


Assuntos
Alérgenos/imunologia , Antígenos de Plantas/química , Reações Cruzadas , Fagus/imunologia , Proteínas de Plantas/química , Antígenos de Plantas/imunologia , Antígenos de Plantas/isolamento & purificação , Betula/imunologia , Epitopos/imunologia , Humanos , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas de Plantas/imunologia , Proteínas de Plantas/isolamento & purificação , Pólen/imunologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Relação Estrutura-Atividade
8.
Clin Exp Allergy ; 48(9): 1206-1213, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29904971

RESUMO

BACKGROUND: The role of sensitization to commercially available allergens of English walnut (Juglans regia) Jug r 1, 2 and 3 in walnut allergy has been previously investigated in walnut allergic adults and was unable to explain all cases of walnut allergy. OBJECTIVES: Identify recognized walnut allergens, other than the ones previously investigated (Jug r 1-3), in walnut allergic adults and determine the sensitization frequency and diagnostic value. METHODS: Three different in-house walnut extracts were prepared and analysed on SDS-PAGE blots to identify allergenic walnut proteins. Immunoblots and immunoprecipitation, followed by LC-MS analysis, were performed to screen for, and confirm, IgE binding to walnut allergens in selected walnut allergic adults. In a cohort of 55 walnut challenged adults, including 33 allergic and 22 tolerant, sensitization to native and recombinant walnut allergen Jug r 4 was assessed using immunoblotting and immuno-line blot (EUROLINE), respectively. RESULTS: Screening of sera of 8 walnut allergic adults identified Jug r 4 as an allergen in our population. In the total cohort of 55 subjects, 5 were positive for Jug r 4 on immunoblot and 10 on EUROLINE. All but one EUROLINE positive subject had a positive food challenge (sensitivity 27%, specificity 95%, PPV 90%, NPV 47%). All 5 subjects positive on immunoblot were also positive on EUROLINE. LC-MS analysis showed a lack of Jug r 4 in the ImmunoCAP extract. Co-sensitization to other 11S albumins (eg hazelnut Cor a 9) was common in Jug r 4 sensitized subjects, potentially due to cross-reactivity. CONCLUSIONS: Walnut 11S globulin Jug r 4 is a relevant minor allergen, recognized by 27% of walnut allergic adults. It has a high positive predictive value of 90% for walnut allergy. Specific IgE against Jug r 4 occurred mostly with concomitant sensitization to other walnut components, mainly Jug r 1.


Assuntos
Antígenos de Plantas/imunologia , Juglans/efeitos adversos , Hipersensibilidade a Noz/imunologia , Proteínas de Plantas/imunologia , Adulto , Antígenos de Plantas/química , Antígenos de Plantas/isolamento & purificação , Cromatografia Líquida , Reações Cruzadas/imunologia , Feminino , Humanos , Imunoensaio , Imunoglobulina E/imunologia , Juglans/química , Masculino , Espectrometria de Massas , Hipersensibilidade a Noz/diagnóstico , Extratos Vegetais/química , Extratos Vegetais/imunologia , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Sensibilidade e Especificidade , Testes Cutâneos , Adulto Jovem
9.
Mol Med Rep ; 17(1): 394-399, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29115430

RESUMO

Platanus acerifolia is one of the major sources of outdoor allergens to humans, and can induce allergic asthma, rhinitis, dermatitis and other allergic diseases. Pla a 2 is a polygalacturonase and represents the major allergen identified in P. acerifolia pollen. The aim of the present study was to express and purify Pla a 2, and to predict B and T cell epitopes of Pla a 2. The gene encoding Pla a 2 was cloned into the pET28a vector and subsequently transfected into ArcticExpress™ (DE3) Escherichia coli cells; purified Pla a 2 was analyzed by western blot analysis. The results of the present study revealed that the Pla a 2 allergen has the ability to bind immunoglobulin E within the sera of patients allergic to P. acerifolia pollen. In addition, the B cell epitopes of Pla a 2 were predicted using the DNAStar Protean system, Bioinformatics Predicted Antigenic Peptides and BepiPred 1.0 software; T cell epitopes were predicted using NetMHCIIpan ­3.0 and ­2.2. In total, eight B cell epitopes (15­24, 60­66, 78­86, 109­124, 232­240, 260­269, 298­306 and 315­322) and five T cell epitopes (62­67, 86­91, 125­132, 217­222 and 343­350) were predicted in the present study. These findings may be used to improve allergen immunotherapies and reduce the frequency of pollen­associated allergic reactions.


Assuntos
Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Mapeamento de Epitopos , Epitopos/genética , Epitopos/imunologia , Expressão Gênica , Pólen/genética , Pólen/imunologia , Adulto , Antígenos de Plantas/química , Antígenos de Plantas/isolamento & purificação , Epitopos/química , Epitopos de Linfócito B , Epitopos de Linfócito T , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Humanos , Imunoglobulina E/imunologia , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Ligação Proteica/imunologia , Conformação Proteica
10.
J Allergy Clin Immunol ; 141(2): 626-631.e7, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28709968

RESUMO

BACKGROUND: Generic immunoassays for peanut cannot discriminate between allergen levels in peanut-derived food products or therapeutics. Clinical trials of oral immunotherapy (OIT) are strengthened by using standardized peanut preparations with defined doses of major allergens. OBJECTIVE: This article describes measurement of Ara h 1, Ara h 2, and Ara h 6 in peanut foods and in peanut flour extracts used for allergy diagnosis and OIT. METHODS: Monoclonal antibody-based enzyme immunoassays for Ara h 1, Ara h 2, and Ara h 6 were used to compare allergen levels in peanut (n = 16) and tree nut (n = 16) butter, peanut flour (n = 11), oils (n = 8), extracts used for diagnosis and OIT (n = 5), and the National Institute for Standards and Technology Peanut Butter Standard Reference Material 2387. RESULTS: Roasted peanut butters contained 991 to 21,406 µg/g Ara h 1 and exceeded Ara h 2 and Ara h 6 levels by 2- to 4-fold. Similarly, National Institute for Standards and Technology Peanut Butter Standard Reference Material 2387 contained 11,275 µg/g Ara h 1, 2,522 µg/g Ara h 2, and 2,036 µg/g Ara h 6. In contrast, peanut flours contained 787 to 14,631 µg/g Ara h 2 and exceeded Ara h 1 levels by 2- to 20-fold. Flour extracts used for OIT contained 394 to 505 µg/mL Ara h 1, 1,187 to 5,270 µg/mL Ara h 2, and 1,104 to 8,092 µg/mL Ara h 6. In most cases specific peanut allergens were not detected in tree nut butters or peanut oils. CONCLUSIONS: The results show marked differences in specific peanut allergen profiles in peanut butter and flour and peanut preparations for clinical use. Roasting can increase Ara h 1 levels in peanut butter. Variability in allergen levels could affect the outcome of clinical trials of peanut OIT, especially with respect to Ara h 1. Specific allergen measurements will improve standardization and provide accurate dosing of peanut preparations that are being used for OIT.


Assuntos
Antígenos de Plantas/química , Antígenos de Plantas/isolamento & purificação , Arachis/química , Análise de Alimentos/métodos , Hipersensibilidade a Amendoim/diagnóstico , Alérgenos , Ensaio de Imunoadsorção Enzimática , Farinha , Humanos , Padrões de Referência
12.
Mol Med Rep ; 16(3): 2887-2892, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28677761

RESUMO

Platanus acerifolia pollen is considered an important source of airborne allergens in numerous cities. Pla a 1 is a major allergen from P. acerifolia pollen. The present study aimed to express and purify Pla a 1, and to prepare its monoclonal antibody. In the present study, the Pla a 1 gene was subcloned into a pET­28a vector and transformed into the ArcticExpress™ (DE3) RP Escherichia coli host strain. The purified Pla a 1 was then used to immunize BALB/c mice. When serum detection was positive, spleen cells were isolated from the mice and fused with SP2/0 myeloma cells at a ratio of 10:1. Hybridoma cells were screened by indirect ELISA and limiting dilution. Positive cells were used to induce the formation of antibody­containing ascites fluid, and the antibodies were purified using protein A­agarose. The results of the present study demonstrated that recombinant Pla a 1 was successfully expressed and purified, and exhibited positive immunoglobulin E­binding to serum from patients allergic to P. acerifolia. A total of 11 hybridomas that steadily secreted anti­Pla a 1 antibody were obtained and an immunoblotting analysis indicated that all of these monoclonal antibodies specifically recognized the Pla a 1 protein. These results suggested that specific anti­Pla a 1 antibodies may be obtained, which can be used for the rapid detection of Pla a 1 allergens and in the preparation of vaccines against P. acerifolia pollen.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Clonagem Molecular/métodos , Adolescente , Adulto , Animais , Antígenos de Plantas/isolamento & purificação , Linhagem Celular , Escherichia coli/genética , Feminino , Vetores Genéticos/genética , Humanos , Imunoglobulina E/imunologia , Magnoliopsida/genética , Magnoliopsida/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pólen , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Rinite Alérgica/imunologia , Rinite Alérgica Sazonal/imunologia , Adulto Jovem
13.
Mol Med Rep ; 16(3): 2851-2855, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28656246

RESUMO

Platanus acerifolia (P. acerifolia) is an important cause of pollinosis in cities. The use of allergen extracts on patients with allergic diseases is the most commonly applied method to attempt to treat pollinosis. Pla a 3, a non­specific lipid transfer protein, is a major allergen present in P. acerifolia pollen extracts. In the present study, the Pla a 3 gene was sub­cloned into a pSUMO­Mut vector using Stu I and Xho I sites and transformed into the Arctic Express™ (DE3) RP E. coli host strain. The purified Pla a 3 allergen was analyzed by western blotting and the results revealed that the Pla a 3 allergen has the ability to bind IgE in the P. acerifolia pollen of allergic patients' sera. Moreover, the authors predicted the potential B cell epitopes of the Pla a 3 allergen using the DNAStar Protean system, the Bioinformatics Predicted Antigenic Peptides system and the BepiPred 1.0 server. In addition, the T cell epitopes were predicted by the SYFPEITHI database and the NetMHCII­2.2 server. As a result, two B cell epitopes (35­45 and 81­86) and four potential T cell epitopes including 2­15, 45­50, 55­61 and 67­73 were predicted in the present study. The current results can be used to contribute to allergen immunotherapies and useful in peptide­based vaccine designs of pollen allergy.


Assuntos
Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Imunoglobulina E/imunologia , Magnoliopsida/imunologia , Rinite Alérgica Sazonal/imunologia , Adolescente , Adulto , Antígenos de Plantas/química , Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Antígenos de Plantas/isolamento & purificação , Clonagem Molecular , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/isolamento & purificação , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/isolamento & purificação , Escherichia coli/genética , Feminino , Humanos , Imunoglobulina E/sangue , Magnoliopsida/química , Magnoliopsida/genética , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Pólen/química , Pólen/genética , Pólen/imunologia , Conformação Proteica , Rinite Alérgica Sazonal/sangue , Adulto Jovem
14.
Int Arch Allergy Immunol ; 173(1): 44-50, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28494467

RESUMO

BACKGROUND: Allergen immunotherapy (AIT) still plays a minor role in the treatment of allergic diseases. To improve the acceptance of AIT by allergic patients, the treatment has to become more convenient and efficacious. One possibility is the oral application of allergens or derivatives thereof. Therefore, we sought to produce a recombinant allergen in the green alga Chlamydomonas reinhardtii as a novel production platform. METHODS: The major birch pollen allergen Bet v 1 was selected as candidate molecule, and a codon-optimized gene was synthesized and stably integrated into the microalga C. reinhardtii FUD50. Positive transformants were identified by PCR, cultured, and thereafter cells were disrupted by sonication. Bet v 1 was purified from algal total soluble protein (TSP) by affinity chromatography and characterized physicochemically as well as immunologically. RESULTS: All transformants showed expression of the allergen with yields between 0.01 and 0.04% of TSP. Algal-derived Bet v 1 displayed similar secondary structure elements as the Escherichia coli-produced reference allergen. Moreover, Bet v 1 produced in C. reinhardtii showed binding comparable to human IgE as well as murine Bet v 1-specific IgG. CONCLUSION: We could successfully produce recombinant Bet v 1 in C. reinhardtii. As microalgae are classified as GRAS (generally recognized as safe), the pilot study supports the development of novel allergy treatment concepts such as the oral administration of allergen-containing algal extracts for therapy.


Assuntos
Alérgenos/genética , Antígenos de Plantas/genética , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Alérgenos/imunologia , Alérgenos/isolamento & purificação , Antígenos de Plantas/imunologia , Antígenos de Plantas/isolamento & purificação , Humanos , Imunoglobulina E/imunologia , Proteínas de Plantas/imunologia , Proteínas de Plantas/isolamento & purificação , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação
15.
Biosci Biotechnol Biochem ; 81(7): 1405-1408, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28471334

RESUMO

An effective method to prepare plant complex type (PCT) N-glycans in large amounts has been required to evaluate their immunological activity. In this study, we found that glycoproteins in bamboo shoots predominantly carry PCT N-glycans including the Lewis a epitope-containing ones, suggesting that bamboo shoot is an excellent source for the plant antigenic glycans to synthesize immunoactive neoglycopolymers.


Assuntos
Antígenos de Plantas/isolamento & purificação , Bambusa/química , Glicoproteínas/isolamento & purificação , Polissacarídeos/isolamento & purificação , Antígenos de Plantas/química , Bambusa/imunologia , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Glicoproteínas/química , Brotos de Planta/química , Brotos de Planta/imunologia , Polissacarídeos/química
16.
J Proteome Res ; 16(2): 988-998, 2017 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-28112517

RESUMO

The hazelnut allergen Cor a 14 belongs to the 2S albumins, a family of heterodimeric seed storage proteins exhibiting a high degree of structural diversity. Given its relevance as an allergen and the potential to elicit severe reactions, elucidation of the sequence heterogeneity of naturally occurring Cor a 14 is essential for the development of reliable diagnostics and risk evaluation. We therefore performed a comprehensive survey on the proteoforms of Cor a 14 and determined their quantitative distribution in three different hazelnut cultivars by a combinatory HPLC-HRMS approach including bottom-up and intact mass analysis. Compared with the Cor a 14 prototype sequence, we identified three sequence polymorphisms, two of the small and one of the large subunit, and elucidated their specific pairing on the protein level. Furthermore, we located a pronounced microheterogeneity on the protein termini and, for the first time, provide data on varying proteoform patterns between different cultivars of an allergenic seed. Together, these data present the basis for a more detailed investigation on the allergenicity of Cor a 14 in different cultivars and constitute, to be best of our knowledge, the largest set of proteoforms so far reported for a 2S albumin.


Assuntos
Alérgenos/química , Antígenos de Plantas/química , Polimorfismo de Nucleotídeo Único , Subunidades Proteicas/química , Proteínas de Armazenamento de Sementes/química , Alérgenos/genética , Alérgenos/isolamento & purificação , Sequência de Aminoácidos , Antígenos de Plantas/genética , Antígenos de Plantas/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Corylus/química , Expressão Gênica , Humanos , Espectrometria de Massas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Proteínas de Armazenamento de Sementes/genética , Proteínas de Armazenamento de Sementes/isolamento & purificação
17.
Mol Immunol ; 83: 100-106, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28126622

RESUMO

Profilins are small actin-binding proteins found in eukaryotes and involved in cell development, cytokinesis, membrane trafficking, and cell motility. From an allergenic point of view, profilins are panallergens usually involved in allergic polysensitization, although they are generally recognized as minor allergens. The objectives of this study were to identify and characterize the profilin from Plantago lanceolata pollen and to investigate the cross-reactivity between profilins from different pollen allergenic sources. Profilins from P. lancelolata (Pla l 2) and palm tree pollen (Pho d 2) were purified by affinity chromatography, deeply characterized and identified by mass spectrometry. Pla l 2 allergenicity was confirmed by immunoblot with serum samples from a patient population sensitized to profilin. Immunoblot inhibition was performed to study IgG reactivity between different pollen profilins. IgE cross-reactivity was demonstrated by ImmunoCAP inhibition. Pla l 2 is the second P. lanceolata allergen included in the IUIS Allergen Nomenclature database. Four peptides from purified Pla l 2 were identified with percentages of homology with other pollen profilins between 73 and 86%. Eighty-six percent (21/24) of the patient population recognized Pla l 2. The allergenic relatedness between Pla l 2, Pho d 2 and six pollen profilins was confirmed, and IgE cross-reactivity of Pla l 2 with rBet v 2 and rPhl p 12 was demonstrated. Pla l 2 is the profilin from P. lanceolata. The demonstrated allergenicity of this protein and its cross-reactivity with other pollen profilins support its use in profilin diagnostic assays.


Assuntos
Alérgenos/imunologia , Glicoproteínas/imunologia , Proteínas de Plantas/imunologia , Plantago/imunologia , Profilinas/imunologia , Adolescente , Adulto , Alérgenos/isolamento & purificação , Animais , Antígenos de Plantas/imunologia , Antígenos de Plantas/isolamento & purificação , Reações Cruzadas , Feminino , Glicoproteínas/isolamento & purificação , Humanos , Immunoblotting , Masculino , Pólen/imunologia , Profilinas/isolamento & purificação , Coelhos , Adulto Jovem
18.
PLoS One ; 12(1): e0169784, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28081194

RESUMO

BACKGROUND: Ragweed (Ambrosia artemisiifolia) and mugwort (Artemisia vulgaris) are the major cause of pollen allergy in late summer. Allergen-specific lymphocytes are crucial for immune modulation during immunotherapy. We sought to generate and pre-clinically characterise highly immunogenic domains of the homologous pectate lyases in ragweed (Amb a 1) and mugwort pollen (Art v 6) for immunotherapy. METHODS: Domains of Amb a 1 (Amb a 1α) and Art v 6 (Art v 6α) and a hybrid molecule, consisting of both domains, were designed, expressed in E. coli and purified. Human IgE reactivity and allergenicity were assessed by ELISA and mediator release experiments using ragweed and mugwort allergic patients. Moreover, T cell proliferation was determined. Blocking IgG antibodies and cytokine production in BALB/c mice were studied by ELISA and ELISPOT. RESULTS: The IgE binding capacity and in vitro allergenic activity of the Amb a 1 and Art v 6 domains and the hybrid were either greatly reduced or abolished. The recombinant proteins induced T cell proliferative responses comparable to those of the natural allergens, indicative of retained allergen-specific T cell response. Mice immunisation with the hypoallergens induced IL-4, IL-5, IL-13 and IFN-γ production after antigen-specific in vitro re-stimulation of splenocytes. Moreover, murine IgG antibodies that inhibited specific IgE binding of ragweed and mugwort pollen allergic patients were detected. CONCLUSION: Accumulation of T cell epitopes and deletion of IgE reactive areas of Amb a 1 and Art v 6, modulated the immunologic properties of the allergen immuno-domains, leading to promising novel candidates for therapeutic approach.


Assuntos
Ambrosia/imunologia , Antígenos de Plantas/metabolismo , Artemisia/imunologia , Epitopos de Linfócito T/imunologia , Proteínas de Plantas/metabolismo , Adolescente , Adulto , Idoso , Alérgenos/imunologia , Ambrosia/química , Sequência de Aminoácidos , Animais , Antígenos de Plantas/genética , Antígenos de Plantas/isolamento & purificação , Artemisia/química , Criança , Dicroísmo Circular , Escherichia coli/metabolismo , Feminino , Humanos , Imunoglobulina E/imunologia , Imunoglobulina E/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Interferon gama/análise , Interleucinas/análise , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Linfócitos T/citologia , Linfócitos T/imunologia , Adulto Jovem
19.
Int J Biometeorol ; 61(1): 23-33, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27317399

RESUMO

One of the key input parameters for numerical pollen forecasts is the distribution of pollen sources. Generally, three different methodologies exist to assemble such distribution maps: (1) plant inventories, (2) land use data in combination with annual pollen counts, and (3) ecological modeling. We have used six exemplary maps for all of these methodologies to study their applicability and usefulness in numerical pollen forecasts. The ragweed pollen season of 2012 in France has been simulated with the numerical weather prediction model COSMO-ART using each of the distribution maps in turn. The simulated pollen concentrations were statistically compared to measured values to derive a ranking of the maps with respect to their performance. Overall, approach (2) resulted in the best correspondence between observed and simulated pollen concentrations for the year 2012. It is shown that maps resulting from ecological modeling that does not include a sophisticated estimation of the plant density have a very low predictive skill. For inventory maps and the maps based on land use data and pollen counts, the results depend very much on the observational site. The use of pollen counts to calibrate the map enhances the performance of the model considerably.


Assuntos
Poluentes Atmosféricos/análise , Alérgenos/análise , Antígenos de Plantas/isolamento & purificação , Modelos Teóricos , Extratos Vegetais/isolamento & purificação , Simulação por Computador , Monitoramento Ambiental , Previsões , França , Reprodutibilidade dos Testes
20.
Ann Allergy Asthma Immunol ; 118(2): 148-153, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-26993170

RESUMO

OBJECTIVE: To review the use of pollen for the production of allergen extracts to diagnose and treat allergic diseases, examine the associated regulations, and highlight candidate areas for improvement. DATA SOURCES: A PubMed search was performed using focused keywords combined with a review of regulatory documents and industry guidelines. STUDY SELECTIONS: The information obtained through literature, documents, and industry was scrutinized and used with personal experience and expertise to write this article. RESULTS: Both genetic and environmental factors affect the allergenic composition of pollen because it is a biologically active pharmaceutical ingredient obtained from nature. The potential effect of airborne contaminants in pollen requires major attention but can be properly addressed through careful collection practices, combined with a proper interpretation of the data on purity obtained for each pollen lot. The regulations associated with pollen used to manufacture allergen extracts in the United States and Europe and the numbers of pollen allergen extracts commercially available in both areas of the world differ. A critical parameter to select the appropriate extracts for diagnosis and allergen immunotherapy is to understand the phenomenon of cross-reactivity among pollen families, genera, and species. CONCLUSION: Physicians should be aware of the factors responsible for the qualitative and quantitative composition of pollen allergen extracts and the associated regulations to produce suitable extracts to diagnose and treat allergic diseases. Collaboration and cooperation among allergen manufacturing companies and regulatory agencies are necessary.


Assuntos
Alérgenos/imunologia , Alérgenos/isolamento & purificação , Extratos Vegetais/imunologia , Extratos Vegetais/isolamento & purificação , Pólen/imunologia , Antígenos de Plantas/imunologia , Antígenos de Plantas/isolamento & purificação , Europa (Continente) , Humanos , Pólen/classificação , Rinite Alérgica Sazonal/diagnóstico , Rinite Alérgica Sazonal/imunologia , Rinite Alérgica Sazonal/terapia , Estados Unidos
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