化学修饰对花生蛋白变应原性的影响。
Effect of chemical modifications on allergenic potency of peanut proteins.
作者信息
Bencharitiwong Ramon, van der Kleij Hanneke P M, Koppelman Stef J, Nowak-Węgrzyn Anna
机构信息
Division of Pediatric Allergy and Immunology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
出版信息
Allergy Asthma Proc. 2015 May-Jun;36(3):185-91. doi: 10.2500/aap.2015.36.3840.
BACKGROUND
Modification of native peanut extracts could reduce adverse effects of peanut immunotherapy.
OBJECTIVE
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.
METHODS
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.
RESULTS
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.
CONCLUSIONS
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.
背景
天然花生提取物的改性可降低花生免疫疗法的不良反应。
目的
我们试图在基于转染人Fcε受体的大鼠嗜碱性白血病（RBL）细胞系的介质释放试验中，比较天然和化学改性的粗花生提取物（CPE）以及主要花生过敏原Ara h 2和Ara h 6。
方法
天然Ara h 2/6被还原并烷基化（RA），有或没有额外的戊二醛处理（RAGA）。CPE被还原并烷基化。花生过敏受试者（16名男性；中位年龄7岁）的血清用于过夜RBL被动致敏。细胞用0.1 pg/mL至10 μg/mL的花生刺激。β-N-乙酰己糖胺酶释放（NHR）用作RBL脱颗粒的标志物，以Triton X引起的总脱颗粒的百分比表示。
结果
花生特异性免疫球蛋白E的中位数为233 kUA/L。19名受试者为反应者，在介质释放试验中NHR≥10%。与天然Ara h 2/6相比，反应者经RA和RAGA处理后NHR降低。改性导致激活的起始浓度提高10至100倍，最大释放降低。在蛋白质浓度为0.1、1和10 ng/mL时，改性的RA-Ara h 2/6和RAGA-Ara h 2/6引起的最大介质释放明显低于天然Ara h 2/6（RA分别为p < 0.001、< 0.001和< 0.001；RAGA分别为< 0.001、0.026和0.041）。在蛋白质浓度为1 ng/mL（p < 0.001）和10 ng/mL（p < 0.002）时，RA-CPE引起的最大NHR明显低于天然CPE。反应者的重组Ara h 2免疫球蛋白E水平较高（平均为61.1 kUA/L；p < 0.001），并且在介质释放试验中对天然Ara h 2/6的NHR高于CPE，这表明Ara h 2/6是这些反应者中最相关的花生过敏原。
结论
纯化的天然Ara h 2和Ara h 6的化学改性在体外试验中使介质释放降低约100倍，这表明变应原性降低，有利于安全花生免疫疗法替代候选物的进一步开发。
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