RESUMEN
Cutting-edge technologies such as genome editing and synthetic biology allow us to produce novel foods and functional proteins. However, their toxicity and allergenicity must be accurately evaluated. It is known that specific amino acid sequences in proteins make some proteins allergic, but many of these sequences remain uncharacterized. In this study, we introduce a data-driven approach and a machine-learning method to find undiscovered allergen-specific patterns (ASPs) among amino acid sequences. The proposed method enables an exhaustive search for amino acid subsequences whose frequencies are statistically significantly higher in allergenic proteins. As a proof-of-concept, we created a database containing 21,154 proteins of which the presence or absence of allergic reactions are already known and applied the proposed method to the database. The detected ASPs in this proof-of-concept study were consistent with known biological findings, and the allergenicity prediction performance using the detected ASPs was higher than extant approaches, indicating this method may be useful in evaluating the utility of synthetic foods and proteins.
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Alérgenos , Aprendizaje Automático , Proteínas , Alérgenos/química , Secuencia de Aminoácidos , Proteínas/químicaRESUMEN
In the Japanese official detection method for unauthorized genetically modified (GM) papayas, one of two types of real-time PCR reagents with DNA polymerase (TaqMan Gene Master Mix [TaqMan Gene] or FastGene QPCR Probe Mastermix w/ROX [FastGene]) is primarily used for measurement. In 2022, we conducted a laboratory performance study on the unauthorized GM papaya line PRSV-YK, and the results revealed that high threshold cycle (Cq) values for the PRSV-YK detection test were obtained using TaqMan Gene with the 7500 Fast & 7500 Real-Time PCR System (ABI7500) and QuantStudio 12K Flex (QS12K), indicating the possibility of false negatives. The possibility of similar problems with all unauthorized GM papaya lines detection tests needs to be evaluated. In this study, we performed detection tests on unauthorized GM papaya lines (PRSV-YK, PRSV-SC, and PRSV-HN), the cauliflower mosaic virus 35S promotor (CaM), and a papaya positive control (Chy), and examined how the limits of detection (LOD) for each test are affected by two types of DNA polymerases (TaqMan Gene and FastGene) and three types of real-time PCR instruments (ABI7500, QS12K, and LightCycler 480 Instrument II [LC480]). In the PRSV-YK and PRSV-SC detection tests using ABI7500 and QS12K, measurement with TaqMan Gene showed a higher LOD than FastGene. In this case, an exponential amplification curve was confirmed on the amplification plot; however, the amplification curve did not cross the ΔRn threshold line and the correct Cq value was not obtained with a threshold line=0.2. The other tests (PRSV-HN, CaM, and Chy with ABI7500 and QS12K, and all detection tests with LC480) showed no important differences in the LOD for each test using either DNA polymerase. Therefore, when performing PRSV-YK and PRSV-SC detection tests with the ABI7500 or QS12K, FastGene should be used to avoid false negatives for foods containing GM papaya lines PRSV-YK and PRSV-SC at low mixing levels.
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Carica , ADN Polimerasa Dirigida por ADN , Plantas Modificadas Genéticamente , Reacción en Cadena en Tiempo Real de la Polimerasa , Carica/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Plantas Modificadas Genéticamente/genética , Alimentos Modificados Genéticamente , Caulimovirus/genética , Potyvirus/genética , Potyvirus/aislamiento & purificaciónRESUMEN
BACKGROUND: Ras homolog gene family H (RhoH) is a membrane-bound adaptor protein involved in proximal T-cell receptor signaling. Therefore RhoH plays critical roles in the differentiation of T cells; however, the function of RhoH in the effecter phase of the T-cell response has not been fully characterized. OBJECTIVE: We sought to explore the role of RhoH in inflammatory immune responses and investigated the involvement of RhoH in the pathogenesis of psoriasis. METHODS: We analyzed effector T-cell and systemic inflammation in wild-type and RhoH-null mice. RhoH expression in T cells in human PBMCs was quantified by using RT-PCR. RESULTS: RhoH deficiency in mice induced TH17 polarization during effector T-cell differentiation, thereby inducing psoriasis-like chronic dermatitis. Ubiquitin protein ligase E3 component N-recognin 5 (Ubr5) and nuclear receptor subfamily 2 group F member 6 (Nr2f6) expression levels decreased in RhoH-deficient T cells, resulting in increased protein levels and DNA binding activity of retinoic acid-related orphan receptor γt. The consequential increase in IL-17 and IL-22 production induced T cells to differentiate into TH17 cells. Furthermore, IL-22 binding protein/Fc chimeric protein reduced psoriatic inflammation in RhoH-deficient mice. Expression of RhoH in T cells was lower in patients with psoriasis with very severe symptoms. CONCLUSION: Our results indicate that RhoH inhibits TH17 differentiation and thereby plays a role in the pathogenesis of psoriasis. Additionally, IL-22 binding protein has therapeutic potential for the treatment of psoriasis.
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Dermatitis/metabolismo , Interleucinas/metabolismo , Psoriasis/metabolismo , Células Th17/inmunología , Factores de Transcripción/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Enfermedad Crónica , Dermatitis/tratamiento farmacológico , Dermatitis/genética , Modelos Animales de Enfermedad , Humanos , Interleucinas/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Psoriasis/tratamiento farmacológico , Psoriasis/genética , Receptores de Interleucina/uso terapéutico , Proteínas Represoras/genética , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/genética , Proteínas de Unión al GTP rho/genética , Interleucina-22RESUMEN
Enzyme-linked immunosorbent assay (ELISA) is commonly used to determine food allergens in food products. However, a significant number of ELISAs give an erroneous result, especially when applied to highly processed food. Accordingly, an improved ELISA, which utilizes an extraction solution comprising the surfactant sodium lauryl sulfate (SDS) and reductant 2-mercaptoethanol (2-ME), has been specially developed to analyze food allergens in highly processed food by enhancing analyte protein extraction. Recently, however, the use of 2-ME has become undesirable. In the present study, a new extraction solution containing a human- and eco-friendly reductant, which is convenient to use at the food manufacturing site, has been established. Among three chemicals with different reducing properties, sodium sulfite, tris(3-hydroxypropyl)phosphine, and mercaptoethylamine sodium sulfite was selected as a 2-ME substitute. The protein extraction ability of SDS/0.1 M sodium sulfite solution was comparable to that of SDS/2-ME solution. Next, the ELISA performance for egg, milk, wheat, peanut, and buckwheat was evaluated by using model-processed foods and commercially available food products. The data showed that the SDS/0.1 M sulfite ELISA significantly correlated with the SDS/2-ME ELISA for all food allergens examined (p < 0.01), thereby establishing the validity of the SDS/0.1 M sulfite ELISA performance. Furthermore, the new SDS/0.1 M sulfite solution was investigated for its applicability to the lateral-flow (LF) test. The result demonstrated the successful analysis of food allergens in processed food, showing consistency with the SDS/0.1 M sulfite ELISA results. Accordingly, a harmonized analysis system for processed food comprising a screening LF test and a quantitative ELISA with identical extraction solution has been established. The ELISA based on the SDS/0.1 M sulfite extraction solution has now been authorized as the revised official method for food allergen analysis in Japan.
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Alérgenos/aislamiento & purificación , Fraccionamiento Químico/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Análisis de los Alimentos/métodos , Sustancias Reductoras/química , Alérgenos/análisis , Animales , Arachis/química , Huevos/análisis , Hipersensibilidad a los Alimentos/diagnóstico , Humanos , Mercaptoetilaminas/química , Leche/química , Fosfinas/química , Sulfitos/química , Triticum/químicaRESUMEN
Salmon roe has a high allergic potency and often causes anaphylaxis in Japan. The major allergic protein of salmon roe is ß'-component, which is a 35kDa vitellogenin fragment consisting of two subunits. To elucidate structural information and immunological characteristics, ß'-component and the subunit components were purified from chum salmon (Onchorhincus keta) roe and vitellogenin-encoding mRNA was used to prepare ß'-component subunit-encoding cDNA. This was PCR-amplified, cloned and sequenced and the deduced amino acid sequence compared with partial sequences of ß'-component obtained by peptide mapping. The recombinant ß'-component subunit was produced by bacterial expression in Escherichia coli and its IgE-binding ability was measured by ELISA using the sera of a patient allergic to salmon roe. This was then compared with that of the native ß'-component with and without carboxymethylation. Following successful cloning of the cDNA encoding the ß'-component subunit, 170 amino acid residues were deduced and matched with the amino acid sequences of 121 and 88 residues in the 16kDa and 18kDa subunits, respectively. The sequences of both ß'-component subunits were almost identical, and the predicted secondary structure of the ß'-component showed a high content of ß-pleated sheets and no α-helices. There was no difference in IgE-binding ability between the native and recombinant ß'-component subunits at the same protein concentration, regardless of carboxymethylation. In conclusion, ß'-component is a homodimer protein composed of two isoform subunits having the same level of IgE-binding ability and, therefore, allergenic identity.
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Alérgenos/metabolismo , Escherichia coli/genética , Hipersensibilidad a los Alimentos/inmunología , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes/metabolismo , Alérgenos/genética , Alérgenos/inmunología , Secuencia de Aminoácidos , Anafilaxia/etiología , Animales , Niño , Preescolar , Clonación Molecular , Proteínas del Huevo/inmunología , Mapeo Epitopo , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/metabolismo , Hipersensibilidad a los Alimentos/complicaciones , Humanos , Inmunoglobulina E/metabolismo , Lactante , Japón , Masculino , Datos de Secuencia Molecular , Oncorhynchus keta/inmunología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
Transgenic plants tolerant to various environmental stresses are being developed to ensure a consistent food supply. We used a transgenic rice cultivar with high saline tolerance by introducing an RNA-binding protein (RBP) from the ice plant (Mesembryanthemum crystallinum); differences in salt-soluble protein expression between nontransgenic (NT) and RBP rice seeds were analyzed by 2D difference gel electrophoresis (2D-DIGE), a gel-based proteomic method. To identify RBP-related changes in protein expression under salt stress, NT and RBP rice were cultured with or without 200 mM sodium chloride. Only two protein spots differed between NT and RBP rice seeds cultured under normal conditions, one of which was identified as a putative abscisic acid-induced protein. In NT rice seeds, 91 spots significantly differed between normal and salt-stress conditions. Two allergenic proteins of NT rice seeds, RAG1 and RAG2, were induced by high salt. In contrast, RBP rice seeds yielded seven spots and no allergen spots with significant differences in protein expression between normal and salt-stress conditions. Therefore, expression of fewer proteins was altered in RBP rice seeds by high salt than those in NT rice seeds.
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Oryza/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Proteínas de Unión al ARN/metabolismo , Semillas/metabolismo , Cloruro de Sodio/análisis , Estrés Fisiológico , Electroforesis en Gel Bidimensional , Oryza/embriología , Oryza/fisiología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en TándemAsunto(s)
Alérgenos/inmunología , Especificidad de Anticuerpos/inmunología , Inmunoglobulina G/inmunología , Proteínas de Vegetales Comestibles/inmunología , Triticum/efectos adversos , Hipersensibilidad al Trigo/inmunología , Adulto , Epítopos , Humanos , Inmunoglobulina E/inmunología , Hipersensibilidad al Trigo/sangreRESUMEN
To assess the risk of food allergies in foods processed under the Japanese food labeling system, estimating exposure to hidden allergens is necessary. We assessed exposure to egg protein in foods processed according to the Japanese food labeling system. First, we estimated the concentration distribution of egg protein by Bayesian methods using data from the literature and the measurement of food products with precautional declarations in the labeling margin. We then estimated the food-intake portion-size distribution under two scenarios: soft drink consumption as an example of single, high-intake consumption, and confections, which are frequently consumed by children, as a realistic example of low-intake consumption. Finally, we estimated the distribution of unexpected intake of egg proteins in the form of single consumption. The mean exposure to egg protein under the high-intake scenario was estimated to be 0.0164 mg for 1-15-year-olds, 0.0171 mg for 4-15-year-olds, 0.0181 mg for 7-15-year-olds, and ≥0.0188 mg for 16-year-olds. The mean exposure to egg protein under the low-intake scenario was estimated to be 0.0018 mg for 1-15-year-olds, 0.0019 mg for 4-15-year-olds, 0.0020 mg for 7-15-year-olds, and ≥0.0022 mg for 16-year-olds. Compared to the reference dose of 2.0 mg proposed by the Joint the Food and Agriculture Organization (FAO)/World Health Organization (WHO) Expert Committee, the risk of onset of food allergies due to egg protein contamination from foods without egg labeling is considered to be extremely low under the current Japanese food labeling system.
RESUMEN
BACKGROUND: We performed an in vitro elicitation test to determine the ability of different types of wheat-allergic patients' IgE to induce humanized mast cell activation after the addition of various time-treated acid-hydrolyzed wheat proteins (HWPs). METHODS: The reactivity of heat- and various time-treated acid-hydrolyzed glutens (acid-HGs) and commercial acid-HWP (HWP1), using serum IgE from wheat allergy accompanied by skin and rhinoconjunctival sensitization to HWP1 in the facial soap, pediatric subjects with food allergy to native wheat, adult wheat-dependent exercise-induced anaphylaxis subjects, and nonatopic healthy subjects, was elucidated by dot blot and a luciferase assay-based in vitro elicitation test (EXiLE test). RESULTS: Serum from subjects sensitized with HWP1 reacted only to acid-HGs (acid-HGs treated for 0.5-3 or 6 h), but not native gluten, in the results of the dot blot. In contrast, sera from pediatric subjects sensitized with native wheat reacted to native gluten more strongly and showed only slight reactions to 0.5- to 1-hour-treated acid-HGs. The results of the in vitro elicitation test showed that acid hydrolyzation of the gluten attenuated antigen-induced luciferase expression in a time-dependent manner for sera from native-wheat-sensitized pediatric subjects. On the other hand, in the sera from HWP1-sensitized subjects, acid hydrolyzation of the gluten for 0.5 h dramatically increased luciferase expression. CONCLUSIONS: Even after prolonged hydrolyzation, acid-HGs still retained the ability to activate mast cells in the case of HWP1-sensitized subjects.
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Alérgenos/inmunología , Anafilaxia/inmunología , Asma Inducida por Ejercicio/inmunología , Conjuntivitis Alérgica/inmunología , Glútenes/inmunología , Mastocitos/inmunología , Proteínas de Plantas/inmunología , Rinitis Alérgica Perenne/inmunología , Piel/inmunología , Hipersensibilidad al Trigo/inmunología , Ácidos/química , Adolescente , Adulto , Anciano , Alérgenos/efectos adversos , Alérgenos/química , Anafilaxia/complicaciones , Asma Inducida por Ejercicio/complicaciones , Degranulación de la Célula , Células Cultivadas , Niño , Preescolar , Conjuntivitis Alérgica/complicaciones , Femenino , Glútenes/química , Humanos , Hidrólisis , Inmunoglobulina E/metabolismo , Lactante , Masculino , Persona de Mediana Edad , Proteínas de Plantas/efectos adversos , Proteínas de Plantas/química , Unión Proteica , Rinitis Alérgica Perenne/complicaciones , Triticum/efectos adversos , Triticum/inmunología , Hipersensibilidad al Trigo/complicaciones , Adulto JovenRESUMEN
Hydrolyzed wheat protein (HWP; hydrolyzed gluten) is used in various types of products worldwide. Several cases of wheat-dependent, exercise-induced anaphylaxis following exposure to HWP (Glupearl 19S) in cosmetics have been reported. Glupearl 19S was produced from the gluten after partial hydrolysis with hydrogen chloride, and its allergenicity is larger than that of gluten (Adachi R., Allergy 2012;67:1392-9.). It is considered that provocation of allergic manifestations is caused by deamidated gluten in food and/or non-food products. Moreover, an increasing number of studies have shown that HWP can induce IgE-mediated hypersensitivity by skin contact and/or food ingestion. However, the essential molecular properties and profiles of HWP are still unknown. In this study, bioinformatic and multivariate analyses using shotgun proteomics have revealed that 27 proteins significantly decreased in Glupearl 19S compared with intact gluten as shown by the ratio of ion signal intensity of tryptic peptides. In contrast, a single protein significantly increased in HWP compared with intact gluten as shown by the ratio of ion signal intensity of tryptic peptides. Furthermore, we have identified six Glupearl 19S-specific peptides using shotgun proteomics, database searches on Mascot Sequence Query, and de novo sequencing. The six peptides were identified as the specific markers of Glupearl 19S.
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Glútenes/química , Glútenes/genética , Péptidos/química , Péptidos/genética , Proteómica/métodos , Triticum/genética , Secuencia de Aminoácidos , Anafilaxia/etiología , Glútenes/efectos adversos , Glútenes/inmunología , Hidrólisis , Espectrometría de Masas , Péptidos/efectos adversos , Péptidos/inmunología , ProteomaRESUMEN
Most drugs contain pharmaceutical excipients. These are pharmacologically inactive substances used as vehicles for the active ingredients of a medication. Some of these pharmaceutical excipients are produced from allergenic foods (e.g., milk, egg, peanut, soybean, and sesame) and removing proteins completely from such excipients is difficult. Therefore, if individuals with food allergy consume drugs containing allergenic food-derived excipients, eliminating the risk of developing specific allergic symptoms induced by them may not be possible. We determined the levels of proteins in pharmaceutical excipients and ethical drugs (inhalants and injections) by spectrophotometric analyses. The level of protein in the pharmaceutical excipient lactose in each sample was approximately 1 mg/g. In the case of oils from soybeans, peanuts, and sesame in pharmaceutical excipients, proteins were detected in the range 7-9 microg/g sample. We also determined levels of allergenic proteins in pharmaceutical excipients and ethical drugs using commercial enzyme-linked immunosorbent assay systems. The milk proteins in lactose were detected in the range 1.39-13.07 microg/g. The results of this study suggest that physicians, patients with food allergies, pharmacists, and healthcare providers must pay attention to presence of potential impurities those may cause allergic symptoms in pharmaceutical products.
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Adyuvantes Farmacéuticos/química , Alérgenos/análisis , Hipersensibilidad a las Drogas/prevención & control , Hipersensibilidad a los Alimentos , Preparaciones Farmacéuticas/química , Proteínas/análisis , Adyuvantes Farmacéuticos/efectos adversos , Alérgenos/efectos adversos , Hipersensibilidad a las Drogas/etiología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Ensayo de Inmunoadsorción Enzimática , Humanos , Lactosa/efectos adversos , Lactosa/análisis , Proteínas de la Leche/efectos adversos , Proteínas de la Leche/análisis , Proteínas/efectos adversos , EspectrofotometríaRESUMEN
BACKGROUND: Chemical leukoderma is a skin depigmentation disorder induced through contact with certain chemicals, most of which have a p-substituted phenol structure similar to the melanin precursor tyrosine. The tyrosinase-catalyzed oxidation of phenols to highly reactive o-quinone metabolites is a critical step in inducing leukoderma through the production of melanocyte-specific damage and immunological responses. OBJECTIVE: Our aim was to find an effective method to evaluate the formation of o-quinone by human tyrosinase and subsequent cellular reactions. METHODS: Human tyrosinase-expressing 293T cells were exposed to various phenolic compounds, after which the reactive o-quinones generated were identified as adducts of cellular thiols. We further examined whether the o-quinone formation induces reductions in cellular GSH or viability. RESULTS: Among the chemicals tested, all 7 leukoderma-inducing phenols/catechol (rhododendrol, raspberry ketone, monobenzone, 4-tert-butylphenol, 4-tert-butylcatechol, 4-S-cysteaminylphenol and p-cresol) were oxidized to o-quinone metabolites and were detected as adducts of cellular glutathione and cysteine, leading to cellular glutathione reduction, whereas 2-S-cysteaminylphenol and 4-n-butylresorcinol were not. In vitro analysis using a soluble variant of human tyrosinase revealed a similar substrate-specificity. Some leukoderma-inducing phenols exhibited tyrosinase-dependent cytotoxicity in this cell model and in B16BL6 melanoma cells where tyrosinase expression was effectively modulated by siRNA knockdown. CONCLUSION: We developed a cell-based metabolite analytical method to detect human tyrosinase-catalyzed formation of o-quinone from phenolic compounds by analyzing their thiol-adducts. The detailed analysis of each metabolite was superior in sensitivity and specificity compared to cytotoxicity assays for detecting known leukoderma-inducing phenols, providing an effective strategy for safety evaluation of chemicals.
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Hipopigmentación , Monofenol Monooxigenasa , Humanos , Monofenol Monooxigenasa/metabolismo , Activación Metabólica , Fenoles/toxicidad , Hipopigmentación/inducido químicamente , Quinonas/análisis , Quinonas/química , Quinonas/metabolismo , Glutatión/metabolismoRESUMEN
In this study, monoclonal antibodies against two major fruit allergens-gibberellin-regulated protein (GRP) and lipid transfer protein (LTP)-were established. Sandwich enzyme-linked immunosorbent assays (ELISAs) for the quantification of peach GRP and LTP were constructed using these antibodies. Both ELISAs reacted with the respective antigens when heated at 100ºC for 20 min, but not when reduced with sodium sulfite, indicating that GRP and LTP are heat-stable, while disulfide bonds play an important role in their native steric structures. GRP and LTP in peaches and peach-containing foods were quantified by these ELISAs. In both cases, there were few differences among peach cultivars normally available on the market; however, concentrations were higher when the peach was ripe. GRP was localized in the pulp of the peach, while LTP was present in the peel. They could be quantified in peach-containing beverages, as well as in dried and canned peaches. GRP in Japanese apricots could also be determined using this ELISA, as its amino acid sequence is the same as that of peach GRP. Then, high concentrations of GRP were detected in umeboshi, a traditional Japanese pickled apricot. Peach leaves were found to have a high LTP content, accordingly, LTP was also observed in lotions containing peach leaf extract. The ability to quantitatively detect GRP and LTP in this study will, therefore, contribute to the improvement of component-resolved diagnoses and quality of life in patients allergic to peaches.
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Hipersensibilidad a los Alimentos , Prunus persica , Alérgenos , Anticuerpos Monoclonales , Antígenos de Plantas , Proteínas Portadoras , Giberelinas , Humanos , Inmunoglobulina E , Proteínas de Plantas , Prunus persica/metabolismo , Calidad de VidaRESUMEN
In the Japanese allergy-labeling system, food labeling is mandated for 7 specific ingredients (egg, cow's milk, wheat, buckwheat, peanut, shrimp, and crab) and recommended for 21 food ingredients in reference to case numbers of actual illness and the degree of seriousness. To monitor the validity of the labeling system, official methods for the detection of specific ingredient proteins in processed foods were developed. The official methods consist of ELISA methods for screening, and western blot methods for egg and milk, and PCR methods for wheat, buckwheat, peanut, shrimp/prawn, and crab as confirmation tests. The official methods consist of ELISA methods for screening, and western blot methods for egg and milk, and PCR methods for wheat, buckwheat, peanut, shrimp/prawn, and crab as confirmation tests. Threshold amounts (a few mg/kg) for labeling were set based on the approach of the analytical detections. Any foods containing protein allergens should be labeled if these contain allergens at greater than 10 ppm (mg/kg). Validation protocol criteria were established to standardize the Japanese official method. Food Safety Commission of Japan conducted a risk assessment of egg as a specific ingredient and judged that current labeling system for foods containing allergens is generally appropriate for "eggs". In the future, it is important to accumulate necessary scientific knowledge in order to carry out food health impact assessment including further refinement. The Japanese experience and knowledge of food allergy-labeling system would contribute to harmonize international labeling guidelines to protect allergic consumers globally.
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Wheat allergy is a pathological event involving immunocompetent cells against ingested wheat allergen and is clearly associated with transdermal sensitization. However, the molecular mechanisms involved in the disease etiology are not completely understood. A complex cellular and tissue network linking to food allergy makes it difficult to understand the molecular mechanism of allergenicity. Animal models are valuable tools to deduce basic principles of human disease without invasive intervention trials. A mouse model of wheat allergy has provided insights into effects of skin exposure to wheat protein; it is a plausible route of human sensitization for wheat anaphylaxis. Further investigation of this model will capture the essential occurrence and flow of events, bringing useful clues to develop effective treatment and control strategies against wheat allergy. Here, we describe a method for analyzing the expression of cell surface molecules in single cells isolated from lymphoid tissue with flow cytometry. Sensitization by wheat extracts significantly increases antigen-specific T cells in the spleen. Collecting information regarding the contribution of immune cells to allergic sensitization in the development of wheat allergy would be useful in preventing and treating food allergies.
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Modelos Animales de Enfermedad , Inmunofenotipificación/métodos , Linfocitos/efectos de los fármacos , Extractos Vegetales/inmunología , Triticum/inmunología , Hipersensibilidad al Trigo/inmunología , Administración Cutánea , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Biomarcadores/metabolismo , Femenino , Harina/análisis , Citometría de Flujo , Expresión Génica , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Interferón gamma/genética , Interferón gamma/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Linfocitos/citología , Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Extractos Vegetales/administración & dosificación , Análisis de la Célula Individual , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunología , Parche Transdérmico , Triticum/química , Hipersensibilidad al Trigo/sangre , Hipersensibilidad al Trigo/genética , Hipersensibilidad al Trigo/patologíaRESUMEN
Statins are 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors that lower atherogenic LDL-cholesterol levels. Statins exert clinically relevant anti-inflammatory effects; however, the underlying molecular mechanism remains unclear. Studies have shown that endogenous and exogenous pathogenic crystals, such as cholesterol and monosodium urate (MSU), and needle-like nanomaterials, such as multi-wall carbon nanotubes (MWCNT), induce the production of IL-1ß and play a critical role in the development of crystal-associated sterile inflammatory pathologies. In this study, we evaluated the effect of statins on crystal-induced IL-1ß production in macrophages. We found that various statins, including pitavastatin, atorvastatin, fluvastatin, and lovastatin, but not squalene synthase inhibitor, repressed IL-1ß release upon MWCNT stimulation. In addition, IL-1ß production induced by cholesterol crystals and MSU crystals, but not by ATP or nigericin, was diminished. MWCNT-stimulated IL-1ß release was dependent on the expression of NLRP3, but not AIM2, NLRC4, or MEFV. Statin-induced repression was accompanied by reduced levels of mature caspase-1 and decreased uptake of MWCNT into cells. Supplementation of mevalonate, geranylgeranyl pyrophosphate, or farnesyl pyrophosphate prevented the reduction in IL-1ß release, suggesting a crucial role of protein prenylation, but not cholesterol synthesis. The statin-induced repression of MWCNT-elicited IL-1ß release was observed in THP-1-derived and mouse peritoneal macrophages, but not in bone marrow-derived macrophages where statins act in synergy with lipopolysaccharide to enhance the expression of IL-1ß precursor protein. In summary, we describe a novel anti-inflammatory mechanism through which statins repress mature IL-1ß release induced by pathogenic crystals and nanoneedles by inhibiting the internalization of crystals by macrophages.
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Colesterol/toxicidad , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Interleucina-1beta/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Nanotubos de Carbono/toxicidad , Animales , Cristalización/métodos , Femenino , Humanos , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Células THP-1RESUMEN
Because food allergens from tree nuts, including walnuts, are a frequent cause of adverse food reactions for allergic patients, the labeling of foods containing ingredients derived from tree nuts is required in numerous countries. According to Japanese regulations, the labeling of food products containing walnuts is recommended. To ensure proper labeling, a novel sandwich ELISA kit for the determination of walnut protein in processed foods (Walnut Protein [2S-Albumin] Kit; Morinaga Institute of Biological Science, Inc.; "walnut kit") has been developed. We prepared seven types of incurred samples (model processed foods: biscuits, bread, sponge cake, orange juice, jelly, chicken meatballs, and rice gruel) containing 10 microg walnut soluble protein/g of food for use in interlaboratory evaluations of the walnut kit. The walnut kit displayed sufficient reproducibility relative standard deviations (interlaboratory precision: 5.8-9.9% RSDR) and a high level of recovery (81-119%) for all the incurred samples. All the repeatability relative standard deviation (RSDr) values for the incurred samples that were examined were less than 6.0%. The results of this interlaboratory evaluation suggested that the walnut kit could be used as a precise and reliable tool for determination of walnut protein in processed foods.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Juglans/química , Proteínas de Plantas/análisis , Calibración , Laboratorios , Juego de Reactivos para Diagnóstico , Reproducibilidad de los ResultadosRESUMEN
The labeling of foods containing ingredients derived from soybean is recommended in Japan because of an increasing number of patients who are allergic to soybeans. To ensure proper labeling, a novel sandwich ELISA kit for the determination of soybean protein in processed foods (FASTKIT Ver. II, "Soybean", Nippon Meat Packers, Inc.; "soy kit") has been developed. Five types of incurred samples (model processed foods: rice gruel, sausage, sweet adzuki bean soup, sweet potato cake, and tomato sauce) containing 10 microg soybean soluble protein/g food were prepared for use in interlaboratory evaluations of the soy kit. The soy kit displayed a sufficient RSD(R) value (interlaboratory precision: 9.3-13.4% RSD(R)) and a high level of recovery (97-114%) for all the incurred samples. The RSD(R) value for the incurred samples was mostly < 4.8%. The results of this interlaboratory evaluation suggest that the soy kit can be used as a precise and reliable tool for the determination of soybean proteins in processed foods.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Análisis de los Alimentos/métodos , Proteínas de Soja/análisis , Ensayo de Inmunoadsorción Enzimática/estadística & datos numéricos , Análisis de los Alimentos/estadística & datos numéricos , Manipulación de Alimentos , Hipersensibilidad a los Alimentos , Humanos , Japón , Laboratorios , Estándares de Referencia , Reproducibilidad de los Resultados , Proteínas de Soja/efectos adversos , Proteínas de Soja/inmunología , Proteínas de Soja/normasAsunto(s)
Glútenes/inmunología , Transglutaminasas/inmunología , Hipersensibilidad al Trigo/inmunología , Adulto , Anciano , Alérgenos/inmunología , Células Cultivadas , Niño , Preescolar , Epítopos de Linfocito B/inmunología , Femenino , Proteínas de Unión al GTP , Glútenes/química , Humanos , Hidrólisis , Inmunoglobulina E/metabolismo , Lactante , Masculino , Persona de Mediana Edad , Proteína Glutamina Gamma Glutamiltransferasa 2 , Pruebas Serológicas/métodos , Triticum , Hipersensibilidad al Trigo/diagnóstico , Adulto JovenRESUMEN
Food scarcity is a serious problem for many developing as well as developed countries. Edible insects have attracted attention recently as a novel food source. Crickets are especially high in nutritional value and easy to breed and harvest. In this study, we evaluated the risk of allergic reactions associated with cricket consumption in individuals with crustacean allergy. We evaluated food allergy risk in the consumption of Gryllus bimaculatus (cricket) in patients with shrimp allergy, using enzyme-linked immunosorbent assay (ELISA) and IgE crosslinking-induced luciferase expression assay (EXiLE). Sera from individuals with shrimp allergy (positive for shrimp-specific IgE by ImmunoCAP (>0.35 UA/mL; n = 9) or without shrimp allergy (negative for shrimp-specific IgE; n = 6) were obtained. There was a strong correlation between shrimp- and Gryllus-specific IgE levels obtained by ELISA (rs = 0.99; P < 0.001). The binding of shrimp-specific IgE on shrimp allergen was dose-dependently inhibited by Gryllus allergen (0-1.0 mg/mL). There was a strong correlation between shrimp- and Gryllus-specific IgE responses, as assessed by EXiLE assays (rs = 0.89; P < 0.001). We determined that a protein of approximately 40 kDa reacted with the positive, but not negative, sera for shrimp-specific IgE by ImmunoCAP. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified the major allergen in shrimp and Gryllus to be tropomyosin. Our data suggest that the cricket allergen has the potential to induce an allergic reaction in individuals with crustacean allergy. Therefore, allergy risk and shrimp-specific IgE levels should be considered before consumption of cricket meal.