Exosome Liberation by Human Neutrophils under L-Amino Acid Oxidase of Calloselasma rhodostoma Venom Action.

L-Amino acid oxidase (LAAO) is an enzyme found in snake venom that has multifaceted effects, including the generation of hydrogen peroxide (H2O2) during oxidative reactions, leading to various biological and pharmacological outcomes such as apoptosis, cytotoxicity, modulation of platelet aggregation, hemorrhage, and neutrophil activation. Human neutrophils respond to LAAO by enhancing chemotaxis, and phagocytosis, and releasing reactive oxygen species (ROS) and pro-inflammatory mediators. Exosomes cellular nanovesicles play vital roles in intercellular communication, including immune responses. This study investigates the impact of Calloselasma rhodostoma snake venom-derived LAAO (Cr-LAAO) on human neutrophil exosome release, including activation patterns, exosome formation, and content. Neutrophils isolated from healthy donors were stimulated with Cr-LAAO (100 µg/mL) for 3 h, followed by exosome isolation and analysis. Results show that Cr-LAAO induces the release of exosomes with distinct protein content compared to the negative control. Proteomic analysis reveals proteins related to the regulation of immune responses and blood coagulation. This study uncovers Cr-LAAO's ability to activate human neutrophils, leading to exosome release and facilitating intercellular communication, offering insights into potential therapeutic approaches for inflammatory and immunological disorders.

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.

Human neutrophils functionality under effect of an Asp49 phospholipase A2 isolated from Bothrops atrox venom.

Bothrops envenomation is associated with a cellular inflammatory response, characterized by pronounced neutrophil infiltration at the site of injury. Neutrophils act as the first line of defence, owing to their ability to migrate to the infected tissue, promoting an acute inflammatory response. At the site of inflammation, neutrophils perform defence functions such as phagocytosis, release of proteolytic enzymes, generation of reactive oxygen species (ROS), and synthesis of inflammatory mediators such as cytokines and lipid mediators. Neutrophils can also form neutrophil extracellular nets (NETs), webs composed of chromatin and granule proteins. This occurs after neutrophil activation and delivers high concentrations of anti-microbial molecules to the site of injury. This study evaluated the impact of BaTX-II, an Asp49 phospholipase A2 (PLA2) isolated from Bothrops atrox snake venom on human neutrophils in vitro. At non-toxic concentrations, BaTX-II induced hydrogen peroxide production by neutrophils, and this was reduced by wortmannin, a PI3K inhibitor. BaTX-II stimulated IL-1ß, IL-8, LTB4, myeloperoxidase (MPO), and DNA content release, consistent with NET formation. This is the first study to show the triggering of relevant pro-inflammatory events by PLA2 Asp49 isolated from secretory venom.

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.

Corrigendum to "Biochemical and Biological Profile of Parotoid Secretion of the Amazonian Rhinella marina (Anura: Bufonidae)".

[This corrects the article DOI: 10.1155/2019/2492315.].

Biochemical and Biological Profile of Parotoid Secretion of the Amazonian Rhinella marina (Anura: Bufonidae).

Skin secretions of frogs have a high chemical complexity. They have diverse types of biomolecules, such as proteins, peptides, biogenic amines, and alkaloids. These compounds protect amphibians' skin against growth of bacteria, fungi, and protozoa and participate in defense system against attack from predators. Therewith, this work performed biochemical and biological profile of macroglands parotoid secretion from cane toad. For poison analysis, we performed molecular exclusion and reverse phase chromatography, electrophoresis, and mass spectrometry. Antimicrobial, antiplasmodial, leishmanicidal, cytotoxicity, genotoxicity, and inflammatory activity of crude and/or fractions of R. marina secretion were also evaluated. Fractionation prior to filtration from poison showed separation of low mass content (steroids and alkaloids) and high molecular mass (protein). Material below 10 kDa two steroids, marinobufagin and desacetylcinobufagin, was detected. Crude extract and fractions were active against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Plasmodium falciparum, Leishmania guyanensis, and Leishmania braziliensis. Crude extract was also active against cancer cells although it was not cytotoxic for normal cells. This extract did not show significant DNA damage but it showed an important inflammatory effect in vivo. The information obtained in this work contributes to the understanding of the constituents of R. marina secretion as well as the bioactive potential of these molecules.

Biochemical and biological profile of parotoid secretion of the amazonian Rhinella marina (Anura: Bufonidae)

Skin secretions of frogs have a high chemical complexity. They have diverse types of biomolecules, such as proteins, peptides, biogenic amines, and alkaloids. These compounds protect amphibians’ skin against growth of bacteria, fungi, and protozoa and participate in defense system against attack from predators. Therewith, this work performed biochemical and biological profile of macroglands parotoid secretion from cane toad. For poison analysis, we performed molecular exclusion and reverse phase chromatography, electrophoresis, and mass spectrometry. Antimicrobial, antiplasmodial, leishmanicidal, cytotoxicity, genotoxicity, and inflammatory activity of crude and/or fractions of R. marina secretion were also evaluated. Fractionation prior to filtration from poison showed separation of low mass content (steroids and alkaloids) and high molecular mass (protein). Material below 10 kDa two steroids, marinobufagin and desacetylcinobufagin, was detected. Crude extract and fractions were active against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Plasmodium falciparum, Leishmania guyanensis, and Leishmania braziliensis. Crude extract was also active against cancer cells although it was not cytotoxic for normal cells. This extract did not show significant DNA damage but it showed an important inflammatory effect in vivo. The information obtained in this work contributes to the understanding of the constituents of R. marina secretion as well as the bioactive potential of these molecules.

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.

Biochemical and biological profile of parotoid secretion of the amazonian Rhinella marina (Anura: Bufonidae)

Skin secretions of frogs have a high chemical complexity. They have diverse types of biomolecules, such as proteins, peptides, biogenic amines, and alkaloids. These compounds protect amphibians’ skin against growth of bacteria, fungi, and protozoa and participate in defense system against attack from predators. Therewith, this work performed biochemical and biological profile of macroglands parotoid secretion from cane toad. For poison analysis, we performed molecular exclusion and reverse phase chromatography, electrophoresis, and mass spectrometry. Antimicrobial, antiplasmodial, leishmanicidal, cytotoxicity, genotoxicity, and inflammatory activity of crude and/or fractions of R. marina secretion were also evaluated. Fractionation prior to filtration from poison showed separation of low mass content (steroids and alkaloids) and high molecular mass (protein). Material below 10 kDa two steroids, marinobufagin and desacetylcinobufagin, was detected. Crude extract and fractions were active against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Plasmodium falciparum, Leishmania guyanensis, and Leishmania braziliensis. Crude extract was also active against cancer cells although it was not cytotoxic for normal cells. This extract did not show significant DNA damage but it showed an important inflammatory effect in vivo. The information obtained in this work contributes to the understanding of the constituents of R. marina secretion as well as the bioactive potential of these molecules.

Role of l-amino acid oxidase isolated from Calloselasma rhodostoma venom on neutrophil NADPH oxidase complex activation.

The action of Cr-LAAO, an l-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, on NADPH oxidase activation in isolated human neutrophil function was investigated. This enzyme has an intrinsic activity of hydrogen peroxide production. Cr-LAAO, in its native form, induces the ROS production in neutrophil and migration of cytosolic NADPH oxidase components p40phox, p47phox and p67phox to the membrane, and Rac, a GTPase protein member, with the involvement of intracellular signaling mediated by phospho PKC-α. In its inactive form, iCr-LAAO does not induce NADPH oxidase activation in neutrophil showing that the intrinsic enzymatic activity does not have a role in this process, suggesting that its primary structure is essential for the cell's stimulation. Accordingly, the data showed for the first time that the Cr-LAAO has a role in NADPH oxidase complex activation triggering relevant proinflammatory events in human neutrophils.

An Update on Potential Molecular Mechanisms Underlying the Actions of Snake Venom L-amino Acid Oxidases (LAAOs).

BACKGROUND: LAAOs (EC 1.4.3.2) are found in concentrations that vary according to each species of snakes; Viperidae, Crotalidae and Elapidae contain 1-9% of this enzyme in their venoms. METHODS: This review focuses on an update on molecular mechanisms, platelet activities, antimicrobial, antiprotozoal, induction of apoptosis and inflammatory potential underlying the actions of SVLAAOs. RESULTS: Snake venom LAAOs (SV-LAAOs) have become an interesting subject for pharmacological, structural and molecular studies. CONCLUSION: Although the mechanisms of action of these enzymes are not well understood they are a subject of a variety of studies, because LAAOs are multifunctional enzymes exhibiting a wide range of pharmacological effects, including the inhibition or induction of platelet aggregation, hemolysis and hemorrhage, in addition to the stimulation of apoptosis, the activation of leukocytes and the formation of edema. Moreover, SV-LAAOs play an important role in bactericidal, cytotoxic, anti-parasitic, anti-tumor, and antiviral activities.

Phospholipase A2 Inhibitor from Crotalus durissus terrificus rattlesnake: Effects on human peripheral blood mononuclear cells and human neutrophils cells.

Crotalus Neutralizing Factor (CNF) is an inhibitor of phospholipase A2 (PLA2), present in the blood plasma of Crotalus durissus terrificus snake. This inhibitor neutralizes the lethal and enzymatic activity of crotoxin, the main neurotoxin from this venom. In this study, we investigated the effects of CNF on the functionality of human peripheral blood mononuclear cells (PBMCs) and human neutrophils. The following parameters were evaluated: viability and proliferation, chemotaxis, cytokines and LTB4 production, cytosolic PLA2s activity, myeloperoxidase (MPO) and superoxide anion (O2-) production. CNF showed no toxicity on PBMCs or neutrophils, and acts by stimulating the release of TNF-α and LTB4, but neither stimulates IL-10 and IL-2 nor affects PBMCs proliferation and O2- release. In neutrophils, CNF induces chemotaxis but does not induce the release of both MPO and O2-. However, it induces LTB4 and IL-8 production. These data show the influence of CNF on PBMCs' function by inducing TNF-α and LTB4 production, and on neutrophils, by stimulating chemotaxis and LTB4 production, via cytosolic PLA2 activity, and IL-8 release. The inflammatory profile produced by CNF is shown for the first time. Our present results suggest that CNF has a role in activation of leukocytes and exert proinflammatory effects on these cell.

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.

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.

The effect of 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene lupane compound isolated from Combretum leprosum Mart. on peripheral blood mononuclear cells.

BACKGROUND: The Combretum leprosum Mart. plant, popularly known as mofumbo, is used in folk medicine for inflammation, pain and treatment of wounds. From this species, it is possible to isolate three triterpenes: (3ß, 6ß, 16ß-trihydroxylup-20(29)-ene) called lupane, arjunolic acid and molic acid. In this study, through preclinical tests, the effect of lupane was evaluated on the cytotoxicity and on the ability to activate cellular function by the production of TNF-α, an inflammatory cytokine, and IL-10, an immuno regulatory cytokine was assessed. The effect of lupane on the enzymes topoisomerase I and II was also evaluated. METHODS: For this reason, peripheral blood mononuclear cells (PBMCs) were obtained and cytotoxicity was assessed by the MTT method at three different times (1, 15 and 24 h), and different concentrations of lupane (0.3, 0.7, 1.5, 6, 3 and 12 µg/mL). The cell function was assessed by the production of TNF-α and IL-10 by PBMCs quantified by specific enzyme immunoassay (ELISA). The activity of topoisomerases was assayed by in vitro biological assays and in silico molecular docking. RESULTS: The results obtained showed that lupane at concentrations below 1.5 µg/mL was not toxic to the cells. Moreover, lupane was not able to activate cellular functions and did not alter the production of IL-10 and TNF-α. Furthermore, the data showed that lupane has neither interfered in the action of topoisomerase I nor in the action of topoisomerase II. CONCLUSION: Based on preclinical results obtained in this study, we highlight that the compound studied (lupane) has moderate cytotoxicity, does not induce the production of TNF-α and IL-10, and does not act on human topoisomerases. Based on the results of this study and taking into consideration the reports about the anti-inflammatory and leishmanicidal activity of 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene, we suggest that this compound may serve as a biotechnological tool for the treatment of leishmaniasis in the future.

Activation of J77A.1 macrophages by three phospholipases A2 isolated from Bothrops atrox snake venom.

In the present study, we investigated the in vitro effects of two basic myotoxic phospholipases A2 (PLA2), BaTX-I, a catalytically inactive Lys-49 variant, and BaTX-II, a catalytically active Asp-49, and of one acidic myotoxic PLA2, BaPLA2, a catalytically active Asp-49, isolated from Bothrops atrox snake venom, on the activation of J774A.1 macrophages. At noncytotoxic concentrations, the toxins did not affect the adhesion of the macrophages, nor their ability to detach. The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis. However, BaTX-I, BaTX-II, and BaPLA2 induced the release of the superoxide anion by J774A.1 macrophages. Additionally, only BaTX-I raised the lysosomal volume of macrophages after 15 min of incubation. After 30 min, all the phospholipases increased this parameter, which was not observed within 60 min. Moreover, BaTX-I, BaTX-II, and BaPLA2 increased the number of lipid bodies on macrophages submitted to phagocytosis and not submitted to phagocytosis. However, BaTX-II and BaPLA2 induced the release of TNF-α by J774A.1 macrophages. Taken together, the data show that, despite differences in enzymatic activity, the three toxins induced inflammatory events and whether the enzyme is acidic or basic does not seem to contribute to these effects.

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.

Local and systemic biochemical alterations induced by Bothrops atrox snake venom in mice.

The local and systemic alterations induced by Bothrops atrox snake venom (BaV) injection in mice were studied. BaV induced superoxide production by migrated neutrophils, mast cell degranulation and phagocytosis by macrophages. Moreover, BaV caused hemorrhage in dorsum of mice after 2hr post- injection. Three hours post-injection in gastrocnemius muscle, we also observed myonecrosis, which was assessed by the determination of serum and tissue CK besides the release of urea, but not creatinine and uric acid, indicating kidney alterations. BaV also induced the release of LDH and transaminases (ALT and AST) indicating tissue and liver abnormalities. In conclusion, the data indicate that BaV induces events of local and systemic importance.

Avaliação farmacocinética de comprimidos contendo lamivudina e zidovudina em plasma humano / Pharmacokinetic evaluation of lamivudine and zidovudine tablets in human plasma

Este estudo se propõe a avaliar o perfil farmacocinético de comprimidos contendo lamivudina e zidovudina associados, produzidos pelo Laboratório Farmacêutico do Estado de Pernambuco ­ LAFEPE. O medicamento foi administrado em 10 voluntários sadios e foram observadas as concentrações plasmáticas dos dois anti-retrovirais durante 12 horas. A partir das concentrações plasmáticas foi possível encontrar os parâmetros farmacocinéticos de ambas os fármacos através de cálculos estatísticos. Os resultados obtidos foram comparados com aqueles resultantes de experimentos realizados por outros autores descritos neste trabalho, em que verificou não haver diferenças significativas entre os valores encontrados e os descritos na literatura e que o fato dos anti-retrovirais estarem associados na mesma formulação não modificou o perfil cinético de nenhuma das substâncias