Food x Immune system –
a daily battle usually with no winners
Marcos Alcocer
Nutritional Sciences
Sutton Bonington campus
University of Nottingham
United Kingdom
IAFP - Campinas 2008
How does food interacts with the IS?
Background
Is food allergy a problem?
Is the prevalence of food allergy increasing?
(What makes an allergen???)
Where does our work fit in this????
Other work
Protein microarray work
Questions
Are we tolerant to all food proteins ?
Are all food (plant) proteins allergenic ?
Which are the characteristics of a molecule that
are responsible for its allergenicity ?
Can we predict when a protein is allergenic ?
Background
The Gut is Bombarded by Foreign Antigens
•Eradication
•Containment
No Response
(Tolerance) • “Disease”
• Crosslinking of IgE by food Ag
• Activation of mucosal mast cells
• Release of inflammatory mediators
Transepithelial fluid loss
Smooth muscle contraction
Response
Vomiting and diarrhoea
(Immune Activation)
Anaphylaxis
mucosal barrier
Mast Cell / basophil degranulation
Video 2
The Th2 response
J ALLERGY CLIN IMMUNOL
VOLUME 121, NUMBER 2
NOVAK AND BIEBER
Is food allergy a problem?
Anaphylaxis Deaths 1992-98
uncertain, 4
peanut
nectarine, 1
walnut
banana, 1 nuts
peanut, 10
milk, 2 chickpea
seafood, 3 seafood
chickpea, 1 milk
walnut, 5
banana
nuts, 10 nectarine
uncertain
Pumphrey 2000
Is the prevalence of food allergy increasing?
Number of cases of anaphylaxis to food
products are increasing…
Gupta & Sheikh, BMJ,2003
Atopic march
Asthma
Incidence
Rhinitis
Eczema
Food allergy
0 1 2 4 8 16 32 64
Age (years)
Redrawn from Durham SR & Church MK, Allergy 2nd edition, 2001, Mosby
Prevalence of IgE mediated food allergy
1992 - USA – 950 cases/year or 0.0004%
1994 - 2% infants – cow’s milk allergy
1998 - USA – 10% of general population= atopic
Europe - 0.3 –7.5%
Europe - 10% atopic = food allergic
2005-7 – USA – 3.5-4% adults
6-8% young children and infants
2007/8– UK, comprehensive House of Lords report - ??????
Where does our work fit in this?
Food allergens work at Nottingham
Protein model system
In vitro DC-studies
Effect of Food matrix
HTP screening
Protein model system
Ber e 1: Brazil nut 2S albumin, well-known allergen, (45,000 tone/year)
SFA 8 : Sunflower 2S albumin, “weak” allergen, (26,852,000 tone/year)
What makes an allergen?
“allergenicity” ????
SFA 8 : Sunflower 2S Ber e 1: Brazil nut 2S albumin
albumin “weak” allergen well-known allergen
IOW67
IOW59
80 GJM 15 IOW52
Lambda (nm) vs Ber e 1 IOW46
60 Lambda (nm) vs SFA8
Lambda (nm) vs pGJM 13 IOW42
Lambda (nm) vs pGJM 10
Lambda (nm) vs pGJM 16 IOW38
40 Lambda (nm) vs pGJM 09
Lambda (nm) vs pGJM 12 IOW37
Lambda (nm) vs pGJM 15
[deg cm2 dmol-1
GJM 13
20
MANe
MANd
0 MANc
MANb
-20
MANa
GJM 12
ITAb
-40
“The major IgE structural epitope
ITAa
ITA06
recognised of Ber e 1 was mapped to a
-60
ITA05
200 210 220 230 240 250
single helix-loop-helix region.”
GJM 16
ITA04
(nm)
ITA03
ITA02
ITA01
GJM 10
GJM 09
SFA8
0.00 20.00 40.00 60.00 80.00 100.00 120.00
I Fluor. x100/I Fluor. Be r e 1
Journal of Molecular Biology, 343:759-769
Protein model system
-All the backbone assigned
EAEAQEECREQMQRQQMLSHCRMYMRQQMEESTYQTMPRRGMEPHMSECCEQLEGMDESCRCE
GLRMMMRMMQQKEMQPRGEQMRRMMRLAENIPSRCNLSPMRCPMGGSIAGF
-Multiple resonances for several residues “Labelled 13C/15N proteins have
been produced and purified. The NMR
3D structure in solution of Ber e 1 is
determined”
Journal of Biomolecular NMR, 32(4):336 (2005)
Food allergens work at Nottingham
“ Early decisions were taken by DC
and ability of Ber e 1 to induce a
Th2 response is depending on the
genetic background”
Protein model system
NON-ATOPIC ATOPIC
In vitro DC-studies
Ber e 1 SFA8 Ber e 1 SFA8
DC CD40 DC DC CD40 DC
ligation ligation
T IL-12 IL-12
IL-12 IL-12 no IL-12 T
Th1 Th1 Th2 Th1
ALLERGY
Food allergens work at Nottingham
1
Dia 0
m 8
Protein model system PC 6
A
rea 4
c.
In vitro DC-studies (m
2
m)
W RE RE REC R RE RE N
Effect of Food matrix
T C C com E C C M
ster tot plex C oth low S
oid al lipids ers mol
lipi wt
ds
NO IgE !!!
“Protein alone is unable to trigger
allergic response. A second natural
signal present in the seed needs to
be present ”
“The allergenic work at Nottingham
Food allergens protein is able to
bind and present natural lipids or
hydrophobic molecules ”
Lipid binding ??
Partial conclusions
Allergenicity of proteins are product of complex signals such as genetic
background, structure of the allergen and food matrix.
Certain cells of the immune system such as DC are the main orchestrators
of the allergic response
How do the non-immune compromised
(normal) population interacts with food?
Protein microarray
B B
G B
Fluorescence B
E
Wheat LTP
Trypsin inhibitor
Trypsin/Chym.inh.
Thyroglobulin
Protein microarray
rSoya 2S Alb1
Soya 2S Alb 3 w t
rSoya 2S Alb 3
Soya 11S
SFA8 w t
rSFA8
Protein A
Pichia extract
Pepstatin
Peanut 05
Peanut 03
Papain
rOV-20
Mouse IgE
lysozyme chicken
Lectin Soya
Lectin Hypogae
hu.sera albumin
human IgG1
human IgE
Gelatine
Concanavalin
Chicken albumin
Carb.anhydr.
Calreticulin w t IoW37 IgE
rCalreticulin IoW52 IgE
Brazilnut total IoW37 IgG4
Bovine albumin
IoW52 IgG4
Beta amylase
Ber e 1 w t
rBer e 1
Ara h 2
Apoferritin
Alc.dehydrog.
0 2000 4000 6000 8000
Fluorescence 532 nm
“All British diet array”
E
G
400 food ingredients “All British diet array”
1.500 “intact” protein groups x 4
Four lasers = IgE/M/A/G -> 20.000 data points/patient
Main applications:
• Specific IgE screening allergic subjects
• Specific IgE screening post-morten
• Specific Ig (A/G/M) screening:
Serum, Milk, saliva.
Vegetables Fruits Herbs and Spices
Alfalfa, Artichoke, Asparagus, Aubergine, Avocado, Baby Chard Leaf, Bamboo Apple, Apricot, Banana, Blackberry, Blackcurrant, Blueberry, Cherry, Arrowroot, Basil, Bay Leaves, Black Pepper, Camomile, Cardamom, Cayenne
Shoot, Beansprout, Beetroot, Bhindi, Borlotti Bean, Broad Bean, Broccoli, Brussels Clementine, Coconut (Desiccated), Coconut Milk, Currants, Dates, Fig, Pepper, Chilli Powder, Cinnamon, Cloves, Coriander (Ground), Cumin, Curry
Sprout, Butternut Squash, Cabbage, Caper, Carrot, Cauliflower, Celeriac, Celery, Gooseberry, Grape, Grapefruit (Red), Guava, Italian Kiwi, Italian Kiwi Leaves, Curry Powder, Custard Powder, Dill, Earl Grey, Fennel Seed, Fragrance
Chervil (Fresh), Chickpea (Cooked), Chilli (Green), Chilli (Red), Chinese Cabbage, (Hayward), Kiwi, Kiwi (Protein), Lemon, Lemon Rind, Lime, Lychee, spice, Garam Masala, Ginger (Ground), Juniper Berry, Lemongrass, Lime Leaves,
Chives (Fresh), Coriander Leaf, Courgette, Cress, Cucumber, Dal Chini, Fennel, Mandarin Peel, Mango, Melon, Nectarine, Orange, Orange Marmalade, Liquorice Root, Marjoram, Mint, Nutmeg, Oregano, Paprika, Parsley, Peppermint,
Ganna (Spain), Garlic, Gherkin, Leek, Lettuce, Lettuce (Lambs), Maize Babycorn, Papaya, Passion Fruit, Paw Paw, Peach, Pear, Physalis, Pineapple, Plum, Pimiento Pepper, Rosemary, Saffron, Sage, Tarragon, Tea Leaves, Thyme,
Mange Tout, Marrow, Mushroom, Mushroom (Oyster), Mushroom (Shitake), Okra, Pomegranate, Prune, Pumpkin, Raisins, Rambutant, Raspberry, Rhubarb, Turmeric.
Olive (Black), Olive (Green), Onion (Red), Onion (White), Pak Choi, Parsnip, Peas, Sharon Fruit, Strawberry, Sultanas, Tamarind (Dried), Tamarind (Fresh),
Pepper (Red), Potato, Radish, Rocket, Runner Bean, Seaweed, Shallot, Spinach, Tangerine, Watermelon.
Spring Onion, Swede, Sweetcorn, Texturised Vegetable protein, Tomato, Turnip,
Water Chestnut, Watercress.
Beans Nuts Cereals
Baked Bean, Black Bean, Black Canna (Chickpea), Black eyed Bean, Black Kidney Almond, Almond, Almond (Blanched), Barley Seed, Brazil Nut, Brazil Nut Basmati Rice, Bread (White), Corn, Corn Starch, Cornflour, Cous Cous, Egg Noodle,
Bean, Butter Bean, Cannalini Bean (Cooked), Chicory, Coffee Bean, Flageolet (Protein), Cashew, Chestnut, Hazelnut, Hazelnut (Organic), Lectin Flour (Chapatti), Lentil (Red), Oats, Pasta, Pearl Barley, Rice (Arborio), Rice
Bean, Green Mung Bean, Haricot Bean, Lectin (Soya), Lentil Yellow (Moog dal), (Peanut), Macadamia, Paleskin Nut, Peanut, Peanut (Blanched), Pecan, (Basmati), Rice (Red), Rye, Sago, Tapioca, Wheat, Wheat Flour, Wheat Germ,
Pinto Bean, Red Bean, Red Kidney Bean, Soya (Total Protein), Soya Bean, Soya Pine Kernel, Pine Nut Kernel, Pistachio, Rye Seed, Walnut, Walnut Wheat LTP.
Bean (UK Soy), Soya Flour, Soya Milk, Soya Milk (Extract), Soya Sprout, Split (Organic).
Chick pea (ChannaDal), Tofu (Extract).
Seeds Dairy Meat
Aniseed, Aniseed Star, Caraway Seeds, Celery Seed, Cocoa, Coriander Seed, Brie, Buttermilk, Cheddar, Cottage Cheese, Cow's Milk, Cow's Milk (Whey), Bacon, Beef, Beef Stock, Chicken, Chicken Liver, Duck, Gelatine, Ham, Kidney (Pig),
Fenugreek, Green Pepper Seeds, Hemp Seed, Linseed, Mustard (Black), Mustard Cream, Edam, Feta, Fromage frais, Goat's Milk, Greek Yogurt, Mozzarella, Lamb Liver, Pepperoni, Pig Liver, Pork, Sausage Casing, Smoked Bacon, Suet,
(Yellow), Mustard Seeds (Black), Pomegranate Seed, Poppy Seed, Rape Seed, Parmesan, Sour Cream, Stilton, Tofu (Paneer), Yogurt (Plain). Turkey, Venison.
Sesame, Sunflower Seed, White Pepper Seed.
Fish Drink and Sauce Others
Anchovy, Cod, Coley Fish, Herring, Kipper, Mackerel, Pilchards, Plaice, Salmon, Ale, Beer, Black Bean Sauce, Black Pepper Sauce, Cranberry Sauce, Baking Powder, Chocolate, Chocolate (Plain), Cream of Tartar, Glutamine (MSG),
Sardine, Smoked Haddock, Sprat, Trout, Tuna. Horseradish Sauce, Malt Vinegar, Marmite, Satay Sauce, Sherry, Soya Pichia, Rock Salt, Sugar (Brown), Sugar (Muscavado), Yeast (Dry).
Sauce, Vegetable Stock, Worcestershire Sauce.
Shellfish Eggs Inhaled Allergens
Cockles, Crab, Crab (Cooked), Mussels, Oyster, Oyster Sauce, Prawn, Scallop, Duck Egg, Duck Egg (White), Duck Egg (Yolk), Duck egg white, Egg Birch Pollen, Grass Pollen, Grass Pollen (Velvet), House dust mite, Nectar.
Squid. Albumin, Egg Ovalbumin, Hen Egg, Hen Egg (White), Hen Egg (Yolk).
“ Normal” population does produce
food specific antibodies in large
quantities.
IgA IgG
60000 60000
50000 50000
532 nm
594 nm
40000 40000
Fluorescence
Fluorescence
30000 30000
20000 20000
400 400
350 350
10000 300 10000 300
ts
ts
“ Some allergic people are
uc
uc
250 250
od
od
200 200
0 0
pr
150 pr 15 14 150
polyspecific, others mono specific.”
15 14
d
od
13 12 13 12
o
11 10 100 11 10 100
Fo
Fo
9 8 9 8
7 6 50 7 6 50
5 4 5 4
Patie 3 0 Patie 3 2 0
nts IgM 2 1 0 nts 1 0
IgE
“ Non allergic control patients also
60000
20000
produce low level of food specific
50000 18000
488 nm
16000
635 nm
IgE.”
40000
14000
Fluorescence
30000 12000
Fluorescence
10000
20000
400 8000
350 6000
10000 300 400
ts
350
uc
250 4000
od
300
cts
200
0
pr
2000 250
du
15 14 150
od
13 12 200
o
11 10 100 0
pr
Fo
9 8 15 14 150
7 6
od
50 13 12
5 4 11 10 100
Fo
Patie 3 2 0 9 8
nts 1 0 7 6 50
5 4
Patie 3 2 0
nts 1 0
Atopic produced less total IgG than
non-atopic.
Atopic Non-Atopic
* Significant Difference (P<0.002) *
* * * * * IgG
Adjusted Relative Scale
*Significant Difference (P<0.050) * *
* * *
IgE
* Significant Difference (P<0.004)
IgA
*
* * *
* Significant Difference (P<0.050)
IgM
*
Some food classes induce food
iry
h
r
h
ts
ce
at
es
ble
s/S it
s
s
ls
gs
s
he
u
ed
an
en
Fis
fis
ria
Nu
Me
Da
Fr
au
Eg
pic
eta
Ot
ell
Se
Be
rg
specific antibodies in large quantities.
Ce
/S
lle
Sh
eg
ink
dA
rb
t/V
Dr
He
ale
an
Pl
Inh
Array, preliminary data – Milk sample
9000
8000
7000
6000
5000
Fluorescence 4000
3000
2000
1000
S15
S13
0
S11
-1000 S9
Days
795
S7
822
1199
998
1299
517
1416
1079
S5
966
904
911
1030
1134
511
S3
1446
838
519
964
933
988
S1
1215
804
426
Samples (ID)
1114
488
1335
Protein microarray+ basophils????
E
G
Activation of RBL 703/21
Light
microscope
GenePix 4000B
scanner Fluorescent intensity (Measured using GenePix software
GPA IgE
491 592 295 344 695 472
E
G
27 47 94 6 5
Anti IgE
Grass pollen
IAFP - Campinas 2008
Our immune system “responds” to our daily load of food proteins
Allergenicity of proteins are product of complex signals such as genetic
background, structure of the allergen and food matrix.
Certain cells of the immune system such as DC are the main orchestrators
of the allergic response
Some food groups are referentially recognised by our immune system.
Novel high-throughput systems are allowing a comprehensive “fingerprinting”
of the immune response to food proteins
Acknowledgements
Zellulaere Allergologie
Forschungszentrum Borstel
Germany
Helmut Haas
Gabi Schramm
School of Pharmacy
University of Nottingham
United kingdom
Franco Falcone
Paul Ehrlich
Institute
Germany
Lothar Vogel
Stefan Vieth
University of Strathclyde
Syngenta Scotland
School of Biosciences
University of Nottingham William Harnett
BBSRC
Universite de Liege
School of Biology Belgium
University of Nottingham Alain Brans
D. Archer