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Introduction of Heated Cow’s Milk Protein in Challenge-Proven Cow’s Milk Allergic Children: The iAGE Study

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The introduction of baked milk products in cow’s milk (CM) allergic children has previously been shown to accelerate induction tolerance in a selected group of children. However, there is no standardized baked milk product on the market. Recently, a new standardized, heated and glycated cow’s milk protein (HP) product was developed. The aim of this study was to measure safety and tolerability of a new, well characterized heated CM protein (HP) product in cow’s milk allergic (CMA) children between the age of 3 and 36 months. The children were recruited from seven clinics throughout the Netherlands. The HP product was introduced in six incremental doses under clinical supervision. Symptoms were registered after introduction of the HP product. Several questionnaires were filled out by parents of the children. Skin prick tests were performed with CM and HP product, sIgE to CM and α-lactalbumin (Bos d4), β-lactoglobulin (Bos d5), serum albumin (Bos d 6), lactoferrin (Bos d7) and casein (Bos d8). Whereas 72% percent (18 out of 25) of the children tolerated the HP product, seven children experienced adverse events. Risk factors for intolerance to the HP product were higher skin prick test (SPT) histamine equivalent index (HEP) results with CM and the HP product, higher specific IgE levels against Bos d4 and Bos d8 levels and Bos d5 levels. In conclusion, the HP product was tolerated by 72% of the CM allergic children. Outcomes of SPT with CM and the HP product, as well as values of sIgE against caseins, α-lactalbumin, and β-lactoglobulin may predict the tolerability of the HP product. Larger studies are needed to confirm these conclusions.
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Citation: de Jong, N.W.; van Splunter,
M.E.; Emons, J.A.M.; Hettinga, K.A.;
Gerth van Wijk, R.; Wichers, H.J.;
Savelkoul, H.F.J.; Sprikkelman, A.B.;
van Neerven, R.J.J.; Liu, L.; et al.
Introduction of Heated Cow’s Milk
Protein in Challenge-Proven Cow’s
Milk Allergic Children: The iAGE
Study. Nutrients 2022,14, 629.
https://doi.org/10.3390/nu14030629
Academic Editor: Elvira Verduci
Received: 27 December 2021
Accepted: 27 January 2022
Published: 31 January 2022
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4.0/).
nutrients
Article
Introduction of Heated Cow’s Milk Protein in Challenge-Proven
Cow’s Milk Allergic Children: The iAGE Study
Nicolette W. de Jong 1,2 ,*, , Marloes E. van Splunter 1 ,† , Joyce A. M. Emons 2, Kasper A. Hettinga 3,
Roy Gerth van Wijk 1, Harry J. Wichers 3, Huub F. J. Savelkoul 4, Aline B. Sprikkelman 5,6,
R. J. Joost van Neerven 4,7 , Liu Liu 8, Gerbrich van der Meulen 9, Irene Herpertz 10, Yvonne C. M. Duijvestijn 11,
Mijke Breukels 12, Marianne I. Brouwer 13 , Jaap Schilperoord 14, Olga van Doorn 14, Berber Vlieg-Boerstra 14 ,
Jochum van den Berg 15, Linette Pellis 16 , Severina Terlouw 1, Astrid I. Hendriks 2, Marco W. J. Schreurs 17,
Frank E. van Boven 1and Nicolette J. T. Arends 2,
1Department of Internal Medicine, Section of Allergology & Clinical Immunology, Erasmus MC, University
Medical Centre Rotterdam, 3015GD Rotterdam, The Netherlands; m.e.vansplunter@erasmusmc.nl (M.E.v.S.);
r.gerthvanwijk@erasmusmc.nl (R.G.v.W.); s.terlouw@erasmusmc.nl (S.T.); f.boven@erasmusmc.nl (F.E.v.B.)
2Depertment of Peadiatric Allergology, Sophia Children Hospital, Erasmus MC, University Medical Centre
Rotterdam, 3015GD Rotterdam, The Netherlands; j.a.m.emons@erasmusmc.nl (J.A.M.E.);
a.hendriks-vantoor@erasmusmc.nl (A.I.H.); n.arends@erasmusmc.nl (N.J.T.A.)
3Food Quality & Design Group, Wageningen University & Research Centre,
6708PB Wageningen, The Netherlands; kasper.hettinga@wur.nl (K.A.H.); harry.wichers@wur.nl (H.J.W.)
4Cell Biology & Immunology Group, Wageningen University & Research Centre,
6708PB Wageningen, The Netherlands; huub.savelkoul@wur.nl (H.F.J.S.); joost.vanneerven@wur.nl (R.J.J.v.N.)
5Department of Peadiatric Pulmonology & Allergology, UMCG, University Medical Center Groningen,
9713GZ Groningen, The Netherlands; a.b.sprikkelman@umcg.nl
6University Medical Center Groningen, GRIAC Research Institute, University of Groningen,
9713GZ Groningen, The Netherlands
7FrieslandCampina, 3811LP Amersfoort, The Netherlands
8Department of Rheumatoloy, Leiden University, 2311BD Leiden, The Netherlands; l.liu@outlook.com
9
Department of Peadiatrics, Martini Hospital, 9728NT Groningen, The Netherlands; g.vander.meulen@mzh.nl
10
Paediatric Allegology Center, VieCuri Medisch Centrum, 5912BL Venlo, The Netherlands; I.Herpertz@mzh.nl
11 Department of Peadiatrics, Noordwest Ziekenhuisgroep, 1815JD Alkmaar, The Netherlands;
y.c.m.duijvestijn@nwz.nl
12 Deparment of Peadiatrics, Elkerliek, 5707HA Helmond, The Netherlands; mbreukels@elkerliek.nl
13 Department of Peadiatrics, Canisius Wilhelmina Ziekenhuis, 6532SZ Nijmegen, The Netherlands;
m.l.brouwer@cwz.nl
14 Department of Peadiatrics, OLVG Hospital, 1091AC Amsterdam, The Netherlands;
j.v.schilperoort@olvg.nl (J.S.); o.vandoorn@olvg.nl (O.v.D.); b.vlieg-boerstra@olvg.nl (B.V.-B.)
15 Department of Peadiatrics, Zuyderland MC, 6419PC Heerlen, The Netherlands;
joc.vandenberg@zuyderland.nl
16 Ausnutria B.V., 8025BM Zwolle, The Netherlands; Linette.Pellis@ausnutria.nl
17 Department of Immunology, Erasmus MC, University Medical Centre Rotterdam,
3015GD Rotterdam, The Netherlands; m.schreurs@erasmusmc.nl
*Correspondence: n.w.dejong@erasmusmc.nl; Tel.: +31-6-21697954
These authors contributed equally to this work.
Abstract:
The introduction of baked milk products in cow’s milk (CM) allergic children has previously
been shown to accelerate induction tolerance in a selected group of children. However, there is no
standardized baked milk product on the market. Recently, a new standardized, heated and glycated
cow’s milk protein (HP) product was developed. The aim of this study was to measure safety and
tolerability of a new, well characterized heated CM protein (HP) product in cow’s milk allergic
(CMA) children between the age of 3 and 36 months. The children were recruited from seven clinics
throughout The Netherlands. The HP product was introduced in six incremental doses under clinical
supervision. Symptoms were registered after introduction of the HP product. Several questionnaires
were filled out by parents of the children. Skin prick tests were performed with CM and HP product,
sIgE to CM and
α
-lactalbumin (Bos d4),
β
-lactoglobulin (Bos d5), serum albumin (Bos d 6), lactoferrin
(Bos d7) and casein (Bos d8). Whereas 72% percent (18 out of 25) of the children tolerated the HP
product, seven children experienced adverse events. Risk factors for intolerance to the HP product
Nutrients 2022,14, 629. https://doi.org/10.3390/nu14030629 https://www.mdpi.com/journal/nutrients
Nutrients 2022,14, 629 2 of 11
were higher skin prick test (SPT) histamine equivalent index (HEP) results with CM and the HP
product, higher specific IgE levels against Bos d4 and Bos d8 levels and Bos d5 levels. In conclusion,
the HP product was tolerated by 72% of the CM allergic children. Outcomes of SPT with CM and
the HP product, as well as values of sIgE against caseins,
α
-lactalbumin, and
β
-lactoglobulin may
predict the tolerability of the HP product. Larger studies are needed to confirm these conclusions.
Keywords: allergy; baked milk; cow’s milk; tolerance
1. Introduction
The prevalence of food allergies varies considerably, depending on self-reported food
allergy (FA) sensitization to food allergens or confirmed food allergy by an open food
challenge (OFC) test, or, preferably, a double-blind, placebo-controlled food challenge
(DBPCFC) test. In Europe, the prevalence of challenge-proven/confirmed cow’s milk
allergy (CMA) in young children (< age 3 year) varies between 0.35 and 2.0% [1,2].
About 70% of CMA children reach clinical tolerance to milk proteins before the age
of two years, and 60% before the age of three years, indicating transient CMA [
3
]. Factors
that are most predictive for spontaneous resolution of CMA are: low milk-specific IgE level
(<2 kU/L), small-wheal CM skin prick test (SPT) (<5 mm) and absence of (or mild) atopic
dermatitis (AD) [4].
Several studies have reported high percentages (59–81%) of tolerance to baked milk in
CMA children [
4
7
]. Children not tolerant towards baked milk products have an increased
risk of developing a persistent CMA compared to children tolerant of baked milk (relative
hazard ratio: 0.28 vs. 4.1) [3].
CMA children are mostly sensitized (presence of specific IgE to cow’s milk proteins)
to multiple cow’s milk proteins. Sensitization can be found to caseins, including
α
s1-,
α
s2-,
β
- and kappa casein (together constituting 80% of cow’s milk proteins (CMP)), and/or to
whey proteins, such as
α
-lactalbumin (Bos d4) and
β
-lactoglobulin (Bos d5) [
8
10
]. To a
lesser extent, sIgE against bovine serum albumin and other whey proteins is also found [
8
].
In particular, high levels of sIgE against
α
s1-,
β
-casein, Bos d4 and Bos d5 are associated
with persistent CMA [
11
13
]. In addition to IgE-mediated cow’s milk allergy, a substantial
part of the children has non-IgE-mediated CMA, resulting in delayed-type reactions
(>2 h)
lacking the typical IgE-mediated symptoms (urticaria, angioedema, respiratory and/or
gastro-intestinal symptoms or anaphylaxis) [
1
,
13
,
14
]. About 90% of patients with CMA
also react to goat’s milk, due to high protein homology (95%) and high protein identity
(>84%) [15].
Consumption of baked milk products in CMA children appeared to accelerate tol-
erance induction in a selected group of children [
6
,
16
]. Moreover, a recent meta-analysis
showed that most studies were observational, lacking an appropriate control group [
17
].
Processing milk proteins changes their immunogenic and allergenic properties and can
lead to both allergy and the induction of tolerance [
18
,
19
]. For example, when milk sugar
lactose and CMP are heated together, glycation takes place, causing sugars to be linked
to the free amino groups of amino acids [
20
]. This can lead to the formation of advanced
glycation end products (AGEs), which can mask existing epitopes but can also create new
immunogenic structures [2022].
In making baked milk products for previous studies on tolerability, both the form
of the product (e.g., cake, bread, or cheese on pizza), and the precise heating process
were found to be highly variable. Especially when baking products such as cakes, the
internal temperature of the product may strongly differ from the surface temperature,
leading to different heat-induced protein modifications throughout the product, which
may even leave some of the CMP intact. For better understanding of the effect of baked
milk on CMA, a standardized, heated and glycated CMP product should be produced,
Nutrients 2022,14, 629 3 of 11
in which the extent of heat-induced modifications, including glycation, have been well
characterized [6,16,2325].
The aim of this study was to measure safety and tolerability of a new, well char-
acterized heated cm protein (HP) product in CMA children between the age of 3 and
36 months.
2. Materials and Methods
2.1. Study Population
This study was performed in seven hospitals throughout The Netherlands: two
university hospitals and five large referral hospitals with expertise in paediatric allergy.
Children between three months and three years of age with cow’s milk allergy diag-
nosed by a doctor (paediatric allergist from participating centres) were approached for
participation in the study.
After inclusion, a DBPCFC or OFC with cow’s milk was performed. Children with
a recent (<6 months) positive CM challenge or a previously severe allergic reaction after
CM consumption reported by the participating clinicians did not undergo a cow’s milk
challenge. Sensitization to cow’s milk was measured in SPT and sIgE. Parents filled out
several questionnaires.
During a whole-day visit at the outpatient clinic, the HP product was introduced to the
diet of the child. Either parents or legal guardians had to understand the Dutch language
and signed informed consent. The medical ethical committee of each participating centre
approved the study. (NL61774.078.17).
2.2. Double-Blind, Placebo Controlled Food challenge (DBPCFC) with CM
A DBPCFC with CM was performed according to the guidelines. Children consuming
extensively hydrolysed formula or formula based on amino acids were challenged using
standardized kits. Children consuming breastmilk or alternative milk (e.g., soy milk or
rice milk) were challenged using the matrix they consumed. The dosage of CMP in the
challenge test remained standard (1-3-10-30-100-300 mg, etc.; Table 1). Symptoms (both
subjective and objective) were scored according PRACTALL [26,27].
Table 1.
Dosages in double-blind, placebo-controlled CM challenge and HP product introduction, as
well as cumulative dosage.
CM DBPCFC CM DBPCFC Open Introduction Open Introduction
Step CM protein (mg)
Cumulative dosage (mg)
Step HP product (mg)
Cumulative dosage (mg)
1 1 1 - -
2 3 4 - -
3 10 14 1 10 10
4 30 44 2 30 40
5 100 144 3 100 140
6 300 444 4 300 440
7 1000 1444 5 1000 1440
8 Age-dependent Age-dependent 6 2000 3440
CM: cow’s milk; DBPCFC: double-blind, placebo-controlled food challenge; mg: milligrams; HP product: heated
cow’s milk product.
2.3. Study Product: Heated Glycated CM Protein (HP)
The HP product was produced by FrieslandCampina (Amersfoort, The Netherlands)
and was a powdered product that contained a mixture of whey (20%) and casein pro-
tein (80%). The HP product was treated at ultra-high temperature (UHT) (120
C) for
20 min,
spray-dried and subsequently canned. The cans were stored at 60
C for 14 days,
resulting in glycation of the CMPs. The products and procedures for making the AGE
products were judged and approved by the Quality assurance/Quality Control (QA/QC)
department of FrieslandCampina to be compliant with IFT guidelines. The amount of
Nutrients 2022,14, 629 4 of 11
carboxymethyllysine (CML) (a measure for glycation) in regular infant formula is
28–81 ng
CML/mg protein [
28
]. CML analysis of the new HP product showed a result of 300 ng
CML/mg protein, which is comparable to evaporated milk.
2.4. Introduction of HP Product
The HP product was introduced in incremental doses in 30 min intervals (10-30-100-
300 mg etc. Table 1) [
27
]. The HP product was dissolved in the participant’s individual
daily milk formula (5% of total protein intake/day). The symptoms were recorded in a
database/chart according to PRACTALL and scored on severity retrospectively by two
independent paediatricians [
29
]. Serious adverse events (SAEs) and adverse events (AEs)
were reported to a data safety monitoring board (DSMB).
2.5. Skin Prick Tests (SPT)
An SPT was performed by applying a drop of skimmed CM (FrieslandCampina),
goat’s milk (Ausnutria B.V., Zwolle, The Netherlands), the HP study product, a positive
(histamine) and a negative (PBS) control. Subsequently, the surface area was measured
with an area scanner and compared with the positive control, which gives the histamine
equivalent prick (HEP) index score as described [
30
]. No threshold values have yet been
defined for the skin prick test (SPT)–HEP index values for cow’s milk allergy. SPTs were
considered positive at values > 3 mmØ [31].
2.6. Serum Collection and sIgE Measurements
Blood samples were collected using either a finger prick (age < 6 months) or a vena
puncture (
6 months of age). Serum was collected and stored at
20
C. ISAC (81-1020-01,
Thermo Fisher Scientific B.V, Breda, The Netherlands) was used to identify specific sIgE
against CM protein. In addition, sIgE against total CMP was performed using Immunocap
(f2) (14-4112-01, Thermo Fisher Scientific B.V., Breda, The Netherlands).
The following specific recombinant allergens were measured by both ISAC and Im-
munoCap:
α
-lactalbumin (Bos d4),
β
-lactoglobulin (Bos d5), bovine serum albumin (Bos
d6), (immunoglobulins/lactoferrin (Bos d7) and whole casein (Bos d8) [32].
2.7. Questionnaires
Validated questionnaires, as used in the “Generation R” study, were used to assess the
medical history of mother, father and child [
33
]. Data collected included date of birth, sex,
race, ethnicity, height, weight and relevant medical history. To measure eczema severity,
POEM and eczema area and severity index (EASI) scores were collected [
34
]. Furthermore,
a questionnaire specifically designed for this study was used to gather information from
the parents about the child’s current situation in relation to, e.g., atopy, dairy consumption,
type of formula, feeding or breastfeeding, introduction of other (solid) food and type of
symptoms. The Food allergy quality of life questionnaire (FAQLQ) was implemented
according to Velde et al. [35].
2.8. Open Clinica Database
All patient-related information of this study (Case report forms (CRFs)) is kept blinded
at Erasmus MC, and data were digitalized in an Open Clinica (OpenClinica, LCC, Waltham,
MA, USA) study database (version 3.12.2).
2.9. Statistical Analysis
This study was originally part of a large long-term clinical trial to test tolerance-
induction of the HP product. As the trial was hampered by a low inclusion rate, we decided
to perform a small exploratory study with the available patients, focusing on measuring
safety and tolerability of the product. For further analyses focused on this aim, the Bayesian
approach is the recommended method for evaluating small samples [
36
]. The Bayes factor
(BF) produces the likelihood ratio of the alternative hypothesis (H1) (difference between
Nutrients 2022,14, 629 5 of 11
groups) and the null hypothesis (H0) (no difference between groups) [
37
]. Evidence for
the alternative hypothesis (H1) was set as BF > 3 (moderate), BF > 10 (strong), BF > 30
(very strong) and BF > 100 (extreme), and evidence for the null hypothesis (H0) was set
asBF<1[
37
]. BF was calculated for proportions of positive sIgE, contingency tables
and two-sample designs of SPT-HEP indexes by the Bayes factor package in R, version
4.0.4/ (https://CRAN.R-project.org/package=BayesFactor, accessed on 15 February 2021).
Priors in the proportions were set to low, mediocre, high or unknown probability. In the
two-sample designs, the prior distribution was set to a Cauchy with rscale = 0.707 [38].
3. Results
In total, 25 CMA children participated in this study: 9 girls and 16 boys, with a mean
age of 14.5 months (range: 6–37 months). A total of 18 children were tolerant to the HP
product (HPt group), and seven children developed an allergic reaction to the HP product
(HPr group). No differences were found in baseline characteristics (e.g., eczema, rhinitis
and asthma) between both groups, as shown in Table 2.
Table 2. Baseline characteristics of the HP-tolerant (HPt) and HP-reactive (HPr) children.
HPt Children (n= 18) HPr Children (n= 7)
Mean Range N Pos (T) % Mean Range N Pos (T) %
Age (months) 14.6 (6.5–22.5) 18 13.3 (6.1–37) 7
Gender: F(tot) 6 (18) 33.3 3 (7) 42.9
Atopy * Eczema 11 (18) 61.1 3 (6) 50.0
EASI 5 (18) 27.8 2 (6) 33.3
POEM 10 (18) 55.6 3 (6) 50.0
Rhinitis 4 (18) 22.2 0/6 0
Asthma-like
symptoms 3 (18) 16.7 1 (6) 16.6
Asthma +
medication 2 (18) 11.1 0 (6) 0
Exclusively
breastfed Period, (months) 5.2 (1–9) 9 (18) 50% 3.2 (2–7) 7 (7)
100%
Formula use at
inclusion visit
eHF 13 (18) 72% 5 (7) 71%
AA 5 (18) 27% 2 (7) 29%
Multiple food
allergy
Egg, peanut
and/or nuts 4 (18) 22% 0 (7) 0%
HPt: HP-product-tolerant; HPr: HP-product-reactive; *: parent reported; EASI: eczema area and severity index, T:
totals; eHF: extra hydrolysed formula; AA: amino acid formula.
Baseline measurements comparing differences in sensitization (SPT, sIgE) to CM and
CM components are shown in Table 3. In the HPt group, 10/17 (59%) children, and in the
HPr group 4/5 (80%) children, had a positive SPT (>3 mmØ) for CM (BF 0.6). Specific
serum IgE to CM was positive in 10/15 (67%) children in the HPt group and in 4/5 (80%)
children in the HPr group (BF 2.4). Most children were sensitized both in SPT and sIgE to
whole CM, but in some cases, only one was positive. Results show that the group does not
contain non-IgE-mediated CMA children, although in some individual cases, symptoms in
DBPCFC occurred > 2 h after the challenge. The HPt group showed lower sIgE levels to
Bos d4 (BF 6,2) and Bos d8 (BF 17,8) in comparison with the HPr group. On the contrary,
the HPr group showed lower Bos d5 sIgE levels (BF 6,2). The SPT with HP product was
found in only half of all children who tested positive (11/22): six (35%) in the HPt group
and four (80%) in the HPr group (BF 6.5). SIgE values measured with ISAC against house
dust mite, grass pollen and birch pollen were negative in all children.
Nutrients 2022,14, 629 6 of 11
Table 3.
Baseline sensitization profiles of the HP-product-tolerant (HPt) and HP-reactive (HPr)
children.
HPt Children (n= 18) HPr Children (n= 7) BF
Mean Range N Pos (T) % Mean Range N Pos (T) %
SPT * CM 0.72 (0–2.98) 10 (17) 58.8 1.17 (0–1.72) 4 (5) 80 0.6
Goat’s milk 0.54 (0–4.23) 6 (15) 40 0.86 (0.22–1.16) 4 (4) 100 0.5
HP product 0.23 (0–1.58) 6 (17) 35.3 1.06 (0–2.33) 4 (5) 80 6.5
sIgE CM (kU/L) 3.08 (0–17.2) 10 (15) 66.7 14.93 (0.01–49.6) 4 (5) 80 2.4
α-lactalbumin Bos
d4 (ISU) 0.41 (0–3.36) 5 (15) 33.3 1.48 (0–5.45) 4 (5) 80 6.2
β-lactoglobulin
Bos d5 (ISU) 1.47 (0–10.7) 5 (15) 33.3 0.42 (0–0.8) 4 (5) 80 6.2
Bovine serum
albumin Bos d6
(ISU)
0.16 (0–0.79) 4 (15) 26.7 0.5 (0–2.5) 1 (5) 20 1.3
Casein Bos d8
(ISU) 0.09 (0–1) 1 (15) 6.7 0.74 (0–2.3) 3 (5) 60 17.8
Lactoferrin (ISU)
Bos d7 (ISU) - - 0/15 0 - - 0 (5) 0 NA
HPt: HP-product-tolerant; HPr: HP-product-reactive; *: HEP index; CM: cow’s milk; BF: Bayes factor theorem;
H0 two values/means are equal; H1 two values/means are not equal. BF < 1 = H0 most likely; BF
3 = H1
most likely.
No differences were observed for characteristics of the parents, households and the
background of children, e.g., familial atopic diseases, between the HPt group and HPr
group. (Table 4) The use of antibiotics in the children was higher in the HPt group versus
the HPr group (BF 9.4). The percentage of children going to a day-care facility was higher
in the HPt group versus the HPr group (BF 3.1), and breastfeeding (ever) was higher in
the HPt group versus the HPr group (BF 29.1). More children in the HPt group received
CM formula feeding in the first week of life compared to the HPr group (61% vs. 28.6%,
respectively).
Table 4. Characteristics of the parents, households and the background of children.
HPt Children (n= 18) HPr Children (n= 7) BF Prior Chance
N Pos (T) % N Pos (T) %
Atopy (parents) Mother 13 (18) 72.2 5 (7) 71.4 1.1 mediocre
Father 11 (18) 61.1 4 (7) 57.1 1.3 mediocre
Both parents 7 (18) 38.9 2 (7) 28.5 2.6 low
Both parents not 1 (18) 5.5 0 (7) 0 NA NA
Background (child) Breastfeeding (ever) 9 (18) 50 7 (7) 100 29.1 high
Antibiotics use (child) 12 (18) 66.7 2 (7) 28.5 9.4 low
Pregnancy Mother Antibiotics use 12 (18) 17.6 0(7) 0 1.3 mediocre
Folic acid 16 (17) 94.1 6 (7) 85.7 0.4 unknown
Vitamin D suppl. 2 (16) 12.5 1 (7) 14.3 1.8 low
-3 fatty acid suppl. 2 (16) 12.5 1 (7) 14.3 1.8 low
fish oil capsules 0 (15) 0 1 (7) 14.3 0.6 unknown
multivitamin suppl. 13/17 76.5 4 (7) 57.1 0.7 unknown
Exposure to smoke During pregnancy * 4 (17) 23.5 2 (7) 28.6 2.3 low
Current smoking 1 (17) 5.9 0 (7) 0 0.7 unknown
Pet keeping Currently 1 (17) 58.8 4 (7) 57.1 1.3 mediocre
Other Day care 11 (18) 61.1 2 (7) 28.6 3.1 mediocre
HPt: HP-product-tolerant; HPr: HP-product-reactive; *: passive smoking + current smoking; n: number;
BF = Bayes factor:
H0 two values/means are equal; H1 two values/means are not equal. BF < 1 = H0 most
likely; BF 3 H1 most likely.
Nutrients 2022,14, 629 7 of 11
Table 5shows the adverse events in the HPr group (n= 7) during introduction of the
HP product. Ref. [
31
] One patient experienced an SAE (ID: 111002), which started 15 min
after the second step (3 mg CM protein). Six patients experienced AEs. An overview of
the category and type of symptoms developed at a certain step of HP product is given in
Supplementary Table S1.
Table 5. Serious adverse events and adverse events (SAE/AE).
Patient
ID
Age Months/
M/F
Allergic
Reactions at
Step
Minutes after
Intake
Medication,
According to National
Anaphylaxis Protocol
Stopped at
Dose/Outcome *
SAE/AE
Sampson
Scale
111002 13/M
Step 2: Step 2: Step 2:
Dose: 2/
After 5 h released
from hospital
SAE
4
Eczema lips
(150diminished) 2 min Adrenaline
auto-injector (0.15 mg),
Xyzal suspension
(2.5 mg),
dexamethason (4 mg)
Step 2 repeated:
Stridor, cough,
crying
Step 2 repeated:
15 min
555004 6/F
Step 6: Step 6:
None Full challenge/
<24 h AE/2
Eczema feet,
back, belly; 15 min;
vomiting, itch,
eczema face,
diarrhea
6–9 h
888004 37/M Dry cough,
stridor
Step 4:
5–10 min
Step 4: Dose: 4/
After 2 h released
from hospital
AE/4
Aerius suspension
(2.5 mL)
888005 8/M
Step Step Step 5:
Dose: 5/
After 2 h released
from hospital
AE/3
1: Sneezing,
erythema chin
1 repeated:
5 min
Aerius-suspension
(2x 2.2 mL)
5: Cough,
redness face,
nausea
5: 10–15 min
999001 11/M
Step:
Step 3: 25 min
Step 3: Aerius
suspension (2.5 mL)
and prednisone
Dose: 3/
15 min no more
wheezing
AE/3
2: increased
eczema
3: Sneezing,
cough, runny
nose, increased
eczema,
wheezing
999002 10/F
Step: Step:
none Dose: 6/
Not specified AE/3
4: Sneezing,
cough, starting
urticaria
4: 15–20 min
5: Runny nose,
redness face,
urticaria
5: directly
6: (after pause):
urticaria 6: after 10 min
999004 10/F Step: Step:
Step 2: Aerius
suspension (2.5 mL)
and 1.6 mL prednisone
Dose: 2/
After 4 h released
from hospital
AE/2
Abbreviations: Step 1 (10 mg), step 2 (30 mg), step 3 (100 mg), step 4 (300 mg), step 5 (1000 mg), step 6 (3000 mg).
* Hours until symptoms disappeared;
SAE: serious adverse events; AE: adverse events; M/F: male/female; mL:
millilitres; min: minutes.
No differences were found in baseline CM challenge (DBPCFC or OFC) between
HPt and HPr groups. In total, 17/25 challenges were double blind. In one child, no
challenge was performed due to two anaphylactic reactions to CM in recent history. In some
individual cases, symptoms in DBPCFC occurred > 2 h after the challenge. The symptoms
Nutrients 2022,14, 629 8 of 11
that occurred in these individuals were skin disorders and vomiting. Epinephrine auto-
injectors had to be used three times in the HPt group, and no epinephrine was administered
in the HPr group (Supplementary Table S2).
In the FAQLQ, no differences were found between groups, except one: we found a
lower parental perception (<2 = very small chance) on appropriate response by others to
allergic reactions in their child in the HPr group (HPt group mean: 2.28; range 0–6; HPr
group mean: 1.5; range 1–2) [6] (Supplementary Table S3).
4. Discussion
This study aimed to investigate the safety and tolerability of a new heated and glycated
cow’s milk protein product, the HP product. This HP product aimed to mimic and to have
tolerance-inducing capacities similar to those of “baked milk” products, with well-defined
production methods, e.g., exact heating time and temperature during glycation process, in
order to achieve a predetermined glycation level.
Due to low inclusion numbers, the primary aim to study tolerance-inducing capacity
of the HP product could not be reached, and planned statistical analyses could not be
performed. Nevertheless, in alternative analyses (e.g., Bayes factor) differences between
HPt and HPr children could be analysed and were, in some cases, significantly high and
should be considered exploratory.
Low inclusion numbers in intervention studies in children with cow’s milk allergy
have been described previously. Many studies on specific food allergy in children are
underpowered, according to a recent Cochrane database systematic review on effects of
eHF use in CMA children [
38
]. Reasons for the low inclusion numbers in the current study
were less motivated parents due to the many planned hospital visits with the child during
the study, following a strictly cow’s-milk-free diet and low numbers of positive cow’s milk
DBPCFC in children with suspected CMA. The latter is not surprising, as recent research
by Vlieg et al. in The Netherlands reported a considerable percentage of overdiagnoses of
CMA in children (66%) when children did not react in a DBPCFC [39].
Eighteen out of the 25 included CMA children (72%) were tolerant of the HP product
(HPt group). Sensitization patterns differed between the two groups. Children in the
HPr group showed a higher sensitization profile. This might be one of the reasons for
allergic reactions to the HP product [
40
]. It is known from literature that, in contrast to
whey proteins, caseins do not denature and aggregate upon heat treatment but can be
glycated [
41
]. These lesser changes due to heat treatment of the caseins may explain the
intolerance of the HP product in this group. Sensitization to caseins was much higher in
the HPr group (60%) in comparison with the HPt group (only one child; 7%).
At the same time, the HPr group had clearly lower sIgE against
β
-lactoglobulin (Bos
d5).
β
-lactoglobulin can be denatured and aggregated after heating, but this HPr group
was borderline sIgE-positive (0.42 ISU) for
β
-lactoglobulin, so that could not affect the
tolerability to the heated cow’s milk protein in this group. Sensitization level in SPT
to goat’s milk and CM was comparable between groups (BF 0.6 and 0.5, respectively).
Apparently, the HP product cannot be tolerated in a substantial number of children with
goat’s milk sensitization.
Regarding baseline characteristics, in the HPr group, the use of antibiotics (BF: 9.4)
and attendance at day care was lower (BF 3.1) Furthermore, all children in the HPr group
received breast feeding (ever), whereas only half of the children in the HPt group did
(BF 29.1). When children attend day care to a lesser extent, a lower use of antibiotics
seems logical, as infections occur less often in “no-day-care” children [
42
], and longer
breastfeeding is easier in practical terms. Although breastfeeding has many benefits, it
does not reduce the risk of CMA [43].
Combining all results of this study, we hypothesize that a possible cause of intolerance
of the HP product (HPr group) lies in an overall lower general exposure to allergens (less
day care and infections) in the first months of life, as previously described by McGowan
et al. [44], and to milk allergens in specific.
Nutrients 2022,14, 629 9 of 11
Seventy-two per cent of the patients could tolerate the HP product. This is comparable
with data from the “baked milk studies” [
4
7
,
16
]. In a more recent study by Agyemang
et al. among 84 children, 72% were tolerant to muffins containing CM [
40
]. Furthermore,
the HPt group showed > 90% negative sIgE to caseins, most likely representing a group
with transient CMA more likely to tolerate baked milk products [8].
In the current study, the safety profile of the HP product was found to be comparable
with larger studies with baked milk challenges [45].
The tolerance-inducing effects of baked milk products are described in many stud-
ies, but the effect of heating and glycation on tolerance-inducing effects is only sparsely
investigated. With this new HP product, we tried to mimic “baked milk” products, while
standardizing its characteristics and production. This is, as far as we could find in the
literature, the first well described “baked milk” product. The powder can be easily added
to the daily formula of very young CMA children, who, in some cases, might not yet be
able to consume baked products, e.g., cake. However, the introduction can cause mild to
severe allergic reactions and should therefore be supervised by a clinician. Sensitization
profiles to CM can be useful to pre-select children who will most likely tolerate the HP
product. Further trials with this promising new HP product should be performed in larger
groups of children to the measure the tolerance-inducing capacity.
In summary, a new HP product was found to be safe and was tolerated by 72% of
challenge-proven CMA children. Outcomes of SPT with CM and the HP product, as
well as values of sIgE against caseins,
α
-lactalbumin and
β
-lactoglobulin, may predict the
tolerability of the HP product. Larger studies are needed to confirm these conclusions.
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10
.3390/nu14030629/s1, Table S1: Characteristics of patients with a reaction during introduction of
the heated cow’s milk protein study product. Table S2: Baseline CM food challenge; comparison
between HP-product-tolerant children (HPt) and HP-product-reactive (HPr) in developed symptoms
and emergency medication. Table S3: Results FAQ-LQ (D-Q1) parents of children in HPt group and
HPr group.
Author Contributions:
Conceptualization, N.W.d.J., A.B.S., K.A.H., H.J.W., J.A.M.E., R.J.J.v.N.,
H.F.J.S., M.E.v.S., N.J.T.A. and R.G.v.W.; methodology, N.W.d.J., J.A.M.E., M.E.v.S. and N.J.T.A.;
formal analysis, F.E.v.B.; investigation, N.W.d.J., M.E.v.S., J.A.M.E., K.A.H., R.G.v.W., A.B.S., L.L.,
G.v.d.M., I.H., Y.C.M.D., M.B., M.I.B., J.S., O.v.D., B.V.-B., J.v.d.B., L.P., S.T., A.I.H., M.W.J.S., F.E.v.B.
and N.J.T.A.; writing—original draft preparation, N.W.d.J., M.E.v.S., J.A.M.E., N.J.T.A., K.A.H.,
R.G.v.W., H.J.W., H.F.J.S., A.B.S., F.E.v.B. and N.J.T.A.; writing—review and editing, N.W.d.J., M.E.v.S.,
J.A.M.E., N.J.T.A., K.A.H., R.G.v.W., H.J.W., H.F.J.S., A.B.S. and F.E.v.B.; funding acquisition, N.W.d.J.,
K.A.H., H.J.W., R.J.J.v.N., H.F.J.S. and A.B.S. All authors have read and agreed to the published
version of the manuscript.
Funding:
This work is part of the iAGE/TTW research programme, project number 14536, which is
(partly) financed by The Netherlands Organisation for Scientific Research (NWO).
Informed Consent Statement:
The medical ethical committee of each participating centre approved
the study. (NL61774.078.17). Written informed consent has been obtained from the patient(s) to
publish this paper.
Acknowledgments:
We would like to thank all the children and their parents for participating in the
iAGE study.
Conflicts of Interest:
R. J. Joost van Neerven is an employee of FrieslandCampina. Linette Pellis is
an employee of Ausnutria. All authors declare no conflict of interest.
Nutrients 2022,14, 629 10 of 11
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... The current study was preceded by a study from the same group in which the introduction of the standardised heated and glycated CM protein product (the iAGE-product) was studied for safety and tolerability in 25 children with challenge-proven CMA [12]. The iAGE product was tolerated by 72% of the 25 included children in the previous study. ...
... As described by de Jong et al. 2022 [12], Friesland Campina (Amersfoort, Netherlands) produced a heated and glycated CM protein powder (iAGE product). This product contained a mixture of casein protein (80%) and whey protein (20%) and was sterilised at 120 • C for 20 min. ...
... The study population in the preceding baseline study consisted of 25 children with challenge-proven CMA and was performed in seven Dutch hospitals specialized in pediatric allergy [12]. Parents of children between three months and three years of age were approached for participation in the study. ...
Article
Full-text available
Citation: van Boven, F.E.; Arends, N.J.T.; Sprikkelman, A.B.; Emons, J.A.M.; Hendriks, A.I.; van Splunter, M.; Schreurs, M.W.J.; Terlouw, S.; Gerth van Wijk, R.; Wichers, H.J.; et al. Abstract: Accelerating the induction of tolerance to cow's milk (CM) reduces the burden of cow's milk allergy (CMA). In this randomised controlled intervention study, we aimed to investigate the tolerance induction of a novel heated cow milk protein, the iAGE product, in 18 children with CMA (diagnosed by a paedriatric allergist). Children who tolerated the iAGE product were included. The treatment group (TG: n = 11; mean age 12.8 months, SD = 4.7) consumed the iAGE product daily with their own diet, and the control group (CG: n = 7; mean age 17.6 months, SD = 3.2) used an eHF without any milk consumption. In each group, 2 children had multiple food allergies. The follow-up procedures consisted of a double-blind placebo-controlled food challenge (DBPCFC) with CM t = 0, t = 1 (8 months), t = 2 (16 months), and t = 3 (24 months). At t = 1, eight (73%) of 11 children in the TG had a negative DBPCFC, versus four out of seven (57%) in the CG (BayesFactor = 0.61). At t = 3, nine of the 11 (82%) children in the TG and five of seven (71%) in the CG were tolerant (BayesFactor = 0.51). SIgE for CM reduced from a mean of 3.41 kU/L (SD = 5.63) in the TG to 1.24 kU/L (SD = 2.08) at the end of intervention, respectively a mean of 2.58 (SD = 3.32) in the CG to 0.63 kU/L (SD = 1.06). Product-related AEs were not reported. CM was successfully introduced in all children with negative DBPCFC. We found a standardised, well-defined heated CM protein powder that is safe for daily OIT treatment in a selected group of children with CMA. However, the benefits of inducing tolerance were not observed.
... However, some parents were reluctant to give milk, but the children could still tolerate products containing traces. They would probably have tolerated baked milk products, as recently shown by the iAGE study [30]. ...
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... Even protein content of the several doses is unknown. FrieslandCampina (Amersfoort, the Netherlands) developed a standardized dry-heated CM protein powder, with an exact baking temperature and time, and the method is accurately described in the article [16]. To test the new baked milk (HP) powder, challenge-proven CMA children were included (3 months-3 years), and the HP powder was introduced in incremental doses by dissolving it in the child's daily milk formula. ...
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Introduction: Cow’s milk allergy (CMA) is a common diagnosis in infants, requiring the exclusion of cow’s milk until tolerance is recovered. In the present study, we aim to determine which factors are associated with the development of tolerance. Methods: Retrospective, observational study of subjects who underwent the same clinical follow-up methodology. We studied 245 cases of CMA (125 IgE-mediated and 120 non-IgE-mediated). The following variables were analysed: age at diagnosis, gender, type of delivery, type of feeding received, feeding during the first months of life, clinical features, and type of feed received as treatment: casein hydrolysates or casein hydrolysates with Lactobacillus rhamnosus GG (LGG). Results: Factors associated with earlier tolerance were non-IgE-mediated CMA (HR = 2.92; 95% CI: 2.20–3.88) and patients receiving casein hydrolysate with LGG (HR = 1.79; 95% CI: 1.33–2.42). Later tolerance was associated with caesarean delivery (HR = 0.78; 95% CI: 0.58–1.05) and breastfeeding for a period of at least 3 days (HR = 0.64; 95% CI: 0.44–0.93). The multivariate study shows that the type of formula (HR = 1.61; 95% CI: 1.19–2.18) and the type of CMA (HR = 2.82; 95% CI: 2.12–3.85) have an effect on the recovery time. Casein hydrolysates with LGG reduces the recovery time in IgE-mediated (HR = 1.88; 95% CI: 1.17–3.01) and non-IgE-mediated CMA (HR = 1.46; 95% CI: 0.98–2.17). Conclusions: Tolerance acquisition is faster in non-IgE-mediated CMA subjects and in those who received casein hydrolysate with LGG.
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