RESUMO
Loxoscelism pose a health issue in the South America. The treatment for these accidents is based on the administration of antivenom produced in animals immunized with Loxosceles venom. In this work, a previously produced non-toxic multiepitopic chimeric protein (rMEPlox), composed of epitopes derived from the main toxins families (sphyngomielinase-D, metalloproteases, and hyaluronidases) of Loxosceles spider venoms, was used as antigen to produce monoclonal antibodies (mAbs). A selected anti-rMEPlox mAb (Lox-mAb3) reacted with metalloprotease from L. intermedia venom and showed cross-reactivity with metalloproteses from Brazilian and Peruvian Loxosceles laeta and Loxosceles gaucho venoms in immunoassays. The sequence recognized by Lox-mAb3 (184ENNTRTIGPFDYDSIMLYGAY205) corresponds to the C-terminal region of Astacin-like metalloprotease 1 and the amino acid sequence IGPFDYDSI, conserved among the homologs metalloproteases sequences, is important for antibody recognition. Lox-mAb3 neutralizes the fibrinogenolytic activity caused by metalloprotease from L. intermedia spider venom in vitro, which may lead to a decrease in hemorrhagic disturbances caused by Loxosceles envenomation. Our results show, for the first time, the use of a non-toxic multiepitopic protein for the production of a neutralizing monoclonal antibody against a metalloprotease of medically important Loxosceles venoms. These results contribute for the production improvement of therapeutic antivenom against loxoscelism.
Assuntos
Anticorpos Monoclonais Murinos/imunologia , Anticorpos Neutralizantes/imunologia , Proteínas de Artrópodes , Epitopos , Metaloendopeptidases , Diester Fosfórico Hidrolases , Venenos de Aranha , Aranhas , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Feminino , Metaloendopeptidases/química , Metaloendopeptidases/genética , Metaloendopeptidases/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/imunologia , Engenharia de Proteínas , Venenos de Aranha/química , Venenos de Aranha/genética , Venenos de Aranha/imunologiaRESUMO
Advanced glycation end products (AGEs) are compounds classified as uremic toxins in patients with chronic kidney disease that have several pro-inflammatory effects and are implicated in the development of cardiovascular diseases. To explore the mechanisms of AGEs-endothelium interactions through the receptor for AGEs (RAGE) in the PKC-ß pathway, we evaluated the production of MCP-1 and VCAM-1 in human endothelial cells (HUVECs), monocytes, and a coculture of both. AGEs were prepared by albumin glycation and characterized by absorbance and electrophoresis. The effect of AGEs on cell viability was assessed with an MTT assay. The cells were also treated with AGEs with and without a PKC-ß inhibitor. MCP-1 and VCAM-1 in the cell supernatants were estimated by ELISA, and RAGE was evaluated by immunocytochemistry. AGEs exposure did not affect cell viability, but AGEs induced RAGE, MCP-1, and VCAM-1 expression in HUVECs. When HUVECs or monocytes were incubated with AGEs and a PKC-ß inhibitor, MCP-1 and VCAM-1 expression significantly decreased. However, in the coculture, exposure to AGEs and a PKC-ß inhibitor produced no significant effect. This study demonstrates, in vitro, the regulatory mechanisms involved in MCP-1 production in three cellular models and VCAM-1 production in HUVECs, and thus mimics the endothelial dysfunction caused by AGEs in early atherosclerosis. Such mechanisms could serve as therapeutic targets to reduce the harmful effects of AGEs in patients with chronic kidney disease.