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Abstract：
Severe equine asthma (SEA) is a common chronic disease of adult horses with characteristic recurrent airway obstruction and similarities to neutrophilic asthma in humans. As an extrinsic stimulus, hay dust exposure is a major risk factor and induces acute exacerbation in susceptible horses. However, single inducing agents of SEA have hardly been identified on a molecular basis. Aspergillus fumigatus (A. fumigatus) is a common mold species in hay and has been described as a major provoking agent of SEA.
Aiming to identify disease-relevant antigens, we analyzed A. fumigatus using an immunoproteomics approach on two-dimensional immunoblots of A. fumigatus protein probed with serum from environmentally matched asthmatic and healthy horses (n=5 pairs). A. fumigatus binding serum immunoglobulins (Pan-Ig), and the isotypes IgG4/7 and IgG3/5 were quantified for each protein spot and then compared between asthmatic and healthy horses.
For 21 out of 289 spots serum immunoglobulin (Ig) binding was different between the two groups for Pan-Ig or the isotypes. If differences were detected, Pan-Ig and IgG4/7 binding to the proteins were lower, while IgG3/5 binding was higher in asthmatic than healthy horse sera. Proteins were extracted from the 21 spots of interest and analyzed by liquid chromatography mass spectrometry. Eight prioritized proteins (candidate antigens) were expressed as recombinant proteins. Some of these have been previously described as major or minor A. fumigatus allergens, alongside other proteins, most with hydrolase activity. Recombinant candidate antigens were tested on 1D immunoblots to confirm their relevance as antigens by serum antibody binding. Four proteins (beta-hexosaminidase, class II aldolase/adducin domain protein, glucoamylase, peptide hydrolase B0XX53) showed different antibody binding characteristics between asthmatic and healthy horses and are likely relevant antigens in SEA. Their identification can provide the basis for innovative diagnostics, prevention, or therapeutic approaches. Additionally, a more profound understanding of SEA and its potential underlying mechanisms can be established. Elevated serum IgG3/5 antibodies correlate with T helper cell 2 responses in other equine pathologies, and the recombinant SEA antigens developed here can become instrumental in analyzing the involvement of SEA-specific T cell responses and Ig responses in future studies.
Aiming to identify disease-relevant antigens, we analyzed A. fumigatus using an immunoproteomics approach on two-dimensional immunoblots of A. fumigatus protein probed with serum from environmentally matched asthmatic and healthy horses (n=5 pairs). A. fumigatus binding serum immunoglobulins (Pan-Ig), and the isotypes IgG4/7 and IgG3/5 were quantified for each protein spot and then compared between asthmatic and healthy horses.
For 21 out of 289 spots serum immunoglobulin (Ig) binding was different between the two groups for Pan-Ig or the isotypes. If differences were detected, Pan-Ig and IgG4/7 binding to the proteins were lower, while IgG3/5 binding was higher in asthmatic than healthy horse sera. Proteins were extracted from the 21 spots of interest and analyzed by liquid chromatography mass spectrometry. Eight prioritized proteins (candidate antigens) were expressed as recombinant proteins. Some of these have been previously described as major or minor A. fumigatus allergens, alongside other proteins, most with hydrolase activity. Recombinant candidate antigens were tested on 1D immunoblots to confirm their relevance as antigens by serum antibody binding. Four proteins (beta-hexosaminidase, class II aldolase/adducin domain protein, glucoamylase, peptide hydrolase B0XX53) showed different antibody binding characteristics between asthmatic and healthy horses and are likely relevant antigens in SEA. Their identification can provide the basis for innovative diagnostics, prevention, or therapeutic approaches. Additionally, a more profound understanding of SEA and its potential underlying mechanisms can be established. Elevated serum IgG3/5 antibodies correlate with T helper cell 2 responses in other equine pathologies, and the recombinant SEA antigens developed here can become instrumental in analyzing the involvement of SEA-specific T cell responses and Ig responses in future studies.
摘要：
■严重马哮喘（SEA）是成年马常见的慢性疾病，具有特征性反复气道阻塞，与人类中性粒细胞性哮喘相似。作为一种外在刺激，干草粉尘暴露是一个主要的危险因素，并导致易感马急性加重。然而,在分子基础上几乎没有鉴定出SEA的单一诱导剂。烟曲霉（A.烟曲霉）是干草中的常见霉菌种，已被描述为SEA的主要激发剂。
■为了识别疾病相关抗原，我们使用免疫蛋白质组学方法对来自环境匹配的哮喘马和健康马(n=5对)的血清中探测的烟曲霉蛋白的二维免疫印迹进行了分析。烟曲霉结合血清免疫球蛋白（Pan-Ig），并对每个蛋白斑点的同种型IgG4/7和IgG3/5进行定量，然后在哮喘和健康马之间进行比较。
■对于289个斑点中的21个，Pan-Ig或同种型的两组之间的血清免疫球蛋白（Ig）结合不同。如果检测到差异，Pan-Ig和IgG4/7与蛋白质的结合较低，而哮喘患者的IgG3/5结合高于健康马血清。从21个感兴趣的点提取蛋白质并通过液相色谱质谱分析。将8种优先排序的蛋白质(候选抗原)表达为重组蛋白质。其中一些以前被描述为主要或次要的烟曲霉过敏原，与其他蛋白质一起，大多数具有水解酶活性。在1D免疫印迹上测试重组候选抗原,以通过血清抗体结合确认它们作为抗原的相关性。四种蛋白质（β-己糖胺酶，II类醛缩酶/内加素结构域蛋白，葡糖淀粉酶,肽水解酶B0XX53）在哮喘马和健康马之间显示出不同的抗体结合特征，并且可能是SEA中的相关抗原。他们的识别可以为创新诊断提供基础，预防,或治疗方法。此外,可以建立对SEA及其潜在潜在潜在机制的更深刻的理解。血清IgG3/5抗体升高与其他马病理中的T辅助细胞2应答相关，在此开发的重组SEA抗原可以在未来的研究中分析SEA特异性T细胞反应和Ig反应的参与。
■为了识别疾病相关抗原，我们使用免疫蛋白质组学方法对来自环境匹配的哮喘马和健康马(n=5对)的血清中探测的烟曲霉蛋白的二维免疫印迹进行了分析。烟曲霉结合血清免疫球蛋白（Pan-Ig），并对每个蛋白斑点的同种型IgG4/7和IgG3/5进行定量，然后在哮喘和健康马之间进行比较。
■对于289个斑点中的21个，Pan-Ig或同种型的两组之间的血清免疫球蛋白（Ig）结合不同。如果检测到差异，Pan-Ig和IgG4/7与蛋白质的结合较低，而哮喘患者的IgG3/5结合高于健康马血清。从21个感兴趣的点提取蛋白质并通过液相色谱质谱分析。将8种优先排序的蛋白质(候选抗原)表达为重组蛋白质。其中一些以前被描述为主要或次要的烟曲霉过敏原，与其他蛋白质一起，大多数具有水解酶活性。在1D免疫印迹上测试重组候选抗原,以通过血清抗体结合确认它们作为抗原的相关性。四种蛋白质（β-己糖胺酶，II类醛缩酶/内加素结构域蛋白，葡糖淀粉酶,肽水解酶B0XX53）在哮喘马和健康马之间显示出不同的抗体结合特征，并且可能是SEA中的相关抗原。他们的识别可以为创新诊断提供基础，预防,或治疗方法。此外,可以建立对SEA及其潜在潜在潜在机制的更深刻的理解。血清IgG3/5抗体升高与其他马病理中的T辅助细胞2应答相关，在此开发的重组SEA抗原可以在未来的研究中分析SEA特异性T细胞反应和Ig反应的参与。
