Seminario: Identification of major rice allergen and their clinical significance in children

Identification of major rice allergen and Identification of major rice allergen and their clinicaltheir clinical

significance in childrensignificance 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 Benítez

III Semestre MedicinaUPB

INTRODUCTIONINTRODUCTION

AllergyAbnormal, inappropriate and exaggerated reaction of immune system to substances that typically are not well tolerated. Is mediated by IgE

Is 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 contact with the allergen.


• 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 atopic dermatitis with IgE-rice positive.

• Is a few reports on the correlation of rice allergen sensitization and their clinical symptoms.

• The immunologic characteristics of rice allergen are unclear.


• 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 as a relevant factor in symptomatic rice allergies.

GENERAL OBJECTIVEGENERAL OBJECTIVE

• Determine the major allergens in rice and their clinical significance.

Objetivo http://freestyle.bligoo.com.co/objetivos

MATERIALES Y MÉTODOSMATERIALES Y MÉTODOS

1. Sujetos y grupos de control

Sujetos


Grupo control44 Pacientes

IgE-Arroz Negativa

2 Grupos

n=18Sujetos sensibilizados, pero

no al arroz

n=26Sujetos no sensibilizados


2. Métodos

1. Análisis de sujetos y grupo control

• Historial médico y pruebas de laboratorio Edad, sexo, síntomas de la alergia, IgE en suero, ECP, IgE específica.

Erupción cutánea, eritema, urticaria, angioedema, el síndrome de alergia oral, rino-conjuntivitis, sibilancias, anafilaxia tras la ingesta de arroz con leche, arroz hervido, galletas de arroz, etc.

* Agravación de la dermatitis atópica se excluyó.


2. Extracción del alérgeno


* Extracto de arroz crudo: 5.6 mg/mL de proteína


1. Sodio dodesil sulfato policrilamida gel electroforesis. (SDS PAGE)

• Muestra + Buffer Se calentó

• Electroforesis durante 90 minutos SDS (amortiguación con el marcador)

• Visualización por tinción con azul de coomassie brillante

• Cuantificación de bandas Densitometría


4. Análisis de las bandas de proteínas de arroz y arroz cocido

Electroforesis:

Comparación y análisis de distribución de bandas

• Arroz crudo• Arroz cocido• Arroz integral cocido• Arroz semicocido

SDS 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

MATERIALES Y MÉTODOSMATERIALES Y MÉTODOS5. SDS PAGE de arroz tratado con calor, fluido gástrico simulado (SGF) y fluido intestinal simulado (SIF)

•Electroforesis en serie

•Proteína de arroz se hirvió a 100°C 10, 30 y 60 minutos Electroforesis

Se trató con enzima digestiva, SGF y SIF 0, 10, 30 y 60 minutos

5 μL de mezcla en tubos de ensayo12 μL de buffer

y 1 μL de 0,2 M NaCO3

Electroforesis

Comparación de bandas de proteínas

6. Inmunoblot de IgE del arroz

En 18 de los 24 pacientes 4 Sueros para el control

Bandas de proteínas Electrotransferidas desde el gel a una membrana nitrocelulosa

Bloqueada con 3% de albúmina de suero bovino-fosfato-buffer-salino por una hora

Lavado 3 veces con PBS-T durante 10 minutos

Adición de suero alérgico y suero control a la membrana

>16 Horas a 4°C

Lavado con PBS-T



Conjugación con anticuerpo biotinilado anti IgE humana

2 horas a temperatura ambiente

Conjugación con avidinfosfatasa por 30 minutos

* Las bandas de proteínas del arroz calentado se electrotransfirieron y se les realizó inmunoblot de forma similar.

RESULTADOSRESULTADOS

Fig. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of (A) raw rice, (B) cooked rice, (C) cooked unpolished rice, and (D) semi-cooked rice (Hatban). M, marker; kDa, kilodalton.


SDS-PAGE para alérgeno del arroz

Arroz crudo

Bandas 9, 14, 16, 18, 20, 22, 25, 28,

31, 33-35, 46, 57, 62, 79, 94, 109 kDa


SDS-PAGE para alérgeno del arroz

Arroz cocido,

Arroz integral cocido

Arroz semicocido

Bandas de proteínas 14-16,

31 - 35 kDa.

intensidad


Fig. 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.


Cambios en las bandas de las proteínas del arroz después del tratamiento con calor y enzimas digestivas

10’ bandas se mantienen.

30’ bandas se mantienen- desvanecimiento.

60’ bandas degradadas, menos 9,14, 31-35 kDa.



Bandas degradadas inmediatamente.

60’ bandas 9, 14, 28, 31, 35 kDa se mantuvieron.



Pocas bandas degradadas.



9, 14, 31 kDa son estables

después del procesamiento con calor

digerido con las enzimas en el estómago y el intestino


Fig. 3. Rice immunoglobulin E immunoblot of patient sera and control. M, marker; kDa, kilodalton.


Inmunoblot de IgE del arroz crudo

Gel de electro-transferencia

Inmunoblot de IgE suero 18 pacientes sensibilizados

grupo control

Proteínas alergénicas

Actividad de unión al IgE


Inmunoblot de IgE del arroz crudo

Proteínas

masa molecular 9 kDa – 109 kDa

14 kDa 88.9%

79 kDa 50%

109 kDa 44.4%

9,33,46 kDa 38.9%

57 kDa 33.3%



Fig. 4. Heated-rice immunoglobulin E immunoblot of patient sera and control. M, marker; kDa, kilodalton.


Inmunoblot de IgE del arroz calentado

Gel de electro-transferencia

Inmunoblot de IgE suero 18 pacientes sensibilizados

control

bandas descoloridas



Proteínas

14 kDa 38,9%

9 kDa 27.8%

31,33 kDa 38.9%



Suero de 4 pacientes con

síntomas alérgicos (5,11,15,16)

Reaccionaron con bandas de proteína

31-33 kDa 75%

9 kDa 50%

14 kDa 50%


DISCUSSIONDISCUSSIONNameName ApproachApproach True or FalseTrue or False

Wu KG, et al.

Shin JE, et al.

There are several studies that serum ECP levels in

patients with atopic dermatitis is related with

disease severity.

False: because in food allergy, the

relationship between serum ECP level

with clinical food allergy, sympton is

not evident.

Figueredo E, et al. An atopic woman, who developed anaphylaxis after

drinking beer with a corn made snack, was sensitized

to not only corn and barley, but also to wheat, rye,

oat and rice.

True: because this suggests the

possibility of latent sensitization by a

common epitope.

DISCUSSIONDISCUSSION

NameName ApproachApproach True or FalseTrue or False

Yamada C, et al.

Untersmayr E, et

al.

Kilshaw PJ, et al.

Heat-processing or digestion can alter antigenicity

of allergen. The rice allergen is weak after the

heat-process. The antigen specific IgE of rice

decreases after heating at 100 for 60 minutes.℃

True: because the allergen rice is

weak after heating and the

bands decreased in intensity and others

disappear in cook rice.

Yum HY, et al. Studies have revealed cross reactivity of rice with

buckwheat, barley or wheat and one report

compared the antigenicity of rice and low

allergenic rice.

False: Because the allergens

that actually cause the allergic symptoms

on the patients with sensitization to rice

allergen are not obvious and an

interpretation of the sensitization to rice

is also unclear.

CONCLUSSIONSCONCLUSSIONS

• Although rice isn't has high allergenicity, in Asian countries like Japan and Korea,

the allergenicity has increased in the numbers of patients sensitized to rice

allergen, because it’s the main food in the meals.

• The allergenicity decrease when the rice is cooked, because the protein

bands decreased in intensity and others disappear in cook rice. Also the allergen

rice is weak after heating.

CONCLUSSIONSCONCLUSSIONS

• Total IgE, 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 .

MAPA CONCEPTUALMAPA CONCEPTUAL

Carolina Giraldo

MAPA CONCEPTUALMAPA CONCEPTUAL

Manuela Colorado

THANKS!THANKS!GRACIAS!GRACIAS!고맙습니다고맙습니다

Health & Medicine

Seminario: Identification of major rice allergen and their clinical significance in children