Immunologic features of infants with milk or egg allergy enrolled in an observational study (Consortium of Food Allergy Research) of food allergy

Article Outline

• Abstract
• Methods
  • Study rationale, design, and enrollment criteria
  • Definitions of atopic diseases and categorization of food allergy
  • SPTs
  • Plasma food-specific IgE assay
  • Mononuclear cell stimulation and PCR analysis
  • Statistics
• Results
  • Demographic characteristics
  • Sensitization
  • Comparison of clinical allergy: mononuclear cell gene expression
• Discussion
• Acknowledgment
• Methods
  • Definition of atopic diseases
  • Categorization of food allergy
    • Confirmed IgE-mediated reaction (approximately 97% certainty)
    • Convincing but not confirmed IgE-mediated reaction (approximately 95% accurate)
    • Serologic diagnosis
    • Potential allergy
    • Not allergic/sensitized
    • Not allergic/not sensitized
    • Not sensitized/never ingested
    • Non-IgE food allergy
  • Skin tests
• Results
  • Additional demographic features
  • Additional results and quality control standards for PCR
• Fig E1. 
• Fig E2. 
• Fig E3. 
• Table E1. 
• Table E2. 
• Table E3. 
• References
• References
• Copyright

Background

Immune features of infants with food allergy have not been delineated.

Objectives

We sought to explore the basic mechanisms responsible for food allergy and identify biomarkers, such as skin prick test (SPT) responses, food-specific IgE levels, and mononuclear cell responses, in a cohort of infants with likely milk/egg allergy at increased risk of peanut allergy.

Methods

Infants aged 3 to 15 months were enrolled with a positive SPT response to milk or egg and either a corresponding convincing clinical history of allergy to milk or egg or moderate-to-severe atopic dermatitis. Infants with known peanut allergy were excluded.

Results

Overall, 512 infants (67% male) were studied, with 308 (60%) having a history of a clinical reaction. Skin test responses, detectable food-specific IgE, or both revealed sensitization as follows: milk, 78%; egg, 89%; and peanut, 69%. SPT responses and food-specific IgE levels were discrepant for peanut (15% for IgE ≥0.35 kU_A/L and negative SPT response vs 8% for positive SPT response and IgE <0.35 kU_A/L, P = .001). Mononuclear cell allergen stimulation screening for CD25, cytokine-inducible SH2-containing protein (CISH), forkhead box protein 3 (FOXP3), GATA3, IL10, IL4, IFNG, and T-box transcription factor (TBET) expression by using casein, egg white, and peanut revealed that only allergen-induced IL4 expression was significantly increased in those with clinical allergy to milk (compared with nonallergic subjects) and in those sensitized to peanut, despite the absence of an increase in GATA3 mRNA expression.

Conclusions

Infants with likely milk/egg allergy are at considerably high risk of having increased peanut-specific IgE levels (potential allergy). Peanut-specific serum IgE levels were a more sensitive indicator of sensitization than SPT responses. Allergen-specific IL4 expression might be a marker of allergic risk. Absence of an increase in GATA3 mRNA expression suggests that allergen-specific IL-4 might not be of T-cell origin.

Key words: Food allergy, sensitization, atopy

Abbreviations used: AD, Atopic dermatitis, Ct, Threshold cycle, SPT, Skin prick test, TBET, T-box transcription factor gene

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Food allergy is estimated to affect approximately 4% to 6% of young children, with egg, milk, and peanut allergy being the most common.¹, ² Food allergy appears to be increasing in prevalence in westernized countries, with specific evidence for an increase in peanut allergy in children within the past decade.³, ⁴, ⁵ Although allergy to egg and milk typically resolves over time, peanut allergy typically persists,⁶ and studies indicate that milk and egg allergies are also becoming more persistent.⁷, ⁸ Although genetic factors clearly predispose a subject to food allergy,⁹ the observed recent increases and persistence of childhood food allergies must be attributable to environmental factors. Food allergy can be life-threatening or fatal¹⁰ and seriously affects quality of life.¹¹ Considering the increasing prevalence and seriousness of this disease, studies to determine risk factors, prevention strategies, better diagnostic tests, and definitive treatments are needed.

The Consortium of Food Allergy Research, funded by the National Institutes of Allergy and Infectious Diseases, is comprised of 5 clinical sites in the United States with established expertise in food-induced allergic disorders that are undertaking observational and treatment studies of food allergy. To address the immunologic, genetic, and environmental factors that affect the course of food allergy, we established a cohort of infants with likely egg allergy, milk allergy, or both who are predicted to be at increased risk to have or develop peanut allergy. These children will be followed longitudinally to determine the course of their egg and milk allergies, as well as the development or resolution of peanut allergy.

Insights into the basic mechanisms responsible for the development of food allergies are lacking. In the current study we sought to determine whether mononuclear cell expression of key cytokine and regulatory genes were markers of current milk/egg allergy or associated with sensitization to peanut in these infants. In what we believe to be the largest and most comprehensive study to date, we present data confirming the expected importance of IL4 but do not detect an associated increase in GATA3 transcripts or a shift in the ratio of GATA3/T-box transcription factor (TBET), findings that raise questions as to the role of T cells in the early development of food allergy. The infants enrolled in the study have demographic features representing common food allergy presentations; we found clinically important observations, including an unexpectedly high rate of peanut sensitization, and we characterize discrepancies in common diagnostic tests in this large cohort, observing that serum testing for peanut sensitization is more sensitive than skin prick tests (SPTs).

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Methods 

Study rationale, design, and enrollment criteria 

We aimed to study markers of development of peanut allergy and the natural course of egg/milk allergy, and therefore we did not intend to enroll children with likely peanut allergy, only those “at risk.” We developed enrollment criteria for this cohort based on the results of various studies of childhood food allergies and atopic dermatitis (AD),¹², ¹³, ¹⁴, ¹⁵, ¹⁶ estimating that children with a convincing clinical reaction to milk, egg, or both with a positive SPT response to the responsible food and/or children with moderate-to-severe AD and a positive SPT response to milk, egg, or both would be likely (approximately 20% to 30%) to have a peanut allergy and have a 25% to 50% chance of resolving their egg or milk allergy by 5 to 7 years of age. We enrolled children aged 3 to 15 months to allow adequate recall of their feeding history and to reduce the chance that they already would have a peanut allergy. Infants known to have a peanut allergy or a peanut-specific IgE level of greater than 5 kU_A/L (representing a more likely current peanut allergy, as described in the Methods section of this article's Online Repository at www.jacionline.org) before screening were therefore excluded from enrollment because they already had evidence of peanut allergy/sensitization and therefore could not be evaluated for the basic question to be addressed prospectively. To maintain uniformity and an observational approach, the study design includes evaluations, care for food allergy, and instructions on dietary management that were uniform among the 5 clinical centers and reflect practice parameters for AD,¹⁷ food allergy,¹⁸ and the American Academy of Pediatrics recommendations for allergy prevention published in 2000, which were current at the initiation of the study.¹⁹

Enrollment required either (1) a history of a convincing immediate allergic reaction to cow's milk (and/or egg), as described further in the Methods section of this article's Online Repository, and a positive SPT (≥3 mm larger than that elicited by the negative control) to cow's milk (and/or egg if the clinical reaction was to egg); (2) moderate-to-severe AD at the time of enrollment (as described further in the Methods section of this article's Online Repository) and a positive SPT response to milk, egg, or both; or (3) both.

Children were excluded if they had chronic disease (other than asthma, AD, or rhinitis), required therapy (eg, heart disease and diabetes), were participating in any interventional study, were unable to discontinue antihistamines for routine tests, had a sibling enrolled in the observational study, or already had confirmed or convincing evidence of peanut allergy (see the Methods section of this article's Online Repository). Study procedures were reviewed and approved by a National Institute of Allergy and Infectious Diseases Data Safety Monitoring Board and by local site institutional review boards, and written signed consent was obtained.

Definitions of atopic diseases and categorization of food allergy 

Atopic diseases (asthma, allergic rhinitis, and AD) were diagnosed and graded by severity as described in the Methods section of this article's Online Repository. Food allergy was diagnosed based on clinical criteria (as described in the Methods section of this article's Online Repository) because as an observational natural history study, repeated scheduled oral food challenges could not be imposed. At enrollment, diagnoses were categorized as confirmed or convincing when there was a clear clinical history and sensitization to the causal food, and in this report these children are categorized as allergic. Those ingesting the food or lacking sensitization are categorized as not allergic. These 2 clinical categories are the primary clinical end points evaluated in the current study. Additional enrollees with potential allergy are described in the Methods section of this article's Online Repository. The total study group was also evaluated with regard to sensitization status, as described below.

SPTs 

SPTs were performed with the GreerPick (Greer Laboratories, Lenoir, NC), with participants avoiding antihistamines for at least 5 half-lives of the specific agent. Tests were performed on the infant's back, and at 15 minutes, the wheal was outlined in pen and transferred by tape to paper. The size of the longest diameter and its longest perpendicular were averaged. Additional details are described in the Methods section of this article's Online Repository.

Plasma food-specific IgE assay 

The concentrations of specific IgE antibody to egg, milk, and peanut were measured from plasma at a single central laboratory by using the ImmunoCAP system (Phadia, Uppsala, Sweden) and reported in kU_A/L. Those at or greater than 0.35 kU_A/L are described as IgE sensitized.

Mononuclear cell stimulation and PCR analysis 

PBMC isolation was performed by means of Ficoll-Paque density gradient centrifugation, and cultures were performed at each clinical site on fresh venous blood samples. The laboratory processing protocol was standardized, and reproducibility across sites was confirmed in pilot studies. Four million cells per condition were cultured for 48 hours in AIM-V serum-free media (Invitrogen, Carlsbad, Calif) with aqueous peanut extract (50 μg/mL); tetanus toxoid (5 μg/mL); purified α, β, and κ caseins (each 50 μg/mL); and egg white protein (50 μg/mL). Control stimulations were performed with medium alone and anti-CD3/anti-CD28 beads (5 μL; DYNAL, Invitrogen). At the end of the culture period, cells expressing CD25 were enriched by means of selection with anti-CD25–coated para-magnetic beads according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). Pilot experiments demonstrated approximately 10-fold enrichment of CD25⁺ cells, with 70% to 80% of selected cells coexpressing CD3, CD4, and CD25, as measured by means of flow cytometry. The entire selected fraction of cells was immediately lysed in RLT buffer (Qiagen, Hilden, Germany) containing approximately 0.14 mol/L β-mercaptoethanol and stored at −80°C until RNA purification. Semiautomated RNA isolation was performed with the RNeasy Micro kit on the QIACube robotic centrifuge (both by Qiagen), according to the manufacturer's protocols, adjusted to the smaller cell lysate volume of approximately 100 μL. A 2-step quantitative RT-PCR protocol was implemented. The first-strand cDNA was synthesized by using the TaqMan kit (Applied Biosystems, Foster City, Calif). The quantitative PCR was carried out according to the in-house established protocol using SYBR Green I fluorescence detection in a 384-well plate on an ABI 7900 (Applied Biosystems).

Raw PCR analysis and annotation were performed on coded samples. The threshold cycle (Ct) number was set by software, with confirmation and adjustment as necessary to define the threshold of linear amplification. On reviewing data and filtering out artifacts (eg, by means of inspection of amplification curves and dissociation analysis), data were exported into Excel for data upload to the central database for further analysis. For the gene expression data, ΔΔCt was calculated by subtracting the RPS9 reporter gene Ct and then normalizing by subtracting the standardized medium control response. Negative values indicate relatively higher activity with fold increase relative to the medium control calculated as 2^-ΔΔCt. Nondetected genes were arbitrarily assigned a Ct of 40. Because of the significant cohort size, the quantitative PCR was performed in one repetition for each gene per sample. Data were compared for those with confirmed/convincing milk allergy (vs those not allergic) and for those sensitized (serum IgE >0.35 kU_A/L) or not for all 3 foods.

Statistics 

Standard descriptive measures and univariate methods were used, including the McNemar test for paired data. Log-transformed IgE values were analyzed with nondetectable values set to 0.001. Relationships among the standardized gene ΔΔCt values were examined with principal component analysis, a multivariate analysis technique that constructs coordinate systems based on linear combinations of the gene expression levels to identify potential important combined influences.²⁰

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Results 

Demographic characteristics 

A total of 512 children were enrolled and included in these analyses. However, blood samples were not obtained for technical reasons from 9 participants whose results are therefore excluded in figures and tables concerning IgE results. Specific food skin tests to milk (n = 1) and egg (n = 1) were not performed in 2 infants because of a recent history of severe anaphylaxis to the trigger food, but these children qualified for enrollment by fulfilling criteria that did not rely on these results and had sensitization to the respective foods by blood tests. There were 287 children screened who were not enrolled because SPT responses to milk and egg were negative. There were 104 subjects excluded from enrollment at the time of screening because of likely peanut allergy (eg, peanut-specific IgE level >5 kU_A/L) having been determined before the enrollment blood test. Overall, 308 (60.2%) were enrolled with clinical reactions to egg, milk, or both.

Table E1 (available in this article's Online Repository at www.jacionline.org) shows the demographic features of the study participants. Among 308 infants enrolled with a clinical reaction, 71.4% reacted to cow's milk, 39.9% reacted to egg, and 11.4% reacted to both foods. Among the 204 enrolled with AD but no acute food-induced allergic reactions, 116 (56.9%) had a positive SPT response to cow's milk, 181 (88.7%) had a positive SPT response to egg, and 93 (45.6%) had skin test sensitivity to both. Table I shows the clinical categories of the enrolled infants, and Table E2 (available in this article's Online Repository at www.jacionline.org) shows sensitization rates to milk, egg, and peanut. Although we specifically avoided enrolling subjects with known clinical allergic symptoms when exposed to peanut, as described above, 69% of the study group had evidence of sensitization to peanut. Of note, 26.6% had a significantly increased peanut IgE level (>5 kU_A/L) at enrollment.

Table I. Food allergy diagnostic categories at the time of enrollment (n = 503)

See the Online Repository at www.jacionline.org for definitions.

Sensitization 

We performed analyses to compare SPT responses and serum IgE measurements with regard to their relative sensitivity in detecting food-specific IgE (not clinical outcomes). Enrollment criteria required subjects to have a positive SPT response to milk, egg, or both, and therefore analysis of peanut is least affected by this bias. Fig E1 (available in this article's Online Repository at www.jacionline.org) shows the relationship of skin test size to serum IgE levels for milk, egg, and peanut. Of note, there are discordances in which one sensitization test response is negative while the other is positive. Table II summarizes the discrepancies for the entire study group by calculating the percentage who are determined to be sensitized based on serum IgE levels among those with a negative SPT response (eg, SPT_negative and IgE_positive/[(SPT_negative and IgE_positive) + (SPT_negative and IgE_negative)]) and the percentage determined to be sensitized by means of SPTs among all of those with undetectable IgE levels. When evaluating only participants with confirmed or convincing milk/egg allergies, test discordance was less pronounced, as shown in Fig E1, A and B (milk: 5.4% for IgE ≥0.35 kU_A/L/negative SPT response vs 20.2% for positive SPT response/IgE <0.35 kU_A/L; egg: 0% for IgE ≥0.35 kUA/L/negative SPT response vs 9.4% for positive SPT response/IgE <0.35 kU_A/L). The pattern of discrepancy for peanut indicates that the serum test was more sensitive than the SPT (P = .001, McNemar) in detecting a sensitized participant. Of note, enrollees were required to have a positive skin test to milk/egg, and therefore only the analysis of peanut sensitization comparing serum with SPT results is unaffected by this type of selection bias. Conversely, despite the exclusion of infants with known peanut-specific IgE levels of greater than 5 kU_A/L before enrollment, the serum test was still more likely to detect sensitization than the skin test.

Table II. Discrepancies in detection of specific IgE by means of SPT versus serum IgE measurement

Comparison of clinical allergy: mononuclear cell gene expression 

Quality control measures for PCR reactions and intersite processing are described in the Results section of this article's Online Repository, including Fig E2, Fig E3 (all available in this article's Online Repository at www.jacionline.org), which demonstrate consistency of PCR products for housekeeping genes and results obtained across the 5 sites.

For purposes of analyzing the outcomes related to gene transcription responses, allergic phenotypes were categorized in 2 ways: (1) allergic (eg, clinical reaction) versus not allergic (eg, clinically tolerant or not sensitized) and (2) sensitized (by serum results) versus not sensitized. The results of stimulation with egg white, caseins, and peanut are shown according to calculated ΔΔCt in Fig 1, and raw data, including controls with tetanus and anti-CD3/anti-CD28, are presented in Table E3 (available in this article's Online Repository at www.jacionline.org). By design, no enrollees had confirmed clinical peanut allergy, and therefore gene expression induced by peanut is categorized by serologic status.

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• Fig 1. 

  Real-time RT-PCR results by stimulant comparing children with confirmed/convincing milk or egg allergy (A) or sensitization status to milk, egg, or peanut (B). Values shown are median ΔΔCt values, as described in the text. Negative values indicate increasing expression. The number of infants in each category is shown alongside the ΔΔCt value. CISH, Cytokine-inducible SH2-containing protein; FOXP3, forkhead box protein 3.

After 48 hours' stimulation in culture, cells expressing CD25⁺ constitutively or as a consequence of activation were selected by using a bead-coupled mAb (CD3⁺CD4⁺CD25⁺ = 70% to 80%), mRNA was isolated, and expression was later analyzed by means of RT-PCR. Consistent with other reports of allergen-induced immune responses in children with food allergy,²¹, ²² significantly higher IL4 expression was associated with both allergy to milk (allergic vs not allergic; ratio of medians, 3.9-fold; P < .001) and with sensitization to milk or peanut (3.9-fold [P < .001] and 1.8-fold [P = .005], respectively). There was no evidence of increased regulatory T-cell responses in any group, as measured by the levels of forkhead box protein 3 (FOXP3) and IL10 expression. Despite the association of increased IL4 expression with clinical allergy and IgE sensitization, there was no evidence of preferential activation (ie, CD25 upregulation) of T_H2-polarized T cells in allergic or sensitized subjects, as indicated by the absence of significant differences in either GATA3 expression or the GATA3/TBET ratio.

Principal component analyses failed to identify other important multivariate relationships that discriminated between the clinical groups. IgE and gene products relationships were examined, and only milk- and peanut-specific IgE levels showed weak correlations with induction of food-specific IL4 (Spearman r = −0.25 [P < .001] and r = −0.14 [P = .003], respectively for ΔΔCt).

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Discussion 

It is estimated that 2.5% of young children have milk allergy, 1% to 2% have egg allergy, and 1% have peanut allergy², ⁵, ²³, ²⁴, ²⁵ and that these allergies are increasing in prevalence and becoming more persistent.³, ⁷, ⁸, ²⁶ However, little is known about the underlying mechanisms resulting in the development of allergy or tolerance to these foods.², ²⁶ The cohort described herein was established to evaluate and elucidate environmental, genetic, and immunologic factors associated with the development or persistence of these common food allergies with the goal of determining better diagnostic and therapeutic modalities. The cohort will be followed longitudinally in this observational study to characterize the outcomes of milk, egg, and peanut allergy, including the conduct of oral food challenges, as clinically indicated. The current study focuses on immune characteristics associated with clinical allergy and sensitization in these infants at enrollment.

We undertook analysis of activated mononuclear cell expression of several key markers of immune regulation and T_H1/T_H2 bias. We found that allergen-induced IL4 expression in peripheral mononuclear cells of this high-risk cohort was associated with clinical allergy to milk and IgE sensitization to milk and peanut. This is consistent with other reports of a T_H2 bias in food allergy, as well as with our current understanding of the role of IL-4 in inducing IgE class switching in activated B cells.²⁷ Differentiated CD4 T_H2 cells have been shown to express high levels of GATA3 and low levels of TBET, master transcriptional regulators for many T_H2 and T_H1 genes, respectively.²⁸ We hypothesized that allergen-specific T cells activated in vitro would be predominantly T_H2 effector memory cells expressing a high GATA3/TBET ratio and that selection of activated cells on the basis of their upregulation of CD25 would be reflected by an increase in the GATA3/TBET ratio of transcripts detectable by means of PCR. Although previous studies have associated T_H2 immune responses with food allergy, the immune phenotype of such young children with early evidence of food allergy or sensitization has not been previously studied on this scale. We did not find evidence of an increased GATA3/TBET ratio with allergen activation. The lack of detectable enrichment for GATA3-expressing cells was not due to a failure of detecting GATA3, which was readily detectable and expressed at higher levels on average than IL4 and several other transcripts (Table E3). Activation of cells, as reflected by an increase in CD25 expression over medium alone, was also detectable (not shown), although it did not differ significantly between clinical groups (Fig 1). This finding suggests that strongly polarized allergen-specific T-cell differentiation might not have occurred in these subjects, even though they have produced IgE in vivo and allergen stimulation in vitro induced IL4. In this study we looked specifically at CD25⁺ cells isolated after stimulation with antigen to capture both potential regulatory (constitutive CD25 expression) and activated (induced CD25 expression) T-cell populations; however, it is known that CD25 is also induced or constitutively expressed on many nonlymphoid cells, including natural killer T cells and basophils, potent potential sources of IL-4.

Basophils constitutively express CD25,²⁹ are enriched with mononuclear cells during density gradient isolation, and produce high levels of IL-4 in sensitized subjects.³⁰ Recently, basophils have been implicated in allergy model systems for playing an important role in priming and enhancing memory T_H2 responses.³¹, ³², ³³ Murine basophils have been shown to express IL4 in the absence of detectable GATA3 or c-maf expression, suggesting that IL-4 might be regulated distinctly in these cells.²⁹ Thus new paradigms are emerging that basophils play a key role in directing T_H2 responses, and our data might provide additional support for this observation. Preliminary studies using flow cytometry revealed that basophils (CD3⁻CD123⁺CD203⁺HLA-DR^dimIL-4⁺, data not shown) are present in the CD25 preparation. Natural killer T cells can also produce significant IL-4, although the role of these cells in allergic sensitization or disease is controversial.

We cannot rule out the possibility that the frequency of T_H2 effector subsets is so low in the peripheral blood that as a fraction of total CD25⁺ cells enriched by means of selection, the overall effect on GATA3 transcript number on the subsequently isolated mRNA is too small to be detected, whereas the activation-induced change in IL4 in those same few cells is detectable. There were not sufficient cell numbers to both phenotype the CD25⁺ fraction and perform RT-PCR in our initial protocol, and therefore we cannot draw clear conclusions regarding the potential role of non-T cells. We are currently investigating the potential role for these populations in the observed in vitro IL-4 response. It will also be interesting to observe in this cohort whether a T-cell signature of T_H2 polarization develops over time and whether the early IL-4 response, potentially from other cells, proves to be important for priming clinical allergy. If so, this would corroborate findings in animal models and suggest new targets for primary prevention of disease.

We observed several differences in gene expression in response to egg stimulation compared with stimulation with milk or peanut. CD25 expression was significantly but only slightly increased for those with egg allergy, but IL4 expression was not increased. The observed increase in CD25 expression after egg stimulation in vitro was marginal and did not reach the magnitude of significance observed for IL4 expression after in vitro stimulation with milk or peanut. The explanation for the difference in response to egg compared with milk and peanut (eg, lack of IL4 expression increase) remains uncertain but might be related to particular proteins used in the stimulation experiments. For example, the antigens used for milk stimulation were comprised of caseins, the major milk allergens, whereas the egg stimulation was performed with whole egg protein extracts. In future studies we will explore whether enhancement with specific egg proteins (eg, Gal d 1) results in different patterns of response.

In addition to the observed immunologic characteristics, several key clinically relevant observations were made. Although children with known peanut allergy or previously known increased (>5 kU_A/L) peanut-specific IgE levels were not eligible for enrollment, there was a strikingly high rate of peanut sensitization and likely peanut allergy: 69% of the cohort were sensitized and 28% of 503 with IgE quantization had IgE concentrations to peanut of greater than 5 kU_A/L, which we estimate is likely associated with clinical peanut allergy (as described in the this article's Online Repository). Although there are no prior studies with precisely our enrollment criteria, of a large international study of infants with active AD in which 68% had moderate-to-severe eczema, 24% were sensitized to peanut.¹⁵ Our observation indicates that infants presenting with likely milk/egg allergy without already known peanut allergy are at high risk of also having peanut sensitization and therefore possible peanut allergy. A recent report concerning the young age of presentation of children with peanut allergy supports the concern that young children with egg or milk allergy or eczematous children are at high risk for exhibiting peanut allergy on their first known ingestion.³⁴

We evaluated the relative sensitivity of SPTs and serum IgE measurements in detecting sensitization (not clinical allergy) in this cohort because there are currently very limited data about these relationships in young infants/children with potential food allergy. We found a strong relationship between SPT wheal size and serum IgE concentration for the 3 foods. As indicated in Table II and Fig E1, when tests were discordant for peanut, the serum IgE test was more sensitive than the skin tests in detecting peanut sensitization (ie, 15% of those tested had detectable peanut-specific serum IgE levels but negative peanut SPT responses compared with 8% who had undetectable peanut IgE levels and a positive peanut SPT response, P < .001), which is in contrast to general conceptions that SPTs are more sensitive than serum tests. The reason for this observation could include our use of a more sensitive serum assay than previous-generation tests,³⁵ reduced skin test reactivity in infants,³⁶ or other factors, such as differences in the proteins being tested in the 2 methods. These findings are important when selecting tests to evaluate infants for possible food hypersensitivity and substantially dispel the long-held notion that skin testing is uniformly more sensitive (more likely to be positive) than serologic testing, especially in younger children.³⁷ Unlike the test results for peanut, those for milk and egg must be interpreted with caution and might be biased because a positive SPT response to milk or egg was required for enrollment into the study. Conversely, we did not enroll infants with serum test results that were strongly positive (>5 kU_A/L) to peanut before their screening, and therefore the bias would have been against our finding the serum test to be a more sensitive indicator of sensitization. We emphasize that sensitization to peanut is not indicative of peanut allergy.³⁸ We are therefore not discussing clinical allergy outcomes but detection of IgE using these methods. As this cohort ages, we will determine whether increased IgE levels in the infants are associated with clinical reactions.

In summary, we have established a cohort of infants with likely egg allergy, milk allergy, or both who will be studied longitudinally for the resolution of egg and milk allergies or the development of peanut allergy. We have focused on relationships of concurrent immune determinants with clinical allergy to milk/egg and sensitization to the 3 most common early childhood food allergens: egg, milk, and peanut. Data about parallel SPTs and serum IgE tests reveal that these tests are highly correlated, but there are also potentially significant discrepancies between them. Mononuclear cell stimulation results indicate that IL4 expression is associated with allergy to milk and sensitization to milk/peanut in the absence of detectable induction of GATA3 in these cells. Lastly, we documented an alarmingly high rate of peanut sensitization (68.8%), including 26.6% with a significantly increased peanut-specific IgE level of greater than 5 kU_A/L. This observation might have clinical implications for pediatricians and allergists evaluating young children with evidence of milk allergy, egg allergy, or both more than mild AD and evidence of sensitization to milk or egg. The results underscore the need for caution in managing these infants' early diets. Specifically, children with these clinical features might represent those for whom testing for food allergy is appropriate before advancing the diet.

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We thank M. Plaut, MD, medical officer for the program, and J. Poyser. We thank the families who kindly participated. Finally, we thank Greer Laboratories (Lenoir, NC) and Phadia (Uppsala, Sweden) for providing reagents.

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Methods 

Definition of atopic diseases 

Atopic disease history in parents and siblings of enrolled infants was based on previously accepted definitions.^E1 A diagnosis of asthma was defined by a minimum of 3 reported episodes (history) of wheezing in addition to respiratory symptoms with response to β-agonists or signs of airway hyperreactivity (wheezing or severe coughing while exercising, cold weather, or disturbed coughing at night) without ongoing upper respiratory tract infection. AD required^E2, ^E3 pruritus and an eczematous rash (acute, subacute, or chronic) with typical morphology and age-specific patterns and a chronic (≥3 weeks) or relapsing history (excluding scabies, seborrheic dermatitis, allergic contact dermatitis, ichthyoses, cutaneous lymphoma, psoriasis, and immune deficiency disease) and atopy (personal history, family history, or both or IgE reactivity) and xerosis. A family history of food allergy required typical symptoms, such as urticaria, angioedema, or asthma, directly after consumption of the food.

For enrolled infants, asthma is graded by severity according to National Heart, Lung, and Blood Institute guidelines, and AD severity is graded by criteria previously described and published by Rajka and Langeland.^E3 Briefly, the AD severity is graded as mild, moderate, or severe by using the following parameters to compute a score summation: (1) extent of disease (by “rule of 9”), (2) course of disease (by history), and (3) intensity of disease (disturbance of night's sleep by itching), each on a 3-point scale. Summation scores of 3 to 4 indicate mild disease, scores of 5 to 7 indicate moderate disease, and scores of 8 to 9 indicate severe AD. A historical grading of AD severity before dietary manipulation (change of formula or maternal exclusion of milk or egg) was allowed to avoid exclusion of milk- or egg-sensitized children who experienced improvement of AD by ongoing milk or egg restriction before consideration for enrollment.

Categorization of food allergy 

Because this is an observational study, repeated diagnostic oral food challenges could not be imposed on infants at enrollment. Therefore the following categorization scheme was developed and designed for longitudinal use.

At enrollment (and for the longitudinal course of the study), we define food allergy according to clinical history and test results, as well as by using oral food challenges when clinically indicated. A clinical history was considered convincing when there were symptoms within an hour of isolated ingestion that included at least urticaria and/or angioedema, difficulty breathing, wheezing, throat tightness, and/or vomiting. Brief ingestion of cow's milk protein formula during the newborn period does not qualify as evidence of tolerance.

Based on available studies for children younger than 2 years, we considered food-specific IgE levels to have diagnostic accuracy of greater than 95% when they were equal to or greater than 5 kU_A/L for milk,^E4 2 kU_A/L for egg,^E5 and 5 kU_A/L for peanut (see below).

We developed a novel classification scheme to categorize each study subject into one of the following food allergy diagnostic categories based on the clinical history and standard IgE levels.

A positive physician-supervised oral food challenge result and sensitization to the food (food-specific IgE ≥0.35 kU_A/L, SPT ≥3 mm, or both), a convincing reaction plus a greater than 95% predictive food-specific IgE test result, or both.

A convincing history with sensitization demonstrated by a positive serologic test result (milk or egg IgE ≥0.35 kU_A/L but less than 95% predictive levels), a positive skin test result, or both or a history of flare of AD on ingestion of the food and a food-specific plasma IgE level greater than 95% of the predictive level.

A food-specific IgE test result that is greater than 95% predictive of a clinical reaction but no ingestion of the food. Based on available studies for children younger than 2 years, we considered food-specific IgE levels to have a diagnostic accuracy of greater than 95% when they were equal to or greater than 5 kU_A/L for milk,^E4 2 kU_A/L for egg,^E5 and 5 kU_A/L for peanut. The predictive value for peanut is derived. Oral food challenges are not typically performed to peanut in this age group, and therefore diagnostic properties of the test have not been determined in infants.^E6, ^E7 The predictive value of serum IgE for clinical reactions varies by age, with younger infants reacting at lower levels than school-aged children. For example, previous studies of egg and milk allergy in infants^E4, ^E5, ^E8 indicate that greater than 95% react at IgE levels to egg and milk that correspond to 50% reaction rates for 5- to 7-year-olds (eg, a level of 2 kU_A/L for egg and milk).^E7 In studies of children at mean ages of 5 to 7 years,^E6, ^E7, ^E9 a level of 5 kU_A/L to peanut is associated with a 70% to 90% clinical reaction rate. Based on these studies of peanut and studies on egg and milk showing that infants react at lower food-specific IgE levels than older children, we estimate that a peanut IgE level of 5 kU_A/L or greater in the infants in this study would indicate a high (>95%) likelihood of current clinical peanut allergy. Based on the above-referenced studies,^E6, ^E7, ^E9 coincident soy allergy affecting a small percentage of children with peanut allergy would not likely influence these predictive values.

No ingestion and an indeterminate positive test result, a convincing history but no sensitization, or a flare of AD and sensitization to the food (but the food-specific IgE level is not in the diagnostic range).

Detectable IgE antibody (sensitized, IgE ≥0.35 kU_A/L, or SPT ≥3 mm) but tolerates eating the food.

No evidence of IgE antibody to the food and food tolerant.

Negative test result and has not ingested the food (does not include exposure to allergen in breast milk).

A positive oral food challenge result but not sensitized (serum IgE < 0.35 kU_A/L and SPT response <3 mm).

As described in the text, we only evaluated mononuclear cell expression of key cytokine and regulatory genes for the clinical end points associated with allergy (confirmed/convincing) or no allergy categories.

Skin tests 

A positive SPT response is defined by a mean wheal diameter of 3 mm or greater after subtraction of the saline control. Tests were considered reliable if the wheal size of the histamine control was at least 3 mm larger than the wheal size elicited by the negative control. All sites used the same lot of reagents, and training was performed to ensure consistency. The following extracts were used (Greer Laboratories catalog no. in parentheses): cow's milk (F293), chicken egg white (F272), and peanut (F171). For additional allergic characterization, SPTs were performed with environmental allergens: standardized cat (TE3), dog epithelia (Canis familiaris, mixed breed; E7), Dermatophagoides pteronyssinus (B70), Dermatophagoides farinae (B64), mold mix no. 1 (Alternaria, Aspergillus, Helminthosporium, Cladosporium, and Penicillium species; MO1), and cockroach mix (American German; B012). Skin tests to a specific agent could be deferred if the infant experienced an unequivocal recent episode of anaphylaxis to the substance.

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Results 

Additional demographic features 

The number of children enrolled at each site were as follows: Denver, Colorado (n = 99); Durham, NC (n = 103); Baltimore, Maryland (n = 109); New York, New York (n = 107); and Little Rock, Arkansas (n = 94). Of the mothers, 79.1% had a college degree or higher, and among fathers, this was 73.0%. Parental atopic disease was reported among 67.4% of mothers and 59.8% of fathers, and 43.6% were families with biparental atopy. The mean birth weight of participants was 3.42 kg (range, 1.16-4.77 kg). Full-term pregnancy (>37 weeks) was reported for 92.4% of the participants, and 13 infants were born prematurely at less than 34 weeks. Pets in the household included dogs (24.8% of households) or cats (13.3%), and 3.1% had both types. Asthma was diagnosed in 28 (5.5%) of the entrants and when present was mild-intermittent in 19, mild-persistent in 8, and moderate-persistent in 1. By parental report, 15.2% of the infants had experienced bronchiolitis. The sites were geographically diverse, and distributions of race, ethnicity, household income, parental education, AD severity, and pets in the home, but not sex, differed significantly (P < .001) by site. For some variables, the range of site-specific characteristics was substantial; for example, the white race rate ranged from 57.5% to 86.0%, incomes of greater than $100,000 from 13.8% to 60.8%, and pets in the home from 21.5% to 50.5%. Table E1 shows the categories of allergy, as described above. Table E2 shows sensitization characteristics.

Additional results and quality control standards for PCR 

Table E3 shows the mean ΔCt and percentage of undetectable stimulations. Overall, 96.8% of the PCRs passed laboratory quality standards, whereas 12.5% of the assays failed to detect the targeted gene (5% for anti-CD3/anti-CD28 and 22% for medium). The 5 clinic sites demonstrated the ability to consistently prepare samples with quality success rates ranging from 95.3% to 97.4% and nondetection rates ranging from 10.7% to 16.1%. As expected, CD25 upregulation after positive control (anti-CD3/anti-CD28) and tetanus toxoid was readily detectable by means of PCR. Transcriptional changes were also evident on allergen stimulation for several target genes in comparison with medium alone. To show quality control, Fig E2 displays a high correlation of housekeeping genes, and Fig E3 shows typical consistent results across study sites for a representative stimulation (peanut-stimulated IL4 gene).

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Fig E1. 

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• Relationship of skin test wheal sizes (in millimeters) to serum IgE antibody levels (in kilounits of antibody per liter) for milk (A), egg (B), and peanut (C) shown on a logarithmic scale. Spearman correlation coefficients are 0.64 for milk and 0.65 for egg and peanut. +, Those with confirmed/convincing milk/egg allergy; Spearman correlation coefficients are 0.47 for milk and 0.48 for egg (P < .001 for this subgroup). SPT responses were recorded as negative if the saline control wheal was larger than the food test wheal.

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Fig E2. 

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• Reproducibility of replicate PCR: correlation of 2 housekeeping gene's threshold cycle (CT) numbers from 2,708 PCR assays using 5 stimulation conditions. Spearman correlation coefficient is 0.84.

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Fig E3. 

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• Reproducibility across study sites: the 5 participating sites (DC, National Jewish Health, Denver, Colo; DU, Duke, Durham, NC; JH, Johns Hopkins, Baltimore, Md; MS, Mount Sinai, New York, NY; and UA, University of Arkansas, Little Rock, Ark) prepared stimulated lymphocytes that were shipped to a central laboratory for quantitative PCR analysis. The site-specific results for the peanut-stimulated IL4 gene ΔΔCt are shown (n ranges from 77-91 per site; there was no significant difference between sites for the detection of IL4, P = .32).

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Table E1. 

Demographic characteristics of the study participants (n = 512)

	No.	Percent
Male sex	345	67.4
Age (mo) at enrollment
3-5	65	12.7
6-8	128	25.0
9-11	182	35.5
12-14	137	26.8
Race
White	378	73.8
Black/African American	79	15.4
Asian	40	7.8
Other	15	2.9
Ethnicity
Hispanic or Latino	36	7.0
Household income
$0-$49,999	86	16.8
$50,000-$99,999	135	26.4
>$100,000	215	42.0
Declined	76	14.8
Caesarian section delivery	178	34.8
Breast-fed
Never	73	14.3
Yes	439	85.7
AD severity
None	40	7.8
Mild	51	10.0
Moderate	258	50.4
Severe	163	31.8

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Table E2. 

Sensitization rates (n = 503)

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Table E3. 

Raw data showing mean ΔCt by stimulation and percentage undetectable

CISH, Cytokine-inducible SH2-containing protein; FOXP3, forkhead box protein 3; RPL41, 60S ribosomal protein L41; RPS9, 40S ribosomal protein S9.

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