A Novel Apilic Antivenom to Treat Massive, Africanized Honeybee Attacks: A Preclinical Study from the Lethality to Some Biochemical and Pharmacological Activities Neutralization

Massive, Africanized honeybee attacks have increased in Brazil over the years. Humans and animals present local and systemic effects after envenomation, and there is no specific treatment for this potentially lethal event. This study evaluated the ability of a new Apilic antivenom, which is composed of F(ab’)2 fraction of specific immunoglobulins in heterologous and hyperimmune equine serum, to neutralize A. mellifera venom and melittin, in vitro and in vivo, in mice. Animal experiments were performed in according with local ethics committee license (UFRJ protocol no. DFBCICB072-04/16). Venom dose-dependent lethality was diminished with 0.25–0.5 μL of intravenous Apilic antivenom/μg honeybee venom. In vivo injection of 0.1–1 μg/g bee venom induced myotoxicity, hemoconcentration, paw edema, and increase of vascular permeability which were antagonized by Apilic antivenom. Cytotoxicity, assessed in renal LLC-PK1 cells and challenged with 10 μg/mL honeybee venom or melittin, was neutralized by preincubation with Apilic antivenom, as well the hemolytic activity. Apilic antivenom inhibited phospholipase and hyaluronidase enzymatic activities. In flow cytometry experiments, Apilic antivenom neutralized reduction of cell viability due to necrosis by honeybee venom or melittin. These results showed that this antivenom is effective inhibitor of honeybee venom actions. Thus, this next generation of Apilic antivenom emerges as a new promising immunobiological product for the treatment of massive, Africanized honeybee attacks.

Amplification of Snake Venom Toxicity by Endogenous Signaling Pathways

The active components of snake venoms encompass a complex and variable mixture of proteins that produce a diverse, but largely stereotypical, range of pharmacologic effects and toxicities. Venom protein diversity and host susceptibilities determine the relative contributions of five main pathologies: neuromuscular dysfunction, inflammation, coagulopathy, cell/organ injury, and disruption of homeostatic mechanisms of normal physiology. In this review, we describe how snakebite is not only a condition mediated directly by venom, but by the amplification of signals dysregulating inflammation, coagulation, neurotransmission, and cell survival. Although venom proteins are diverse, the majority of important pathologic events following envenoming follow from a small group of enzyme-like activities and the actions of small toxic peptides. This review focuses on two of the most important enzymatic activities: snake venom phospholipases (svPLA2) and snake venom metalloproteases (svMP). These two enzyme classes are adept at enabling venom to recruit homologous endogenous signaling systems with sufficient magnitude and duration to produce and amplify cell injury beyond what would be expected from the direct impact of a whole venom dose. This magnification produces many of the most acutely important consequences of envenoming as well as chronic sequelae. Snake venom PLA2s and MPs enzymes recruit prey analogs of similar activity. The transduction mechanisms that recruit endogenous responses include arachidonic acid, intracellular calcium, cytokines, bioactive peptides, and possibly dimerization of venom and prey protein homologs. Despite years of investigation, the precise mechanism of svPLA2-induced neuromuscular paralysis remains incomplete. Based on recent studies, paralysis results from a self-amplifying cycle of endogenous PLA2 activation, arachidonic acid, increases in intracellular Ca2+ and nicotinic receptor deactivation. When prolonged, synaptic suppression supports the degeneration of the synapse. Interaction between endothelium-damaging MPs, sPLA2s and hyaluronidases enhance venom spread, accentuating venom-induced neurotoxicity, inflammation, coagulopathy and tissue injury. Improving snakebite treatment requires new tools to understand direct and indirect effects of envenoming. Homologous PLA2 and MP activities in both venoms and prey/snakebite victim provide molecular targets for non-antibody, small molecule agents for dissecting mechanisms of venom toxicity. Importantly, these tools enable the separation of venom-specific and prey-specific pathological responses to venom.

The Effect of Adjunctive Fresh Frozen Plasma Administration on Coagulation Parameters and Survival in a Canine Model of Antivenom-treated Brown Snake Envenoming

This study aimed to assess the effects of dugite envenoming on blood coagulation and platelet count in a canine model, and the efficacy of fresh frozen plasma (FFP) in reversing the clotting disorder after both adequate and inadequate venom neutralization. Following initial dosing and administration studies, an intravenous venom dose of 1μg/kg was administered to eleven dogs. This was followed 30 minutes later by antivenom in either adequate or inadequate doses. A further 30 minutes later, the animals were given either two units of their own FFP or saline. Fibrinogen, aPTT and platelet levels were monitored for eight hours. Of the six study dogs given antivenom plus FFP, two died at around 60 to 90 minutes post envenoming, at the end of the FFP infusions, and all but one of the survivors had persistent afibrinogenaemia. Of the five study dogs given antivenom and no FFP, all but one had return of detectable fibrinogen at eight hours after envenoming. The platelet count fell in all animals with recovery independent of antivenom dose, administration of FFP, or regeneration of fibrinogen. Post mortem examinations of dogs that died during dosage and administration studies showed massive intracardiac clots. We conclude that early death from Brown Snake envenoming may be due to massive intravascular clotting. FFP administration was associated with persistent afibrinogenaemia regardless of antivenom dose. In the absence of any evidence for its efficacy, this study suggests that the role of FFP after Brown Snake envenoming should be reconsidered.