研究背景

蒿花粉是我国北方地区夏秋季最重要的致敏花粉。除具有典型季节性鼻炎和/或哮喘症状之外，部分蒿花粉症患者进食蔬菜、水果后会发生食物过敏反应。目前诊断食物过敏主要依靠病史和过敏原检测。由于花粉和食物变应原之间存在交叉反应，虽然临床有很多花粉症患者花粉相关食物血清特异性IgE（sIgE）阳性，但仅部分患者进食相关食物会出现过敏症状。因此，食物sIgE检测在诊断花粉相关食物过敏中的临床价值有限。近年，过敏原致敏蛋白组分诊断技术能更精确地诊断与临床症状相关的食物过敏。磷脂转移蛋白（lipid transfer protein，LTP）是蒿花粉和植物类食物的重要致敏蛋白组分，也是地中海地区部分成人食物较严重过敏反应的主要致敏蛋白。目前，尚未见到有关LTP致敏与中国蒿花粉相关食物过敏人群临床症状相关性的研究。本课题希望通过比较中国蒿花粉症合并桃过敏人群血清sIgE水平、LTP组分检测，主要致敏蛋白组分分析和嗜碱性粒细胞活化试验，探讨精准诊断中国蒿花粉相关食物过敏人群严重过敏反应的方法。

研究方法

本研究对北京协和医院变态反应科门诊就诊的107例蒿花粉症合并食物过敏患者临床资料进行分析。采用国外重组的LTP组分检测3种常见致敏食物LTP（桃Pru p 3、花生Ara h 9和榛子Cor a 8）sIgE水平。

以蒿花粉症合并桃过敏作为研究蒿花粉相关食物过敏反应的代表。采用本土桃变应原对42例蒿花粉症合并桃过敏患者血清进行免疫印迹分析。

检测蒿花粉相关桃过敏患者（n=89）、单纯蒿花粉症患者（n=52）和健康对照组（n=10）血清桃及其组分sIgE水平。采用流式细胞术，分别以桃总蛋白和主要致敏蛋白Pru p 3作为刺激物进行嗜碱性粒细胞活化试验（basophil activation test, BAT）。

采用多肽筛选的方法对蒿花粉相关桃过敏反应中Pru p 3的主要T细胞表位进行初步鉴定。

研究结果

蒿花粉症合并食物过敏患者的桃Pru p 3、花生Ara h 9和榛子Cor a 8 致敏率为80-90%。Pru p 3与Ara h 9、Cor a 8 sIgE水平明显正相关（r分别为0.912和0.857）；Pru p 3 sIgE水平与致敏食物的数量呈正相关（r=0.606，P <0.01）。全身过敏反应（systemic reaction, SR）患者Pru p 3 sIgE水平高于口腔变态反应综合征（oral allergy syndrome, OAS）患者（P =0.028）。

通过免疫印迹方法发现12KD蛋白是桃的主要致敏组分；全身过敏反应患者该蛋白致敏率明显高于OAS患者（100% vs 55%，P <0.001）。质谱分析结果显示，12KD蛋白与变应原库中桃组分Pru p 3.0101的氨基酸序列符合率为100%。

蒿花粉相关桃过敏组桃及其组分Pru p 3 sIgE水平均高于单纯花粉症和健康对照组（P <0.01）；表现为系统症状（SR）组Pru p 3 sIgE水平高于OAS组（P =0.036），桃sIgE水平在两组间无明显差别。分别以桃总蛋白和主要致敏蛋白Pru p 3作为刺激物进行BAT：以桃变应原为刺激物时，桃过敏患者CD63⁺嗜碱性粒细胞比例高于花粉症患者，明显高于正常对照（P <0.01）；但在OAS和SR两组间无明显差别。而以Pru p 3作为刺激物时，其嗜碱性粒细胞活化程度可以预测过敏反应发生的风险以及症状严重程度。BAT曲线下面积明显大于桃sIgE（AUC 0.744, 95%CI 0.550-0.937,P =0.039）和Pru p 3 sIgE（AUC 0.865, 95%CI 0.705-1.000,P =0.002）。其中Pru p 3作为刺激物的BAT曲线下面积（AUC 0.981, 95%CI 0.825-1.000,P <0.001）大于桃变应原活化的BAT（100ng/ml，AUC 0.942, 95%CI 0.762-1.000,P <0.001）。

Pru p 3_23-38和Pru p 3_67-82是引起我国蒿花粉相关桃过敏反应的主要T细胞表位，55-70%蒿花粉相关桃过敏患者T细胞能识别该表位，发生增殖反应。Pru p 3_67-82和Art v 3_90-105氨基酸序列存在81%一致性。

结论

LTP是我国蒿花粉相关食物过敏反应的主要致敏蛋白，LTP致敏与食物诱发的过敏反应严重程度相关；桃变应原组分Pru p 3.0101是我国蒿花粉症合并桃过敏反应的的主要致敏蛋白，与进食桃诱发的全身过敏反应相关；BAT在蒿花粉相关桃过敏诊断中的价值优于桃及其组分的血清sIgE检测；其中Pru p 3活化的BAT具有最佳的灵敏度和特异度，是诊断蒿花粉相关桃过敏反应和预测严重程度的最佳指标；推测Pru p 3_67-82可能是参与蒿花粉相关桃过敏交叉反应的T细胞表位。

创新点

本研究首次证实LTP是我国蒿花粉相关食物过敏反应的主要致敏蛋白。以蒿花粉相关桃过敏反应为例，从血清学和细胞功能水平，证实Pru p 3是中国蒿花粉症合并桃过敏反应的主要致敏蛋白，与进食桃诱发的全身症状相关。Pru p 3活化的BAT是诊断蒿花粉相关桃过敏和预测症状严重程度的最佳指标。明确了中国人群Pru p 3致敏发生的关键T细胞表位，为将来肽段免疫治疗奠定基础。

Background

Mugwort  pollen is the one of the most common causes of rhinoconjunctivitis and allergic asthma in northern China. It has long been known that patients with mugwort pollen allergy may develop immediate reactions to fruits and vegetables. The diagnosis of food allergy relies mainly on medical history and the allergen detection. As cross-reaction between pollen and plant foods, IgE reactivity to plant foods can be detected in patients with pollinosis, and most of the food-sensitized patients do not have clinical food allergy. Thus, IgE is an unreliable test to diagnose pollen-associated food allergy. Allergen component-resolved diagnostics have the potential to provide a more accurate assessment in diagnosing food allergy. Lipid transfer proteins (LTPs) are important airborne and food allergens, and LTPs represent a major cause of systemic food allergic reactions in the Mediterranean area. Little data about the relationship between LTP sensitization and the clinical symptoms of  mugwort pollen related food allergy are available. In this study, by comparing the utility of sIgE, component-specific IgE and basophil activation test (BAT) in predicting outcome and severity of  mugwort pollen-related peach allergy, we sought to find a test that could accurately diagnose  mugwort pollen-related food allergy.

Methods 

1.   107 individuals with mugwort pollen-related food allergy completed a standardized questionnaire, skin test and sIgE to recombinant food allergen.

2.  We investigated mugwort pollen-related peach allergy as a representative of  mugwort pollen-related food allergy. The allergenic components of peach were detected by SDS-PAGE and immunoblotting.

3.   Peach allergic (n=89), peach-sensitized but tolerant (n=52) and non-peach-sensitized nonallergic (n=10) patients underwent sIgE to peach and its components. BAT was performed using flow cytometry.

4.   Peripheral blood mononuclear cells (PBMCs) from 20 peach-allergic patients were cultured with  Pru p 3 and with a panel of 8 derived of peptides (16-mer overlapping in 5 amino acids representing the full sequence of Pru p 3).

Results

1.   IgE reactivity to Art v 3, Pru p 3, Ara h 9 and Cor a 8 were prevalent (80-90%) among individuals with plant food allergy. Pru p 3 showed a strong positive relationship with Ara h 9 (r=0.912) and Cor a 8 (r=0.857); the level of IgE to Pru p 3 was related to the number of plant food sensitizations (r=0.606, P <0.01). Pru p 3 sIgE were significantly higher in patients with systemic reactions than in patients with oral allergy syndrome (P =0.028). 

2.  The major peach allergen for mugwort pollen-related peach allergy was a 12kDa protein, which was identified as Pru p 3.0101 by matrix-assisted laser desorption/ionization mass spectrometry analysis.

3.  The patients with peach allergy had higher IgE levels for peach and Pru p 3 than peach sensitized population (P <0.01); participants with a history of systemic reaction  showed higher IgE values for Pru p 3 than patients with oral allergy syndrome (P <0.01). By stimulation with peach extract, BAT in peach-allergic patients showed a significant dose-dependent upregulation of CD63 compared with peach sensitized but tolerant and non-peach-sensitized nonallergic patients, but did not correlated with severity of peach-induced allergic reaction. While stimulated with Pru p 3, BAT could discriminate between peach allergy and tolerance, and basophil reactivity was associated with severity of allergic reaction to peach. Receiver operating characteristic curves showed basophil reactivity had larger area under the curve than IgE to peach (AUC 0.744, 95%CI 0.550-0.937, P =0.039) and Pru p 3 (AUC 0.865, 95%CI 0.705-1.000, P =0.002); while BAT stimulated with Pru p 3 had the largest area at 0.981 and stimulation with peach extract (100ng/ml) at 0.942.

4.   Two immunodominant T-cell-reactive regions of  Pru p 3 were identified: Pru p 3_67-82 was detected by 70% of PBMCs from peach-allergic patients, and Pru p 3_23-38 activated 55% of PBMCs from peach-allergic patients. 81% of sequence identity  was found between Mugwort Art v 3_90-105 and peach Pru p 3_67-82.

Conclusion

 LTPs are major food allergens for mugwort pollen-related food allergy in China, and associated with systemic reactions. Pru p 3.0101 is the major allergen responsible for mugwort pollen-related peach allergy in China. BAT stimulated with Pru p 3 were superior to other diagnostic test in discriminating between peanut allergy and tolerance, and could serve as the best tool to predict clinical severity. Pru p 3_67-82 may be the relevant  epitope cross-reacting with mugwort pollen.

Innovation

      This is the first study to identify LTPs as major food allergens for mugwort pollen-related food allergy in Cnina. Represented by mugwort pollen-related peach allergy, we confirm that Pru p 3 is the major allergen, and responsible for systemic reactions from the level of serology and cellular function. BAT stimulated with Pru p 3 is superior to other diagnostic test in discriminating between peanut allergy and tolerance, and predicting clinical severity. Identification of the dominant Pru p 3 T-cell epitopes will be helpful in the development of the hypoallergenic immunotherapy.