Optimization of Xenografting Methods for Generating Human Skeletal Muscle in Mice.

Xenografts of human skeletal muscle generated in mice can be used to study muscle pathology and to test drugs designed to treat myopathies and muscular dystrophies for their efficacy and specificity in human tissue. We previously developed methods to generate mature human skeletal muscles in immunocompromised mice starting with human myogenic precursor cells (hMPCs) from healthy individuals and individuals with facioscapulohumeral muscular dystrophy (FSHD). Here, we examine a series of alternative treatments at each stage in order to optimize engraftment. We show that (i) X-irradiation at 25Gy is optimal in preventing regeneration of murine muscle while supporting robust engraftment and the formation of human fibers without significant murine contamination; (ii) hMPC lines differ in their capacity to engraft; (iii) some hMPC lines yield grafts that respond better to intermittent neuromuscular electrical stimulation (iNMES) than others; (iv) some lines engraft better in male than in female mice; (v) coinjection of hMPCs with laminin, gelatin, Matrigel, or Growdex does not improve engraftment; (vi) BaCl2 is an acceptable replacement for cardiotoxin, but other snake venom preparations and toxins, including the major component of cardiotoxin, cytotoxin 5, are not; and (vii) generating grafts in both hindlimbs followed by iNMES of each limb yields more robust grafts than housing mice in cages with running wheels. Our results suggest that replacing cardiotoxin with BaCl2 and engrafting both tibialis anterior muscles generates robust grafts of adult human muscle tissue in mice.

Skeletal muscle fiber hypercontraction induced by Bothrops asper myotoxic phospholipases A2 ex vivo does not involve a direct action on the contractile apparatus.

Myonecrosis is a frequent clinical manifestation of envenomings by Viperidae snakes, mainly caused by the toxic actions of secreted phospholipase A2 (sPLA2) enzymes and sPLA2-like homologs on skeletal muscle fibers. A hallmark of the necrotic process induced by these myotoxins is the rapid appearance of hypercontracted muscle fibers, attributed to the massive influx of Ca2+ resulting from cell membrane damage. However, the possibility of myotoxins having, in addition, a direct effect on the contractile machinery of skeletal muscle fibers when internalized has not been investigated. This question is here addressed by using an ex vivo model of single-skinned muscle fibers, which lack membranes but retain an intact contractile apparatus. Rabbit psoas skinned fibers were exposed to two types of myotoxins of Bothrops asper venom: Mt-I, a catalytically active Asp49 sPLA2 enzyme, and Mt-II, a Lys49 sPLA2-like protein devoid of phospholipolytic activity. Neither of these myotoxins affected the main parameters of force development in striated muscle sarcomeres of the skinned fibers. Moreover, no microscopical alterations were evidenced after their exposure to Mt-I or Mt-II. In contrast to the lack of effects on skinned muscle fibers, both myotoxins induced a strong hypercontraction in myotubes differentiated from murine C2C12 myoblasts, with drastic morphological alterations that reproduce those described in myonecrotic tissue in vivo. As neither Mt-I nor Mt-II showed direct effects upon the contractile apparatus of skinned fibers, it is concluded that the mechanism of hypercontraction triggered by both myotoxins in patients involves indirect effects, i.e., the large cytosolic Ca2+ increase after sarcolemma permeabilization.

SDS-induced hexameric oligomerization of myotoxin-II from Bothrops asper assessed by sedimentation velocity and nuclear magnetic resonance.

We report the solution behavior, oligomerization state, and structural details of myotoxin-II purified from the venom of Bothrops asper in the presence and absence of sodium dodecyl sulfate (SDS) and multiple lipids, as examined by analytical ultracentrifugation and nuclear magnetic resonance. Molecular functional and structural details of the myotoxic mechanism of group II Lys-49 phospholipase A2 homologues have been only partially elucidated so far, and conflicting observations have been reported in the literature regarding the monomeric vs. oligomeric state of these toxins in solution. We observed the formation of a stable and discrete, hexameric form of myotoxin-II, but only in the presence of small amounts of SDS. In SDS-free medium, myotoxin-II was insensitive to mass action and remained monomeric at all concentrations examined (up to 3 mg/ml, 218.2 µM). At SDS concentrations above the critical micelle concentration, only dimers and trimers were observed, and at intermediate SDS concentrations, aggregates larger than hexamers were observed. We found that the amount of SDS required to form a stable hexamer varies with protein concentration, suggesting the need for a precise stoichiometry of free SDS molecules. The discovery of a stable hexameric species in the presence of a phospholipid mimetic suggests a possible physiological role for this oligomeric form, and may shed light on the poorly understood membrane-disrupting mechanism of this myotoxic protein class.

Effects of inhibition of 5-lipoxygenase and 12-lipoxygenase pathways on skeletal muscle fiber regeneration.

We investigated the effect of inhibition of 5-lipoxigenase (LOX) and 12-LOX pathways on the regeneration of skeletal muscle fibers after injury induced by a myotoxin (MTX) phospholipase A2 from snake venom in an in vivo experimental model. Gastrocnemius muscles of mice injected with MTX presented an increase in 5-LOX protein expression, while 12-LOX was found to be a constitutive protein of skeletal muscle. Animals that received oral treatments with 5-LOX inhibitor MK886 or 12-LOX inhibitor baicalein 30 min and 48 h after MTX-induced muscle injury showed a reduction in the inflammatory process characterized by a significant decrease of cell influx and injured fibers in the degenerative phase (6 and 24 h after injury). In the beginning of the regeneration process (3 days), mice that received MK886 showed fewer new basophilic fibers, suggesting fewer proliferative events and myogenic cell fusion. Furthermore, in the progression of tissue regeneration (14-21 days), the mice treated with 5-LOX inhibitor presented a lower quantity of central nucleus fibers and small-caliber fibers, culminating in a muscle that is more resistant to the stimulus of fatigue during muscle regeneration with a predominance of slow fibers. In contrast, animals early treated with the 12-LOX inhibitor presented functional fibers with higher diameters, less resistant to fatigue and predominance of fast heavy-chain myosin fibers as observed in control animals. These effects were accompanied by an earlier expression of myogenic factor MyoD. Our results suggest that both 5-LOX and 12-LOX pathways represent potential therapeutic targets for muscle regeneration. It appears that inhibition of the 5-LOX pathway represses only the degenerative process by reducing tissue inflammation levels. Meanwhile, inhibition of the 12-LOX pathway also favors the anticipation of maturation and earlier recovery of muscle fiber activity function after injury.

Lys49 myotoxins, secreted phospholipase A2-like proteins of viperid venoms: A comprehensive review.

Muscle necrosis is a potential clinical complication of snakebite envenomings, which in severe cases can lead to functional or physical sequelae such as disability or amputation. Snake venom proteins with the ability to directly damage skeletal muscle fibers are collectively referred to as myotoxins, and include three main types: cytolysins of the "three-finger toxin" protein family expressed in many elapid venoms, the so-called "small" myotoxins found in a number of rattlesnake venoms, and the widespread secreted phospholipase A2 (sPLA2) molecules. Among the latter, protein variants that conserve the sPLA2 structure, but lack such enzymatic activity, have been increasingly found in the venoms of many viperid species. Intriguingly, these sPLA2-like proteins are able to induce muscle necrosis by a mechanism independent of phospholipid hydrolysis. They are commonly referred to as "Lys49 myotoxins" since they most often present, among other substitutions, the replacement of the otherwise invariant residue Asp49 of sPLA2s by Lys. This work comprehensively reviews the historical developments and current knowledge towards deciphering the mechanism of action of Lys49 sPLA2-like myotoxins, and points out main gaps to be filled for a better understanding of these multifaceted snake venom proteins, to hopefully lead to improved treatments for snakebites.

Solving the microheterogeneity of Bothrops asper myotoxin-II by high-resolution mass spectrometry: Insights into C-terminal region variability in Lys49-phospholipase A2 homologs.

Myotoxin-II, a phospholipase A2 (PLA2)-like protein found in Bothrops asper venom, causes rapid necrosis of muscle fibers in spite of lacking enzymatic activity. This toxic action maps to its C-terminal region, within a segment known as "115-129" (consensus numbering) that displays a combination of cationic and hydrophobic amino acids, capable of destabilizing membranes. Although myotoxin-II is found in B. asper from both the Caribbean and Pacific regions of Costa Rica, this work shows that in the latter, position 124 is occupied by phenylalanine, instead of leucine reported for the Caribbean variant (UniProt P24605), thus solving the ambiguity described in the original sequencing of this toxin. A comparative inspection of sequences in the C-terminal region of 70 PLA2-like proteins showed that, with few exceptions, position 124 is occupied by either leucine or phenylalanine with equal frequencies. In line with earlier observations on primary and three-dimensional structural data, this comparison supports the notion that the disruptive mechanism of PLA2-like myotoxins toward membranes is not dependent on a fixed amino acid sequence motif across members of this subfamily, but instead on a spatial array of physicochemical properties which can be provided by variable combinations of cationic and hydrophobic residues. This plasticity bears resemblance to features of many antimicrobial peptides acting upon membranes. From a practical point of view, it is recommended to define the identity of the many isoforms of PLA2s and PLA2-like proteins found in viperid venoms by relying on the accurate determination of their intact mass, as these proteins are not known to present post-translational modifications.

Mutagenic and genotoxic activities of Phospholipase A2 Bothropstoxin-I from Bothrops jararacussu in Drosophila melanogaster and human cell lines.

Phospholipase A2 Bothropstoxin-I (PLA2 BthTX-I) is a myotoxic Lys49-PLA2 from Bothrops jararacussu snake venom. In order to evaluate the DNA damage caused by BthTX-I, we used the Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster and Comet assay in HUVEC and DU-145 cells. For SMART, different concentrations of BthTX-I (6.72 to 430 µg/mL) were used and no significant changes in the survival rate were observed. Significant frequency of mutant spots was observed for the ST cross at the highest concentration of BthTX-I due to recombinogenic activity. In the HB cross, BthTX-I increased the number of mutant spots at intermediate concentrations, being 53.75 µg/mL highly mutagenic and 107.5 µg/mL predominantly recombinogenic. The highest concentrations were neither mutagenic nor recombinogenic, which could indicate cytotoxicity in the wing cells of D. melanogaster. In vitro, all BthTX-I concentrations (1 to 50 µg/mL) induced decrease in HUVEC cell viability, as well as in DU-145 cells at concentrations of 10, 25, and 50 µg/mL. The comet assay showed that in HUVEC and DU-145 cells, all BthTX-I concentrations promoted increase of DNA damage. Further studies should be performed to elucidate the mechanism of action of PLA2 BthTX-I and its possible use in therapeutic strategies against cancer.

Localization of Myotoxin I and Myotoxin II from the venom of Bothrops asper in a murine model.

Phospholipases A2 (PLA2s) and PLA2-like proteins are significant components of snake venoms. Some of these proteins act as potent toxins causing muscle necrosis, which may lead to amputation in severe envenomings. Fundamental aspects of the mechanism of action of these toxins are still not completely known. Myotoxin-I is a catalytically active Asp49 PLA2 from the venom of Bothrops asper, a medically relevant pit viper from Central America. Myotoxin-II is a catalytically inactive Lys49 PLA2-homolog also present in the venom of this snake. For the first time, the in vivo cellular localization of these myotoxins was studied in mouse skeletal muscle using immunofluorescence. Results showed that after 5 min of injection in the gastrocnemius muscle, both toxins initially interacted with the sarcolemma, and some colocalization with nuclei was already evident, especially for Mt-II. After 3 h of injection, a significant colocalization with the nuclei was observed for both toxins. These in vivo results confirm the importance of the initial interaction of these toxins with the sarcolemma and furthermore highlight the internalization and interaction of the toxins with nuclei during their pathophysiological activities, as observed in recent studies using cell culture.

Biological activities of a new crotamine-like peptide from Crotalus oreganus helleri on C2C12 and CHO cell lines, and ultrastructural changes on motor endplate and striated muscle.

Crotamine and crotamine-like peptides are non-enzymatic polypeptides, belonging to the family of myotoxins, which are found in high concentration in the venom of the Crotalus genus. Helleramine was isolated and purified from the venom of the Southern Pacific rattlesnake, Crotalus oreganus helleri. This peptide had a similar, but unique, identity to crotamine and crotamine-like proteins isolated from other rattlesnakes species. The variability of crotamine-like protein amino acid sequences may allow different toxic effects on biological targets or optimize the action against the same target of different prey. Helleramine was capable of increasing intracellular Ca2+ in Chinese Hamster Ovary (CHO) cell line. It inhibited cell migration as well as cell viability (IC50 = 11.44 µM) of C2C12, immortalized skeletal myoblasts, in a concentration dependent manner, and promoted early apoptosis and cell death under our experimental conditions. Skeletal muscle harvested from mice 24 h after helleramine injection showed contracted myofibrils and profound vacuolization that enlarged the subsarcolemmal space, along with loss of plasmatic and basal membrane integrity. The effects of helleramine provide further insights and evidence of myotoxic activities of crotamine-like peptides and their possible role in crotalid envenomings.

Muscle proteolysis via ubiquitin-proteasome system (UPS) is activated by BthTx-I Lys49 PLA2 but not by BthTx-II Asp49 PLA2 and Bothrops jararacussu venom.

Bites by viperid snakes belonging to Bothrops genus produce fast and intense local edema, inflammation, bleeding and myonecrosis. In this study, we investigated the role of Myogenic Regulatory Factors (MRFs: MyoD; Myog), negatively regulated by GDF-8 (Myostatin), and ubiquitin-proteasome system pathway (UPS: MuRF-1; Fbx-32) in gastrocnemius muscle regeneration after Bothrops jararacussu snake venom (Bjussu) or its isolated phospholipase A2 myotoxins, BthTx-I (Lys-49 PLA2) and BthTx-II (Asp-49 PLA2) injection. Male Swiss mice received a single intra-gastrocnemius injection of crude Bjussu, at a dose/volume of 0.83 mg/kg/20 µl, and BthTx-I or BthTx-II, at a dose/volume of 2.5 mg/kg/20 µl. Control mice (Sham) received an injection of sterile saline solution (NaCl 0.9%; 20 µl). At 24, 48, 72 and 96 h post injection, right gastrocnemius was collected for protein expression analyses. Based on the temporal expressional dynamics of MyoD, Myog and GDF-8/Myostatin, it was possible to propose that the myogenesis pathway was impacted most badly by BthTx-II followed by BthTx-I and lastly by B. jararacussu venom, thus suggesting that catalytic activity has likely inhibitory role on the satellite cells-mediated reparative myogenesis pathway. Inversely, the catalytic activity seems to be not a determinant for the activation of proteins ubiquitination by MuRF-1 and Fbx-32/Atrogin-1 E3 proteasome ligases, given proteolysis pathway through UPS was activated neither after Bjussu, nor after BthTx-II, but just after the catalytically-inactive BthTx-I Lys-49 PLA2-homologue exposure. The findings of this study disclose interesting perspective for further mechanistic studies about pathways that take part in the atrophy and repair after permanent damage induced by bothropic snakebites.

Isolation and structural characterization of bioactive compound from Aristolochia sprucei aqueous extract with anti-myotoxic activity.

A bioactive compound isolated from the stem extract of Aristolochia sprucei through High Performance Liquid Chromatography (HPLC) was identified via Nuclear Magnetic Resonance (NMR) as the aristolochic acid (AA). This compound showed an inhibitory effect over the myotoxic activity of Bothrops jararacussu and Bothrops asper venoms, being also effective against the indirect hemolytic activity of B. asper venom. Besides, AA also inhibited the myotoxic activity of BthTX-I and MTX-II with an efficiency greater than 60% against both myotoxins. Docking predictions revealed an interesting mechanism, through which the AA displays an interaction profile consistent with its inhibiting abilities, binding to both active and putative sites of svPLA2. Overall, the present findings indicate that AA may bind to critical regions of myotoxic Asp 49 and Lys49-PLA2s from snake venoms, highlighting the relevance of domains comprising the active and putative sites to inhibit these toxins.

ACP-TX-I and ACP-TX-II, Two Novel Phospholipases A2 Isolated from Trans-Pecos Copperhead Agkistrodon contortrix pictigaster Venom: Biochemical and Functional Characterization.

This work reports the purification and biochemical and functional characterization of ACP-TX-I and ACP-TX-II, two phospholipases A2 (PLA2) from Agkistrodon contortrix pictigaster venom. Both PLA2s were highly purified by a single chromatographic step on a C18 reverse phase HPLC column. Various peptide sequences from these two toxins showed similarity to those of other PLA2 toxins from viperid snake venoms. ACP-TX-I belongs to the catalytically inactive K49 PLA2 class, while ACP-TX-II is a D49 PLA2, and is enzymatically active. ACP-TX-I PLA2 is monomeric, which results in markedly diminished myotoxic and inflammatory activities when compared with dimeric K49 PLA2s, confirming the hypothesis that dimeric structure contributes heavily to the profound myotoxicity of the most active viperid K49 PLA2s. ACP-TX-II exhibits the main pharmacological actions reported for this protein family, including in vivo local myotoxicity, edema-forming activity, and in vitro cytotoxicity. ACP-TX-I PLA2 is cytotoxic to A549 lung carcinoma cells, indicating that cytotoxicity to these tumor cells does not require enzymatic activity.

Neutralizing properties of LY315920 toward snake venom group I and II myotoxic phospholipases A2.

A need exists to develop specific and clinically useful inhibitors of toxic enzymes present in snake venoms, responsible for severe tissue damage and life-threatening effects occurring in thousands of people suffering envenomations globally. LY315920 (Varespladib, S-5920, A-001), a low molecular weight drug developed to inhibit several human secreted phospholipases A2 (PLA2s), was recently shown to also inhibit PLA2s in whole snake venoms with high potency, yet no studies have examined its direct effect on purified snake venom PLA2s. This work evaluated the ability of LY315920 to neutralize the enzymatic and toxic activities of three isolated PLA2 toxins of structural groups I (pseudexin) and II (crotoxin B and myotoxin I), and their corresponding whole venoms. In vitro, LY315920 inhibited the catalytic activity of these three enzymes upon a synthetic substrate. The drug also blocked their cytotoxic effect on cultured murine myotubes. In mice, preincubation of the toxins or venoms with LY315920, followed by their intramuscular injection, resulted in significant inhibition of muscle damage. Finally, immediate, independent injection of LY315920 at the site of toxin or venom inoculation also resulted in a large reduction of myonecrosis in the case of pseudexin and myotoxin-I, and of Pseudechis colletti and Bothrops asper whole venoms, suggesting a possible method of drug delivery in emergency situations. Present findings add evidence to suggest the possibility of using LY315920 as a field antidote in snakebites, aiming to limit the myonecrosis induced by many venom PLA2s in the clinical setting.

A novel pentameric phospholipase A2 myotoxin (PophPLA2) from the venom of the pit viper Porthidium ophryomegas.

The first toxin isolated from the venomous pit viper Porthidium ophryomegas is a basic pentameric phospholipase A2 (PophPLA2). Elucidation of its amino acid sequence showed that it belongs to the group IIA of secreted PLA2s, with the presence of all 14 conserved cysteine positions. The toxin displayed catalytic activity, in agreement with the presence of Asp49 in its sequence of 121 residues. SDS-PAGE analysis revealed that this toxin is pentameric in non-reducing conditions, a structural organization that has not been described for any viperid PLA2. PophPLA2 displayed moderate myotoxic (in vivo) and cytotoxic (in vitro) activities, as well as anticoagulant activity on human plasma (in vitro). PophPLA2 was not lethal, and did not induce signs of toxicity or distress in mice, when administered intravenously at a dose of up to 100 µg (5.9 µg/g). The toxin showed highest sequence identity with other PLA2s from the venoms of ancestral Asian pit viper species.

A myotoxic Lys49 phospholipase A2-homologue is the major component of the venom of Bothrops cotiara from Misiones, Argentina.

Bothrops cotiara is a pitviper found in Southeastern Brazil and, scarcely, in the Misiones province of Argentina. In contrast to considerable information available on the venom of the Brazilian snake population, that of Misiones has received little attention. While exploring the chromatographic venom profile of Argentinean B. cotiara, a major protein peak was found which, according to a previous study, is not present in the venom of Brazilian origin. The corresponding protein was isolated by RP-HPLC, and characterized by electrophoresis, mass spectrometry, phospholipase A2 (PLA2) assay, and myotoxic activities. Representing nearly 15% of B. cotiara venom from Misiones, this protein was identified as a Lys49 PLA2 homologue. In accordance with the characteristics of this toxin family, the protein induced myotoxicity in mice and was devoid of PLA2 activity. Since previous work reported that no PLA2 or PLA2-homologues occur in B. cotiara venom of Brazilian origin, the presence of an abundant Lys49 PLA2 homologue in the venom from Misiones highlights a striking phenotypic variation in toxin expression within two populations of a single snake species inhabiting different geographic areas. The considerable proportion of B. cotiara Lys49 PLA2 homologue myotoxin in the venom alerts that skeletal muscle necrosis might be a potentially relevant consequence of eventual envenomings by this species in Misiones.

Phenotypic Variation in Mojave Rattlesnake (Crotalus scutulatus) Venom Is Driven by Four Toxin Families.

Phenotypic diversity generated through altered gene expression is a primary mechanism facilitating evolutionary response in natural systems. By linking the phenotype to genotype through transcriptomics, it is possible to determine what changes are occurring at the molecular level. High phenotypic diversity has been documented in rattlesnake venom, which is under strong selection due to its role in prey acquisition and defense. Rattlesnake venom can be characterized by the presence (Type A) or absence (Type B) of a type of neurotoxic phospholipase A 2 (PLA 2 ), such as Mojave toxin, that increases venom toxicity. Mojave rattlesnakes (Crotalus scutulatus), represent this diversity as both venom types are found within this species and within a single panmictic population in the Sonoran Desert. We used comparative venom gland transcriptomics of nine specimens of C. scutulatus from this region to test whether expression differences explain diversity within and between venom types. Type A individuals expressed significantly fewer toxins than Type B individuals owing to the diversity of C-type lectins (CTLs) and snake venom metalloproteinases (SVMPs) found in Type B animals. As expected, both subunits of Mojave toxin were exclusively found in Type A individuals but we found high diversity in four additional PLA 2 s that was not associated with a venom type. Myotoxin a expression and toxin number variation was not associated with venom type, and myotoxin a had the highest range of expression of any toxin class. Our study represents the most comprehensive transcriptomic profile of the venom type dichotomy in rattlesnakes and C. scutulatus. Even intra-specifically, Mojave rattlesnakes showcase the diversity of snake venoms and illustrate that variation within venom types blurs the distinction of the venom dichotomy.

Evaluation of pro-inflammatory events induced by Bothrops alternatus snake venom.

Inflammation is a major local feature of envenomation by bothropic snakes being characterized by a prominent local edema, pain, and extensive swelling. There are reports demonstrating that whole Bothrops snake venoms and toxins isolated from them are able to activate macrophages functions, such as phagocytosis, production of reactive oxygen, cytokines and eicosanoids, however, little is known about the effects of Bothrops alternatus (B.a.) venom on macrophages. In this work, we evaluated the proinflammatory effects of B.a. venom with in vivo and in vitro experiments using the Raw 264.7 cell line and mouse peritoneal macrophages. We detected that B.a. venom augments cell permeability (2-fold), and cellular extravasation (mainly neutrophils), increase proinflammatory cytokines IL1 (∼300-fold), IL12 (∼200-fold), and TNFα (∼80-fold) liberation and induce the expression of enzymes related to lipid signaling, such as cPLA2α and COX-2. Additionally, using lipidomic techniques we detected that this venom produces a release of arachidonic acid (∼10 nMol/mg. Protein) and other fatty acids (16:0 and 18:1 n-9c). Although much of these findings were described in inflammatory processes induced by other bothropic venoms, here we demonstrate that B.a. venom also stimulates pro-inflammatory pathways involving lipid mediators of cell signaling. In this sense, lipidomics analysis of macrophages stimulated with B.a. venom evidenced that the main free fatty acids are implicated in the inflammatory response, and also demonstrated that this venom, is able to activate lipid metabolism even with a low content of PLA2.

Evaluation of pro-inflammatory events induced by Bothrops alternatus snake venom

Inflammation is a major local feature of envenomation by bothropic snakes being characterized by a prominent local edema, pain, and extensive swelling. There are reports demonstrating that whole Bothrops snake venoms and toxins isolated from them are able to activate macrophages functions, such as phagocytosis, production of reactive oxygen, cytokines and eicosanoids, however, little is known about the effects of Bothrops alternatus (B.alpha.) venom on macrophages. In this work, we evaluated the proinflammatory effects of B.alpha. venom with in vivo and in vitro experiments using the Raw 264.7 cell line and mouse peritoneal macrophages. We detected that B.alpha. venom augments cell permeability (2-fold), and cellular extravasation (mainly neutrophils), increase proinflammatory cytokines IL1 (similar to 300-fold), IL12 (similar to 200-fold), and TNF alpha (similar to 80-fold) liberation and induce the expression of enzymes related to lipid signaling, such as cPLA(2 alpha) and COX-2. Additionally, using lipidomic techniques we detected that this venom produces a release of arachidonic acid (similar to 10 nMol/mg. Protein) and other fatty acids (16:0 and 18:1 n-9c). Although much of these findings were described in inflammatory processes induced by other bothropic venoms, here we demonstrate that B.alpha. venom also stimulates pro-inflammatory pathways involving lipid mediators of cell signaling. In this sense, lipidomics analysis of macrophages stimulated with B.alpha. venom evidenced that the main free fatty acids are implicated in the inflammatory response, and also demonstrated that this venom, is able to activate lipid metabolism even with a low content of PLA(2).

Lys49 myotoxin from the Brazilian lancehead pit viper elicits pain through regulated ATP release.

Pain-producing animal venoms contain evolutionarily honed toxins that can be exploited to study and manipulate somatosensory and nociceptive signaling pathways. From a functional screen, we have identified a secreted phospholipase A2 (sPLA2)-like protein, BomoTx, from the Brazilian lancehead pit viper (Bothrops moojeni). BomoTx is closely related to a group of Lys49 myotoxins that have been shown to promote ATP release from myotubes through an unknown mechanism. Here we show that BomoTx excites a cohort of sensory neurons via ATP release and consequent activation of P2X2 and/or P2X3 purinergic receptors. We provide pharmacological and electrophysiological evidence to support pannexin hemichannels as downstream mediators of toxin-evoked ATP release. At the behavioral level, BomoTx elicits nonneurogenic inflammatory pain, thermal hyperalgesia, and mechanical allodynia, of which the latter is completely dependent on purinergic signaling. Thus, we reveal a role of regulated endogenous nucleotide release in nociception and provide a detailed mechanism of a pain-inducing Lys49 myotoxin from Bothrops species, which are responsible for the majority of snake-related deaths and injuries in Latin America.

Alpha-type phospholipase A2 inhibitors from snake blood.

It is of popular and scientific knowledge that toxins from snake venom (among them the PLA2 and myotoxins) are neutralized by various compounds, such as antibodies and proteins purified from animal blood. Venomous and nonvenomous snakes have PLA2 inhibitory proteins, called PLIs, in their blood serum. One hypothesis that could explain the presence of these PLIs in the serum of venomous snakes would be self-protection against the enzymes of their own venom, which eventually could reach the circulatory system. However, the presence of PLIs in non-venomous snakes suggests that their physiological role might not be restricted to protection against PLA2 toxins, but could be extended to other functions, as in the innate immune system and local regulation of PLA2s. The present study aimed to review the currently available literature on PLA2 and myotoxin alpha inhibitors present in snake plasma, thus helping to improve the research on these molecules. Furthermore, this review includes current information regarding the mechanism of action of these inhibitors in an attempt to better understand their application, and proposes the use of these molecules as new models in snakebite therapy. These molecules may help in the neutralization of different types of phospholipases A2 and myotoxins, complementing the conventional serum therapy.