- Email: [email protected]
Induction of oral tolerance to cow's milk proteins in rats fed with a whey protein hydrolysate
Nuhition Research, Vol. 18. No. 8, pp. 1335-1341.1998 Copyright 0 1998 EIsevier Science Inc. Printed in the USA. AII rights reserved 0271-5317198 $19.00+ .CO ELSEVIER
INDUCTION
PI1 SO271-5317(98)OOll2-2
OF ORAL TOLERANCE TO COW’S MILK PROTEINS WITH A WHEY PROTEIN HYDROLYSATE
IN RATS FED
Rodolphe Fritsche, PhD’ Nestle Research Center, Lausanne, Switzerland
ABSTRACT
Animal models have shown that oral administration of an antigen induces a specific immunologic tolerance to this antigen. Induction of such an oral tolerance has been well documented with intact proteins. The purpose of our study was to determine whether oral tolerance to cow’s milk proteins (CMP) can be induced also with protein peptides contained in either partially hydrolysed or extensively hydrolysed cow’s milk formulas. Five-week-old Sprague-Dawley rats were fed cow’s milk formulas ad libitum before they were parenterally challenged with CMP. The modulation of specific IgE anti-CMP antibodies and intestinal mast cell stimulation were measured. Results showed that preventive feeding of rats with a partially hydrolysed cow’s milk formula suppresses specific IgE anti-CMP antibodies as well as IgE mediated mediator release (rat mast cell protease RMCPII) from intestinal mast cells. An extensively hydrolysed cow’s milk formula was unable to achieve the induction of such an oral tolerance. Our experiments show that selected peptides of CMP may induce a specific oral tolerance to CMP. 0199aElaNicr.%imccInc. Oral tolerance, Cow’s milk proteins, Partially/Extensively KEY WORDS: hydrolysed formulas, IgE, Mast cells
INTRODUCTION
Immune regulation by the induction of oral tolerance to food antigens is thought to prevent food allergy (1). Animal models have shown that specific immunological oral tolerance can be induced with intact proteins (2, 3, 4). Age of the host and dosis of antigen administered
‘Correspondence: Dr.R.Fritsche, Nestec ResearchCenter, CH-1000 Lausamie 26, Switzerland. Phone: +41/21 785 86 83, Fax: +41/21 785 89 25
Vers-chez-les-Blanc,
P.O.Box 44,
R. FRITSCHE
1336
are important parameters (5) but few studies with antigen fragments (6) or digests (7) have been done. In this work we studied whether enzymatic hydrolysates of cow’s milk proteins are able to induce oral tolerance to these proteins when administered preventively ad libitum in a rat model. We selected S-lactoglobulin (PLG) as a representative protein for these studies because it is a potent allergen of cow’s milk and represemts the main whey protein fraction.
MATERIAL
AND METHODS
Milk Formulas The following commercial products were fed to rats : NAN, a standard formula based on intact cow’s milk proteins ; BEBA-HA, a partially hydrolysed (trypsin) whey formula; ALFARE, an extensively hydrolysed (pancreatin) whey formula.
Experimental
Protocol
Groups of rats were given different experimental liquid milk formulas or water,(control) ad libitum in their drinking bottles and a solid (
ELISA Determination
of Suecific IaE Antibodies
Specific IgE anti+lactoglobulin (or anti-Ovalbumin) antibodies were determined (8) by coating microtitration plates with l3LG (or OVA) for 24 hours at 4”C, blocking with fish gelatin 90 minutes at room temperature (RT), washing with PBS-Tween buffer and adding test sera dilutions. Plates were then incubated at RT for 2 hours, washed and incubated with a sheep antirat IgE antibody for 1 hour at RT. After another washing step, plates are incubated with a peroxidase labelled second antibody for 1 hour at RT. After a final washing, o-phenylene-diamine substrate is added and optical density read after 15 minutes at 492 nm. Concentrations of antibodies were expressed as log5 titers of maximum sera dilutions above a 1 :5 diluted normal rat sera pool taken as negative control. The specificity of the anti-rat IgE antiserum was tested with the rat IgE myeloma IR-2 and polyclonal rat IgG.
ORAL TOLERANCE
3H-Serotonin
INDUCTION
1337
Release Assay
This assay was used for the determination of cow’s milk protein allergenicity in milk products. Peritoneal mast cells from normal rats are sensitized passively with rat IgE anti-PLG antibodies, labelled with 3H-Serotonin and triggered for mediator release with dilutions of l3LG or cow’s milk formulas according to a published method (8). Briefly, peritoneal lavages were obtained with Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum. Cells were washed in this medium and kept overnight at 4°C. After two washes in phosphate-HEPES-gelatin buffer (PHG) pH 7.0, cells were resuspended at 500’000 / ml. 3H-Serotonin (10 &%/ml) and 1 ml of rat serum rich in IgE anti-PLG antibodies were added. This mixture was then incubated at 37°C for 2 hours, washed three times in PHG and resuspended in 2 ml PHG. Triggering for mediator release was set up in microtiter plates by mixing 0.05 ml of PLG or cow’s milk formula dilution with 0.1 ml of sensitized mast cells. The mixture was incubated for 60 minutes at 37°C and centrifuged. An aliquot (0.05 ml) of the supemataut was counted for radioactivity on a p counter.
Determination
of Rat Mast Cell Protease RMCPII
RMCPII is released into blood following IgE mediated triggering of intestinal mast cells. Oral challenge for release of RMCPII is a measure of IgE sensitization or tolerization at the intestinal mast cell level. RMCPII levels are determined with a commercial ELISA kit (Moredun Animal Health Ltd., Edinburgh, Scotland) based on the sandwich test principle in which the plate coating is made with a monoclonal anti-RMCPII antibody, followed by the addition of test serum and a second sheep anti-RMCPII polyclonal antibody coupled to horseradish peroxidase.
RESULTS
Residual Allergenicitv
of Cow’s Milk Formulas
The level of PLG specific IgE mediated allergenicity of NAN, BEBA-HA and ALFARE was determined with the 3H-Serotonin release assay. Figure 1 shows that different dose-dependent release curves are obtained for each formula. One can see that 1000 times more BEBA-HA than NAN, calculated on a protein equivalent basis, is needed for triggering release of the same amounts of 3H-Serotonin. Calculated values of residual PLG specific allergenicities in NAN, BEBA-HA and ALFARE are 100,O.l and 0.0002 mg l3LG / gm protein equivalent, respectively.
Snecific Antibody
Sunnression
in Orally Tolerized Rats
The three cow’s milk formulas were compared for their capacity to induce antigen-specific oral tolerance. Despite the fact that BEBA-HA is 1000 times less allergenic than NAN (according to above results) its oral tolerizing capacity has been fully preserved, as BEBA-HA and NAN
1338
FL FRITSCHC
suppress similarly the specific IgE response to P-lactoglobulin. An over 100 fold IgE anti-j3LG titer suppression compared to control rats is observed (figure 2) when these formulas are given preventively ad libitum to rats. In contrast, ALFARE, the extensively hydrolysed formula, has no tolerizing activity : specific IgE anti+LG antibodies are not suppressed by prefeeding with ALFARE. The specific IgE anti-PLG response of animals fed NAN or BEBA-HA was significantly different (p
In Vivo Suvvression
of InE Mediated Intestinal Mast Cell Resvonse
Figure 3 shows that specific release of intestinal mucosal mast cell protease (RMCPII) of sensitized rats could be suppressed by prefeeding with intact (NAN) or partially hydrolysed (BEBA-HA) cow’s milk proteins. Prefeeding with an extensively hydrolysed (ALFARE) formula does not suppress intestinal mast cell sensitization and therefore RMCPII release upon challenge with whey proteins as this is the case with control rats (given water only during prefeeding). The specific levels of RMCPII released from intestinal mast cells of rats tolerized with NAN or BEBA-HA were significantly different (p
Z g0) 8
1
25OOS
er c ._ ;
2oooo --
g
lOOo--
3 zi
+
BEBA-HA
15ooo --
5000 -0 X-X i 0 lE-04 0.001 0.01
0.1
1
10
loo
100010000
mcg protein equival./ml Fig. 1 Residual PLG specific allergenicity in cow’s milk formulas by 3H-Serotonin release from rat mast cells sensitized passively with rat IgE anti-PLG antibodies. Dose-dependent release curves obtained by triggering sensitized mast cells with PLG or cow’s milk formulas are shown.
ORAL TOLERANCE
6
.F
1
1339
I
5
RATsFEDwlTw
ua
!z
INDUCTION
•unNAN I BEBA-HA
4
% i
%LARE
W
IgE a-BLG
IgE a-OVA
Fig. 2 Antibody suppression by oral tolerance induction with cow’s milk formulas. ELISA log5 titers of IgE anti$LG or anti-Ovalbumin antibodies in sera from rats orally prefed with NAN, BEBA-HA, ALFARE or water for tolerance induction.
T
?? NAN ?? BEBA-HI ?? ALFARE I-JH20
I
Expl
Exp2
Exp3
Fig. 3 In vivo suppression of mast cell response. FUvICPII release from intestinal mast cells of rats prefed with NAN, BEBA-HA, ALFAFE or water for oral tolerance.
1340
R. FRITSCHE
induction. Serum levels of RMCPII after challenge with whey proteins different experiments in which the same protocole was used.
are shown for three
DISCUSSION
We show with the help of a rat model that a partially hydrolysed cow’s milk formula with low allergenicity (1000 times reduced compared to an intact formula) is able to induce specific oral tolerance to cow’s milk proteins when administered preventively.In contrast, an extensively hydrolysed cow’s milk formula does not induce such an oral tolerance. We have shown in this study that specific IgE anti-cow’s milk protein antibodies are strongly suppressed in rats orally tolerized with a partially hydrolysed cow’s milk formula (over 100 times specific IgE titer reduction). This suppression is antigen specific because IgE anti-Ovalbumin antibodies are not suppressed by preventive feeding with the partially hydrolysed cow’s milk protein formula. Our results show further that the suppression of the allergic response occurs also at the intestinal level when rats were orally tolerized with the partially hydrolysed formula. The release of the specific intestinal mast cell protease RMCPII is a good indicator of in vivo mast cell sensitization and activation (10). Its modulation by oral tolerance induction demonstrates that tolerizing peptides of the partially hydrolyzed formula exert their specific action also at the intestinal level, a common target of the cow’s milk allergic reaction. Here again, the partially hydrolysed formula is able to suppress RMCPII release whereas the extensively hydrolysed formula is unable to achieve this. Our experiments show that as far as IgE antibody formation and the intestinal mast cell response are concerned, oral tolerance to cow’s milk proteins has been induced by selected BOW’Smilk protein peptides present in the partial hydrolysate and absent in the extensively hydrolysed formula. We are characterizing now a tolerogenic peptide fraction of the partial hydrolysate which could be shown to be devoid of intact proteins and has a molecular weight range of 2 to 5 kD. It is generally accepted that breastfeeding is the best choice for prevention of cow’s milk allergy in infants of atopic parents. However, when breastfeeding is not possible in such (( at risk )) infants, and according to our results, the administration of a partially hydrolysed formula should be preferred to an extensively hydrolysed formula for inducing actively a long-term oral tolerance to cow’s milk proteins.
ORAL TOLERANCE
INDUCTION
1341
REFERENCES
1.
Mowat AM. The regulation Today 1987 ;8:93-98.
2.
Wells HG, Osborne TB. The biological reactions of the vegetable proteins. J Infect Dis 1911 ;8 :66-124.
3.
Thomas HC, Parrot DMV. The induction of tolerance to a soluble protein antigen by oral administration. Immunology 1974 ;27 :631-9.
4.
Hanson DG, Vaz NM, Maia L, Hombrook M, Lynch J, Roy C. Inhibition of specific immune responses by feeding protein antigens. Int Arch Allergy Appl Immunol 1977 ;55 :526-32.
5.
Strobe1 S, Ferguson 1984 ;18 :588-94.
6.
Michael JG. The role of digestive Invest 1989 ;18 :1049-54.
7.
Hachimura S, Takahashi Y, Fujikawa Y, Tsumori C, Enomoto A, Yoshino U, Kaminogawa S. Suppression of the systemic immune response to casein by oral administration of a tryptic digest of casein. Biosci Biotech Biochem 1993 ;57 :1674-77.
8.
Fritsche R, Bonzon M. Determination of cow milk formula allergenicity in the rat model by in vitro mast cell triggering and in vivo IgE induction. Int Arch Allergy Appl Immunol 1990 ;93 :289-93.
9.
Fritscht R, Pahud JJ, Pecquet S, Pfeifer A. Induction of systemic immunologic tolerance to P-lactoglobulin by oral administration of a whey protein hydro1ysate.J Allergy Clin Immunol 1997 ;lOO :266-73.
10.
Turner MW, Bamett GE, Strobe1 S. Mucosal mast cell activation patterns in the rat following repeated feeding of antigen. Clin Exp Allergy 1990 ;20 :421-27.
Accepted
for
publication
of immune responses to dietary protein antigens. Imnnmol
S. Immune responses to fed protein antigens in mice. Pediatr Res
February
enzymes in orally induced immune tolerance. Immunol
25,
1998.
INDUCTION
PI1 SO271-5317(98)OOll2-2
OF ORAL TOLERANCE TO COW’S MILK PROTEINS WITH A WHEY PROTEIN HYDROLYSATE
IN RATS FED
Rodolphe Fritsche, PhD’ Nestle Research Center, Lausanne, Switzerland
ABSTRACT
Animal models have shown that oral administration of an antigen induces a specific immunologic tolerance to this antigen. Induction of such an oral tolerance has been well documented with intact proteins. The purpose of our study was to determine whether oral tolerance to cow’s milk proteins (CMP) can be induced also with protein peptides contained in either partially hydrolysed or extensively hydrolysed cow’s milk formulas. Five-week-old Sprague-Dawley rats were fed cow’s milk formulas ad libitum before they were parenterally challenged with CMP. The modulation of specific IgE anti-CMP antibodies and intestinal mast cell stimulation were measured. Results showed that preventive feeding of rats with a partially hydrolysed cow’s milk formula suppresses specific IgE anti-CMP antibodies as well as IgE mediated mediator release (rat mast cell protease RMCPII) from intestinal mast cells. An extensively hydrolysed cow’s milk formula was unable to achieve the induction of such an oral tolerance. Our experiments show that selected peptides of CMP may induce a specific oral tolerance to CMP. 0199aElaNicr.%imccInc. Oral tolerance, Cow’s milk proteins, Partially/Extensively KEY WORDS: hydrolysed formulas, IgE, Mast cells
INTRODUCTION
Immune regulation by the induction of oral tolerance to food antigens is thought to prevent food allergy (1). Animal models have shown that specific immunological oral tolerance can be induced with intact proteins (2, 3, 4). Age of the host and dosis of antigen administered
‘Correspondence: Dr.R.Fritsche, Nestec ResearchCenter, CH-1000 Lausamie 26, Switzerland. Phone: +41/21 785 86 83, Fax: +41/21 785 89 25
Vers-chez-les-Blanc,
P.O.Box 44,
R. FRITSCHE
1336
are important parameters (5) but few studies with antigen fragments (6) or digests (7) have been done. In this work we studied whether enzymatic hydrolysates of cow’s milk proteins are able to induce oral tolerance to these proteins when administered preventively ad libitum in a rat model. We selected S-lactoglobulin (PLG) as a representative protein for these studies because it is a potent allergen of cow’s milk and represemts the main whey protein fraction.
MATERIAL
AND METHODS
Milk Formulas The following commercial products were fed to rats : NAN, a standard formula based on intact cow’s milk proteins ; BEBA-HA, a partially hydrolysed (trypsin) whey formula; ALFARE, an extensively hydrolysed (pancreatin) whey formula.
Experimental
Protocol
Groups of rats were given different experimental liquid milk formulas or water,(control) ad libitum in their drinking bottles and a solid (
ELISA Determination
of Suecific IaE Antibodies
Specific IgE anti+lactoglobulin (or anti-Ovalbumin) antibodies were determined (8) by coating microtitration plates with l3LG (or OVA) for 24 hours at 4”C, blocking with fish gelatin 90 minutes at room temperature (RT), washing with PBS-Tween buffer and adding test sera dilutions. Plates were then incubated at RT for 2 hours, washed and incubated with a sheep antirat IgE antibody for 1 hour at RT. After another washing step, plates are incubated with a peroxidase labelled second antibody for 1 hour at RT. After a final washing, o-phenylene-diamine substrate is added and optical density read after 15 minutes at 492 nm. Concentrations of antibodies were expressed as log5 titers of maximum sera dilutions above a 1 :5 diluted normal rat sera pool taken as negative control. The specificity of the anti-rat IgE antiserum was tested with the rat IgE myeloma IR-2 and polyclonal rat IgG.
ORAL TOLERANCE
3H-Serotonin
INDUCTION
1337
Release Assay
This assay was used for the determination of cow’s milk protein allergenicity in milk products. Peritoneal mast cells from normal rats are sensitized passively with rat IgE anti-PLG antibodies, labelled with 3H-Serotonin and triggered for mediator release with dilutions of l3LG or cow’s milk formulas according to a published method (8). Briefly, peritoneal lavages were obtained with Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum. Cells were washed in this medium and kept overnight at 4°C. After two washes in phosphate-HEPES-gelatin buffer (PHG) pH 7.0, cells were resuspended at 500’000 / ml. 3H-Serotonin (10 &%/ml) and 1 ml of rat serum rich in IgE anti-PLG antibodies were added. This mixture was then incubated at 37°C for 2 hours, washed three times in PHG and resuspended in 2 ml PHG. Triggering for mediator release was set up in microtiter plates by mixing 0.05 ml of PLG or cow’s milk formula dilution with 0.1 ml of sensitized mast cells. The mixture was incubated for 60 minutes at 37°C and centrifuged. An aliquot (0.05 ml) of the supemataut was counted for radioactivity on a p counter.
Determination
of Rat Mast Cell Protease RMCPII
RMCPII is released into blood following IgE mediated triggering of intestinal mast cells. Oral challenge for release of RMCPII is a measure of IgE sensitization or tolerization at the intestinal mast cell level. RMCPII levels are determined with a commercial ELISA kit (Moredun Animal Health Ltd., Edinburgh, Scotland) based on the sandwich test principle in which the plate coating is made with a monoclonal anti-RMCPII antibody, followed by the addition of test serum and a second sheep anti-RMCPII polyclonal antibody coupled to horseradish peroxidase.
RESULTS
Residual Allergenicitv
of Cow’s Milk Formulas
The level of PLG specific IgE mediated allergenicity of NAN, BEBA-HA and ALFARE was determined with the 3H-Serotonin release assay. Figure 1 shows that different dose-dependent release curves are obtained for each formula. One can see that 1000 times more BEBA-HA than NAN, calculated on a protein equivalent basis, is needed for triggering release of the same amounts of 3H-Serotonin. Calculated values of residual PLG specific allergenicities in NAN, BEBA-HA and ALFARE are 100,O.l and 0.0002 mg l3LG / gm protein equivalent, respectively.
Snecific Antibody
Sunnression
in Orally Tolerized Rats
The three cow’s milk formulas were compared for their capacity to induce antigen-specific oral tolerance. Despite the fact that BEBA-HA is 1000 times less allergenic than NAN (according to above results) its oral tolerizing capacity has been fully preserved, as BEBA-HA and NAN
1338
FL FRITSCHC
suppress similarly the specific IgE response to P-lactoglobulin. An over 100 fold IgE anti-j3LG titer suppression compared to control rats is observed (figure 2) when these formulas are given preventively ad libitum to rats. In contrast, ALFARE, the extensively hydrolysed formula, has no tolerizing activity : specific IgE anti+LG antibodies are not suppressed by prefeeding with ALFARE. The specific IgE anti-PLG response of animals fed NAN or BEBA-HA was significantly different (p
In Vivo Suvvression
of InE Mediated Intestinal Mast Cell Resvonse
Figure 3 shows that specific release of intestinal mucosal mast cell protease (RMCPII) of sensitized rats could be suppressed by prefeeding with intact (NAN) or partially hydrolysed (BEBA-HA) cow’s milk proteins. Prefeeding with an extensively hydrolysed (ALFARE) formula does not suppress intestinal mast cell sensitization and therefore RMCPII release upon challenge with whey proteins as this is the case with control rats (given water only during prefeeding). The specific levels of RMCPII released from intestinal mast cells of rats tolerized with NAN or BEBA-HA were significantly different (p
Z g0) 8
1
25OOS
er c ._ ;
2oooo --
g
lOOo--
3 zi
+
BEBA-HA
15ooo --
5000 -0 X-X i 0 lE-04 0.001 0.01
0.1
1
10
loo
100010000
mcg protein equival./ml Fig. 1 Residual PLG specific allergenicity in cow’s milk formulas by 3H-Serotonin release from rat mast cells sensitized passively with rat IgE anti-PLG antibodies. Dose-dependent release curves obtained by triggering sensitized mast cells with PLG or cow’s milk formulas are shown.
ORAL TOLERANCE
6
.F
1
1339
I
5
RATsFEDwlTw
ua
!z
INDUCTION
•unNAN I BEBA-HA
4
% i
%LARE
W
IgE a-BLG
IgE a-OVA
Fig. 2 Antibody suppression by oral tolerance induction with cow’s milk formulas. ELISA log5 titers of IgE anti$LG or anti-Ovalbumin antibodies in sera from rats orally prefed with NAN, BEBA-HA, ALFARE or water for tolerance induction.
T
?? NAN ?? BEBA-HI ?? ALFARE I-JH20
I
Expl
Exp2
Exp3
Fig. 3 In vivo suppression of mast cell response. FUvICPII release from intestinal mast cells of rats prefed with NAN, BEBA-HA, ALFAFE or water for oral tolerance.
1340
R. FRITSCHE
induction. Serum levels of RMCPII after challenge with whey proteins different experiments in which the same protocole was used.
are shown for three
DISCUSSION
We show with the help of a rat model that a partially hydrolysed cow’s milk formula with low allergenicity (1000 times reduced compared to an intact formula) is able to induce specific oral tolerance to cow’s milk proteins when administered preventively.In contrast, an extensively hydrolysed cow’s milk formula does not induce such an oral tolerance. We have shown in this study that specific IgE anti-cow’s milk protein antibodies are strongly suppressed in rats orally tolerized with a partially hydrolysed cow’s milk formula (over 100 times specific IgE titer reduction). This suppression is antigen specific because IgE anti-Ovalbumin antibodies are not suppressed by preventive feeding with the partially hydrolysed cow’s milk protein formula. Our results show further that the suppression of the allergic response occurs also at the intestinal level when rats were orally tolerized with the partially hydrolysed formula. The release of the specific intestinal mast cell protease RMCPII is a good indicator of in vivo mast cell sensitization and activation (10). Its modulation by oral tolerance induction demonstrates that tolerizing peptides of the partially hydrolyzed formula exert their specific action also at the intestinal level, a common target of the cow’s milk allergic reaction. Here again, the partially hydrolysed formula is able to suppress RMCPII release whereas the extensively hydrolysed formula is unable to achieve this. Our experiments show that as far as IgE antibody formation and the intestinal mast cell response are concerned, oral tolerance to cow’s milk proteins has been induced by selected BOW’Smilk protein peptides present in the partial hydrolysate and absent in the extensively hydrolysed formula. We are characterizing now a tolerogenic peptide fraction of the partial hydrolysate which could be shown to be devoid of intact proteins and has a molecular weight range of 2 to 5 kD. It is generally accepted that breastfeeding is the best choice for prevention of cow’s milk allergy in infants of atopic parents. However, when breastfeeding is not possible in such (( at risk )) infants, and according to our results, the administration of a partially hydrolysed formula should be preferred to an extensively hydrolysed formula for inducing actively a long-term oral tolerance to cow’s milk proteins.
ORAL TOLERANCE
INDUCTION
1341
REFERENCES
1.
Mowat AM. The regulation Today 1987 ;8:93-98.
2.
Wells HG, Osborne TB. The biological reactions of the vegetable proteins. J Infect Dis 1911 ;8 :66-124.
3.
Thomas HC, Parrot DMV. The induction of tolerance to a soluble protein antigen by oral administration. Immunology 1974 ;27 :631-9.
4.
Hanson DG, Vaz NM, Maia L, Hombrook M, Lynch J, Roy C. Inhibition of specific immune responses by feeding protein antigens. Int Arch Allergy Appl Immunol 1977 ;55 :526-32.
5.
Strobe1 S, Ferguson 1984 ;18 :588-94.
6.
Michael JG. The role of digestive Invest 1989 ;18 :1049-54.
7.
Hachimura S, Takahashi Y, Fujikawa Y, Tsumori C, Enomoto A, Yoshino U, Kaminogawa S. Suppression of the systemic immune response to casein by oral administration of a tryptic digest of casein. Biosci Biotech Biochem 1993 ;57 :1674-77.
8.
Fritsche R, Bonzon M. Determination of cow milk formula allergenicity in the rat model by in vitro mast cell triggering and in vivo IgE induction. Int Arch Allergy Appl Immunol 1990 ;93 :289-93.
9.
Fritscht R, Pahud JJ, Pecquet S, Pfeifer A. Induction of systemic immunologic tolerance to P-lactoglobulin by oral administration of a whey protein hydro1ysate.J Allergy Clin Immunol 1997 ;lOO :266-73.
10.
Turner MW, Bamett GE, Strobe1 S. Mucosal mast cell activation patterns in the rat following repeated feeding of antigen. Clin Exp Allergy 1990 ;20 :421-27.
Accepted
for
publication
of immune responses to dietary protein antigens. Imnnmol
S. Immune responses to fed protein antigens in mice. Pediatr Res
February
enzymes in orally induced immune tolerance. Immunol
25,
1998.