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Abstract: Snakebite envenoming by Bothrops caribbaeus, an endemic viperid from the Lesser Antillean island of Saint Lucia, is clinically characterized by local tissue damage and systemic thrombosis that can lead to cerebral, myocardial or pulmonary infarctions and venous thromboses. Systemic effects (lethality, pulmonary hemorrhage, thrombocytopenia and coagulopathy) induced by intravenous (i.v.) administration of B. caribbaeus venom were studied in mice. The role of snake venom metalloproteinases (SVMPs) in these systemic alterations was assessed by inhibition with the chelating agent calcium disodium ethylenediaminetetraacetic acid (CaNa2EDTA). A snake C-type lectin-like (snaclec) and a type P-III hemorrhagic SVMP were isolated and characterized from this venom, and the effect of venom and the isolated snaclec on human platelet aggregation was studied in vitro. Results indicate that SVMPs play an important role in the overall toxicity of B. caribbaeus venom, being responsible for systemic hemorrhage and lethality, but not thrombocytopenia, whereas the isolated snaclec is involved in the thrombocytopenic effect. Both venom and snaclec induce platelet aggregation/agglutination. Moreover, the snaclec binds directly to glycoprotein Ib (GPIb) and induces agglutination in washed fixed platelets. On the other hand, B. caribbaeus venom hydrolyzed fibrinogen in vitro and induced a partial drop of fibrinogen levels with an increase in fibrin/fibrinogen degradation products (FDP) levels in vivo. The negative result for D-dimer (DD) in plasma is consistent with the lack of microscopic evidence of pulmonary thrombosis and endothelial cell damage. Likewise, no increments in plasma sE-selectin levels were detected. The absence of thrombosis in this murine model suggests that this effect may be species-specific. [Copyright &y& Elsevier]

Abstract: Non-enzymatic proteins from snake venoms play important roles in the immobilization of prey, and include some large and well-recognized families of toxins. The study of such proteins has expanded not only our understanding of venom toxicity, but also the knowledge of normal and disease states in human physiology. In many cases their characterization has led to the development of powerful research tools, diagnostic techniques, and pharmaceutical drugs. They have further yielded basic understanding of protein structure–function relationships. Therefore a number of studies on these non-enzymatic proteins had major impact on several life science and medical fields. They have led to life-saving therapeutics, the Nobel prize, and development of molecular scalpels for elucidation of ion channel function, vasoconstriction, complement system activity, platelet aggregation, blood coagulation, signal transduction, and blood pressure regulation. Here, we identify research papers that have had significant impact on the life sciences. We discuss how these findings have changed the course of science, and have also included the personal recollections of the original authors of these studies. We expect that this review will provide impetus for even further exciting research on novel toxins yet to be discovered. [Copyright &y& Elsevier]

Abstract: Phospholipases A2 (PLA2s) are abundant components of snake venoms, where they play toxic and digestive roles. Despite having a similar three-dimensional structure, venom PLA2s exert an amazing variety of toxic and pharmacological effects, which include neurotoxic, myotoxic, hemolytic, edematogenic, hyperalgesic, pro-inflammatory, hypotensive, platelet-aggregation inhibitory, anticoagulant, cytotoxic, and bactericidal activities. Toxinologists have made significant contributions to deciphering the structure, molecular evolution, mechanisms of action, receptors, role of enzymatic activity for toxicity, structural determinants of toxicity and selectivity, and the impact of these enzymes in the overall pathophysiology of snakebite envenoming. The present work highlights some of the most relevant contributions in the study of venom PLA2s, including the personal accounts of the authors of these studies. [Copyright &y& Elsevier]

Secretory phospholipasesA2 (sPLA2s) form a large family of structurally related enzymes widespread in nature. Herein, we studied the inhibitory effects of sPLA2s from Vipera lebetina (VLPLA2), Vipera berus berus (VBBPLA2), and Naja naja oxiana (NNOPLA2) venoms on (i) human platelets, (ii) four different bacterial strains (gram-negative Escherichia coli and Vibrio fischeri; gram-positive Staphylococcus aureus and Bacillus subtilis) and (iii) five types of cancer cells (PC-3, LNCaP, MCF-7, K-562 and B16-F10) in vitro. sPLA2s inhibited collagen-induced platelet aggregation: VBBPLA2 IC50 = 0.054, VLPLA2 IC50 = 0.072, NNOPLA2 IC50 = 0.814 μM. p-Bromophenacylbromide-inhibited sPLA2 had no inhibitory action on platelets. 36.17 μM VBBPLA2 completely inhibited the growth of gram-positive Bacillus subtilis whereas no growth inhibition was observed towards gram-negative Escherichia coli. The inhibitory action of sPLA2s (~0.7 μM and ~7 μM) towards cancer cells depended on both venom and cell type. VBBPLA2 (7.2 μM) inhibited significantly the viability of K-562 cells and the cell death appeared apoptotic. The sPLA2s exhibited no inhibitory effect towards LNCaP cells and some effect (8%-20%) towards other cells. Thus, already sub-μM concentrations of sPLA2s inhibited collagen-induced platelet aggregation and from the current suite of studied svPLA2s and test cells, VBBPLA2 was the most growth inhibitory towards Bacillus subtilis and K-562 cells. [ABSTRACT FROM AUTHOR]

Venoms consist of a complex mixture of toxic components that are used by a variety of animal species for defense and predation. Envenomation of mammalian species leads to an acute inflammatory response and can lead to the development of IgE-dependent venom allergy. However, the mechanisms by which the innate immune system detects envenomation and initiates inflammatory and allergic responses to venoms remain largely unknown. Here we show that bee venom is detected by the NOD-like receptor family, pyrin domain-containing 3 inflammasome and can trigger activation of caspase-1 and the subsequent processing and unconventional secretion of the leaderless proinflammatory cytokine IL-1β in macrophages. Whereas activation of the inflammasome by bee venom induces a caspase-1-dependent inflammatory response, characterized by recruitment of neutrophils to the site or envenomation, the inflammasome is dispensable for the allergic response to bee venom. Finally, we find that caspase-1-deficient mice are more susceptible to the noxious effects of bee and snake venoms, suggesting that a caspase-1-dependent immune response can protect against the damaging effects of envenomation. [ABSTRACT FROM AUTHOR]

Abstract: Recently, P2X7 receptor (P2X7R) has been found to contribute to the development of inflammatory pain, however, the role of spinal P2X7R is not clear. The present study was designed to determine the roles of spinal P2X7R in the bee venom (BV) model, characterized by multiple pain-related behaviors and obvious inflammatory edema. We determined the effects of P2X7R antagonist A438790 on BV-induced PSN, mechanical allodynia and inflammatory swelling. Pre-treatment with intrathecal administration of A438079 significantly inhibited BV-induced PSN and mechanical allodynia in a dose-dependent manner, but had no effect on BV-induced inflammatory swelling. These data suggest that the activation of spinal P2X7Rs may play a key role in BV-induced nociception, but not inflammation. [Copyright &y& Elsevier]

Envenomation by snakes of the genus Bothrops is characterized by local pain, systemic bleeding, tachycardia, acute renal failure, as well as swelling and redness caused by the enzymatic and proteolytic action of venom. The toxicological and histological effects of the venom of Bothrops itapetiningae, one of the smallest pit vipers in Brazil, were characterized using BALB/c male mice as models. Venom impacts on liver, kidney, and plasma enzymes that were evaluated at six concentrations. We observed lethality, toxicity, edema forming, and hemorrhagic symptoms such as polymorphonuclear infiltration, hydropic degeneration, and ischemic necrosis. Moreover, increases of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatise, and creatinine levels were observed in the presence of venom. We assessed LD50, minimal hemorrhagic dose, minimal edematogenic activity, and platelet aggregation, improving our knowledge of B. itapetiningae venom toxic activity. [ABSTRACT FROM AUTHOR]

Abstract: Hyaluronidase “venom spreading factor” is a common component of snake venoms and indirectly potentiates venom toxicity. It may cause permanent local tissue destruction at the bite site/systemic collapse of the envenomated victim. The present study was performed to assess the benefits of inhibiting the hyaluronidase activity of Egyptian horned viper, Cerastes cerastes (Cc). The aqueous extracts of some medicinal plants were screened for their inhibitory effect on hyaluronidase activity of Cc venom. The results revealed that the Rosmarinus officinalis (Ro) extract is the most potent hyaluronidase inhibitor among the tested extracts. The Ro extract is more potent inhibitory effect on the hyaluronidase activity than the prepared rabbit monoclonal antiserum of previously purified hyaluronidase enzyme from Cc venom (anti-CcHaseII). In addition, the Ro extract is efficiently inhibited the activity of hemorrhagic toxin previously purified from Cc venom, and it also neutralized the edema inducing activity of the Cc venom in vivo. Furthermore, the Ro extract markedly increased the survival time of experimental mice injected with lethal dose of Cc venom up to 7h in compared to mice injected with venom alone or with venom/anti-CcHaseII (15±5, 75±4min), respectively. Our findings imply the significance of plant-derived hyaluronidase inhibitor in the neutralization of local effects of Cc venom and retardation of death time. Therefore, it may use as a therapeutic value in complementary snakebite therapy. [Copyright &y& Elsevier]

This review will focus on a description of the three-dimensional structures of two β-neurotoxins, the monomeric PLA 2 ammodytoxin from Vipera ammodytes ammodytes , and heterodimeric crotoxin from Crotalus durissus terrificus , and a detailed structural analysis of their multiple functional sites. We have recently determined at high resolution the crystal structures of two natural isoforms of ammodytoxin (AtxA and AtxC) ( Saul et al., 2010 ) which exhibit different toxicity profiles and different anticoagulant properties. Comparative structural analysis of these two PLA 2 isoforms, which differ only by two amino acid residues, allowed us to detect local conformational changes and delineate the role of critical residues in the anticoagulant and neurotoxic functions of these PLA 2 ( Saul et al., 2010 ). We have also determined, at 1.35 Å resolution, the crystal structure of heterodimeric crotoxin ( Faure et al., 2011 ). The three-dimensional structure of crotoxin revealed details of the binding interface between its acidic (CA) and basic (CB) subunits and allowed us to identify key residues involved in the stability and toxicity of this potent heterodimeric β-neurotoxin ( Faure et al., 2011 ). The precise spatial orientation of the three covalently linked polypeptide chains in the mature CA subunit complexed with CB helps us to understand the role played by critical residues of the CA subunit in the increased toxicity of the crotoxin complex. Since the CA subunit is a natural inhibitor of the catalytic and anticoagulant activities of CB, identification of the CA–CB binding interface describes residues involved in this inhibition. We propose future research directions based on knowledge of the recently reported 3D structures of crotoxin and ammodytoxin. [ABSTRACT FROM AUTHOR]

Peptides isolated from animal venoms have shown the ability to regulate pancreatic beta cell function. Characterization of wasp venoms is important, since some components of these venoms present large molecular variability, and potential interactions with different signal transduction pathways. For example, the well studied mastoparan peptides interact with a diversity of cell types and cellular components and stimulate insulin secretion via the inhibition of ATP dependent K + (K ATP ) channels, increasing intracellular Ca 2+ concentration. In this study, the insulin secretion of isolated pancreatic islets from adult Swiss mice was evaluated in the presence of synthetic Agelaia MP-I (AMP-I) peptide, and some mechanisms of action of this peptide on endocrine pancreatic function were characterized. AMP-I was manually synthesized using the Fmoc strategy, purified by RP-HPLC and analyzed using ESI-IT-TOF mass spectrometry. Isolated islets were incubated at increasing glucose concentrations (2.8, 11.1 and 22.2 mM) without (Control group: CTL) or with 10 μM AMP-I (AMP-I group). AMP-I increased insulin release at all tested glucose concentrations, when compared with CTL ( P < 0.05). Since molecular analysis showed a potential role of the peptide interaction with ionic channels, insulin secretion was also analyzed in the presence of 250 μM diazoxide, a K ATP channel opener and 10 μM nifedipine, a Ca 2+ channel blocker. These drugs abolished insulin secretion in the CTL group in the presence of 2.8 and 11.1 mM glucose, whereas AMP-I also enhanced insulin secretory capacity, under these glucose conditions, when incubated with diazoxide and nifedipine. In conclusion, AMP-I increased beta cell secretion without interfering in K ATP and L-type Ca 2+ channel function, suggesting a different mechanism for this peptide, possibly by G protein interaction, due to the structural similarity of this peptide with Mastoparan-X, as obtained by modeling. [ABSTRACT FROM AUTHOR]

Nerve growth factor (NGF) is a dimeric molecule that modulates the survival, proliferation, and differentiation of nervous cells and is also known to act on cells of the immune system and endocrine system. NGFs extracted from mouse submaxillary gland and cobra venom have different immunological behaviors, yet the underlying mechanism remains unclear. Here we report the crystal structure of the NGF purified from Chinese cobra Naja naja atra (cNGF), which unexpectedly reveals a 2-tailed lipid molecule that is embedded between the two protomers of the NGF homodimer. In addition, crystallographic analysis indicated that the purified mouse NGF(mNGF) is free from lipid but can bind lysophosphatidylserine (lyso- PS) in the same pocket as cNGF. Bioassays indicated that the binding of lipid molecules to cNGF and mNGF are essential for their mast cell activation activity and abates their p75NTR binding capacity. Taken together, these results suggest a new mechanism for the regulation of the function of NGF. [ABSTRACT FROM AUTHOR]

Abstract: An investigation into the cardiotoxic effects in human cardiomyocytes of different fractions (as produced from an FPLC) of the venom from Chironex fleckeri showed that whole venom caused cardiac cell death in minutes, measured as cell detachment using xCELLigence technology. However, only one fraction of the venom was responsible for this effect. When all extracted venoms were recombined a similar result was seen for the toxic fraction, however these effects were slower than unfractionated venom alone even though the concentrations were similar. The difference in the results between fractioned and unfractionated venom may have been caused by compounds remaining in the FPLC column, which may interact with the toxic fraction to cause rapid cell detachment or death. [Copyright &y& Elsevier]

The cardiovascular responses to Bothrops alternatus snake venom in anesthetized dogs were investigated. Venom (0.3 mg/kg, i.v.) markedly decreased arterial blood pressure, coronary perfusion pressure, and cardiac output (CO) after 5 min, with progressive recovery of the first two parameters to pre-venom levels after 3 h; CO showed little recovery. There was an abrupt, sustained decrease in left and right ventricular systolic work and stroke volume but no significant changes in heart rate, electrocardiogram, and pulmonary hemodynamics; systemic vascular resistance increased from 1 h onwards. A venom dose of 1 mg/kg produced more pronounced cardiovascular alterations, with a progressive decrease to death. There were no significant changes in blood gas (pO, pCO, HCO, SBC, and SBE) and metabolic (pH, lactate, glucose, creatine kinase activity, Na, and K) parameters, although there was a transitory increase in plasma lactate dehydrogenase 2 min after the lower venom dose. There were no cardiac histological alterations, but microaneurysms and epithelial desquamation were seen in renal tubules. Circulating venom concentrations (determined by ELISA) decreased rapidly after administration, but venom was still detectable after 4 h. These results show that in dogs, B. alternatus venom produces marked hypotension and a direct cardiac action, with few metabolic alterations. [ABSTRACT FROM AUTHOR]

Background Diagnostic tests in patients with Hymenoptera venom allergy are frequently positive to venoms of both honey bee and wasp ( Vespula). Component-resolved analysis with recombinant species-specific major allergens (r SSMA) may help to distinguish true double sensitization from crossreactivity. Methods Included were 121 patients with systemic allergic reactions to Hymenoptera stings, 76 with double positivity of serum-specific Ig E (s Ig E) to both venoms, 45 with single positivity to bee or wasp venom, and 32 controls without history of systemic reactions to Hymenoptera stings and no s Ig E to whole venoms. In venom-allergic patients and controls, s Ig E to r SSMA Api m 1 of bee venom and to Ves v 1 and Ves v 5 of wasp venom were tested by Immuno CAP. Results Only 47% of 76 patients with double positivity to whole venoms reacted also to r SSMA of both species. Specificity of s Ig E to the 3 r SSMA was very high, with no s Ig E to r SSMA of the other species in single-positive venom-allergic patients and only one control with low s Ig E to Ves v 1. All wasp-allergic single-positive patients had s Ig E to Ves v 5 and/or Ves v 1, and 78.3% of single-positive bee venom-allergic patients had s Ig E to Api m 1. Conclusion Specificity of s Ig E to r SSMA of both species is excellent. Sensitivity of s Ig E to r SSMA was optimal for wasp venom. Sensitivity of bee venom Api m 1 could be increased by adding r SSMA of other important bee venom allergens. [ABSTRACT FROM AUTHOR]

Abstract: Neoplastic transformation is the abnormal proliferation of cells. These transformations are often related to changes in cell surface glycoconjugates which can be detected by lectins. We evaluated the anti-tumor potential of BlL, a galactoside-binding lectin isolated from Bothrops leucurus venom as well as its cytotoxicity and hemolysis activity. The phosphatidylserine externalization and mitochondrial membrane potential were also determined. BlL exhibited cytotoxic activity against all tumor cell lines tested by induced phosphatidylserine externalization and mitochondrial depolarization, indicating cell death by apoptosis. [Copyright &y& Elsevier]

Androctonus australis is primarily involved in envenomations in North Africa, notably in Tunisia and Algeria, and constitutes a significant public health problem in this region. The toxicity of the venom is mainly due to various neurotoxins that belong to two distinct structural and immunological groups, group I (the AahI and AahIII toxins) and group II (AahII). Here, we report the use of a diabody mixture in which the molar ratio matches the characteristics of toxins and polymorphism of the venom. The mixture consists of the Db9C2 diabody (anti-group I) and the Db4C1op diabody (anti-AahII), the latter being modified to facilitate in vitro production and purification. The effectiveness of the antivenom was tested in vivo under conditions simulating scorpion envenomation. The intraperitoneal injection of 30 μg of the diabody mixture protected almost all the mice exposed to 3 LD50 s.c. of venom. We also show that the presence of both diabodies is necessary for the animals to survive. Our results are the first demonstration of the strong protective power of small quantities of antivenom used in the context of severe envenomation with crude venom. [ABSTRACT FROM AUTHOR]

In this article, the cytotoxicity of the venom from the nematocysts of jellyfish Cyanea nozakii Kishinouye on human hepatoma cells (Bel-7402, SMMC-7721) and human colon cancer cells (H630) was investigated first. Of the three kinds of cells, the venom had the strongest cytotoxicity on H630 cells with the 50% lethal concentration (IC50) of 5.1 μg/ml. However, the IC50 on Bel-7402 and SMMC-7721 was approximate 17.9 and 24.3 μg/ml, respectively. The cytotoxicity of the venom was affected by pH, temperature and storage conditions. At the pH 4.5–8.5, the venom displayed obvious cytotoxicity and the percentage of survival was about 50%. When pre-incubated at temperatures over 60°C for as short as 10 min, the percentage of survival sharply improved from 4.6% up to 80%. The venom was stored in a more stable condition at −80°C and in lyophilized state compared to other storage conditions used in this study. Lactate dehydrogenase release assay performed on H630 cells indicated that exposure to the venom could result in damage to the cell membrane. [ABSTRACT FROM PUBLISHER]

Black widow spiders can cause variable clinical scenarios from local damage to very serious conditions including death. Acute myocardial damage is rarely observed and its prognostic significance is not known. We report a rare case of a 35-year-old man who developed an acute myocarditis with cardiogenic pulmonary edema requiring mechanical ventilation caused by black widow spider's envenomation. The patient was previously healthy. The clinical course was associated with systemic and cardiovascular complaints. His electrocardiogram revealed ST-segment elevation with T-wave amplitude. The plasma concentrations of cardiac enzymes were elevated. His first echocardiography showed hypokinesis of the left ventricle (left ventricle ejection fraction 48%). Magnetic resonance imaging showed also focal myocardial injury of the LV. There was progressive improvement in cardiac traces, biochemical and echocardiographical values (second left ventricle ejection fraction increased to 50%). Myocardial involvement after a spider bite is rare and can cause death. The exact mechanism of this myocarditis is unknown. We report a rare case of acute myocarditis with cardiogenic pulmonary edema requiring mechanical ventilation caused by black widow spider's envenomation. We objectively documented progressive clinical and electrical improvement. [ABSTRACT FROM AUTHOR]

Background: A clear diagnosis of deep venous thrombosis (DVT) is still challenging: many patients with unclear compression/duplex-sonography undergo a trial of anticoagulative treatment with the immanent risk of systemic hemorrhagic complications. As contrast enhanced ultrasound (CEUS) has the potential to visualize the blood pool, we conducted this pilot study to determine its potential in the characterization of the deep venous system of the lower limb. Material and Methods: CEUS was performed with a 9-3 MHz broadband linear transducer (iU22®, Philips, USA) after the standard-application of a second-generation contrast agent (SonoVue®, Bracco, Italy) in three healthy volunteers. Transverse US-scans were performed for depiction of the fibular-, posterior tibial-vein group, the popliteal, femoral, the external iliacal and the inferior caval vein at defined levels. Results: On our three volunteers the intended segments of the deep venous system of the lower limb were visualized clearly between 45 and 350 seconds by CEUS. The continuous proximal ward scanning demonstrated the venous topography up to the external iliac veins. Discussion and Conclusions: These preliminary results show that the use of CEUS is at least promising in the detection and characterization of the deep venous system of the lower limb. This should be pathbreaking, especially in patients with e.g. high Body Mass Index, local edema, diffuse inflammation etc. undergoing sonographic assessment for suspected deep vein thrombosis not definable by complete compression venous ultrasound or duplex ultrasound. [ABSTRACT FROM AUTHOR]

Natural products that elicit discomfort or pain represent invaluable tools for probing molecular mechanisms underlying pain sensation. Plant-derived irritants have predominated in this regard, but animal venoms have also evolved to avert predators by targeting neurons and receptors whose activation produces noxious sensations. As such, venoms provide a rich and varied source of small molecule and protein pharmacophores that can be exploited to characterize and manipulate key components of the pain-signalling pathway. With this in mind, here we perform an unbiased in vitro screen to identify snake venoms capable of activating somatosensory neurons. Venom from the Texas coral snake (Micrurus tener tener), whose bite produces intense and unremitting pain, excites a large cohort of sensory neurons. The purified active species (MitTx) consists of a heteromeric complex between Kunitz- and phospholipase-A2-like proteins that together function as a potent, persistent and selective agonist for acid-sensing ion channels (ASICs), showing equal or greater efficacy compared with acidic pH. MitTx is highly selective for the ASIC1 subtype at neutral pH; under more acidic conditions (pH?100-fold) proton-evoked activation of ASIC2a channels. These observations raise the possibility that ASIC channels function as coincidence detectors for extracellular protons and other, as yet unidentified, endogenous factors. Purified MitTx elicits robust pain-related behaviour in mice by activation of ASIC1 channels on capsaicin-sensitive nerve fibres. These findings reveal a mechanism whereby snake venoms produce pain, and highlight an unexpected contribution of ASIC1 channels to nociception. [ABSTRACT FROM AUTHOR]

Aim:To assess the cytotoxic effect of crotoxin (CrTX), a potent neurotoxin extracted from the venom of the pit viper Crotalus durissus terrificus, in human lung adenocarcinoma A549 cells and investigated the underlying mechanisms.Methods:A549 cells were treated with gradient concentrations of CrTX, and the cell cycle and apoptosis were analyzed using a flow cytometric assay. The changes of cellular effectors p53, caspase-3 and cleaved caspase-3, total P38MAPK and pP38MAPK were investigated using Western blot assays. A549 xenograft model was used to examine the inhibition of CrTX on tumor growth in vivo.Results:Treatment of A549 cells with CrTX (25-200 μg/mL) for 48 h significantly inhibited the cell growth in a dose-dependent manner (IC50=78 μg/mL). Treatment with CrTX (25 μg/mL) for 24 h caused G1 arrest and induced cell apoptosis. CrTX (25 μg/mL) significantly increased the expression of wt p53, cleaved caspase-3 and phospho-P38MAPK. Pretreatment with the specific P38MAPK inhibitor SB203580 (5 μmol/L) significantly reduced CrTX-induced apoptosis and cleaved caspase-3 level, but G1 arrest remained unchanged and highly expressed p53 sustained. Intraperitoneal injection of CrTX (10 μg/kg, twice a week for 4 weeks) significantly inhibited A549 tumor xenograft growth, and decreased MVD and VEGF levels.Conclusion:CrTX produced significant anti-tumor effects by inducing cell apoptosis probably due to activation of P38MAPK and caspase-3, and by cell cycle arrest mediated by increased wt p53 expression. In addition, CrTX displayed anti-angiogenic effects in vivo. [ABSTRACT FROM AUTHOR]

Background: Among scorpion species, the Buthidae produce the most deadly and painful venoms. However, little is known regarding the venom components that cause pain and their mechanism of action. Using a paw-licking assay (Mus musculus), this study compared the pain-inducing capabilities of venoms from two species of New World scorpion (Centruroides vittatus, C. exilicauda) belonging to the neurotoxin-producing family Buthidae with one species of non-neurotoxin producing scorpion (Vaejovis spinigerus) in the family Vaejovidae. A pain-inducing a-toxin (CvIV4) was isolated from the venom of C. vittatus and tested on five Na+ channel isoforms. Principal Findings: C. vittatus and C. exilicauda venoms produced significantly more paw licking in Mus than V. spinigerus venom. CvIV4 produced paw licking in Mus equivalent to the effects of whole venom. CvIV4 slowed the fast inactivation of Nav1.7, a Na+ channel expressed in peripheral pain-pathway neurons (nociceptors), but did not affect the Nav1.8-based sodium currents of these neurons. CvIV4 also slowed the fast inactivation of Nav1.2, Nav1.3 and Nav1.4. The effects of CvIV4 are similar to Old World a-toxins that target Nav1.7 (AahII, BmK MI, LqhIII, OD1), however the primary structure of CvIV4 is not similar to these toxins. Mutant Nav1.7 channels (D1586A and E1589Q, DIV S3-S4 linker) reduced but did not abolish the effects of CvIV4. Conclusions: This study: 1) agrees with anecdotal evidence suggesting that buthid venom is significantly more painful than non-neurotoxic venom; 2) demonstrates that New World buthids inflict painful stings via toxins that modulate Na+ channels expressed in nociceptors; 3) reveals that Old and New World buthids employ similar mechanisms to produce pain. Old and New World &agr;-toxins that target Nav1.7 have diverged in sequence, but the activity of these toxins is similar. Pain-inducing toxins may have evolved in a common ancestor. Alternatively, these toxins may be the product of convergent evolution. [ABSTRACT FROM AUTHOR]

Abstract: Alpha-KTxs are a diverse group of scorpion short-chain peptide toxins that affect animal potassium channels. We report the biochemical purification, gene cloning, and functional characterization of a new α-KTx named MeuTx3B, from venom of the scorpion Mesobuthus eupeus. MeuTx3B is an orthologue of BmTx3B/Martentoxin (α-KTx16 subfamily) from Mesobuthus martensii that differs by three amino acid substitutions. We found that despite their orthologous relationship, MeuTx3B and BmTx3B exhibit different post-transcriptional processing patterns due to nucleotide mutations in their untranslated regions (UTRs). Our results show that MeuTx3B also differs functionally from BmTx3B in that it lacks inhibitory activity on large conductance calcium-activated potassium channels (BK), implicating the amino acids of difference in conferring the inhibitory activity of BmTx3B. Furthermore, we show that MeuTx3B (2μM) partially inhibits human voltage-gated potassium channel Kv1.3. By using codon-substitution models, we detected signals of positive selection that could drive adaptive evolution of MeuTx3B and related toxins in the functional region associated with pharmacological diversification of toxins in the α-KTx 1 and 16 subfamilies. [Copyright &y& Elsevier]

Abstract: Background: Pressure immobilization bandages have been shown to delay onset of systemic toxicity after Eastern coral snake (Micrurus fulvius) envenomation to the distal extremity. Objectives: To assess the efficacy of a novel compression device in delaying onset of systemic toxicity after truncal envenomations with Eastern coral snake (Micrurus fulvius) venom in a porcine model. Methods: With University approval, nine juvenile pigs (11 kg to 22 kg) were sedated, anesthetized, and intubated but not paralyzed to ensure continuous spontaneous respirations in a university animal laboratory. Each animal was injected subcutaneously with 10 mg of M. fulvius venom in a pre-selected area of the trunk. After 1 min, six animals had the application of a novel, localizing circumferential compression (LoCC) device applied to the bite site (treatment group) and three animals had no treatment (control group). The device was composed of a rigid polymer clay form molded into a hollow fusiform shape with an internal dimension of 8 × 5 × 3 cm and an elastic belt wrapped around the animal securing the device in place. Vital signs were recorded at 30-min intervals. End points included a respiratory rate below 3 breaths/min, oxygen saturation < 80%, or survival to 8 h. Survival to 8 h was analyzed using Fisher''s exact test, with p < 0.05 indicating significance. Survival analysis was performed using the Mantel-Cox test to assess time to death with outcomes represented in a Kaplan-Meier Cumulative survival plot. Results: Five of the six pigs in the treatment group survived 8 h (293–480 min). None of the control pigs survived to 8 h (Fisher''s exact p = 0.04), with mean time of respiratory failure 322 min (272–382 min). Survival analysis showed a significant delay in time to event in the treatment group compared to the control group (p = 0.04). Conclusions: The LoCC device used in this study delayed the onset of systemic toxicity and significantly increased survival time after artificial truncal envenomation by Eastern coral snake venom. [Copyright &y& Elsevier]

Context. The local effects of Crotalinae envenomation can cause significant tissue destruction, pain, paresthesias, and deformity of the limb, which mimic findings of compartment syndrome, despite rare subfascial penetration of the fangs. Complicating this are the various techniques and ideas about what determines compartment syndrome combined with the fact that elevated intracompartmental pressures have been documented after Crotalinae envenomation, without clear evidence of compartment syndrome or tissue hypoperfusion. Objective. The purpose of this review is to evaluate the North American literature to provide an evidenced-based conclusion about the indications for fasciotomy in Crotalinae envenomations. Methods. The search was conducted with studies published only in the English language. The search included all human and animal publications, regardless of the format of study. The Cochrane Central Register, MEDLINE/ Pub Med, Scopus, and Biological Science databases were searched. Citations from all the articles were also cross-referenced if they were pertinent to the review. Major toxicology and emergency medicine and surgical textbooks were also referenced. Abstracts from the North American Congress of Clinical Toxicology, Poisonidex®, and personal articles were also scanned to complete the process, resulting in a total of 640 sources. Papers were excluded if they were duplicates, non-North American, involved excisional therapy, or did not discuss fasciotomy. This left 99 publications applicable to our study. Findings. No randomized controlled trials, 8 animal experiments, 1 human prospective observational study, 24 retrospective reviews, 32 review articles, 10 case reports, 15 textbooks references, 2 abstracts, and 7 editorials were included in the analysis. Controlled animal experiments show that crotaline Fab antivenin reduces intracompartmental pressure and increases tissue perfusion, while fasciotomy either has no beneficial effect or worsens myonecrosis. The case reports and opinions supporting fasciotomy come from the surgical literature and precede the modern crotaline Fab antivenin. Conclusion. The current evidence does not support the use of fasciotomy or dermotomy following Crotalinae envenomation with elevated intracompartmental pressures. At present, early and adequate administration of crotaline Fab antivenin is the treatment of choice. Fasciotomy cannot be recommended until further well-designed investigations are completed. [ABSTRACT FROM AUTHOR]

It has been demonstrated that rattlesnakes can discriminate between envenomed and nonenvenomed rodent prey based on venom-related cues deposited during the strike. This behavior is crucial to the snake's ability to choose the chemical trail left by an envenomed rodent fleeing the strike area and aids in the snake's ability to relocate the rodent. The purpose of the current study was to assess the importance of the vomeronasal system (VNS) in mediating a rattlesnake's ability to distinguish between envenomed and nonenvenomed prey items. The VNS in adult rattlesnakes was temporarily blocked with Xylocaine ointment, and the rattlesnakes were presented with two prey items, one envenomated and one manually killed by the experimenter and not envenomed. Rattlesnakes in the Xylocaine condition failed to discriminate between envenomed and nonenvenomed prey items, whereas this discrimination was evident in the control condition. These data provide direct evidence that the VNS plays a critical role in mediating post-strike prey discrimination in rattlesnakes. [ABSTRACT FROM AUTHOR]

In humans and other mammals, Tityus discrepans ( Td) scorpion envenomation produces a variety of systemic effects including respiratory distress, a generalized inflammatory reaction, modulation of blood pressure, fibrin formation, and platelet activation. For many of these effects, the venom components and underlying mechanisms are not known. In the present study, we demonstrate that Td venom ( TdV) stimulates integrin ααIIbββ3-dependent aggregation of washed human and mouse platelets downstream of Src kinase activation. The pattern of increase in tyrosine phosphorylation induced by TdV in human platelets is similar to that induced by the collagen receptor GPVI, and includes FcR γγ-chain, Syk, and PLC γγ 2. Confirmation of GPVI activation by TdV was achieved by expression of human GPVI in chicken DT40 B cells and use of a reporter assay. To our surprise, TdV was able to activate mouse platelets deficient in the GPVI-FcR γγ-chain complex through a pathway that was also dependent on Src kinases. TdV therefore activates platelets through GPVI and a second, as yet unidentified Src kinase-dependent pathway. [ABSTRACT FROM AUTHOR]

Abstract: Allergy to Myrmecia pilosula (Jack Jumper Ant) venom is common in Australia, affecting ∼2.7% of some communities. Venom immunotherapy is a highly effective treatment, but for the venom to be widely distributed for clinical use, the stability and shelf-life of formulated Jack Jumper Ant venom must be demonstrated. HPLC–UV, ELISA Inhibition, SDS-PAGE and SDS-PAGE Immunoblot were used to assess venom stability under conditions of varying temperature, pH and in the presence of various stabilising agents. Optimal stability occurred between pH 8 and 10, however the presence of benzyl alcohol within this pH range resulted in a cloudy appearance within 3 days, so a pH of 6 was used. Increasing polysorbate 80 concentrations accelerated the degradation of allergenic peptides in 100μg/mL venom, but improved stability at concentrations of 1μg/mL or less. Sucrose reduced degradation of allergens Myr p 1 and Myr p 3, whilst glycerol was destabilising. In the presence of 22% sucrose, 1.1mg/mL Jack Jumper Ant venom was stable at −18°C and 4°C for 12 months; following dilution to 100μg/mL with 0.9% sodium chloride, 10mM phosphate (pH 6), 0.05% polysorbate 80 and 0.9% benzyl alcohol (giving 2% sucrose), venom was stable for 7 days when stored at 4°C. Concentrated Jack Jumper Ant venom can be stored in 22% sucrose for 12 months, and after dilution to 100μg/mL for clinical use, it should be discarded after 7 days. [ABSTRACT FROM AUTHOR]

Abstract: Metabolic expenditure has been shown to increase abruptly in several snake species directly after venom expenditure, while the later stages of venom replenishment seem to involve minor costs. This study examines the dependence of increases in metabolic rate following venom expenditure on the stage of venom replenishment that the venom producing tissue is in at the time of venom extraction in the Common Death Adder, Acanthophis antarcticus. Potential changes in venom composition during venom replenishment are also explored to elucidate whether replenishment is achieved via low rates of synthesis of all venom components or by non-parallel protein production, i.e. initial production of some venom components and subsequent synthesis of others. The results of this study indicate that venom expenditure is followed by a sudden increase in metabolic rate when snakes have previously not expended venom for at least two days, suggesting that repetitive venom expenditure does not further increase the activity of venom gland tissue in this initial time period but that a second upregulation occurs when the tissue is past the initial activation stage. In addition, venom composition appears to remain constant during replenishment within an individual, while substantial variations can be observed even between siblings. [ABSTRACT FROM AUTHOR]

Abstract: Caterpillar envenomation has been an emergent health issue. Lonomia obliqua is a medically important animal that causes a hemorrhagic syndrome that can progress to acute renal failure, intracranial hemorrhage and death. In the past few years the molecular characterization of L. obliqua venom in addition to experimental models has provided fundamental information to the understanding of the envenomation syndrome. Herein studies from several authors which characterized the complex toxic-pharmacological actions of whole venom are reviewed. [ABSTRACT FROM AUTHOR]

Abstract: In order to better understand the relationship between the primary structure of TsHpt-I – a bradykinin-potentiating peptide (BPP) isolated from the venom of the yellow scorpion Tityus serrulatus, with a non-canonical Lys residue prior to the conservative Pro-Pro doublet – and its cardiovascular effects, a series of ladder peptides were synthesized using the C-terminal portion of TsHpt-I as a template. All synthetic peptides having the Pro-Pro doublet at their C-terminal were able to potentiate the hypotensive effect of bradykinin. Conversely, only those analogues having Lys residue could induce a transient hypotension when intravenously administrated in male rats, indicating that the positive charge located toward the radical of this amino acid residue is crucial for this cardiovascular effect. Differently from all known BPPs, TsHpt-I acts as an agonist of the B2 receptor and does not inhibit angiotensin-converting enzyme. The capacity of this peptide to activate this subtype of kinin receptor, releasing NO, was also affected by the absence of Lys’ side-chain positive charge. Moreover, this study has demonstrated that the minimization of the primary structure of TsHpt-I does not significantly alter the biological effects of this native peptide, which could be of interest for biotechnological purposes. [ABSTRACT FROM AUTHOR]

Abstract: Loxoscelism is caused by envenomation by spiders from Loxosceles genus. Clinical symptoms only appear a few hours after envenomation and can evolve in local reactions, such as dermonecrosis, and systemic reactions, such as intravascular haemolysis, intravascular coagulation and renal failure. Current therapies are not effective, often not based in scientific research and can be even detrimental. A lack of understanding of the mechanism of action of the venom of the Loxosceles spider had thus far prevented development of effective therapies. In this review we aim to give an overview of our contributions to the understanding of the mechanism of action of the Loxosceles venom and propose targets and therapeutics for medical intervention. [ABSTRACT FROM AUTHOR]

Abstract: Muscarinic toxins (MTs) are three-finger folded peptides isolated from mamba snake venoms. In this report we describe a selective antagonistic interaction of MTα with the human α2B-adrenoceptor. In a functional assay, measuring the α2B-adrenoceptor-induced increase in intracellular [Ca2+], we found that both venomous MTα and synthetic MTα inhibited the response in a concentration-dependent way. MTα did not affect the responses of α2A-, α2C-, α1A- or α1B-adrenoceptors. To further explore the binding of MTα to the α2B-adrenoceptor, we performed ligand binding experiments on Sf9 cell homogenates with [3H]RX821002 as reporter ligand. MTα bound to the receptor rather slowly requiring about 60 min to reach equilibrium. In equilibrium binding experiments, MTα displaced the radioligand with an IC50 of 3.2 nM, but was not able to displace all bound radioligand. Using a saturation binding protocol, we found that MTα suppressed the maximum binding without any greater impact on the affinity of the radioligand, indicating a non-competitive mode of inhibition. The toxin bound reversibly to α2B-adrenoceptor, but extensive washing was needed for full recovery of binding sites at high toxin concentrations. Surprisingly, MTα did not affect [3H]-N-methylscopolamine binding to the muscarinic receptor subtypes at concentrations found to fully block α2B-adrenoceptors, showing that the toxin is a more potent antagonist for the α2B-adrenoceptor than for muscarinic receptors. These findings should open up new views in terms of selective adrenoceptor drug design as well as in elucidation of α2-adrenoceptor physiology. [Copyright &y& Elsevier]

Abstract: Camelid IgG has been reported to be less immunogenic, less able to activate the complement cascade and more thermostable than IgG from other mammals, and has the ability to bind antigens that are unreactive with other mammalian IgGs. We are investigating whether these attributes of camelid IgG translate into antivenom with immunological and venom-neutralising efficacy advantages over conventional equine and ovine antivenoms. The objective of this study was to determine the preclinical venom-neutralising effectiveness of IgG from camels immunised with venoms, individually or in combination, of the saw-scaled viper, Echis ocellatus, the puff adder, Bitis arietans and the spitting cobra, Naja nigricollis – the most medically-important snake species in West Africa. Neutralisation of the pathological effects of venoms from E. ocellatus, B. arietans and N. nigricollis by IgG from the venom-immunised camels, or commercial antivenom, was compared using assays of venom lethality (ED50), haemorrhage (MHD) and coagulopathy (MCD). The E. ocellatus venom ED50, MHD and MCD results of the E. ocellatus monospecific camel IgG antivenom were broadly equivalent to comparable ovine (EchiTAbG®, MicroPharm Ltd, Wales) and equine (SAIMR Echis, South African Vaccine Producer, South Africa) antivenoms, although the equine antivenom required half the amount of IgG. The B. arietans monospecific camel IgG neutralised the lethal effects of B. arietans venom at one fourth the concentration of the SAIMR polyspecific antivenom (a monospecific B. arietans antivenom is not available). The N. nigricollis camel IgG antivenom was ineffective (at the maximum permitted dose, 100 μl) against the lethal effects of N. nigricollis venom. All the equine polyspecific antivenoms required more than 100 μl to be effective against this venom. The polyspecific camel IgG antivenom, prepared from five camels, was effective against the venom-induced effects of E. ocellatus but not against that of B. arietans and N. nigricollis venoms. No direct correlation was evident between either camel IgG relative avidity or titre and the effectiveness of venom neutralisation in preclinical assays. [Copyright &y& Elsevier]

Abstract: Crotalus durissus terrificus envenomation has been associated with direct nephrotoxicity, rhabdomyolysis, hyperuricemia, urinary hypoosmolality, alterations in aminopeptidase activities (AP) and oxidative stress. This study evaluated the effects of lipoic acid (LA) on renal function, lethality, AP and GSSG/GSH in mice injected with C. d. terrificus venom (vCdt). The doses and routes of administration of LA and vCdt promoted no systemic myotoxicity. LA did not alter significantly the lethality of vCdt. In nonenvenomed, LA induced hypercreatinemia, urinary hyperosmolality, decrease of urinary urea and creatinine, increase of protein in plasma and in soluble fraction (SF) and decrease in membrane-bound fraction (MF) of cortex and medulla. Decreased levels of all AP (except neutral-AP in MF-medulla) were also induced by LA in nonenvenomed. LA associated with vCdt decreased plasma osmolality, hematocrit, urinary uric acid, but increased urinary and SF-medullar protein. LA mitigated the increase of protein in SF-cortex and corrected hyperuricemia, GSSG/GSH and protein in MF-cortex and MF-medulla, as well as decreased plasma neutral AP and acid AP in MF-medulla of envenomed mice. Data suggest that LA contributes to the solubilization/remotion of proteins in MF with impairment of most AP, but it could be beneficial for the treatment of the direct nephrotoxicity of vCdt. [Copyright &y& Elsevier]

Abstract: The Macrovipera lebetina venom consists of a complex mixture of proteins belonging to a few main families according to their enzymatic and pharmacological activity. Given the serious pathophysiological effects caused by M. lebetina bites mainly induced by muscle degeneration, we decided to investigate the myotoxic activity of some venom fractions. In the present study we describe the purification and characterization of a 22.600 kDa protein, named in the following Mlp4.2, that shares myotoxic but not haemorrhagic activity in vivo. Herein we report that Mlp4.2 is a metalloproteinase belonging to the PI-SVMPS family able, in vitro, to proteolyse extracellular matrix proteins as laminin and fibronectin. Histological observations of mouse anterior tibialis Mlp4.2-treated muscle, demonstrate that this protein induces a massive degeneration of myofibers but not haemorrhage. The immunofluorescence analysis of protein-treated anterior tibialis, demonstrates that Mlp4.2 is able to disarray the laminin network surrounding muscle fibers. Finally Mlp4.2 did not show any direct cytolytic activity towards the myogenic cell line C2C12 in culture. The data reported herein suggest that the myotoxicity of Mlp4.2 is primarily linked to the disruption of the muscle fibers interaction with extracellular matrix proteins. [Copyright &y& Elsevier]

Abstract: Snake envenoming is a significant cause of mortality and morbidity in sub-Saharan Africa. The only effective treatment, antivenom, has been in short supply since the 1990s. Whilst the humanitarian response by some antivenom producers has significantly improved the situation, strategies to ensure the long term stability of antivenom supply are still necessary. We are investigating whether the potential safety and logistic advantages of camel IgG antivenom can be exploited to improve antivenom provision in many countries where snakebite is endemic. This study assessed the IgG titre, specificity and avidity of camels immunised with either individual venom or a mixture of venoms from the three most medically important snakes of West Africa, the saw-scale viper (Echis ocellatus), the puff adder (Bitis arietans) and the spitting cobra (Naja nigricollis). Seven of the eight immunised camels generated IgG titres and avidities comparable to, or exceeding, that of commercial equine and ovine antivenoms that are highly effective in envenomed patients. In this, the first of a series of reports on the potential utility of camelid IgG antivenom, we describe an immunisation protocol that induced potent, sustained serological response of very high antibody avidity. These attributes suggest, from an immunological perspective, that camel IgG antivenoms should be as efficacious as current equine and ovine antivenoms. [Copyright &y& Elsevier]

In geographically isolated populations where intensive medical care or serum therapy is not easily accessible snake envenomation is a major cause for concern. The aim of the present study was to test Camellia sinensis extracts, theaflavin and epigallocatechin (two of the main C. sinensis components) against the irreversible neuromuscular blockade induced by Crotalus durissus terrificus venom in mouse phrenic-nerve diaphragm preparations. A quantitative histological study was also performed. The venom (20µg/ml) completely decreased twitch tension after 70min and 5µg/ml venom abolished 50% of twitch amplitude after 60min. C. sinensis extract induced intense facilitatory effect in the preparation activity at 0.2mg/ml and slightly facilitatory effect at 0.05mg/ml. Both 0.05mg/ml C. sinensis extract and 0.05µg/ml commercial theaflavin maintained partial muscular activity in presence of 5µg/ml venom. The histological data confirms that Cs is able to protect the muscle from the myotoxic activity of the venom. Commercial epigallocatechin gallate did not show pre-synaptic nor post-synaptic activities. C. sinensis extract was able to protect the mouse phrenic-nerve diaphragm against the irreversible neuromuscular blockade induced by C. durissus terrificus venom. [ABSTRACT FROM AUTHOR]

Mouse P19 embryonic carcinoma (EC) cells are pluripotent and can differentiate into a population consisting largely of neurons and glia cells using a concentration of 5x10-7M of retinoic acid (RA). Thus, P19 EC cells are a good model system to study events occurring during the critical phases of neuronal differentiation, in vitro . Honey bee venom (BV) consists of mellitin, phospholipase A2, apamin and several other bioactive substances. Previous studies have shown that mellitin and phospholipase A2 - two major components of BV - play an important role in the differentiation of neurons. The purpose of this study was to examine effects of BV and RA on the differentiation of cholinergic neuron in P19 cell line. Preliminary results obtained from morphological examination showed that six days after treatment with 5x10-7M RA, P19 cells produced processes, and gradually obtained neuronal phenotype at approximately day-10. All cells then died at day-11. P19 cells treated with 1.3µg/ml BV produced processes on day-6 and neurons appeared in the next four days. They then proceeded to total size until day-10 and produced elongated processes; however, all cells died on day-11. Using BV and RA together had the same effect but more pronounced differentiating results. It can be concluded that applying BV with RA has an additive effect on cell differentiation and proliferation. The presence of acetylcholinesterase (AChE), frequently used as a marker for neuronal differentiation, was also determined and found using DTNB. [ABSTRACT FROM AUTHOR]

Testing whether venoms may aid in digestion of the prey, eleven snake venoms were compared for the presence of proteases and endopeptidases that function in alkaline pH conditions. In vitro experiments examined the relative protease and endopeptidase activity of the venoms, which involved combining bovine muscle and snake venom in a buffered solution, encased within dialysis tubing. This mixture was then incubated at room temperature (~20°C) for 24hr, with constant shaking. Bicinchoninic acid (BCA) assay and ninhydrin assay were used to determine peptide and amino acid concentrations. Histological and immunohistochemical investigations using N. kaouthia venom confirmed in vitro findings. Results show that B. arietans venom generated the highest amount of protein/peptides and amino acids in the dialysates, while O. scutellatus, N. ater niger and P . textilis venom did not show any significant protein degradation under alkaline conditions. Histological examination revealed varying degrees of muscle cell damage for each of the venom investigated, and the immunohistochemical study on N. kaouthia venom showed that the venom penetrated the muscle tissue to a significant degree. In vitro assays and histological results indicate that particular venoms may possess the ability to enhance digestion of bovine muscle tissue. [ABSTRACT FROM AUTHOR]

The venomous viper Vipera palaestinae (Vp) is responsible for most envenomations in humans and animals in Israel. Its venom contains proteases, haemorrhagins, L-amino acid oxidase and phospholipase A2 but its effects on haemostasis have yet to be characterised. This prospective study aimed to characterise haemostatic abnormalities in Vp-envenomed dogs from presentation to discharge or death, and their association with mortality. Samples from 39 Vp-envenomed dogs were collected periodically and examined for haematology, prothrombin time (PT), activated partial thromboplastin time (aPTT), antithrombin activity (ATA), fibrinogen level and D-dimer concentration. All dogs presented with severe local signs and most (79%) had systemic signs. Six dogs (15%) died. Haemostatic abnormalities were present in 37/39 dogs. Increased D-dimer concentrations were detected in 28/31 dogs. Disseminated intravascular coagulation was diagnosed in 10 dogs and in all non-survivors. Platelet and leucocyte counts at presentation, maximum PT and aPTT, and minimum ATA during hospitalisation were significantly different between survivors and non-survivors and were good predictors of the outcome. The results show that hypercoagulability, consumption and derangement of haemostasis are common in Vp-envenomed dogs and are associated with mortality. Haemostasis should be closely monitored in such dogs. [ABSTRACT FROM AUTHOR]

The immunological cross-reactivity among the Elapidae, Viperidae and Buthidae venoms was detected in dot blot and western blot assays and quantified in ELISA as percentage of absorbance of heterologous versus homologous reaction. Mutual cross-reactivity between the Elapidae and Viperidae venoms was recognized, reflecting high inter-family relationship whereas only weak crossreactions were observed between the scorpion and snake venoms. The anti-scorpion antivenom exceptionally binds a 30-35 kDa Cerastes cerastes venom component, revealing common antigenic components between scorpions and C. cerastes venom. Many of the moderate and weak cross reactivity in ELISA got unnoticed in the dot blot technique. This work primarily describes the commonality among venoms from remote source animals which is potentially important in the designation of venom detection kits and also in exploring common allergens responsible for cross-sensitization of patients to heterologous bites or stings leading to immediate hypersensitivity reactions. [ABSTRACT FROM AUTHOR]

Abstract: Australo-papuan death adders (Acanthophis spp.) are a cause of serious envenomations in Papua New Guinea and northern Australia often resulting in neurotoxic paralysis. Furthermore, victims occasionally present with delayed-onset neurotoxicity that sometimes responds poorly to antivenom or anticholinesterase treatment. This clinical outcome could be explained by the presence of potent snake presynaptic phospholipase A2 neurotoxin (SPAN) complexes and monomers, in addition to long- and short-chain postsynaptic α-neurotoxins, that bind irreversibly, block neurotransmitter release and result in degeneration of the nerve terminal. The present study therefore aimed to determine within-genus variations in expression of high molecular mass SPAN complexes in the venoms of six major species of Acanthophis, four geographic variants of Acanthophis antarcticus. Venoms were separated by size-exclusion liquid chromatography under non-denaturing conditions and fractions corresponding to proteins in the range of 22 to >60 kDa were subjected to pharmacological characterization using the isolated chick biventer cervicis nerve-muscle (CBCNM) preparation. All venoms, except Acanthophis wellsi and Acanthophis pyrrhus, contained high mass fractions with phospholipase A2 activity that inhibited twitch contractions of the CBCNM preparation. This inhibition was of slow onset, and responses to exogenous nicotinic agonists were not blocked, consistent with the presence of SPAN complexes. The results of the present study indicate that clinicians may need to be aware of possible prejunctional neurotoxicity following envenomations from A. antarcticus (all geographic variants except perhaps South Australia), Acanthophis praelongus, Acanthophis rugosus and Acanthophis. laevis species, and that early antivenom intervention is important in preventing further development of toxicity. [Copyright &y& Elsevier]

Abstract: Bothrops marajoensis is found in the savannah of Marajó Island in the State of Pará and regions of Amapá State, Brazil. The aim of the work was to study the renal and cardiovascular effects of the B. marajoensis venom and phospholipase A2 (PLA2). The venom was fractionated by Protein Pack 5PW. N-terminal amino acid sequencing of sPLA2 showed amino acid identity with other lysine K49 sPLA2s of snake venom. B. marajoensis venom (30μg/mL) decreased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate and sodium tubular transport. PLA2 did not change the renal parameters. The perfusion pressure of the mesenteric bed did not change after infusion of venom. In isolated heart, the venom decreased the force of contraction and increased PP but did not change coronary flow. In the arterial pressure, the venom and PLA2 decreased mean arterial pressure and cardiac frequency. The presence of atrial flutter and late hyperpolarisation reversed, indicating QRS complex arrhythmia and dysfunction in atrial conduction. In conclusion, B. marajoensis venom and PLA2 induce hypotension and bradycardia while simultaneously blocking electrical conduction in the heart. Moreover, the decrease in glomerular filtration rate, urinary flow and electrolyte transport demonstrates physiological changes to the renal system. [Copyright &y& Elsevier]

We report the clinical case of a 2 year old girl, who developed nausea, vomiting, increased salivation and arterial hypertension, after a scorpion sting in her left arm, despite the early administration of scorpion antivenom and captopril. Next day, due to a hypertensive emergency, the patient developed generalized tonic seizures, refractory to diazepam, which were controlled with an infusion of dyphenilhydantoine for 24 hours. The hypertensive emergency extended for 10 days and was treated with oral captopril, nifedipine and carvedilol. This clinical picture was accompanied with myocarditis, pancreatitis and an adverse drug reaction to the anti venom. Other drugs administered were corticosteroids, midazolam and fentanyl. The Outcome was satisfactory and the girl was discharged in good general conditions, two weeks after admission. This case appears with uncommon features, such as the occurrence of myocardic and cardiovascular patterns, severity of vascular pattern, the duration of the hypertensive emergency, ineffectiveness of captopril and the scorpion antivenom, and the adverse reaction to antivenin. [ABSTRACT FROM AUTHOR]

Abstract: Snakebites are still a critical public health problem in developing countries or isolated areas. In Brazil, the North Region has a high distribution coefficient worsened by the significant number of eventually unreported cases, due to difficulties in access to health services, to the natural geographic barriers and the vast territory. In the Rio Negro area, the species Bothrops atrox, Bothrops brazili, Lachesis muta muta and Bothriopsis taeniata are thought to be the major species responsible for snakebites. The aim of this study was to qualitatively and quantitatively determine the antigenic cross-reactivity and expression of toxins and the immunogenicity of Bothrops venom species of the Amazon and to evaluate the general efficacy of the therapeutic sera. The in vivo assays demonstrated that the defibrinating activity of B. taeniata venom was absent but that the lethal and hemorrhagic properties were more intense than in the B. atrox venom. The results evidence venom variability among the two B. atrox populations from two distinct Amazonian regions, which may reveal a subjacent speciation process. The results point to new aspects that may guide the improvement of anti-Bothropic therapeutic serum. [Copyright &y& Elsevier]

Abstract: The aims of this study were to devise a process for raising antibodies against Brazilian Bothrops venom in chicken egg yolks, to determine the best delipidation method for the preparation of the aqueous extract and to define the best purification conditions for IgY bothropic antivenom produced in eggs from hens immunized with Brazilian standard bothropic antigen. A group of nine Single Comb White Leghorn laying hens were immunized with venom from five different species of pit vipers of the genus Bothrops. The immunization process was carried out in three cycles, each performed six weeks apart. For extraction, the egg yolk was diluted 1:10 in distilled water, adjusted to a pH of 5.0, subjected to a freeze–thaw cycle, centrifuged and filtered before being precipitated with 20%(w/v) ammonium sulfate salt. This methodology retrieved 2.57 mg of IgY/ml of yolk from eggs. This preparation yielded antibodies capable of neutralizing lethal toxic activity of the pool of Bothrops sp venoms from five species, with an effective dose (ED50) of 365 μL/2 LD50 and, 1.0 mL of IgY antivenom neutralized 0.154 mg of venom. [Copyright &y& Elsevier]

Abstract: The aim of this study was to identify candidate antivenoms with specific activity against the venom of the saw-scaled or carpet viper (Echis ocellatus) in northern Nigeria, where bites by this species cause great morbidity and mortality but where effective antivenoms have become scarce and unaffordable. Selected antivenoms were destined to be compared by randomised controlled clinical trials (RCTs). Standard pre-clinical neutralisation assays were carried out in rodents. We included two licensed antivenoms of established clinical efficacy and 6 candidate antivenoms. Although 6 of the tested antivenoms showed promising efficacy, all but 3 were excluded from further study because of inadequate pre-clinical efficacy or because they were unavailable or unaffordable for the anticipated RCTs. Median effective doses (ED50) of the remaining three candidate antivenoms suggested that the following doses might neutralise the maximum observed venom yield of 24.8mg (dry weight) of venom milked from captive E. ocellatus: 10ml of MicroPharm “EchiTAb G” (ET-G) antivenom; 30ml of Instituto Clodomiro Picado “EchiTAb-Plus-ICP” (ET-Plus) antivenom; 50ml of VacSera, Cairo “EgyVac” antivenom. A preliminary clinical dose-finding and safety study of these three antivenoms was carried out in 24 patients with incoagulable blood after E. ocellatus bites who were not severely envenomed. A 3+3 dose escalation design was employed. Initial doses of 10ml ET-G and 30ml ET-Plus restored blood coagulability in groups of 6 patients with early mild reactions (pruritus only) in not more than one third of them. EgyVac antivenom did not fulfil efficacy or safety criteria in 12 patients. On the basis of these results, ET-G and ET-Plus were selected for comparison in a RCT. [Copyright &y& Elsevier]

Abstract: Three venom peptides (OdVP1, OdVP2 and OdVP3) were isolated from the venom of the solitary wasp Orancistrocerus drewseni (Hymenoptera: Eumenidae). The mature venom peptide sequences were determined via ESI-Q-TOF MS/MS and by searching the O. drewseni venom gland/sac-specific EST library. All of the OdVPs shared the typical characteristics of amidated C-termini proteins and contained a high content of hydrophobic and positively charged amino acids, suggesting that they adopt an amphipathic α-helical secondary structure, as is the case for mastoparan from Vespula lewisii. The cDNA sequence of the OdVP1 precursor was obtained by 5′- and 3′-rapid amplification of cDNA ends (RACE), and the OdVP2 and OdVP3 precursor transcripts were identified from the venom gland/sac-specific EST library. While the mature peptide sequences were distinct from one another, the overall transcript structure of the OdVPs showed a high homology to that of mastoparan-B from Vespa basalis in that they contained a signal sequence, a prosequence, a mature peptide and a C-terminal glycine. The OdVPs, particularly OdVP2 and OdVP2L (an analog of OdVP2), exhibited strong antifungal activities, but poor antibacterial activities. OdVP2L, which possessed additional Glu–Pro residues, did not have antimicrobial activity against bacteria or Gram-positive yeast but retained activity against Botrytis cinerea. [Copyright &y& Elsevier]

Abstract: An understanding of the cross-neutralisation of snake venoms by antibodies is important for snake antivenom development. We investigated the cross-neutralisation of brown snake (Pseudonaja textilis) venom, taipan (Oxyuranus scutellatus) venom and death adder (Acanthophis antarcticus) with commercial antivenoms and monovalent anti-snake IgG, using enzyme immunoassays, in vitro clotting and neurotoxicity assays. Each commercial antivenom bound all three venoms, and neutralised clotting activity of brown snake and taipan venoms and neurotoxicity of death adder venom. The ‘in-house’ monovalent anti-snake venom IgG raised against procoagulant brown snake and taipan venoms, did not neutralise the neurotoxic effects of death adder venom. However, they did cross-neutralise the procoagulant effects of both procoagulant venoms. This supports the idea of developing antivenoms against groups of snake toxins rather than individual snake venoms. [Copyright &y& Elsevier]