Laboratório de Biologia Molecular de Parasitas e Fungos, Centro de Parasitologia e Micologia, Instituto Adolfo Lutz, Sao Paulo, Brazil.
Programa de Pós-graduação em Ciências da Coordenadoria de Controle de Doenças da Secretaria de Estado da Saúde de São Paulo, Sao Paulo, Brazil.
Parasite Immunol. 2023 Sep;45(9):e13004. doi: 10.1111/pim.13004. Epub 2023 Jul 20.
This retrospective cohort study analysed extracellular vesicles (EVs) and microRNAs (miRNAs) excreted in canine sera from dogs with canine visceral leishmaniasis (CanVL). A total of 56 canine sera were divided into Group I (28, from healthy dogs) and Group II (28, from the same dogs, but already with CanVL). CanVL was determined by clinical and laboratory diagnoses. Canine sera were ultra-centrifuged to recover EVs (Can-EVs). Analyses by transmission electron microscopy, nanoparticle tracking analysis (NTA), sodium dodecyl sulfate-poli-acrylammide gel eletroforesis (SDS-PAGE) and, Immunoblot confirmed the presence of (i) microvesicles/exosomes and (ii) the tetraspanins CD63 and CD9. EVs secreted by Leishmania (Leishmania) infantum-EVs were reactive against sera from dogs with CanVL (performed by ELISA and Immunoblot). NTA analyses exhibited that concentrations of Can-EVs from dogs with CanVL (7.78 × 10 Can-EVs/mL) were higher (p < .0001) than the non-infected dogs (mean: 1.47 × 10 Can-EVs/mL). These results suggested that concentrations of Can-EVs were able to distinguish dogs with CanVL from healthy dogs. The relative expressions of 11 miRNAs species (miR-21-5p, miR-146a-5p, miR-125b-5p, miR-144-3p, miR-194-5p, miR-346, miR-29c-3p, miR-155-5p, miR-24-3p, miR-181a-5p, and miR-9-5p) were estimated in purified miRNAs of 30 canine sera. Dogs with CanVL up-expressed miR-21-5p and miR-146a-5p when compared with healthy dogs. The other miRNA species were poorly or not expressed in canine sera. In conclusion, this study suggests that CanVL induces changes in size and concentration of Can-EVs, as well as, the up-expression of miR-21-5p and miR-146a-5p in infected dogs.
本回顾性队列研究分析了犬内脏利什曼病（CanVL）犬血清中分泌的细胞外囊泡（EVs）和 microRNAs（miRNAs）。总共收集了 56 份犬血清，分为 I 组（28 份，来自健康犬）和 II 组（28 份，来自同一犬，但已患有 CanVL）。CanVL 通过临床和实验室诊断确定。犬血清经超速离心回收 EVs（Can-EVs）。透射电子显微镜、纳米颗粒跟踪分析（NTA）、十二烷基硫酸钠-聚丙酰胺凝胶电泳（SDS-PAGE）和免疫印迹分析证实存在（i）微囊泡/外泌体和（ii）四跨膜蛋白 CD63 和 CD9。利什曼原虫（Leishmania）感染犬的 EVs 可与 CanVL 犬血清发生反应（通过 ELISA 和免疫印迹进行）。NTA 分析显示，CanVL 犬的 Can-EVs 浓度（7.78×10 Can-EVs/mL）高于未感染犬（平均值：1.47×10 Can-EVs/mL）（p<.0001）。这些结果表明，Can-EVs 浓度能够区分 CanVL 犬和健康犬。在 30 份犬血清纯化的 miRNAs 中，估计了 11 种 miRNA 物种（miR-21-5p、miR-146a-5p、miR-125b-5p、miR-144-3p、miR-194-5p、miR-346、miR-29c-3p、miR-155-5p、miR-24-3p、miR-181a-5p 和 miR-9-5p）的相对表达。与健康犬相比，CanVL 犬表达 miR-21-5p 和 miR-146a-5p。其他 miRNA 物种在犬血清中表达水平较低或不表达。总之，本研究表明，CanVL 可引起 Can-EVs 大小和浓度的变化，以及感染犬中 miR-21-5p 和 miR-146a-5p 的上调。
