小鼠过敏性哮喘模型的研究进展及评价

支气管哮喘（简称哮喘）是常见的慢性病,随着过敏患者的增加,小鼠过敏性哮喘模型的研究越来越重要。本文通过对近年来国内外小鼠过敏性哮喘的实验研究文献进行总结,从实验小鼠的选择、制备模型的方法及模型的评价指标等方面进行综合分析,为进一步开展哮喘研究提供帮助。     

Two new variants of the lipocalin allergen Bos d 2

Allergens from various sources have been shown to comprise several isoforms. In the present study, a series of chromatographic steps was carried out to separate the lipocalin allergen Bos d 2 isoforms present in cow dander. Subsequent HPLC-MS–MS analyses revealed two new Bos d 2 variants. In one of the proteins, tyrosine (Y83) was substituted by aspartic acid, and in the other protein valine (V102) was replaced by alanine. We propose the three Bos d 2 variants be named as Bos d 2.0101 (previously sequenced Bos d 2), Bos d 2.0102 and Bos d 2.0103. Our results suggest that molecular polymorphism is a common property among lipocalin allergens. Since allergen isoforms may show variation in their IgE binding and/or T-cell reactivity, all of the many allergen forms should be taken into account when planning preparations for immunotherapy.     

Recombinant expression of Ole e 6, a Cys-enriched pollen allergen, in Pichia pastoris yeast: detection of partial oxidation of methionine by NMR

Olive pollen is one of the main causes of allergy in Mediterranean countries. Ole e 6, an olive pollen allergen, is a small (5.8 kDa) and acidic protein (pI 4.2) and no homologous proteins have been isolated or characterized so far. Ole e 6 has been efficiently expressed in the methylotrophic yeast Pichia pastoris. The cDNA encoding Ole e 6 was inserted into the plasmid vector pPIC9 and overexpressed in GS115 yeast cells. The recombinant product was purified by size-exclusion chromatography followed by reverse-phase HPLC. N-terminal sequencing, amino acid composition analysis, CD, NMR, and IgG-binding experiments were employed to characterize the purified protein. NMR data revealed the oxidation of the methionine at position 28 in approximately 50% of the recombinant protein but, although this alters its electrophoretic behavior, it did not affect folding or IgG-binding properties of rOle e 6. The recombinant form of Ole e 6 expressed in P. pastoris can be employed for structural and biochemical studies.     

Performance of the Coriolis air sampler,a high-volume aerosol-collection system for quantification of airborne spores and pollen grains

The Coriolis δ air sampler manufactured by Bertin Technologies (France) is a continuous air sampler, dedicated to outdoor monitoring of airborne spores and pollen grains. This high-volume sampler is based on patented Coriolis technology delivering a liquid sample. The air is drawn into a conical vial in a whirling type motion using suction; particles are pulled against the wall by centrifugal force. Airborne particles are separated from the air and collected in a liquid medium. This innovative solution allows rapid analysis by several techniques including PCR assay and serological assay in order to measure the antigenicity/allergenicity of pollen grains and fungal spores. Also, traditional counting of pollen grains or taxa identification by optical microscopy can be done. A study has been carried out by the Health Protection Agency (HPA), Porton Down, UK, to measure the physical efficiency of the Coriolis air sampler. The physical efficiency of the sampler for collection of micro-organism-laden particles of various sizes has been compared with that of membrane filter samplers using the techniques described by ISO 14698-1. The Coriolis was operated simultaneously with membrane filter samplers in a controlled room where they were challenged with uniform-sized particles of different diameters containing bacterial spores. For the larger particle sizes, it was found that the physical efficiency of the Coriolis was 92% for 10-μm particles. The biological performance of the Coriolis in the collection of airborne fungal spores and pollen grains was evaluated in comparison with a Hirst spore trap (one-week tape-on-drum type sampler) which is one of the most frequently used traps in the measurement of outdoor pollen grain concentrations. The advantages and limitations of both technologies are discussed. The Coriolis was operated simultaneously with a Hirst spore trap in the sampling station of Réseau National de Surveillance Aérobiologique, France (RNSA); the pollen grain and fungal spore counts were analysed by optical microscopy. The pollen grain count m⁻³ collected was compared for both devices. The dispersion values were obtained and statistical analysis was carried out. This study shows that the Coriolis air sampler provided equivalent recovery of pollen grain and fungal spores compared with the volumetric trap standard method (not significantly different, W test, α = 0.05). Nowadays, the French-led project, acronym MONALISA, with financial support from the European Commission––Life-Environment (LIFE05 ENV/F/000068), is testing this innovative air sampler in order to measure the antigenicity/allergenicity of the main aeroallergen particles, i.e. Betula (birch), Poaceae (grasses), Parietaria (pellitory), Olea spp (olive tree), and Artemisia (mugwort) pollen grains, and Alternaria (fungal spores) to validate a new approach of monitoring instead of quantifying pollen grains by their morphology. The robustness and efficiency of the MONALISA system is being demonstrated at a national level throughout Europe in eight different countries with different bio-climatic and topography characteristics: France, UK, Finland, Poland, Spain, Portugal, Switzerland, and Italy.     

特异性免疫不同方式给药治疗变应性鼻炎的临床效果观察

目的:研究特异性免疫不同方式给药治疗变应性鼻炎的临床效果及安全性。方法:选取2013年4月-2015年4月我院收治的80例变应性鼻炎患者为研究对象,按照给药方式的不同分为对照组与观察组,对照组给予变应原疫苗皮下免疫治疗,观察组给予变应原滴剂舌下免疫治疗,比较两组患者的治疗效果、症状积分、体征积分、RQLQ评分的改善情况与不良反应的发生情况。结果:观察组患者治疗1年后症状积分、体征积分、RQLQ评分均显著低于对照组(P0.05),但总有效率显著高于对照组(P0.01),两组患者均无严重不良反应发生,对照组患者局部不良反应的发生率明显高于观察组(P0.01)。结论:采用皮下免疫法对变应性鼻炎的治疗效果明显优于舌下免疫治疗,可有效控制患者症状和体征,且安全性更高。     

Molecular cloning and characterization of Cup a 4, a new allergen from Cupressus arizonica

Sensitization to Cupressaceae pollen has become one of the most important causes of pollinosis in Western countries during winter and early spring. However, the characterization of the extracts, the allergens involved and the cross-reactivity with other pollen sources still remain poorly studied; in the case of Cupressus arizonica only two allergens have been described so far. A new allergen from C. arizonica pollen, Cup a 4, was cloned and expressed in Escherichia coli as an N-terminally His-tag recombinant protein that was characterized biochemically, immunologically and by circular dichroism spectroscopy. The new allergen has high sequence identity with Prickly Juniper allergen Jun o 4 and contains four EF-hand domains. The recombinant protein has structural similarities with other calcium binding allergens such as Ole e 3, Ole e 8 and Phl p 7. Cup a 4 is expressed in mature pollen grains and shares antigenic properties with the recombinant form. Sera from 9.6% C. arizonica allergic patients contain specific IgE antibodies against recombinant Cup a 4.     

Characterization of Phleum pratense pollen extracts by 2-D DIGE and allergen immunoreactivity

The allergen content of standardized pollen material is crucial for an effective diagnosis and treatment. However, variations in IgE reactivities of allergic patients to different preparations of Phleum pratense pollen have been reported. In order to define and directly compare the allergen composition of pollen preparations provided by different suppliers, a comprehensive proteome analysis of three different timothy grass pollen extracts was performed. More than 140 proteins were annotated comprising the pollen proteome/allergome in a global 2-D map. With regard to the individual pollen preparations, several major differences in the overall protein composition were detected that also affected known Phleum allergens and their isoforms. Importantly, these differences were also reflected at the level of antibody reactivities in 1-D and 2-D immunoblots. As a consequence, it is suggested that the observed differences should be taken into consideration aiming for a standardized diagnosis and therapy of grass pollen allergies as recommended by international medical agencies.     

Crystal structure and some properties of a major house dust mite allergen, Derf 2

Pyroglyphid house dust mites are a major source of allergens in house dust. Mite allergens sensitize and induce asthma, rhinitis, and eczema in a large portion of patients with allergic diseases. Here, the crystal structure of a major mite allergen, Derf 2, derived from Dermatophagoides farinae was solved by single isomorphous replacement method with anomalous scattering (SIRAS) at 2.1A resolution. The present study also demonstrated that the conformation of the allergen was critical in the determination of Th1/Th2 shift based on physicochemical and immunological analyses. This indicates that rigidly folded and singly dispersed structure is essentially required for the generation of Th2 type cells by the allergen, while conformational variant protein leads to Th1 skewing, irrespective of the same amino acid sequence. This structure/function relationship may allow us to develop a novel strategy for hyposensitization therapy in patients with allergic diseases triggered by house dust mite allergens.     

实验动物致敏研究进展

实验动物致敏（laboratory Animal Allergy,LAA）,是一种职业过敏性疾病,造成人的呼吸道及皮肤发生炎症。该病在国外研究较多,过敏源是动物皮毛、尿液、唾液中的一类酸性小分子蛋白质;可以通过皮肤试验、放射过敏吸附试验及ELISA等方法检测易感人员。对易感人员可以通过控制环境中的过敏原来保护。目前国内尚无专门机构研究此病症,笔者综述了该职业性疾病的症状、机理、控制方法及国内外对该病症认识上的差异。     

花生主要变应原Ara h2的克隆及原核表达

目的:在原核载体中克隆、表达花生主要变应原Ara h2,为其重组变应原应用研究奠定基础。方法:合成花生主要变应原Ara h2基因,设计特异性引物行PCR扩增,经Eco RⅠ、HindⅢ双酶切后与做相应酶切的pET-28a载体连接,转化大肠杆菌BL21,提取质粒进行双酶切鉴定及测序分析,使用IPTG诱导融合蛋白表达,使用Ara h2特异性多克隆抗体对表达产物进行免疫印迹鉴定。结果:成功扩增了Ara h2基因,重组质粒双酶切见目的条带,基因测序显示Ara h2在正确开放阅读框中,基因长423bp,编码140个氨基酸,预测的等电点为5.3,分子量约16660.17 Da,基因比对分析显示其与相关报道的核苷酸序列一致性达100%。重组pET-28a-Ara h2/BL21经0.6 mmol/L IPTG诱导表达可见重组融合蛋白在相应分子量大量表达,使用Ara h2多克隆抗体免疫印迹法能检测到目的蛋白。结论:成功克隆、表达了花生主要变应原Ara h2,为其重组变应原应用研究奠定了基础。