title: Enzymatic characterization, antigenic cross-reactivity and neutralization of dermonecrotic activity of five Loxosceles spider venoms of medical importance in the Americas.
abstract: Loxosceles spiders have a wide distribution in the temperate and tropical regions of the world. Loxoscelism is characterized by necrotic skin ulceration at the bite site and, less commonly, a systemic illness that may be fatal. The purpose of this study was to characterize and compare aspects of the major medically important Loxosceles spider venoms in a standardized manner, particularly considering their neutralization by two Brazilian antivenoms. By SDS-PAGE (12% acrylamide), Loxosceles deserta, Loxosceles gaucho, Loxosceles intermedia, Loxosceles laeta and Loxosceles reclusa venoms had similar electrophoretic profiles, with the major protein bands of 32-35 kDa. All venoms exhibited gelatinolytic, caseinolytic and fibrinogenolytic activities in vitro with a large array of proteases, mainly between 18.1 and 31.8 kDa. Most of these enzymes were metalloproteases as this activity was abolished by 1,10-phenanthroline. Hyaluronidase activity was detected in a protein band of approximately 44 kDa in all venoms. Sphingomyelinase activity was demonstrated in all five venoms. Antigenic cross-reactivity, by Western blotting, was also observed among all venoms studied using commercial equine antivenoms produced in Brazil (Institute Butantan and CPPI). These antivenoms recognized mainly components between 25 and 40 kDa in all venoms with several minor components of >89 kDa. Strong cross-reactivity was also seen among all venoms through the ELISA technique (titre range: 64,000-512,000). All venoms (5 microg doses) induced a similar local reaction when injected intradermally into the flank of rabbits, demonstrating dermonecrosis, hemorrhage, vasoconstriction, edema, and erythema. However, no reaction was observed when each venom was pre-incubated (1 h, 37 degrees C) with Brazilian commercial sera prior to injection. The antivenoms also abolished the sphingomyelinase activity in vitro, suggesting the venoms of the major medically important Loxosceles spider species have generally similar toxic and enzymatic characteristics. Thus, as Brazilian commercial antivenoms are able to neutralize the dermonecrosis induced by Loxosceles venoms of diverse geographical origin, clinical studies should be undertaken on the potential for a single global Loxosceles antivenom.
authors.affiliation: Laboratory of Immunopathology, Butantan Institute, Av Vital Brazil 1500, 05503-900, São Paulo, SP, Brazil. kbarbaro@usp.br
authors.forename: Katia Cristina
authors.initials: KC
authors.lastname: Barbaro
authors.forename: Irene
authors.initials: I
authors.lastname: Knysak
authors.forename: Rosana
authors.initials: R
authors.lastname: Martins
authors.forename: Christopher
authors.initials: C
authors.lastname: Hogan
authors.forename: Ken
authors.initials: K
authors.lastname: Winkel
mesh_terms: D000818:Animals; D000997:Antivenins / Q000378:metabolism*; D015153:Blotting, Western; D001938:Brazil; D002364:Caseins / Q000378:metabolism; D003429:Cross Reactions / Q000187:drug effects* / Q000276:immunology; D004487:Edema / Q000139:chemically induced; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004890:Erythema / Q000139:chemically induced; D005340:Fibrinogen / Q000378:metabolism; D005780:Gelatin / Q000378:metabolism; D006820:Hyaluronic Acid / Q000378:metabolism; D009336:Necrosis / Q000139:chemically induced; D009500:Neutralization Tests; D011817:Rabbits; D012867:Skin / Q000473:pathology*; D013045:Species Specificity; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013111:Spider Venoms / Q000037:antagonists & inhibitors / Q000201:enzymology / Q000276:immunology / Q000633:toxicity*; D013112:Spiders / Q000737:chemistry*
chemical_list: D000997:Antivenins; D002364:Caseins; D013111:Spider Venoms; D005780:Gelatin; D005340:Fibrinogen; D006820:Hyaluronic Acid; D013108:Sphingomyelin Phosphodiesterase