Bothrops Alternatus Research Articles

Resource partitioning in a snake assemblage from east-central Argentina.

Two dimensions of the ecological niche (diet and habitat) of a snake assemblage from an endemic rich area in east-central Argentina, the Sierras de Ventania mountain chain, were analyzed. Field data collection was performed in 15-week study periods between 2010 and 2014. Snakes were hand-captured using transect surveys. Field observations on diet were analyzed together with stomach content data from museum specimens. Our results supported the partitioning of the snake assemblage by both habitat use and diet into at least three functional groups: species restricted to microhabitats under rocks and with a diet composed exclusively of ants (Epictia australis); species found mostly in stream microhabitats and feeding mainly upon anurans (Erythrolamprus poecilogyrus and Lygophis elegantissimus); and species found mostly in grassland microhabitats, with specialized diets of terrestrial prey items (Philodryas patagoniensis and Bothrops alternatus). Consistent with previous work, diet was more important than habitat in explaining ecological niche partitioning of this snake assemblage. Our results showed that high overlap values of microhabitat use were compensated by low overlap values of the trophic niche dimension, thus matching the traditional complementary niches hypothesis.

A complex biogeographic history of diversification in Neotropical lancehead pitvipers (Serpentes, Viperidae)

AbstractBased on the literature, we had predicted that the diversification within the Neotropical snake genusBothropsoccurred along a latitudinal gradient from north to south, with diversification into unoccupied niches through ecological opportunity, not correlated with geoclimatic events. Using a dated phylogeny and estimating likelihoods of ancestral states at cladogenesis events, we reconstructed ancestral areas and assessed major events of the diversification ofBothropsclades, and we also discuss systematic implications for this group. Based on the phylogeny we produced,B. lojanuswas not considered as part of the genusBothropssince the results recovered this species nested within theBothrocophiasclade. We infer that the diversification of the MioceneBothrops pictusandBothrops alternatusclades may be related to the uplift of the western slopes of the Andes and the Argentinian Patagonian Andes, respectively. The PlioceneBothrops taeniatusandBothrops osborneiclades may be related to the uplift of the eastern and northern Andes, respectively. The Plio‐PleistoceneBothrops neuwiediclade may be related to the habitat transitions from a warmer and forested environment to a cooler and open landscape; theBothrops jararaca(i.e. island endemic species) andBothrops lanceolatusclades to over‐water dispersal with island speciation; andBothrops atroxclade to the appearance of the Panamanian land bridge. We found that a multitemporal and multidirectional history of diversification may be correlated with geoclimatic and dispersalist events. We argue that the vacant niche hypothesis by itself does not explainBothropsdiversification.

Bothrops alternatus Snake Venom Induces Cytokine Expression and Oxidative Stress on Renal Function.

Envenomation caused by Bothrops alternatus is common in Southern Brazil. Acute Kidney Injury occurs after Bothrops snakebite and more information is necessaryrequired to understand its mechanism. The objective was to evaluate the effect of Bothrops alternatus venom (BaV) on renal cells and rat isolated kidney function. Wistar rats (n = 6, weighing 260-320 g) were perfused with a Krebs-Henseleit solution containing 6 g 100 mL-1 of bovine serum albumin. After 30 minutes, the kidneys were perfused with BaV to a final concentration of 1 and 3 μgmL-1; and subsequently were evaluated for Perfusion Pressure (PP), Renal Vascular Resistance (RVR), Urinary Flow (UF), Glomerular Filtration Rate (GFR), and percentage of electrolyte tubular transport. Renal histological analysis, cytokine release, oxidative stress and cytotoxicity in renal proximal tubular cells were assessed. BaV reduced PP, RVR, GFR, UF, total and proximal sodium transport (%TNa+), and chloride (%TCl-) in the isolated kidney perfusion model. Histological analysis of perfused kidneys disclosed the presence of proteinaceous material in the glomeruli and renal tubules, vacuolar tubular epithelial cell degeneration, Bowman's capsule degeneration, swelling of glomerular epithelial cells, glomerular atrophy and degeneration, and the presence of intratubular protein. Cytokine release (TNF-α, IL-1β, IL-10) and oxidative stress were increased in the kidneys. The viability of LLC-MK2 cells (IC50: 221.3 μg/mL) was decreased by BaV and necrosis was involved in cell death. These findings indicate that BaV modifies functional parameters in an isolated perfused kidney model and has cytotoxic effects on renal lineage cells.

Inhibition by essential oils of Mentha viridis and Mentha pulegium (Lamiaceae) in proteolysis, fibrinogenolysis and coagulation caused by venomous snakes

Snake venom are widely used as laboratory tools for studies of physiological, pharmacological and toxicological mechanisms. Venoms used here are rich sources of several classes of proteases that act on factors of the coagulation cascade, fibrinogenolysis and fibrinolysis, altering the hemostatic processes, and phospholipases A2 which are involved mainly in inflammatory and clotting processes since they act hydrolyzing membrane phospholipids and may result in the release of arachidonic acid whose structure is a precursor of eicosanoids by cyclooxygenase and lipoxygenase pathways. Natural products such as essential oils are made up of active ingredients with wide application in the food, pharmaceutical and cosmetic industries. Thus, in this study evaluate the essential oils from Mentha viridis and Mentha pulegium on coagulation, fibrinogenolysis and degradation of azocasein, induced by Bothrops sp and Lachesis muta muta venoms. These oils were achieved by hydrodistillation and presented, respectively, as the main constituents linalool (40.70%), carvone (13.52%) and α-terpinene (8.56%); pulegone (50.01%), menthol (31.90%) and menthone (16.56%). The essential oils were previously incubated with Bothrops alternatus venom, for two different times, plasma was added and timing. The M. pulegium and M. viridis oils in the volume of 0.30 μL (10 min of incubation) presented greater anticlotting potential. Meanwhile, 0.15 μL the M. pulegium oil presented proclotting activity. In 20 min of incubation, both oils presented anticlotting activity with 0.15 and 0.30 μL. At azocaseinolytic assay the oil from M. pulegium reduced the activity for all evaluated venoms. The highest inhibitions were 34.33% and 39.99% for 0.6 and 1.2 μL of oil, respectively; on activity induced by B. jararacussu, M. viridis with 0.6 and 1.2 μL reduced the activity in 40.93% and 57.72%, respectively. On B. moojeni, the same volumes were responsible for inhibitions of 74.67% and 47.4%, respectively. The fibrinogenolysis induced by B. moojeni venom was totally inhibited by both oils in the evaluated proportions. The results show the presence in oils of protease inhibitors, considering metalloproteases (mainly with thrombin-like and hemorrhagic activity) and serineproteases (actuating on clotting factors), as well as phospholipase A2­ inhibitors (involved in inflammation and clotting processes) of wide application in medical and biotechnology areas.

Lecanicillium aphanocladii: snake venom phospholipases A2 and proteases as tools to prospect enzymatic inhibitors.

Natural enzyme inhibitors have been widely described in literature because of its pharmacological and cosmetic applications. Fungi found in caves represent a promising source of bioactive substances that are still little explored scientifically. Thus, the present work evaluated the presence of enzymatic modulators in a filtrate obtained from the cultivation of the cave fungus Lecanicillium aphanocladii (Family: Cordycipitaceae). Snake venoms from Bothrops alternatus and Bothrops atrox were used as an enzymatic source for the induction of the phospholipase, proteolytic, thrombolytic, cytotoxic and coagulant activities. Compounds present in the fungal filtrate inhibited 50, 23·8, 26·6, 50·9 and 52·5% of the proteolytic, phospholipase, haemolytic, thrombolytic and coagulant activities respectively. The filtrate was not cytotoxic on erythrocytes, but induced partial dissolution of thrombi. Fungal enzyme inhibitors that have low or no toxicity and can be obtained on a large scale and at low cost are relevant in the medical-scientific context. Therefore, the inhibition of phospholipases A2 and proteases observed in the present work highlights the potential of fungal metabolites for the development of drugs that can be used in the treatment of haemostasis and inflammation-related disorders. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, secondary metabolites synthesized by Lecanicillium aphanocladii, a fungus isolated from caves, demonstrated modulating action on proteases and phospholipases A2 present in snake venoms of the Bothrops genus, widely used as tools for the study of pathophysiology processes related to haemostasis and inflammation. The results suggest the possibility of future applications for these metabolites in the development of pharmaceuticals of medical-scientific interest.

Molecular interactions between p-coumaric acid and snake venom toxins.

A large number of natural compounds, such as phenolic compounds, have been scientifically evaluated in the search for enzyme inhibitors. The interactions between the phenolic compound p-coumaric acid and the enzymes present in snake venoms (used as research tools) were evaluated in vitro and in silico. The p-coumaric acid was able to inhibit 31% of the phospholipase activity induced by Bothrops alternatus venom, 27% of the hemolytic activity induced by B. moojeni, 62.5% of the thrombolytic activity induced by B. jararacussu, and approximately 27% of the activity thrombosis induced by Crotalus durissus terrificus. Previous incubation of p-coumaric acid with the venoms of B. atrox and B. jararacussu increased the coagulation time by 2.18 and 2.16-fold, respectively. The activity of serine proteases in B. atrox and B. jararacussu venoms was reduced by 60% and 66.34%, respectively. Computational chemistry analyses suggests the specific binding of p-coumaric acid to the active site of proteases through hydrogen and hydrophobic interactions. The phenolic compound evaluated in this work has great potential in therapeutic use to both prevent and treat hemostatic alterations, because the venom proteins inhibited by the p-coumaric acid have high homology with human proteins that have a fundamental role in several pathologies.

IgY-based antivenom against Bothrops alternatus: Production and neutralization efficacy

Antivenom for the treatment of bothropic snakebite is a priority for public health institutions from Latin America. An alternative to the conventional antivenom production is based on the use of egg yolk antibodies – IgY-technology – by immunizing laying hens. In this study, we produced, characterized and assessed the efficacy of IgY-based antivenoms against B. alternatus venom. Immunochemical studies (reactivity, avidity and antigen recognition pattern) as well as antivenom efficacy assays were performed. After the 3rd immunization, levels of specific IgY reached a maximum that was maintained throughout the observation period, while avidity indexes of the extracts increased after the successive immunizations. Furthermore, IgY against B. alternatus recognized protein complexes of the venom with high (>40 kDa), medium (20–40 kDa) and low (<20 kDa) molecular weights. IgY antivenoms obtained after 8 immunizations neutralized 35.65 μg of B. alternatus venom per mg of antivenom, while specific activities values ranged from 0.28 to 0.42. In conclusion, we produced and characterized IgY antivenoms capable of neutralizing the lethal activity of B. alternatus venom at a preclinical level. Thus, IgY-technology may allow the production of effective and affordable antivenoms fulfilling the urgent needs of many countries where conventional manufacture is unable to provide enough availability of antivenoms.

Genomic features of a multidrug-resistant Enterobacter cloacae ST279 producing CTX-M-15 and AAC(6')-Ib-cr isolated from fatal infectious stomatitis in a crossed pit viper (Bothrops alternatus).

The widespread dissemination of extended-spectrum β-lactamase-producing Enterobacteriaceae has become a major issue in veterinary medicine. However, until now, there has been no report of bacteria with such a phenotype in infected snakes. The aim of this study was to report the first draft genome sequence of an Enterobacter cloacae isolate (SERP1) recovered from a snake with infectious stomatitis. The whole genome of E. cloacae strain SERP1 was sequenced on an Illumina NextSeq platform and was de novo assembled using CLC NGS Cell v.10. Data analysis was performed using online tools from the Center of Genomic Epidemiology. The genome size was calculated at 4966856bp, containing a total of 4796 protein-coding sequences. The strain was assigned to sequence type 279 (ST279) and, besides the clinically relevant blaCTX-M-15 and aac(6')-Ib-cr genes, it also presented resistance genes to β-lactams, aminoglycosides, phenicols, sulphonamides, tetracyclines, trimethoprim, quinolones and fosfomycin. These data offer novel information regarding multidrug-resistant E. cloacae dissemination in wild animals and might contribute to further comparative genomic analysis.

Species distribution models and empirical test: Comparing predictions with well-understood geographical distribution of Bothrops alternatus in Argentina.

Species distribution models (SDMs) estimate the geographical distribution of species although with several limitations due to sources of inaccuracy and biases. Empirical tests arose as the most important steps in scientific knowledge to assess the efficiency of model predictions, which are poorly rigorous in SDMs. A good approach to the empirical distribution (ED) of a species can be obtained from comprehensive empirical knowledge, that is, well‐understood distributions gathered from large amount of data generated with appropriate spatial and temporal samples coverage. The aims of this study were to (a) compare different SDMs predictions with an ED; and (b) evaluate if fuzzy global matching (FGM) could be used as an index to compare SDMs predictions and ED. Six algorithms with 5 and 20 variables were used to assess their accuracy in predicting the ED of the venomous snake Bothrops alternatus (Viperidae). Its entire distribution is known, thanks to thorough field surveys across Argentina, with 1,767 records. ED was compared with SDMs predictions using Map Comparison Kit. SDMs predictions showed important biases in all methods used, from 70% sub‐estimation to 40% over‐estimation of ED. BIOCLIM predicted ≈31% of B. alternatus ED. DOMAIN predicted 99% of ED, but over‐estimated 40% of the area. GLM with five variables calculated 75% of ED, while Genetic Algorithm for Rule‐set Prediction showed ≈60% of ED; the last two presenting overpredictions in areas with favorable climatic conditions but not inhabited by the species. MaxEnt and RF were the only methods to detect isolated populations in the southern distribution of B. alternatus. Although SDMs proved useful in making predictions about species distribution, predictions need validation with expert maps knowledge and ED. Moreover, FGM showed a good performance as an index with values similar to True Skill Statistic, so that it could be used to relate ED and SDMs predictions.

Use of EDTA in the treatment of local tissue damage caused by the Bothrops alternatus venom

ABSTRACT Twelve adult rabbits were distributed in three groups and received on the femoral biceps region, via intradermal injection (ID), 25µg of Bothrops alternatus venom dissolved in NaCl 0.9% and diluted in 0.25mL of phosphate buffered saline (PBS). Thirty minutes later, the group G1 received 0.25mL of phosphate buffered saline (PBS) ID while to G2 and G3 25mg of ethylenediamine tetraacetic acid (EDTA) dissolved in 0.25mL of PBS were administered via intramuscular (IM) and intravenous (IV) injection, respectively. Evaluations included local lesion and blood profile of all animals, before (time zero) and at 1, 2, 3, 4, 5, 6, 12, 18 and 24h after venom administration. All animal treated with PBS (G1) and EDTA IV (G3) presented increase of nociceptive stimuli at the site of inoculation of the venom, followed by moderate edema that persisted for 24h. Animals treated with IM EDTA (G2) only manifested increase of nociceptive stimuli at the site of injection 1h after treatment with discrete local edema between 12 and 24h. In relation to the local hemorrhagic halo no differences were found amongst the studied groups. Blood profile revealed significant decrease of segmented neutrophils in all groups. There was also increase in triglycerides and decrease in total protein and albumin in all groups. The local lesion was not altered by the treatments.

Could mesenchymal stem cell therapy help in the treatment of muscle damage caused by Bothrops alternatus venom?

ABSTRACT: The aim of this study was to evaluate the use of mesenchymal stem cells (MSC) in the treatment of myonecrosis induced by Bothrops alternatus venom in rats. Seventy-five male adult Wistar rats were divided into three experimental groups. G1 and G2 were injected in the gastrocnemius muscle with 120μg of B. alternatus venom, while G3 received 200μL of PBS only. Three days after the venom injection, 12 rats from G1 were treated with 5.0 x 106 MSC in PBS, whereas G2 and G3 rats received PBS. Every three days, blood and muscle samples of five animals from each group were taken for serum biochemical and pathological analyses. Histological examinations showed more intense muscle lesions following MSC treatment, characterized by disorganization and loss of muscle fibers, with focal necrosis and inflammatory infiltration by mononuclear cells. In conclusion, the use of MSC for the treatment of local damage caused by inoculation of B. alternatus venom impaired muscle regeneration and interfered in the healing process.

Purification of a fragment obtained by autolysis of a PIIIb-SVMP from Bothrops alternatus venom

Snake Venom Metalloproteinases (SVMPs) represent 43.1% of the components in Bothrops alternatus venom and play an important role in envenomation. Disintegrins and disintegrin-like domains are released by proteolytic processing of PII and PIII classes of SVMPs respectively and are potent inhibitors of integrin–ligand interaction. Baltergin is a PIIIb-SVMP isolated from this venom and able to undergo autolysis in vitro, giving rise to a stable disintegrin-like/cystein-rich fragment (baltergin-DC). Conditions of baltergin autolysis were adjusted in order to carry out the purification of baltergin-DC and its effect on cell adhesion was studied. Autolysis was maximal at 37°C and a pH range of 7.0–8.0. Baltergin-DC amino-terminal sequence begins with IISPPVCGNELLEVGEECDCGTPENCQNECCDAATC, which shows a high degree of homology with other disintegrin-like proteins. Baltergin and purified baltergin-DC were both able to inhibit C2C12 adhesion to fetal bovine serum (FBS) coated plates, indicating that a non-catalytic process is involved, probably mediated by binding to membrane integrins. Baltergin-DC, lacking proteolytic action, becomes an attractive molecule for future studies on blocking integrin–ligand interactions.

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.

BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom

BackgroundSnake venoms are a complex mixture of proteins, organic and inorganic compounds. Some of these proteins, enzymatic or non-enzymatic ones, are able to interact with platelet receptors, causing hemostatic disorders. The possible therapeutic potential of toxins with antiplatelet properties may arouse interest in the pharmacological areas. The present study aimed to purify and characterize an antiplatelet DC protein from Bothrops alternatus snake venom.MethodsThe protein, called BaltDC (DC protein from B. alternatus snake venom), was purified by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. The molecular mass was estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The amino acid sequence of the N-terminal region was carried out by Edman degradation method. Platelet aggregation assays were performed in human platelet-rich plasma (PRP). Infrared (IR) spectroscopy was used in order to elucidate the interactions between BaltDC and platelet membrane.ResultsBaltDC ran as a single protein band on SDS-PAGE and showed apparent molecular mass of 32 kDa under reducing or non-reducing conditions. The N-terminal region of the purified protein revealed the amino acid sequence IISPPVCGNELLEVGEECDCGTPENCQNECCDA, which showed identity with other snake venom metalloproteinases (SVMPs). BaltDC was devoid of proteolytic, hemorrhagic, defibrinating or coagulant activities, but it showed a specific inhibitory effect on platelet aggregation induced by ristocetin and epinephrine in PRP. IR analysis spectra strongly suggests that PO32− groups, present in BaltDC, form hydrogen bonds with the PO2− groups present in the non-lipid portion of the membrane platelets.ConclusionsBaltDC may be of medical interest since it was able to inhibit platelet aggregation.

Prospection of Effects of Guava Leaves Infusion: Antigenotoxic Action and Enzymatic Inhibition

Several forms of Psidium guajava L. (guava) leaf preparations have been widely used for prevention and treatment of illnesses. However, researches about the protective action of guava's natural products on the genetic material of animal cells is scarce. Accordingly, the aim of this study was to evaluate the antigenotoxic potential of infusions of guava leaves from three cultivars (Pedro Sato, Paluma and Roxa) against DNA damage induced by Doxorubicin (DXR) in human leukocytes, and their effects on enzymatic inhibition. To assess the antigenotoxic potential of these infusions, a comet assay (single cell electrophoresis) was conducted. In addition, their inhibitory potential on phospholipase and hemolytic activities was assessed. The evaluated infusion volumes (25, 50 and 300μL) presented inhibitions around 75% of the DXR-induced damage frequencies. Their protective effect was demonstrated by arbitrary unit calculations, with values between 52.51 and 66.60 for all treatments, 250.51 for positive control (DXR), and 45.49 for phosphate buffered saline (negative control). The infusions significantly inhibited phospholipase and hemolytic activities induced by Bothrops alternatus and B. moojeni venoms, with inhibitions ≥ 50% for both activities induced by B. alternatus. The infusions of P. guajava from the cultivars analyzed present antigenotoxic potential, but also demonstrate potential of enzymatic inhibition on A2 phospholipases and proteases.

Effects of Bothrops alternatus venom in zebrafish: a histopathological study.

Zebrafish is an excellent model organism for studying tissue alterations caused by Bothrops alternatus venom (BAV) and for screening new anti-venom drugs. To study tissue alterations following exposure to BAV and the roles that glucocorticoids play in these tissue reactions, zebrafish were randomly divided into five groups: the free injection control group (FIC), the phosphate-buffered saline injection control group (PIC), the venom injected group (VI), the group treated with dexamethasone 1h before venom injection (D1hBVI) and the group treated with dexamethasone 1h after venom injection (D1hAVI). The concentration of BAV injected was 0.13mg/mL and each fish received an injection of 20 μL. Body weight measurements and histopathological characteristics of the gills, kidneys, liver, and intestine were determined. Histopathological analyses showed necrosis, inflammation and weight gain in animals that received BAV. The histological alteration indices of the gills, liver, kidneys, and intestines were statistically higher in the animal groups treated with BAV. These alteration indices were lower in the D1hBVI and D1hAVI groups compared to the group treated with BAV alone. The D1hBVI group is presented with minor alterations. A significant difference in the histological alterations index was observed in the intestinal tissue of the FIC group compared to the PIC group. Cumulatively, zebrafish may serve as a useful biomarker for alterations induced by BAV. Interestingly, dexamethasone reduced the damage caused by BAV in the organs studied, which suggests that zebrafish might be useful for screening new drugs that can mitigate tissue damage caused by snakebites.

Apoptosis induced by a snake venom metalloproteinase from Bothrops alternatus venom in C2C12 muscle cells.

In this study, the apoptosis inducing effects of baltergin as well as its influence on cell adhesion and migration on muscles cells in vitro were studied. Morphological analysis made by scanning electron and phase contrast microscopy demonstrated typical futures of programmed cell death, apoptosis. This mechanism was confirmed by fluorescence staining, molecular analysis of endonuclease activity and increased mRNA expression level of two representative genes (p53 and bax). On the other hand, baltergin exert an inhibition effect on myoblast cell adhesion and migration in vitro probably through a mechanism that involves the interaction of this enzyme with cell integrins. In conclusion, our results suggest that the absence of appropriate extracellular matrix contacts triggers anoikis. Therefore, this is the first report that demonstrated the mechanism of programmed cell death triggered by baltergin, a PIII metalloprotease isolated from Bothrops alternatus venom, in a myoblast cell line.

RBaltMIP, a recombinant alpha-type myotoxin inhibitor from Bothrops alternatus (Rhinocerophis alternatus) snake, as a potential candidate to complement the antivenom therapy

Phospholipase A2 inhibitors (PLIs) are important targets in the search and development of new drugs. This study aimed at evaluating the potential of an alpha-type phospholipase A2 inhibitor from Bothrops alternatus (Rhinocerophis alternatus) snake in its recombinant form (rBaltMIP) to complement the conventional antivenom therapy. Biochemical experiments showed that rBaltMIP presented pI 5.8 and molecular masses of ∼21 kDa by SDS-PAGE and 19.57 kDa by MALDI/TOF MS. After tryptic peptides sequencing, the results were compared with other PLIs available in databases, showing 100% identity between rBaltMIP and its native inhibitor BaltMIP and from 92% to 96% identity with other inhibitors. Myotoxic activities of BthTX-I and BthTX-II toxins were measured via plasma CK levels, showing myotoxic effective concentrations (EC50) of 0.1256 μg/μL and 0.6183 μg/μL, respectively. rBaltMIP neutralized the myotoxicity caused by these two toxins up to 65%, without promoting primary antibody response against itself. Nevertheless, this recombinant PLI was immunogenic when standard immunization protocol with Freud's adjuvant was used. In paw edema assays, EC50 of 0.02581 μg/μL and 0.02810 μg/μL, respectively, were observed with edema reductions of up to 40% by rBaltMIP, suggesting its use as an additional antivenom. In addition, myotoxicity neutralization experiments with the myotoxin BthTX-I showed that rBaltMIP was more effective in inhibiting muscle damage than the conventional antivenom. Thus, considering the severity of envenomations due to Bothrops alternatus (Rhinocerophis alternatus) and the low neutralization of their local effects (such as myotoxicity) by the current antivenoms, rBaltMIP is a promising molecule for the development of novel therapeutic strategies for clinical applications.

Acción local de vacunas elaboradas con veneno de &lt;i&gt;Bothrops alternatus&lt;/i&gt; en equinos productores de suero antiofídico

El objetivo del trabajo consistió en estudiar los efectos locales causados por sucesivas inoculaciones de veneno de Bothrops alternatus, en un grupo de tres equinos inmunizados para la producción de suero antiofídico monovalente. El adyuvante de Freund causó una respuesta importante para el estímulo inmunológico, en tanto que la inmunización con el veneno entero indujo sangrado local, edema, dolor y escape de enzimas del tejido muscular. La reacción local varió con las sucesivas vacunas, siendo constantes la hemorragia local y la inflamación, con formación de abscesos en las últimas inoculaciones. La actividad AST reveló elevaciones significativas de su tasa sérica, en tanto que LDH se incrementó de manera altamente significativa a partir de las 3 h con acmé a las 6 h y posterior descenso a las 24 h. En la primera vacunación, CPK se elevó significativamente a las 3 h y registró un segundo pico a las 24 h. Se observó una reducción de la magnitud de elevación de las actividades enzimáticas en las últimas inoculaciones (con altas dosis de veneno), quizás debido a que los anticuerpos preformados hayan conferido protección y reducido la destrucción de fibras musculares. Se concluye que los intensos efectos locales (dolor, hemorragia y posterior fibrosis local) imponen ensayar nuevas técnicas de inmunización a fin de minimizar los efectos adversos causados a los equinos.

Efficient muscle regeneration after highly haemorrhagic Bothrops alternatus venom injection

Bothrops alternatus snake venom is particularly characterized for inducing a prominent haemorrhage and affecting hemostasis as a consequence of 43.1% of metallo-proteinases and less than 10% of PLA2 (almost all non-myotoxic phospholipases) in its venomics. In addition, myonecrosis is the major local effect in viper envenoming which might lead to permanent sequela. Then, the rebuilding of the microvasculature at the local injured site acquires significance since represents one of the pivotal stages for subsequent skeletal muscle regeneration either at morphological or functional aspects. Due to the significance played by vasculature in this process, it is important to study by histology and immunohistochemical techniques, the muscular damage and the sequence of skeletal muscle reconstruction (degree of damage, reconstitution of muscle fibres and capillaries). In this work, we injected intramuscularly 50 or 100 μg per mouse of B. alternatus venom in gastrocnemius muscles. We provided a complete description and characterization of the different stages of myogenesis after mild (50 µg) and severe (100 µg) local injury induced by B. alternatus venom toxins. The regeneration was evaluated 24 h, 3, 7, 14 and 28 days after receiving venom injection. Finally, both doses induced an extended necrosis at the site of injection where, when critical steps in the regenerative process are taking place, an efficient tissue rebuilding is achieved. B. alternatus venom is characterized by the high percentage of exclusively class P-III metalloproteinases, and by the lack of class P-I metalloproteinases in its venom composition. This could explain the effectiveness of muscle regeneration after venom injection despite the severity of the initial phase of envenoming.