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1.
Food Chem ; 378: 132061, 2022 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-35032803

RESUMO

Whole peptide-displayed phage particles are promising alternatives to antibodies in sensor development; however, greater control and functionalization of these particles are required. In this study, we aimed to identify and create highly sensitive and selective phage-based electrochemical biosensors for detecting ovomucoid, a known food allergen. Phage display was performed using two different phage libraries (cyclic and linear form of peptides), which displayed affinity peptides capable of binding specifically to ovomucoid. Throughout the biopanning, two phage clones that displayed both peptides (CTDKASSSC and WWQPYSSAPRWL) were selected. After the characterization of their binding affinities, both whole phage particles were covalently attached to a gold electrode using crosslinking chemistry (MUA-EDC/NHS and Sulfo-LC/SPDP); the developed phage sensor was characterized using cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS). The cyclic peptide-displayed phage sensor modified using EDC/NHS chemistry exhibited significantly better binding affinity (Kd = 2.36 ± 0.44 µg/mL) and limit of detection (LOD, 0.12 µg/mL) for ovomucoid than the linear phage sensor, resulting in good reproducibility and recovery, even in an actual egg and white wine samples. This approach may provide an alternative and more efficient way of sensing food allergens with desirable sensitivity, selectivity, and feasibility in food diagnostic applications.


Assuntos
Bacteriófagos , Técnicas Biossensoriais , Bacteriófagos/genética , Técnicas Eletroquímicas , Eletrodos , Limite de Detecção , Ovomucina , Reprodutibilidade dos Testes
2.
Food Chem ; 366: 130596, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34293545

RESUMO

Clarification of the mechanism of heat-induced gel formation by proteins under natural food systems could provide important references for the regulation of food texture. In the present study, the proteins involved in the early stage (heating at 72 °C for 8 min) of egg-white thermal gel (EWG) formation were studied quantitatively through comparative proteomic analysis. We discovered that the abundance of ovalbumin and ovomucoid increased significantly (p < 0.01), whereas that of ovotransferrin, lysozyme, ovomucin (mucin 5B and mucin 6) decreased significantly (p < 0.01), in the supernatant of EWG. If the initial interaction of egg white proteins was altered by ultrasonic pretreatment, the abundance of ovomucin and lysozyme in the supernatant of EWG increased, and was accompanied by the change from a solid gel to a fluid gel. Based on these results, we hypothesize that ovomucin has a key role in the formation and regulation of EWG properties.


Assuntos
Ovomucina , Proteômica , Animais , Galinhas , Conalbumina , Proteínas do Ovo , Ovalbumina
3.
Nutrients ; 13(7)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202741

RESUMO

We investigated the effects of different types of heat treatments on hen's egg white (HEw) and quail egg white (QEw) proteins and their cross-reactivity in young children. Crude extracts of raw and water-boiled HEw and QEw and commercially developed stone-baked HEw were prepared. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was then performed. Patients diagnosed with HEw allergy were enrolled, and pooled sera were tested with each extract in an enzyme-linked immunosorbent assay (ELISA)-inhibition test. A skin prick test (SPT) and oral food challenge (OFC) were also performed. The median age of 12 patients was 2.5 years. SDS-PAGE results revealed strongly stained bands for the ovomucoid of boiled HEw and QEw, while stone-baked HEw displayed remarkable changes for all protein fractions. In the ELISA-inhibition test, pre-incubation of the sera led to a profound decrease, moderate decrease, and minimal decrease in the amount of IgE binding to boiled and raw HEw, boiled and raw QEw, and stone-baked HEw proteins, respectively. SPTs and OFC demonstrated cross-reactivity values of 41.7% (5/12) and 16.7% (2/12) for boiled QEw and stone-baked HEw, respectively. We observed moderate cross-reactivity between QEw and HEw. Boiling had a limited effect on altering egg allergenicity. Commercially developed, stone-baked HEw can be an alternative food for children with HE allergy.


Assuntos
Alérgenos/imunologia , Reações Cruzadas/imunologia , Hipersensibilidade a Ovo/imunologia , Proteínas do Ovo/imunologia , Temperatura Alta , Animais , Galinhas , Pré-Escolar , Culinária , Eletroforese em Gel de Gradiente Desnaturante/métodos , Ovos/análise , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Ovomucina/imunologia , Estudos Prospectivos , Codorniz , Testes Cutâneos
4.
J Food Sci ; 86(7): 3014-3022, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34151424

RESUMO

Ovomucoid (OVM), known as the major allergen in egg white, has gained increasing concerns in industrialized countries. Here, we found the deglycosylation and Maillard reaction with galactooligosaccharide (GOS) and fructooligosaccharide (FOS) can induce conformational transformation of OVM from other structures (ß-turn, strang, and random coils) to α-helix. We also introduced an approach to reduce the allergenicity of Gallus domesticus OVM by Maillard reaction with GOS and FOS. However, the OVM glycated by mannosan (MOS) and deglycosylated OVM exhibited higher allergenicity than native OVM. Therefore, GOS and FOS, especially GOS, could be applied in the reduction of the potential allergenicity of OVM through glycation. Furthermore, these findings may provide new insights into the development of hypoallergenic egg products. PRACTICAL APPLICATION: In this study, the allergenicity and conformation of OVM treated with deglycosylation and glycation (GOS, FOS, and MOS) were investigated. The results would provide a better understanding of the effects of deglycosylation and Maillard reaction with different reducing sugars on the molecular characteristics of OVM and further provide new insights into the development of hypoallergenic egg products.


Assuntos
Clara de Ovo/química , Ovomucina/química , Ovomucina/imunologia , Alérgenos/química , Alérgenos/imunologia , Animais , Galinhas , Hipersensibilidade a Ovo/imunologia , Ovos/análise , Manipulação de Alimentos , Glicosilação , Humanos , Reação de Maillard , Masculino , Conformação Molecular , Oligossacarídeos/química
5.
J Agric Food Chem ; 69(21): 5887-5896, 2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34013725

RESUMO

Egg white ovomucin (OVM) is homologically related to MUC2, the key component of colonic mucous layer. This study investigated the effects of orally administered OVM from egg white on the colonic mucosal barrier and the development of colitis using a colitis C57BL/6J mice model. The results showed that daily supplementation of 125 and 250 mg/kg BW of OVM partially relieved the villous destruction and loss of intestinal barrier integrity, and hence decreased the epithelial barrier permeability. The supplementation also reduced the secretion of proinflammatory cytokines TNF-α and IL-6. Besides, OVM administration significantly increased the relative abundance of intestinal beneficial bacteria including Lactobacilli, Faecalibaculum, Ruminococcus, etc. and further upregulated the production of bacterial metabolites such as short-chain fatty acids (SCFAs), which is a direct source of energy for the proliferation of epithelia and goblet cells. In conclusion, OVM from egg white ameliorates colitis by enhancing the intestinal barrier function and abundance of intestinal bacteria, thereby increasing the number of SCFAs.


Assuntos
Colite , Ovomucina , Animais , Bactérias/genética , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colo , Sulfato de Dextrana , Modelos Animais de Doenças , Mucosa Intestinal , Camundongos , Camundongos Endogâmicos C57BL
7.
Allergol Immunopathol (Madr) ; 49(2): 6-14, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33641288

RESUMO

INTRODUCTION AND OBJECTIVES: It was urgent to explain the role of egg yolk allergen sensitization to the egg allergic population and we would evaluate the diagnostic value of allergen components in whole eggs, including egg white and egg yolk. MATERIALS AND METHODS: Firstly, we collected 99 positive and 21 negative sera against egg allergy. Then we used modified enzyme linked immunosorbent assay (ELISA) to survey specific IgE (sIgE) to all-proven and single component in eggs, Ovomucoid (Gal d 1), Ovalbumin (Gal d 2), Ovotransferrin (Gal d 3), Lysozyme C (Gal d 4), Serum Albumin (Gal d 5), and YGP42(Gal d 6) in allergic and non-allergic populations. Last but not least, we studied the sIgE reactivities to egg allergen components by receiver operating characteristic (ROC) analysis. RESULTS: Among egg-allergic individuals, nearly 10% were sensitized to five of six egg allergen components, and the cross-reaction frequency between two egg yolk allergens with Gal d 1 was about 30% in the groups diagnosed with egg allergy or non-allergy. The best component-combination diagnosis in egg allergy of Gal d 1+ Gal d 6 demonstrated the largest area under curve (AUC) of 0.994. CONCLUSIONS: Our results suggested that there were individual differences in allergenicity of different egg allergen components, especially in the samples negative to egg allergy diagnosed but sensitive to egg yolk components. It was indicated that component resolved diagnosis of egg yolk improved the value for egg allergy management indispensably.


Assuntos
Alérgenos/efeitos adversos , Hipersensibilidade a Ovo/diagnóstico , Gema de Ovo/efeitos adversos , Adolescente , Adulto , Alérgenos/imunologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Conalbumina/efeitos adversos , Conalbumina/imunologia , Hipersensibilidade a Ovo/sangue , Hipersensibilidade a Ovo/imunologia , Clara de Ovo/efeitos adversos , Gema de Ovo/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Lactente , Masculino , Muramidase/efeitos adversos , Muramidase/imunologia , Ovalbumina/efeitos adversos , Ovalbumina/imunologia , Ovomucina/efeitos adversos , Ovomucina/imunologia , Adulto Jovem
8.
Org Lett ; 23(5): 1904-1909, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33570417

RESUMO

Meleagrin B is a terpene-alkaloid hybrid natural product that contains both the conidiogenone and meleagrin scaffold. Their derivatives show diverse biological activities. We characterized the biosynthesis of (-)-conidiogenone B (1), which involves a diterpene synthase and a P450 monooxygenase. In addition, an α,ß-hydrolase (Con-ABH) was shown to catalyze an aza-Michael addition between 1 and imidazole to give 3S-imidazolyl conidiogenone B (6). Compound 6 was more potent than 1 against Staphylococcus aureus strains.


Assuntos
Diterpenos/química , Imidazóis/química , Ovomucina/biossíntese , Estrutura Molecular , Ovomucina/química
9.
Poult Sci ; 100(2): 452-460, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33518096

RESUMO

Ovomucoid is a major egg white protein which is considered as the most dominant allergen in chicken eggs. Owing to the difficulty of separating ovomucoid from egg whites, researchers have adopted genetic deletion for development of hypoallergenic eggs. Previously, we used CRISPR/Cas9 to establish chickens with ovomucoid gene (OVM) mutations, but it remained unknown whether such hens could produce eggs at maturity. Here, we have reported on eggs laid by OVM-targeted hens. Except for watery egg whites, the eggs had no evident abnormalities. Real-time PCR revealed alternative splicing of OVM mRNA in hens, but their expression was limited. Immunoblotting detected neither mature ovomucoid nor ovomucoid-truncated splicing variants in egg whites. Sixteen chicks hatched from 28 fertilized eggs laid by OVM-targeted hens, and fourteen of the sixteen chicks demonstrated healthy growth. Taken together, our results demonstrated that OVM knockout could almost completely eliminate ovomucoid from eggs, without abolishing fertility. Thus, the eggs developed in this study have potential as a hypoallergenic food source for most patients with egg allergies.


Assuntos
Galinhas/genética , Ovos/normas , Mutação , Ovomucina/genética , Alérgenos/genética , Animais , Galinhas/crescimento & desenvolvimento , Galinhas/fisiologia , Clara de Ovo/efeitos adversos , Clara de Ovo/química , Clara de Ovo/normas , Feminino , Deleção de Genes , Masculino , Oviposição/genética , Ovomucina/efeitos adversos , Óvulo
10.
Nat Prod Res ; 35(21): 3830-3838, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32189530

RESUMO

The crystal structure and unambiguous absolute configuration of meleagrin (1) isolated from fungus Emericella dentata Nq45 is reported herein to first time on the bases of single crystal X-ray diffraction. Together with 1, haenamindole (2), isorugulosuvine (3), secalonic acid D (4), ergosterol (5) and cerebroside A (6) were obtained and their structures were determined by ESI MS and NMR data analysis. Diverse biological activity of meleagrin (1) was investigated. Compound 1 pronounced potent cytotoxicity against the human cervix carcinoma cell line KB-3-1 and its multidrug resistant sub-clone KB-V1 of IC50 3.07 and 6.07 µM, respectively, in comparison with the reference (+) - griseofulvin (IC50 19, 19.5 µM). Based on the antibiofilm activity, compound 1 displayed as well potent activity against Staphylococcus aureus with an MIC of 0.25 mg/mL. Isolation of the producing fungus and taxonomical characterization is stated as well.


Assuntos
Emericella , Ovomucina/farmacologia , Organismos Aquáticos/química , Linhagem Celular Tumoral , Emericella/química , Humanos , Estrutura Molecular , Ovomucina/química , Staphylococcus aureus/efeitos dos fármacos
11.
Clin Exp Allergy ; 50(12): 1415-1423, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32909666

RESUMO

BACKGROUND: Although hen's egg allergy is more prevalent in children, up to 0.6% of adults from different European countries suffer from a persistent or newly onset hen's egg allergy, making accurate diagnosis in adults necessary. However, sensitization to hen's egg extracts, components and linear epitopes is solely studied in children. METHODS: Hen's egg allergic (n = 16) and tolerant (n = 19) adults were selected by sensitization towards recombinant components rGal d 1 and/or 3. Sensitization profiles towards egg white and yolk extract and the native components Gal d 1, 2, 3 and 4 were respectively evaluated with the ImmunoCAP or the EUROLINE system. Characterization of linear epitopes was performed with a peptide microarray containing 15mer peptides representing the entire sequence of mature Gal d 1 and 3. RESULTS: Overall, sIgE titres against hen's egg extracts and single components overlapped largely between allergic and tolerant adults. Although the median sIgE/sIgG4 ratio to Gal d 1 was increased in allergic adults, the range was comparable between both groups. Clinically relevant sensitization to Gal d 1 was confirmed by sIgE-binding to the linear epitopes aa30-41, aa39-50 or aa84-95 in 6/13 allergic adults, mainly suffering from objective symptoms. In comparison, these epitopes were recognized by 1/15 tolerant patient. Only a few linear epitopes were detected for Gal d 3, suggesting a greater importance of conformational epitopes for the recognition of Gal d 3. CONCLUSION AND CLINICAL RELEVANCE: Specific IgE-binding to linear epitopes of Gal d 1 is highly specific in identifying hen's egg allergic adults with objective symptoms.


Assuntos
Hipersensibilidade a Ovo/diagnóstico , Proteínas do Ovo/administração & dosagem , Epitopos Imunodominantes , Imunoglobulina E/sangue , Testes Imunológicos , Ovomucina/administração & dosagem , Adulto , Idoso , Biomarcadores/sangue , Hipersensibilidade a Ovo/sangue , Hipersensibilidade a Ovo/imunologia , Proteínas do Ovo/imunologia , Feminino , Humanos , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Ovomucina/imunologia , Valor Preditivo dos Testes , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Estudos Retrospectivos , Adulto Jovem
12.
Int J Biol Macromol ; 162: 385-393, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32569696

RESUMO

Intestinal dysfunction, which may cause a series of metabolic diseases, has become a worldwide health problem. In the past few years, studies have shown that consumption of poultry eggs has the potential to prevent a variety of metabolic diseases, and increasing attention has been directed to the bioactive proteins and their peptides in poultry eggs. This review mainly focused on the biological activities of an important egg-derived protein named ovomucin. Ovomucin and its derivatives have good anti-inflammatory, antioxidant, immunity-regulating and other biological functions. These activities may affect the physical, biological and immune barriers associated with intestinal health. This paper reviewed the structure and the structure-activity relationship of ovomucin,the potential role of ovomucin and its derivatives in modulation of intestinal health are also summarized. Finally, the potential applications of ovomucin and its peptides as functional food components to prevent and assist in the pretreatment of intestinal health problems are prospected.


Assuntos
Anti-Inflamatórios/metabolismo , Antioxidantes/metabolismo , Galinhas/metabolismo , Proteínas do Ovo/metabolismo , Microbioma Gastrointestinal , Mucosa Intestinal/metabolismo , Ovomucina/metabolismo , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Anti-Inflamatórios/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Ovos , Mucosa Intestinal/imunologia , Ácido N-Acetilneuramínico/metabolismo , Ovomucina/química , Ovomucina/imunologia , Peptídeos/química , Peptídeos/metabolismo , Aves Domésticas , Relação Estrutura-Atividade
13.
Allergy ; 75(10): 2633-2643, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32391917

RESUMO

BACKGROUND: Egg-white ovomucoid, that is, Gal d 1, is associated with IgE-mediated allergic reactions in most egg-allergic children. Epitope-specific IgE levels have been correlated with the severity of egg allergy, while emerging evidence suggests that other antibody isotypes (IgG1 , IgG4 , IgA, and IgD) may have a protective function; yet, their epitope-specific repertoires and associations with atopic comorbidities have not been studied. METHODS: Bead-based epitope assay (BBEA) was used to quantitate the levels of epitope-specific (es)IgA, esIgE, esIgD, esIgG1 , and esIgG4 antibodies directed at 58 (15-mer) overlapping peptides, covering the entire sequence of ovomucoid, in plasma of 38 egg-allergic and 6 atopic children. Intraclass correlation (ICC) and coefficient of variation (CV) were used for the reliability assessment. The relationships across esIgs were evaluated using network analysis; linear and logistic regressions were used to compare groups based on egg allergy status and comorbidities. RESULTS: BBEA had high reliability (ICC >0.75) and low variability (CV <20%) and could detect known IgE-binding epitopes. Egg-allergic children had lower esIgA1 (P = .010) and esIgG1 (P = .016) and higher esIgE (P < .001) and esIgD (P = .015) levels compared to the atopic controls. Interestingly, within the allergic group, children with higher esIgD had decreased odds of anaphylactic reactions (OR =0.48, P = .038). Network analysis identified most associations between esIgE with either esIgG4 or esIgD; indicating that IgE-secreting plasma cells could originate from either sequential isotype switch from antigen-experienced intermediate isotypes or directly from the IgD+ B cells. CONCLUSIONS: Collectively, these data point toward a contribution of epitope-specific antibody repertoires to the pathogenesis of egg allergy.


Assuntos
Imunoglobulina E , Ovomucina , Alérgenos , Criança , Epitopos , Humanos , Imunoglobulina A , Imunoglobulina D , Imunoglobulina G , Reprodutibilidade dos Testes
15.
Food Chem ; 315: 126207, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31991252

RESUMO

Five major proteins from egg white were separated using a successive extraction/precipitation protocol. The yield and purity of the separated proteins were measured. The separated proteins were confirmed by MALDI-TOF-MS, and their structures were characterized by CD spectrum. Lysozyme was first separated using FPC 3500 resin and then ovomucin from the lysozyme-free egg white. Ammonium sulfate and citric acid were added to the resulting lysozyme- and ovomucin-free egg white solution to precipitate ovotransferrin. Ovomucoid and ovalbumin were separated from the resulting supernatant using ethanol. The separated proteins were further purified and the optimal conditions for the further purifications were suggested. The purity and yield of lysozyme, ovotransferrin, ovalbumin, and ovomucoid were higher than 90% and 77%, while those of ovomucin were about 72% and 75%, respectively. This study separated five major proteins in egg white successively using resin adsorption, pH adjustment, salt/ethanol precipitation, and ultrafiltration.


Assuntos
Fracionamento Químico/métodos , Proteínas do Ovo/análise , Proteínas do Ovo/isolamento & purificação , Clara de Ovo/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sulfato de Amônio/química , Animais , Precipitação Química , Conalbumina/análise , Conalbumina/isolamento & purificação , Proteínas do Ovo/química , Clara de Ovo/análise , Etanol/química , Muramidase/análise , Muramidase/isolamento & purificação , Ovalbumina/análise , Ovalbumina/isolamento & purificação , Ovomucina/análise , Ovomucina/isolamento & purificação , Estrutura Secundária de Proteína
16.
J Pharm Biomed Anal ; 180: 113034, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-31838281

RESUMO

Official method in Ph. Eur. for evaluation of timolol enantiomeric purity is normal-phase high performance liquid chromatography (NP-HPLC) method. Compared to other HPLC modes, NP is depicted as quite expensive with high consumption of organic solvents which leads to chronic exposure of analysts to toxic and carcinogenic effects. In order to overcome above-mentioned drawbacks, the aim of this study was to develop new method with better eco-friendly features. This was enabled by using protein type Chiral Stationary Phase (CSP) in reversed-phase mode that required up to 10 % (v/v) of organic solvent. Therefore, an enantioselective HPLC method was developed and validated for quantification of (S)-timolol and its chiral impurity, (R)-isomer. Optimized separation conditions on ovomucoid column were set using Analytical Quality by Design (AQbD) approach in method development. Optimization step was performed following the Box-Behnken experimental plan and the influence of three critical method parameters (CMPs) towards enantioseparation of the above-mentioned peak pair was examined. CMPs included variation of acetonitrile content in the mobile phase (5-10 %, v/v), pH value of the aqueous phase (6.0-7.0) and ammonium chloride concentration in the aqueous part of the mobile phase (10-30 mmol L-1). The most relevant critical method attributes (CMAs) in this case were the separation criterion between studied critical pair and retention factor of the second eluting peak, (S)-timolol. Qualitative Design Space (DS) was defined by Monte Carlo simulations providing adequate assurance of method's qualitative robustness (π = 95 %). The selected working point situated in the middle of the DS was characterized by following combination of CMPs: acetonitrile content in the mobile phase 7 % (v/v), pH value of the aqueous phase 6.8 and concentration of ammonium chloride in aqueous phase 14 mmol L-1. In the next step, the quantitative robustness was tested by Plackett-Burman experimental design. The validation studies confirmed adequacy of the proposed method for its intended purpose. Finally, Analytical Eco-Scale metric tool was applied to confirm that developed method represents excellent green analytical method compared to the official one.


Assuntos
Ovomucina/química , Timolol/análise , Timolol/isolamento & purificação , Cloreto de Amônio/química , Cromatografia Líquida de Alta Pressão , Limite de Detecção , Modelos Lineares , Modelos Moleculares , Estrutura Molecular , Reprodutibilidade dos Testes , Solventes/química , Estereoisomerismo , Relação Estrutura-Atividade
17.
J Pediatr (Rio J) ; 96(6): 725-731, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31513760

RESUMO

OBJECTIVE: To assess the frequency of baked egg tolerance in IgE-mediated egg allergy patients through the oral food challenge and to assess the tolerance predictability of different skin prick tests, as well as specific serum IgE measurement to egg proteins. METHODS: In this cross-sectional study, 42 patients with a diagnosis of egg allergy were submitted to different skin prick tests with egg (in natura, boiled, muffin, ovalbumin, and ovomucoid), and specific IgE to egg white, ovalbumin, and ovomucoid; as well as to the oral food challenge with food containing egg, extensively baked in a wheat matrix. RESULTS: Of the total, 66.6% of patients tolerated the ingestion of egg-containing foods in the oral food challenge. A comparative analysis with positive and negative oral food challenge found no significant differences regarding age, gender, other food allergies, or even specific skin prick tests and IgE values between the groups. CONCLUSIONS: The study demonstrated an elevated frequency of baked egg food-tolerant individuals among egg allergy patients. None of the tested markers, skin prick tests, or specific IgE, were shown to be good predictors for identifying baked egg-tolerant patients. The oral food challenge with egg baked in a matrix is central to demonstrate tolerance and the early introduction of baked foods, improving patients' and families' quality of life and nutrient intake.


Assuntos
Culinária , Hipersensibilidade a Ovo , Qualidade de Vida , Alérgenos , Estudos Transversais , Hipersensibilidade a Ovo/diagnóstico , Ovos , Humanos , Tolerância Imunológica , Imunoglobulina E , Ovomucina , Testes Cutâneos
19.
Appl Biochem Biotechnol ; 189(2): 680-689, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31102127

RESUMO

Ovomucoid (OVM) is a protein found in chicken egg white. When it is hydrolyzed by a protease, subtilisin A from Bacillus licheniformis, it possesses Cu2+-chelating activity. In the present work, we demonstrate that the resulting OVM hydrolysates bind to reverse-phase chromatography media in pipette tips and can be applied to remove Cu2+ within microdroplets. 1.4 nmol of purified OVM was digested in the presence of 17 pmol of subtilisin A at 55 °C for 3 h. The OVM hydrolysates efficiently removed 2.1 and 2.4 nmol of Cu2+ in the droplets by binding to the C4 and C18 chromatography media, respectively. Conversely, 0.6 and 1.0 nmol of Cu2+ were removed by the non-digested OVM bound to the C4 and C18 media, respectively. The removal ratio of Cu2+ increased as more OVM was digested by subtilisin A. The digested OVM polypeptides were stained with Cu2+ after they were separated by non-denaturing electrophoresis. These results indicate that OVM hydrolysates bound to chromatography media in a pipette tip can be applied to remove Cu2+ within microdroplets of biological samples.


Assuntos
Bacillus licheniformis/enzimologia , Proteínas de Bactérias/química , Cromatografia de Fase Reversa , Cobre/química , Ovomucina/química , Subtilisinas/química
20.
J Toxicol Sci ; 44(4): 283-297, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30944281

RESUMO

We previously developed a test for detecting naturally occurring protein-induced skin sensitization based on the markers and criteria of the human cell-line activation test (h-CLAT) and showed that the h-CLAT was useful for assessing the allergenic potency of proteins. However, test proteins were contaminated with varying amounts of lipopolysaccharide (LPS), which might have contributed to the stimulation of CD86 and CD54 expression. In this study, we developed a method to exclude the effects of LPS in the assessment of skin sensitization by naturally occurring proteins. We tested two inhibitors [the caspase-1 inhibitor acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-cmk; hereafter referred to as YVAD), which can mitigate the LPS-induced increases in CD54 expression, and polymyxin B (PMB), which suppresses the effect of LPS by binding to its lipid moiety (i.e., the toxic component of LPS)]. After a 24 hr exposure, YVAD and PMB reduced LPS-induced CD86 and CD54 expression. In particular, the effect of PMB was dependent upon pre-incubation time and temperature, with the most potent effect observed following pre-incubation at 37°C for 24 hr. Moreover, only pre-incubation with cell-culture medium (CCM) at 37°C for 24 hr showed an inhibitory effect similar to that of PMB, with this result possibly caused by components of CCM binding to LPS. Similar effects were observed in the presence of ovalbumin (with 1070 EU/mg LPS) and ovomucoid, and lysozyme (with 2.82 and 0.234 EU/mg LPS, respectively) in CCM. These results indicated that PMB and CCM effectively eliminated the effects of LPS during assessment of protein allergenicity, thereby allowing a more accurate evaluation of the potential of proteins to induce skin sensitization.


Assuntos
Clorometilcetonas de Aminoácidos/farmacologia , Hipersensibilidade/etiologia , Hipersensibilidade/imunologia , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/imunologia , Polimixina B/farmacologia , Proteínas/efeitos adversos , Proteínas/imunologia , Testes Cutâneos/métodos , Pele/imunologia , Clorometilcetonas de Aminoácidos/administração & dosagem , Clorometilcetonas de Aminoácidos/metabolismo , Antígeno B7-2/genética , Antígeno B7-2/imunologia , Antígeno B7-2/metabolismo , Meios de Cultura , Expressão Gênica/efeitos dos fármacos , Humanos , Imunização , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Molécula 1 de Adesão Intercelular/metabolismo , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/metabolismo , Muramidase , Ovalbumina , Ovomucina , Polimixina B/administração & dosagem , Polimixina B/metabolismo , Ligação Proteica , Células THP-1 , Temperatura , Fatores de Tempo
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