Asper Venom Research Articles

Antagonization of TNF attenuates systemic hemodynamic manifestations of envenomation in a rat model of Vipera aspis snakebite.

Tumor necrosis factor (TNF) has been reported as a mediator of local tissue injury following snake envenomation in an intact rat model. We investigated whether systemic release of TNF occurs following Vipera aspis envenomation. We further analyzed the possible connection between envenomation-related hemodynamic depression and TNF antagonization (TNF antibodies or soluble TNF receptor). A prospective, randomized, controlled experimental study using a rat model for snake envenomation. A medical university hospital research laboratory. Eighty rats (300-400 g) were divided into four groups (n = 20): control and three experimental groups. Intramuscular injection of V. asis 500 microg/kg was administered to the three experimental groups: venom only (group 1), venom and 40 microg anti-TNF antibodies (group 2), venom and 250 microg soluble TNF receptor (p55-R; group 3). Hemodynamic parameters were monitored up to 4 h following venom injection. A significant hemodynamic deterioration (reduction in heart rate and blood pressure) occurred 30 min following venom injection in group 1 compared to groups 2 and 3, where hemodynamic parameters remained stable throughout the 4 h observation period. Serum levels of TNF were detected 15 min after venom injection and peaked after 2 h at 485+/-12 pg/ml. The hemodynamic consequences of intramuscular injection of V. aspis venom can be blunted in a rat by systemic antagonization of TNF activity prior to venom injection. The poisonous hemodynamic effects of the V. aspis venom might be caused by systemic release of TNF.

Comparison between IgG and F(ab′) 2 polyvalent antivenoms: neutralization of systemic effects induced by Bothrops asper venom in mice, extravasation to muscle tissue, and potential for induction of adverse reactions

Whole IgG and F(ab′) 2 equine-derived polyvalent (Crotalinae) antivenoms, prepared from the same batch of hyperimmune plasma, were compared in terms of neutralization of the lethal and defibrinating activities induced by Bothrops asper venom, their ability to reach the muscle tissue compartment in envenomated mice, and their potential for the induction of adverse reactions. Both preparations were adjusted to the same potency against the lethal effect of B. asper venom in experiments involving preincubation of venom and antivenom. Then, “rescue” experiments were performed, i.e. antivenom was administered either intravenously or intramuscularly at various times after envenomation. IgG and F(ab′) 2 antivenoms were equally effective in the neutralization of lethality, both being more effective when administered i.v. than after i.m. injection. Neutralization decreased as the time lapse between envenomation and treatment increased. No significant differences were observed in the ability of antivenoms to neutralize defibrinating activity of B. asper venom in experiments involving independent injection of venom and antivenoms. There was a much higher accumulation of equine antibodies in muscle tissue that had been injected with B. asper venom than in non-envenomated tissue, indicating that venom-induced microvessel damage probably favors a prominent and similar extravasation of both IgG and F(ab′) 2 antibodies. This may explain the similar effectiveness of both types of antivenom in previously reported studies on the neutralization of venom-induced local tissue damage. Both IgG and F(ab′) 2 antivenoms activate human complement in vitro and induce an anti-equine immunoglobulin response in mice, indicating that Fc removal per se does not eliminate the potential for inducing adverse reactions. However, IgG antivenom had higher anticomplementary activity and induced a stronger anti-immunoglobulin response than F(ab′) 2 antivenom.

Inhibition of local hemorrhage and dermonecrosis induced by Bothrops asper snake venom: effectiveness of early in situ administration of the peptidomimetic metalloproteinase inhibitor batimastat and the chelating agent CaNa2EDTA.

The effectiveness of the chelating agent CaNa2EDTA and the peptidomimetic matrix metalloproteinase inhibitor batimastat (BB-94) to inhibit local tissue damage induced by Bothrops asper snake venom was studied in mice. Both compounds totally inhibited proteolytic, hemorrhagic, and dermonecrotic effects, and partially reduced edema-forming activity, when incubated with venom prior to injection. Much lower concentrations of batimastat than of CaNa2EDTA were required to inhibit these effects. In addition, batimastat, but not CaNa2EDTA, partially reduced myotoxic activity of the venom. When the inhibitors were administered at various time intervals after envenomation at the same site of venom injection, both compounds were effective in neutralizing local hemorrhage and dermonecrosis if administered rapidly after venom. Inhibition was not as effective as the time lapse between venom and inhibitor injections increased. Owing to the relevance of metalloproteinases in the pathogenesis of local tissue damage induced by B. asper and other pit viper venoms, it is suggested that administration of peptidomimetic metalloproteinase inhibitors or CaNa2EDTA at the site of venom injection may represent a useful alternative to complement antivenoms in the neutralization of venom-induced local tissue damage.

Leukocyte recruitment induced by Bothrops asper venom (BaV). Participation of complement system

Leukocyte recruitment induced by Bothrops asper venom (BaV). Participation of complement system

The ability of polyvalent antivenom to neutralize hyperalgesia induced by Bothrops asper venom

The ability of polyvalent antivenom to neutralize hyperalgesia induced by Bothrops asper venom

Increments in serum cytokine and nitric oxide levels in mice injected with Bothrops asper and Bothrops jararaca snake venoms

Changes in serum levels of several cytokines and nitric oxide were studied in BALB/c mice injected intraperitoneally with one median lethal dose ( ld 50) of the venoms of Bothrops asper and Bothrops jararaca, two of the medically most important poisonous snakes of Latin America. Despite differences observed in the time-course of cytokine increments and in serum cytokine levels, both venoms induced prominent elevations of TNF-α, IL-1, IL-6, IL-10 and IFN-γ. There was an early increase in TNF-α and IL-1, followed by a more pronounced increment by 18 h. IL-6 levels peaked between 4 and 6 h, and this cytokine probably modulates the secretion of TNF-α and IL-1 and the synthesis of acute-phase proteins. Both venoms induced an early increment in serum IL-10, whereas IFN-γ levels reached higher values in mice injected with B. jararaca venom than in those receiving B. asper venom. Serum nitric oxide concentration increased in mice injected with both venoms rapidly after envenomation, remaining elevated for 24 h. It is concluded that a complex pattern of cytokine and nitric oxide synthesis and secretion occurs in severe experimental envenomation by B. asper and B. jararaca venoms. Furthermore, it is suggested that some of these mediators, particularly TNF-α, IL-1 and nitric oxide, might play a relevant role in the pathophysiology of systemic alterations induced by these venoms.

Horse IgG isotypes and cross-neutralization of two snake antivenoms produced in Brazil and Costa Rica

Horse IgG isotypes and cross-neutralization of two snake antivenoms produced in Brazil and Costa Rica. Toxicon 000–000. This work compared the specificity, ELISA titers and IgG subclass content of the polyvalent antivenom (anti- Bothrops asper, Crotalus durissus durissus and Lachesis muta stenophrys) of Instituto Clodomiro Picado (Costa Rica) and the bothropic antivenom (anti- Bothrops jararaca, B. jararacussu, B. moojeni, B. neuwiedi and B. alternatus) of Instituto Butantan (Brazil). The role of IgG(T) and IgGa subclasses in neutralization of some venom toxic activities and the cross neutralization of the antivenoms against B. jararaca and B. asper venoms were also evaluated. Both antivenoms were able to recognize B. asper and B. jararaca venoms by immunoblotting and presented similar antibody titers when assayed by ELISA. IgG(T) was highest, followed by IgGa, IgGb and IgGc. IgGa and IgG(T) isotypes isolated from both antivenoms by affinity chromatography were tested for neutralization of lethal, hemorrhagic, coagulant and phospholipase A 2 activities of the homologous venoms. In both antivenoms, IgG(T) was the major isotype responsible for neutralization of all the tested activities, followed by IgGa. These results suggest that Instituto Butantan and Instituto Clodomiro Picado antivenoms have the same IgG profile and their neutralizing ability is due mostly to the IgG(T) isotype. Also, they neutralize lethality in mice induced by homologous and heterologous venoms, the bothropic antivenom of Instituto Butantan being more effective.

Purification and characterization of BaH4, a hemorrhagic metalloproteinase from the venom of the snake Bothrops asper

A hemorrhagic metalloproteinase, named BaH4, was isolated from the venom of the snake Bothrops asper by a combination of ion-exchange chromatography on DEAE-Sepharose and gel filtration on Sephacryl S-200. BaH4 is a 69 kDa protein with a pI of 5.3. It was recognized by antibodies raised against hemorrhagic metalloproteinase BaH1 isolated from B. asper venom, with a reaction of partial immunologic identity. BaH4 shows proteolytic activity on biotinylated casein, hide powder azure and fibrin, although having lower activity than crude B. asper venom and metalloproteinase BaP1 isolated from the same venom. BaH4 hydrolyzed fibronectin, laminin and type IV collagen in vitro, albeit at a relatively high enzyme:substrate ratio. Proteolytic activity was inhibited by chelating agents and 2-mercaptoethanol, but not by soybean trypsin inhibitor. Prominent hemorrhage developed in gastrocnemius and cremaster muscles after administration of BaH4. Moreover, it induced lethality in mice after intravenous injection, with an LD 50 of 0.37 μg/g. Histological observations showed conspicuous pulmonary hemorrhage when the enzyme was injected intravenously. BaH4 is a hemorrhagic metalloproteinase which may play a relevant role in local and systemic bleeding characteristic of B. asper envenomations.

Comparative study on the ability of IgG and Fab sheep antivenoms to neutralize local hemorrhage, edema and myonecrosis induced by Bothrops asper (terciopelo) snake venom

The ability of sheep antivenoms, consisting of whole IgG molecules or Fab fragments, to neutralize local hemorrhage, edema and myonecrosis induced by Bothrops asper venom was comparatively studied in mice. The two antivenoms were produced from the same batch of hyperimmune plasma and were adjusted to the same neutralizing potency against these effects in assays where venom and antivenoms were incubated prior to injection. Thus, if differences are observed in experiments involving independent injection of venom and antivenoms, they would depend on the pharmacokinetic profiles of the products. Despite the observation that both antivenoms neutralized the three effects if preincubated with venom, neutralization was only partial when antivenoms were administered i.v. at various time intervals after envenomation. No significant differences were observed between IgG and Fab antivenoms concerning neutralization of hemorrhagic and edema-forming activities, whereas IgG antivenom was slightly more effective in neutralizing myotoxic activity in experiments involving independent injection of venom and antivenom. These results do not support the hypothesis that Fab fragments are more effective than whole IgG molecules in the neutralization of locally-acting toxins from B. asper venom.

Characterization of a basic phospholipase A 2-homologue myotoxin isolated from the venom of the snake Bothrops neuwiedii (yarará chica) from Argentina

A basic protein was isolated by CM-Sephadex C-25 chromatography from the venom of Bothrops neuwiedii from Argentina, and named B. neuwiedii myotoxin I. This protein exerted local myotoxic and edema-forming effects in mice, with potencies comparable to other myotoxins isolated from Bothrops spp. venoms. When injected by i.v. route at doses up to 4.7 mg/kg of body weight, the toxin was not lethal. In vitro, the toxin had no detectable phospholipase A 2 activity on egg yolk phospholipids. B. neuwiedii myotoxin I appeared as a homodimer in sodium dodecylsulphate–polyacrylamide gel electrophoresis, with a subunit molecular weight of 15 kD. Gel immunodiffusion revealed a pattern of partial antigenic identity between the newly isolated myotoxin and myotoxin II from Bothrops asper venom. The sequence of B. neuwiedii myotoxin I was determined for the first 40 amino acid residues, showing high homology to several class II phospholipase A 2 myotoxins of the Lys-49 family from crotalids. Altogether, results suggest that this toxin is a new member of the Lys-49 phospholipase A 2-homologues with myotoxic, cytolytic, and edema-inducing activities.

Vascular endothelial growth factor VEGF-like heparin-binding protein from the venom of Vipera aspis aspis (Aspic viper).

The heparin-binding dimeric hypotensive factor (HF) was purified from Vipera aspis aspis (Aspic viper) venom [Komori, Y. and Sugihara, H. (1990) Toxicon 28, 359-369]. In this study, the amino acid sequence, and structure and function of HF, were elucidated. By electrospray ionization mass spectrometry (ESI-MS), the molecular weight of HF was determined to be 25 072.1. The complete amino acid sequence of HF was determined by Edman sequencing of the S-pyridylethylated HF and its peptides derived from enzymatic digestion. The theoretical molecular mass calculated from the primary structure agrees well with the molecular weight determined by ESI-MS. HF consists of two homogeneous monomers bound covalently. The monomer with an N-terminal blocked by pyroglutamic acid contains 110 amino acid residues, including eight cysteine residues, two of which are considered to be involved in intermolecular disulfide bonds. Sequential homology search revealed that the primary structure of HF is similar to that of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) with a sequential homology of 45 and 22%, respectively. When injected intradermally into a rat, an increase in capillary permeability was observed with HF or VEGF. On the other hand, only HF exerted a strong hypotensive effect after intravenous injection of samples into a rat. Purified HF has a mitogenic effect on endothelial cells. Through the use of bovine aortic endothelial cells (BAEC), the half-maximal mitogenic concentration of HF was determined to be 5-5. 5 nM (125-138 ng/mL). Similarly, VEGF had a mitogenic concentration at 0.5-1 nM. When incubated with HF and cycloheximide or HF and heparin, the cell growth was inhibited, suggesting that the mechanism of action of HF is similar to that of VEGF.

Citrate inhibition of snake venom proteases

Thirty snake venoms had a citrate content of 2.3 to 12.9%, dry basis, by an aconitase–isocitric dehydrogenase coupled enzyme assay. This is a venom concentration range of approximately 30 to 150 mM citrate assuming 25% venom solids content. Inhibition of snake venom protease activity by the addition of exogenous citrate was obtained using azure blue hide powder and azocasein as substrates. Protease inhibitions of 7.5% for Crotalus atrox venom to 78% for Bothrops picadoi venom were observed with citrate. Complete inhibition of snake venom protease activity by citrate was not observed. Bothrops asper (Pacifico) venom showed a 41% protease inhibition by citrate with azocasein as the substrate and 46% inhibition of Bothrops asper (Alantico) venom protease with azure blue hide power as a substrate. Trypsin was not inhibited in this system. Citrate may inhibit some venom protease activity by forming a complex with the zinc of zinc-dependent enzymes.

Pharmacological modulation of edema induced by Lys-49 and Asp-49 myotoxic phospholipases A 2 isolated from the venom of the snake Bothrops asper (terciopelo)

The pharmacological modulation of edema-forming activity of Bothrops asper myotoxins II and III, Lys-49 and Asp-49 phospholipases A 2, respectively, was studied plethysmographically in the mouse foot pad model. Myotoxin III had phospholipase A 2 activity, whereas myotoxin II was devoid of enzymatic activity when tested on egg yolk phosphatidylcholine. Both toxins induced a dose-dependent edema of rapid onset. Chemical modification of myotoxin III with p-bromophenacyl bromide abrogated enzymatic activity and significantly reduced edema-forming activity, although a residual effect remained. Pretreatment of animals with diphenhydramine, dexamethasone, indomethacin and prazosin significantly reduced the effect of both myotoxins. It is concluded that (a) these myotoxins are important edema-forming components of B. asper venom, (b) enzymatic activity is not a strict requirement to exert this effect, although in the case of myotoxin III it contributes to its development, and (c) several inflammatory mediators participate in mouse foot pad edema induced by these myotoxins.

Neutralization of local tissue damage induced by Bothrops asper (terciopelo) snake venom

Local tissue damage represents a serious consequence of Bothrops asper envenomations. It encompasses a complex series of alterations, including myonecrosis, dermonecrosis, hemorrhage and edema. Due to its rapid development it is difficult to neutralize by antivenoms, especially if there is a delay in serotherapy. Experimental studies with this venom and the polyvalent (Crotalinae) antivenom produced in Costa Rica indicate that antivenom is effective in neutralizing these toxic activities when incubated with the venom prior to injection. However, if venom and antivenom are injected independently in mice, neutralization of these effects is only partial. Moreover, neutralization is not complete even if homologous or heterologous antibodies are present in the circulation before venom is injected. Despite differences in their pharmacokinetic profiles, equine whole IgG and F(ab′) 2 antivenoms show similar efficacy in the neutralization of edema, hemorrhage and myonecrosis induced by B. asper venom, suggesting that the use of antivenoms made of antibody fragments may not improve neutralization of these effects. This is due, at least in part, to the fact that microvessel disruption by venom components favors a similar antibody concentration in the affected tissues. Recent advances in the development of neutralizing substances of rapid diffusion, that could be injected locally in the field, may contribute to the neutralization of metalloproteinases and phospholipases A 2. In addition, the rapid administration of antivenoms with high antibody titers against locally-acting toxins is very important in the treatment of these effects.

Inhibition by CaNa 2EDTA of local tissue damage induced by Bothrops asper (terciopelo) venom: Application in horse immunization for antivenom production

The ability of the chelating agent CaNa 2EDTA to inhibit local tissue damage induced by Bothrops asper venom was studied in mice and in horses used for polyvalent (Crotalinae) antivenom production. CaNa 2EDTA was devoid of toxicity when injected i.m. or s.c. inducing only a mild edema. Preincubation of B. asper venom with CaNa 2EDTA inhibited hemorrhagic and dermonecrotic activities, but did not reduce edema-forming and myotoxic effects. A group of horses initially immunized with native venoms developed less severe local tissue reactions when injected with booster doses of venom and CaNa 2EDTA than when receiving booster injections of venom alone, although they showed a similar antivenom response. Moreover, antivenoms produced from plasmas of horses that received booster injections of either venom alone or venom plus CaNa 2EDTA had similar neutralizing activity against lethal, hemorrhagic and coagulant effects induced by B. asper venom. The similar antibody response was corroborated by Western blotting using crude venom and by an ELISA that estimates anti-myotoxin titer. It is concluded that the chelating agent CaNa 2EDTA reduces the extent of local tissue damage induced by B. asper venom, without affecting the immune response of horses against pharmacologically-relevant venom components.

Immunoglobulin G and F(ab′) 2 polyvalent antivenoms do not differ in their ability to neutralize hemorrhage, edema and myonecrosis induced by Bothrops asper (terciopelo) snake venom

The ability of whole immunoglobulin G (IgG) and F(ab')2 polyvalent (Crotalinae) antivenoms to neutralize the hemorrhagic, edema-forming and myotoxic activities of Bothrops asper venom was studied. Both antivenoms were adjusted to the same neutralizing potency against lethal and hemorrhagic activities in experiments where venom and antivenoms were incubated before injection. Thus, in these experimental conditions, differences in the neutralizing ability in experiments involving independent injection of venom and antivenoms would depend mainly on the different pharmacokinetic profiles of whole IgG and F(ab')2 antivenoms. Experiments involving local injection of venom followed by intravenous administration of antivenom at either 0, 15 or 30 min after envenomation did not reveal any significant difference between whole IgG and F(ab')2 products concerning neutralization of hemorrhage, edema and myonecrosis induced by B. asper venom. The three effects were neutralized by antivenoms only to a partial extent and neutralization decreased as the time-lapse between envenomation and antivenom administration increased. Moreover, with the exception of one time-interval, no significant differences in the neutralization of hemorrhage were observed when antivenom was administered by the intramuscular or intraperitoneal route. The results do not support the assumption that F(ab')2 antivenom is more effective than whole IgG antivenom in the neutralization of local hemorrhage, edema and myonecrosis induced by B. asper venom in mice.

Crystal structure of myotoxin-11: a myotoxic phospholipase A2 - homologue from bothrops moojenivenom

Abstract: The crystal structure ofMyotoxin-II (MjTX-II), a Lys49 PLAi-homologue from Bothrops moojeni venom has been determined and refined at 2.0 A to a crystallographic residual of 19.7% (Ru.0=28.1%). MjTX-II is a dimer in the crystal, with the monomers in the asymmetric unit related by a two-fold symmetry axis running through the dimer interface. The dimers of MjTX-II and the Lys49 PLA2 from B. asper venom are similar, however the relative orientations of the monomers suggests a flexible dimer interface, which serves as a hinge between the two molecules.

Inhibition of the hemorrhagic activity of Bothrops asper venom by a novel neutralizing mixture

This study screened 25 sera, 19 synthetic products and five antivenoms obtained after immunization for their ability to neutralize the hemorrhagic activity of venom from the snake Bothrops asper. Among the sera screened, the homologous serum of B. asper itself was found to possess the highest neutralizing capacity, abolishing the hemorrhagic effect of the venom at weight ratio of 3:1. It was more efficient than the antisera obtained by immunization. Among the synthetic compounds tested, only O-phenanthroline and EDTA salts inhibited the hemorrhagic activity at concentrations of 0.5–10 mM; however, only CaNa 2EDTA was non-toxic at the concentrations studied. Intravenous injections and in situ administration of the non-toxic inhibitors revealed that a fraction of B. asper serum, the horse polyvalent antivenom and CaNa 2EDTA were the most potent antihemorrhagic materials against B. asper venom, especially when administered in situ as a mixture. This work suggests that this neutralizing mixture could be highly useful in the neutralization of local and systemic hemorrhage developing after B. asper envenomation.

Immunoreactivity of a new generation of horse F(ab′) 2 preparations against European viper venoms and the tetanus toxin

The immunoreactivity of the current and the new more purified, pasteurized preparations of horse F(ab′) 2 against the tetanus toxin and Vipera aspis venom was investigated with a biosensor based on technology using the optical phenomenon of surface plasmon resonance. Immunoreactivity data were compared with seroneutralization titres to investigate immunoreactivity-immunoprotection efficacy relationships. The association-dissociation rate and affinity constants of the current and the new tetanus toxin-specific F(ab′) 2 preparations were similar, at about 10 4 M −1 sec −1, 10 −4 sec −1 and 10 8 M −1, respectively. Similar values were found using a solid immunoradiometric assay. To assess the immunoreactivity of V. aspis venom-specific horse F(ab′) 2, the mol. wt and percentage of the antigenic fractions of V. aspis venom were determined. Western blotting of electrophoresis gels showed four antigenic fractions of V. aspis venom (mol. wts 17,500, 28,500, 32,000 and 60,000), which represented 6, 3.4, 17.7 and 5% of total venom, respectively. Association and dissociation rate constants were in the same range as those of the tetanus toxin-F(ab′) 2 interactions for each of the four antigenic fractions. Seroneutralization of both tetanus toxin and V. aspis by the corresponding specific F(ab′) 2 showed that the ld 50 mg −1 protein was 1.76-fold and 1.51-fold higher with the new than with the current preparations, respectively. These improvements in efficacy were in close agreement with the higher immunoreactive fraction ratios, which were 2-fold and 1.8-fold higher with the new preparations. These results demonstrate that the removal of non-IgG T immunoglobulins and the pasteurization treatment have no overall influence on F(ab′) 2 affinity but improve the specific activity of these new antitoxin horse F(ab′) 2.

Chemical modification of histidine and lysine residues of myotoxic phospholipases A 2 isolated from Bothrops asper and Bothrops godmani snake venoms: Effects on enzymatic and pharmacological properties

Lysine and histidine residues of two myotoxic phospholipases A 2, Bothrops asper myotoxin III and Bothrops godmani myotoxin I, were chemically modified in order to study the effects of these treatments on enzymatic and pharmacological properties. After lysine acetylation the overall basicity of these toxins was lost and their enzymatic activity was significantly reduced, although a residual effect remained, which corresponded to 25% of the activity of native toxins. This treatment abolished both myotoxic and anticoagulant effects, and partially reduced liposome-disrupting activity. Histidine alkylation with P-bromophenacyl bromide affected phospholipase A 2, myotoxic and anticoagulant effects in a parallel way. After 24 hr of incubation with the alkylating reagent, these three activities were totally inhibited, in contrast to the liposome-disrupting effect which was only partially affected by this treatment. It is suggested that: (1) catalytic activity plays a role in the pharmacological effects of these myotoxins; (2) lysine residues are relevant for the toxic effects induced by these phospholipases A 2; and (3) despite the apparent relevance of enzymatic activity to the pharmacological properties of these toxins, the dissociation observed in lysine acetylation experiments suggests that these myotoxins have a molecular region, different from the catalytic site, which might be also involved in the toxic effects observed.