A new 3D model of L929 fibroblasts microtissues uncovers the effects of Bothrops erythromelas venom and its antivenom.

In Brazil, around 80% of snakebites are caused by snakes of the genus Bothrops. A three-dimensional culture model was standardized and used to perform treatments with Bothrops erythromelas venom (BeV) and its antivenom (AV). The MRC-5 and L929 cell lines were cultured at increasing cell densities. Morphometric parameters were evaluated through images obtained from an inverted microscope: solidity, circularity, and Feret diameter. L929 microtissues (MT) showed better morphometric data, and thus they were used for further analysis. MT viability was assessed using the acridine orange and ethidium bromide staining method, which showed viable cells in the MT on days 5, 7, and 10 of cultivation. Histochemical and histological analyses were performed, including hematoxylin/eosin staining, which showed a good structure of the spheroids. Alcian blue staining revealed the presence of acid proteoglycans. Immunohistochemical analysis with ki-67 showed different patterns of cell proliferation. The MT were also subjected to pharmacological tests using the BeV, in the presence or absence of its AV. The results showed that the venom was not cytotoxic, but it caused morphological changes. The MT showed cell detachment, losing their structure. The antivenom was able to partially prevent the venom activities.

Effect of combined red and infrared wavelengths on inflammation, hemorrhage, and muscle damage caused by Bothrops leucurus snake venom.

To evaluate the effects of red and infrared wavelengths, separately and combined, on the inflammatory process and collagen deposition in muscle damage caused by B. leucurus venom. 112 mice were inoculated with diluted venom (0.6mg/kg) in the gastrocnemius muscle. The animals were divided into four groups: one control (CG) and three treatments, namely: 1) red laser (λ=660 nm) (RG), 2) infrared laser (λ=808 nm) (IG) and 3) red laser (λ=660 nm) + infrared (λ=808 nm) (RIG). Each group was subdivided into four subgroups, according to the duration of treatment application (applications every 24 hours over evaluation times of up to 144 hours). A diode laser was used (0.1 W, CW, 1J/point, ED: 10 J/cm2). Both wavelengths reduced the intensity of inflammation and the combination between them significantly intensified the anti-inflammatory response. Photobiomodulation also changed the type of inflammatory infiltrate observed and RIG had the highest percentage of mononuclear cells in relation to the other groups. Hemorrhage intensity was significantly lower in treated animals and RIG had the highest number of individuals in which this variable was classified as mild. As for collagen deposition, there was a significant increase in RG in relation to CG, in RIG in relation to CG and in RIG in relation to IG. Photobiomodulation proved to be effective in the treatment of inflammation and hemorrhage caused by B. leucurus venom and stimulated collagen deposition. Better results were obtained with the combined wavelengths.

Pit Viper Envenomation in Two Pregnant Bitches.

Snake envenomation is relatively common in small animals, particularly in endemic areas. Effects and outcomes of envenomation during pregnancy are poorly described in humans and more so in veterinary patients. Two young pregnant female dogs presented to a university teaching hospital with a history of acute soft tissue swelling and bleeding. History, physical examination findings, and diagnostics were consistent with envenomation by crotalid snakes. Medical management of one of the dogs included administration of antivenin. Both dogs survived envenomation with minimal complications and went on to whelp without complications, and all fetuses survived. This is the first description of the management of pit viper envenomation in pregnant dogs.

Fab Antivenom Reversal of Neurotoxicity Caused by a Juvenile Crotalus horridus Lacking Canebrake Toxin.

We present a case of neurotoxic effects in a pediatric patient after envenomation by a timber rattlesnake (Crotalus horridus) in the Appalachian upstate of South Carolina. Though some members of this species are capable of primarily neurotoxic envenomation, there is heterogeneity in venom composition, and neurotoxic timber rattlesnakes are not endemic to the Appalachian region. However, neurotoxic effects caused by C horridus species lacking typical neurotoxins have been suspected, though not previously confirmed in the medical literature. This case presents a patient who was envenomated by a genotypically confirmed non-neurotoxic C horridus but who nevertheless presented with symptoms consistent with primary neurotoxicity. Neurotoxic effects can be variable in their response to traditional antivenom, though this patient demonstrated rapid response to treatment, representing a novel case in the literature of neurotoxic effects from a snake lacking typical neurotoxins with documented improvement with traditional antivenom.

Sequence Divergence in Venom Genes Within and Between Montane Pitviper (Viperidae: Crotalinae: Cerrophidion) Species is Driven by Mutation-Drift Equilibrium.

Snake venom can vary both among and within species. While some groups of New World pitvipers-such as rattlesnakes-have been well studied, very little is known about the venom of montane pitvipers (Cerrophidion) found across the Mesoamerican highlands. Compared to most well-studied rattlesnakes, which are widely distributed, the isolated montane populations of Cerrophidion may facilitate unique evolutionary trajectories and venom differentiation. Here, we describe the venom gland transcriptomes for populations of C. petlalcalensis, C. tzotzilorum, and C. godmani from Mexico, and a single individual of C. sasai from Costa Rica. We explore gene expression variation in Cerrophidion and sequence evolution of toxins within C. godmani specifically. Cerrophidion venom gland transcriptomes are composed primarily of snake venom metalloproteinases, phospholipase A[Formula: see text]s (PLA[Formula: see text]s), and snake venom serine proteases. Cerrophidion petlalcalensis shows little intraspecific variation; however, C. godmani and C. tzotzilorum differ significantly between geographically isolated populations. Interestingly, intraspecific variation was mostly attributed to expression variation as we did not detect signals of selection within C. godmani toxins. Additionally, we found PLA[Formula: see text]-like myotoxins in all species except C. petlalcalensis, and crotoxin-like PLA[Formula: see text]s in the southern population of C. godmani. Our results demonstrate significant intraspecific venom variation within C. godmani and C. tzotzilorum. The toxins of C. godmani show little evidence of directional selection where variation in toxin sequence is consistent with evolution under a model of mutation-drift equilibrium. Cerrophidion godmani individuals from the southern population may exhibit neurotoxic venom activity given the presence of crotoxin-like PLA[Formula: see text]s; however, further research is required to confirm this hypothesis.

RESUMEN: El veneno de las serpientes puede variar entre y dentro de las especies. Mientras algunos grupos de viperidos del Nuevo Mundo­como las cascabeles­han sido bien estudiadas, muy poco se sabe acerca del veneno de las nauyacas de frío (Cerrophidion) que se encuentran en las zonas altas de Mesoamérica. Comparadas con las extensamente estudiadas cascabeles, que estan ampliamente distribuidas, las poblaciones de Cerrophidion, aisladas en montañas, pueden poseer trayectorias evolutivas y diferenciación en su veneno unicos. En el presente trabajo, describimos el transcriptoma de las glándulas de veneno de poblaciones de C. petlalcalensis, C. tzotzilorum, y C. godmani de México, y un individuo de C. sasai de Costa Rica. Exploramos la variación en la expresión de toxinas en Cerrophidion y la evolución en las secuencias geneticas en C. godmani específicamente. El transcriptoma de la glándula de veneno de Cerrophidion esta compuesto principalmente de Metaloproteinasas de Veneno de Serpiente, Fosfolipasas A[Formula: see text] (PLA[Formula: see text]s), y Serin Proteasas de Veneno de Serpiente. Cerrophidion petlalcalensis presenta poca variación intraespecífica; sin embargo, los transcriptomas de la glandula de veneno de C. godmani y C. tzotzilorum difieren significativamente entre poblaciones geográficamente aisladas. Curiosamente, la variación intraespecífica estuvo atribuida principalmente a la expresión de las toxinas ya que no encontramos señales de selección en las toxinas de C. godmani. Adicionalmente, encontramos miotoxinas similares a PLA[Formula: see text] en todas las especies excepto C. petlalcalensis, y PLA[Formula: see text]s similares a crotoxina en la población sureña de C. godmani. Nuestros resultados demuestran la presencia de variacion intraespecífica presente en el veneno de C. godmani y C. tzotzilorum. Las toxinas de Cerrophidion godmani muestran poca evidencia de selección direccional, y la variación en la secuencias de las toxinas es consistente con evolucion bajo un modelo de equilibrio de mutación-deriva. Algunos individuos de C. godmani de la población del sur potencialmente tienen un veneno neurotóxico dada la presencia de PLA[Formula: see text]s similares a la crotoxina, sin embargo, se necesita más evidencia para corroborar esta hipótesis.

The early inhibition of the COX-2 pathway in viperid phospholipase A2-induced skeletal muscle myotoxicity accelerates the tissue regeneration.

The administration of non-steroidal anti-inflammatory drugs in the treatment of injury and muscle regeneration is still contradictory in effectiveness, especially regarding the timing of their administration. This can interfere with the production of prostaglandins originating from inflammatory isoform cyclooxygenase-2 (COX-2), which is essential to modulate tissue regeneration. The phospholipases A2 (PLA2) from viperid venoms cause myotoxicity, therefore constituting a tool for the study of supportive therapies to improve skeletal muscle regeneration. This study investigated the effect of early administration of lumiracoxib (selective inhibitor of COX-2) on the degeneration and regeneration stages of skeletal muscle after injury induced by a myotoxic PLA2. After 30 min and 48 h of intramuscular injection of PLA2, mice received lumiracoxib orally and histological, functional, and transcriptional parameters of muscle were evaluated from 6 h to 21 days. Inhibition of COX-2 in the early periods of PLA2-induced muscle degeneration reduced leukocyte influx, edema, and tissue damage. After the second administration of lumiracoxib, in regenerative stage, muscle showed increase in number of basophilic fibers, reduction in fibrosis content and advanced recovery of functionality characterized by the presence of fast type II fibers. The expression of Pax7 and myogenin were increased, indicating a great capacity for storing satellite cells and advanced mature state of tissue. Our data reveals a distinct role of COX-2-derived products during muscle degeneration and regeneration, in which early administration of lumiracoxib was a therapeutic strategy to modulate the effects of prostaglandins, providing a breakthrough in muscle tissue regeneration induced by a myotoxic PLA2.

Mouse skin peptidomic analysis of the hemorrhage induced by a snake venom metalloprotease.

Hemorrhage induced by snake venom metalloproteases (SVMPs) results from proteolysis, capillary disruption, and blood extravasation. HF3, a potent SVMP of Bothrops jararaca, induces hemorrhage at pmol doses in the mouse skin. To gain insight into the hemorrhagic process, the main goal of this study was to analyze changes in the skin peptidome generated by injection of HF3, using approaches of mass spectrometry-based untargeted peptidomics. The results revealed that the sets of peptides found in the control and HF3-treated skin samples were distinct and derived from the cleavage of different proteins. Peptide bond cleavage site identification in the HF3-treated skin showed compatibility with trypsin-like serine proteases and cathepsins, suggesting the activation of host proteinases. Acetylated peptides, which originated from the cleavage at positions in the N-terminal region of proteins in both samples, were identified for the first time in the mouse skin peptidome. The number of peptides acetylated at the residue after the first Met residue, mostly Ser and Ala, was higher than that of peptides acetylated at the initial Met. Proteins cleaved in the hemorrhagic skin participate in cholesterol metabolism, PPAR signaling, and in the complement and coagulation cascades, indicating the impairment of these biological processes. The peptidomic analysis also indicated the emergence of peptides with potential biological activities, including pheromone, cell penetrating, quorum sensing, defense, and cell-cell communication in the mouse skin. Interestingly, peptides generated in the hemorrhagic skin promoted the inhibition of collagen-induced platelet aggregation and could act synergistically in the local tissue damage induced by HF3.

Novel Toxicodynamic Model of Subcutaneous Envenomation to Characterize Snake Venom Coagulopathies and Assess the Efficacy of Site-Directed Inorganic Antivenoms.

Venomous snake bite adversely affects millions of people yearly, but few animal models allow for the determination of toxicodynamic timelines with hemotoxic venoms to characterize the onset and severity of coagulopathy or assess novel, site-directed antivenom strategies. Thus, the goals of this investigation were to create a rabbit model of subcutaneous envenomation to assess venom toxicodynamics and efficacy of ruthenium-based antivenom administration. New Zealand White rabbits were sedated with midazolam via the ear vein and had viscoelastic measurements of whole blood and/or plasmatic coagulation kinetics obtained from ear artery samples. Venoms derived from Crotalus scutulatus scutulatus, Bothrops moojeni, or Calloselasma rhodostoma were injected subcutaneously, and changes in coagulation were determined over three hours and compared to samples obtained prior to envenomation. Other rabbits had ruthenium-based antivenoms injected five minutes after venom injection. Viscoelastic analyses demonstrated diverse toxicodynamic patterns of coagulopathy consistent with the molecular composition of the proteomes of the venoms tested. The antivenoms tested attenuated venom-mediated coagulopathy. A novel rabbit model can be used to characterize the onset and severity of envenomation by diverse proteomes and to assess site-directed antivenoms. Future investigation is planned involving other medically important venoms and antivenom development.

Evaluation of crotamine based probes as intracellular targeted contrast agents for magnetic resonance imaging.

Crotamine is a lysine and cysteine rich 42 amino acids long bio-active polypeptide, isolated from the venom of a South American rattlesnake, that can also be used as cell penetrating peptide. A facile synthetic scheme for coupling cargo molecules like fluorophores (carboxyfluorescein) or MRI probes (Gd-DO3A-based macrocycle) is presented. The toxicity, cellular internalization and steady-state accumulation after long-term incubation for 18 h, as well as magnetic resonance relaxivities and cellular relaxation rates of crotamine based probes were evaluated and compared to its shorter synthetic fragment CyLoP-1. The longitudinal relaxivity (r1) of the conjugates of CyLoP-1 and crotamine is significantly lower in medium than in water indicating to the lower contrast enhancement efficacy of DO3A-based probes in biological samples. Carboxyfluorescein labeled crotamine did not exhibit toxicity up to a concentration of 2.5 µM. CyLoP-1 accumulated about four times better within the cells compared to crotamine. Fluorescence microscopy suggests different predominant uptake mechanisms for crotamine and CyLoP-1 in 3T3 cells. While crotamine is predominantly localized in vesicular structures (most likely endosomes and lysosomes) within the cell, CyLoP-1 is mainly homogeneously distributed in the cytosol. The cellular relaxation rate (R1, cell) of the crotamine based probe was not significantly increased whereas the corresponding CyLoP-1-derivative showed a slightly elevated R1, cell. This study indicates the potential of crotamine and in particular the shorter fragment CyLoP-1 to be useful for an efficient transmembrane delivery of agents directed to intracellular (cytosolic) targets. However, the applicability of the conjugates synthesized here as contrast agents in MR imaging is limited. Further improvement is needed to prepare more efficient probes for MRI applications, i.e., by replacing the DO3A- with a DOTA-based chelate.

Crotalus atrox venom-induced cellular toxicity: Early wound progression involves reactive oxygen species.

Understanding the mechanisms that produce cellular cytotoxicity is fundamental in the field of toxicology. Cytotoxic stimuli can include organic toxins such as hemorrhagic snake venom, which can lead to secondary complications such as the development of necrotic tissue and profuse scarring. These clinical manifestations mimic cytotoxic responses induce by other organic compounds such as organic acids. We used hemorrhagic snake venom and human embryonic kidney cells (HEK 293T) as a model system to better understand the cellular responses involved in venom induced cytotoxicity. Cells stimulated with Crotalus atrox (CA) (western diamondback) venom for 4 or 10 h demonstrated significant cytotoxicity. Results from 2',7'-Dichlorodihydrofluorescein diacetate (H2 DCF-DA) assays determine CA venom stimulation induces a robust production of reactive oxygen species (ROS) over a 3-h time course. In contrast, pretreatment with polyethylene glycol (PEG)-catalase or N-acetyl cysteine (NAC) prior to CA venom stimulation significantly blunted H2 DCFDA fluorescence fold changes and showed greater cytoprotective effects than cells stimulated with CA venom alone. Pre- incubating HEK293T cells with the NADPH oxidase (NOX) pan-inhibitor VAS2870 prior venom stimulation significantly minimized the venom-induced oxidative burst at early timepoints (≤2 h). Collectively, our experiments show that pre-application of antioxidants reduces CA venom induce cellular toxicity. This result highlights the importance of ROS in the early stages of cytotoxicity and suggests muting ROS production in noxious injuries may increase positive clinical outcomes.

Inhibitory Effects of Varespladib, CP471474, and Their Potential Synergistic Activity on Bothrops asper and Crotalus durissus cumanensis Venoms.

Snakebite is a neglected tropical disease that causes extensive mortality and morbidity in rural communities. Antivenim sera are the currently approved therapy for snake bites; however, they have some therapeutic limitations that have been extensively documented. Recently, small molecule toxin inhibitors have received significant attention as potential alternatives or co-adjuvant to immunoglobulin-based snakebite therapies. Thus, in this study, we evaluated the inhibitory effects of the phospholipase A2 inhibitor varespladib and the metalloproteinase inhibitor CP471474 and their synergistic effects on the lethal, edema-forming, hemorrhagic, and myotoxic activities of Bothrops asper and Crotalus durissus cumanensis venoms from Colombia. Except for the preincubation assay of the lethal activity with B. asper venom, the mixture showed the best inhibitory activity. Nevertheless, the mix did not display statistically significant differences to varespladib and CP471474 used separately in all assays. In preincubation assays, varespladib showed the best inhibitory activity against the lethal effect induced by B. asper venom. However, in independent injection assays, the mix of the compounds partially inhibited the lethal activity of both venoms (50%). In addition, in the assays to test the inhibition of edema-forming activity, the mixture exhibited the best inhibitory activity, followed by Varespladib, but without statistically significant differences (p > 0.05). The combination also decreased the myotoxic activity of evaluated venoms. In these assays, the mix showed statistical differences regarding CP471474 (p < 0.05). The mixture also abolished the hemorrhagic activity of B. asper venom in preincubation assays, with no statistical differences to CP471474. Finally, the mixture showed inhibition in studies with independent administration in a time-dependent manner. To propose a mode of action of varespladib and CP471474, molecular docking was performed. PLA2s and SVMPs from tested venoms were used as targets. In all cases, our molecular modeling results suggested that inhibitors may occupy the substrate-binding cleft of the enzymes, which was supported by specific interaction with amino acids from the active site, such as His48 for PLA2s and Glu143 for the metalloproteinase. In addition, varespladib and CP471474 also showed interaction with residues from the hydrophobic channel in PLA2s and substrate binding subsites in the SVMP. Our results suggest a synergistic action of the mixed inhibitors and show the potential of varespladib, CP471474, and their mixture to generate new treatments for snakebite envenoming with application in the field or as antivenom co-adjuvants.

Diagnostic Tests for Hypofibrinogenemia Resulting from Green Pit Viper (Trimeresurus albolabris) Envenomation: A Simulated In Vitro Study.

INTRODUCTION: The green pit viper (GPV) Trimeresurus albolabris is found in Southeast Asia. Its venom has a thrombin-like activity that can cause hypofibrinogenemia. Fibrinogen measurement is not always available. We aimed to establish a more available diagnostic tool indicating hypofibrinogenemia caused by GPV envenomation. METHODS: This was an in vitro study, in which healthy subjects aged 20 to 45 y were enrolled. There were 2 experiments. In Experiment 1, blood samples from 1 subject had varying amounts of T albolabris venom added to determine its effect on the fibrinogen level (FL). In Experiment 2, 3 sets of blood samples were obtained from another 25 subjects. The 2 venom doses established in Experiment 1 were used on 2 sets of the samples to simulate severe (FL <1.0 g·L-1) and mild hypofibrinogenemia (FL 1.0-1.7 g·L-1). The third set of samples was venom-free. All samples were used for platelet counts, prothrombin time (PT)/international normalized ratio (INR)/activated partial thromboplastin time (aPTT), and 2 bedside clotting tests. Diagnostic parameters were calculated against the target FL of <1.0 g·L-1 and <1.7 g·L-1. RESULTS: Twenty-five subjects were enrolled in Experiment 2. On referencing normal cutoff values (platelet count >150,000 cells/mm3, venous clotting time <15 min, normal 20-min whole blood clotting time, INR <1.2, aPTT <30), we found abnormalities of 5, 0, 0, 3, and 22%, respectively. The highest correlation with hypofibrinogenemia was provided by PT/INR. For an FL of <1.0 g·L-1, PT and INR revealed the highest areas under the receiver operating characteristic curve, 0.76 (95% CI, 0.55-0.97) and 0.76 (95% CI, 0.57-0.97), respectively. The highest accuracy and the highest sensitivity were provided by PT/INR. CONCLUSIONS: PT/INR could be used as a diagnostic test for severe hypofibrinogenemia in GPV envenomation because of its high accuracy and area under the receiver operating characteristic curve.

Inflammatory effect of Bothropstoxin-I from Bothrops jararacussu venom mediated by NLRP3 inflammasome involves ATP and P2X7 receptor.

Muscle tissue damage is one of the local effects described in bothropic envenomations. Bothropstoxin-I (BthTX-I), from Bothrops jararacussu venom, is a K49-phospholipase A2 (PLA2) that induces a massive muscle tissue injury, and, consequently, local inflammatory reaction. The NLRP3 inflammasome is a sensor that triggers inflammation by activating caspase 1 and releasing interleukin (IL)-1ß and/or inducing pyroptotic cell death in response to tissue damage. We, therefore, aimed to address activation of NLRP3 inflammasome by BthTX-I-associated injury and the mechanism involved in this process. Intramuscular injection of BthTX-I results in infiltration of neutrophils and macrophages in gastrocnemius muscle, which is reduced in NLRP3- and Caspase-1-deficient mice. The in vitro IL-1ß production induced by BthTX-I in peritoneal macrophages (PMs) requires caspase 1/11, ASC and NLRP3 and is dependent on adenosine 5'-triphosphate (ATP)-induced K+ efflux and P2X7 receptor (P2X7R). BthTX-I induces a dramatic release of ATP from C2C12 myotubes, therefore representing the major mechanism for P2X7R-dependent inflammasome activation in macrophages. A similar result was obtained when human monocyte-derived macrophages (HMDMs) were treated with BthTX-I. These findings demonstrated the inflammatory effect of BthTX-I on muscle tissue, pointing out a role for the ATP released by damaged cells for the NLRP3 activation on macrophages, contributing to the understanding of the microenvironment of the tissue damage of the Bothrops envenomation.

Exploring the ability of low-level laser irradiation to reduce myonecrosis and increase Myogenin transcription after Bothrops jararacussu envenomation.

Envenoming caused by snakebites is a very important neglected tropical disease worldwide. The myotoxic phospholipases present in the bothropic venom disrupt the sarcolemma and compromise the mechanisms of energy production, leading to myonecrosis. Photobiomodulation therapy (PBMT) has been used as an effective tool to treat diverse cases of injuries, such as snake venom-induced myonecrosis. Based on that, the aim of this study was to analyze the effects of PBMT through low-level laser irradiation (904 nm) on the muscle regeneration after the myonecrosis induced by Bothrops jararacussu snake venom (Bjssu) injection, focusing on myogenic regulatory factors expression, such as Pax7, MyoD, and Myogenin (MyoG). Male Swiss mice (Mus musculus), 6-8-week-old, weighing 22 ± 3 g were used. Single sub-lethal Bjssu dose or saline was injected into the right mice gastrocnemius muscle. At 3, 24, 48, and 72 h after injections, mice were submitted to PBMT treatment. When finished the periods of 48 and 72 h, mice were euthanized and the right gastrocnemius were collected for analyses. We observed extensive inflammatory infiltrate in all the groups submitted to Bjssu injections. PBMT was able to reduce the myonecrotic area at 48 and 72 h after envenomation. There was a significant increase of MyoG mRNA expression at 72 h after venom injection. The data suggest that beyond the protective effect promoted by PBMT against Bjssu-induced myonecrosis, the low-level laser irradiation was able to stimulate the satellite cells, thus enhancing the muscle repair by improving myogenic differentiation.

Bothrops lanceolatus snake (Fer-de-lance) venom triggers inflammatory mediators' storm in human blood.

Systemic increased inflammatory mediators' levels are a hallmark in a plethora of pathological conditions, including thrombotic diseases as the envenomation by Bothrops lanceolatus snake. Multiple organ infarctions, which are not prevented by anticoagulant therapy, are the main cause of death on this envenomation. However, the potential mechanisms involved in these systemic reactions are underexplored. This study aimed to explore the potential systemic events which could contribute to thrombotic reactions on the envenomation by B. lanceolatus in an ex vivo human whole-blood model. B. lanceolatus venom elicited an inflammatory reaction, which was characterized by a strong complement activation, since we detected high C3a, C4a and C5a anaphylatoxins levels. Besides, the venom promoted soluble Terminal Complement Complex (sTCC) assembly. Complement activation was accompanied by intense lipid mediators' release, which included LTB4, PGE2 and TXB2. In addition, in the blood exposed to B. lanceolatus venom, we detected IL-1ß, IL-6 and TNF-α interleukins production. Chemokines, including CCL2, CCL5 and CXCL8 were upregulated in the venom presence. These outcomes show that B. lanceolatus venom causes a strong inflammatory reaction in the blood favoring a potential setting to thrombi formation. Thus, inhibiting inflammatory mediators or their receptors may help in the envenomed patients' management.

A lipidomics approach reveals new insights into Crotalus durissus terrificus and Bothrops moojeni snake venoms.

Snakebite envenomation causes > 81,000 deaths and incapacities in another 400,000 people worldwide every year. Snake venoms are complex natural secretions comprised of hundreds of different molecules with a wide range of biological functions that after injection cause local and systemic manifestations. Although several studies have investigated snake venoms, the majority have focused on the protein portion (toxins), without significant attention paid to the lipid fraction. Therefore, an untargeted lipidomic approach based on liquid chromatography with high-resolution mass spectrometry (LC-HRMS) was applied to investigate the lipid constituents of venoms of the snake species Crotalus durissus terrificus and Bothrops moojeni. Phosphatidylcholines (PC), Lyso-PCs, phosphatidylethanolamines (PE), Lyso-PE, phosphatidylserine (PS), phosphatidylinositol (PI), ceramides (Cer), and sphingomyelin (SM) species were detected in the analyzed snake venoms. The identified lipids included bioactive compounds such as platelet-activating factor (PAF) precursor, PAF-like molecules, plasmalogens, ceramides, and sphingomyelins with long fatty acid chain lengths, which may be associated with the systemic responses triggered by C. d. terrificus and B. moojeni envenomation. These responses include platelet aggregation, activation of intercellular adhesion molecule 1 (ICAM1), apoptosis, as well as the production of pro-inflammatory lipid mediators, cytokines, and reactive species. The newly proposed lipidomics strategy provided valuable information regarding the lipid profiles of viperid venoms, which could lead to increased understanding of the complex pathology promoted by snakebite envenomation.

A general model for cell death and biomarker release from injured tissues.

Cellular response to insults may result in the initiation of different cell death processes. For many cases the cell death process will result in an acute release of cellular material that in some circumstances provides valuable information about the process (i.e. may represent a biomarker). The characteristics of the biomarker release is often informative and plays critical roles in clinical practice and toxicology research. The aim of this study is to develop a general, semi-mechanistic model to describe cell turnover and biomarker release by injured tissue that can be used for estimation in pharmacokinetic and (toxicokinetic)-pharmacodynamic studies. The model included three components: (1) natural tissue turnover, (2) biomarker release from cell death and its movement from the cell through the tissue into the blood, (3) different target insult mechanisms of cell death. We applied the general model to biomarker release profiles for four different cell insult causes. Our model simulations showed good agreements with reported data under both delayed release and rapid release cases. Additionally, we illustrate the use of the model to provide different biomarker profiles. We also provided details on interpreting parameters and their values for other researchers to customize its use. In conclusion, our general model provides a basic structure to study the kinetic behaviour of biomarker release and disposition after cellular insult.

Actions of Cannabis sativa L. fixed oil and nano-emulsion on venom-induced inflammation of Bothrops moojeni snake in rats.

BACKGROUND: Bothrops moojeni snake venom (VBm) has toxins that cause pronounced tissue damage and exacerbated inflammatory reaction. Cannabis sativa L. is a plant species that produces an oil (CSO) rich in unsaturated fatty acids. Nano-emulsions have several advantages, such as better stability and higher penetrating power in membranes. Therefore, this study evaluated the effect of a nano-emulsion based on this herbal derivative (NCS) against VBm-induced inflammation in Wistar rats. METHODS: The CSO and NCS were submitted to physicochemical characterization. The inflammatory process was induced by the VBm (0.10 mg/kg) as follows: rat paw edema, peritonitis, analysis of leukocyte infiltrate in gastrocnemius muscle of rats and formation of granulomatous tissue. RESULTS: No significant changes were observed when the NCS was submitted to the centrifugation and thermal stress tests. There was no phase separation, changes in density (0.978 ± 0.01 g/cm3) and viscosity (0.889 ± 0.15). The droplet diameter ranged from 119.7 ± 065 to 129.3 ± 0.15 nm and the polydispersity index ranged from 0.22 ± 0.008 to 0.23 ± 0.011. The results showed that treatments with CSO (200 and 400 mg/kg) and NCS (100 mg/kg) were able to decrease significantly (p < 0.001) the formation of edema and granulomatous tissue. The CSO and NCS groups significantly attenuated (p < 0.001) the recruitment of inflammatory cells in the tests for peritonitis and leukocyte infiltrate. The histopathological analysis of the gastrocnemius muscle showed a reduction in tissue damage caused by VBm. CONCLUSION: The results obtained in this study showed anti-inflammatory activity of the CSO which may be due to a high UFA content. The nanosizing, as evidenced by the incorporation of the CSO in the NCS improved the effect and opens the perspective for the obtainment of a nanomedicine in which a kinetic stable phytotherapic can be used at low doses.

Catechin and epicatechin as an adjuvant in the therapy of hemostasis disorders induced by snake venoms.

Snake toxins, such as phospholipases A2 and proteases, are used as research tools to evaluate biological activities and to understand physiopathological processes of natural compounds better. In the present study, the phenolic compounds catechin and epicatechin were incubated with snake venoms to evaluate their inhibition against different substrates. Catechin and epicatechin exerted inhibitions between 20% and 95% on the activity of phospholipases A2 present in the venom of Bothrops alternatus. In the hemolytic activity, catechin exerted inhibitions between 20% and 25% in all proportions evaluated on the B. jararacussu venom, whereas epicatechin inhibited 20% of the venom activity. Coagulation induced by B. atrox and B. jararacussu venoms was significantly inhibited by catechin and epicatechin, where the time for coagulation was two to three times higher after previous incubation of the venoms with the compounds. The most significant inhibitions for the proteolytic activity on casein were 17% and 27%, respectively, by both compounds. Catechin inhibited serine protease activity induced by B. atrox venom by 64% and epicatechin by 65%. Regarding B. atrox-induced thrombolysis, catechin exerted 40% inhibition and epicatechin around 30%. The fibrinogen proteolysis was completely inhibited by catechin acting on the B. atrox venom in the proportion of 1:1 and by epicatechin on B. jararacussu venom. Catechin and epicatechin showed promising inhibitory action on proteases and phospholipases A2 . Therefore, these compounds can be explored as an adjuvant for serum therapy or pharmaceutical purposes, once they act on homologous enzymes that are present in humans.

Photobiomodulation reduces cell death and cytokine production in C2C12 cells exposed to Bothrops venoms.

Snakebites caused by the genus Bothrops are often associated with severe and complex local manifestations such as edema, pain, hemorrhage, and myonecrosis. Conventional treatment minimizes the systemic effects of venom; however, their local action is not neutralized. The purpose of this study was to evaluate the effect of photobiomodulation (PBM) on C2C12 muscle cells exposed to B. jararaca, B. jararacussu, and B. moojeni venoms on events involved in cell death and the release of inflammatory mediators. Cells were exposed to venoms and immediately irradiated with low-level laser (LLL) application in continuous wave at the wavelength of 660 nm, energy density of 4.4 J/cm2, power of 10 mW, area of 0.045 cm2, and time of 20 s. Cell integrity was analyzed by phase contrast microscope and cell death was performed by flow cytometry. In addition, interleukin IL1-ß, IL-6, and IL-10 levels were measured in the supernatant. Our results showed that the application of PBM increases cell viability and decreases cell death by apoptosis and necrosis. Moreover, the release of pro-inflammatory interleukins was also reduced. The data reported here indicate that PBM resulted in cytoprotection on myoblast C2C12 cells after venom exposure. This protection involves the modulation of cell death mechanism and decreased pro-inflammatory cytokine release.