A potent vasoactive cytolysin isolated from Scorpaena plumieri scorpionfish venom

A new vasoactive cytolytic toxin, referred to as Sp-CTx, has been purified from the venom of the scorpionfish Scorpaena plumieri by a combination of gel filtration and anion exchange chromatographies. An estimation of Sp-CTx native molecular mass, performed by size exclusion chromatography, demonstrated that it is a 121 kDa protein. Further physicochemical studies revealed its glycoproteic nature and dimeric constitution, comprising subunits of approximately 65 kDa (MALDI-TOF-MS). Such protein has proved to possess a potent hemolytic activity on washed rabbit erythrocytes (EC₅₀ 0.46 nM), whose effect was strongly reduced after treatment with antivenom raised against stonefish venom - Synanceja trachynis (SFAV). This cross-reactivity has been confirmed by western blotting. Like S. plumieri whole venom (100 μg/mL), Sp-CTx (1-50 nM) caused a biphasic response on phenylephrine pre-contracted rat aortic rings, characterized by an endothelium- and dose-dependent relaxation phase followed by a contractile phase. The vasorelaxant activity has been abolished by l-NAME, demonstrating the involvement of nitric oxide on the response. We report here the first isolation of a cytolytic/vasoactive protein from scorpionfish venom and the data provided suggest structural and functional similarities between Sp-CTx and previously published stonefish hemolytic toxins.     

Systemic response induced by Scorpaena plumieri fish venom initiates acute lung injury in mice

Scorpaena plumieri venomous fish inflicted severe injuries in humans characterized by systemic effects and cardiovascular abnormalities. Although cardiotoxic and hypotensive effects induced in rats by this venom have been studied, little is known about their effect on bronchial epithelial permeability and airway inflammation in mice. The primary goal of this study was to determine whether the intraplantar or intraperitoneal injection of S. plumieri venom results in systemic response, and whether this event initiates acute lung injury. We found that BALB/c mice developed neutrophilic infiltrates, areas of lung hemorrhage and alveolar macrophage activation within 24h after injection with S. plumieri venom. These histopathological changes were associated with an early increase in BAL fluid protein and early induction of cytokines, chemokines and matrix metalloproteinases, followed by a later increase in BAL fluid neutrophils. These findings provide clear evidence that the injection of S. plumieri venom in footpad or peritoneal cavity of mice results in venom deposition in the airway and initiates a sustained inflammatory response in the lungs.     

Biological properties of the venom from the scorpionfish (Scorpaena plumieri) and purification of a gelatinolytic protease.

In this work we describe some biological properties and a partial biochemical characterization of the Scorpanea plumieri crude venom. The fresh venom induced a decrease in blood pressure, cardiac and respiratory frequency, and exhibited hemorrhagic, hemolytic and proteolytic activities. The LD(50) (i.v. mouse) was 0.28 mg/kg. The pharmacological activities were found to be very unstable and this fact could be associated with proteolytic activity. Enzymes which hydrolyze casein and gelatin were found in this venom. A gelatinolytic protease (Sp-GP) was purified to homogeneity from S. plumieri venom through a combination of three chromatographic steps: gel filtration on Sephacryl S-200; ion exchange on DEAE-cellulose and reverse-phase/HPLC on a Vydac C4 column. The purified protease was approximately 2% of the whole protein in the soluble crude venom. The molecular mass of the Sp-GP scorpionfish gelatinase estimated by SDS-PAGE was around 80,000 Da under reducing conditions and 72,000 Da under non-reducing conditions. Attempts to determine the N-terminal sequence by automatic Edman degradation were unsuccessful, probably due to blockage of the N-terminal group. Gelatinolytic activity was optimal at pH 7-8. This is the first report of the isolation and characterization of a scorpionfish venom protease.     

Local inflammatory events induced by Bothrops atrox snake venom and the release of distinct classes of inflammatory mediators

Bothrops atrox is responsible for most accidents involving snakes in the Brazilian Amazon and its venom induces serious systemic and local effects. The local effects are not neutralized effectively by commercial antivenoms, resulting in serious sequelae in individuals bitten by this species. This study investigates the local inflammatory events induced in mice by B. atrox venom (BaV), such as vascular permeability, leukocyte influx and the release of important inflammatory mediators such as cytokines, eicosanoids and the chemokine CCL-2, at the injection site. The effect of BaV on cyclooxygenase (COX-1 and COX-2) expression was also investigated. The results showed that intraperitoneal (i.p.) injection of BaV promoted a rapid and significant increase in vascular permeability, which reached a peak 1 h after venom administration. Furthermore, BaV caused leukocyte infiltration into the peritoneal cavity between 1 and 8 h after i.p. injection, with mononuclear leukocytes (MNs) predominating in the first 4 h, and polymorphonuclear leukocytes (PMNs) in the last 4 h. Increased protein expression of COX-2, but not of COX-1, was detected in leukocytes recruited in the first and fourth hours after injection of BaV. The venom caused the release of eicosanoids PGD₂, PGE₂, TXA₂ and LTB₄, cytokines TNF-α, IL-6, IL-10 and IL-12p70, but not IFN-γ, and chemokine CCL-2 at different times. The results show that BaV is able to induce an early increase in vascular permeability and a leukocyte influx to the injection site consisting mainly of MNs initially and PMNs during the later stages. These phenomena are associated with the production of cytokines, the chemokine CCL-2 and eicosanoids derived from COX-1 and COX-2.     

Histopathological changes induced by Hemiscorpius lepturus scorpion venom in mice

Envenomation by Hemiscorpius lepturus (H. lepturus) is associated with local necrosis, followed by systemic manifestations. In this work the LD₅₀ of H. lepturus venom were determined by subcutaneous (SC) injection in white Balb/c mice (5 mg/kg). Histopathological alterations in organs such as kidney, heart, liver, lungs, stomach and intestine were determined in 3, 6, 12 and 24 h following experimental (SC) envenoming injection of one LD₅₀ of the venom in Balb/c mice. Histological studies showed degenerative changes in the kidney with disorganized glomeruli and necrotic tubular in 3 h and reached to its climax in 6 h. Myocardium showed massive myocytolysis with interstitial necrosis in 3 h and reached to its peak after 6 h past envenoming. Bowels showed edema of lamina propria and slight villous necrosis. The enzymatic activities of creatine kinase (CK) and lactate dehydrogenase (LDH) were significantly increased in the serum in 9 h. No necrotic lesion observed in lungs and liver. The results indicate that the venom of H. lepturus is a highly cytotoxic, and induces massive tissue damages in specific organs, starting from the heart and kidney as the first target in 3 h and ends to the bowels in 6 h post envenomation.     

Effects induced by Apis mellifera venom and its components in experimental models of nociceptive and inflammatory pain

The effects induced by Apis mellifera venom (AMV), melittin-free AMV, fraction with molecular mass < 10 kDa (F_<10) or melittin in nociceptive and inflammatory pain models in mice were investigated. Subcutaneous administration of AMV (2, 4 or 6 mg/kg) or melittin-free AMV (1, 2 or 4 mg/kg) into the dorsum of mice inhibited both phases of formaldehyde-induced nociception. However, F_<10 (2, 4 or 6 mg/kg) or melittin (2 or 3 mg/kg) inhibited only the second phase. AMV (4 or 6 mg/kg), but not F_<10, melittin-free AMV or melittin, induced antinociception in the hot-plate model. Paw injection of AMV (0.05 or 0.10 mg), F_<10 (0.05 or 0.1 mg) or melittin (0.025 or 0.050 mg) induced a nociceptive response. In spite of inducing nociception after paw injection, scorpion (Tityus serrulatus) or snake (Bothrops jararaca) venom injected into the dorsum of mice did not inhibit formaldehyde-induced nociception. In addition, AMV (6 mg/kg), but not F_<10 (6 mg/kg) or melittin (3 mg/kg), inhibited formaldehyde paw oedema. Concluding, AMV, F_<10 and melittin induce two contrasting effects: nociception and antinociception. AMV antinociception involves the action of different components and does not result from non-specific activation of endogenous antinociceptive mechanisms activated by exposure to noxious stimuli.     

Characterization of inflammatory reaction induced by neuwiedase, a P-I metalloproteinase isolated from Bothrops neuwiedi venom

The Snake Venom Metalloproteinases (SVMPs) play a relevant role in the multifactorial inflammatory response induced by Bothrops envenomations. Neuwiedase, an SVMP isolated from Bothrops neuwiedi venom, is devoid of hemorrhagic activity on skin tests, but is able to induce myonecrosis and degrade fibrinogen, fibrin, type I collagen, fibronectin and laminin. In this study, we analyzed the inflammatory reaction induced by neuwiedase in gastrocnemius muscle, with special focus on cytokines release. Our results showed clear evidence of inflammatory infiltrate in the gastrocnemius muscle and an increase of MMP-9, and the cytokines KC, IL-1β and IL-6 in the early periods after toxin injection. The cytokine release was also evaluated in inflammatory and muscular cell culture. Both murine peritoneal adherent cells (MPACs) and muscle cells (C2C12) released pro-inflammatory cytokines after stimulus with neuwiedase. MPACs showed increased production of KC, IL-1β and IL-6 in the cell culture supernatant while in C2C12, the predominant chemokine expressed was KC. These data reinforce the importance of SVMPs in the inflammatory response caused by envenomation and point out the role of muscle cells in this event by releasing pro-inflammatory mediators able to attract leukocytes to the muscle, thus starting and amplifying the setting of the inflammatory reaction.     

Inflammation induced by Bothrops asper venom

Inflammation is a major characteristic of envenomation by snakes from viperine and crotaline species. Bothrops asper snake venom elicits, among other alterations, a pronounced inflammatory response at the site of injection both in humans and experimental animals. This review describes the current status of our understanding of the inflammatory reaction, including pain, triggered by Bothrops asper venom. The experimental studies on the action of this venom as well as the complex network of chemical mediators involved are summarized. Moreover, aspects of the molecular mechanisms orchestrating this important response to envenomation by Bothrops asper are presented. Considering that isolated toxins are relevant tools for understanding the actions of the whole venom, studies dealing with the mechanisms of inflammatory and nociceptive properties of phospholipases A₂, a metalloproteinase and serine-proteases isolated from Bothrops asper venom are also described.     

Correlation between blood pressure, cytokines and nitric oxide in conscious rabbits injected with Leiurus quinquestriatus quinquestriatus scorpion venom

Activation of the inflammatory response with the release and activation of pro-inflammatory cytokines is among the factors thought to be important in the pathogenesis of many deleterious inflammatory effects seen in case of scorpion envenomation. The released inflammatory mediators interact in the body with a large number of proteins and receptors; this interaction determines the eventual inflammatory effect of the venom. Thus, in the present study an attempt was made to map the time course of scorpion envenomation and correlate the effects observed on the cardiovascular and respiratory systems with the changes that could take place in the levels of selected cytokines and nitric oxide during the course of experimental envenomation. New Zealand white male conscious rabbits were prepared for blood pressure recording. Arterial blood pressure was measured from the left central ear artery while a cannula was inserted into the right central ear artery and blood samples collected at different time interval after venom injection for biochemical and hematological analyses. In general, subcutaneous injection of Leiurus quinquestriatus quinquestriatus venom caused a significant (P ± 0.05) triphasic effect on BP consisting of an initial transient reduction, followed by an increase that peaked 2 h after venom injection, and a gradual terminal hypotensive phase. The significantly high serum level of IL8, TNFα (P < 0.001) and nitric oxide (P < 0.0001) observed in the present study supports the evidence for the role of these potent vasodilators in the terminal hypotension that is usually observed in humans and animals after envenomation.     

Inflammatory mediators generated at the site of inoculation of Loxosceles gaucho spider venom

Patients bitten by Loxosceles spiders generally manifest marked local inflammatory reaction and dermonecrosis. This report evaluated edema formation, leukocyte infiltration and release of inflammatory mediators at the injection site of Loxosceles gaucho venom. BALB/c mice were i.d. injected with venom and thereafter paws were disrupted and homogenized to obtain differential counts of migrated cells, as well to assay the levels of cytokines, chemokines and lipid mediators. Increased footpad thickness was detected as soon as 30 min after venom injection, and 24 h later was similar to that of the control group. Loxosceles venom mildly augmented the recruitment of leukocytes to the footpad in comparison with PBS-injected mice. Moreover, it stimulated the release of IL-6, MCP-1 and KC at 2 and 24 h after venom injection. In addition, higher levels of PGE₂ were detected 30 min after venom injection in comparison with control group. However, the venom failed to increase levels of IL-1β, TNF-α, TXB₂ and LTB₄. Our results demonstrate that L. gaucho venom evokes an early complex inflammatory reaction, stimulating the secretion of pro-inflammatory cytokines and lipid mediators (PGE₂), and recruiting leukocytes to the footpad which contribute to the local reaction induced by L. gaucho venom.     

Biochemical characterization of the Micrurus pyrrhocryptus venom

Snake venom toxicity is the consequence of a combination of peptides and proteins whose identification and characterization are of great importance to understand envenomation and develop new clinical treatments. The Elapinae subfamily includes coral snakes whose bite causes mainly neurotoxic effects which disable muscle contraction and paralyse the heart as well as inhibit respiration. However, the structure-function relationship of venom toxins has been investigated only for a few species. We herein study biological aspects of the Micrurus pyrrhocryptus venom such as LD₅₀, hemorrhagic, necrotic, coagulant, myotoxic and hemolytic activity as well as the ability of venom components to compete with alpha-Bungarotoxin for the ligand-binding site of the nicotinic acetylcholine receptor. Besides, we report the determination of the molecular mass and N-terminal sequence of toxins including PLA2s, short, long and weak neurotoxins. The complete sequence of one of the short neurotoxins has also been obtained, this being the first sequence of an alpha-neurotoxin determined in the M. pyrrhocryptus venom and one of the few fully determined in members of the Micrurus genus.     

A pro-inflammatory profile of endothelial cell in Lonomia obliqua envenomation

Lonomia obliqua envenomation is characterized by intense local inflammatory reaction, which, dependent on the severity of the case, is followed by severe clinical manifestations related to hemorrhagic disorders that can lead to fatal outcome. These effects were imputed to several toxins present in L. obliqua venom, which are responsible for pro-coagulant, anticoagulant as well as antithrombotic activities, being also able to interfere with vascular cells functions. In this work, the intravital microscopy analysis show that after administration of low doses of L. obliqua venom (1-3 μg/ml) on hamster cheek pouch, there was no alterations neither on arterioles or venules caliber nor in the vascular permeability up to 30 min. However, after 10 min in contact with venom occurred a clear activation in the vascular bed, characterized by an increase in leukocyte rolling and adhesion on endothelium of hamster cheek pouch venules. A confocal analysis of vascular beds, confirmed these results showing an increase in endothelial E-selectin and VCAM-1 expression. The effects of L. obliqua venom on human endothelial cell (EC) in vitro were also investigated. The treatment of EC with venom (1-3 μg/ml) did not affect cell viability. However, at concentrations as low as 3 μg/ml of L. obliqua venom modifies actin cytoskeleton dynamics, and increases focal adhesion contacts, inducing stress fiber formation, focal adhesion kinase (FAK) phosphorylation and its subsequent association to actin. These effects are followed by the activation of NF-κB pathway, a critical signaling in several events associated to vascular inflammation. Accordingly, L. obliqua venom leads to a significant increase in COX-2, NOS-2, HO-1, MMP-2 and MMP-9 expression. Taken together the data show that, even at low concentrations, L. obliqua venom can activate endothelial cells, which assume a pro-inflammatory profile, contributing for local effects and probably also for systemic disturbances due to its ability to modulate the properties of the vascular system.     

Influence of sphingomyelin and TNF-alpha release on lethality and local inflammatory reaction induced by Loxosceles gaucho spider venom in mice.

It is well known that Loxosceles venom induces local dermonecrosis in rabbits, guinea pigs and humans but not in mice, although, depending on the dose, Loxosceles venom can be lethal to mice. In this work we demonstrate that mice injected intradermally in the dorsal area of the back can survive a lethal dose of Loxosceles gaucho venom and also develop an inflammatory reaction (with infiltration of leukocytes shown by histological analysis) at the local injection site when the venom is co-administered with sphingomyelin. It was observed that more venom was retained for a longer period of time at the local injection site when venom was co-administered with sphingomyelin. The presence of exogenous sphingomyelin did not influence significantly the release of TNF-alpha induced by L. gaucho venom. These results suggest that the action of venom on sphingomyelin, producing ceramide phosphate, causes the development of an inflammatory reaction, which in turn traps the venom in the local area for a long period of time and does not allow it to disperse systemically in a dose sufficient to cause death. Our findings also indicate that the size and availability of the local sphingomyelin pool may be important in determining the outcome of Loxosceles envenoming in different mammalian species.     

Mediators involved in the febrile response induced by Tityus serrulatus scorpion venom in rats.

Tityus serrulatus venom (Tsv) was intraperitoneally (ip) injected at doses of 75, 150 and 300mug/kg and IL-1beta (2.0 microg/kg) was given intravenously (iv) to male Wistar rats. Rectal temperature was measured by radiotelemetry. Vagotomy was performed according to Bluthe et al. [1994. Lipopolysaccharide induces sickness behaviour in rats by a vagal mediated mechanism. C R Acad. Sci. 317(6), 499-503]. Cerebrospinal fluid (CSF) and peritoneal fluid (PF) levels of bradykinin (BK) were measured by ELISA. B(1) (des-Arg(9)-[Leu(8)]-BK; DALBK) and B(2) kinin receptor (icatibant) antagonists (1.0 mg/kg each), the induced nitric oxide synthase inhibitor aminoguanidine (50.0 mg/kg), the neuronal nitric oxide synthase inhibitor 7-nitroindazole (30.0 mg/kg), the dual cyclooxygenase inhibitor ibuprofen (10.0 mg/kg), the selective interleukin-1 receptor antagonist IL-ra (2.0 mg/kg) and dipyrone (120 mg/kg) were given ip. Celecoxib (5 mg/kg) was given per os (po). Tsv at doses of 75 microg/kg evoked no change in rectal temperature while at doses of 150 and 300 microg/kg it promoted long-lasting fever (2 degrees C+/-0.1). Tsv (150 microg/kg) increased by nearly 3 and 5 times, respectively BK concentration in the CSF and in the PF. Subdiaphragmatic vagotomy or 7-nitroindazole reduced, icatibant, DALBK, IL-1ra, aminoguanidine and dipyrone abolished, while ibuprofen and celecoxib failed to affect Tsv-induced fever. These results suggest that PGs do not play a relevant role, whereas, kinins via their B(1) and B(2) receptors, IL-1, nitric oxide and vagal neurotransmission are involved in Tsv-induced fever.     

Effect of low-level laser therapy in the inflammatory response induced by Bothrops jararacussu snake venom.

This article reports the effect of low-level laser therapy (LLLT) on the edema formation and leukocyte influx caused by Bothrops jararacussu snake venom as an alternative treatment for Bothrops snakebites. The inflammatory reaction was induced by injection of 0.6 mg/kg of B. jararacussu venom, in gastrocnemius muscle. Cell influx and edema were evaluated at 3 or 24h after venom injection. Mice were irradiated at the site of injury by a low-level laser (685 nm) with a dose of 4.2J/cm(2). A therapy that combines LLLT and antivenom was also studied. B. jararacussu venom caused a significant edema formation 3 and 24h after its injection, and a prominent leukocyte infiltrate composed predominantly of neutrophils at 24h after venom inoculation. LLLT significantly reduced edema formation by 53% and 64% at 3 and 24h, respectively, and resulted in a reduction of neutrophils accumulation (P<0.05). The combined therapy showed to be more efficient than each therapy acting separately. In conclusion, LLLT significantly reduced the edema and leukocyte influx into the envenomed muscle, suggesting that LLLT should be considered as a potentially therapeutic approach for the treatment of the local effects of Bothrops species.     

Hemostatic properties of Venezuelan Bothrops snake venoms with special reference to Bothrops isabelae venom

In Venezuela, Bothrops snakes are responsible for more than 80% of all recorded snakebites. This study focuses on the biological and hemostatic characteristics of Bothrops isabelae venom along with its comparative characteristics with two other closely related Bothrops venoms, Bothrops atrox and Bothrops colombiensis. Electrophoretic profiles of crude B. isabelae venom showed protein bands between 14 and 100 kDa with the majority in the range of 14-31 kDa. The molecular exclusion chromatographic profile of this venom contains five fractions (F1-F5). Amidolytic activity evaluation evidenced strong thrombin-like followed by kallikrein-like activities in crude venom and in fractions F1 and F2. The fibrinogenolytic activity of B. isabelae venom at a ratio of 100:1 (fibrinogen/venom) induced a degradation of Aα and Bβ chains at 15 min and 2 h, respectively. At a ratio of 100:10, a total degradation of Aα and Bβ chains at 5 min and of γ chains at 24 h was apparent. This current study evidences one of rarely reported for Bothrops venoms, which resembles the physiologic effect of plasmin. B. isabelae venom as well as F2 and F3 fractions, contain fibrinolytic activity on fibrin plate of 36, 23.5 and 9.45 mm²/μg, respectively using 25 μg of protein. Crude venom and F1 fraction showed gelatinolytic activity. Comparative analysis amongst Venezuelan bothropoid venoms, evidenced that the LD₅₀ of B. isabelae (5.9 mg/kg) was similar to B. atrox-Puerto Ayacucho 1 (6.1 mg/kg) and B. colombiensis-Caucagua (5.8 mg/kg). B. isabelae venom showed minor hemorrhagic activity, whereas B. atrox-Parguasa (Bolivar state) was the most hemorrhagic. In this study, a relative high thrombin-like activity was observed in B. colombiensis venoms (502-568 mUA/min/mg), and a relative high factor Xa-like activity was found in B. atrox venoms (126-294 mUA/min/mg). Fibrinolytic activity evaluated with 10 μg protein, showed that B. isabelae venom contained higher specific activity (50 mm²/μg) than B. colombiensis and B. atrox venoms, which should encourage the isolation of these fibrinolytic molecules to improve the quality of immunotherapy.     

Histophatological changes and inflammatory response induced by Tityus discrepans scorpion venom in rams

Anesthetized rams envenomed s.c. with 40 μg/kg Tityus discrepans scorpion venom developed fasciculation, hypothermia, polyuria, pulmonary wet rales, tachypnea, respiratory distress and arrhythmia. Rams developed a cascade of inflammation reactions, characterized by activation of macrophages, fibroblasts and neutrophils, neutrophil infiltration and aggregation, vasculitis, arteritis and abundant fibrin deposition. At the inoculation site, venom was detected by immunohistochemistry in the extra cellular matrix, lymphatic vessels’ and venules’ lumen, inside macrophages and surrounding nerves. Extra cellular matrix was degraded at the inoculation site perhaps by activated neutrophils. Envenoming produced hepatocytes with Mallory body-like vacuoles which may be due to the increased plasmatic levels of TNF-α and IL6. Venom produced degranulation and vacuolization of acinary cells as well as interstitial swelling and necrosis. Necrosis of the Langerhan's islets occurred occasionally. Lungs showed the most deleterious effects developing wall collapse and necrosis, diffuse injury of the alveolar capillary barrier, interstitial and alveolar fibrin deposits with strong neutrophil infiltration. Massive infiltration of lymphocytes and macrophage occurred in the intestinal submucose, to the point that it modified villi and intestinal folding morphology. Envenomation developed a marked leukocyte aggregation surrounding nerves at the inoculation site. This study reveals that beyond its neurotoxicity, Tityus venom produces a severe and widespread inflammatory syndrome, expressed as histopathological changes at the site of inoculation, as well as in remote organs such as pancreas, lungs, intestine and liver. Our results suggest that not all remote targets are directly affected by the venom but that, as proposed earlier, are modified by inflammation by products produced elsewhere.     

Comparison of the effect of Crotalus simus and Crotalus durissus ruruima venoms on the equine antibody response towards Bothrops asper venom: Implications for the production of polyspecific snake antivenoms

Antivenoms are preparations of immunoglobulins purified from the plasma of animals immunized with snake venoms. Depending on the number of venoms used during the immunization, antivenoms can be monospecific (if venom from a single species is used) or polyspecific (if venoms from several species are used). In turn, polyspecific antivenoms can be prepared by purifying antibodies from the plasma of animals immunized with a mixture of venoms, or by mixing antibodies purified from the plasma of animals immunized separately with single venom. The suitability of these strategies to produce polyspecific antibothropic-crotalic antivenoms was assessed using as models the venoms of Bothrops asper, Crotalus simus and Crotalus durissus ruruima. It was demonstrated that, when used as co-immunogen, C. simus and C. durissus ruruima venoms exert a deleterious effect on the antibody response towards different components of B. asper venom and in the neutralization of hemorrhagic and coagulant effect of this venom when compared with a monospecific B. asper antivenom. Polyspecific antivenoms produced by purifying immunoglobulins from the plasma of animals immunized with venom mixtures showed higher antibody titers and neutralizing capacity than those produced by mixing antibodies purified from the plasma of animals immunized separately with single venom. Thus, despite the deleterious effect of Crotalus sp venoms on the immune response against B. asper venom, the use of venom mixtures is more effective than the immunization with separate venoms for the preparation of polyspecific bothropic-crotalic antivenoms.     

Inflammatory oedema induced by Lachesis muta muta (Surucucu) venom and LmTX-I in the rat paw and dorsal skin

The ability of crude venom and a basic phospholipase A₂ (LmTX-I) from Lachesis muta muta venom to increase the microvascular permeability in rat paw and skin was investigated. Crude venom or LmTX-I were injected subplantarly or intradermally and rat paw oedema and dorsal skin plasma extravasation were measured. Histamine release from rat peritoneal mast cell was also assessed. Crude venom or LmTX-I induced dose-dependent rat paw oedema and dorsal skin plasma extravasation. Venom-induced plasma extravasation was inhibited by the histamine H₁ antagonist mepyramine (6 mg/kg), histamine/5-hydroxytriptamine antagonist cyproheptadine (2 mg/kg), cyclooxygenase inhibitor indomethacin (5 mg/kg), nitric oxide synthesis inhibitor l-NAME (100 nmol/site), tachykinin NK₁ antagonist SR140333 (1 nmol/site) and bradykinin B₂ receptor antagonist Icatibant (0.6 mg/kg). Platelet-activating factor (PAF) antagonist PCA4248 (5 mg/kg) had no effect. LmTX-I-induced skin extravasation was inhibited by cyproheptadine, mepyramine, indomethacin and PCA4248, while l-NAME and SR140333 had no effect. Additionally, both Lachesis muta muta venom and LmTX-I concentration-dependently induced histamine release from rat mast cells. In conclusion, Lachesis muta muta venom and LmTX-I increase microvascular permeability by mechanisms involving in vivo mast cell activation and arachidonic acid metabolites. Additionally, crude venom-induced responses also involve substance P, nitric oxide and bradykinin release, whether LmTX-I-induced responses involve PAF.     

BthMP: a new weakly hemorrhagic metalloproteinase from Bothrops moojeni snake venom

In this work, a new weakly hemorrhagic metalloproteinase (BthMP) was purified from Bothrops moojeni snake venom. This enzyme was homogeneous by native and SDS-PAGE. It showed a polypeptide chain of 23.5 kDa, pI = 7.1, and N-terminal blocked. BthMP is comprised of high proteolytic activity on casein, fibrin and bovine fibrinogen, with no coagulating, esterase or phospholipase A₂ activities; it was inhibited by EDTA, EGTA and 1,10-phenanthroline and maintained its activity on pH from 7.0 to 9.0 and temperature from 5-40 °C. Assays with metal ions showed that Ca²⁺ is an activator, whereas Zn²⁺ and Hg²⁺ inhibited about 50 and 80% of its activity, respectively. The edema evidenced the important role of the toxin in the inflammatory activity of the venom. BthMP also caused unclotting, and provoked histological alterations in the gastrocnemius muscle of mice inducing hemorrhage, necrosis and leukocytic infiltrate. The molecular mass and the inhibition assays suggest that the metalloproteinase BthMP belongs to class P-I of SVMPs.