Bothrops atrox mice experimental envenoming treatment using light-emitting diode (led) as an adjunct therapy to conventional serum therapy.

The use of anti-venom is one of the main control measures for snakebite envenoming when applied immediately after the snakebite. Systemic effects of the envenoming are usually reversed; however, neutralization of local effects is hardly achieved. The need for adjuvant therapies associated with serum therapy can improve the treatment for local effects of envenoming, with greater effectiveness in preventing or delaying the progression of damage, reducing the clinical signs and symptoms of victims of snakebites. The purpose of the study was to evaluate the photobiomodulation therapy using LED and/or dexamethasone associated with conventional serum therapy for the treatment of local damage caused by Bothrops atrox envenomation in a murine model. For this, experimental envenoming was carried out in the gastrocnemius muscle of male Swiss mice weighing 18 to 22 g divided into 8 groups of animals, distributed in groups non-treat, treated with anti-bothropic serum, dexamethasone, and LED, or the associated treatments, by intramuscular inoculation of 50 µg of venom or sterile PBS (control). After 30 min, the proposed treatments were administered alone or in combination. After 3 h, blood and muscle samples were collected for myotoxicity, cytotoxicity, histological analysis, and IL-1ß assays. The evaluation of the treatment alone showed that serum therapy is not effective for the treatment of local damage and photobiomodulation demonstrated to be an effective therapy to reduce leukocyte infiltration, hemorrhage, and myotoxicity in experimental envenoming; dexamethasone proved to be a good resource for the treatment of the inflammatory process reducing the leukocyte infiltration. The association of serum therapy, LED, and dexamethasone was the best treatment to reduce the local effects caused by Bothrops atrox venom. All in all, the association of photobiomodulation therapy using LED with conventional serum therapy and the anti-inflammatory drug is the best treatment for reducing the undesirable local effects caused by snakebite accidents involving B. atrox species.

Reactive oxygen species-dependent-NLRP3 inflammasome activation in human neutrophils induced by l-amino acid oxidase derived from Calloselasma rhodostoma venom.

l-Amino acid oxidase isolated from Calloselasma rhodostoma (Cr-LAAO) snake venom is a potent stimulus for neutrophil activation and production of inflammatory mediators, contributing to local inflammatory effects in victims of envenoming. Cr-LAAO triggered the activation of nicotinamide adenine dinucleotide phosphatase (NADPH) oxidase complex and protein kinase C (PKC)-α signaling protein for reactive oxygen species (ROS) production. This study aims to evaluate the ROS participation in the NLRP3 inflammasome complex activation in human neutrophil. Human neutrophils were isolated and stimulated for 1 or 2 h with RPMI (negative control), LPS (1 µg/mL, positive control) or Cr-LAAO (50 µg/mL). The neutrophil transcriptome was examined using the microarray technique, and RT-qPCR for confirmation of gene expression. Immunofluorescence assays for NLRP3, caspase-1, IL-1ß and GSDMD proteins was performed by Western blot in the presence and/or absence of Apocynin, an inhibitor of NADPH oxidase. IL-1ß release was also detected in the presence and/or absence of NLRP3, caspase-1 and NADPH oxidase inhibitors. Results showed that Cr-LAAO upregulated the expression of genes that participate in the NADPH oxidase complex formation and inflammasome assembly. NLRP3 was activated and accumulated in the cytosol forming punctas, indicating its activation. Gasdermin D was not cleaved but lactate dehydrogenase was released. Furthermore, ROS inhibition decreased the expression of NLRP3 inflammasome complex proteins, as observed by protein expression in the presence and/or absence of apocynin, an NADPH oxidase inhibitor. IL-1ß was also released, and pharmacological inhibition of NLRP3, caspase-1, and ROS reduced the amount of released cytokine. This is the first report demonstrating the activation of the NLRP3 inflammasome complex via ROS generation by Cr-LAAO, which may lead to the development of local inflammatory effects observed in snakebite victims.

Cytosolic phospholipase A2-α participates in lipid body formation and PGE2 release in human neutrophils stimulated with an L-amino acid oxidase from Calloselasma rhodostoma venom.

Cr-LAAO, an L-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, has been demonstrated as a potent stimulus for neutrophil activation and inflammatory mediator production. However, the mechanisms involved in Cr-LAAO induced neutrophil activation has not been well characterized. Here we investigated the mechanisms involved in Cr-LAAO-induced lipid body (also known as lipid droplet) biogenesis and eicosanoid formation in human neutrophils. Using microarray analysis, we show for the first time that Cr-LAAO plays a role in the up-regulation of the expression of genes involved in lipid signalling and metabolism. Those include different members of phospholipase A2, mostly cytosolic phospholipase A2-α (cPLA2-α); and enzymes involved in prostaglandin synthesis including cyclooxygenases 2 (COX-2), and prostaglandin E synthase (PTGES). In addition, genes involved in lipid droplet formation, including perilipin 2 and 3 (PLIN 2 and 3) and diacylglycerol acyltransferase 1 (DGAT1), were also upregulated. Furthermore, increased phosphorylation of cPLA2-α, lipid droplet biogenesis and PGE2 synthesis were observed in human neutrophils stimulated with Cr-LAAO. Treatment with cPLA2-α inhibitor (CAY10650) or DGAT-1 inhibitor (A922500) suppressed lipid droplets formation and PGE2 secretion. In conclusion, we demonstrate for the first time the effects of Cr-LAAO to regulate neutrophil lipid metabolism and signalling.

Increase in the risk of snakebites incidence due to changes in humidity levels: A time series study in four municipalities of the state of Rondônia.

INTRODUCTION: Snakebites represent a serious global public health problem, especially in tropical countries. In Brazil, the incidence of snakebites ranges from 19 to 22 thousand cases per 100000 persons annually. The state of Rondônia, in particular, has had an increasing incidence of snakebites. METHODS: A retrospective cross-sectional study on snakebites was conducted from January 2007 to December 2018. Brazil's Information System for Notifiable Diseases was queried for all snakebites reported in Porto Velho, Ariquemes, Cacoal, and Vilhena. Data on land surface temperatures during the day and night, precipitation, and humidity were obtained using the Google Earth Engine. A Bayesian time series model was constructed to describe the pattern of snakebites and their relationship with climate data. RESULTS: In total, 6326 snakebites were reported in Rondônia. Accidents were commonly caused by Bothrops sp. (n=2171, 81.80%). Snakebites most frequently occurred in rural areas (n=2271, 85.5%). Men, with a median age of 34 years (n=2101, 79.1%), were the most frequent bitten. Moderate clinical manifestation was the most common outcome of an accident (n=1101, 41.50%). There were clear seasonal patterns with respect to rainfall, humidity, and temperature. Rainfall and land surface temperature during the day or night did not increase the risk of snakebites in any city; however, changes in humidity increased the risk of snakebites in all cities. CONCLUSION: This study identified the population exposed to snakes and the influence of anthropic and climatic factors on the incidence of snakebites. According to climate data, changes in humidity increased the risk of snakebites.

Increase in the risk of snakebites incidence due to changes in humidity levels: A time series study in four municipalities of the state of Rondônia

Abstract INTRODUCTION: Snakebites represent a serious global public health problem, especially in tropical countries. In Brazil, the incidence of snakebites ranges from 19 to 22 thousand cases per 100000 persons annually. The state of Rondônia, in particular, has had an increasing incidence of snakebites. METHODS: A retrospective cross-sectional study on snakebites was conducted from January 2007 to December 2018. Brazil's Information System for Notifiable Diseases was queried for all snakebites reported in Porto Velho, Ariquemes, Cacoal, and Vilhena. Data on land surface temperatures during the day and night, precipitation, and humidity were obtained using the Google Earth Engine. A Bayesian time series model was constructed to describe the pattern of snakebites and their relationship with climate data. RESULTS: In total, 6326 snakebites were reported in Rondônia. Accidents were commonly caused by Bothrops sp. (n=2171, 81.80%). Snakebites most frequently occurred in rural areas (n=2271, 85.5%). Men, with a median age of 34 years (n=2101, 79.1%), were the most frequent bitten. Moderate clinical manifestation was the most common outcome of an accident (n=1101, 41.50%). There were clear seasonal patterns with respect to rainfall, humidity, and temperature. Rainfall and land surface temperature during the day or night did not increase the risk of snakebites in any city; however, changes in humidity increased the risk of snakebites in all cities. CONCLUSION: This study identified the population exposed to snakes and the influence of anthropic and climatic factors on the incidence of snakebites. According to climate data, changes in humidity increased the risk of snakebites.

Local and systemic effects caused by Crotalus durissus terrificus, Crotalus durissus collilineatus, and Crotalus durissus cascavella snake venoms in swiss mice.

INTRODUCTION: Crotalus envenomations cause serious complications and can be fatal without appropriate treatment. Venom isoforms present and inter/intraspecific variations in the venom composition can result in different symptoms presented by bites by snakes from the same species but from different geographical regions. We comparatively evaluated the local and systemic effects caused by Crotalus durissus terrificus (Cdt), C.d. collilineatus (Cdcolli), and C.d. cascavella (Cdcasc) envenomation. METHODS: Venom chromatography was performed. Proteolytic, phospholipase, and LAAO activities were analyzed. Edema, myotoxicity, hepatotoxicity, nephrotoxicity, and coagulation alterations were evaluated. RESULTS: The venom SDS-PAGE analyses found the presence of convulxin, gyroxin, crotoxin, and crotamine in Cdt and Cdcolli venoms. Crotamine was not present in the Cdcasc venom. Cdt, Cdcollli, and Cdcasc venoms had no proteolytic activity. Only Cdcasc and Cdt venoms had phospholipase activity. LAAO activity was observed in Cdcolli and Cdcasc venoms. Cdcolli and Cdcasc venoms caused 36.7% and 13.3% edema increases, respectively. Cdt venom caused a 10% edema induction compared to those by other venoms. All venoms increased TOTAL-CK, MB-CK, and LDH levels (indicating muscle injury) and ALT, AST, GGT, and ALP levels (markers of liver damage) and were able to induce a neuromuscular blockade. Urea and creatinine levels were also altered in both plasma and urine, indicating kidney damage. Only Cdcolli and Cdcasc venoms increased TAPP and TAP. CONCLUSIONS: Together, these results allow us to draw a distinction between local and systemic effects caused by Crotalus subspecies, highlighting the clinical and biochemical effects produced by their respective venoms.

Local and systemic effects caused by Crotalus durissus terrificus, Crotalus durissus collilineatus, and Crotalus durissus cascavella snake venoms in swiss mice

Abstract INTRODUCTION: Crotalus envenomations cause serious complications and can be fatal without appropriate treatment. Venom isoforms present and inter/intraspecific variations in the venom composition can result in different symptoms presented by bites by snakes from the same species but from different geographical regions. We comparatively evaluated the local and systemic effects caused by Crotalus durissus terrificus (Cdt), C.d. collilineatus (Cdcolli), and C.d. cascavella (Cdcasc) envenomation. METHODS: Venom chromatography was performed. Proteolytic, phospholipase, and LAAO activities were analyzed. Edema, myotoxicity, hepatotoxicity, nephrotoxicity, and coagulation alterations were evaluated. RESULTS: The venom SDS-PAGE analyses found the presence of convulxin, gyroxin, crotoxin, and crotamine in Cdt and Cdcolli venoms. Crotamine was not present in the Cdcasc venom. Cdt, Cdcollli, and Cdcasc venoms had no proteolytic activity. Only Cdcasc and Cdt venoms had phospholipase activity. LAAO activity was observed in Cdcolli and Cdcasc venoms. Cdcolli and Cdcasc venoms caused 36.7% and 13.3% edema increases, respectively. Cdt venom caused a 10% edema induction compared to those by other venoms. All venoms increased TOTAL-CK, MB-CK, and LDH levels (indicating muscle injury) and ALT, AST, GGT, and ALP levels (markers of liver damage) and were able to induce a neuromuscular blockade. Urea and creatinine levels were also altered in both plasma and urine, indicating kidney damage. Only Cdcolli and Cdcasc venoms increased TAPP and TAP. CONCLUSIONS: Together, these results allow us to draw a distinction between local and systemic effects caused by Crotalus subspecies, highlighting the clinical and biochemical effects produced by their respective venoms.

Local and systemic effects of BdipTX-I, a Lys-49 phospholipase A2 isolated from Bothrops diporus snake venom.

The present work aimed to isolate a basic phospholipase A2 (PLA2) from Bothrops diporus snake venom (BdV), evaluate and compare the myotoxic and oedema-inducing activities, as well as the systemic effects caused by both the isolated PLA2 and BdV on Swiss mice. A Lys-49 PLA2 (BdipTX-I) was obtained through two chromatographic steps: an ion-exchange and a reverse phase. The local (oedema and myotoxicity) and systemic (hepatic and renal functions) effects were then assessed for BdipTX-I and BdV. Results showed that the oedema-inducing activity was significant in all tested doses (5 and 20 µg/paw) for both BdipTX-I and BdV. Myotoxicity was evaluated by the increase of serum CK, CK-MB and LDH, and results showed that BdV effect is more prominent than BdipTX-I effect. The systemic effects were evaluated by determining specific laboratory markers: AST, ALT, GGT, ALP, urea, creatinine, protein and calcium. BdipTX-I and BdV were able to induce renal changes in the experimental model, leading to proteinuria (induced both by BdipTX-I and by BdV) and uremia (induced only by BdV). Thus, it is concluded that the systemic effects of BdV and BdipTX-I occur differently.

Effect of BjcuL, a lectin isolated from Bothrops jararacussu, on human peripheral blood mononuclear cells.

BjcuL is a C-type lectin with specificity for the binding of ß-d-galactose units isolated from Bothrops jararacussu venom. It triggers cellular infiltration in post capillary venules, increases edema and vascular permeability in murine models, contributes to in vitro neutrophil activation and modulates macrophage functional activation towards an M1 state. The purpose of this study was to investigate the effect of BjcuL on human peripheral blood mononuclear cells (PBMCs) activation with a focus on PBMCs proliferation and inflammatory mediators release. Results showed that BjcuL is not toxic to PBMCs, that BjcuL inhibits PBMCs proliferation and that it stimulates PBMCs to produce superoxide anion and hydrogen peroxide, primarily via lymphocyte stimulation, but does not stimulate the production of nitric oxide and PGE2. These results demonstrate that BjcuL has an immunomodulatory effect on PBMCs. Further studies are needed to confirm the immunomodulatory effect of BjcuL, to elucidate the molecular mechanisms of action responsible for its effects and to determine its potential application as an immunopharmacological and biotechnological tool.

An overview of Bothrops erythromelas venom.

This review discusses studies on the venom of Bothrops erythromelas published over the past 36 years. During this period, many contributions have been made to understand the venomous snake, its venom, and its experimental and clinical effects better. The following chronological overview is based on 29 articles that were published between 1979 and 2015, with emphasis on diverse areas. The complexity of this task demands an integration of multidisciplinary research tools to study toxinology. This science is in need of renewed conceptual and experimental platforms aimed at obtaining a profound understanding of the highly complex pathophysiology of snakebite envenoming and toxins isolated from snakes.

An overview of Bothrops erythromelas venom

Abstract This review discusses studies on the venom of Bothrops erythromelas published over the past 36 years. During this period, many contributions have been made to understand the venomous snake, its venom, and its experimental and clinical effects better. The following chronological overview is based on 29 articles that were published between 1979 and 2015, with emphasis on diverse areas. The complexity of this task demands an integration of multidisciplinary research tools to study toxinology. This science is in need of renewed conceptual and experimental platforms aimed at obtaining a profound understanding of the highly complex pathophysiology of snakebite envenoming and toxins isolated from snakes.

p38 MAPK is involved in human neutrophil chemotaxis induced by L-amino acid oxidase from Calloselasma rhodosthoma.

The action of LAAO, an L-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, on isolated human neutrophil function was investigated. Cr-LAAO showed no toxicity on neutrophils. Cr-LAAO in its native form induced the neutrophil chemotaxis, suggesting that its primary structure is essential for stimulation the cell. p38 MAPK and PI3K have a role as signaling pathways of CR-LAAO induced chemotaxis. This toxin also induced the production of hydrogen peroxide and stimulated phagocytosis in neutrophils. Furthermore, Cr-LAAO was able to stimulate neutrophils to release IL-6, IL-8, MPO, LTB4 and PGE2. Together, the data showed that the Cr-LAAO triggers relevant proinflammatory events.

Effect of L-amino acid oxidase from Calloselasma rhodosthoma snake venom on human neutrophils.

The in vitro effects of LAAO, an l-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, on isolated human neutrophil function were investigated. LAAO showed no toxicity on neutrophils. At non-cytotoxic concentrations, LAAO induced the superoxide anion production by isolated human neutrophil. This toxin, in its native form, is also able to stimulate the production of hydrogen peroxide in neutrophils, suggesting that its primary structure is essential for stimulation the cell. Moreover, the incubation of LAAO and phenol red medium did not induce the production of hydrogen peroxide. Furthermore, LAAO was able to stimulate neutrophils to release proinflammatory mediators such as IL-8 and TNF-α as well as NETs liberation. Together, the data showed that the LAAO triggers relevant proinflammatory events. Particular regions of the molecule distinct from the LAAO catalytic site may be involved in the onset of inflammatory events.

Effect of Bothrops bilineata snake venom on neutrophil function.

The aim of the study was to evaluate the in vitro effects of Bothrops bilineata crude venom (BbV) on isolated human neutrophil function. We proved that BbV isn't toxic towards human neutrophils. During an incubation of human neutrophils with BbV hydrogen peroxide was produced. Moreover, BbV was able to stimulate neutrophil release of proinflammatory mediators such as IL-8 and IL-6 as well as PGE2 and NETs liberation. There is no data in the literature showing the effect of BbV on the production of IL-6 and IL-8 or NETs liberation by isolated human neutrophils. Taken together our results testify that BbV triggers relevant proinflammatory events in human neutrophils.