RESUMO
The study of venom components is an important step toward understanding the mechanism of action of such venoms and is indispensable for the development of new therapies. This work aimed to investigate the venom of Lachesis muta rhombeata and evaluate enzymes related to its toxicity. Phospholipase A2 (PLA(2)), L-amino acid oxidase (LAAO), and proteinase activities were measured, and the molecular weights were estimated. We found the venom to contain one PLA(2) (17 kDa), one LAAO (132 kDa), and three serine proteinases (40, 31, and 20 kDa). Although only serine proteinases were observed in the zymogram, metalloproteinases were found to contribute more to the total proteolytic activity than did serine proteinases. The work confirmed the presence of highly active enzymes; and, moreover, we proposed a novel method for confirming the presence of LAAOs by zymography. We also suggested a simple step to increase the sensitivity of proteinase assays.
Assuntos
L-Aminoácido Oxidase/química , Peptídeo Hidrolases/química , Fosfolipases A2/química , Venenos de Víboras/enzimologia , Viperidae , Animais , Caseínas/química , Embrião de Galinha , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Leucina/química , Camundongos , Peso Molecular , Oxirredução , Fosfolipases A2/farmacologia , Inibidores de Proteases/química , Proteólise , Soroalbumina Bovina/química , Venenos de Víboras/química , Venenos de Víboras/farmacologiaRESUMO
In this work, we present recent advances in the use of phage display technology for the preparation of antivenoms for animal toxin neutralization. Even though classical antivenoms have been used since the early 20th century, envenomation remains a global public health problem. Recently, the phage display technique has been used in an attempt to circumvent some of the difficulties associated with traditional preparations of antivenom. Here, we review studies that developed antibody fragments with potential inhibitory effects against animal toxins and discuss the most current technical issues and perspectives regarding phage display technology in this field.
Assuntos
Anticorpos Neutralizantes/uso terapêutico , Antivenenos/uso terapêutico , Técnicas de Visualização da Superfície Celular/métodos , Modelos Imunológicos , Mordeduras de Serpentes/tratamento farmacológico , HumanosRESUMO
Approximately 20,000 snakebites are registered each year in Brazil. The classical treatment for venomous snakebite involves the administration of sera obtained from immunized horses. Moreover, the production and care of horses is costly, and the use of heterologous sera can cause hypersensitivity reactions. The production of human antibody fragments by phage display technology is seen as a means of overcoming some of these disadvantages. The studies here attempted to test human monoclonal antibodies specific to Bothrops jararacussu against other Bothrops sp. venoms, using the Griffin.1 library of human single-chain fragment-variable (scFv) phage antibodies. Using the Griffin.1 phage antibody library, this laboratory previously produced scFvs capable of inhibiting the phospholipase and myotoxic activities of Bothrops jararacussu venom. The structural and functional similarities of the various forms of phospholipase A2 (PLA2) in Bothrops venom served as the basis for the present study wherein the effectiveness of those same scFvs were evaluated against B. jararaca, B. neuwiedi, and B. moojeni venoms. Each clone was found to recognize all three Bothrops venoms, and purified scFvs partially inhibited their in vitro phospholipase activity. In vivo assays demonstrated that the scFv clone P2B7 reduced myotoxicity and increased the survival of animals that received the test venoms. The results here indicate that the scFv P2B7 is a candidate for inclusion in a mixture of specific antibodies to produce a human anti-bothropic sera. This data demonstrates that the human scFv P2B7 represents an alternative therapeutic approach to heterologous anti-bothropic sera available today.
Assuntos
Anticorpos Monoclonais/imunologia , Antivenenos/imunologia , Bothrops/imunologia , Venenos de Crotalídeos/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Anticorpos Monoclonais/química , Antivenenos/química , Técnicas de Visualização da Superfície Celular , Creatina Quinase/sangue , Venenos de Crotalídeos/química , Feminino , Técnica de Placa Hemolítica , Humanos , Camundongos , Fosfolipases A2/química , Especificidade da Espécie , Homologia Estrutural de ProteínaRESUMO
In Brazil, the species Tityus serrulatus is responsible for the most severe cases of scorpion envenomation. There is currently a need for new scorpion anti-venoms that are more effective and less harmful. This study attempted to produce human monoclonal antibodies capable of inhibiting the activity of T. serrulatus venom (TsV), using the Griffin.1 library of human single-chain fragment-variable (scFv) phage antibodies. Four rounds of phage antibody selection were performed, and the round with the highest phage antibody titer was chosen for the production of monoclonal phage antibodies and for further analysis. The scFv 2A, designated serrumab, was selected for the production and purification of soluble antibody fragments. In a murine peritoneal macrophage cell line (J774.1), in vitro assays of the cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-10 were performed. In male BALB/c mice, in vivo assays of plasma urea, creatinine, aspartate transaminase, and glucose were performed, as well as of neutrophil recruitment and leukocyte counts. It was found that serrumab inhibited the TsV-induced increases in the production of IL-6, TNFα, and IL-10 in J774.1 cells. The in vivo inhibition assay showed that serrumab also prevented TsV-induced increases in the plasma levels of urea, creatinine, aspartate transaminase, and glucose, as well as preventing the TsV-induced increase in neutrophil recruitment. The results indicate that the human monoclonal antibody serrumab is a candidate for inclusion in a mixture of specific antibodies to the various toxins present in TsV. Therefore, serrumab shows promise for use in the production of new anti-venom.