Mast cell alpha-chymase reduces IgE recognition of birch pollen profilin by cleaving antibody-binding epitopes.

In sensitized individuals birch pollen induces an allergic response characterized by IgE-dependent mast cell degranulation of mediators, such as alpha-chymase and other serine proteases. In birch and other plant pollens, a major allergen is profilin. In mammals, profilin homologues are found in an intracellular form bound to cytoskeletal or cytosolic proteins or in a secreted form that may initiate signal transduction. IgE specific to birch profilin also binds human profilin I. This cross-reactivity between airborne and endogenous proteins may help to sustain allergy symptoms. The current work demonstrates that cultured mast cells constitutively secrete profilin I, which is susceptible to degranulation-dependent proteolysis. Coincubation of chymase-rich BR mastocytoma cells with Ala-Ala-Pro-Phe-chloromethylketone (a chymase inhibitor) blocks profilin cleavage, which does not occur in degranulated HMC-1 mast cells, which are rich in tryptase, but chymase deficient. These data implicate chymase as the serine protease cleaving secreted mast cell profilin. Sequencing of chymase-cleaved profilins reveals hydrolysis at Tyr(6)-Val(7) and Trp(35)-Ala(36) in birch profilin and at Trp(32)-Ala(33) in human profilin, with all sites lying within IgE-reactive epitopes. IgE immunoblotting studies with sera from birch pollen-allergic individuals demonstrate that cleavage by chymase attenuates binding of birch profilin to IgE. Thus, destruction of IgE-binding epitopes by exocytosed chymase may limit further mast cell activation by this class of common plant allergens, thereby limiting the allergic responses in sensitized individuals.     

红星梭子蟹变应原原肌球蛋白基因的克隆、表达及其免疫学鉴定

克隆、表达红星梭子蟹蟹肉中变应原原肌球蛋白（Tropomyosin）,并对其免疫学特性进行鉴定。RT-PCR克隆红星梭子蟹（Portunus sanguinolentus）蟹肉中变应原原肌球蛋白的全长基因,根据序列设计带有酶切位点的特异性引物,扩增蟹Tropomyosin的完整开放阅读框,与pET-28a载体连接并转化大肠杆菌Escherichia. coli BL21(DE3),诱导表达后,Ni2+亲和层析柱纯化重组蛋白,Western-blot检测其免疫学活性,胰酶消化后进行MALDI-TOF-MS质谱分析鉴定。克隆所得蟹Tropomyosin基因包括一个编码285个氨基酸的开放阅读框。序列分析结果显示所克隆得到的基因与已知虾、螨、蟑螂等的Tropomyosin变应原基因有较高的同源性（>80%）。根据变应原的命名规则,将其命名为Pors 1,并提交GenBank数据库,登录号为EF143836。重组蟹Tropomyosin在大肠杆菌中能高效的表达,表达产物经Ni2+亲和层析柱纯化后进行Western-blot检测,结果显示该重组蛋白具有良好的免疫学活性,MALDI-TOF-MS质谱分析进一步证实了该重组变应原的正确性。本研究成功克隆和表达了红星梭子蟹变应原Tropomyosin,为蟹过敏性疾病的诊断和治疗奠定了基础。     

Purification,characterization and immunoreactivity of tropomyosin,the allergen in Octopus fangsiao

Octopus is one of the foods that can cause food allergies. In this study, tropomyosin (TM) – the primary allergen in octopus – was purified from Octopus fangsiao following different food processing treatments. The changes in the allergenicity and digestibility of different TM samples were estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunological experiments. The results showed that TM was resistant to pepsin but not to trypsin or α-chymotrypsin. Compared with crude TM, the TM treated by ultrasound-heat (UH) and ultraviolet radiation (UVR) showed no significant change in IgG/IgE-binding ability, except high pressure steaming (HP). And the resistance of the treated TM to digestive enzymes was reduced, with HP-treated TM showing the most significant change in stability. It was different that the resistance of TM to the digestive enzymes was increased after treatment with the Maillard reaction (MR); the IgG/IgE-binding activities of the MR products were almost completely eliminated. Thus, all of these results show that the immunoreactivity of octopus TM can be reduced by HP and MR treatment.     

Fine structural recognition specificities of IgE antibodies distinguishing amoxicilloyl and amoxicillanyl determinants in allergic subjects.

IgE antibodies in the sera of subjects allergic to beta-lactam antibiotics detect a spectrum of specificities ranging from side-chain groups to an entire penicillin or cephalosporin molecule. In addition to such structural heterogeneity of allergenic determinants, IgE antibodies in the sera of different allergic subjects show heterogeneous recognition responses. Detailed immunochemical studies were carried out on the sera of penicillin-allergic subjects that showed selective and unexpected reactions with the frequently prescribed penicillin, amoxicillin. Antibodies from one subject reacted only with the amoxicilloyl determinant while IgE from another subject showed multiple reactivity with penicilloyl and penicillanyl determinants of different penicillins but not with the amoxicilloyl determinant. Quantitative hapten inhibition studies revealed that the combining sites of the former antibodies were complementary to amoxicillin in a form that permits binding to the hydroxyaminobenzyl side-chain and the thiazolidine ring carboxyl. These conditions are satisfied with the drug in the '-oyl' but not in the '-anyl' form which involves linkage through the 2-carboxyl of the thiazolidine ring. With the second serum, adsorption studies showed that the wide-ranging reactivity of IgE was due to a single population of antibodies that detected a common specificity on the different penicillins. Combining site studies revealed clear recognition of the benzyl portion of the side-chain of benzylpenicilloyl, benzylpenicillanyl, ampicilloyl, ampicillanyl and amoxicillanyl determinants when free antibody access to the side-chain was possible but little or no recognition of the ring hydroxyl of amoxicillin. Such uninhibited access may not occur, however, when amoxicillin is conjugated in the '-oyl' form since opening the beta-lactam ring allows increased flexibility and rotation of the molecule and the possibility of close association of the hydroxyaminobenzyl side-chain of amoxicillin with the linked peptide carrier. In such close steric association, H-bonding involving the ring hydroxyl and amino acids of the carrier may prevent antibody access to the side-chain region of the amoxicilloyl determinant.     

文昌鱼tropomyosin基因的克隆、进化分析及其胚胎发育与成体中的表达模式

Tropomyosin是一种分布广泛而且在进化上十分保守的蛋白,是肌肉形成和收缩过程中重要的调节蛋白质。通过RT-PCR和RACE技术得到文昌鱼tropomyosin基因全长,编码一个含284个氨基酸残基的蛋白质,将文昌鱼Tropomyosin和在其他物种中的同源物进行比对建树,发现其在功能域上高度保守并且只有一个拷贝,符合动物分类学中各物种的进化地位。胚胎整体原位杂交实验得知,tropomyosin在文昌鱼早期发育的表达,最早从原肠胚末期神经胚早期开始,定位于分化中的中内胚层。到神经胚期,tropomyosin的表达出现在发育中的体节和脊索中。随着发育的进行,tropomyosin的表达稳定地集中在体节、脊索处。到72h幼虫阶段,tropomyosin的表达仍然在肌节内。成体的切片原位杂交结果显示,tropomyosin在肌节中的表达大幅度下调,而在神经管细胞、脊索和腮区腮瓣处仍然可以检测到明显的表达,在外胚层和表皮内没有发现杂交信号。研究结果表明,tropomyosin的表达与文昌鱼肌节、肌肉以及神经索的发生相关,参与文昌鱼胚胎躯体模式的构建,而且在成体的生命活动中发挥重要作用。     

Rapamycin attenuates airway hyperreactivity, goblet cells, and IgE in experimental allergic asthma

The mammalian target of rapamycin (mTOR) signaling pathway integrates environmental cues, promotes cell growth/differentiation, and regulates immune responses. Although inhibition of mTOR with rapamycin has potent immunosuppressive activity, mixed effects have been reported in OVA-induced models of allergic asthma. We investigated the impact of two rapamycin treatment protocols on the major characteristics of allergic asthma induced by the clinically relevant allergen, house dust mite (HDM). In protocol 1, BALB/c mice were exposed to 10 intranasal HDM doses over a period of 24 d and treated with rapamycin simultaneously during the sensitization/exposure period. In protocol 2, rapamycin was administered after the mice had been sensitized to HDM (i.p. injection) and prior to initiation of two intranasal HDM challenges over 4 d. Airway hyperreactivity (AHR), IgE, inflammatory cells, cytokines, leukotrienes, goblet cells, and activated T cells were assessed. In protocol 1, rapamycin blocked HDM-induced increases in AHR, inflammatory cell counts, and IgE, as well as attenuated goblet cell metaplasia. In protocol 2, rapamycin blocked increases in AHR, IgE, and T cell activation and reduced goblet cell metaplasia, but it had no effect on inflammatory cell counts. Increases in IL-13 and leukotrienes were also blocked by rapamycin, although increases in IL-4 were unaffected. These data demonstrated that rapamycin can inhibit cardinal features of allergic asthma, including increases in AHR, IgE, and goblet cells, most likely as a result of its ability to reduce the production of two key mediators of asthma: IL-13 and leukotrienes. These findings highlight the importance of the mTOR pathway in allergic airway disease.     

Strain-dependent induction of allergic sensitization caused by peanut allergen DNA immunization in mice

To investigate the potential application of allergen gene immunization in the modulation of food allergy, C3H/HeSn (C3H) mice received i.m. injections of pAra h2 plasmid DNA encoding one of the major peanut allergens, Ara h2. Three weeks following pDNA immunization, serum Ara h2-specific IgG2a, IgG1, but not IgE, were increased significantly in a dose-dependent manner. IgG1 was 30-fold higher in multiply compared with singly immunized mice. Ara h2 or peanut protein injection of immunized mice induced anaphylactic reactions, which were more severe in multiply immunized mice. Heat-inactivated immune serum induced passive cutaneous anaphylaxis, suggesting that anaphylaxis in C3H mice was mediated by IgG1. IgG1 responses were also induced by intradermal injection of pAra h2, and by i.m. injection of pOMC, the plasmid DNA encoding the major egg allergen protein, ovomucoid. To elucidate whether the pDNA immunization-induced anaphylaxis was a strain-dependent phenomenon, AKR/J and BALB/c mice also received multiple i.m. pAra h2 immunizations. Injection of peanut protein into these strains at weeks 3 or 5 following immunization did not induce reactions. Although IgG2a was increased significantly from week 2 in AKR/J mice and from week 4 in BALB/c mice and remained elevated for at least 6 wk, no IgG1 or IgE was detected. These results indicate that the type of immune responses to pDNA immunization in mice is strain dependent. Consequently, models for studying human allergen gene immunization require careful selection of suitable strains. In addition, this suggests that similar interindividual variation is likely in humans.     

过敏性鼻炎患者鼻腔菌群特征及其 与血清IgE和黏膜嗜酸性粒细胞水平的关系

目的探究过敏性鼻炎(AR)患者鼻腔菌群特征及其与血清IgE和黏膜嗜酸性粒细胞(Eos)水平的关系。方法选取2019年3月至6月在首都医科大学北京友谊医院平谷医院诊断及治疗的AR患者28例作为过敏性鼻炎组,选取同时期体检的健康个体28例作为对照组,检测两组患者鼻腔微生物特征,同时对比两组血清IgE及粘膜Eos%,使用Pearson相关性分析探究各指标间的相关性。结果两组样本微生物构成存在显著差异,样本PCA1及PCA2分别为27.67%及14.23%,过敏性鼻炎组链球菌属、金黄色葡萄球菌、嗜血杆菌属、梭杆菌属、差异球菌属、变形菌门的相对丰度显著高于对照组,对照组痤疮丙酸杆菌、放线菌门、丙酸杆菌属、气球菌属、棒杆菌属、嗜胨菌属的相对丰度显著高于过敏性鼻炎组(Ps0.05),过敏性鼻炎组IgE水平(Z=6.005,P=0.000)及Eos%(t=10.776,P=0.000)显著高于对照组,变形菌门与IgE水平呈显著正相关(r=0.391,P=0.017),痤疮丙酸杆菌与IgE(r=-0.421,P=0.009)及Eos%(r=-0.328,P=0.048)呈显著负相关。结论 AR患者鼻腔变形菌门、痤疮丙酸杆菌分别与IgE水平、鼻粘膜Eos水平具有相关性。     

Gain of structure and IgE epitopes by eukaryotic expression of the major Timothy grass pollen allergen, Phl p 1

Approximately 400 million allergic patients are sensitized against group 1 grass pollen allergens, a family of highly cross-reactive allergens present in all grass species. We report the eukaryotic expression of the group 1 allergen from Timothy grass, Phl p 1, in baculovirus-infected insect cells. Domain elucidation by limited proteolysis and mass spectrometry of the purified recombinant glycoprotein indicates that the C-terminal 40% of Phl p 1, a major IgE-reactive segment, represents a stable domain. This domain also exhibits a significant sequence identity of 43% with the family of immunoglobulin domain-like group 2/3 grass pollen allergens. Circular dichroism analysis demonstrates that insect cell-expressed rPhl p 1 is a folded species with significant secondary structure. This material is well behaved and is adequate for the growth of crystals that diffract to 2.9 A resolution. The importance of conformational epitopes for IgE recognition of Phl p 1 is demonstrated by the superior IgE recognition of insect-cell expressed Phl p 1 compared to Escherichia coli-expressed Phl p 1. Moreover, insect cell-expressed Phl p 1 induces potent histamine release and leads to strong up-regulation of CD203c in basophils from grass pollen allergic patients. Deglycosylated Phl p 1 frequently exhibits higher IgE binding capacity than the recombinant glycoprotein suggesting that rather the intact protein structure than carbohydrate moieties themselves are important for IgE recognition of Phl p 1. This study emphasizes the important contribution of conformational epitopes for the IgE recognition of respiratory allergens and provides a paradigmatic tool for the structural analysis of the IgE allergen interaction.     

Total and specific serum IgE decreases with age in patients with allergic rhinitis,asthma and insect allergy but not in patients with atopic dermatitis

Concerning allergic diseases, the incidence of allergic symptoms, as well as their severity, seems to decrease with age. The decline of onset of allergic symptoms observed in ageing might result from a decrease of serum total and specific IgE. Atopic disorders are complex diseases that involve interactions among several physiological systems, e.g. skin, lung, mucosae, and the immune system. It was the aim of this study to compare the effects of age on total and specific IgE in patients with atopic dermatitis (AD), allergic rhinitis or asthma, and insect allergy, respectively.     

Purification and structural analysis of the novel glycoprotein allergen Cyn d 24, a pathogenesis-related protein PR-1, from Bermuda grass pollen

Bermuda grass pollen (BGP) contains a very complex mixture of allergens, but only a few have been characterized. One of the allergens, with an apparent molecular mass of 21 kDa, has been shown to bind serum IgE from 29% of patients with BGP allergy. A combination of chromatographic techniques (ion exchange and reverse phase HPLC) was used to purify the 21 kDa allergen. Immunoblotting was performed to investigate its IgE binding and lectin-binding activities, and the Lysyl-C endopeptidase digested peptides were determined by N-terminal sequencing. The cDNA sequence was analyzed by RACE PCR-based cloning. The protein mass and the putative glycan structure were further elucidated using MALDI-TOF mass spectrometry. The purified 21 kDa allergen was designated Cyn d 24 according to the protocol of International Union of Immunological Societies (IUIS). It has a molecular mass of 18,411 Da by MALDI-TOF analysis and a pI of 5.9. The cDNA encoding Cyn d 24 was predicted to produce a 153 amino acid mature protein containing tow conserved sequences seen in the pathogen-related protein family. Carbohydrate analysis showed that the most abundant N-linked glycan is a alpha(3)-fucosylated pauci-mannose (Man3GlcNAc2) structure, without a Xyl beta-(1,2)-linked to the branching beta-Man. Thus, Cyn d 24 is a glycoprotein and the results of the sequence alignment indicate that this novel allergen is a pathogenesis-related protein 1. To the best of our knowledge, this is the first study to identify any grass pollen allergen as a pathogenesis-related protein 1.     

Nuclear magnetic resonance structure and IgE epitopes of Blo t 5, a major dust mite allergen

A high incidence of sensitization to Blomia tropicalis, the predominant house dust mite species in tropical regions, is strongly associated with allergic diseases in Singapore, Malaysia, and Brazil. IgE binding to the group 5 allergen, Blo t 5, is found to be the most prevalent among all B. tropicalis allergens. The NMR structure of Blo t 5 determined represents a novel helical bundle structure consisting of three antiparallel alpha-helices. Based on the structure and sequence alignment with other known group 5 dust mite allergens, surface-exposed charged residues have been identified for site-directed mutagenesis and IgE binding assays. Four charged residues, Glu76, Asp81, Glu86, and Glu91 at around the turn region connecting helices alpha2 and alpha3 have been identified to be involved in the IgE binding. Using overlapping peptides, we have confirmed that these charged residues are located on a major putative linear IgE epitope of Blo t 5 from residues 76-91 comprising the sequence ELKRTDLNILERFNYE. Triple and quadruple mutants have been generated and found to exhibit significantly lower IgE binding and reduced responses in skin prick tests. The mutants induced similar PBMC proliferation as the wild-type protein but with reduced Th2:Th1 cytokines ratio. Mass screening on a quadruple mutant showed a 40% reduction in IgE binding in 35 of 42 sera of atopic individuals. Findings in this study further stressed the importance of surface-charged residues on IgE binding and have implications in the cross-reactivity and use of Blo t 5 mutants as a hypoallergen for immunotherapy.     

Patient-tailored cloning of allergens by phage display: Peanut (Arachis hypogaea) profilin, a food allergen derived from a rare mRNA

A peanut cDNA phage surface display library was constructed and screened for the presence of IgE-binding proteins. We used a serum from a peanut-sensitized individual with a low specific IgE level to peanut extract and suffering from mild symptoms after peanut ingestion. A total of 10¹¹ cDNA clones were screened by affinity selection towards serum IgE immobilized to solid-phase supports. After five rounds of selective enrichment, sequence determination of 25 inserts derived from different clones revealed presence of a single cDNA species. The cDNA-encoded gene product, formally termed Ara h 5, shows up to 80% amino acid sequence identity to the well-known plant allergen profilin, a 14 kD protein present only in low amount in peanut extracts. Immunoblot analysis of fifty sera from individuals sensitized to peanut showed that 16% had mounted a detectable IgE response to the newly identified peanut profilin. High-level expression as non-fusion protein in BL21 (DE3) was carried under control of the inducible T7 promoter. Peanut profilin was purified by affinity chromatography on poly-( -proline)-Sepharose and yielded 30 mg l⁻¹ culture of highly pure recombinant allergen. In spite of the high level of up to 80% amino acid identity to other plant profilins, inhibition experiments with recombinant profilins of peanut, cherry, pear, celery and birch revealed marked differences regarding their IgE-binding capacity.     

变应性鼻炎患者血清IL-17和IL-17R表达及其与IgE的相关性

目的:探讨变应性鼻炎患者血清中IL-17及其受体的表达,及其与血清中总IgE的关系.方法:采用酶联免疫法检测51例健康志愿者(对照组)和37例变应性鼻炎患者(实验组)血清中IL-17,IL-17R及IgE的含量.结果:与对照组相比,变应性鼻炎患者血清IL-17,IL-17R及IgE的含量均明显升高.IL-17与变应性鼻炎患者血清中IgE成正相关(r=0.9678,P＜0.05),而IL-17R与IgE无明显相关性.结论:IL-17和IL-17R均参与变应性鼻炎的发病过程,可作为诊断变应性鼻炎的新指标,且降低IL-17的含量有助于变应性鼻炎的临床治疗.     

Role of mast cells and basophils in IgE responses and in allergic airway hyperresponsiveness

We established a diphtheria toxin (DT)-based conditional deletion system using Il4 enhancer elements previously shown to be specific for IL-4 production in mast cells (MCs) or basophils (Mas-TRECK and Bas-TRECK mice). DT treatment of Bas-TRECK mice resulted in specific deletion of basophils, whereas both MCs and basophils were deleted in Mas-TRECK mice. DT-treated Mas-TRECK mice had impaired passive cutaneous anaphylaxis, IgE-mediated passive systemic anaphylaxis, and IgE-mediated chronic allergic inflammation, whereas DT-treated Bas-TRECK mice had impaired IgE-mediated chronic allergic inflammation. Using these mice, we also sought to tease out the role of MCs and basophils in airway hyperresponsiveness (AHR). Although MC deletion resulted in a slight increase in basal Ag-specific IgE levels and significant increases in basal IgE levels, we found that this deletion markedly impaired the AHR effector phase and was accompanied by decreased histamine levels. By contrast, basophil deletion had no effect on the AHR effector phase or on IgE production induced by systemic OVA immunization. Our results, using these newly established Mas-TRECK and Bas-TRECK models, demonstrated an indispensable role for MCs as effector cells in AHR.     

Molecular cloning, expression, sequence analyses of dust mite allergen Der f 6 and its IgE-binding reactivity with mite allergic asthma patients in southeast China

We report the cloning and molecular characterization of a full-length cDNA encoding house dust mite allergen, Der f 6 from D. farinae isolated in China. The full-length Der f 6 cDNA was obtained with 840 nucleotides long. Nucleotide sequencing analyses showed a total of 36 mutations in five Der f 6 cDNA clones, corresponding to 23 incompatible amino acid residues. Recombinant Der f 6 (rDer f 6) protein was successfully expressed in and purified from E. coli BL21. Among 20 asthmatic patients, 45% was positive to rDer f 6 by ELISA. Bioinformatics analyses revealed that the mature Der f 6 was a hydrophobic and extracellular protein with chymotrypsin-like serine protease activity, its secondary structure was composed of alpha helix (7.69%), extended strand (34.62%), random coils (57.69%), and the similarity of Der f 6 to Blo t 6, Sui m 6, Der f 3 and Der f 9 was 64, 65, 35, and 38%, respectively.     

IFN-gamma-inducing factor (IL-18) increases allergic sensitization, serum IgE, Th2 cytokines, and airway eosinophilia in a mouse model of allergic asthma

We investigated the effects of IFN-gamma-inducing factor (IL-18) in a ragweed (RW) mouse model of allergic asthma. Administration of IL-18 in conjunction with allergic sensitization and challenge in wild-type, but not IFN-gamma -/- mice, inhibited the bronchoalveolar lavage (BAL) eosinophilia induced by RW challenge, and increased serum levels of RW-specific IgG2a and production of IFN-gamma from splenocytes cultured with RW, indicating a critical role for IFN-gamma in mediating these effects. Paradoxically, the same treatment schedule in WT mice increased serum levels of RW-specific IgE and IgG1, and production of IL-4 and IL-5 from splenocytes cultured with RW. When the effects of the same IL-18 treatment schedule were allowed to mature for 3 wk, the inhibition of lung eosinophil recruitment was replaced by augmentation of lung eosinophil recruitment. In another experiment, IL-18 administered only with allergic sensitization increased BAL eosinophilia and lung expression of IL-5 and IFN-gamma, while IL-18 administered only with RW challenge decreased BAL eosinophilia and increased lung IFN-gamma expression, while lung expression of IL-5 remained unchanged. IL-18 administered without RW or adjuvant to naive mice increased total serum IgE levels. Finally, intrapulmonary administrations of IL-18 plus RW in naive mice dramatically increased Th2 cytokine production, IgE levels, eosinophil recruitment, and airway mucus, demonstrating induction of allergic sensitization. This is the first report demonstrating that IL-18 promotes a Th2 phenotype in vivo, and potently induces allergic sensitization. These results suggest that IL-18 may contribute to the pathogenesis of allergic asthma.     

PR-10 protein is induced by copper stress in roots and leaves of a Cu/Zn tolerant clone of birch, Betula pendula

The response of a Cu- and Zn-tolerant birch ( Betula pendula ) clone to copper stress was investigated. The plants were exposed to control and EC50 concentrations of Cu (0·3 and 30 μ M CuSO₄, respectively) for 7 d in hydroponic culture. Total proteins were extracted from the roots and leaves and separated by two-dimensional polyacrylamide gel electrophoresis. The differences in protein patterns on silver- or Coomassie-stained gels were analysed. The most apparent quantitative difference was the increase in the amount of a 17 kDa polypeptide caused by Cu stress in both roots and leaves. The protein was identified as Bet v 1-Sc3 (according to the current nomenclature PR-10c) using N-terminal amino acid sequencing and on-line high-performance liquid chromatography/electrospray ionization/ion trap mass spectrometry. The present results indicate that PR-10 is not only activated by pathogens but also by excessive amounts of copper ions. PR-10 proteins in birch have been reported earlier not to be induced by Ag, Li or Cd in birch suspension culture, but Cu has not been previously tested.