Fabio Carlos Magnoli

Mechanism of induction of complement susceptibility of erythrocytes by spider and bacterial sphingomyelinases

We have recently shown that the sphingomyelinase toxins P1 and P2 from the venom of the spider Loxosceles intermedia induce complement (C)‐dependent lysis of autologous erythrocytes by induction of the cleavage of cell surface glycophorins through activation of an endogenous metalloproteinase facilitating the activation of the alternative pathway of C. Phospholipase D (PLD) from Corynebacterium pseudotuberculosis shows some degree of homology with the spider sphingomyelinases and can induce similar clinical symptoms to those observed after spider envenomation. The aim of this study was to investigate if the bacterial PLD‐induced haemolysis of human erythrocytes was C dependent and if cleavage of glycophorins occurred. We show here that haemolysis of both PLD‐ and P1‐treated human erythrocytes was C dependent, but while PLD‐mediated haemolysis was dependent on activation of the classical pathway of C, P1 induced lysis via both the classical and alternative pathways. P1, but not PLD, induced cleavage of glycophorins and no change in expression of complement regulators was induced by either of the toxins. In both cases, annexin V binding sites were exposed, suggesting that the membrane asymmetry had been disturbed causing exposure of phosphatidylserine to the cell surface. Our results suggest that C susceptibility induced by L. intermedia and C. pseudotuberculosis PLD is a result of exposure of phosphatidylserine, and the higher potency of P1 toxin can be explained by its additional effect of cleavage of glycophorins.

Endotoxemic-like shock induced by Loxosceles spider venoms: pathological changes and putative cytokine mediators

The systemic symptoms, tissue lesions and release of cytokines were analysed in four isogenic mouse strains with distinct haplotypes injected with various doses of Loxosceles intermedia spider venom. The estimated LD50 were 24.5 microg for C57Bl/6, 17.6 microg for BALB/c, 6.3 microg for C3H/HeJ and 4.6 microg for A/Sn mice. Prostration, acute cachexia, hypothermia, neurological disorders and hemoglobinuria were the signals preceding death. Accumulation of eosinophilic material inside the proximal and distal renal tubules and acute tubular necrosis were the most common histopathological findings. Death was prevented by previous treatment of venom with specific antivenom serum. The protein F35 purified from the whole venom retained the ability to induce the symptoms of the whole venom. The cytokines tumor necrosis factor (TNF), interleukins IL-6 and IL-10 and the radical nitric oxide were detected in serum at different levels after venom injection. These findings indicate that the state of shock produced in mice by whole endotoxin-free L. intermedia venom or by its purified fraction, protein F35, mimics the endotoxemic shock, that susceptibility to the systemic effects of the venom varies among mice of different haplotypes and that the pattern of in vivo cytokine release resembles that of endotoxemic shock.

Loxosceles spider venom induces metalloproteinase mediated cleavage of MCP/CD46 and MHCI and induces protection against C-mediated lysis

We have recently shown that sphingomyelinase D toxins from the spider Loxosceles intermedia induce Complement (C) ‐dependent haemolysis of autologous erythrocytes by the induction of cleavage of cell‐surface glycophorins through activation of a membrane‐bound metalloproteinase. The aim of this study was to investigate the effects of these toxins on C‐regulator expression and the C‐resistance of nucleated cells. Cells were incubated with Loxosceles venom/toxins and the expression of C‐regulators was assessed by flow cytometry. A reduced expression of membrane co‐factor protein (MCP) was observed, while expression of decay‐accelerating factor (DAF) and CD59 was not affected. Analysis of other cell‐surface molecules showed a reduced expression of major histocompatibility complex I (MHCI). Western blotting showed that a truncated form of MCP was released into the supernatant. Release could be prevented by inhibitors of metalloproteinases of the adamalysin family but not by inhibitors specific for matrix metalloproteinases. Cleavage of MCP was induced close to or within the membrane as demonstrated by the cleavage of transmembrane chimeras of CD59 and MCP. Although the venom/toxins induced a release of MCP, the C‐susceptibility was decreased. The mechanism of this induction of resistance may involve a change in membrane fluidity induced by the sphingomyelinase activity of the toxin/venom and/or involvement of membrane‐bound proteases. The soluble forms of MCP found in tissues and body under pathological conditions like cancer and autoimmune diseases may be released by a similar mechanism. The identity of the metalloproteinase(s) activated by the spider venom and the role in pathology of Loxoscelism remains to be established.

Loxosceles spider venom induces the release of thrombomodulin and endothelial protein C receptor: implications for the pathogenesis of intravascular coagulation as observed in loxoscelism

Summary.  Background: The venom of the spider Loxosceles can cause both local and systemic effects including disseminated intravascular coagulation. Aim: The aim of this study was to investigate the effects of the venom of Loxosceles intermedia (L. intermedia) and the purified Sphingomyelinase D (SMaseD) toxin upon the Protein C (PC) natural anticoagulant pathway. Results: Both the venom and e purified SMaseD reduced the cell surface expression of thrombomodulin (TM) and Endothelial PC Receptor on endothelial cells in culture. The reduction of cell surface expression was caused by cleavage from the cell surface mediated by activation of an endogenous metalloproteinase. Reduction of TM and Endothelial PC Receptor on the surface of these cells resulted in an impaired ability of the cells to assist in the thrombin‐induced activation of PC. Conclusion: This novel observation gives further insight into the mechanisms of the pathology induced by venom from Loxosceles spiders and may aid the development of a suitable therapy.

P-I snake venom metalloproteinase is able to activate the complement system by direct cleavage of central components of the cascade.

Background Snake Venom Metalloproteinases (SVMPs) are amongst the key enzymes that contribute to the high toxicity of snake venom. We have recently shown that snake venoms from the Bothrops genus activate the Complement system (C) by promoting direct cleavage of C-components and generating anaphylatoxins, thereby contributing to the pathology and spread of the venom. The aim of the present study was to isolate and characterize the C-activating protease from Bothrops pirajai venom. Results Using two gel-filtration chromatography steps, a metalloproteinase of 23 kDa that activates Complement was isolated from Bothrops pirajai venom. The mass spectrometric identification of this protein, named here as C-SVMP, revealed peptides that matched sequences from the P-I class of SVMPs. C-SVMP activated the alternative, classical and lectin C-pathways by cleaving the α-chain of C3, C4 and C5, thereby generating anaphylatoxins C3a, C4a and C5a. In vivo, C-SVMP induced consumption of murine complement components, most likely by activation of the pathways and/or by direct cleavage of C3, leading to a reduction of serum lytic activity. Conclusion We show here that a P-I metalloproteinase from Bothrops pirajai snake venom activated the Complement system by direct cleavage of the central C-components, i.e., C3, C4 and C5, thereby generating biologically active fragments, such as anaphylatoxins, and by cleaving the C1-Inhibitor, which may affect Complement activation control. These results suggest that direct complement activation by SVMPs may play a role in the progression of symptoms that follow envenomation.

Complement Activation by Animal Venoms

AbstractDuring activation of complement (C) system, C4b and C3b attach covalently to targets such as cell membranes. Deposited C4b serve both as the focus for the assembly of the C3 convertases and as ligands for C3b receptors or inactivators. C4a, C3a and C5a, mediators of the early events of inflammation, are released into the fluid phase while C5b serves to organize the cytolytical complex C5b-C9. Among the Arthropoda the venom from some spiders (Loxosceles) contain activators of the mammalian C system, although the exact mechanism and point of the activation cascade in where they act were not yet elucidated. In the insect venoms as honeybee, yellow jacket, yellow hornet, white-facet hornet, caterpillars and wasp there are some components capable of reducing total haemolytic and serum levels. Interestingly, mellitin, a soluble haemolytic peptide present in bee venom shares structural homology with human C9. Scorpion venom apparently are not active on the C system. Snakes, belonging to ELAPIDAE, CROTALI...

Characterization of anti-crotalic antibodies

Crotalus durissus terrificus, C. d. collilineatus, C. d. cascavella and C. d. marajoensis are responsible minor but severe snake bites in Brazil. The venoms of these snakes share the presence of crotoxin, a neurotoxin comprising of two associated components, crotapotin and phospholipase A2 (PLA2). Treatment of the victims with specific antiserum is the unique effective therapeutic measure. The ability of anti-Crotalus antisera produced by the routine using crude venom to immunize horses or purified crotoxin and PLA2 as individual immunogens was compared. Antisera obtained from horses immunized with C. durissus terrificus crude venom were able to recognize and neutralize not only the toxins presents in C. durissus terrificus, but also the ones present in the venoms from C. d. collilineatus, C. d. cascavella and C. d. marajoensis. Antisera from horses immunized with individual crotoxin or PLA2, although in lesser titers, were also able of recognizing the toxins in all four Crotalus species and neutralize the lethality of the C. d. terrificus venom.

Development of IgY antibodies against anti-snake toxins endowed with highly lethal neutralizing activity

Abstract Snakebite envenoming is a major neglected disease related to poverty in developing countries. Treatment involves the administration of a specific antivenom serum and auxiliary therapies, if necessary. The improvement of antibodies is of great importance for the technological advancement of antivenom therapy and to reduce the morbidity and mortality associated with this medical burden. In the present study, adult hens were immunized nine times with 20 &mgr;g of B. arietans or C. d. terrificus venoms at three‐week intervals between immunizations. Developing antibodies presented increasing avidity and affinity to antigenic toxin epitopes along immunization, attaining a plateau after the seventh immunization. Pooled egg yolk‐purified IgY antivenom antibodies, subjected to in vitro–in vivo lethality assay using Swiss adult mice, exhibited potent venom lethal neutralizing activity. Taken together, chickens under the described immunization schedule were considered alternative candidates for antivenom production. Lower maintenance costs, a simple antibody manufacturing process and immunization suffering restrictions are additional advantages. Graphical abstract Figure. No Caption available.

A serine protease isolated from the bristles of the Amazonic caterpillar, Premolis semirufa, is a potent complement system activator.

Background The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called “Pararamose”, characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa’s bristles extract could interfere with the human complement system. Results The bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly interfere with the activity of the Ps82, whereas the presence of PMSF, serine protease inhibitor, totally blocked the activity. Conclusion These data show that a serine protease present in the Premolis semirufa’s bristles extract has the ability to activate the complement system, which may contribute to the inflammatory process presented in humans after envenomation.

Phoneutria nigriventer spider toxin Tx2-6 induces priapism in mice even after cavernosal denervation

&NA; The Phoneutria nigriventer spider toxin Tx2‐6 causes priapism in humans and mice. This toxin produces a delay in Sodium channel inactivation, generalized vascular congestion and death by respiratory failure. NO‐Synthase inhibitors seem to abolish toxin‐induced priapism. The understanding of the ultimate molecular mechanism involved in toxin‐induced priapism may shed light on aspects of erectile function/dysfunction. This study investigates if cavernosal denervation can abolish the toxin‐induced priapism. Surgical cavernosal nerve excision/denervation was performed in mice and confirmed by infertility, histological assessment of fibrosis and immunohistochemical staining for synaptophysin. Denervated mice showed intense fibrosis of the cavernosal tissue as well as absence of synaptophysin IHC staining; surprisingly mice showed toxin‐induced priapism when tested 15, 30 or 60 days after denervation. While sham‐operated mice presented full priapism, denervated animals showed only partial priapism possibly due to the fibrosis. These results reveal that erection caused by Tx2‐6 toxin may not depend on cavernosal nerves integrity. The effect of this toxin on sodium channels seem not directly involved in priapism as many toxins have identical effects but do not induce priapism. Discussion approaches the many different potential sites of intervention listed in the signaling cascades of NO/cGMP, RhoA/Rho‐Kinase, as well as the emerging new gasotransmitter H2S. The pharmacological inhibition of Rho‐kinase and toxin Tx2‐6 have similar effects in vivo. HighlightsPhoneutria nigriventer spider toxin Tx2‐6 causes priapism.Toxin effect are reverted by NOS inhibitors.Penile erection depends on cavernosal nerve integrity.Tx2‐6 effects were evaluated after cavernosal nerve ablation.Tx2‐6 induced priapism even after cavernosal nerve ablation.