Single-tube nested real-time PCR versus normalised real-time PCR for the quantification of allergenic cashew nut in foods: Impact of thermal processing and matrix.

In this work, three allergen-encoding genes (Ana o 1, Ana o 2, Ana o 3) were investigated for the detection of cashew nut as an allergenic food. Normalised and single-tube nested real-time PCR approaches targeting the Ana o 2 or Ana o 3 genes are proposed and compared. Normalised real-time PCR detected 10 pg, while single-tube nested real-time PCR achieved 1 pg of cashew nut DNA. Single-tube nested real-time PCR targeting Ana o 3 allowed the best relative sensitivities (10 mg/kg cashew nut in dough/biscuit), being successfully validated regarding precision/accuracy. The normalised real-time PCR did not show acceptable accuracy for both targets. Sensitivity of single-tube nested real-time PCR was affected by the matrix (pasta), but not by thermal processing (dough/biscuit). Herein, two highly sensitive and specific single-tube nested real-time PCR targeting allergen-encoding genes are proposed for the first time as quantitative/validated tools for cashew nut analysis as an allergenic food.

Ana o 1 and Ana o 2 cashew allergens share cross-reactive CD4(+) T cell epitopes with other tree nuts.

BACKGROUND: Allergies to cashew are increasing in prevalence, with clinical symptoms ranging from oral pruritus to fatal anaphylactic reaction. Yet, cashew-specific T cell epitopes and T cell cross-reactivity amongst cashew and other tree nut allergens in humans remain uncharacterized. OBJECTIVES: In this study, we characterized cashew-specific T cell responses in cashew-allergic subjects and examined cross-reactivity of these cashew-specific cells towards other tree nut allergens. METHODS: CD154 up-regulation assay was used to determine immunodominance hierarchy among cashew major allergens at the T cell level. The phenotype, magnitude and functionality of cashew-specific T cells were determined by utilizing ex vivo staining with MHC class II tetramers. Dual tetramer staining and proliferation experiments were used to determine cross-reactivity to other tree nuts. RESULTS: CD4(+) T cell responses were directed towards cashew allergens Ana o 1 and Ana o 2. Multiple Ana o 1 and Ana o 2 T cell epitopes were then identified. These epitopes elicited either TH 2 or TH 2/TH 17 responses in allergic subjects, which were either cashew unique epitope or cross-reactive epitopes. For clones that recognized the cross-reactive epitope, T cell clones responded robustly to cashew, hazelnut and/or pistachio but not to walnut. CONCLUSIONS: Phylogenetically diverse tree nut allergens can activate cashew-reactive T cells and elicit a TH 2-type response at an epitope-specific level. CLINICAL RELEVANCE: Lack of cross-reactivity between walnut and cashew suggests that cashew peptide immunotherapy approach may not be most effective for walnut.

Human leucocyte antigen polymorphisms in nut-allergic patients in South Wales.

BACKGROUND: Peanuts and tree nuts are among the most common foods provoking severe allergic reactions including fatal anaphylaxis. However, little is known of the underlying genetic and immunological mechanisms involved. OBJECTIVE: Based on findings in other allergic diseases, we have investigated whether specific human leucocyte antigens (HLA) are associated with nut allergy. METHOD: Eighty-four patients presenting at the allergy clinic with symptoms of nut allergy were typed for the HLA Class I (HLA-A and B) and Class II (HLA-DRB1 and DQB1) loci by PCR using sequence-specific primers. Carriage frequencies were compared with 82 atopic non-nut-allergic subjects and 1798 random blood donors. RESULTS: The frequency of HLA-B(*)07 (28.57%) and DRB1(*)11 (15.48%) was increased in the nut-allergic patients compared to the atopic controls (12.20% and 3.66%, respectively) but not when compared to the blood donors (28.86% and 10.12%). DRB1(*)13 and DQB1(*)06 were both increased in frequency in the nut allergy patients over both the atopic and blood donor controls. However, none of these increased frequencies were significant when corrected for the number of comparisons undertaken. CONCLUSION: At HLA '2-digit resolution' and with undifferentiated patients with nut allergy, there are no major disturbances in the frequency of HLA-A, B, DRB1 or DQB1 types. However, the difference in frequency of HLA-DRB1(*)11 between the nut allergy patients and the atopic controls merits further investigation as this may represent an important phenotypic relationship.

Polymorphism in the STAT6 gene encodes risk for nut allergy.

Nut allergy is an important and potentially life threatening food allergy with a prevalence of one in 150 children in the UK population. STAT6 (signal transducer and activator of transcription) is an important molecule in the induction and regulation of an allergic response, which maps to chromosome 12q in a region previously linked with total serum IgE concentration and atopy in different populations. We have examined the frequency of a single nucleotide polymorphism (SNP) in the 3'UTR region of STAT6 gene in 71 UK Caucasoid patients diagnosed with nut allergy and 45 atopic patients without nut allergy using PCR-RFLP and compared these with 184 UK healthy controls. The STAT6 G allele frequency was significantly increased in nut allergy patients compared with blood donor controls (P < 0.0001, OR = 2.9, 95% CI: 1.7-4.9), which was under a recessive model (GG vs GA+AA, P = 0.0001, OR = 3.2, 95% CI: 1.7-5.8) but not in atopic patients without nut allergy. The G allele was most frequent in the severe cases and GG homozygosity was associated with the increased risk of severe reaction (OR = 3.9, 95% CI: 1.9-8.3). We conclude that STAT6 3'UTR polymorphism is associated with susceptibility and severity in nut allergic patients in our population.