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Introduction: Complement activation split products are signatures of many immunopathological disorders. Among the laboratory findings observed in these diseases, a reduction in the level of circulating intact complement components can be mentioned, and this change has also been detected in envenomation by multiple Africanized honeybee (Apis mellifera) stings. Although envenomation by these animals elicits diverse life-threatening reactions, the capacity of bee venom (AmV) to activate the human complement system remains elusive., Methods and Findings: By coupling immunochemical and functional approaches, it was observed that AmV strongly consumes components of the alternative pathway (AP) of the complement system in normal human serum (NHS). Additionally, AmV interfered with classical (CP) and lectin pathways (LP) activities. In parallel, a high increase in Ba fragment levels was detected, suggesting that the changes in AP activity were due to its activation. Furthermore, an increase in the level of the C1s-C1INH complex and a decrease in the physiological level of MASP1-C1INH suggested that CP and LP were also activated in the presence of AmV. Strikingly, NHS exposed to increasing AmV concentrations varying from 5 to 1000 µg/mL presented a high generation of C3a, C4a and C5a anaphylatoxins, and sC5b-9 complexes assembly, thus reinforcing that AmV triggers complement activation., Conclusion: These results show that AmV is a strong complement activator. This activation presents a mixed profile, with a predominance of AP activation. This suggests that complement split products can play important roles in the envenomation by Africanized honeybee, as they could induce diverse immunopathological events observed in patients and may also dictate patient clinical prognosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Silva de França, de Oliveira Orsi, Fernandes, Leonel and Tambourgi.)

The systemic increase in inflammatory mediator levels can induce diverse pathological disorders, including potentially thrombus formation, which may be lethal. Among the clinical conditions in which the formation of thrombi dictates the patient's prognosis, envenomation by Bothrops lanceolatus should be emphasized, as it can evolve to stroke, myocardial infarction and pulmonary embolism. Despite their life-threatening potential, the immunopathological events and toxins involved in these reactions remain poorly explored. Therefore, in the present study, we examined the immunopathological events triggered by a PLA 2 purified from B. lanceolatus venom, using an ex vivo human blood model of inflammation. Our results showed that the purified PLA 2 from the venom of B. lanceolatus damages human erythrocytes in a dose dependent way. The cell injury was associated with a decrease in the levels of CD55 and CD59 complement regulators on the cell surface. Moreover, the generation of anaphylatoxins (C3a and C5a) and the soluble terminal complement complex (sTCC) indicates that human blood exposure to the toxin activates the complement system. Increased production of TNF-α, CXCL8, CCL2 and CCL5 followed complement activation. The venom PLA 2 also triggered the generation of lipid mediators, as evidenced by the detected high levels of LTB 4 , PGE 2 and TXB 2 . The scenario here observed of red blood cell damage, dysfunctions of the complement regulatory proteins, accompanied by an inflammatory mediator storm, suggests that B. lanceolatus venom PLA 2 contributes to the thrombotic disorders present in the envenomed individuals.

Snake venom enzymes have a broad range of molecular targets in plasma, tissues, and cells, among which hyaluronan (HA) is outstanding. HA is encountered in the extracellular matrix of diverse tissues and in the bloodstream, and its different chemical configurations dictate the diverse morphophysiological processes in which it participates. Hyaluronidases are highlighted among the enzymes involved in HA metabolism. This enzyme has been detected along the phylogenetic tree, suggesting that hyaluronidases exert multiple biological effects on different organisms. Hyaluronidases have been described in tissues, blood and snake venoms. Snake venom hyaluronidases (SVHYA) contribute to tissue destruction in envenomations and are called spreading factors since their action potentiates venom toxin delivery. Interestingly, SVHYA are clustered in Enzyme Class 3.2.1.35 together with mammalian hyaluronidases (HYAL). Both HYAL and SVHYA of Class 3.2.1.35 act upon HA, generating low molecular weight HA fragments (LMW-HA). LMW-HA generated by HYAL becomes a damage-associated molecular pattern that is recognized by Toll-like receptors 2 and 4, triggering cell signaling cascades culminating in innate and adaptive immune responses that are characterized by lipid mediator generation, interleukin production, chemokine upregulation, dendritic cell activation and T cell proliferation. In this review, aspects of the structures and functions of HA and hyaluronidases in both snake venoms and mammals are presented, and their activities are compared. In addition, the potential immunopathological consequences of HA degradation products generated after snakebite envenoming and their use as adjuvant to enhance venom toxin immunogenicity for antivenom production as well as envenomation prognostic biomarker are also discussed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Silva de França and Tambourgi.)

Systemic complement activation drives a plethora of pathological conditions, but its role in snake envenoming remains obscure. Here, we explored complement's contribution to the physiopathogenesis of Naja annulifera envenomation. We found that N. annulifera venom promoted the generation of C3a, C4a, C5a, and the soluble Terminal Complement Complex (sTCC) mediated by the action of snake venom metalloproteinases. N. annulifera venom also induced the release of lipid mediators and chemokines in a human whole-blood model. This release was complement-mediated, since C3/C3b and C5a Receptor 1 (C5aR1) inhibition mitigated the effects. In an experimental BALB/c mouse model of envenomation, N. annulifera venom promoted lipid mediator and chemokine production, neutrophil influx, and swelling at the injection site in a C5a-C5aR1 axis-dependent manner. N. annulifera venom induced systemic complementopathy and increased interleukin and chemokine production, leukocytosis, and acute lung injury (ALI). Inhibition of C5aR1 with the cyclic peptide antagonist PMX205 rescued mice from these systemic reactions and abrogated ALI development. These data reveal hitherto unrecognized roles for complement in envenomation physiopathogenesis, making complement an interesting therapeutic target in envenomation by N. annulifera and possibly by other snake venoms., Competing Interests: JL is the founder of Amyndas Pharmaceuticals, which is developing complement inhibitors for therapeutic purposes. JL is inventor of patents or patent applications that describe the use of complement inhibitors for therapeutic purposes, some of which are developed by Amyndas Pharmaceuticals. JL is also the inventor of the compstatin technology licensed to Apellis Pharmaceuticals (i.e., 4(1MeW)7W/POT-4/APL-1 and PEGylated derivatives such as APL-2/pegcetacoplan). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Silva de França, Villas-Boas, Cogliati, Woodruff, Reis, Lambris and Tambourgi.)

Systemic increased inflammatory mediators' levels are a hallmark in a plethora of pathological conditions, including thrombotic diseases as the envenomation by Bothrops lanceolatus snake. Multiple organ infarctions, which are not prevented by anticoagulant therapy, are the main cause of death on this envenomation. However, the potential mechanisms involved in these systemic reactions are underexplored. This study aimed to explore the potential systemic events which could contribute to thrombotic reactions on the envenomation by B. lanceolatus in an ex vivo human whole-blood model. B. lanceolatus venom elicited an inflammatory reaction, which was characterized by a strong complement activation, since we detected high C3a, C4a and C5a anaphylatoxins levels. Besides, the venom promoted soluble Terminal Complement Complex (sTCC) assembly. Complement activation was accompanied by intense lipid mediators' release, which included LTB 4 , PGE 2 and TXB 2 . In addition, in the blood exposed to B. lanceolatus venom, we detected IL-1β, IL-6 and TNF-α interleukins production. Chemokines, including CCL2, CCL5 and CXCL8 were upregulated in the venom presence. These outcomes show that B. lanceolatus venom causes a strong inflammatory reaction in the blood favoring a potential setting to thrombi formation. Thus, inhibiting inflammatory mediators or their receptors may help in the envenomed patients' management.

Background: Naja annulifera is a medically important venomous snake occurring in some of the countries in Sub-Saharan Africa. Accidental bites result in severe coagulation disturbances, systemic inflammation and heart damage, as reported in dogs, and death, by respiratory arrest, in humans. Despite the medical importance of N. annulifera, little is known about its venom composition and the pathogenesis of envenomation. In this paper, the toxic, inflammatory and immunogenic properties of N. annulifera venom were analyzed., Methodology/principal Findings: Venom proteomic analysis identified 79 different proteins, including Three Finger Toxins, Cysteine Rich Secretory Proteins, Metalloproteinases, Phospholipases A2 (PLA2), Hyaluronidase, L-amino-acid oxidase, Cobra Venom Factor and Serine Proteinase. The presence of PLA2, hyaluronidase, fibrinogenolytic and anticoagulant activities was detected using functional assays. The venom was cytotoxic to human keratinocytes. In an experimental murine model of envenomation, it was found that the venom induced local changes, such as swelling, which was controlled by anti-inflammatory drugs. Moreover, the venom caused death, which was preceded by systemic inflammation and pulmonary hemorrhage. The venom was shown to be immunogenic, inducing a strong humoral immune response, with the production of antibodies able to recognize venom components with high molecular weight and to neutralize its lethal activity., Conclusions/significance: The results obtained in this study demonstrate that N. annulifera venom contains toxins able to induce local and systemic inflammation, which can contribute to lung damage and death. Moreover, the venom is immunogenic, an important feature that must be considered during the production of a therapeutic anti-N. annulifera antivenom., Competing Interests: The authors have declared that no competing interests exist.