桃和蒿草花粉交叉过敏研究

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1 Component-Resolved Diagnosis of Peach and Mugwort Allergy and Cross-Reactivity in China 中国蒿草花粉和桃果实过敏原组分诊断和交叉反应蛋白分析 GAO Zhong-shan Allergy Research Center,Zhejiang University 高中山, 浙江大学过敏研究中心 ( 农业与生物技术学院 ) Tel 联系电话 : gaozhongshan@zju.edu.cn

2 Contents 报告内容 Background 研究背景 Research design 研究方案 Results and conclusion 结果和结论 New progress and Plan 新进展和计划

3 Background 研究背景 (1-Europe) Pollen-fruit allergy syndrome 花粉 - 水果交叉过敏 ( 北欧 ) Birch-Apple 桦树花粉 - 苹果桃梨芹菜胡萝卜等 Mild OAS 症状轻微, 口腔过敏综合症 (OAS ) Central and Northern Europe 与地理位置 / 环境相关 / 种群 ( 欧洲中北部 ) Mediterranean peach allergy 地中海区域桃过敏 True food allergy 桃等核果类为主, 少与花粉症相关 Systematic and OAS symptoms 口腔综合过敏以及较严重全身性过敏症状 Peach LTP is the primary sensitizer 桃果实中的脂质转移蛋白是初始激发原 Cross-reactivity molecular basis 过敏原分子同源性 Bet v 1 --Mal d 1, Pru p 1 etc. 桦树花粉 - 苹果桃樱桃 PR-10 protein 植物病程相关蛋白第 10 家族 LTP, Pru p 3 Mal d 3, Pru av 3, 水果中的脂质转移蛋白 Mugwort pollen allergens ( 蒿草花粉过敏原分子鉴定 ) South Europe, Austria (Ferreira F group)

4 Peach allergen identification 桃过敏原分子鉴定 Allergen components 桃过敏原组分 Pru p 1: PR-10, 与桦树花粉中 Bet v 1 交叉 Pru p 2: PR-5, 类甜蛋白, 较少, 但可以引起严重休克 ( 西班牙, 意大利 ) Pru p 3: PR-14, 脂质转移蛋白 (LTP, 欧洲地中海地区, 初始致敏原 ) Pru p 4: profilin 肌动蛋白结合 ( 少, 一般与其它混合出现 )

5 Mugwort pollen allergens 已鉴定蒿花粉过敏原分子 Art v 1: defensin-like ( 类防护蛋白 ) Art v 2: PR-1 ( 病程相关 1 蛋白 ) Art v 3: PR-14(LTP, 脂质转移蛋白,9 kda) Art v 4: Profilin, actin-binding ( 肌动蛋白结合蛋白, 抑制蛋白 ) Art v 5:EF-hand calcium-binding protein( 钙结合蛋白 ) Art v 6:Pectate lyase ( 果胶酸裂解酶 )

6 Background 研究背景 (2-China) Fruit allergy in China ( 中国水果过敏问题 ) Subtropical fruits, like mango, lychee, pineapple, Chinese bayberry( 热带和亚热带水果 ) Stone fruits, peach, plum,some severe cases report ( 核果类, 桃等, 严重休克报道 ) Pome fruits: apple, pear, loquat ( 仁果类 ) Grapes ( 葡萄 ) Vegetable fruits (tomato, cucumber, 番茄, 黄瓜 ) Due to consumption increase,diversity and availability ( 水果种类多样充足性和消费量 Food allergy associated mugwort allergy 花粉与食物过敏交叉 First report by Wen and Ye, 2002 ( 文昭明, 叶世泰 ) Beans, peach, sunflower seeds, peanut 豆科蔬菜植物桃葵花籽花生 70% Food allergy occurred later or at the same time 食物过敏迟于或者同于花粉过敏 Systematic accompanying OAS symptoms 以全身性伴随口腔综合反应 Allergic rhinitis associated food allergy in Beijing 过敏性鼻炎伴发食物过敏 (Zhang et al.2011) 70% are mugwort pollen allergy 蒿草花粉过敏 Food allergy source curry, shrimp, peach, pineapple, peanut, spinach. 食物过敏 : 咖喱 ( 茴香?), 虾, 桃, 凤梨, 花生, 菠菜

7 Mechanism of Allergy( 过敏机理 ) allergen IL-13 Primary sensitizer Cross-reactive

8 Hypothesis on molecules involved in Mugwort-Peach allergy in China Peach mugwort 桃蒿草花粉共同的过敏原组分 LTP 脂质转移蛋白家族 Pru p 3 Art v 3 Profilin? which one is the primary sensitizer 谁是初始致敏原? Peach 桃 Mugwort 蒿

Research Design 研究方案 History 患者病史 Patients peach 招募患者

Extracts Single components P 桃单一致敏 P/M 交叉过敏 M 艾蒿花粉单一致敏

9 Research Design 研究方案 History 患者病史 Patients peach 招募患者桃过敏交叉过敏 both 艾蒿花粉过敏 mugwort 体外诊断 (ImmunoCAP) 过敏原组分 P. 桃果实提取物 M. 艾蒿花粉提取物 + Pru p 1 Pru p 3 Pru p 4 Art v 3 Art v 1 + Extracts Single components P 桃单一致敏 P/M 交叉过敏 M 艾蒿花粉单一致敏 sige values comp. 比值 Art v 3 /Pru p 3 Art v 3/ Pru p 3 IgE Inhibition 抑制实验 Corresponding to different primary sensitizer

10 Patients info 患者信息 Places 血清采集地 Zhejiang( 杭州 ),Shanxi( 太原大同 ) 88 samples (5 negatives, 含 5 份阴性对照 ) 13 excluded 排除 13 例阴性血清 ( 只有桃毛反应, Neg. sige) Allergy group Nr. M/F ( 男 / 女 ) N 北方 S 南方 P. and M. 桃和艾蒿花粉 24 13/ Peach 桃 15 11/ Mugwort 艾蒿花粉 31 17/ Total 总计 70 41/

11 Symptoms Peach 桃过敏症状 OAS 口腔症状 (25/39), Urt. 荨麻疹 (16/39), G-I 消化道症状 (7/39) Conj. 结膜炎,Dys. 呼吸困难,Ana. 过敏性休克 Mugwort 蒿草花粉过敏症状 Rhinitis 季节性鼻炎伴随打喷嚏 (49/55) Asthma 哮喘 (27/55)

12 IgE (ImmunoCAP) test 检测结果 sige levels of peach allergenic subjects sige levels of mugwort allergenic subjects 100 n=39 n=3 n=37 n= n=55 n=47 n=36 IgE Value (KU A /l) IgE Value (KU A /l) Extract rpru p 1 rpru p 3 rpru p Extract nart v 1 nart v 3 Peach UniCAPs 桃过敏患者 Mugwort UniCAPs 蒿花粉过敏患者 Main allergens 主要过敏原 :Pru p 3, Art v 1 and Art v 3 sige 浓度 : 蒿提取物 > 桃提取物,Art v 3 (20kU A /L)> Pru p 3 (8kU A /L)

13 Extract and Single component sige Positive distribution 按患者单独过敏 / 交叉过敏分组 ( 提取物和单一组分 IgE 阳性数量 ) 过敏组 Group Nr. Peach 桃 Mugwort 蒿 E 提取物 rpru p 1 rpru p 3 rpru p 4 E 提取物 nart v 1 nart v 3 P&M 桃和蒿 P 桃 M 蒿 Tot 总计 Some positive Art v 3 or Pru p 3 IgE,no symptom yet, only IgE binding crossreactive 有些桃或者蒿草 LTP sige 阳性担并没有表现症状, 仅仅是 IgE 结合交叉反应

14 Pru p 3 and Art v 3 sige values Pru p 3 Art v 3 Large group: Art v 3 sige is greater Small group: Pru p 3 sige is greater

15 LTP sige inhibition method 过敏原组分免疫抑制 Sera: two pools and individuals Inhibitors 抑制剂 : Both rpru p 3, rart v 3 同源和异源过敏原组分 Concentration 浓度 (0,0.25,2.5,25,250μg/ml ) ImmunoCAPs 测试血清 sige 1: Inhibitor and serum 75 μl mix, incubation 2 hours 2: sige measuring by Art v 3 and Pru p 3 CAPs respectively

16 H 高 Art v 3/ Pru p 3 L 低 Art v 3 /Pru p 3 Pru p 3 CAP Inhibition sera: high Art v 3/ low Pru p 3 Pru p 3 CAP Inhibition sera: lower Art v 3/ positive Pru p 3 Pru p 3 CAP 异源抑制效果接近同源抑制 % Inhibition (+/-SE) inhibitor mg/ml 250 Pru p 3 Art v 3 % Inhibition (+/-SE) inhibitor mg/ml 250 *** Pru p 3 Art v 3 Pru p 3 CAP 异源抑制效果弱于同源抑制 Art v 3 CAP Inhibition sera: high Art v 3/ low Pru p 3 Art v 3 CAP Inhibition sera: lower Art v 3/ positive Pru p 3 Art v 3 CAP 异源抑制效果弱于同源抑制 % Inhibition (+/-SE) *** Pru p 3 Art v 3 % Inhibition (+/-SE) Pru p 3 Art v 3 Art v 3 CAP 异源抑制效果接近同源抑制 Pru p 3 Art v 3 inhibitor mg/ml inhibitor mg/ml Art v 3 Pru p 3

17 Conclusions 结论 Identify main allergens 明确主要过敏原 peach 桃 :Pru p 3,Mugwort 蒿草 :Art v 1, -3 Primary sensitizer for peach 初始致敏原 S China 南方 : 桃患者以 Pru p 3 为主 N China 北方 : 蒿花粉 Art v 3 为主, 与桃 Pru p 3 交叉致敏 Lipid transfer protein is potential risk food allergen in China, environmental pollen attributing occurrence of food allergy 脂质转移蛋白是中国食物过敏的潜在危险组分

18 Recent progress and plan Antibody against Peach LTP (Pru p 3) mab (IgG1, IgG2) ( 桃主要致敏蛋白的单抗 ) Measurement of LTP content of various peach cultivars ( 不同桃品种果实过敏蛋白含量测试 ) Genome survey of putative LTP sequences from mugwort species in China Putative LTP amino acids sequences Recombinant isoallergens and variants 重要过敏原异构体的重组蛋白 Cell biological analysis of allergenicity of mugwort and peach LTP ( 蒿和桃 LTP 过敏原致敏性的细胞生物学机理 )

19 Diversity of the lipid transfer proteins in peach and mugwort pollen All peaches (including nectarine, flat peach) have the same amino acid sequences by genome survey China has different mugwort species and putative different LTP sequences by homologous gene sequencing

20 Protein analysis of pollens from different Artemisia spp. Collection of pollens Protein extract mrna and proteomics Allergens /isoallergens Variants

21 Recent papers 论文 Gao ZS, Yang ZW, Wu SD, Wang HY, Liu ML, Mao WL, Wang J, Gadermaier G, Ferreira F, Zheng M, van Ree R, Peach allergy in China: A dominant role for mugwort pollen lipid transfer protein as a primary sensitizer,j Allergy Clinical Immunology,2013,131,1: Wang HY, Gao ZS, Yang ZW, Shao JX, Zhao XZ, van Ree R. Anaphylaxis and generalized urticaria in a woman eating Chinese bayberry fruit. Zhejiang University Sciences B 2012, 13(10)

22 Acknowledgement 致谢杨朝崴 ( 浙大农学院 ) 吴善东 ( 浙大生物基因公司 ) 汪慧英 ( 浙二过敏科 ) 毛卫林 ( 浙一检验科 ) 郑敏 ( 浙二皮肤科 ) Gabriele Gadermaier ( 奥地利萨尔斯堡大学过敏诊断和治疗研究所奥地利 ) Fatima Ferreira ( 奥地利萨尔斯堡大学过敏诊断和治疗研究所 ) 刘美玲 ( 山西大同大学附属医院 ) 王健 ( 山西商业职工医院 ) Ronald van Ree ( 荷兰阿姆斯特丹大学医学中心 ) Grants 项目资助 : 国家自然科学基金 (NSFC,2010;2013)

by mild (22.7%). Inhaled corticosteroids, systemic corticosteroids, and antibiotics were applied to 94.8% (292 cases), 74.7% (230 cases), and 90.9% (2

by mild (22.7%). Inhaled corticosteroids, systemic corticosteroids, and antibiotics were applied to 94.8% (292 cases), 74.7% (230 cases), and 90.9% (2 论著临床研究 doi: 0.7499/j.issn.008-8830.204.2.007 郑州市城区儿童哮喘的流行病学调查及发病相关因素分析赵坤宋桂华古华倩 2 刘爽 3 张岩郭彦荣 (. 河南中医学院第一附属医院儿科医院, 河南郑州 450000; 2. 杭州市余杭区