Soheila J. Maleki

Early consumption of peanuts in infancy is associated with a low prevalence of peanut allergy

BACKGROUND Despite guidelines recommending avoidance of peanuts during infancy in the United Kingdom (UK), Australia, and, until recently, North America, peanut allergy (PA) continues to increase in these countries. OBJECTIVE We sought to determine the prevalence of PA among Israeli and UK Jewish children and evaluate the relationship of PA to infant and maternal peanut consumption. METHODS A clinically validated questionnaire determined the prevalence of PA among Jewish schoolchildren (5171 in the UK and 5615 in Israel). A second validated questionnaire assessed peanut consumption and weaning in Jewish infants (77 in the UK and 99 in Israel). RESULTS The prevalence of PA in the UK was 1.85%, and the prevalence in Israel was 0.17% (P < .001). Despite accounting for atopy, the adjusted risk ratio for PA between countries was 9.8 (95% CI, 3.1-30.5) in primary school children. Peanut is introduced earlier and is eaten more frequently and in larger quantities in Israel than in the UK. The median monthly consumption of peanut in Israeli infants aged 8 to 14 months is 7.1 g of peanut protein, and it is 0 g in the UK (P < .001). The median number of times peanut is eaten per month was 8 in Israel and 0 in the UK (P < .0001). CONCLUSIONS We demonstrate that Jewish children in the UK have a prevalence of PA that is 10-fold higher than that of Jewish children in Israel. This difference is not accounted for by differences in atopy, social class, genetic background, or peanut allergenicity. Israeli infants consume peanut in high quantities in the first year of life, whereas UK infants avoid peanuts. These findings raise the question of whether early introduction of peanut during infancy, rather than avoidance, will prevent the development of PA.

Structure of the Major Peanut Allergen Ara h 1 May Protect IgE-Binding Epitopes from Degradation

In the past decade, there has been an increase in allergic reactions to peanut proteins, sometimes resulting in fatal anaphylaxis. The development of improved methods for diagnosis and treatment of peanut allergies requires a better understanding of the structure of the allergens. Ara h 1, a major peanut allergen belonging to the vicilin family of seed storage proteins, is recognized by serum IgE from >90% of peanut-allergic patients. In this communication, Ara h 1 was shown to form a highly stable homotrimer. Hydrophobic interactions were determined to be the main molecular force holding monomers together. A molecular model of the Ara h 1 trimer was constructed to view the stabilizing hydrophobic residues in the three dimensional structure. Hydrophobic amino acids that contribute to trimer formation are at the distal ends of the three dimensional structure where monomer-monomer contacts occur. Coincidentally, the majority of the IgE-binding epitopes are also located in this region, suggesting that they may be protected from digestion by the monomer-monomer contacts. On incubation of Ara h 1 with digestive enzymes, various protease-resistant fragments containing IgE-binding sites were identified. The highly stable nature of the Ara h 1 trimer, the presence of digestion resistant fragments, and the strategic location of the IgE-binding epitopes indicate that the quaternary structure of a protein may play a significant role in overall allergenicity.

Characterization of lymphocyte responses to peanuts in normal children, peanut-allergic children, and allergic children who acquired tolerance to peanuts

Comparing lymphocyte responses to allergenic and nonallergenic foods could reveal the differences between pathogenic and normal immune responses to foods. Defining the cytokine-producing phenotypes of peanut-specific lymphocytes from peanut-allergic children, children who outgrew peanut allergy, and children who have always tolerated peanuts may be useful for understanding the mechanisms of food tolerance. Investigating immune responses against foods is hindered, however, by the fact that circulating food antigen-specific lymphocytes are very rare. In a novel approach we used carboxyfluorescein succinimidyl ester to detect peanut-specific lymphocytes by flow cytometry. We confirmed that these cells are indeed peanut specific by cloning. Peanut-allergic donors show Th2 polarization of cytokine production by peanut-specific cells (IFN-gamma (low), TNF-alpha (low), IL-4 (high), IL-5 (high), IL-13 (high)). Conversely, nonallergic children and children who have outgrown their allergy show Th1 skewing to peanut antigens (IFN-gamma(high), TNF-alpha (high), IL-4 (low), IL-5 (low), IL-13(low)), similarly to nonallergenic food antigens (beta-lactoglobulin, OVA). This finding suggests that peanut antigens do not intrinsically induce Th2 skewing, but that the type of response depends upon the donors allergic status. In conclusion, food allergic status is characterized by a Th2 response whereas Th1-skewed responses underlie oral tolerance.

Staphylococcus aureus AgrA Binding to the RNAIII-agr Regulatory Region

The control of virulence gene expression in the human pathogen Staphylococcus aureus is under the partial control of the two-component quorum-sensing system encoded by genes of the agr locus. The product of the agrA gene has been shown by amino acid sequence similarity to be the putative response regulator; however, binding of AgrA to promoters under its control has not yet been demonstrated. In this study, we isolated and purified soluble AgrA by expression under osmotic shock conditions and ion-exchange chromatography. Purified AgrA showed high-affinity binding to the RNAIII-agr intergenic region by electrophoretic mobility shift assays. Binding was localized by DNase I protection assays to a pair of direct repeats in the P2 and P3 promoter regions of the agr locus. We found that this binding was enhanced by the addition of the small phosphoryl donor, acetyl phosphate. The difference in binding affinity between these two promoters was found to result from a 2-bp difference between the downstream direct repeats of the P2 and P3 sites. Mutation of these base pairs in the P3 site to match those found in the P2 site increased the affinity of AgrA for the P3 site relative to that for the P2 site. These results are consistent with the function of AgrA as a response regulator with recognition sites in the promoter regions of RNAIII and the agr locus.

Processing Can Alter the Properties of Peanut Extract Preparations

As peanut allergy is an increasing public health risk, affecting over 1% of the United States and United Kingdom school children, it is important that methods and reagents for accurate diagnosis of food allergy and detection of allergenic foods are reliable and consistent. Given that most current experimental, diagnostic, and detection tests rely on the presence of soluble allergens in food extracts, we investigated the effects of thermal processing on the solubility and IgE binding of the major peanut allergens, Ara h 1 and Ara h 2. The soluble and insoluble fractions of peanuts that were boiled, fried, and roasted were subjected to electrophoresis and Western blot analysis using anti-Ara h 1 and anti-Ara h 2 antibodies and serum IgE from peanut allergic individuals. Overall protein solubility is reduced with processing and IgE binding increases in the insoluble fractions, due mostly to the increase in the amount of insoluble proteins, with increased time of heating in all processes tested. Therefore, it can be concluded that thermal processing of peanuts alters solubility, and the differences in protein solubility within various extract preparations may contribute to inconsistent skin prick test and immunoassay results, particularly when nonstandardized reagents are used.

Skin exposure promotes a Th2-dependent sensitization to peanut allergens

Sensitization to foods often occurs in infancy, without a known prior oral exposure, suggesting that alternative exposure routes contribute to food allergy. Here, we tested the hypothesis that peanut proteins activate innate immune pathways in the skin that promote sensitization. We exposed mice to peanut protein extract on undamaged areas of skin and observed that repeated topical exposure to peanut allergens led to sensitization and anaphylaxis upon rechallenge. In mice, this epicutaneous peanut exposure induced sensitization to the peanut components Ara h 1 and Ara h 2, which is also observed in human peanut allergy. Both crude peanut extract and Ara h 2 alone served as adjuvants, as both induced a bystander sensitization that was similar to that induced by the atopic dermatitis-associated staphylococcal enterotoxin B. In cultured human keratinocytes and in murine skin, peanut extract directly induced cytokine expression. Moreover, topical peanut extract application induced an alteration dependent on the IL-33 receptor ST2 in skin-draining DCs, resulting in Th2 cytokine production from T cells. Together, our data support the hypothesis that peanuts are allergenic due to inherent adjuvant activity and suggest that skin exposure to food allergens contributes to sensitization to foods in early life.

Structural and Immunologic Characterization of Ara h 1, a Major Peanut Allergen

Allergic reactions to peanuts and tree nuts are major causes of anaphylaxis in the United States. We compare different properties of natural and recombinant versions of Ara h 1, a major peanut allergen, through structural, immunologic, and bioinformatics analyses. Small angle x-ray scattering studies show that natural Ara h 1 forms higher molecular weight aggregates in solution. In contrast, the full-length recombinant protein is partially unfolded and exists as a monomer. The crystal structure of the Ara h 1 core (residues 170–586) shows that the central part of the allergen has a bicupin fold, which is in agreement with our bioinformatics analysis. In its crystalline state, the core region of Ara h 1 forms trimeric assemblies, while in solution the protein exists as higher molecular weight assemblies. This finding reveals that the residues forming the core region of the protein are sufficient for formation of Ara h 1 trimers and higher order oligomers. Natural and recombinant variants of proteins tested in in vitro gastric and duodenal digestion assays show that the natural protein is the most stable form, followed by the recombinant Ara h 1 core fragment and the full-length recombinant protein. Additionally, IgE binding studies reveal that the natural and recombinant allergens have different patterns of interaction with IgE antibodies. The molecular basis of cross-reactivity between vicilin allergens is also elucidated.

TILLING for allergen reduction and improvement of quality traits in peanut (Arachis hypogaea L.)

BackgroundAllergic reactions to peanuts (Arachis hypogaea L.) can cause severe symptoms and in some cases can be fatal, but avoidance is difficult due to the prevalence of peanut-derived products in processed foods. One strategy of reducing the allergenicity of peanuts is to alter or eliminate the allergenic proteins through mutagenesis. Other seed quality traits could be improved by altering biosynthetic enzyme activities. Targeting Induced Local Lesions in Genomes (TILLING), a reverse-genetics approach, was used to identify mutations affecting seed traits in peanut.ResultsTwo similar copies of a major allergen gene, Ara h 1, have been identified in tetraploid peanut, one in each subgenome. The same situation has been shown for major allergen Ara h 2. Due to the challenge of discriminating between homeologous genes in allotetraploid peanut, nested PCR was employed, in which both gene copies were amplified using unlabeled primers. This was followed by a second PCR using gene-specific labeled primers, heteroduplex formation, CEL1 nuclease digestion, and electrophoretic detection of labeled fragments. Using ethyl methanesulfonate (EMS) as a mutagen, a mutation frequency of 1 SNP/967 kb (3,420 M2 individuals screened) was observed. The most significant mutations identified were a disrupted start codon in Ara h 2.02 and a premature stop codon in Ara h 1.02. Homozygous individuals were recovered in succeeding generations for each of these mutations, and elimination of Ara h 2.02 protein was confirmed. Several Ara h 1 protein isoforms were eliminated or reduced according to 2D gel analyses. TILLING also was used to identify mutations in fatty acid desaturase AhFAD2 (also present in two copies), a gene which controls the ratio of oleic to linoleic acid in the seed. A frameshift mutation was identified, resulting in truncation and inactivation of AhFAD2B protein. A mutation in AhFAD2A was predicted to restore function to the normally inactive enzyme.ConclusionsThis work represents the first steps toward the goal of creating a peanut cultivar with reduced allergenicity. TILLING in peanut can be extended to virtually any gene, and could be used to modify other traits such as nutritional properties of the seed, as shown in this study.

Resolution and identification of major peanut allergens using a combination of fluorescence two-dimensional differential gel electrophoresis, Western blotting and Q-TOF mass spectrometry

Peanut allergy is triggered by several proteins known as allergens. In this study, the complexity the peanut allergome is investigated with proteomic tools. The strength of this investigation resides in combining the high-resolving power and reproducibility of fluorescence two-dimensional differential gel electrophoresis with specific immunological detection as well as polypeptide sequencing by high-resolution mass spectrometry. Matching of the peanut proteins in 2D gels was achieved by differential labelling whereby peanut proteins and purified allergens (Ara h 1, Ara h 2 or Ara h 3/4) were run on the same gel. Ten protein spots on a mass line of ca. 63-68 kDa were likely to correspond to Ara h 1. Two doublets on two different mass lines at ca. 16 and 18 kDa matched with purified allergen Ara h 2. The basic and acidic sub-units of Ara h 3/4 were observed at masses of ca. 25 kDa and 40-45 kDa, respectively. Subsequently the antibody-binding capacity of spots corresponding to peanut allergens was investigated by Western blotting of 2D gels using antibodies (IgY) raised against Ara h 1, Ara h 2 and the recombinant 40 kDa sub-unit of Ara h 3/4. Final confirmation of the identity of the protein spots matched after 2D electrophoresis and identified by Western blotting was obtained by in-gel digestion of protein spots and analysis by quadrupole time-of-flight mass spectrometry. By using the method developed in our work, the location and identification of two different isoforms of the allergen Ara h 1, the allergen Ara h 2 and six isoforms of the allergen Ara h 3/4 in 2D peanut protein maps was established.

Reduction of IgE Binding and Nonpromotion of Aspergillus flavus Fungal Growth by Simultaneously Silencing Ara h 2 and Ara h 6 in Peanut

The most potent peanut allergens, Ara h 2 and Ara h 6, were silenced in transgenic plants by RNA interference. Three independent transgenic lines were recovered after microprojectile bombardment, of which two contained single, integrated copies of the transgene. The third line contained multiple copies of the transgene. Ara h 2 expression was significantly suppressed in all three lines, whereas Ara h 6 was reduced in two lines. Expression of peanut allergens Ara h 1 and Ara h 3 was not noticeably affected. Significant reduction of human IgE binding to Ara h 2 and Ara h 6 also was observed. Seed weight and germination data from transgenic and nontransgenic segregants showed no significant differences. Data collected from in vitro Aspergillus flavus infection indicate no significant difference in fungal growth between the transgenic lines and the nontransgenic controls. These data suggest that silencing Ara h 2 and Ara h 6 is a feasible approach to produce hypoallergenic peanut.