Yvonne M. Vissers

Effect of Heating and Glycation on the Allergenicity of 2S Albumins (Ara h 2/6) from Peanut

Background Peanut allergy is one of the most common and severe food allergies, and processing is known to influence the allergenicity of peanut proteins. We aimed to establish the effect of heating and glycation on the IgE-binding properties and biological activity of 2S albumins (Ara h 2/6) from peanut. Methodology/Principal Findings Native Ara h 2/6 was purified from raw peanuts and heated in solution (15 min, 110°C) in the presence or absence of glucose. Ara h 2 and 6 were also purified from roasted peanut. Using PBMC and sera from peanut-allergic patients, the cellular proliferative potency and IgE reactivity (reverse EAST inhibition) and functionality (basophil degranulation capacity) of allergens were assessed. Heating Ara h 2/6 at 110°C resulted in extensive denaturation, hydrolysis and aggregation of the protein, whilst Ara h 2 and 6 isolated from roasted peanut retained its native conformation. Allergen stimulation of PBMC induced proliferation and Th2 cytokine secretion which was unaffected by thermal processing. Conversely, IgE reactivity and functionality of Ara h 2/6 was decreased by heating. Whilst heating-glycation further reduced the IgE binding capacity of the proteins, it moderated their loss of histamine releasing capacity. Ara h 2 and 6 purified from roasted peanut demonstrated the same IgE reactivity as unheated, native Ara h 2/6. Conclusions/Significance Although no effect of processing on T-cell reactivity was observed, heat induced denaturation reduced the IgE reactivity and subsequent functionality of Ara h 2/6. Conversely, Ara h 2 and 6 purified from roasted peanut retained the structure and IgE reactivity/functionality of the native protein which may explain the allergenic potency of this protein. Through detailed molecular study and allergenicity assessment approaches, this work then gives new insights into the effect of thermal processing on structure/allergenicity of peanut proteins.

Boiling peanut Ara h 1 results in the formation of aggregates with reduced allergenicity

SCOPE Roasting rather than boiling and Maillard modifications may modulate peanut allergenicity. We investigated how these factors affect the allergenic properties of a major peanut allergen, Ara h 1. METHODS AND RESULTS Ara h 1 was purified from either raw (N-Ara h 1) or roasted (R-Ara h 1) peanuts. Boiling (100°C 15 min; H-Ara h 1) resulted in a partial loss of Ara h 1 secondary structure and formation of rod-like branched aggregates with reduced IgE-binding capacity and impaired ability to induce mediator release. Glycated Ara h 1 (G-Ara h 1) formed by boiling in the presence of glucose behaved similarly. However, H- and G-Ara h1 retained the T-cell reactivity of N-Ara h 1. R-Ara h 1 was denatured, comprised compact, globular aggregates, and showed no evidence of glycation but retained the IgE-binding capacity of the native protein. CONCLUSION Ara h 1 aggregates formed by boiling were morphologically distinct from those formed by roasting and had lower allergenic activity. Glycation had no additional effect on Ara h 1 allergenicity compared with heating alone. Taken together with published data on the loss of Ara h 2/6 from boiled peanuts, this supports the hypothesis that boiling reduces the allergenicity of peanuts.

Effect of roasting on the allergenicity of major peanut allergens Ara h 1 and Ara h 2/6: the necessity of degranulation assays

Background Peanuts are often consumed after roasting, a process that alters the three‐dimensional structure of allergens and leads to Maillard modification. Such changes are likely to affect their allergenicity.

Differential effects of Lactobacillus acidophilus and Lactobacillus plantarum strains on cytokine induction in human peripheral blood mononuclear cells

Lactic acid bacterial strains have received interest for their immunomodulating activities and potential use in probiotic products. A wide variety of strain-dependent properties have been reported, but comparative studies at the species level are scarce. The objective of this study was to assess the immunomodulatory effect of Lactobacillus species on the cytokine profiles and proliferative response of human peripheral blood mononuclear cells (hPBMC), and in particular, on the comparison between the species Lactobacillus acidophilus and Lactobacillus plantarum. hPBMC from healthy donors were stimulated in the presence or absence of the lactic acid bacteria, and cytokine production, surface marker staining, proliferation and cell death were determined after 1 and 4 days of culture. All Lactobacillus strains tested were capable of inducing the production of interleukin (IL)-1beta, IL-10, interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). The bacterial strains did not differentially influence the amount of proliferating, viable, apoptotic and necrotic cells. Generally, L. plantarum showed a significantly higher induction capacity of IFN-gamma, IL-12 and TNF-alpha compared with L. acidophilus. We conclude that the variation in immunomodulatory effects between species is even larger than the variation between the strains of the same species. In addition, we demonstrate that L. plantarum strains are most potent in skewing the T-cell differentiation toward a putative Th1 response.

T cell responses in fresh and cryopreserved peripheral blood mononuclear cells: Kinetics of cell viability, cellular subsets, proliferation, and cytokine production☆

Polyclonal stimuli like phorbol myristate acetate (PMA) plus calcium ionophore (Ca-I), concanavalin A (ConA) or anti-CD3 plus anti-CD28 (alphaCD3/alphaCD28) are widely used T cell stimuli. All three stimuli act at different sites and in different ways to activate the T cell receptor pathway and are widely used in different concentrations, stimulation durations and read-out systems. This study was designed to establish the most suitable polyclonal stimulus in human peripheral blood mononuclear cells (PBMC) experiments by assessing the kinetics of cell viability, present immunophenotypes, proliferation, and cytokine production of the PBMC. In addition, changes in these read-out parameters due to cryopreservation have been investigated by comparing fresh and cryopreserved PBMC cultures at days 1, 3, 5, and 7. This study showed a reduction in the cytokine levels after cryopreservation of PMA/Ca-I stimulated PBMC, whereas no significant differences due to the cryopreservation were observed in ConA or alphaCD3/alphaCD28 stimulated PBMC. Cryopreservation did not alter the maximal proliferation capacity of ConA or alphaCD3/alphaCD28 stimulated PBMC, whereas it did delay the proliferation. Although cryopreservation had no effect on the CD3+CD4+ or CD3+CD8+ T cell subsets, PMA/Ca-I significantly reduced the amount of both T cell subsets over time. In conclusion, PMA/Ca-I is suitable as a positive control in experiments where high cytokine production is expected and only fresh PBMC are used. Proliferation and effects on the T cell subsets in long-term PBMC cultures should use ConA or alphaCD3/alphaCD28 as positive control.

Impact of Maillard Reaction on Immunoreactivity and Allergenicity of the Hazelnut Allergen Cor a 11

Few studies exist on the influence of processing methods on structural changes and allergenic potential of hazelnut proteins. This study focused on the effect of glycation (Maillard reaction) on the immunoreactivity and degranulation capacity of the purified hazelnut 7S globulin, Cor a 11. After heating, the extent of the Maillard reaction, sensitivity to proteolysis, binding of human IgE or rabbit IgG, and degranulation capacity were analyzed. Changes in electrophoretic mobility, amount of free amino groups, and contents of bound sugar and fructosamine indicated that glycation of Cor a 11 occurred at all conditions. Glycation at 37 °C did not influence the specific IgG or IgE binding and was decreased after heating at 60 and 145 °C. Heating, with or without glucose, at 145 °C increased basophil degranulation capacity. The results suggest that glycation of Cor a 11 at 60 and 145 °C may decrease the IgE/IgG binding properties but not the degranulation capacity of basophils. This is possibly related to aggregation of the proteins as a result of the Maillard reaction.

Lactobacillus strains differentially modulate cytokine production by hPBMC from pollen allergic patients

The objective of this study was to assess the potential immunomodulatory effect of six Lactobacillus strains on human peripheral blood mononuclear cells (hPBMC) isolated from allergic patients. hPBMC from patients allergic to birch pollen or grass pollen were cultured in vitro in the presence or absence of selective bacterial strains. Cultures were left unstimulated or stimulated with αCD3/αCD28 or Bet v 1. After 1, 4 and 8 days, cells and culture supernatants were harvested and the effect on cellular proliferation and the supernatant levels of several cytokines was assessed. All strains had the ability to repress IL-13 production but did show a differential effect on IFN-γ induction. Both strains B223 and B1697 showed a lower IFN-γ, IL-12 and TNF-α induction as compared with the other tested strains. Strain B633 showed the best proliferation-suppressive properties in αCD3/αCD28-stimulated cells. Suppression of the T-helper type 2 (Th2) cytokine induction and induction of the Th1 cytokine production by specific strains might be beneficial for allergic patients having a disturbed Th1/Th2 immune balance. Furthermore, hPBMC of patients with seasonal allergy outside the pollen season can be used to determine the immunomodulatory activities of probiotic bacteria.

Influence of Food Processing, Digestion and the Food Matrix on Allergenicity & Cellular Measures of Allergenicity

The degree of processing and digestion and the impact of embedding in food matrix components could influence the way in which allergens are presented to the immune system, and thus contribute to the severity of an allergic reaction. In addition, processing, digestion and matrix effects are also of importance in diagnostic procedures, in particular when food challenges are part of the diagnostic process. Food processing, for example, thermal processing, could alter the nature of the epitopes of a protein; however, there is no general rule on how proteins respond and the type of allergen, type of food structure, and type of thermal processing may all contribute to the impact of processing. As most proteins are digested in the stomach and the intestine, the immune system is mostly exposed to digested protein. However, digestion, even with a variety of gastric enzymes, is not always sufficient to abolish the allergo-reactivity of allergens. Recently developed in vitro models of digestion (e.g., including surfactants) are improving our knowledge on digestion. Furthermore, matrix compounds such as the fat content and proteolytic and oxidative enzymes could have an effect on the allergenicity. Enzyme immunoassays and immunoblotting are often used to assess IgE binding. However, these methods do not determine whether the allergen can cause cross-linking of FcɛR1 bound IgE antibodies; for this a cellular assay is needed. Histamine release tests, CAST, BAT and mediator release from RBL cells are examples of such cellular measurements looking at cross linking of allergens while T-cell polarization assays can be used to assess the potency of an allergen to activate CD4+ helper T-cells and skew them towards a Th2 like profile. A few cellular assays are discussed more extensively and are considered crucial to providing a comprehensive analysis of the effects of food matrix and processing methods on individual allergenic proteins.

Roasting or heating increases elicitation capacity of peanut allergens but does not affect their sensitisation potential in a brown Norway rat model for food allergy

Background Allergenic potential of processed food allergens has primarily been studied by their IgE-binding capacity (elicitation). Roasting of peanuts has been shown to increase IgE-binding capacity. In this study we examined whether processing of whole peanuts or of the major peanut allergen Ara h 1 influenced the sensitisation potential. Methods Brown Norway rats were either dosed orally by gavage each day for 42 days with finely ground whole peanut products (blanched or roasted peanuts or peanut butter) mixed with water [~2 mg Ara h 1/rat/day] or immunised i.p. three times with 200 μg of native, heated or heat glycated Ara h 1. Sera obtained at sacrifice were analysed for specific IgG and IgE by ELISA and for biological functionality of IgE by rat basophilic leukaemia (RBL) assay. Results Processing was found to decrease solubility and thus extractability of Ara h 1 from peanut products. Aggregation state and secondary structure changes induced by heating of purified Ara h 1 were identical to those observed when Ara h 1 was heated in the presence of glucose. Although a significant anti-Ara h 1 IgE response was only found when dosing rats with roasted peanuts, examination of functional specific IgE by RBL assay showed that processing of peanuts did not influence sensitisation potential. However, extract from roasted peanuts was found to be a superior elicitor compared to extract from blanched peanuts irrespective of the peanut product used for sensitisation. Processing of purified native Ara h 1 did not influence the sensitisation capacity. Nonetheless, ELISA results indicated that new epitopes are formed or disclosed by heating of Ara h 1. Furthermore, IgG1-binding capacity was found to reflect whether rats were sensitised to native or processed Ara h 1 or dosed with blanched or roasted peanut products. Conclusion Roasted peanuts, either as such or as peanut butter, do not have a higher sensitisation capacity than blanched peanuts. This is supported by the finding that processmodified Ara h 1 has a similar sensitisation capacity as native Ara h 1. Yet, our results show that roasting increases elicitation capacity.

Effect of heating and glycation on the allergenicity of Ara h 2/6

Results Heating Ara h 2/6 at 110°C resulted in extensive denaturation whilst Ara h 2/6 extracted from roasted peanut retained its native conformation. Allergen stimulation of PBMC from peanut allergic patients induced proliferation of mainly CD4+ T-cells and induction of Th2 cytokine secretion which was unaffected by thermal processing. IgE reactivity and functionality of Ara h 2/6 was decreased by heating. Whilst heating-glycation further reduced the IgE binding capacity of the proteins, it moderated their loss of histamine releasing capacity. Ara h 2/6 purified from roasted peanut demonstrated the same IgE reactivity as unheated, native Ara h 2/6.