RESUMO
BACKGROUND: Severe asthma in horses, known as severe equine asthma (SEA), is a prevalent, performance-limiting disease associated with increased allergen-specific immunoglobulin E (IgE) against a range of environmental aeroallergens. OBJECTIVE: To develop a protein microarray platform to profile IgE against a range of proven and novel environmental proteins in SEA-affected horses. ANIMALS: Six SEA-affected and 6 clinically healthy Warmblood performance horses. METHODS: Developed a protein microarray (n = 384) using protein extracts and purified proteins from a large number of families including pollen, bacteria, fungi, and arthropods associated with the horses, environment. Conditions were optimized and assessed for printing, incubation, immunolabeling, biological fluid source, concentration techniques, reproducibility, and specificity. RESULTS: This method identified a number of novel allergens, while also identifying an association between SEA and pollen sensitization. Immunolabeling methods confirmed the accuracy of a commercially available mouse anti-horse IgE 3H10 source (R2 = 0.91). Biological fluid source evaluation indicated that sera and bronchoalveolar lavage fluid (BALF) yielded the same specific IgE profile (average R2 = 0.75). Amicon centrifugal filters were found to be the most efficient technique for concentrating BALF for IgE analysis at 40-fold. Overnight incubation maintained the same sensitization profile while increasing sensitivity. Reproducibility was demonstrated (R2 = 0.97), as was specificity using protein inhibition assays. Arthropods, fungi, and pollens showed the greatest discrimination for SEA. CONCLUSIONS AND CLINICAL IMPORTANCE: We have established that protein microarrays can be used for large-scale IgE mapping of allergens associated with the environment of horses. This technology provides a sound platform for specific diagnosis, management, and treatment of SEA.
Assuntos
Asma/veterinária , Líquido da Lavagem Broncoalveolar/imunologia , Doenças dos Cavalos/imunologia , Imunoglobulina E/sangue , Análise Serial de Proteínas/veterinária , Animais , Artrópodes/imunologia , Asma/sangue , Asma/imunologia , Estudos de Casos e Controles , Fungos/imunologia , Doenças dos Cavalos/sangue , Cavalos , Imunoglobulina E/imunologia , Camundongos , Pólen/imunologia , Análise Serial de Proteínas/métodosRESUMO
The centipede Scolopendra subspinipes mutilans is an environmentally beneficial and medically important arthropod species. Although this species is increasingly applied as a reliable source of new antimicrobial peptides, the transcriptome of this species is a prerequisite for more rational selection of antimicrobial peptides. In this report, we isolated total RNA from the whole body of adult centipedes, S. subspinipes mutilans, that were nonimmunized and immunized against Escherichia coli, and we generated a total of 77,063 pooled contigs and singletons using high-throughput sequencing. To screen putative antimicrobial peptides, in silico analyses of the S. subspinipes mutilans transcriptome were performed based on the physicochemical evidence of length, charge, isoelectric point, and in vitro and in vivo aggregation scores together with the existence of continuous antimicrobial peptide stretches. Moreover, we excluded some transcripts that showed similarity with both previously known antimicrobial peptides and the human proteome, had a proteolytic cleavage site, and had downregulated expression compared with the nonimmunized sample. As a result, we selected 17 transcripts and tested their antimicrobial activity with a radial diffusion assay. Among them, ten synthetic peptides experimentally showed antimicrobial activity against microbes and no toxicity to mouse erythrocytes. Our results provide not only a useful set of antimicrobial peptide candidates and an efficient strategy for novel antimicrobial peptide development but also the transcriptome data of a big centipede as a valuable resource.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Artrópodes/farmacologia , Artrópodes/genética , Medicamentos de Ervas Chinesas/metabolismo , Transcriptoma , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/genética , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/genética , Artrópodes/imunologia , Artrópodes/microbiologia , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Mapeamento de Sequências Contíguas , Alcaloides Diterpenos , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Escherichia coli/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Hemólise/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunização , Camundongos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Alinhamento de Sequência , Técnicas de Síntese em Fase Sólida , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Major allergens may have special aerobiological properties and allergenic structures. It would also be instructive to consider the properties of nonallergens and nonallergenic responses. In some cases, nonallergenic responses appear to result from a lack of antigenicity and in others from regulation. Proteolytic activity has been proposed as an adjuvant for allergenicity, but lipid binding is far more common and is found for more than 50% of the major allergens. Such structures can enhance allergenicity via Toll-like receptor (TLR) or CD1 pathways. TLR signaling can enhance both Th1 and Th2 responses and be induced by peptides as well as nonproteinaceous ligands.