Crude Peanut Extract Research Articles

Formulation and in-vitro characterization of fast-disintegrating herbal extract sublingual immunotherapy tablet for peanut-induced allergic asthma

BackgroundAllergen immunotherapy (AIT) involves the regimen of gradually incrementing doses of the allergen, thereby inducing desensitization and tolerance. Sublingual Immunotherapy tablets (SLIT-tablets) have been formulated for several allergies and had manifested efficacy for allergic rhinitis and allergic asthma. SLIT promises an alternative method to other routes of AIT enabling patients to self-administer AIT. ObjectiveThe study aimed to formulate fast disintegrating SLIT containing crude peanut extract for peanut-induced allergic asthma. MethodsThe crude peanut extract was prepared by a simple extraction method and was subjected to quantitative and qualitative analysis. The extract was also characterised for its physical properties. The preformulation study for the extract and excipients of the tablet was performed using FT-IR spectroscopy and Differential scanning calorimetry. The tablet powder blends were characterised for pre-compression properties. The SLIT tablets were developed by direct compression and the post-compression evaluation was performed. ResultsThe results of the quantitative and qualitative analysis of extract confirmed the presence of peanut proteins in the extract. The preformulation studies using FT-IR spectroscopy and Differential Scanning Calorimetry revealed that there is no significant interaction between the CPE and excipients. The pre-compression characterisation showed that the powder blends had good flowproperties. Three doses of SLIT tablets were formulated with each dose containing four batches and the tablet of each dose was optimized by studying the effect of varying concentrations of super disintegrants on disintegration time and dissolution rate. The post compression characterization of the tablets was performed and the optimized batch of the three doses with the concentration of 5% crospovidone and 2% croscarmellose sodium showed less wetting time and high-water absorption ratio, shorter disintegration time of 14secs and maximum drug release of >90% within 2–3 min. ConclusionThe results indicated the suitability of formulated SLIT tablets for peanut induced allergic asthma.

Fructo-Oligosaccharides Modify Human DC Maturation and Peanut-Induced Autologous T-Cell Response of Allergic Patients In Vitro

BackgroundDendritic cells (DCs) play an important role in antigen presentation, and are an interesting target for immune-modulation in allergies. Short- and long-chain fructo-oligosaccharides (scFOS/lcFOS, FF) have immunomodulatory capacities, and may influence the outcome of DC antigen presentation.ObjectiveThis study investigated the effect of FF during DC maturation and allergen presentation using cells of peanut-allergic patients in an autologous DC-T cell assay.MethodsCD14+ and CD4+ T cells were isolated from peanut-allergic patients. CD14+ monocytes were differentiated into immature DCs (imDCs), and matured (matDCs) in the presence or absence of crude peanut-extract (CPE) and/or FF, and co-cultured in an autologous DC-T cell assay. T cell polarization, proliferation and cytokine production were measured.ResultsExpression of maturation surface molecule markers on matDCs was not affected by CPE and/or FF. By contrast, the IL-10 secretion by matDCs increased compared to imDCs, upon exposure to CPE and FF compared to CPE alone. Also the IP-10 secretion increased in CPE/FF-matDCs compared to imDC. CPE-matDCs enhanced IL-13 release in the DC-T-cell assay and Treg polarization in presence or absence of FF. CPE/FF-DCs tended to increase the Treg/Th1 and Treg/Th2 ratios compared to matDCs. The proliferation of both Treg and Th2 cells tended to increase when T cells were co-cultured with CPE-matDCs compared to matDCs, which became significant when CPE-matDCs were also exposed to FF and a same tendency was shown for Th1 proliferation.ConclusionOnly in the presence of FF, CPE-matDCs produced increased regulatory and Th1-related mediators. CPE-matDCs modified T cell polarization and proliferation, and additional exposure to FF tended to enhance Treg/Th2 and Treg/Th1 ratios instructed by CPE/FF-matDCs. However this effect was not strong enough to suppress CPE-matDCs induced IL-13 release by Th-cells. This indicates the ability of FF to modify DC maturation in the presence of an allergen supporting a more Treg/Th1 prone direction of the successive allergen specific Th2 cell response.

Moringa leaf extract potential in the incidence of allergy in experimental rats

The incidence of food allergy out break worldwide is becoming a public health concern. Food allergy is growing at an epidemic proportion affecting all age groups of human population especially such occurrence in young children is worrisome. Management of food allergy without side effect is still a challenge to the medical community. The objective of this study was to investigate the effectiveness of moringa leaf extract in the treatment of food allergy without side effects in experimental rats. Sixty (60) Wistar male rats, (4-6) weeks old, weighing between (100-120) g were grouped into five (5) sections. They were grouped under: Positive Control group, Negative Control group, Epinephrine control group, Chemotherapeutic and Chemo-preventive groups. The animals were allowed to acclimatize for two weeks where the rats were given free food ration and distilled water. After 15 days of consecutive administration of 1 ml crude peanut extract and 10µlcholera toxin as a potent adjuvant in rats, made food allergy established. Results from the study indicated that the means of rectal temperatures after 20th day of allergy induction were declined from (37.17- 34.47) ?C, (37.0-34.67) ?C, and (37.52-3542) ?C in Positive control group, Chemopreventive and Chemotherapeutic groups respectively. The reductions in rectal temperatures confirmed that food allergy was established in experimental rats. Also the study showed that moringa leaf extract reduced serum Immunoglobulin E levels from (324.50-275.4)ng/ml, (181.20-170.50)ng/ml, (279.50-44.38)ng/ml respectively under Epinephrine treatment, Chemopreventive and Chemotherapeutic experimental group treatments. This suggested that moringa leaf extract had potential to prevent and control food allergy.

066 Skin-targeted delivery of crude peanut extracts using dissolving microneedle arrays

Peanut allergy is a common IgE-mediated hypersensitivity that has no curative option. Allergen-specific immunotherapy (ASIT) has recently emerged as a promising desensitization approach for several hypersensitivities. True potential of ASIT for peanut allergy has yet to be achieved due to the concerns of efficacy, safety, cost, and convenience of prevailing investigational ASIT methods. Skin-targeted ASIT offers an attractive solution to treatment of peanut allergy since the skin is an easily accessible organ that contains high-density antigen-presenting cells (APCs) and immune-accessory cells. However, it is very challenging to strategically and consistently deliver hydrophilic macromolecule peanut allergens to immune-cell-rich skin microenvironments due to the physical barrier imposed by stratum corneum. Here, we described skin-targeted delivery of crude peanut extracts (CPEs) using dissolving microneedle array(MNA)s toward a safe and effective cutaneous immunotherapy for peanut allergy. Dissolving MNAs integrating CPEs as an alternative to single peanut protein antigens were reproducibly manufactured from a biomaterial composition of Carboxymethlycellulose and Trehalose which are considered safe by FDA. The fabricated MNAs consisted of high-fidelity micron-scale needles incorporating CPEs at their tips that maintained their protein integrity. These MNAs successfully and reproducibly penetrated the skin in mice in vivo and humans ex vivo, dissolved upon penetration, and delivered their biocargoes to APC-rich cutaneous microenvironments. The MNA-delivered CPEs resided in the mouse skin microenvironments for at least 8 hrs in vivo and were present in the skin-draining lymph nodes 48 hr after application. Together, these studies suggest that MNAs could obviate limitations of conventional topical transdermal patches and imprecise hypodermic needle injections to enable the development of an effective cutaneous immunotherapy for peanut allergy.

Expression of a codon-optimised recombinant Ara h 2.02 peanut allergen in Escherichia coli

Current diagnostic tools for peanut allergy using crude peanut extract showed low predictive value and reduced specificity for detection of peanut allergen-specific immunoglobulin E (IgE). The Ara h 2.02, an isoform of the major peanut allergen Ara h 2, contains three IgE epitope recognition sequence of 'DPYSPS' and may be a better reagent for component resolve diagnosis. This research aimed to generate a codon-optimised Ara h 2.02 gene for heterologous expression in Escherichia coli and allergenicity study of this recombinant protein. The codon-optimised gene was generated by PCR using overlapping primers and cloned into the pET-28a (+) expression vector. Moderate expression of a 22.5 kDa 6xhistidine-tagged recombinant Ara h 2.02 protein (6xHis-rAra h 2.02) in BL21 (DE3) host cells was observed upon induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The insoluble recombinant protein was purified under denaturing condition using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography and refolded by dialysis in decreasing urea concentration, amounting to a yield of 74 mg/l of expression culture. Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) and immunoblot analysis confirmed the production of the recombinant 6xHis-rAra h 2.02. The refolded recombinant 6xHis-rAra h 2.02, with or without adjuvant, was able to elicit comparable level of allergen-specific IgE and IgG1 in sensitised Balb/c mice. In addition, the specific IgE antibodies raised against the recombinant protein were able to recognise the native Ara h 2 protein, demonstrating its allergenicity and potential as a reagent for diagnosis and therapeutic study.

Standardization of RP-HPLC methods for the detection of the major peanut allergens Ara h 1, Ara h 2 and Ara h 3

Crude peanut extract (CPE) was analyzed for three major allergens (Ara h 1, h 2, and h 3) using a C12 and a C18 column at two wavelengths (280 and 220nm) and under different solvent conditions. HPLC profiles were compared for retention time, resolution, and peak heights. CPE samples were spiked with pure allergens to identify the peaks corresponding to allergens. The HPLC fractions of corresponding allergens were collected and freeze-dried in order to perform SDS–PAGE and immunoblotting tests. The best method was identified the one with a shorter retention time, better resolution, and greater peak height as compared with the other methods. In general, the peak heights were greater at 220nm than at 280nm. The major disadvantage of the C12 column was the need for two sets of conditions to identify the allergens as compared to the C18 column where all three allergens could be identified in one run.

Effect of chemical modifications on allergenic potency of peanut proteins.

Modification of native peanut extracts could reduce adverse effects of peanut immunotherapy. We sought to compare native and chemically modified crude peanut extract (CPE) and major peanut allergens Ara h 2 and Ara h 6 in a mediator-release assay based on the rat basophilic leukemia (RBL) cell line transfected with human Fcε receptor. Native Ara h 2/6 was reduced and alkylated (RA), with or without additional glutaraldehyde treatment (RAGA). CPE was reduced and alkylated. Sera of subjects with peanut allergy (16 males; median age 7 years) were used for overnight RBL-passive sensitization. Cells were stimulated with 0.1 pg/mL to 10 μg/mL of peanut. β-N-acetylhexosaminidase release (NHR) was used as a marker of RBL degranulation, expressed as a percentage of total degranulation caused by Triton X. Median peanut-specific immunoglobulin E was 233 kUA/L. Nineteen subjects were responders, NHR ≥ 10% in the mediator release assay. Responders had reduced NHR by RA and RAGA compared with the native Ara h 2/6. Modification resulted in a later onset of activation by 10- to 100-fold in concentration and a lowering of the maximum release. Modified RA-Ara h 2/6 and RAGA-Ara h 2/6 caused significantly lower maximum mediator release than native Ara h 2/6, at protein concentrations 0.1, 1, and 10 ng/mL (p < 0.001, < 0.001, and < 0.001, respectively, for RA; and < 0.001, 0.026, and 0.041, respectively, for RAGA). RA-CPE caused significantly lower maximum NHR than native CPE, at protein concentration 1 ng/mL (p < 0.001) and 10 ng/mL (p < 0.002). Responders had high rAra h 2 immunoglobulin E (mean, 61.1 kUA/L; p < 0.001) and higher NHR in mediator release assay to native Ara h 2/6 than CPE, which indicates that Ara h 2/6 were the most relevant peanut allergens in these responders. Chemical modification of purified native Ara h 2 and Ara h 6 reduced mediator release in an in vitro assay ∼100-fold, which indicates decreased allergenicity for further development of the alternative candidate for safe peanut immunotherapy.

444. One-Time Gene Therapy to Prevent Peanut-Induced Anaphylaxis

Peanut allergy is a major public health problem that accounts for the majority of fatal food-induced anaphylactic reactions. There are no curative or preventative therapies. Therapy with omalizumab, a human anti-IgE monoclonal antibody, is transiently effective, but it must be administered parenterally, has a short half-life and is costly, thus it is challenging to use as a preventative for peanut allergy. To circumvent this challenge, we hypothesized that a one-time administration of an adeno-associated virus (AAV) gene transfer vector expressing the genetic sequence of omalizumab would provide persistent high circulating levels of anti-IgE sufficient to protect against repeated peanut exposure in the peanut allergic host. To assess this hypothesis, we created a humanized murine model of peanut allergy by reconstituting NOD-scid IL2rgnull mice with 3 x107 blood mononuclear cells (PBMC) derived from peanut allergic individuals or non-allergic controls and treating the mice with a non-human primate serotype rh.10 AAV expressing omalizumab (AAVrh.10anti-IgE) or control vector. Administration of a single dose (1×1011 gc) of AAVrh.10anti-IgE resulted in persistent high levels of anti-IgE in both wild type and humanized mouse models, with anti-IgE levels > 400 mg/ml at 6 wk and remained at similar levels to the last time point evaluated at 24 wk in wild type mice and levels above 220 mg/ml were achieved in NSG mice at wk 8. No anti-IgE was detected in serum from mice that received a non-relevant control vector or PBS alone. The humanized NOD-scid IL2rgnull mice were sensitized intraperitoneally with crude peanut extract (CPE) weekly for 4 wk and then challenged with CPE via oral gavage. Total serum human IgG, IgE and peanut-specific IgE were measured weekly. After challenge, allergic phenotype was assessed using an anaphylaxis score, histamine levels, rectal temperature and behavior. All NSG mice were successfully reconstituted with mononuclear cells as measured by persistent levels of human IgG. Mice treated with AAVrh.10anti-IgE had elevated total and peanut-specific IgE from day 28 onwards with total IgE lower in the AAVrh.10anti-IgE treated mice. Strikingly, after peanut challenge, AAVrh.10anti-IgE treated mice displayed a less severe allergic phenotype compared to those that received the control vector as defined by decreased histamine release, a lower anaphylaxis score, no drop in rectal temperature and suppressed ambulation. These results demonstrate that a single treatment with AAVrh.10anti-IgE provides long term protection from peanut allergen challenge in the peanut sensitized population, representing a paradigm shift in current therapeutic approaches.

Peanut allergy: a mouse model comparison (HYP4P.309)

Abstract Peanut induced anaphylaxis often results in a bi-phasic response, associated with a second anaphylaxis once current treatment interventions have been removed. Previous reports have suggested that this response is associated with elevated levels of platelet activating factor. Our studies have focused on refining the murine model of peanut anaphylaxis with the hypothesis that we can generate a model that recapitulates the anaphylactic response to peanuts. We used BALB/c and C3H/HeJ mice (N=20 for each study) mice were sensitized weekly for 6 weeks with crude peanut extract in cholera toxin. Mice were then grouped for crude peanut extract or saline challenge by intra-peritoneal injection. In all mice, temperatures and clinical scores were accessed both early post injection as well as 2 hours later. Serum, lungs, spleen, and liver were stored from all animals for future studies. The BALB/c strain had a mean temperature change from baseline of 0.2 C⁰ for the controls initial post IP versus the 1.89 C⁰ for the challenged mice. The C3H/HeJ strain had a mean temperature change from baseline of 0.1 C⁰ for the controls at the initial post IP versus the 4.2 C⁰ change for the challenged mice. Similar results were noted at the 2 hour mark with the C3H/HeJ strain with the greater temperature change. Both strains had notable changes in clinical scores between the control and challenge groups. This was more pronounced in the C3H/HeJ mice both clinically and by temperature measurement.

Characterization of peanut-reactive monoclonal antibodies (HYP7P.265)

Abstract Peanut allergies are a rising concern in Western society. Incidence and prevalence of affected children has tripled from 1997 to 2010. The exact cause for this increase is unknown, but lack of exposure to pathogens and increasing hygienic conditions has been implicated. Exposure to peanuts causes the immune system to become sensitized to peanut proteins. Subsequent exposure to peanuts causes hypersensitivity reactions which may escalate to anaphylaxis and death. The only course of prevention is complete avoidance. Limiting peanut exposure is difficult as even trace amounts of peanut (100µg-50mg; 1 peanut ≈ 1g) consumed can cause a reaction. Immunotherapies aim to increase the tolerance of these severely allergic individuals. Current approaches are limited to certain populations and are potentially dangerous. Better therapeutics require thorough characterization of peanut proteins responsible for peanut allergies. In this study, C3H/HeJ mice were sensitized to crude peanut extract (CPE). The mice were then exposed to CPE to induce a hypersensitive reaction. Hybridomas were generated , characterized for antibody production, subtype, and screened for peanut-specific immunoreactivity, as well as specific peanut proteins believed to be involved with peanut allergies. Characterization of peanut-specific monoclonal antibodies (MAb) may aid in the identification of immunodominant epitopes and lead to/aid in the development of immunotherapeutic interventions.

Memory B Cells Are Necessary for the Adoptive Transfer of Murine Peanut Allergy

We hypothesize that memory B cells are necessary and sufficient for the adoptive transfer of murine peanut allergy (PNA). Peanut-allergic donor C3H/HeJ mice (with similar anti-peanut IgE levels, symptom scores (SS) and temperature drops (dT) following intraperitoneal (IP) challenge with crude peanut extract (CPE)) were treated intravenously with either anti-CD20 or isotype control (IC) antibody for 18 weeks. Naïve, lethally irradiated recipients were given bone marrow (BM) and splenocytes (SPL) from either anti-CD20 or IC-treated donors. In a separate experiment, B cells (negatively selected, >96% pure) from SPL of peanut-allergic or naïve donors were injected into naïve, irradiated recipients with or without the addition of naive SPL. Sera were obtained from recipients on days 7 and 17 post-reconstitution, and recipients were challenged IP with CPE on days 8 and 18. Treatment of donor mice with anti-CD20 for 18 weeks significantly reduced B cell numbers in the blood (p<0.0001, >95% reduction) and the spleen (p<0.0001, >98% reduction) but did not affect IgE levels. Recipients (n=15) given BM and SPL from anti-CD20 treated donors were protected from developing anti-peanut IgE (p<0.0001), SS (p<0.0001), and dT (p<0.0001) compared to recipients (n=14) given cells from IC-treated donors. Recipients (n=9) given purified B cells from peanut-allergic donors plus naïve SPL developed significantly elevated anti-peanut IgE (p<0.001), SS (p<0.002), and dT (p<0.003) compared to controls. These results demonstrate that memory B cells are required but not sufficient for the adoptive transfer of murine PNA. Help from a yet-to-be-defined cell population(s) found in naïve SPL is required.

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.

Relationship of IgE to basophil phenotypes in peanut-sensitized adults

Relationship of IgE to basophil phenotypes in peanut-sensitized adults

Sublingual versus oral immunotherapy for peanut-allergic children: A retrospective comparison

Sublingual versus oral immunotherapy for peanut-allergic children: A retrospective comparison

In silico prediction of Ara h 2 T cell epitopes in peanut‐allergic children

Despite the frequency and severity of peanut allergy, the only approved treatment is strict avoidance. Different types of immunotherapy with crude peanut extract are not universally effective and have been associated with relatively high adverse reaction rates. We sought to determine whether in silico predictive algorithms were useful in identifying candidate peptides for an Ara h 2 peptide-based vaccine using peanut-allergic patients' peripheral blood mononuclear cells (PBMCs) in vitro. A human leucocyte antigen (HLA) distribution analysis was also performed. Major histocompatibility complex (MHC)-class II-binding peptides were predicted using NetMHCIIpan-2.0 and NetMHCII-2.2 algorithms. PBMCs from 80 peanut-allergic patients were stimulated with overlapping 20-mer Ara h 2 peptides. Cell supernatant cytokine profiles were evaluated by multiplex assays. HLA-DRB1* and HLA-DQB1* typing were performed. Four regions of overlapping sequences induced PBMC proliferation and predominant Th2 cytokine production. HLA genotyping showed 30 different DRB1* allele specificities and eight DQ serological specificities. The in silico analysis revealed similar relevant regions and predicted identical or similar core 9-mer epitopes to those identified in vitro. If relevant peptides, as determined by either in vitro or in silico analysis (15 peptides and 9 core epitopes respectively), were used in a peptide-based vaccine, they would cover virtually all subjects in the cohort studied. Four dominant regions in Ara h 2 have been identified, containing sequences that could serve as potential candidates for peptide-based immunotherapy. MHC-class II-based T cell epitope prediction algorithms for HLA-DR and -DQ loci accurately predicted Ara h 2 T cell epitopes in peanut-allergic subjects, suggesting their potential utility in a peptide-based vaccine design for food allergy.

Blockade of peanut allergy with a novel Ara h 2–Fcγ fusion protein in mice

Conventional immunotherapy for peanut allergy using crude peanut extracts is not recommended because of the unacceptably high risk of anaphylaxis. Allergen-specific immunotherapy is not currently undertaken for peanut allergy. The objective of this study was to develop a novel peanut-human fusion protein to block peanut-induced anaphylaxis. We genetically designed and expressed a novel plant-human fusion protein composed of the major peanut allergen Ara h 2 and human IgG Fcγ1. We tested the Ara h 2-Fcγ fusion protein (AHG2)'s function in purified human basophils. Transgenic mice expressing human FcεRIα and a murine peanut allergy model were used. AHG2 inhibited histamine release induced by whole peanut extract (WPE) from basophils of patients with peanut allergy, whereas the fusion protein itself did not induce mediator release. AHG2 inhibited the WPE-induced, peanut-specific, IgE-mediated passive cutaneous anaphylaxis in hFcεRIα transgenic mice. AHG2 also significantly inhibited acute anaphylactic reactivity, including the prototypical decrease in body temperature in WPE-sensitized mice challenged with crude peanut extract. Histologic evaluation of the airways showed that AHG2 decreased peanut-induced inflammation, whereas the fusion protein itself did not induce airway inflammation in peanut-sensitized mice. AHG2 did not exert an inhibitory effect in mice lacking FcγRII. AHG2 inhibited peanut-specific IgE-mediated allergic reactions in vitro and in vivo. Linking specific peanut allergen to Fcγ can provide a new approach for the allergen immunotherapy of peanut allergy.

Ara h 2 and Ara h 6 Have Similar Allergenic Activity and Are Substantially Redundant

Background: The moderately homologous (approx. 60%) proteins Ara h 2 and Ara h 6 are the most potent peanut allergens. This study was designed to define the relative individual contributions of Ara h 2 and Ara h 6 to the overall allergenic activity of a crude peanut extract (CPE). Methods: Ara h 2 and Ara h 6 were removed from CPE by gel filtration chromatography. Ara h 2.01, Ara h 2.02 and Ara h 6 were further purified (>99%). The potency of each allergen and the ability of these allergens to reconstitute the allergenic activity of CPE depleted of Ara h 2 and Ara h 6 was measured with RBL SX-38 cells sensitized with IgE from sensitized peanut allergic patients. Results: The potency of the native proteins were significantly different (p < 0.0001) although not dramatically so, with a rank order of Ara h 2.01 > Ara h 2.02 > Ara h 6. The addition of either purified Ara h 2 or Ara h 6 independently at their original concentration to CPE depleted of both Ara h 2 and Ara h 6 restored 80–100% of the original CPE allergenic activity. Addition of both Ara h 2 and Ara h 6 consistently completely restored the allergenic activity of CPE. Conclusions: These studies indicate that either Ara h 2 or Ara h 6 independently can account for most of the allergenic activity in a CPE and demonstrate important redundancy in the allergenic activity of these related molecules.

Redefining the major peanut allergens.

Food allergy has become a major public health concern in westernized countries, and allergic reactions to peanuts are particularly common and severe. Allergens are defined as antigens that elicit an IgE response, and most allergenic materials (e.g., pollens, danders, and foods) contain multiple allergenic proteins. This has led to the concept that there are "major" allergens and allergens of less importance. "Major allergens" have been defined as allergens that bind a large amount of IgE from the majority of patients and have biologic activity. However, the ability of an allergen to cross-link complexes of IgE and its high-affinity receptor FcεRI (IgE/FcεRI), which we have termed its allergic effector activity, does not correlate well with assays of IgE binding. To identify the proteins that are the most active allergens in peanuts, we and others have employed in vitro model assays of allergen-mediated cross-linking of IgE/FcεRI complexes and have demonstrated that the most potent allergens are not necessarily those that bind the most IgE. The importance of a specific allergen can be determined by measuring the allergic effector activity of that allergen following purification under non-denaturing conditions and by specifically removing the allergen from a complex allergenic extract either by chromatography or by specific immunodepletion. In our studies of peanut allergens, our laboratory has found that two related allergens, Ara h 2 and Ara h 6, together account for the majority of the effector activity in a crude peanut extract. Furthermore, murine studies demonstrated that Ara h 2 and Ara h 6 are not only the major elicitors of anaphylaxis in this system, but also can effectively desensitize peanut-allergic mice. As a result of these observations, we propose that the definition of a major allergen should be based on the potency of that allergen in assays of allergic effector activity and demonstration that removal of that allergen from an extract results in loss of potency. Using these criteria, Ara h 2 and Ara h 6 are the major peanut allergens.

IL-7Rα and L-selectin, but not CD103 or CD34, are required for murine peanut-induced anaphylaxis

BackgroundAllergy to peanuts results in severe anaphylactic responses in affected individuals, and has dramatic effects on society and public policy. Despite the health impacts of peanut-induced anaphylaxis (PIA), relatively little is known about immune mechanisms underlying the disease. Using a mouse model of PIA, we evaluated mice with deletions in four distinct immune molecules (IL7Rα, L-selectin, CD34, CD103), for perturbed responses.MethodsPIA was induced by intragastric sensitization with peanut antigen and cholera toxin adjuvant, followed by intraperitoneal challenge with crude peanut extract (CPE). Disease outcome was assessed by monitoring body temperature, clinical symptoms, and serum histamine levels. Resistant mice were evaluated for total and antigen specific serum IgE, as well as susceptibility to passive systemic anaphylaxis.ResultsPIA responses were dramatically reduced in IL7Rα−/− and L-selectin−/− mice, despite normal peanut-specific IgE production and susceptibility to passive systemic anaphylaxis. In contrast, CD34−/− and CD103−/− mice exhibited robust PIA responses, indistinguishable from wild type controls.ConclusionsLoss of L-selectin or IL7Rα function is sufficient to impair PIA, while CD34 or CD103 ablation has no effect on disease severity. More broadly, our findings suggest that future food allergy interventions should focus on disrupting sensitization to food allergens and limiting antigen-specific late-phase responses. Conversely, therapies targeting immune cell migration following antigen challenge are unlikely to have significant benefits, particularly considering the rapid kinetics of PIA.

The 2S albumin allergens of Arachis hypogaea, Ara h 2 and Ara h 6, are the major elicitors of anaphylaxis and can effectively desensitize peanut‐allergic mice

Ara h 2 and Ara h 6, co-purified together in a 13-25kD fraction (Ara h 2/6; 20kD fraction) on gel filtration chromatography, account for the majority of effector activity in a crude peanut extract (CPE) when assayed with RBL SX-38 cells sensitized with IgE from human peanut allergic sera. To determine if Ara h 2/6 are the primary peanut allergens responsible for allergic reactions in vivo and to determine if Ara h 2/6 would be sufficient to prevent allergic reactions to a complete CPE. An oral sensitization mouse model of peanut allergy was used to assess the activity of Ara h 2/6 (20kD) and CPE without the 20kD fraction (CPE w/o 20kD) for allergic provocation challenge and immunotherapy. The activity of these preparations was also tested in an assay of histamine release from human basophils in whole blood. Compared with mice challenged with control CPE, mice challenged with CPE w/o 20kD experienced reduced symptoms (P<0.05) and a smaller decrease in body temperature (P<0.01). Results with the basophil histamine release assay corroborated these findings (P<0.01). The mouse model was also used to administer Ara h 2/6 (20kD) in an immunotherapy protocol, in which peanut-allergic mice treated with the 20kD fraction experienced significantly reduced symptoms, changes in body temperature, and mast cell protease (MMCP-1) release compared with placebo (P<0.01 for all parameters). Importantly, immunotherapy with the 20kD fraction was just as effective as treatment with CPE, whereas CPE w/o 20kD was significantly less effective for higher dose peanut challenges. Ara h 2/6 are the most potent peanut allergens in vivo and can be used to desensitize peanut-allergic mice. These results have potential implications for clinical research in the areas of diagnosis and immunotherapy for peanut allergy.