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1.
Mol Immunol ; 118: 210-221, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31901836

RESUMO

Eggplant or brinjal (Solanum melongena L.) is widely consumed worldwide and thought to trigger allergic reactions in sensitive individuals. So far, no molecular information is available on the allergy-eliciting components of eggplant. In this study, a 17 kDa profilin, Sola m 1, was identified from eggplant by employing an immunoproteomic approach. Based on MALDI-TOF/TOF derived sequences, the full-length cDNA of Sola m 1 was PCR amplified and then cloned. Recombinant (r) Sola m 1 was expressed in E. coli and then purified by metal affinity and gel filtration. rSola m 1 reacted with IgE-antibodies in the sera from all eggplant allergic patients. rSola m 1 also displayed allergenic activity by stimulating histamine release. rSola m 1 was monomeric, and the CD spectra revealed it to be folded with a mixture of α-helices and ß-strands. In the melting curve, rSola m 1 exhibited an irreversible denaturation where no refolding took place. Sola m 1 was found to share >80 % sequence identity with Bet v 2, which was further validated by confirming the presence of significant cross-reactivity with Bet v 2 in IgE-inhibition assay. IgE-cross reactivity was also observed between rSola m 1 and profilins from six other foods. In SGF assay, no rSola m 1-derived fragments exhibited IgE-reactivity after prolonged digestion suggesting the association of rSola m 1 with the oral allergy syndromes. Immunofluorescence localization revealed a high abundance of Sola m 1 allergen in eggplant seeds as compared to other edible parts. Taken together, Sola m 1 is the first major eggplant allergen reported in this study, which has the potential of being used as a candidate antigen in component-resolved diagnosis and immunotherapy.


Assuntos
Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Escherichia coli/genética , Profilinas/imunologia , Solanum melongena/imunologia , Adolescente , Adulto , Idoso , Reações Cruzadas/imunologia , DNA Complementar/genética , Feminino , Hipersensibilidade Alimentar/imunologia , Liberação de Histamina/imunologia , Humanos , Imunoglobulina E/imunologia , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Adulto Jovem
2.
J Appl Microbiol ; 128(3): 862-874, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31758869

RESUMO

AIM: To study the prophylactic effect of recombinant Lactococcus lactis (rLl) harbouring Ara h 2.02 peanut allergen, in sensitized and challenged mice. METHODS AND RESULTS: Ara h 2.02 cDNA was cloned into pNZ8048 for heterologous expression in L. lactis. The purified recombinant allergen showed IgE binding comparable with native Ara h 2. Balb/c mice were fed with either recombinant (rLl), nonrecombinant L. lactis (Ll) or NaHCO3 (Sham) prior to sensitization and challenged with rAra h 2.02, whereas the baseline group was only fed with Ll. Allergen-specific immunoglobulin and splenocyte cytokines responses were determined for each mouse. Mice fed with either Ll or rLl showed significant alleviation of IgE and IgG1 compared to the Sham group. Despite no significant decrease in Th2 (IL-4, IL-13, IL-6) or increase in Th1 (IFN-γ) cytokines, both groups showed lower IL-10 level, while the IL-4 : IFN-γ ratio was significantly lower for rLl compared to Ll group. CONCLUSIONS: Oral administration of rLl harbouring Ara h 2.02 demonstrated alleviation of Th2-associated responses in allergen-challenged mice and a possible added allergen-specific prophylactic effect. SIGNIFICANCE AND IMPACT OF THE STUDY: Ara h 2.02 coupled with the intrinsic properties of probiotic L. lactis as a delivery vehicle can be explored for the development of a commercially scalable vaccine.


Assuntos
Albuminas 2S de Plantas/imunologia , Antígenos de Plantas/imunologia , Lactococcus lactis/genética , Lactococcus lactis/imunologia , Hipersensibilidade a Amendoim/prevenção & controle , Albuminas 2S de Plantas/genética , Administração Oral , Animais , Antígenos de Plantas/genética , Citocinas/imunologia , Feminino , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Hipersensibilidade a Amendoim/imunologia , Probióticos/administração & dosagem
3.
J Agric Food Chem ; 68(5): 1207-1212, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31755264

RESUMO

Human noroviruses (HuNoVs) are among the main pathogens causing acute nonbacterial gastroenteritis. Histo-blood group antigens (HBGAs) are widely accepted receptors for HuNoV specific binding. HBGA-like substances in produce are also considered as the critical ligands for capture of HuNoVs. However, the composition of viral ligands from food substrates remains unknown. In this study, an oligosaccharide (H2N2F2) was captured and isolated from romaine lettuce extract by a bacterial surface display system. Using electrospray ionization mass spectrometry and tandem mass spectrometry, it was shown that H2N2F2 was most likely to be a chimera of type A, H, and Lewis a HBGAs. The composition was consistent with our ELISA results using a panel of monoclonal antibodies against HBGAs. Our results revealed a possible interaction mechanism between HuNoVs and romaine lettuce. Better understanding of the interaction of HuNoVs with easily contaminated produce will ultimately aid in the control of and reduction in disease outbreaks.


Assuntos
Antígenos de Plantas/metabolismo , Antígenos de Grupos Sanguíneos/metabolismo , Alface/virologia , Norovirus/fisiologia , Receptores Virais/metabolismo , Ligação Viral , Antígenos de Plantas/química , Antígenos de Plantas/genética , Antígenos de Grupos Sanguíneos/química , Antígenos de Grupos Sanguíneos/genética , Infecções por Caliciviridae/genética , Infecções por Caliciviridae/metabolismo , Infecções por Caliciviridae/virologia , Humanos , Alface/química , Alface/genética , Alface/metabolismo , Espectrometria de Massas , Norovirus/genética , Oligossacarídeos/química , Oligossacarídeos/genética , Oligossacarídeos/metabolismo , Ligação Proteica , Receptores Virais/química , Receptores Virais/genética
4.
J Agric Food Chem ; 67(47): 13127-13138, 2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31682438

RESUMO

Allergen-specific immunotherapy is the only available curative treatment for IgE-mediated allergen diseases. A safe hypoallergenic allergen derivative with high efficiency is required as a tolerogen to induce immune tolerance to the causitive allergens. In this study, to generate a rice-based oral allergy vaccine for Japanese cedar (JC) pollinosis, the tertiary structures of major JC pollen allergens, Cry j 1 and Cry j 2, were more completely destructed by shuffling than the previous ones without losing immunogenicity and then were specifically expressed in the endosperm of transgenic rice seed. They accumulated at high levels and were deposited in endoplasmic reticulum (ER) and ER-derived protein bodies. The low allergenicity of these deconstructed Cry j 1 and Cry j 2 allergens was evaluated by examining their binding activities to the specific IgE antibody and by the basophil degranulation test.


Assuntos
Antígenos de Plantas/imunologia , Cryptomeria/imunologia , Hipersensibilidade/imunologia , Oryza/genética , Plantas Geneticamente Modificadas/genética , Animais , Antígenos de Plantas/genética , Cryptomeria/genética , Humanos , Hipersensibilidade/terapia , Imunoterapia , Camundongos , Oryza/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Pólen/genética , Pólen/imunologia , Ratos , Sementes/genética , Sementes/imunologia , Vacinas/administração & dosagem , Vacinas/genética , Vacinas/imunologia
5.
Int J Mol Sci ; 20(23)2019 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-31771269

RESUMO

The lily (Lilium spp.) anther contains a lot of pollen. It is not known if lily pollen contains allergens, and therefore screening pollen allergy-related proteins and genes is necessary. The pollen development period of lily 'Siberia' was determined by microscope observation. Early mononuclear microspores and mature pollens were used as sequencing materials. The analysis of the pollen transcriptome identified differentially expressed genes (DEGs), e.g., Profilin, Phl p 7 (Polcalcin), Ole e 1, and Phl p 11, which are associated with pollen allergens. The proteome analysis positively verified a significant increase in pollen allergenic protein content. The expression levels of LoProfiilin and LoPolcalcin, annotated as allergen proteins, gradually increased in mature pollen. LoProfiilin and LoPolcalcin were cloned and their open reading frame lengths were 396 bp and 246 bp, which encoded 131 and 81 amino acids, respectively. Amino acid sequence and structure alignment indicated that the protein sequences of LoProfilin and LoPolcalcin were highly conserved. Subcellular localization analysis showed that LoProfilin protein was localized in the cell cytoplasm and nucleus. LoProfilin and LoPolcalcin were highly expressed in mature pollen at the transcriptional and protein levels. A tertiary structure prediction analysis identified LoProfilin and LoPolcalcin as potential allergens in lily pollen.


Assuntos
Alérgenos/metabolismo , Lilium/metabolismo , Pólen/metabolismo , Proteoma/metabolismo , Transcriptoma , Alérgenos/química , Alérgenos/genética , Sequência de Aminoácidos , Antígenos de Plantas/química , Antígenos de Plantas/genética , Antígenos de Plantas/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulação da Expressão Gênica de Plantas , Lilium/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pólen/genética , Estrutura Secundária de Proteína , Alinhamento de Sequência
6.
Int J Mol Sci ; 20(20)2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31614967

RESUMO

Rubber particles are a specific organelle for natural rubber biosynthesis (NRB) and storage. Ethylene can significantly improve rubber latex production by increasing the generation of small rubber particles (SRPs), regulating protein accumulation, and activating many enzyme activities. We conducted a quantitative proteomics study of different SRPs upon ethylene stimulation by differential in-gel electrophoresis (DIGE) and using isobaric tags for relative and absolute quantification (iTRAQ) methods. In DIGE, 79 differentially accumulated proteins (DAPs) were determined as ethylene responsive proteins. Our results show that the abundance of many NRB-related proteins has been sharply induced upon ethylene stimulation. Among them, 23 proteins were identified as rubber elongation factor (REF) and small rubber particle protein (SRPP) family members, including 16 REF and 7 SRPP isoforms. Then, 138 unique phosphorylated peptides, containing 129 phosphorylated amino acids from the 64 REF/SRPP family members, were identified, and most serine and threonine were phosphorylated. Furthermore, we identified 226 DAPs from more than 2000 SRP proteins by iTRAQ. Integrative analysis revealed that almost all NRB-related proteins can be detected in SRPs, and many proteins are positively responsive to ethylene stimulation. These results indicate that ethylene may stimulate latex production by regulating the accumulation of some key proteins. The phosphorylation modification of REF and SRPP isoforms might be crucial for NRB, and SRP may act as a complex natural rubber biosynthetic machine.


Assuntos
Antígenos de Plantas/genética , Hevea/genética , Látex/biossíntese , Proteínas de Plantas/genética , Sequência de Aminoácidos , Etilenos/metabolismo , Hevea/metabolismo , Proteoma/genética , Proteômica , Borracha/química , Borracha/metabolismo
7.
Int J Biol Macromol ; 141: 1287-1292, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31499107

RESUMO

ß-conglycinin is one of the most allergenic proteins, and its constituent subunits α', α, and ß are all potential allergens to humans. In the present study, we concentrated on the destructed antigenic sites of ß subunit of ß-conglycinin after high hydrostatic pressure (HHP) treatment. In this paper, the overlapping gene fragments of the ß subunit of ß-conglycinin were amplified by polymerase chain reaction (PCR) and cloned into T7 phage vectors. After being packaged in vitro, the recombinant T7 phage was constructed, and the overlapping fragments of the ß subunit were displayed on the phage surface. The recombinant phages that expressed the overlapping fragments of the ß subunit were used to react with specific antiserum by indirect ELISA to identify the HHP destructed antigenic sites. After three rounds of expression and identification, we used synthetic peptide technology to identify that the obtained fragment was a conformational epitope. We further confirmed that HHP treatment changed the conformational structure of ß-conglycinin, which reduced the antigenicity of the protein.


Assuntos
Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Epitopos/genética , Engenharia Genética , Globulinas/genética , Globulinas/imunologia , Subunidades Proteicas/genética , Subunidades Proteicas/imunologia , Proteínas de Armazenamento de Sementes/genética , Proteínas de Armazenamento de Sementes/imunologia , Proteínas de Soja/genética , Proteínas de Soja/imunologia , Sequência de Aminoácidos , Antígenos de Plantas/química , Bacteriófagos/genética , DNA Recombinante/genética , Globulinas/química , Pressão Hidrostática , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química , Proteínas de Armazenamento de Sementes/química , Proteínas de Soja/química
8.
J Agric Food Chem ; 67(40): 11219-11229, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31408330

RESUMO

Peanut allergy is a major health problem worldwide. Detection of food allergens is a critical aspect of food safety. The VHH domain of single chain antibody from camelids, also known as nanobody (Nb), showed its advantages in the development of biosensors because of its high stability, small molecular size, and ease of production. However, no nanobody specific to peanut allergens has been developed. In this study, we constructed a library with random triplets (NNK) in its CDR regions of a camel nanobody backbone. We screened the library with peanut allergy Ara h 3 and obtained several candidate nanobodies. One of the promising nanobodies, Nb16 was further biochemical characterization by gel filtration, isothermal titration calorimetry (ITC), cocrystallization, and Western blot in terms of its interaction with Ara h 3. Nb16 specifically binds to peanut major allergen Ara h 3 with a dissociation constant of 400 nM. Furthermore, we obtained the Ara h 3-Nb16 complex crystals. Structure analysis shows the packing mode is completely different between the Ara h 3-Nb16 complex crystal and the native Ara h 3 crystal. Structural determination of Ara h 3-Nb16 will provide the necessary information to understand the allergenicity of this important peanut allergen. The nanobody Nb16 may have application in the development of biosensors for peanut allergen detection.


Assuntos
Antígenos de Plantas/imunologia , Arachis/imunologia , Proteínas de Armazenamento de Sementes/imunologia , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Sequência de Aminoácidos , Antígenos de Plantas/química , Antígenos de Plantas/genética , Arachis/química , Arachis/genética , Bacteriófagos/genética , Bacteriófagos/metabolismo , Western Blotting , Técnicas de Visualização da Superfície Celular , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/genética , Anticorpos de Domínio Único/análise
9.
J Immunol ; 203(7): 1693-1700, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31462504

RESUMO

An allergic reaction is rapidly generated when allergens bind and cross-link IgE bound to its receptor FcεRI on effector cells, resulting in cell degranulation and release of proinflammatory mediators. The extent of effector cell activation is linked to allergen affinity, oligomeric state, valency, and spacing of IgE-binding epitopes on the allergen. Whereas most of these observations come from studies using synthetic allergens, in this study we have used Timothy grass pollen allergen Phl p 7 and birch pollen allergen Bet v 4 to study these effects. Despite the high homology of these polcalcin family allergens, Phl p 7 and Bet v 4 display different binding characteristics toward two human patient-derived polcalcin-specific IgE Abs. We have used native polcalcin dimers and engineered multimeric allergens to test the effects of affinity and oligomeric state on IgE binding and effector cell activation. Our results indicate that polcalcin multimers are required to stimulate high levels of effector cell degranulation when using the humanized RBL-SX38 cell model and that multivalency can overcome the need for high-affinity interactions.


Assuntos
Alérgenos/imunologia , Afinidade de Anticorpos , Antígenos de Plantas/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Degranulação Celular , Imunoglobulina E/imunologia , Modelos Imunológicos , Proteínas de Plantas/imunologia , Alérgenos/genética , Antígenos de Plantas/genética , Proteínas de Ligação ao Cálcio/genética , Epitopos/genética , Epitopos/imunologia , Células HEK293 , Humanos , Proteínas de Plantas/genética , Multimerização Proteica/genética , Multimerização Proteica/imunologia
10.
Immunology ; 158(2): 94-103, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31323138

RESUMO

Transgenic rice seeds that contain genetically modified Cry j 1 and Cry j 2, the two major allergens of Cryptomeria japonica (Japanese cedar; JC), have been developed as immunotherapeutic candidates for JC pollinosis. Because the transgenic rice (TG-rice) seeds express allergens containing whole amino acid sequences of Cry j 1 and Cry j 2 in the endosperm tissue (edible part of rice grain), they can potentially target all Cry j 1- and Cry j 2-specific T-cells. However, it was unknown whether antigenicity of Cry j 1 and Cry j 2 could be completely preserved in TG-rice seeds. We verified the antigenicity of TG-rice seeds to T-cells through the analysis of the proliferative responses of T-cells in Cry j 1- or Cry j 2-immunized mice or T-cell lines to TG-rice seed extract. First, four mouse strains were immunized with Cry j 1 or Cry j 2. T-cells in the immunized mice proliferated on treatment with TG-rice seed extract, but not non-transgenic wild-type rice (WT-rice) seed extract. Furthermore, T-cell lines were established from the spleen cells of the immunized mice. Each T-cell line resulted in a proliferative response to TG-rice seed extract, but not to WT-rice seed extract, suggesting that TG-rice seeds certainly express T-cell epitopes corresponding to T-cell lines. Considering the modified amino acid sequences of Cry j 1 and Cry j 2 in TG-rice seeds, the expression of specific T-cell epitopes suggested that TG-rice seeds express all possible T-cell epitope repertoires of Cry j 1 and Cry j 2.


Assuntos
Alérgenos/farmacologia , Antígenos de Plantas/imunologia , Epitopos de Linfócito T/imunologia , Oryza/química , Proteínas de Plantas/imunologia , Rinite Alérgica Sazonal/imunologia , Linfócitos T/efeitos dos fármacos , Alérgenos/genética , Alérgenos/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Plantas/química , Antígenos de Plantas/genética , Proliferação de Células/efeitos dos fármacos , Cryptomeria/genética , Cryptomeria/imunologia , Modelos Animais de Doenças , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Expressão Gênica , Imunização , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos , Oryza/genética , Oryza/imunologia , Mapeamento de Peptídeos , Extratos Vegetais/imunologia , Extratos Vegetais/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Pólen/genética , Pólen/imunologia , Cultura Primária de Células , Rinite Alérgica Sazonal/induzido quimicamente , Rinite Alérgica Sazonal/genética , Rinite Alérgica Sazonal/patologia , Sementes/química , Baço/efeitos dos fármacos , Baço/imunologia , Baço/patologia , Linfócitos T/imunologia , Linfócitos T/patologia , Transgenes
11.
J Agric Food Chem ; 67(31): 8626-8631, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31287307

RESUMO

An almond allergen with two known short peptide sequences was reported as the almond 2S albumin but was later suspected to be almond vicilin. However, this allergen was not designated by the World Health Organization/International Union of Immunological Societies. This study aimed to determine the true identity of this elusive almond allergen. cDNAs were synthesized from total RNA of the Nonpareil almond. The complete sequence of the previously reported almond allergen was determined from its coding sequence. The deduced protein was produced recombinantly and was confirmed to be a food allergen by testing with 18 almond-allergic sera. The allergen is a potential cysteine-rich antimicrobial protein with characteristic C[X]3C-[X]10-12-C[X]3C motifs of the hairpinin antimicrobial protein. This first member of a novel family of food allergens was named Pru du 8. The signature motif of the hairpinin antimicrobial protein can be found in the N-terminal region of some vicilin allergens (e.g., Ara h 1). It can also be found in the signal peptide of other vicilin allergens (e.g., Car i 2). In many species, however, vicilins do not contain such a motif, indicating that the presence of the signature motifs of the hairpinin antimicrobial protein in vicilins might be a result of translocation during evolution.


Assuntos
Alérgenos/imunologia , Antígenos de Plantas/imunologia , Prunus dulcis/imunologia , Alérgenos/química , Alérgenos/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Antígenos de Plantas/química , Antígenos de Plantas/genética , DNA Complementar/genética , Hipersensibilidade Alimentar/imunologia , Humanos , Prunus dulcis/química , Prunus dulcis/genética , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/genética , Proteínas de Armazenamento de Sementes/imunologia , Alinhamento de Sequência , Análise de Sequência de DNA
12.
Int J Biol Macromol ; 138: 97-105, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31302128

RESUMO

The inhalation of Chenopodium album (C. album) pollen, especially polcalcin (Che a 3), has been reported as a significant source of allergic respiratory symptoms. This study was conducted to characterize biophysical differences of recombinant polcalcin come from three different Escherichia coli strains using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), optimize recombinant polcalcin expression, and linear B-cell epitopes identification using in silico methods. ATR-FTIR results of purified proteins showed spectra intensity variance in the amide I region, while there were no changes in pick position and shape of the bands. SHuffle® T7 Express was selected for subsequent optimization due to ability in the correction of the mis-oxidized bonds and promotes proper folding which was validated by ATR-FTIR analysis results. Then, Response Surface Methodology was performed to optimize critical factors including induction temperature, duration of induction, and concentration of inducer. The best partitioning conditions were achieved in 1.5 mM IPTG for 10.97 h at 29.9 °C. Finally, prediction of polcalcin B-cell epitopes was carried out which indicated the presence of 4 different epitopes. Together, the results may help to the development of diagnostic approaches and also vaccine manufacture for desensitization and modulation of the allergic response in patients.


Assuntos
Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Simulação por Computador , Escherichia coli/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Espectroscopia de Infravermelho com Transformada de Fourier , Antígenos de Plantas/química , Sítios de Ligação , Proteínas de Ligação ao Cálcio/química , Mapeamento de Epitopos , Expressão Gênica , Imunoglobulina E/imunologia , Peso Molecular , Proteínas Recombinantes/química
13.
Int J Mol Sci ; 20(10)2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31096561

RESUMO

Pollinosis is sub-diagnosed and rarely studied in tropical countries. Cashew tree pollen has been reported as an allergen source although the knowledge of its immunoglobulin E (IgE)-reactive molecules is lacking. Therefore, this work aimed to identify IgE-reactive molecules and provide a proteomic profile of this pollen. From the 830 proteins identified by shotgun analysis, 163 were annotated to gene ontology, and a list of 39 proteins filtered for high confidence was submitted to the Allfam database where nine were assigned to allergenic families. Thus, 12 patients from the northeast of Brazil with persistent allergic rhinitis and aggravation of symptoms during cashew flowering season were selected. Using a 2D-based approach, we identified 20 IgE-reactive proteins, four already recognized as allergens, including a homolog of the birch isoflavone-reductase (Bet v 6). IgE-reactivity against the extract in native form was confirmed for five patients in ELISA, with three being positive for Bet v 6. Herein, we present a group of patients with rhinitis exposed to cashew tree pollen with the first description of IgE-binding proteins and a proteomic profile of the whole pollen. Cashew tree pollen is considered an important trigger of rhinitis symptoms in clinical practice in the northeast of Brazil, and the elucidation of its allergenic molecules can improve the diagnostics and treatment for allergic patients.


Assuntos
Alérgenos/imunologia , Anacardium/química , Imunoglobulina E/imunologia , Pólen/efeitos adversos , Pólen/química , Rinite Alérgica Sazonal/induzido quimicamente , Adolescente , Adulto , Idoso , Alérgenos/química , Animais , Antígenos de Plantas/efeitos adversos , Antígenos de Plantas/química , Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Betula/metabolismo , Brasil , Proteínas de Transporte/análise , Proteínas de Transporte/imunologia , Criança , Pré-Escolar , Reações Cruzadas/imunologia , Dermatophagoides farinae , Dermatophagoides pteronyssinus , Feminino , Humanos , Masculino , Proteínas de Plantas/efeitos adversos , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Pólen/genética , Proteômica , Rinite Alérgica Sazonal/imunologia , Testes Cutâneos
14.
Cells ; 8(4)2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991709

RESUMO

Fusion proteins incorporating the TLR5-ligand flagellin are currently undergoing clinical trials as vaccine candidates for many diseases. We recently reported a flagellin:allergen fusion protein containing the TLR5-ligand flagellin A (FlaA) from Listeria monocytogenes and the major birch pollen allergen Bet v 1 (rFlaA:Betv1) to prevent allergic sensitization in an experimental mouse model. This study analyzes the signaling pathways contributing to rFlaA:Betv1-mediated pro- and anti-inflammatory cytokine secretion and cell metabolism in myeloid dendritic cells (mDCs) in vitro. The influence of mammalian target of rapamycin (mTOR)-, NF?B-, and MAP kinase (MAPK)-signaling on cytokine secretion and metabolic activity of bone marrow (BM)-derived mDCs stimulated with rFlaA:Betv1 were investigated by pre-treatment with either mTOR- (rapamycin), NF?B- (dexamethason, BMS-345541, TPCA-1, triptolide, or BAY-11) or MAPK- (SP600125, U0126, or SB202190) inhibitors, respectively. rFlaA:Betv1-mediated IL-10 secretion as well as activation of mDC metabolism, rather than pro-inflammatory cytokine secretion, were inhibited by rapamycin. Inhibition of NFκB-signaling suppressed rFlaA:Betv1-induced IL-12, while inhibition of MAPK-signaling dose-dependently suppressed rFlaA:Betv1-induced IL-10 as well as pro-inflammatory IL-6 and TNF-α production. Notably, with the exception of a partial JNK-dependency, rFlaA:Betv1-mediated effects on mDC metabolism were mostly NF?B- and MAPK-independent. Therefore, MAPK-mediated activation of both NFκB- and mTOR-signaling likely is a key pathway for the production of pro- and anti-inflammatory cytokines by flagellin fusion protein vaccines.


Assuntos
Antígenos de Plantas/efeitos adversos , Células Dendríticas/imunologia , Hipersensibilidade a Drogas/imunologia , Flagelina/efeitos adversos , Sistema de Sinalização das MAP Quinases/imunologia , NF-kappa B/imunologia , Proteínas Recombinantes de Fusão/efeitos adversos , Animais , Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Citocinas/imunologia , Células Dendríticas/efeitos dos fármacos , Hipersensibilidade a Drogas/etiologia , Flagelina/imunologia , Flagelina/metabolismo , Interleucina-10/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Serina-Treonina Quinases TOR/imunologia , Vacinas/imunologia
15.
Int J Food Sci Nutr ; 70(6): 688-700, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30704309

RESUMO

The present work studied the effect of the year of harvest, the genotype and the cultivation method on the nutritional quality and the allergen content of three plum cultivars. The common quality parameters and the phytochemical content strongly varied with the year and the cultivar, while the system of cultivation had a minor influence. In particular, ascorbic acid greatly decreased in 2016 compared to 2015, while polyphenols were higher in 2016. The health-promoting compounds, and particularly phenolics, were significantly correlated with the antioxidant capacity. Finally, the allergen content was strongly dependent on the content of flavan-3-ols, suggesting that this class of phenolics is determinant in influencing the allergen content in plums. Results showed that the major factor affecting the quality and the concentration of natural metabolites of plum, in addition to the diversity among genotypes, is the year-to-year variation, whereas the system of cultivation plays a marginal role.


Assuntos
Alérgenos/análise , Genótipo , Agricultura Orgânica , Prunus domestica/química , Prunus domestica/crescimento & desenvolvimento , Antígenos de Plantas/análise , Antígenos de Plantas/genética , Antioxidantes/análise , Ácido Ascórbico/análise , Catecol Oxidase/análise , Frutas/química , Compostos Fitoquímicos/análise , Extratos Vegetais/química , Proteínas de Plantas/análise , Polifenóis/análise , Prunus domestica/genética , Estações do Ano , Tempo (Meteorologia)
16.
Talanta ; 196: 64-70, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30683412

RESUMO

Allergen genes of the peanut and soybean were selected as model targets. Four hairpin DNA probes, H1, H2, H3, H4 were designed. Cy3-labeled H1 and H2 were used to detect peanut DNA, while FAM-labeled H3 and H4 were used to detect soybean DNA. Graphene oxide (GO) was used as the adsorption material for capturing the hairpin probes, and as a selective fluorescence quencher to reduce the background signal. To develop an allergen gene detection system with a GO-based paper chip format, we integrated the hybridization chain reaction (HCR) with fluorescence resonance energy transfer (FRET) in our design. The results showed that in the absence of peanut DNA (TP) and soybean DNA (TS), the detection probes attached to the GO surface, which quenched their fluorescence. In the presence of TP or TS, however, complementary probe binding to the targets initiated HCR, producing long double-stranded DNA products that could not be absorbed onto the GO surface. Hence, a strong red or green fluorescent signal was generated. The detection limit for both peanut and soybean DNA was 1 nM using this method, indicating the high sensitivity of our approach. This method also exhibited good specificity and a single chip could be used to simultaneously detect two different targets.


Assuntos
Alérgenos/genética , Arachis/genética , Sondas de DNA/química , DNA de Plantas/análise , Grafite/química , Óxidos/química , Soja/genética , Antígenos de Plantas/genética , Transferência Ressonante de Energia de Fluorescência , Papel
17.
Mucosal Immunol ; 12(1): 132-144, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30242254

RESUMO

Allergic poly-sensitization affects a large number of allergic patients and poses a great challenge for their treatment. In this study we evaluated the effects of the probiotic Escherichia coli Nissle 1917 (EcN) expressing a birch and grass pollen allergen chimera 'Bet v 1, Phl p 1 and Phl p 5' (EcN-Chim) on allergy prevention after oral or intranasal application in poly-sensitized mice. In contrast to oral application, intranasal pretreatment with EcN-Chim prior to poly-sensitization led to a significant reduction of lung inflammation (eosinophils, IL-5, and IL-13 in bronchoalveolar lavage) along with suppressed levels of allergen-specific serum IgE. The suppression was associated with increased levels of allergen-specific IgA in lungs and serum IgG2a along with increased Foxp3, TGF-ß, and IL-10 mRNA in bronchial lymph nodes. In vitro EcN induced high levels of IL-10 and IL-6 in both lung and intestinal epithelial cells. Importantly, using in vivo imaging techniques we demonstrated that intranasally applied EcN do not permanently colonize nose, lung, and gut and this strain might therefore be a safe delivery vector against allergy in humans. In conclusion, our data show that intranasal application of recombinant EcN expressing a multiallergen chimera presents a novel and promising treatment strategy for prevention of allergic poly-sensitization.


Assuntos
Escherichia coli/genética , Vetores Genéticos/genética , Hipersensibilidade/imunologia , Membrana Mucosa/fisiologia , Alérgenos/genética , Alérgenos/imunologia , Animais , Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Betula/imunologia , Reações Cruzadas , Citocinas/genética , Citocinas/metabolismo , Feminino , Humanos , Imunização , Imunoglobulina E/sangue , Camundongos , Camundongos Endogâmicos BALB C , Microrganismos Geneticamente Modificados , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Poaceae/imunologia , Pólen/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinação
19.
J Agric Food Chem ; 67(1): 425-432, 2019 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-30512943

RESUMO

Almond is one of the tree nuts listed by U.S. FDA as a food allergen source. A food allergen identified with patient sera has been debated to be the 2S albumin or the 7S vicilin. However, neither of these proteins has been defined as a food allergen. The purpose of this study was to clone, express, and purify almond vicilin and test whether it is a food allergen. Western blot experiment was performed with 18 individual sera from patients with double-blind, placebo-controlled clinical almond allergy. The results showed that 44% of the sera contained IgE antibodies that recognized the recombinant almond vicilin, indicating that it is an almond allergen. Identifying this and additional almond allergens will facilitate the understanding of the allergenicity of seed proteins in tree nuts and their cross-reactivity.


Assuntos
Antígenos de Plantas/imunologia , Hipersensibilidade Alimentar/imunologia , Prunus dulcis/imunologia , Proteínas de Armazenamento de Sementes/imunologia , Sequência de Aminoácidos , Antígenos de Plantas/química , Antígenos de Plantas/genética , Reações Cruzadas , Hipersensibilidade Alimentar/sangue , Humanos , Imunoglobulina E/imunologia , Dados de Sequência Molecular , Prunus dulcis/química , Prunus dulcis/genética , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/genética , Sementes/química , Sementes/genética , Sementes/imunologia , Alinhamento de Sequência
20.
J Plant Physiol ; 233: 1-11, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30572279

RESUMO

Abiotic and biotic stress situations cause the upregulation of the transcription of a number of plant defence genes. They code for so-called pathogenesis-related (PR) proteins such as PR proteins of class-10 (PR-10), whose biological functions are still unclear. PR10 proteins are members of the Bet v 1 (major birch pollen allergen) superfamily including related proteins from the cultivated strawberry Fragaria × ananassa (Fra a 1 proteins). Here, we analyzed the expression of 21 Fra a 1 genes in different tissues of the strawberry plant by quantitative real-time PCR. Thirteen members were mainly expressed in roots, three in stems, two in red fruits and leaves, and one in flowers. Five genes (Fra a 1.04-1.08) were selected based on their expression profiles, heterologously expressed in Escherichia coli, and their recombinant proteins functionally characterized. Ribonuclease activity, demonstrated by in-solution and in-gel RNA degradation assays, indicated complete hydrolysis of RNA only by Fra a 1.06. Moreover, phosphorylation assays showed that except for Fra a 1.06, the remaining four recombinant proteins were phosphorylated. Consequently, we investigated whether the phosphorylation status of the proteins affects their ribonuclease activity. Using an in-solution as well as an in-gel RNase activity assay, results demonstrated that the four recombinant proteins, dephosphorylated with phosphatases, exhibited ribonucleolytic activity against total RNA. Thus, the PR10 related proteins characterized in this study harbour a phosphorylation-dependent RNase activity. The results shed new light on the assumed function of PR10 proteins in plant defence.


Assuntos
Antígenos de Plantas/metabolismo , Ribonucleases/metabolismo , Antígenos de Plantas/genética , Escherichia coli , Flores/metabolismo , Fragaria/genética , Fragaria/metabolismo , Frutas/metabolismo , Genes de Plantas , Microrganismos Geneticamente Modificados , Fosforilação , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Caules de Planta/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes
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