Bencharitiwong Ramon, van der Kleij Hanneke P M, Koppelman Stef J, Nowak-Węgrzyn Anna
Division of Pediatric Allergy and Immunology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
Allergy Asthma Proc. 2015 May-Jun;36(3):185-91. doi: 10.2500/aap.2015.36.3840.
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.
天然花生提取物的改性可降低花生免疫疗法的不良反应。
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。
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。
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。
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。
