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1.
Viruses ; 16(7)2024 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-39066287

RESUMO

Food allergy (FA) is estimated to impact up to 10% of the population and is a growing health concern. FA results from a failure in the mucosal immune system to establish or maintain immunological tolerance to innocuous dietary antigens, IgE production, and the release of histamine and other mediators upon exposure to a food allergen. Of the different FAs, peanut allergy has the highest incidence of severe allergic responses, including systemic anaphylaxis. Despite the recent FDA approval of peanut oral immunotherapy and other investigational immunotherapies, a loss of protection following cessation of therapy can occur, suggesting that these therapies do not address the underlying immune response driving FA. Our lab has shown that liver-directed gene therapy with an adeno-associated virus (AAV) vector induces transgene product-specific regulatory T cells (Tregs), eradicates pre-existing pathogenic antibodies, and protects against anaphylaxis in several models, including ovalbumin induced FA. In an epicutaneous peanut allergy mouse model, the hepatic AAV co-expression of four peanut antigens Ara h1, Ara h2, Ara h3, and Ara h6 together or the single expression of Ara h3 prevented the development of a peanut allergy. Since FA patients show a reduction in Treg numbers and/or function, we believe our approach may address this unmet need.


Assuntos
Dependovirus , Vetores Genéticos , Hipersensibilidade a Amendoim , Hipersensibilidade a Amendoim/terapia , Hipersensibilidade a Amendoim/imunologia , Animais , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Humanos , Dependovirus/genética , Dependovirus/imunologia , Terapia Genética/métodos , Linfócitos T Reguladores/imunologia , Camundongos , Imunoterapia/métodos , Modelos Animais de Doenças , Dessensibilização Imunológica/métodos , Alérgenos/imunologia , Arachis/imunologia
2.
J Allergy Clin Immunol ; 154(2): 492-497.e1, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38593870

RESUMO

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin-3 (Siglec-3 [CD33]) is a major Siglec expressed on human mast cells and basophils; engagement of CD33 leads to inhibition of cellular signaling via immunoreceptor tyrosine-based inhibitory motifs. OBJECTIVE: We sought to inhibit human basophil degranulation by simultaneously recruiting inhibitory CD33 to the IgE-FcεRI complex by using monoclonal anti-IgE directly conjugated to CD33 ligand (CD33L). METHODS: Direct and indirect basophil activation tests (BATs) were used to assess both antigen-specific (peanut) and antigen-nonspecific (polyclonal anti-IgE) stimulation. Whole blood from donors with allergy was used for direct BAT, whereas blood from donors with nonfood allergy was passively sensitized with plasma from donors with peanut allergy in the indirect BAT. Blood was incubated with anti-IgE-CD33L or controls for 1 hour or overnight and then stimulated with peanut, polyclonal anti-IgE, or N-formylmethionyl-leucyl-phenylalanine for 30 minutes. Degranulation was determined by measuring CD63 expression on the basophil surface by flow cytometry. RESULTS: Incubation for 1 hour with anti-IgE-CD33L significantly reduced basophil degranulation after both allergen-induced (peanut) and polyclonal anti-IgE stimulation, with further suppression after overnight incubation with anti-IgE-CD33L. As expected, anti-IgE-CD33L did not block basophil degranulation due to N-formylmethionyl-leucyl-phenylalanine, providing evidence that this inhibition is IgE pathway-specific. Finally, CD33L is necessary for this suppression, as monoclonal anti-IgE without CD33L was unable to reduce basophil degranulation. CONCLUSIONS: Pretreating human basophils with anti-IgE-CD33L significantly suppressed basophil degranulation through the IgE-FcεRI complex. The ability to abrogate IgE-mediated basophil degranulation is of particular interest, as treatment with anti-IgE-CD33L before antigen exposure could have broad implications for the treatment of food, drug, and environmental allergies.


Assuntos
Basófilos , Degranulação Celular , Imunoglobulina E , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Humanos , Basófilos/imunologia , Imunoglobulina E/imunologia , Degranulação Celular/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Tetraspanina 30/imunologia , Tetraspanina 30/metabolismo , Receptores de IgE/imunologia , Receptores de IgE/metabolismo , Hipersensibilidade a Amendoim/imunologia , Teste de Degranulação de Basófilos , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Anti-Idiotípicos/farmacologia
3.
Front Immunol ; 15: 1299484, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38380329

RESUMO

Introduction: Peanut allergy is an immunoglobulin E (IgE) mediated food allergy. Rubia cordifolia L. (R. cordifolia), a Chinese herbal medicine, protects against peanut-induced anaphylaxis by suppressing IgE production in vivo. This study aims to identify IgE-inhibitory compounds from the water extract of R. cordifolia and investigate the underlying mechanisms using in vitro and in vivo models. Methods: Compounds were isolated from R. cordifolia water extract and their bioactivity on IgE production was assessed using a human myeloma U266 cell line. The purified active compound, xanthopurpurin (XPP), was identified by LC-MS and NMR. Peanut-allergic C3H/HeJ mice were orally administered with or without XPP at 200µg or 400µg per mouse per day for 4 weeks. Serum peanut-specific IgE levels, symptom scores, body temperatures, and plasma histamine levels were measured at challenge. Cytokines in splenocyte cultures were determined by ELISA, and IgE + B cells were analyzed by flow cytometry. Acute and sub-chronic toxicity were evaluated. IL-4 promoter DNA methylation, RNA-Seq, and qPCR analysis were performed to determine the regulatory mechanisms of XPP. Results: XPP significantly and dose-dependently suppressed the IgE production in U266 cells. XPP significantly reduced peanut-specific IgE (>80%, p <0.01), and plasma histamine levels and protected the mice against peanut-allergic reactions in both early and late treatment experiments (p < 0.05, n=9). XPP showed a strong protective effect even 5 weeks after discontinuing the treatment. XPP significantly reduced the IL-4 level without affecting IgG or IgA and IFN-γ production. Flow cytometry data showed that XPP reduced peripheral and bone marrow IgE + B cells compared to the untreated group. XPP increased IL-4 promoter methylation. RNA-Seq and RT-PCR experiments revealed that XPP regulated the gene expression of CCND1, DUSP4, SDC1, ETS1, PTPRC, and IL6R, which are related to plasma cell IgE production. All safety testing results were in the normal range. Conclusions: XPP successfully protected peanut-allergic mice against peanut anaphylaxis by suppressing IgE production. XPP suppresses murine IgE-producing B cell numbers and inhibits IgE production and associated genes in human plasma cells. XPP may be a potential therapy for IgE-mediated food allergy.


Assuntos
Anafilaxia , Hipersensibilidade Alimentar , Hipersensibilidade a Amendoim , Camundongos , Humanos , Animais , Hipersensibilidade a Amendoim/terapia , Anafilaxia/prevenção & controle , Histamina , Interleucina-4 , Medula Óssea , Camundongos Endogâmicos C3H , Imunoglobulina E , Água
4.
Int J Biol Macromol ; 258(Pt 1): 128340, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38000575

RESUMO

Interactions between plant polyphenols and food allergens may be a new way to alleviate food allergies. The non-covalent interactions between the major allergen from peanut (Ara h 2) with procyanidin dimer (PA2) were therefore characterized using spectroscopic, thermodynamic, and molecular simulation analyses. The main interaction between the Ara h 2 and PA2 was hydrogen bonding. PA2 statically quenched the intrinsic fluorescence intensity and altered the conformation of the Ara h 2, leading to a more disordered polypeptide structure with a lower surface hydrophobicity. In addition, the in vitro allergenicity of the Ara h 2-PA2 complex was investigated using enzyme-linked immunosorbent assay (ELISA) kits. The immunoglobulin E (IgE) binding capacity of Ara h 2, as well as the release of allergenic cytokines, decreased after interacting with PA2. When the ratio of Ara h 2-to-PA2 was 1:50, the IgE binding capacity was reduced by around 43 %. This study provides valuable insights into the non-covalent interactions between Ara h 2 and PA2, as well as the potential mechanism of action of the anti-allergic reaction caused by binding of the polyphenols to the allergens.


Assuntos
Hipersensibilidade a Amendoim , Proantocianidinas , Arachis/química , Antígenos de Plantas/química , Alérgenos/química , Proantocianidinas/metabolismo , Glicoproteínas/química , Imunoglobulina E/metabolismo , Polifenóis/metabolismo , Proteínas de Plantas/química
5.
Allergy ; 79(2): 445-455, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37916710

RESUMO

BACKGROUND: Conventional basophil activation tests (BATs) measure basophil activation by the increased expression of CD63. Previously, fluorophore-labeled avidin, a positively-charged molecule, was found to bind to activated basophils, which tend to expose negatively charged granule constituents during degranulation. This study further compares avidin versus CD63 as basophil activation biomarkers in classifying peanut allergy. METHODS: Seventy subjects with either a peanut allergy (N = 47), a food allergy other than peanut (N = 6), or no food allergy (N = 17) were evaluated. We conducted BATs in response to seven peanut extract (PE) concentrations (0.01-10,000 ng/mL) and four control conditions (no stimulant, anti-IgE, fMLP (N-formylmethionine-leucyl-phenylalanine), and anti-FcεRI). We measured avidin binding and CD63 expression on basophils with flow cytometry. We evaluated logistic regression and XGBoost models for peanut allergy classification and feature identification. RESULTS: Avidin binding was correlated with CD63 expression. Both markers discriminated between subjects with and without a peanut allergy. Although small by percentage, an avidin+ /CD63- cell subset was found in all allergic subjects tested, indicating that the combination of avidin and CD63 could allow a more comprehensive identification of activated basophils. Indeed, we obtained the best classification accuracy (97.8% sensitivity, 96.7% specificity) by combining avidin and CD63 across seven PE doses. Similar accuracy was obtained by combining PE dose of 10,000 ng/mL for avidin and PE doses of 10 and 100 ng/mL for CD63. CONCLUSIONS: Avidin and CD63 are reliable BAT activation markers associated with degranulation. Their combination enhances the identification of activated basophils and improves the classification accuracy of peanut allergy.


Assuntos
Teste de Degranulação de Basófilos , Hipersensibilidade a Amendoim , Humanos , Hipersensibilidade a Amendoim/diagnóstico , Hipersensibilidade a Amendoim/metabolismo , Avidina/metabolismo , Imunoglobulina E/metabolismo , Basófilos/metabolismo , Citometria de Fluxo , Arachis , Tetraspanina 30/metabolismo
6.
J Allergy Clin Immunol Pract ; 12(2): 310-315, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38151118

RESUMO

This article explores the multifaceted approach of food allergy (FA) advocacy, research, and education to address the diverse challenges associated with FA, such as disparities in socioeconomic status, food security, quality of life, and the overall burden of the disease. Advocacy initiatives are instrumental in driving policy changes, raising public awareness, and directing substantial research funding, with a focus on reducing disparities. They have influenced allergen labeling regulations and improved access to epinephrine, emphasizing the importance of school-based management plans, especially in underserved communities. Research in FA informs medical practices and offers them hope for improved treatments. Recent breakthroughs in peanut allergy prevention and oral immunotherapy trials exemplify the potential for advancements while highlighting the need to address disparities in health care access. Education is a critical tool for prevention, raising awareness, and reducing the risk of allergic reactions. Efforts should be tailored to reach marginalized communities, particularly in schools where education on FA management is essential. Collaborating directly with communities is imperative to ensure inclusivity and address disparities. Barriers such as mistrust, language and cultural differences, and lack of diversity among researchers must be overcome to encourage diverse participation in research studies. This article concludes by emphasizing the significance of a comprehensive approach to FA research that prioritizes equity and inclusivity. The call to action highlights the need for global initiatives to reshape the landscape of FA care and address disparities in health care access and outcomes.


Assuntos
Hipersensibilidade Alimentar , Hipersensibilidade a Amendoim , Humanos , Qualidade de Vida , Hipersensibilidade Alimentar/terapia , Hipersensibilidade Alimentar/prevenção & controle , Escolaridade , Epinefrina , Hipersensibilidade a Amendoim/epidemiologia , Hipersensibilidade a Amendoim/prevenção & controle
7.
Front Immunol ; 14: 1293158, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38022648

RESUMO

Introduction: Peanut allergy is one of the most prevalent food allergies globally. Currently, most research into the mechanisms involved in protein allergy focuses on the protein allergens under investigation, and information on the function of accompanying compounds, such as lipids, is scarce. Thus, this research investigates the role of peanut-associated lipids and invariant natural killer T (iNKT) cells in peanut allergy using a novel, human, in vitro assay. Methods: PBMCs from non-allergic and peanut-allergic subjects were stimulated with the glycolipid, α-Galactosylceramide (α-GalCer), over 14 days for iNKT cell expansion. Autologous dendritic cells (DCs) were stimulated with either peanut oil, the lipid-binding peanut allergen, Ara h 8, or both peanut oil and Ara h 8. The expanded iNKT cells were then immunomagnetically isolated and co-cultured for 5 h with autologous DCs, and cytokine expression was measured by flow cytometry. Results: A 5-fold higher iNKT cell population was observed in peanut-allergic subject peripheral blood compared to non-allergic controls. In all subjects, conventional flow analysis highlighted iNKTs co-cultured with autologous α-GalCer-pulsed DCs displayed increased IL-4 and IFN-y secretion within 5 hours of co-culture. A 10-parameter unsupervised clustering analysis of iNKT phenotype found significantly more CD3+CD8+CD25+IL-4+IL-5+IL-10+IFNγ+ cells in non-allergic adults following culture with peanut oil. Conclusion: For the first time, we show iNKT cells are more abundant in peanut-allergic adults compared to non-allergic adults, and peanut lipid-exposed iNKT cells resulted in the identification of a subset of CD8+ iNKT cells which was significantly lower in peanut-allergic adults. Thus, this study proposes a role for iNKT cells and peanut allergen-associated lipids in peanut allergy.


Assuntos
Células T Matadoras Naturais , Hipersensibilidade a Amendoim , Humanos , Adulto , Óleo de Amendoim , Arachis , Interleucina-4 , Linfócitos T CD8-Positivos , Alérgenos
8.
Mol Nutr Food Res ; 67(22): e2300134, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37706599

RESUMO

SCOPE: The unstructured region of Ara h 2, referred to as epitope 3, contains a repeated motif, DYPSh (h = hydroxyproline) that is important for IgE binding. METHODS AND RESULTS: IgE binding assays to 20mer and shorter peptides of epitope 3, defines a 16mer core sequence containing one copy of the DPYSh motif, DEDSYERDPYShSQDP. This study performs alanine scanning of this and a related 12mer mimotope, LLDPYAhRAWTK. IgE binding, using a pool of 10 sera and with individual sera, is greatly reduced when alanine is substituted for aspartate at position 8 (D8; p < 0.01), tyrosine at position 10 (Y10; p < 0.01), and hydroxyproline at position 12 (h12; p < 0.001). IgE binding to alanine-substituted peptides of a mimotope containing the DPY_h motif confirm the critical importance of Y (p < 0.01) and h (p < 0.01), but not D. Molecular modeling of the core and mimotope suggests an h-dependent conformational basis for the recognition of these sequences by polyclonal IgE. CONCLUSIONS: IgE from pooled sera and individual sera differentially bound amino acids throughout the sequences of Epitope 3 and its mimotope, with Y10 and h12 being most important for all sera. These results are highly significant for designing hypoallergenic forms of Ara h 2.


Assuntos
Aminoácidos , Hipersensibilidade a Amendoim , Humanos , Sequência de Aminoácidos , Antígenos de Plantas/química , Alanina , Hidroxiprolina , Epitopos , Proteínas de Plantas/química , Peptídeos , Imunoglobulina E/metabolismo , Albuminas 2S de Plantas , Alérgenos/química
9.
Immunobiology ; 228(6): 152731, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37607433

RESUMO

Food allergies, which lead to life-threatening acute symptoms, are considered an important public health problem. Therefore, it is essential to develop efficient preventive and treatment measures. We developed a crude peanut protein extract (PPE)-induced allergy mouse model to investigate the effects of lycopene on peanut allergy. Mice were divided into four groups: 5 mg/kg lycopene, 20 mg/kg lycopene, no treatment, and control groups. Serum inflammatory factors were detected using enzyme-linked immunosorbent assay. In addition, pathology and immunohistochemistry analyses were used to examine the small intestine of mice. We found that lycopene decreased PPE-specific immunoglobulin E (IgE) and IL-13 levels in the serum, relieved small intestine inflammation, attenuated the production of histamine and mouse mast cell protease-1, and downregulated PI3K and AKT1 expression in the small intestine tissues of mice allergic to peanuts. Our results suggest that lycopene can ameliorate allergy by attenuating the PI3K/AKT pathway and the anaphylactic reactions mediated by PPE-specific IgE.


Assuntos
Hipersensibilidade Alimentar , Hipersensibilidade a Amendoim , Camundongos , Animais , Arachis/metabolismo , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Licopeno , Camundongos Endogâmicos BALB C , Hipersensibilidade Alimentar/tratamento farmacológico , Hipersensibilidade a Amendoim/tratamento farmacológico , Hipersensibilidade a Amendoim/patologia , Imunoglobulina E , Alérgenos
10.
J Allergy Clin Immunol Pract ; 11(11): 3485-3492.e2, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37495080

RESUMO

BACKGROUND: A precise diagnosis of peanut allergy is extremely important. We identified 4 Ara h 2 peptides that improved Ara h 2-specific IgE (sIgE) diagnostic accuracy. OBJECTIVE: To assess the diagnostic utility of sIgE to the mixture of these peptides and their role in mast cell response to peanut allergens. METHODS: sIgE to the peptide mix was determined using ImmunoCAP. Its diagnostic utility was compared with Ara h 2-sIgE and sIgE to the individual peptides. The functional relevance of the peptides was tested on the mast cell activation test using laboratory of allergic diseases 2 cell line and flow cytometry. RESULTS: A total of 52 peanut-allergic (PA), 36 peanut-sensitized but tolerant, and 9 nonsensitized nonallergic children were studied. Peptide mix-sIgE improved the diagnostic performance of Ara h 2-sIgE compared with Ara h 2-sIgE alone (area under the receiver operating characteristic curve .92 vs .89, respectively; P = .056). The sensitivity and specificity of Ara h 2-sIgE combined with the peptide mix were 85% and 96%, respectively. sIgE to individual peptides had the highest specificity (91%-96%) but the lowest sensitivity (10%-52%) compared with Ara h 2-sIgE (69% specificity and 87% sensitivity) or with peptide mix-sIgE (82% specificity and 63% sensitivity). Peptide 3 directly induced mast cell activation, and the peptide mix inhibited Ara h 2-induced activation of mast cells sensitized with plasma from Ara h 2-positive PA patients. CONCLUSIONS: sIgE to the peptide mix improved the diagnostic performance of Ara h 2-sIgE similarly to sIgE to individual peptides. The peptides interfered with Ara h 2-induced mast cell activation, confirming its relevance in peanut allergy.


Assuntos
Hipersensibilidade a Amendoim , Criança , Humanos , Hipersensibilidade a Amendoim/diagnóstico , Mastócitos , Antígenos de Plantas , Imunoglobulina E , Albuminas 2S de Plantas , Arachis , Alérgenos , Peptídeos
11.
J Clin Invest ; 133(16)2023 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-37384412

RESUMO

BACKGROUNDIgE-mediated anaphylaxis is a potentially fatal systemic allergic reaction for which there are no currently FDA-approved preventative therapies. Bruton's tyrosine kinase (BTK) is an essential enzyme for IgE-mediated signaling pathways and is an ideal pharmacologic target to prevent allergic reactions. In this open-label trial, we evaluated the safety and efficacy of acalabrutinib, a BTK inhibitor that is FDA approved to treat some B cell malignancies, in preventing clinical reactivity to peanut in adults with peanut allergy.METHODSAfter undergoing graded oral peanut challenge to establish their baseline level of clinical reactivity, 10 patients had a 6-week rest period, then received 4 standard doses of 100 mg acalabrutinib twice daily and underwent repeat food challenge. The primary endpoint was the change in patients' threshold dose of peanut protein to elicit an objective clinical reaction.RESULTSAt baseline, patients tolerated a median of 29 mg of peanut protein before objective clinical reaction. During subsequent food challenge on acalabrutinib, patients' median tolerated dose significantly increased to 4,044 mg (range 444-4,044 mg). 7 patients tolerated the maximum protocol amount (4,044 mg) of peanut protein with no clinical reaction, and the other 3 patients' peanut tolerance increased between 32- and 217-fold. 3 patients experienced a total of 4 adverse events that were considered to be possibly related to acalabrutinib; all events were transient and nonserious.CONCLUSIONAcalabrutinib pretreatment achieved clinically relevant increases in patients' tolerance to their food allergen, thereby supporting the need for larger, placebo-controlled trials.TRIAL REGISTRATIONClinicalTrials.gov NCT05038904FUNDINGAstraZeneca Pharmaceuticals, the Johns Hopkins Institute for Clinical and Translational Research, the Ludwig Family Foundation, and NIH grants AI143965 and AI106043.


Assuntos
Anafilaxia , Hipersensibilidade a Amendoim , Adulto , Humanos , Anafilaxia/prevenção & controle , Tirosina Quinase da Agamaglobulinemia , Benzamidas/farmacologia , Pirazinas/efeitos adversos , Hipersensibilidade a Amendoim/tratamento farmacológico , Hipersensibilidade a Amendoim/prevenção & controle , Alérgenos , Arachis
12.
J Agric Food Chem ; 71(23): 9110-9119, 2023 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-37256970

RESUMO

Given that roasting changes the structure and allergenicity of peanut allergens, the structural information of peanut allergens must be expounded to explain the alteration in their allergenicity. This work focused on allergen aggregations (AAs) in roasted peanuts. IgE recognition capability was assessed via western blot analysis. The disulfide bond (DB) rearrangement and chemical modification in AAs were identified by combining mass spectroscopy and software tools, and structural changes induced by cross-links were displayed by molecular dynamics and PyMOL software. Results showed that AAs were strongly recognized by IgE and cross-linked mainly by DBs. The types of DB rearrangement in AAs included interprotein (98 peptide pairs), intraprotein (22 peptide pairs), and loop-linked (6 peptides) DBs. Among allergens, Ara h 2 and Ara h 6 presented the most cysteine residues to cross-linkf with others or themselves. DB rearrangement involved IgE epitopes and induced structural changes. Ara h 1 and Ara h 3 were predominantly chemically modified. Moreover, chemical modification altered the local structures of proteins, which may change the allergenic potential of allergens.


Assuntos
Arachis , Hipersensibilidade a Amendoim , Arachis/química , Alérgenos/química , Proteínas de Plantas/química , Antígenos de Plantas/química , Imunoglobulina E/metabolismo , Dissulfetos , Albuminas 2S de Plantas
13.
Clin Exp Allergy ; 53(6): 636-647, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37038893

RESUMO

BACKGROUND: Clinical and experimental analyses indicate a pathognomonic role for allergen IgE crosslinking through epitope-paratope interactions as a major initial step in the cascade leading to effector cell activation and clinical manifestations of lgE-mediated food allergies. We aimed to undertake the initial development and assessment of Ara h 2-specific IgE epitope-like peptides that can bind to allergen-specific IgE paratopes and suppress effector cell activation. METHODS: We performed biopanning, screening, IgE binding, selection and mapping of peptides. We generated synthetic peptides for use in all functional experiments. ImmunoCAP inhibition, basophil and mast cell activation tests, with LAD2 cells, a human mast cell line were performed. Twenty-six children or young adults who had peanut allergy were studied. RESULTS: We identified and selected three linear peptides (DHPRFNRDNDVA, DHPRYGP and DHPRFST), and immunoblot analyses revealed binding to lgE from peanut-allergic individuals. The peptide sequences were aligned to the disordered region corresponding to the loop between helices 2 and 3 of Ara h 2, and conformational mapping showed that the peptides match the surface of Ara h 2 and h 6 but not other peanut allergens. In ImmunoCAP inhibition experiments, the peptides significantly inhibit the binding of IgE to Ara h 2 (p < .001). In basophil and mast cell activation tests, the peptides significantly suppressed Ara h 2-induced effector cell activation (p < .05) and increased the half-maximal Ara h 2 effective concentration (p < .05). Binding of the peptides to specific IgEs did not induce activation of basophils or mast cells. CONCLUSIONS: These studies show that the indicated peptides reduce the allergenic activity of Ara h 2 and suppress lgE-dependent basophil and mast cell activation. These observations may suggest a novel therapeutic strategy for food allergy based on epitope-paratop blocking.


Assuntos
Hipersensibilidade Alimentar , Hipersensibilidade a Amendoim , Criança , Adulto Jovem , Humanos , Epitopos , Antígenos de Plantas , Glicoproteínas , Peptídeos , Imunoglobulina E , Alérgenos , Arachis , Albuminas 2S de Plantas
14.
J Allergy Clin Immunol ; 152(2): 436-444.e6, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37028524

RESUMO

BACKGROUND: Surprisingly, IgE cross-reactivity between the major peanut allergens Ara h 1, 2, and 3 has been reported despite very low sequence identities. OBJECTIVE: We investigated the unexpected cross-reactivity between peanut major allergens. METHODS: Cross-contamination of purified natural Ara h 1, 2, 3, and 6 was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot test, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and sandwich enzyme-linked immunosorbent assay (ELISA). IgE cross-reactivity was studied with sera of peanut-allergic patients (n = 43) by ELISA and ImmunoCAP inhibition using both intact natural and recombinant allergens and synthetic peptides representing postulated Ara h 1 and Ara h 2 cross-reactive epitopes. RESULTS: Both purified nAra h 1 and nAra h 3 were demonstrated to contain small but significant amounts of Ara h 2 and Ara h 6 (<1%) by sandwich ELISA, SDS-PAGE/Western blot analysis, and LC-MS/MS. IgE cross-inhibition between both 2S albumins and Ara h 1 and Ara h 3 was only observed when using natural purified allergens, not recombinant allergens or synthetic peptides. Apparent cross-reactivity was lost when purified nAra h 1 was pretreated under reducing conditions, suggesting that Ara h 2 and Ara h 6 contaminations may be covalently bound to Ara h 1 via disulfide interactions. CONCLUSION: True cross-reactivity of both peanut 2S albumins with Ara h 1 and Ara h 3 could not be demonstrated. Instead, cross-contamination with small quantities was shown to be sufficient to cause significant cross-inhibition that can be misinterpreted as molecular cross-reactivity. Diagnostic tests using purified nAra h 1 and nAra h 3 can overestimate their importance as major allergens as a result of the presence of contaminating 2S albumins, making recombinant Ara h 1 and Ara h 3 a preferred alternative.


Assuntos
Alérgenos , Hipersensibilidade a Amendoim , Humanos , Alérgenos/química , Proteínas de Plantas/química , Arachis , Antígenos de Plantas/metabolismo , Cromatografia Líquida , Imunoglobulina E , Espectrometria de Massas em Tandem , Albuminas 2S de Plantas , Peptídeos/metabolismo , Albuminas/metabolismo , Hipersensibilidade a Amendoim/diagnóstico
15.
Food Res Int ; 164: 112297, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36737898

RESUMO

Peanuts are prone to trigger allergic reactions with high mortality rate. There is currently no effective way to prevent peanut allergy. In order to reduce the allergy risk of peanuts, it's significant to reduce sensitization of peanut prior to ingestion. In this study, the effects of five major apple polyphenols (epicatechin, phlorizin, rutin, chlorogenic acid, and catechin) -peanut protein on the sensitization of peanut allergens were studied by BALB/c peanut allergy model to access the contribution of each polyphenol in apple to peanut allergen sensitization reduction. Then, the mechanism was explored in terms of the effect of polyphenols on the simulated gastric digestion of peanut protein and the changes in structure of Ara h 1. The results showed that polyphenol binding could alleviate allergencitiy of peanut and regulate MAPK related signaling pathway. Among the five major apple polyphenols, epicatechin had the strongest inhibitory effect. The binding of epicatechin to the constitutive epitopes arginine led to changes in the spatial structure of Ara h 1, which resulted in the effective linear epitopes reduction. Modification of peanut allergens with polyphenols could effectively reduce the sensitization of peanut protein.


Assuntos
Catequina , Hipersensibilidade a Amendoim , Arachis , Hipersensibilidade a Amendoim/prevenção & controle , Polifenóis , Alérgenos/metabolismo , Imunoglobulina E/metabolismo , Epitopos
18.
Allergy ; 78(2): 500-511, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36377289

RESUMO

BACKGROUND: Food allergy affects up to 10% of the pediatric population. Despite ongoing efforts, treatment options remain limited. Novel models of food allergy are needed to study response patterns downstream of IgE-crosslinking and evaluate drugs modifying acute events. Here, we report a novel human ex vivo model that displays acute, allergen-specific, IgE-mediated smooth muscle contractions using precision cut intestinal slices (PCIS). METHODS: PCIS were generated using gut tissue samples from children who underwent clinically indicated surgery. Viability and metabolic activity were assessed from 0 to 24 h. Distribution of relevant cell subsets was confirmed using single nucleus RNA sequencing. PCIS were passively sensitized using plasma from peanut allergic donors or peanut-sensitized non-allergic donors, and exposed to various stimuli including serotonin, histamine, FcɛRI-crosslinker, and food allergens. Smooth muscle contractions and mediator release functioned as readouts. A novel program designed to measure contractions was developed to quantify responses. The ability to demonstrate the impact of antihistamines and immunomodulation from peanut oral immunotherapy (OIT) was assessed. RESULTS: PCIS viability was maintained for 24 h. Cellular distribution confirmed the presence of key cell subsets including mast cells. The video analysis tool reliably quantified responses to different stimulatory conditions. Smooth muscle contractions were allergen-specific and reflected the clinical phenotype of the plasma donor. Tryptase measurement confirmed IgE-dependent mast cell-derived mediator release. Antihistamines suppressed histamine-induced contraction and plasma from successful peanut OIT suppressed peanut-specific PCIS contraction. CONCLUSION: PCIS represent a novel human tissue-based model to study acute, IgE-mediated food allergy and pharmaceutical impacts on allergic responses in the gut.


Assuntos
Hipersensibilidade Alimentar , Hipersensibilidade a Amendoim , Humanos , Criança , Histamina , Hipersensibilidade a Amendoim/terapia , Alérgenos , Imunoglobulina E , Arachis
19.
Nutrients ; 16(1)2023 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-38201880

RESUMO

BACKGROUND: Data suggest that food allergies greatly impact a child's health and growth due to inadequate nutrient intake. Our study aimed to establish the long-term outcome of children with food allergies compared to a control group. METHODS: This study was a retrospective cohort study with longitudinal follow-up with a mean period of 4.85 years from the diagnosis to the last study visit. The patients' nutritional intake was assessed using a three-day food diary and analysed by a dietitian. Patients (61 boys and 33 girls, mean age 6.9 years) had a single food allergy including 21 patients with cow's milk, 34 with egg, and 39 with peanut allergies. The control group included 36 children (19 boys and 17 girls, mean age 8.03 years). Blood analysis was performed on all participants. RESULTS: Data from our study showed that patients with cow's milk, egg or peanut allergies had normal growth and achieved catch-up growth from the diagnosis until the last study visit. In the cow's milk allergy group, the allergy was shown to affect calcium intake (p < 0.05), while egg and peanut allergies did not impact the dietary intake of nutrients. None of the investigated food allergies affected blood results (p < 0.05). CONCLUSIONS: In the present study, we showed that single food allergies do not compromise growth in children if they are provided with appropriate support and that the affected children reach catch-up growth from the diagnosis.


Assuntos
Hipersensibilidade a Ovo , Hipersensibilidade a Amendoim , Masculino , Criança , Animais , Bovinos , Feminino , Humanos , Seguimentos , Estado Nutricional , Leite , Estudos Retrospectivos
20.
Front Immunol ; 13: 974374, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36248809

RESUMO

Background: 10% of US residents have food allergies, including 2% with peanut allergy. Mast cell mediators released during the allergy effector phase drive allergic reactions. Therefore, targeting sensitized mast cells may prevent food allergy symptoms. Objective: We used novel, human, allergen-specific, IgE monoclonal antibodies (mAbs) created using human hybridoma techniques to design an in vitro system to evaluate potential therapeutics targeting sensitized effector cells. Methods: Two human IgE mAbs specific for peanut, generated through human hybridoma techniques, were used to sensitize rat basophilic leukemia (RBL) SX-38 cells expressing the human IgE receptor (FcϵRI). Beta-hexosaminidase release (a marker of degranulation), cytokine production, and phosphorylation of signal transduction proteins downstream of FcϵRI were measured after stimulation with peanut. Degranulation was also measured after engaging inhibitory receptors CD300a and Siglec-8. Results: Peanut-specific human IgE mAbs bound FcϵRI, triggering degranulation after stimulation with peanut in RBL SX-38 cells. Sensitized RBL SX-38 cells stimulated with peanut increased levels of phosphorylated SYK and ERK, signal transduction proteins downstream of FcϵRI. Engaging inhibitory cell surface receptors CD300a or Siglec-8 blunted peanut-specific activation. Conclusion: Allergen-specific human IgE mAbs, expressed from human hybridomas and specific for a clinically relevant food allergen, passively sensitize allergy effector cells central to the in vitro models of the effector phase of food allergy. Peanut reproducibly activates and induces degranulation of RBL SX-38 cells sensitized with peanut-specific human IgE mAbs. This system provides a unique screening tool to assess the efficacy of therapeutics that target allergy effector cells and inhibit food allergen-induced effector cell activation.


Assuntos
Hipersensibilidade Alimentar , Hipersensibilidade a Amendoim , Alérgenos , Animais , Anticorpos Monoclonais/farmacologia , Arachis , Degranulação Celular , Citocinas , Humanos , Imunoglobulina E , Ratos , Receptores de IgE/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , beta-N-Acetil-Hexosaminidases
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