RESUMO
Digestive diseases caused by flagellated bacteria are a huge public health problem worldwide and rapid detection methods are needed for contaminated environments. In this study, we propose a method to detect patterns associated with pathogens based on the properties of the innate immune system. Specifically, we use Toll-like receptor 5 (TLR5), a transmembrane protein that specifically recognizes flagellin (the structural protein of bacterial flagella). TLR5, which was obtained by recombinant production in insect cells, was immobilized into liposomes to form TLR5-proteoliposomes. Through surface plasmon resonance (SPR) and competition flow cytometry assays, the sensitivity of proteoliposomes to recognize Escherichia coli and Salmonella typhimurium flagellin was evaluated. In addition, we compared the results obtained by immobilizing anti-flagellin antibodies into liposomes. The results of the flagellin-affinity tests, expressed as an SPR kinetic rate constant ratio in the equilibrium equation K(D) = k(d)/k(a), showed values of 13.8 × 10(-9) and 7.73 × 10(-9) M for the TLR5-proteoliposomes and anti-flagellin antibodies, respectively, against S. typhimurium. The anti-flagellin affinity results for E. coli showed K(D) of 84.1 × 10(-8) M for SPR assays and K (D) of 3.5 × 10(-8) M for competitive flow cytometry, which was used as a detection system without the immobilization of proteoliposomes. This research demonstrates the practical possibility of using proteoliposomes as recognition elements in the generation of systems for the rapid detection of flagellated bacteria, which could help avoid consumption of contaminated food by humans and thereby prevent intestinal infections.
Assuntos
Flagelina/análise , Citometria de Fluxo/métodos , Ressonância de Plasmônio de Superfície/métodos , Receptor 5 Toll-Like/metabolismo , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Flagelina/metabolismo , Humanos , Lipossomos/química , Ligação Proteica , Salmonella typhimurium/isolamento & purificação , Salmonella typhimurium/metabolismo , Receptor 5 Toll-Like/química , Receptor 5 Toll-Like/genéticaRESUMO
Para conocer el efecto del Factor de Transferencia (FT) en la respuesta leucocitaria de bovino lactantes, se estudiaron 30 becerras lactantes, divididos en dos grupos. Un grupo se trató con FT, posteriormente a su nacimiento por vía subcutánea, y otro se dejó como testigo. Se observaron durante 30 días de lactación, obteniéndose los siguientes resultados: Hubo en los animales tratados un incremento en la respuesta leucocitaria entre los días 8 y 15 de la investigación; este evento no sucedió en el grupo testigo. La incidencia de diarreas y neumonías fue menor entre los 10 y 34 días en becerras con FT respectivamente. Asimismo en el grupo experimental los porcentajes de morbilidad y mortalidad fueron de 80 porciento y 6.7 porciento respectivamente, a diferencias de los del grupo testigo, de 100 y 13.4 porciento.