RESUMEN

BACKGROUND: The venom of Bothrops lanceolatus, a viperid species endemic to the Lesser Antillean Island of Martinique, induces thrombosis in a number of patients. Previous clinical observations indicate that thrombotic events are more common in patients bitten by juvenile specimens. There is a need to develop an experimental model of this effect in order to study the mechanisms involved. METHODOLOGY/PRINCIPAL FINDINGS: The venoms of juvenile and adult specimens of B. lanceolatus were compared by (a) describing their proteome, (b) assessing their ability to induce thrombosis in a mouse model, and (c) evaluating their in vitro procoagulant activity and in vivo hemostasis alterations. Venom proteomes of juvenile and adult specimens were highly similar, albeit showing some differences. When injected by the intraperitoneal (i.p.) route, the venom of juvenile specimens induced the formation of abundant thrombi in the pulmonary vasculature, whereas this effect was less frequent in the case of adult venom. Thrombosis was not abrogated by the metalloproteinase inhibitor Batimastat. Both venoms showed a weak in vitro procoagulant effect on citrated mouse plasma and bovine fibrinogen. When administered intravenously (i.v.) venoms did not affect classical clotting tests (prothrombin time and activated partial thromboplastin time) but caused a partial drop in fibrinogen concentration. The venom of juvenile specimens induced partial alterations in some rotational thromboelastometry parameters after i.v. injection. When venoms were administered i.p., only minor alterations in classical clotting tests were observed with juvenile venom, and no changes occurred for either venom in rotational thromboelastometry parameters. Both juvenile and adult venoms induced a marked thrombocytopenia after i.p. injection. CONCLUSIONS/SIGNIFICANCE: An experimental model of the thrombotic effect induced by B. lanceolatus venom was developed. This effect is more pronounced in the case of venom of juvenile specimens, despite the observation that juvenile and adult venom proteomes are similar. Adult and juvenile venoms do not induce a consumption coagulopathy characteristic of other Bothrops sp venoms. Both venoms induce a conspicuous thrombocytopenia. This experimental model reproduces the main clinical findings described in these envenomings and should be useful to understand the mechanisms of the thrombotic effect.

Asunto(s)

Bothrops , Venenos de Crotálidos , Modelos Animales de Enfermedad , Trombosis , Animales , Ratones , Trombosis/inducido químicamente , Venenos de Crotálidos/toxicidad , Masculino , Proteoma , Coagulación Sanguínea/efectos de los fármacos , Inyecciones Intraperitoneales , Serpientes Venenosas

RESUMEN

Venom-induced hemorrhage analysis usually is performed by Minimum Hemorrhagic Dose (MHD), however a similar method can be used to compare venoms with fewer laboratory animals. Our work compared the MHD of five different venoms, with the size of hemorrhagic spot, finding good correlations in the results. Considering the 3Rs principle, we propose the use of the hemorrhagic spot method to compare hemorrhagic activity of snake venoms, rather than using the MHD method, since the first one needs 5 times less animals than the other.

Asunto(s)

Hemorragia , Venenos de Serpiente , Animales , Hemorragia/inducido químicamente , Venenos de Serpiente/toxicidad , Ratones , Alternativas a las Pruebas en Animales , Venenos Elapídicos/toxicidad , Venenos de Crotálidos/toxicidad , Mordeduras de Serpientes

RESUMEN

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.

Asunto(s)

Antivenenos , Bothrops , Supervivencia Celular , Venenos de Crotálidos , Fibroblastos , Animales , Venenos de Crotálidos/toxicidad , Antivenenos/farmacología , Supervivencia Celular/efectos de los fármacos , Línea Celular , Fibroblastos/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ratones , Humanos , Técnicas de Cultivo de Célula , Serpientes Venenosas

RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: Hymenaea eriogyne Benth (Fabaceae) is popularly known as "Jatobá". Despite its use in folk medicine to treat inflammatory disorders, there are no descriptions that show its anti-inflammatory potential. AIM OF THE STUDY: In this sense, this study aimed to evaluate the anti-inflammatory and antivenom action of bark and leaves extract of H. eriogyne. MATERIALS AND METHODS: The in vivo anti-inflammatory activity was conducted by carrageenan-induced paw edema and zymosan-induced air pouch models, evaluating the edematogenic effect, leukocyte migration, protein concentration, levels of pro-inflammatory cytokines, malondialdehyde (MDA) and myeloperoxidase (MPO) activity. The antivenom potential was investigated in vitro on the enzymatic action (proteolytic, phospholipase and hyaluronidase) of Bothrops brazili and B. leucurus venom, as well as in vivo on the paw edema model induced by B. leucurus. Furthermore, the influence of its markers (astilbin and rutin) on MPO activity was investigated in silico. For molecular docking, AutodockVina, Biovia Discovery Studio, and Chimera 1.16 software were used. RESULTS: The extracts and bark and leaves of H. eriogyne revealed a high anti-inflammatory effect, with a reduction in all inflammatory parameters evaluated. The bark extract showed superior results when compared to the leaf extract, suggesting the influence of the astilbin concentration, higher in the bark, on the anti-inflammatory action. In addition, only the H. eriogyne bark extract was able to reduce MDA, indicating an associated antioxidant effect. Regarding the in vitro antivenom action, the extracts (bark and leaves) revealed the ability to inhibit the proteolytic, phospholipase and hyaluronidase action of both bothropic venom, with a greater effect against B. leucurus venom. In vivo, extracts from the bark and leaves of H. eriogyne (50-200 mg/kg) showed antiedematogenic activity, reducing the release of MPO and pro-inflammatory cytokines, indicating the presence of bioactive components useful in controlling the inflammatory process induced by the venom. In the in silico assays, astilbin and rutin showed reversible interactions of 9 possible positions and orientations towards MPO, with affinities of -9.5 and -10.4 kcal/mol and interactions with Phe407, Gln91, His95 and Arg239, important active pockets of MPO. Rutin demonstrated more effective types of interactions with MPO. CONCLUSION: This approach reveals for the first time the anti-inflammatory action of H. eriogyne bark and leaf extracts in vivo, as well as its antiophidic potential. Moreover, the distinct effect of pharmacogens as antioxidant agents and distinct effect of astilbin and rutin under MPO sheds light on the different anti-inflammatory mechanisms of bioactive compounds present in H. eriogyne extracts, with high potential for the prospection of new pharmacological agents.

Asunto(s)

Antiinflamatorios , Carragenina , Edema , Simulación del Acoplamiento Molecular , Corteza de la Planta , Extractos Vegetales , Hojas de la Planta , Antiinflamatorios/farmacología , Antiinflamatorios/química , Antiinflamatorios/aislamiento & purificación , Animales , Extractos Vegetales/farmacología , Extractos Vegetales/química , Edema/tratamiento farmacológico , Edema/inducido químicamente , Hojas de la Planta/química , Corteza de la Planta/química , Masculino , Relación Estructura-Actividad , Peroxidasa/metabolismo , Fabaceae/química , Antivenenos/farmacología , Antivenenos/química , Ratas Wistar , Venenos de Crotálidos/toxicidad , Ratones , Bothrops , Citocinas/metabolismo , Zimosan , Biomarcadores/metabolismo , Rutina/farmacología

RESUMEN

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.

Asunto(s)

Bothrops , Colágeno , Venenos de Crotálidos , Hemorragia , Inflamación , Terapia por Luz de Baja Intensidad , Músculo Esquelético , Animales , Ratones , Terapia por Luz de Baja Intensidad/métodos , Músculo Esquelético/efectos de la radiación , Músculo Esquelético/efectos de los fármacos , Hemorragia/patología , Colágeno/metabolismo , Colágeno/análisis , Venenos de Crotálidos/toxicidad , Rayos Infrarrojos , Masculino , Láseres de Semiconductores/uso terapéutico , Mordeduras de Serpientes/radioterapia

RESUMEN

This case report presents an exotic envenomation by a Chinese snake, Protobothrops mangshanensis. Its venom exhibited potent activity against plasma and fibrinogen, among other enzymatic activities. The patient initially presented with edema of the right upper limb, without tissue necrosis. There were no signs of bleeding; however, severe hypofibrinogenemia was observed (nadir value at 0.4 g/L), with a marked increase in fibrinogen degradation products and D-dimers, without any other coagulation disturbances. In the absence of a specific antivenom available against Asian Crotalinae venoms, the patient was treated at the 29th hour after bite with six vials of Antivipmyn™ TRI (Instituto Bioclon, Mexico, Mexico), a Mexican antivenom initially intended for American Crotalinae venoms, i.e., Bothrops asper, Lachesis muta and Crotalus durissus. Fibrinogen began to rise 6 hours after the antivenom infusion and was within the normal range 38 hours later. The report also underscores the utility of ClotPro® (Haemonetics ®USA), a viscoelastic test, for real-time monitoring of the snakebite-related coagulopathy. The clotting time was extended to 188 seconds on the EX-test while the MCF was decreased to 31 mm on the EX-test and the AP-test and was not measurable on the FIB-test, confirming severe hypofibrinogenemia. In order to confirm the paraspecificity of antivenom on the venom of P. mangshanensis, we studied the experimental neutralization of the venom procoagulant effect by Antivipmyn TRI and Green Pit Viper antivenom, which has been used in previous published clinical cases of P. mangshanensis envenomation. Both Antivipmyn™ TRI and Green Pit Viper antivenom corrected the procoagulant effect induced by P. mangshanensis venom. These findings suggest that Antivipmyn™ TRI cross-reacts with Protobothrops mangshanensis venom. In the absence of antivenom covering Asian Crotalinae, Antivipmyn TRI should be considered to treat an envenomation by Protobothrops spp.

Asunto(s)

Antivenenos , Venenos de Crotálidos , Mordeduras de Serpientes , Antivenenos/uso terapéutico , Animales , Mordeduras de Serpientes/tratamiento farmacológico , Humanos , Venenos de Crotálidos/toxicidad , Masculino , México , Francia , Crotalinae , Coagulación Sanguínea/efectos de los fármacos , Fibrinógeno

RESUMEN

Bothrops atrox envenomations in the Brazilian Amazon are responsible for a number of local and systemic effects. Among these, stroke presents the worst prognosis for the patient since it may evolve into disabilities and/or premature death. This complication is caused by coagulation disorders and generates hemorrhagic and thrombotic conditions. This study presents a case report of a 54-year-old female patient who presented extensive cerebral ischemia after a B. atrox envenomation that occurred in the state of Amazonas, Brazil. The patient was hospitalized for 102 days, which included a stay in the intensive care unit. Clinical and laboratory findings indicated a thrombogenic coagulopathy. On discharge, the patient had no verbal response, partial motor response, and right hemiplegia. The assessment carried out four years after discharge evidenced incapacitation, global aphasia and bilateral lower and upper limbs showed hypotrophy with a global decrease in strength. Ischemic stroke is a possible complication of B. atrox snakebites even after antivenom treatment, with the potential to cause debilitating long-term consequences.

Asunto(s)

Antivenenos , Bothrops , Mordeduras de Serpientes , Mordeduras de Serpientes/complicaciones , Femenino , Persona de Mediana Edad , Animales , Humanos , Brasil , Antivenenos/uso terapéutico , Accidente Cerebrovascular Isquémico/etiología , Venenos de Crotálidos/toxicidad , Venenos de Crotálidos/envenenamiento , Isquemia Encefálica/etiología , Bothrops atrox

RESUMEN

BACKGROUND: Photobiomodulation has exhibited promise in mitigating the local effects induced by Bothrops snakebite envenoming; however, the mechanisms underlying this protection are not yet fully understood. Herein, the effectiveness of photobiomodulation effects on regenerative response of C2C12 myoblast cells following exposure to Bothrops jararacussu venom (BjsuV), as well as the mechanisms involved was investigated. METHODOLOGY/PRINCIPAL FINDINGS: C2C12 myoblast cells were exposed to BjsuV (12.5 µg/mL) and irradiated once for 10 seconds with laser light of 660 nm (14.08 mW; 0.04 cm2; 352 mW/cm2) or 780 nm (17.6 mW; 0.04 cm2; 440 mW/ cm2) to provide energy densities of 3.52 and 4.4 J/cm2, and total energies of 0.1408 and 0.176 J, respectively. Cell migration was assessed through a wound-healing assay. The expression of MAPK p38-α, NF-Кß, Myf5, Pax-7, MyoD, and myogenin proteins were assessed by western blotting analysis. In addition, interleukin IL1-ß, IL-6, TNF-alfa and IL-10 levels were measured in the supernatant by ELISA. The PBM applied to C2C12 cells exposed to BjsuV promoted cell migration, increase the expression of myogenic factors (Pax7, MyF5, MyoD and myogenin), reduced the levels of proinflammatory cytokines, IL1-ß, IL-6, TNF-alfa, and increased the levels of anti-inflammatory cytokine IL-10. In addition, PBM downregulates the expression of NF-kB, and had no effect on p38 MAKP. CONCLUSION/SIGNIFICANCE: These data demonstrated that protection of the muscle cell by PBM seems to be related to the increase of myogenic factors as well as the modulation of inflammatory mediators. PBM therapy may offer a new therapeutic strategy to address the local effects of snakebite envenoming by promoting muscle regeneration and reducing the inflammatory process.

Asunto(s)

Bothrops , Venenos de Crotálidos , Citocinas , Terapia por Luz de Baja Intensidad , Mioblastos , Miogenina , Animales , Mioblastos/efectos de los fármacos , Mioblastos/efectos de la radiación , Mioblastos/metabolismo , Ratones , Terapia por Luz de Baja Intensidad/métodos , Citocinas/metabolismo , Línea Celular , Venenos de Crotálidos/toxicidad , Miogenina/metabolismo , Miogenina/genética , Factor de Transcripción PAX7/metabolismo , Factor de Transcripción PAX7/genética , FN-kappa B/metabolismo , Proteína MioD/metabolismo , Proteína MioD/genética , Movimiento Celular/efectos de los fármacos , Movimiento Celular/efectos de la radiación , Factor 5 Regulador Miogénico/metabolismo , Factor 5 Regulador Miogénico/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Mordeduras de Serpientes/radioterapia , Serpientes Venenosas

RESUMEN

Our study presents the anticancer potential of crotamine from Crotalus durissus terrificus in human prostate cancer cell line DU-145. Crotamine isolation was conducted through RP-FPLC, its molecular mass analyzed by MALDI-TOF was 4881.4 kDa, and N-terminal sequencing confirmed crotamine identity. Crotamine demonstrated no toxicity and did not inhibit migration in HUVEC cells. Although no cell death occurred in DU-145 cells, crotamine inhibited their migration. Thus, crotamine presented potential to be a prototype of anticancer drug.

Asunto(s)

Antineoplásicos , Movimiento Celular , Venenos de Crotálidos , Neoplasias de la Próstata , Humanos , Masculino , Neoplasias de la Próstata/tratamiento farmacológico , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Venenos de Crotálidos/toxicidad , Antineoplásicos/farmacología , Crotalus , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Animales

RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: Snakebite envenomation (SBE) is the world's most lethal neglected tropical disease. Bothrops jararaca is the species that causes the greatest number of SBEs in the South and Southeastern of Brazil. The main symptoms are local (inflammation, edema, hemorrhage, and myonecrosis) and systemic (hemorrhage, hemostatic alterations with consumptive coagulopathy, and death) effects. Species of the genus Siparuna, Siparunaceae, are used in folk and traditional medicine to treat SBE. However, limited information is available concerning Brazilian Siparuna species against SBE. AIM OF THE STUDY: To investigate the correlation between the compounds present in the extracts of five Siparuna species as potential agents against proteolytic activity, plasma coagulation, and phospholipase A2 (PLA2) activity caused by B. jararaca venom, using data obtained by UHPLC-MS/MS, biological activity, and multivariate statistics. MATERIALS AND METHODS: The ethanol extracts from leaves of S. ficoides, S. decipiens, S. glycycarpa, S. reginae, and S. cymosa were fractionated by liquid-liquid extraction using different solvents of increasing polarity (hexane, dichloromethane, ethyl acetate, and n-butanol), affording their respective extracts, totaling 25 samples that were assayed through in vitro plasma coagulation and proteolytic activity assays. Moreover, the extracts were analyzed by UHPLC-MS/MS, using electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI) in negative and positive ionization modes. The data was processed in MZmine v. 2.53 and evaluated by multivariate statistical tests (PLS) using the software UnscramblerX v. 10.4. These data were also used to build molecular networks (GNPS), and some ions of interest could be annotated using the library of molecules on the GNPS platform. RESULTS: A total of 19 extracts inhibited B. jararaca-induced plasma coagulation, with emphasis on S. cymosa and S. reginae (800 s). The inhibition of the proteolytic activity was also promising, ranging from 16% (S. glycycarpa) to 99% (S. cymosa, S. decipiens, and S. reginae). In addition, most extracts from S. cymosa and S. reginae inhibited 70-90% of PLA2 activity. Based on data from positive mode APCI analyses, it was possible to obtain a statistic model with reliable predictive capacity which exhibited an average R2 of 0.95 and a Q2 of 0.88, indicating a robust fit. This process revealed five ions, identified as the alkaloids: coclaurine (1), stepholidine (2) O-methylisopiline (3), nornantenine (4) and laurolitsine (5). This is the first study to evidence the potential antivenom of alkaloids from Siparuna species. CONCLUSIONS: Altogether, our results give support to the popular use of Siparuna extracts in SBE accidents, suggesting their potential as an alternative or complementary strategy against envenoming by B. jararaca venom. The predicted ions in the chemometric analysis for the assayed activities can also be correlated with the blocking activity and encourage the continuation of this study for possible isolation and testing of individual compounds on the used models.

Asunto(s)

Alcaloides , Coagulación Sanguínea , Bothrops , Venenos de Crotálidos , Extractos Vegetales , Animales , Coagulación Sanguínea/efectos de los fármacos , Venenos de Crotálidos/toxicidad , Extractos Vegetales/farmacología , Extractos Vegetales/química , Alcaloides/farmacología , Alcaloides/aislamiento & purificación , Alcaloides/química , Brasil , Proteolisis/efectos de los fármacos , Fosfolipasas A2/metabolismo , Inhibidores de Fosfolipasa A2/farmacología , Inhibidores de Fosfolipasa A2/aislamiento & purificación , Hojas de la Planta/química , Antivenenos/farmacología , Antivenenos/aislamiento & purificación , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/aislamiento & purificación , Espectrometría de Masas en Tándem , Bothrops jararaca

RESUMEN

Rattlesnakes belonging to the genus Crotalus are widely distributed throughout the Americas. In Brazil, symptoms commonly associated with envenomation by Crotalus durissus collilineatus include myalgia, rhabdomyolysis, renal failure, neurotoxicity, and progressive paralysis, which are related to the protein composition of this venom. Snake venom composition exhibits compositional variability that may reflect geographic distribution, age, captivity, diet, sex, and even individual genetics. Although seasonality is also considered a possible source of variation, there are few reports of such variability in snake venom. In this work, venoms of the same eight C. durissus collilineatus were extracted every three months for two years, to analyze seasonal changes in composition and activities. To this end, venom composition was analyzed by protein quantification, SDS-PAGE, and HPLC, and the LAAO, PLA2 and coagulant activities were measured. Venoms of these C. d. collilineatus showed minor seasonal differences in venom activities and no composition differences were found. LAAO and coagulant activities displayed a pattern of seasonal change, while PLA2 activity seemed to have no seasonality tendency. Also, there are sexual differences, in which males seem to be more stable than females in regard to some activities. Individual variability occurs even in seasonal variation of activities, highlighting the importance of controlling circumstances of venom extraction before comparing results between groups of snakes.

Asunto(s)

Venenos de Crotálidos , Crotalus , Estaciones del Año , Animales , Venenos de Crotálidos/toxicidad , Venenos de Crotálidos/química , Masculino , Femenino , Brasil , Cromatografía Líquida de Alta Presión , Fosfolipasas A2 , Serpientes Venenosas

RESUMEN

Despite a recent surge in high-throughput venom research that has enabled many species to be studied, some snake venoms remain understudied. The long-tailed rattlesnakes (Crotalus ericsmithi, C. lannomi, and C. stejnegeri) are one group where such research lags, largely owing to the rarity of these snakes and the hazardous areas, ripe with drug (marijuana and opium) production, they inhabit in Mexico. To fill this knowledge gap, we used multiple functional assays to examine the coagulotoxic (including across different plasma types), neurotoxic, and myotoxic activity of the venom of the long-tailed rattlesnakes. All crude venoms were shown to be potently anticoagulant on human plasma, which we discovered was not due to the destruction of fibrinogen, except for C. stejnegeri displaying minor fibrinogen destruction activity. All venoms exhibited anticoagulant activity on rat, avian, and amphibian plasmas, with C. ericsmithi being the most potent. We determined the mechanism of anticoagulant activity by C. ericsmithi and C. lannomi venoms to be phospholipid destruction and inhibition of multiple coagulation factors, leading to a net disruption of the clotting cascade. In the chick biventer assay, C. ericsmithi and C. lannomi did not exhibit neurotoxic activity but displayed potential weak myotoxic activity. BIRMEX® (Faboterápico Polivalente Antiviperino) antivenom was not effective in neutralising this venom effect. Overall, this study provides an in-depth investigation of venom function of understudied long-tailed rattlesnakes and provides a springboard for future venom and ecology research on the group.

Asunto(s)

Anticoagulantes , Venenos de Crotálidos , Crotalus , Animales , Venenos de Crotálidos/toxicidad , Humanos , Anticoagulantes/farmacología , Cannabis/química , Ratas , Coagulación Sanguínea/efectos de los fármacos , México

RESUMEN

Camelid single-domain antibodies (VHH) represent a promising class of immunobiologicals for therapeutic applications due to their remarkable stability, specificity, and therapeutic potential. To enhance the effectiveness of antivenoms for snakebites, various methods have been explored to address limitations associated with serum therapy, particularly focusing on mitigating local damage and ensuring sustainable production. Our study aimed to characterize the pharmacological profile and neutralization capacity of anti-Phospholipase A2 (PLA2) monomeric VHH (Genbank accessions: KC329718). Using a post-envenoming mouse model, we used intravital microscopy to assess leukocyte influx, measured CK and LDH levels, and conducted a histopathology analysis to evaluate VHH KC329718's ability to neutralize myotoxic activity. Our findings demonstrated that VHH KC329718 exhibited heterogeneous distribution in muscle tissue. Treatment with VHH KC329718 reduced leukocyte influx caused by BthTX-I (a Lys-49 PLA2) by 28 %, as observed through intravital microscopy. When administered at a 1:10 ratio [venom or toxin:VHH (w/w)], VHH KC329718 significantly decreased myotoxicity, resulting in a 35-40 % reduction in CK levels from BthTX-I and BthTX-II (an Asp-49 PLA2) and a 60 % decrease in CK levels from B. jararacussu venom. LDH levels also showed reductions of 60%, 80%, and 60% induced by BthTX-I, BthTX-II, and B. jararacussu venom, respectively. Histological analysis confirmed the neutralization potential, displaying a significant reduction in tissue damage and inflammatory cell count in mice treated with VHH KC329718 post B. jararacussu venom inoculation. This study underscores the potential of monomeric anti-PLA2 VHH in mitigating myotoxic effects, suggesting a promising avenue for the development of new generation antivenoms to address current therapeutic limitations.

Asunto(s)

Antivenenos , Bothrops , Fosfolipasas A2 , Anticuerpos de Dominio Único , Mordeduras de Serpientes , Animales , Anticuerpos de Dominio Único/inmunología , Mordeduras de Serpientes/tratamiento farmacológico , Mordeduras de Serpientes/inmunología , Antivenenos/farmacología , Antivenenos/uso terapéutico , Ratones , Fosfolipasas A2/metabolismo , Venenos de Crotálidos/inmunología , Venenos de Crotálidos/toxicidad , Masculino , Modelos Animales de Enfermedad , Músculo Esquelético/patología , Músculo Esquelético/efectos de los fármacos , Leucocitos/efectos de los fármacos , Leucocitos/inmunología , Humanos , Creatina Quinasa/sangre

RESUMEN

BACKGROUND: Snakebite envenomation (SBE) causes diverse toxic effects in humans, including disability and death. Current antivenom therapies effectively prevent death but fail to block local tissue damage, leading to an increase in the severity of envenomation; thus, seeking alternative treatments is crucial. METHODS: This study analyzed the potential of two fucoidan sulfated polysaccharides extracted from brown seaweeds Fucus vesiculosus (FVF) and Undaria pinnatifida (UPF) against the fibrinogen or plasma coagulation, proteolytic, and phospholipase A2 (PLA2) activities of Bothrops jararaca, B. jararacussu, and B. neuwiedi venom. The toxicity of FVF and UPF was assessed by the hemocompatibility test. RESULTS: FVF and UPF did not lyse human red blood cells. FVF and UPF inhibited the proteolytic activity of Bothrops jararaca, B. jararacussu, and B. neuwiedi venom by approximately 25%, 50%, and 75%, respectively, while all venoms led to a 20% inhibition of PLA2 activity. UPF and FVF delayed plasma coagulation caused by the venoms of B. jararaca and B. neuwiedi but did not affect the activity of B. jararacussu venom. FVF and UPF blocked the coagulation of fibrinogen induced by all these Bothropic venoms. CONCLUSION: FVF and UPF may be of importance as adjuvants for SBE caused by species of Bothrops, which are the most medically relevant snakebite incidents in South America, especially Brazil.

Asunto(s)

Coagulación Sanguínea , Venenos de Crotálidos , Fucus , Fosfolipasas A2 , Polisacáridos , Undaria , Animales , Antivenenos/farmacología , Coagulación Sanguínea/efectos de los fármacos , Bothrops , Bothrops jararaca , Venenos de Crotálidos/toxicidad , Venenos de Crotálidos/enzimología , Algas Comestibles/química , Fucus/química , Fosfolipasas A2/metabolismo , Polisacáridos/farmacología , Polisacáridos/aislamiento & purificación , Proteolisis/efectos de los fármacos , Algas Marinas/química , Undaria/química , Serpientes Venenosas

RESUMEN

Snake venom metalloproteases (SVMPs) are hydrolytic enzymes dependent on metal binding, primarily zinc (Zn2+), at their catalytic site. They are classified into three classes (P-I to P-III). BjussuMP-II, a P-I SVMP isolated from Bothrops jararacussu snake venom, has a molecular mass of 24 kDa. It exhibits inhibitory activity on platelet aggregation and hydrolyzes fibrinogen. TNF-α upregulates the expression of adhesion molecules on endothelial cell surfaces, promoting leukocyte adhesion and migration during inflammation. Literature indicates that SVMPs may cleave the TNF-α precursor, possibly due to significant homology between metalloproteases from mammalian extracellular matrix and SVMPs. This study aimed to investigate BjussuMP-II's effects on human umbilical vein endothelial cells (HUVEC), focusing on viability, detachment, adhesion, release, and cleavage of TNF-α, IL-1ß, IL-6, IL-8, and IL-10. HUVEC were incubated with BjussuMP-II (1.5-50 µg/mL) for 3-24 h. Viability was determined using LDH release, MTT metabolization, and 7AAD for membrane integrity. Adhesion and detachment were assessed by incubating cells with BjussuMP-II and staining with Giemsa. Cytokines were quantified in HUVEC supernatants using EIA. TNF-α cleavage was evaluated using supernatants from PMA-stimulated cells or recombinant TNF-α. Results demonstrated BjussuMP-II's proteolytic activity on casein. It was not toxic to HUVEC at any concentration or duration studied but interfered with adhesion and promoted detachment. PMA induced TNF-α release by HUVEC, but this effect was not observed with BjussuMP-II, which cleaved TNF-α. Additionally, BjussuMP-II cleaved IL-1ß, IL-6, and IL-10. These findings suggest that the zinc metalloprotease BjussuMP-II could be a valuable biotechnological tool for treating inflammatory disorders involving cytokine deregulation.

Asunto(s)

Adhesión Celular , Citocinas , Células Endoteliales de la Vena Umbilical Humana , Metaloproteasas , Humanos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Citocinas/metabolismo , Metaloproteasas/metabolismo , Adhesión Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Bothrops/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Venenos de Crotálidos/metabolismo , Venenos de Crotálidos/toxicidad , Proteolisis/efectos de los fármacos

RESUMEN

Accidents caused by Bothrops jararaca (Bj) snakes result in several local and systemic manifestations, with pain being a fundamental characteristic. The inflammatory process responsible for hyperalgesia induced by Bj venom (Bjv) has been studied; however, the specific roles played by the peripheral and central nervous systems in this phenomenon remain unclear. To clarify this, we induced hyperalgesia in rats using Bjv and collected tissues from dorsal root ganglia (DRGs) and spinal cord (SC) at 2 and 4 h post-induction. Samples were labeled for Iba-1 (macrophage and microglia), GFAP (satellite cells and astrocytes), EGR1 (neurons), and NK1 receptors. Additionally, we investigated the impact of minocycline, an inhibitor of microglia, and GR82334 antagonist on Bjv-induced hyperalgesia. Our findings reveal an increase in Iba1 in DRG at 2 h and EGR1 at 4 h. In the SC, markers for microglia, astrocytes, neurons, and NK1 receptors exhibited increased expression after 2 h, with EGR1 continuing to rise at 4 h. Minocycline and GR82334 inhibited venom-induced hyperalgesia, highlighting the crucial roles of microglia and NK1 receptors in this phenomenon. Our results suggest that the hyperalgesic effects of Bjv involve the participation of microglial and astrocytic cells, in addition to the activation of NK1 receptors.

Asunto(s)

Bothrops , Venenos de Crotálidos , Ganglios Espinales , Hiperalgesia , Receptores de Neuroquinina-1 , Animales , Hiperalgesia/inducido químicamente , Hiperalgesia/metabolismo , Venenos de Crotálidos/toxicidad , Masculino , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Receptores de Neuroquinina-1/metabolismo , Minociclina/farmacología , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Microglía/efectos de los fármacos , Microglía/metabolismo , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Ratas , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas de Unión al Calcio/metabolismo , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Proteínas de Microfilamentos/metabolismo , Antagonistas del Receptor de Neuroquinina-1/farmacología , Ratas Sprague-Dawley

RESUMEN

Crotalus culminatus is a medically significant species of rattlesnake in Mexico [1]. While the proteomic composition of its venom has been previously reported for both juvenile and adult specimens, there has been limited research into its functional properties, with only a few studies, including one focusing on coagulotoxicity mechanisms. In this study, we aimed to compare the biochemical and biological activities of the venom of juvenile and adult snakes. Additionally, we assessed antibody production using the venoms of juveniles and adults as immunogens in rabbits. Our findings reveal lethality and proteolytic activity differences between the venoms of juveniles and adults. Notably, juvenile venoms exhibited high proportions of crotamine, while adult venoms displayed a reduction of this component. A commercially available antivenom demonstrated effective neutralization of lethality of both juvenile and adult venoms in mice. However, it failed to neutralize the paralytic activity induced by crotamine, which, in contrast, was successfully inhibited by antibodies obtained from hyperimmunized rabbits. These results suggest the potential inclusion of C. culminatus venom from juveniles in commercial antivenom immunization schemes to generate antibodies targeting this small myotoxin.

Asunto(s)

Antivenenos , Venenos de Crotálidos , Conejos , Animales , Ratones , Antivenenos/farmacología , Crotalus , Proteómica , Venenos de Crotálidos/toxicidad , Venenos de Crotálidos/química , Neurotoxinas , México

RESUMEN

Snakebite envenomings most frequently reported in Colombia are caused by snakes of the genera Bothrops, Bothriechis, Bothrocophias, and Porthidium. Their venoms induce local and systemic pathophysiological effects, sometimes leading to permanent sequelae such as reduced mobility of the limbs, amputations, besides the risk of death. The genus Bothrocophias includes nine species, among which B. campbelli has a distribution restricted to the department of Nariño in Colombia. In this work we determined the toxinological profile its venom, by performing assays for the lethal, hemorrhagic, edematogenic, and myotoxic activities in mouse models, as well as for in vitro coagulant activity on human plasma. The lethal toxicity of the venom was 142.7 µg venom/mouse (111.4-179.8 µg/mouse; 6.6-10.6 µg/g body weight) by intraperitoneal route. Its hemorrhagic activity (minimum hemorrhagic dose: 12.7 ± 2.3 µg) is generally weaker compared to other South American vipers, but edematogenic (minimum edematogenic dose 1.0 ± 0.3 µg), and myotoxic (minimum myotoxic dose 3.9 ± 2.5 µg) activities are very potent. Histopathological examination of the injected mouse gastrocnemius muscle showed prominent disorganization of the myofibrils, myonecrosis, and an intense inflammatory leukocyte infiltrate. In vitro, the minimal coagulant dose was 12.3 ± 0.5 µg. Overall, this toxinological profile would predict that the clinical picture of envenomings by B. campbelli might be characterized by moderate disturbances in the coagulation cascade, mild local hemorrhage, and, conversely, severe myonecrosis and edema, which could potentially lead to compartment syndrome and gangrene.

Asunto(s)

Bothrops , Venenos de Crotálidos , Humanos , Animales , Ratones , Colombia , Venenos de Crotálidos/toxicidad , Hemorragia/inducido químicamente , Serpientes , Antivenenos/efectos adversos

RESUMEN

The present work addressed the abilities of two L-amino acid oxidases isolated from Bothrops moojeni (BmooLAAO-I) and Bothrops jararacussu (BjussuLAAO-II) snake venoms to control the growth and prevent the biofilm formation of clinically relevant bacterial pathogens. Upon S. aureus (ATCC BAA44) and S. aureus (clinical isolates), BmooLAAO-I (MIC = 0.12 and 0.24 µg/mL, respectively) and BjussuLAAO-II (MIC = 0.15 µg/mL) showed a potent bacteriostatic effect. Against E. coli (ATCC BAA198) and E. coli (clinical isolates), BmooLAAO-I (MIC = 15.6 and 62.5 µg/mL, respectively) and BjussuLAAO-II (MIC = 4.88 and 9.76 µg/mL, respectively) presented a lower extent effect. Also, BmooLAAO-I (MICB50 = 0.195 µg/mL) and BjussuLAAO-II (MICB50 = 0.39 µg/mL) inhibited the biofilm formation of S. aureus (clinical isolates) in 88% and 89%, respectively, and in 89% and 53% of E. coli (clinical isolates). Moreover, scanning electron microscopy confirmed that the toxins affected bacterial morphology by increasing the roughness of the cell surface and inhibited the biofilm formation. Furthermore, analysis of the tridimensional structures of the toxins showed that the surface-charge distribution presents a remarkable positive region close to the glycosylation motif, which is more pronounced in BmooLAAO-I than BjussuLAAO-II. This region may assist the interaction with bacterial and biofilm surfaces. Collectively, our findings propose that venom-derived antibiofilm agents are promising biotechnological tools which could provide novel strategies for biofilm-associated infections.

Asunto(s)

Bothrops , Venenos de Crotálidos , Serpientes Venenosas , Animales , Venenos de Crotálidos/toxicidad , L-Aminoácido Oxidasa/farmacología , L-Aminoácido Oxidasa/química , Staphylococcus aureus , Escherichia coli , Venenos de Serpiente/química , Bacterias , Biopelículas

RESUMEN

Snakebite envenomation is classified as a Neglected Tropical Disease. Bothrops jararaca venom induces kidney injury and coagulopathy. HF3, a hemorrhagic metalloproteinase of B. jararaca venom, participates in the envenomation pathogenesis. We evaluated the effects of HF3 in mouse kidney and blood plasma after injection in the thigh muscle, mimicking a snakebite. Transcriptomic analysis showed differential expression of 31 and 137 genes related to kidney pathology after 2 h and 6 h, respectively. However, only subtle changes were observed in kidney proteome, with differential abundance of 15 proteins after 6 h, including kidney injury markers. N-terminomic analysis of kidney proteins showed 420 proteinase-generated peptides compatible with meprin specificity, indicating activation of host proteinases. Plasma analysis revealed differential abundance of 90 and 219 proteins, respectively, after 2 h and 6 h, including coagulation-cascade and complement-system components, and creatine-kinase, whereas a semi-specific search of N-terminal peptides indicated activation of endogenous proteinases. HF3 promoted host reactions, altering the gene expression and the proteolytic profile of kidney tissue, and inducing plasma proteome imbalance driven by changes in abundance and proteolysis. The overall response of the mouse underscores the systemic action of a hemorrhagic toxin that transcends local tissue damage and is related to known venom-induced systemic effects.

Asunto(s)

Bothrops , Venenos de Crotálidos , Ratones , Animales , Proteoma , Multiómica , Metaloproteasas/metabolismo , Venenos de Serpiente/toxicidad , Péptidos , Plasma/metabolismo , Riñón/metabolismo , Bothrops/metabolismo , Venenos de Crotálidos/toxicidad , Venenos de Crotálidos/metabolismo