seminario: identification of major rice allergen and their clinical significance in children

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  • 1. Identification of major rice allergen and their clinical significance in children You Hoon Jeon, MD, Se Jo Oh, PhD, Hyeon Jong Yang, MD, Soo Young Lee, MD, Bok Yang Pyun, MD Manuela ColoradoCarolina Giraldo Bentez III Semestre MedicinaUPB

2. INTRODUCTIONAbnormal, inappropriate and exaggerated Allergyreaction of immune system to substances thattypically are not well tolerated. Is mediated byIgEIs produce by allergens Food allergy is different from food intolerance Allergen: is a substance that can induce a hypersensitivity (allergic)reaction in susceptible people, who have previously been in contactwith the allergen. 3. INTRODUCTION Rice is known to be a relatively low allergenic food, but have become a problem. A study shows that not eat rice has helped at 76% of 25 patients with atopicdermatitis with IgE-rice positive. Is a few reports on the correlation of rice allergen sensitization and their clinicalsymptoms. The immunologic characteristics of rice allergen are unclear. 4. INTRODUCTION Some allergens have been mentioned as possible major allergens of rice:-Globulin in the embryonic bud of rice-Albumin with protein bands of 14-16 kDa Protein bands of 26, 33, 56 kDa are the major IgE-binding components in rice. Some studies reported that rice is less allergenic if it is heating first. Heat- and acid-stable protein, lipid transfer protein (LTP) has emerged recently asa relevant factor in symptomatic rice allergies. 5. GENERAL OBJECTIVE Determine the major allergens in rice and theirclinical significance.Objetivohttp://freestyle.bligoo.com.co/objetivos 6. MATERIALES Y MTODOS1. Sujetos y grupos de control Sujetos 7. MATERIALES Y MTODOS 44 PacientesGrupo controlIgE-Arroz Negativa 2 Grupos n=18n=26Sujetos sensibilizados, pero Sujetos no sensibilizadosno al arroz 8. MATERIALES Y MTODOS2. Mtodos1. Anlisis de sujetos y grupo control Historial mdico y pruebas de laboratorioEdad, sexo,sntomas de la alergia, IgE en suero, ECP, IgE especfica.Erupcin cutnea, eritema, urticaria, angioedema, el sndrome de alergia oral, rino-conjuntivitis, sibilancias, anafilaxia tras la ingesta de arroz con leche, arroz hervido, galletas de arroz, etc. * Agravacin de la dermatitis atpica se excluy. 9. MATERIALES Y MTODOS2. Extraccin del alrgeno 10. MATERIALES Y MTODOS* Extracto de arrozcrudo: 5.6 mg/mL deprotena 11. MATERIALES Y MTODOS1. Sodio dodesil sulfato policrilamida gel electroforesis. (SDS PAGE) Muestra + Buffer Se calent Electroforesis durante 90 minutos SDS (amortiguacin con el marcador) Visualizacin por tincin con azul de coomassie brillante Cuantificacin de bandas Densitometra 12. MATERIALES Y MTODOS4. Anlisis de las bandas de protenas de arroz y arrozcocido Arroz crudo Arroz cocidoElectroforesis: Arroz integral cocido Arroz semicocidoComparacin y anlisis de distribucin de bandasSDS PAGE Electroforesishttp://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Protein-Expression-and-Analysis/Protein-Gel-Electrophoresis/Protein-Gels/Novex-NuPAGE-SDS-PAGE-Gels.html 13. MATERIALES Y MTODOS5. SDS PAGE de arroz tratado con calor, fluido gstrico simulado (SGF) y fluidointestinal simulado (SIF)Electroforesis en serieProtena de arroz se hirvi a 100C 10, 30 y 60 minutosElectroforesis Se trat con enzima digestiva, SGF y SIF 0, 10, 30 y 60 minutos5 L de mezcla en 12 L de buffertubos de ensayo y 1 L de 0,2 M NaCO3 ElectroforesisComparacin de bandasde protenas 14. MATERIALES Y MTODOS6. Inmunoblot de IgE del arrozEn 18 de los 24 pacientes4 Sueros para el controlBandas de protenasElectrotransferidas desde Bloqueada con 3% de albmina el gel a una membrana de suero bovino-fosfato-buffer- nitrocelulosa salino por una hora>16 Horas a 4C Adicin desueroLavado 3 veces con PBS-Talrgico y suero control durante 10 minutosa la membranaLavado con PBS-T 15. MATERIALES Y MTODOSConjugacincon2 horas a Conjugacin conanticuerpo biotiniladotemperatura avidinfosfatasa poranti IgE humana ambiente30 minutos* Las bandas de protenas del arroz calentadose electrotransfirieron y se les realizinmunoblot de forma similar. 16. RESULTADOSFig. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of (A) raw rice, (B) cooked rice, (C) cookedunpolished rice, and (D) semi-cooked rice (Hatban). M, marker; kDa, kilodalton. 17. RESULTADOSSDS-PAGE para alrgeno del arrozArroz crudoBandas 9, 14, 16, 18, 20, 22, 25, 28,31, 33-35, 46, 57, 62, 79, 94, 109 kDa 18. RESULTADOSSDS-PAGE para alrgeno del arroz Arroz cocido, Arroz integral cocido intensidad Arroz semicocidoBandas de protenas 14-16, 31 - 35 kDa. 19. RESULTADOSFig. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of rice treated with (A) heat, (B) simulated gastric fluid (SGF), and (C) simulated intestinal fluid (SIF). M, marker; kDa, kilodalton. 20. RESULTADOSCambios en las bandas de las protenas del arroz despus del tratamientocon calor y enzimas digestivas 10 bandas se mantienen. 30 bandas se mantienen- desvanecimiento. 60 bandas degradadas, menos 9,14, 31-35 kDa. 21. RESULTADOSCambios en las bandas de las protenas del arroz despus del tratamientocon calor y enzimas digestivas Bandas degradadas inmediatamente.60 bandas 9, 14, 28, 31, 35 kDa se mantuvieron. 22. RESULTADOSCambios en las bandas de las protenas del arroz despus del tratamientocon calor y enzimas digestivas Pocas bandas degradadas. 23. RESULTADOSCambios en las bandas de las protenas del arroz despus del tratamientocon calor y enzimas digestivas9, 14, 31 kDa son estables despus del procesamiento con calor digerido con las enzimas en el estmago y el intestino 24. RESULTADOSFig. 3. Rice immunoglobulin E immunoblot of patient sera and control. M, marker; kDa, kilodalton. 25. RESULTADOSInmunoblot de IgE del arroz crudoGel de electro-transferenciaInmunoblot de IgE suero 18 pacientessensibilizadosgrupo controlProtenas alergnicasActividad de unin al IgE 26. RESULTADOS Inmunoblot de IgE del arroz crudoProtenasmasa molecular9 kDa 109 kDa14 kDa88.9%79 kDa50%109 kDa 44.4%9,33,46 kDa 38.9%57 kDa33.3% 27. RESULTADOS 28. RESULTADOSFig. 4. Heated-rice immunoglobulin E immunoblot of patient sera and control. M, marker; kDa, kilodalton. 29. RESULTADOSInmunoblot de IgE del arroz calentadoGel de electro-transferenciaInmunoblot de IgE suero 18 pacientes sensibilizados controlbandas descoloridas 30. RESULTADOSInmunoblot de IgE del arroz calentadoProtenas14 kDa 38,9%9 kDa 27.8%31,33 kDa 38.9% 31. RESULTADOS Inmunoblot de IgE del arroz calentadoSuero de 4 pacientes con sntomas alrgicos (5,11,15,16)Reaccionaron con bandas de protena31-33 kDa 75%9 kDa 50%14 kDa50% 32. RESULTADOS 33. DISCUSSION Name ApproachTrue or FalseWu KG, et al. There are several studies that serum ECP levels in False: because in food allergy, theShin JE, et al. patients with atopic dermatitis is related withrelationship between serum ECP leveldisease severity.with clinical food allergy, sympton is not evident.Figueredo E, et al. An atopic woman, who developed anaphylaxis after True: because this suggests thedrinking beer with a corn made snack, was sensitized possibility of latent sensitization by ato not only corn and barley, but also to wheat, rye, common epitope.oat and rice. 34. DISCUSSIONNameApproachTrue or FalseYamada C, et al. Heat-processing or digestion can alter antigenicity True: becausethe allergenriceisUntersmayr E, et of allergen. The rice allergen is weak after theweak after heating andtheal.heat-process. The antigen specific IgE of ricebands decreased in intensity and othersKilshaw PJ, et al. decreases after heating at 100 for 60 minutes. disappear in cook rice.Yum HY, et al. Studies have revealed cross reactivity of rice with False: Because the allergens buckwheat, barley or wheat and one report that actually cause the allergic symptoms compared the antigenicity of rice and low on the patients with sensitization to rice allergenic rice.allergen are not obvious and an interpretation of the sensitization to rice is also unclear. 35. CONCLUSSIONS Although rice isnt has high allergenicity, in Asian countries like Japan and Korea,the allergenicity has increased in the numbers of patients sensitized to riceallergen, because its the main food in the meals. The allergenicity decrease when the rice is cooked, because the proteinbands decreased in intensity and others disappear in cook rice. Also the allergenrice is weak after heating. 36. CONCLUSSIONS TotalIgE, is an important factor in terms of sensitivity to rice, because in these cases the IgE is increased along with the ECP (eosinophil cationic protein). This investigation is very important, because this area is poorly studied, and also help to find the inmunologic characteristics of rice allergen . 37. MAPA CONCEPTUALCarolina Giraldo 38. MAPA CONCEPTUALManuela Colorado 39. THANKS! GRACIAS!

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