the rat brown norway model to asses the oral...
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The rat Brown Norway model to asses the oral sensitizing properties of food proteins André Penninks
13 April 2012 ILSI-HESI-PATC, Prague, Czech Republic
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Special acknowledgement
Léon Knippels, PhD
Present address: Danone Research-Centre for Specialised Nutrition
Wageningen The Netherlands
and colleagues involved in the studies conducted in the BN
rat model.
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Why Should We Use Animal Models?
Sensitization studies in humans not possible, only challenge reaction can be studied in patients
Mechanistic research of IgE-mediated food allergy
Development of methods for prevention or therapy of food allergy
Prediction of potential allergenicity of new food proteins (e.g. GMO’s), ranking of the relative allergenicity
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Prediction of Food Allergy
More attention for animal models predicting potential
allergenicity was raised following the introduction of GMO’s:
Is a protein that was not previously common in the human diet likely to be allergenic if introduced in commodity crops? Will production of biotechnology (GM) crops alter the allergenicity of endogenous proteins?
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IFBC Allergenicity Assessment for GM crops
Source of Gene (Allergenic)
Sequence 1 Similarity
Solid Phase Immunoassay
Commonly Allergenic
Less Commonly Allergenic
Stability to Digestion/
Processing Skin Prick Test
DBPCFC (IRB)
STOP or label
Consult with Reg. Agency
for appropriate further tests
Reg. Approval / Market
Yes No
Yes
No Yes Yes
Yes No
No
Yes
No No (<5 sera)
Yes No
No (>5 sera)
Further testing = animal models, or ex-vivo testing ?
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Assessment of the Allergenic Potential of Foods Derived from Biotechnology
FAO/WHO 2001 Decission Tree
Source of gene allergenic
Sequence Homology
Sequence Homology
Specific Serum Screen
Targeted Serum Screen
Likely Allergenic
Pepsin Resistance
Animal Models
+/+ +/- -/-
High Low
Probability of
Allergenicity
Yes No
No Yes
No Yes No
No Yes
Yes
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Key factors for animal models (1)
Simple protocols for sensitization and challenges which are reproducible between laboratories and over time, and which take into account:
Genetic predisposition (species/strain-high/low responder/atopic prevalence)
Unscheduled dietary pre-exposure of test animals to test protein (diet, parents)
Sensitization age – neonate, adolescent, adult route – oral/gavage/i.p./dermal/subcutaneous. test material – whole foods/purified proteins dose frequency- daily, twice weekly, weekly etc Dose amount – high dose (tolerance)/low dose (sensitization) Use of adjuvant [no adjuvant, cholera toxin (oral), Alum (i.p.)]
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Key factors for animal models(2)
Tolerant to most food proteins Comparable allergenicity – strong/week/non-allergenic
IgE response to comparable proteins as found in patients
Clinical reactions upon challenge
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Relative Food Allergenicity - Animal Models (severity) -
Alle
rgen
icity
Proteins
Ara h 2
ovomucoid b-lactoglobulin
Sol t 1 (Potato)
shrimp tropomyosin Ara h1
6 – 12 soybean proteins
Wheat
Celery
Rubisco
BSA Ara h3
ovalbumin a-lactalbumin caseins
10 10 10 10 10
Animal Models
Predictive models - Brown Norway rat - oral/i.p. (Knippels et al, from 1998) - Balb/c mice - i.p., no adjuvant (Dearman et al, from 2000) - Balb/c mice - oral, CT (Adel-Patient et al, 2005) - B10A mice - i.p./nasal (Akiyama et al, 2001) - Guinea pig - oral/gavage/drinking water/i.p. (Kitagawa et al,
1995) - Dog - food with adjuvant (Ermel et al, 1997)
Mechanistic models - C3H/HeJ mice - oral with CT (Li et al, from 1999, 2000; van Wijk
et al, from 2004) - DBA/2 mice (Ito et al, 1997) - Swine (Helm et al, 2002, 2003) Each model has pro’s and con’s and potential problems
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Brown Norway rat food allergy model Several dosing protocols studied in BN rats:
- Ad libitum via drinking water; 0.002, 0.02, 0.2, 2 and 20 mg/mL OVA for 6 weeks
- Gavage dosing of 1 mg/kg OVA, daily, once a week, twice a week, once every two week
Knippels et al, Clin Exp Allergy, 1998, 28, 368-375.
Several rat strains studied with OVA 1mg/rat for 6 wks:
- BN rats - Hooded Lister (HL) - Piebold Viral Glaxo (PVG) - Wistar IgG in all strains from d 7, IgE only in BN rats
Knippels et al, Fd Chem Toxicol, 1999, 37, 881-888.
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Brown Norway rat food allergy model
Young male Brown Norway rats (4-6 wks), bred and raised on a test protein free diet (e.g. OVA, egg white, cow’s milk free diets). 1 mg protein/ml/rat/day by gavage dosing for 42 days no adjuvants Specific IgG1, IgG2a and IgE responses Specific T cell proliferation Clinical symptoms after oral challenge Comparison of antibody responses to allergic food proteins in BN rats to those in allergic patients
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Sensitization and challenge in the BN rat allergy model
0 42 50
Sensitization Rest Challenge
Daily gavage 1 mg protein
•Blood samples weekly
•IgG and IgE
protein by gavage • Blood pressure
• Respiratory effects
• Gut permeability
• RMCPII
• T cell proliferation
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Ovalbumin-specific IgG and IgE responses
0
2
4
6
8
10
12
14
0 7 14 21 28 35 42 49 56 63
2Log
IgG
tite
r
0
2
4
6
8
10
12
14
0 7 14 21 28 35 42 49 56 63 2L
og Ig
E tit
er
Ovalbumin-specific IgG response Ovalbumin-specific IgE response
Days Days
Knippels et al, Allergy, 55, 251-258
15 15 15 15
Hen’s egg white and cow’s milk-specific IgG responses
14
Days
0
4
8
12
16
20
2log
IgG
tite
r
6 5 6 4 4 5 4 3 4 2 4 2 4 2 3 2 2
7 0 21 28 35 42 49 56 63 0 7 14 21 28 35 42 49 56 63
Days
0
3
6
9
12
15
2log
IgG
tite
r 2/5 4/5 4/5 5/5 5/5
Milk Egg
Knippels et al, Allergy, 55, 251-258
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Immune-mediated clinical effects (OVA sensitized and challenged)
Blood pressure - decreased 10-20% of the sensitized animals Respiratory effects - decreased 10-20% of the sensitized animals Gut permeability - increased uptake bystander protein RMCP-II - increased release in serum
T cell proliferation - increased
Knippels et al, Toxicol Appl Pharmacol, 1999, 156, 161-169
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Airway responsiveness control animals
0 20 40 60 80 100 120
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
Brea
thin
g Fr
eque
ncy
(Hz)
0 20 40 60 80 100 120
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
Brea
thin
g Fr
eque
ncy
(Hz)
Time (min) Time (min)
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Airway responsiveness sensitized animals
0 20 40 60 80 100 120
Time (min)
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
Brea
thin
g Fr
eque
ncy
(Hz)
0 20 40 60 80 100 120
Time (min)
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
Brea
thin
g Fr
eque
ncy
(Hz)
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β-lactoglobulin levels in sera from control and test animals
Time (hr) 0 0.5 1 2 3 5 8
ip sens. <LOD 1.7-4.6* 0.9-2.4* <LOD-1.6 <LOD <LOD <LOD
oral sens <LOD 0.05-0.13* 0.07-0.09 <LOD-0.05 <LOD <LOD <LOD
control <LOD <LOD-0.02 <LOD <LOD <LOD <LOD <LOD
LOD: Limit of detection (0.01 µg/ml)
Knippels et al, Toxicol Appl Pharmacol, 156, 161-169
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Unscheduled dietary pre-exposure!
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Soy-protein specific IgG responses
i.p.
0
5
10
15
20
2log
IgG
tite
r
rats bred on a soy-protein containing diet
42 28 28 RB RB RB RB
ad lib ad lib Soy i.p.
Soy Soy
0
5
10
15
20
2log
IgG
tite
r
rats bred on a soy-protein free diet
F4 F4
28 28 28 28 42 42
F4
ad lib
F4
ad lib
F4 F4 Soy
ad lib
F4 Soy
ad lib USF USF USF Soy
i.p.
Knippels et al., JACI, 1998;101:815-20
i.p.
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Dietary Control Soy-protein specific IgG titers
RB F0 F1 F2 F3 F4 0
2
4
6
8
2log
IgG
tite
r
Knippels et al., JACI, 1998;101:815-20
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Soy-protein specific IgG responses
i.p.
0
5
10
15
20
2log
IgG
tite
r
rats bred on a soy-protein containing diet
42 28 28 RB RB RB RB
ad lib ad lib Soy i.p.
Soy Soy
0
5
10
15
20
2log
IgG
tite
r
rats bred on a soy-protein free diet
F4 F4
28 28 28 28 42 42
F4
ad lib
F4
ad lib
F4 F4 Soy
ad lib
F4 Soy
ad lib USF USF USF Soy
i.p.
Knippels et al., JACI, 1998;101:815-20
i.p.
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25 25 25 25 25
Peanut specific IgG2a responses
0
2
4
6
8
10
12
Day 7 Day 35 Day 42
2log
IgG
2a ti
ters
20 10 40 89 50 100 60 40 20 70 70 50 80 40
I.P
100
0,01 mg/day 135°C
0,1 mg/day 135°C
1,0 mg/day 135°C
1,0 mg/day 160°C
10 mg/day 135°C
Ladics et al, Toxicol Sci, 2003; 73, 8-16 Kimber et al, Env Health Persp, 2003, 111, 8, 1125-1130
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Rat mast cell protease II (RMCP-II) levels in serum after oral challenge with CPE or RPE
0
5
10
15
20
25
30
35
40
0 0,5 1 2 3
Time after challenge (hours)
RM
CPI
I con
cent
ratio
n (n
g/m
l)
0 5
10 15 20 25 30 35 40 45
Group
RM
CPI
I con
cent
ratio
n (n
g/m
l)
t = 0 t = 3
** **
C C 1 10 1 10 1
CPE RPE
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Specific IgG2a responses against Ara h1, Ara h2, and Ara h3
Serum Dilution
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1\8 1\16 1\32 1\64 1\128 1\256 1\512 1\1024 0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1\8 1\16 1\32 1\64 1\128 1\256 1\512 1\1024
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1\8 1\16 1\32 1\64 1\128 1\256 1\512 1\1024
Serum Dilution
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
1\8 1\16 1\32 1\64 1\128 1\256 1\512 1\1024
Serum Dilution
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
1\8 1\16 1\32 1\64 1\128 1\256 1\512 1\1024
Serum Dilution
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
1\8 1\16 1\32 1\64 1\128 1\256 1\512 1\1024
Serum Dilution
Ara h1 Ara h2 Ara h3
I.P.
Oral
Ladics et al, Toxicol Sci, 2003: 73, 8-16; Ladics et al, Reg Tox Pharmacol, 2010, 56, 212-224
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Dietary control in the development of a peanut allergy model in BN rats
BN rats bred for 3 generations on a peanut- and soy- free diet (3G PE/soy-
free) and rats raised on a commercially peanut-and whey-protein free diet
(PE-W free) were daily i.g. dosed with 1 or 10 mg PE without adjuvant.
In 3G PE/soy-free BN rats IgE titers > than those of the PE-W free rats
In 3G PE/soy-free BN rats 100% IgE responders both to 1 and 10 PE.
In PE-W free BN rats only 10% IgE responders (2/20) sensitized with 1 mg
PE and no IgE responders when sensitized with 10 mg PE
In 3G PE/soy-free BN rats 100% IgG1 and IgG2a responders and higher
levels than in PE-W free BN rats
In PE-W free BN rats 19/20 IgG1 and 17/20 IgG2a responders sensitized with
1 mg PE and 9/10 IgG1 and IgG2a responders sensitized with 10 mg PE
De Jonge et al, Methods 41,99-111, 2007
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Protein Source Molecular weight (by gel-migration)
Purity
Sol t 1 (Patatin0 Weak
Potato tuber
Ara h 1 strong
Peanut
Shrimp tropomyosin Intermediate
Raw brown Shrimp
Beef Tropomyosin No allergen
Raw beef
94.6%
99.3%
92.6%
88%
37-38 kDa
35-39 kDa
60-67 kDa
40-43 kDa
Purified Proteins
Ladics et al, Reg Toxicol Pharmacol 2010, 56, 212-224
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Brown Norway - IgE results – 2nd Exp.
Day 42 IgE Responders IgG2a RespondersELISA PCA ELISA ELISA
Titre(#rats) # rats Titre # ratsAra h11 mg/ml 4.7 (3/6) 1/6 11.3 6/61 mg/ml + CT 4.3 (3/4) 0/4 4.5 2/4I.p. + Alum 4.2 (6/6) 1/6 12.2 6/6
Pen a 11 mg/ml 9.5 (2/6) 1/6 12.2 6/61 mg/ml + CT 0 (0/4) 0/4 10.5 4/4I.p. + Alum 10.2 (6/6) 3/6 12.5 6/6
Sol t 11 mg/ml 9 (1/6) 1/6 15 6/61 mg/ml + CT 8.5 (2/4) 0/4 14.5 4/4I.p. + Alum 10.8 (6/6) 5/6 13.8 6/6
Beef1 mg/ml 0 (0/6) 0/6 3.75 4/61 mg/ml + CT 0 (0/4) 0/4 4 4/4I.p. + Alum 0 (0/6) 0/6 3 3/6
Human allergen ranking high high low-med. non-
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2S albumin form Brazil nut (Ber e1): The role of -S-S- bridges
Hypothesis: “Brazil nut 2S albumin is a potent allergen due to its stability
towards digestion. This stability is supported by -S-S- bridges”
Testing the hypothesis:
prepare 2 forms of Brazil nut 2S albumin and test for: structure, stability, allergenicity
Native Brazil nut 2S albumin (N-2S) Reduced-alkylated Brazil nut 2s albumin (RA-2S)
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(RA)-2S albumin (Ber e1)-specific antibody responses (day 42)
0
5
10
15
20
2log
ant
ibod
y tit
er
water 0.1 mg 1 mg 0.1 mg 1 mg
2S albumin RA-2S albumin
50 50
50
70 70
50 50
60
IgG1
IgG2a
IgE
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IgG and IgE responses in BN rat model upon oral exposure to whole foods
Protein 2log IgG Number of responders IgG IgE PE (1 mg) 10 10/10 4/10 HEW (2.5 mg) 7.2 5/6 1/6 HEW (10 mg) 14 4/6 2/6 CM (10 mg) 2.5 4/5 2/5 Knippels and Penninks, Toxicol Appl Pharmacol, 2005
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IgG and IgE responses in BN rat model upon oral exposure to purified proteins
Protein 2log IgG Number of responders IgG IgE i.p. OVA 12.3 8/10 6/10 6/6 Ber e1 6.9 7/10 5/10 6/6 Ara h1 11.3 6/6 3/6 6/6 Pen a1 12.2 6/6 2/6 6/6 Sol t1 15 6/6 1/6 6/6 Beef tropo 3.8 4/6 0/6 0/6
Knippels and Penninks, Toxicol Appl Pharmacol, 2005
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Results obtained with BN rats in other labs Atkinson et al, Toxicology 91, 281-288, 1994 Atkinson et al, Fd Chem Toxicol 34, 27-34, 1996 Miller et al, Clin Exp Allergy 29, 1696-1704, 1999
Diet, gavage and i.p. exposures in the presence of adjuvant Akiyama et al, Immunology Letters 78, 1-5, 2001
OVA-specific IgE upon gavage > drinking water (1 mg OVA/day) Jia et al, World J Gastroenterol 11, 5381-5384, 2005
IgE responders OVA (8/10) > BSA (2/10) > PAP (0/10) Madsen and Pilegaard, Int Arch Allergy Immunol 130, 66-72, 2003
No priming of the IR in newborn BN rats dosed with OVA in mouth Pilegaard and Madsen, Toxicology 196, 247-257, 2004
Female BN rats > IgE/IgG titers against OVA/egg white than males Bogh et al, Clin Exp Allergy 39, 1611-1621
Digested Ara h1 has sensitizing capacity in BN rats
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Further Food Allergy Research (1)
Currently at TNO no further development of the BN rat model for prediction of the potential allergenicity of new proteins. At TNO special attention will be given to the a slightly adapted
protocol of the mice model of Li et al (1999, 2000) for assessment of potental allergenicity of new proteins. In collaboration with the Utrecht Centre of Food Allergy (UCFA)
a new mice model is currently validated to test for hypo-allergenicity of partial and extensively hydrolysed infant formula (to replace the guinea pig ASA/PCA test).
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Further Food Allergy Research (2) Mechanistic research in animal models for food allergy is further
focussed on improvement of strategies for prevention (tolerance induction) and therapy. Special attention is given at TNO to the collection of physical
and biological data of proteins (strong-, week-, and non allergenic proteins) for development of a toolbox to predict potential allergenicity of new proteins. Special attention is given at TNO to quantitative risk
assessment in food allergy.
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Dept Toxicology and Applied Risk Assessment TNO Triskelion BV
Zeist
Thank you for your attention
André Penninks
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