Effective Pre-Clinical Treatment of Fish Envenoming with Polyclonal Antiserum.

Envenomation by venomous fish, although not always fatal, is capable of causing damage to homeostasis by activating the inflammatory process, with the formation of edema, excruciating pain, necrosis that is difficult to heal, as well as hemodynamic and cardiorespiratory changes. Despite the wide variety of pharmacological treatments used to manage acute symptoms, none are effective in controlling envenomation. Knowing the essential role of neutralizing polyclonal antibodies in the treatment of envenoming for other species, such as snakes, this work aimed to produce a polyclonal antiserum in mice and test its ability to neutralize the main toxic effects induced by the venoms of the main venomous Brazilian fish. We found that the antiserum recognizes the main toxins present in the different venoms of Thalassophryne nattereri, Scorpaena plumieri, Potamotrygon gr. Orbignyi, and Cathorops spixii and was effective in pre-incubation trials. In an independent test, the antiserum applied immediately to the topical application of T. nattereri, P. gr orbygnyi, and C. spixii venoms completely abolished the toxic effects on the microcirculation, preventing alterations such as arteriolar contraction, slowing of blood flow in postcapillary venules, venular stasis, myofibrillar hypercontraction, and increased leukocyte rolling and adherence. The edematogenic and nociceptive activities induced by these venoms were also neutralized by the immediate application of the antiserum. Importantly, the antiserum prevented the acute inflammatory response in the lungs induced by the S. plumieri venom. The success of antiserum containing neutralizing polyclonal antibodies in controlling the toxic effects induced by different venoms offers a new strategy for the treatment of fish envenomation in Brazil.

Effects of Mauritia flexuosa L. f. buriti oil on symptoms induced by Bothrops moojeni snake envenomation.

ETHNOPHARMACOLOGICAL RELEVANCE: In Brazil, there are species of snakes that become involved in accidents and cause serious health problems to the inhabitants, highlighting the genus Bothrops for being responsible for approximately 90% of accidents reported annually. In the northern region of the country, this genus is responsible for the largest number of accidents, especially among rural dwellers. These populations invest in alternative treatments for with the purpose of improving the symptoms caused by snakebites. The species Mauritia flexuosa L. f., known as buriti, is traditionally used for the treatment of envenomation by snakes. AIM OF THE STUDY: This study aimed to evaluate the antiophidic potential of the oil of Mauritia flexuosa L. f. for Bothrops moojeni H. venom, confronting cultural and scientific knowledge. MATERIALS AND METHODS: The physicochemical properties were determined, and the components present in the oil, extracted from fruit pulp, were analyzed by Gas Chromatography Coupled with Mass Spectrometry. The in vitro inhibitory capacity of the oil for phospholipase, metalloprotease and serine protease activities was investigated. In the in vivo studies, male Swiss mice were used to evaluate the effect of oil on lethality and toxicity, and hemorrhagic, myotoxic and edematogenic activities were assessed. RESULTS: GC‒MS analysis identification of 90.95% of the constituents of the oil, with the main components being 9-eicosenoic acid, (Z)- (34.54%), n-hexadecanoic acid (25.55%) and (E)-9-octadecenoic acid ethyl ester (12.43%). For the substrates, the outcomes indicate that the oil inhibited the activity of the main classes of toxins present in Bothrops moojeni H. venom (VBm) at the highest dose tested (0.5 µL), with inhibition of 84% for the hydrolysis of the selective substrate for serine protease and inhibition of 60% for the hydrolysis of substrates for PLA2 and metalloproteases. The antiophidic activity in vivo was evaluated with two concentrations of the oil: 1.5 mg, the dosage the population, diluted in mineral oil to a volume of 1 tablespoon and 15 mg, administered by gavage 30 min before poisoning and at time zero (concomitant to poisoning), and both concentrations administered by gavage in combination with topical use at time zero. The bleeding time in the group treated with oil at a concentration of 15 mg administered at time zero was significantly lower than that in the control group (p < 0.05). However, a greater inhibition of bleeding time was observed when local application was combined with the gavage treatment at both concentrations tested at time zero (p < 0.05). In the myotoxicity test, oil was efficient in reducing the myotoxic effects induced by the venom at the two concentrations tested, with gavage administration at time zero and gavage plus topical administration at time zero (p < 0.05). CONCLUSIONS: The data obtained show that the oil is safe to use at the concentrations studied and contains fatty acids that may collaborate for cellular-level repair of the injuries caused by Bm poisoning. The in vitro and in vivo experiments showed that oil inhibits the main proteolytic enzymes present in the venom and that it has important activities to control the local effects caused by bothropic venom.

Effects of Mauritia flexuosa L. f. buriti oil on symptoms induced by Bothrops moojeni snake envenomation

In Brazil, there are species of snakes that become involved in accidents and cause serious health problems to the inhabitants, highlighting the genus Bothrops for being responsible for approximately 90% of accidents reported annually. In the northern region of the country, this genus is responsible for the largest number of accidents, especially among rural dwellers. These populations invest in alternative treatments for with the purpose of improving the symptoms caused by snakebites. The species Mauritia flexuosa L. f., known as buriti, is traditionally used for the treatment of envenomation by snakes. Aim of the study This study aimed to evaluate the antiophidic potential of the oil of Mauritia flexuosa L. f. for Bothrops moojeni H. venom, confronting cultural and scientific knowledge. Materials and methods The physicochemical properties were determined, and the components present in the oil, extracted from fruit pulp, were analyzed by Gas Chromatography Coupled with Mass Spectrometry. The in vitro inhibitory capacity of the oil for phospholipase, metalloprotease and serine protease activities was investigated. In the in vivo studies, male Swiss mice were used to evaluate the effect of oil on lethality and toxicity, and hemorrhagic, myotoxic and edematogenic activities were assessed. Results GC‒MS analysis identification of 90.95% of the constituents of the oil, with the main components being 9-eicosenoic acid, (Z)- (34.54%), n-hexadecanoic acid (25.55%) and (E)-9-octadecenoic acid ethyl ester (12.43%). For the substrates, the outcomes indicate that the oil inhibited the activity of the main classes of toxins present in Bothrops moojeni H. venom (VBm) at the highest dose tested (0.5 μL), with inhibition of 84% for the hydrolysis of the selective substrate for serine protease and inhibition of 60% for the hydrolysis of substrates for PLA2 and metalloproteases. The antiophidic activity in vivo was evaluated with two concentrations of the oil: 1.5 mg, the dosage the population, diluted in mineral oil to a volume of 1 tablespoon and 15 mg, administered by gavage 30 min before poisoning and at time zero (concomitant to poisoning), and both concentrations administered by gavage in combination with topical use at time zero. The bleeding time in the group treated with oil at a concentration of 15 mg administered at time zero was significantly lower than that in the control group (p < 0.05). However, a greater inhibition of bleeding time was observed when local application was combined with the gavage treatment at both concentrations tested at time zero (p < 0.05). In the myotoxicity test, oil was efficient in reducing the myotoxic effects induced by the venom at the two concentrations tested, with gavage administration at time zero and gavage plus topical administration at time zero (p < 0.05). Conclusions The data obtained show that the oil is safe to use at the concentrations studied and contains fatty acids that may collaborate for cellular-level repair of the injuries caused by Bm poisoning. The in vitro and in vivo experiments showed that oil inhibits the main proteolytic enzymes present in the venom and that it has important activities to control the local effects caused by bothropic venom.

Effective pre-clinical treatment of fish envenoming with polyclonal antiserum

Envenomation by venomous fish, although not always fatal, is capable of causing damage to homeostasis by activating the inflammatory process, with the formation of edema, excruciating pain, necrosis that is difficult to heal, as well as hemodynamic and cardiorespiratory changes. Despite the wide variety of pharmacological treatments used to manage acute symptoms, none are effective in controlling envenomation. Knowing the essential role of neutralizing polyclonal antibodies in the treatment of envenoming for other species, such as snakes, this work aimed to produce a polyclonal antiserum in mice and test its ability to neutralize the main toxic effects induced by the venoms of the main venomous Brazilian fish. We found that the antiserum recognizes the main toxins present in the different venoms of Thalassophryne nattereri, Scorpaena plumieri, Potamotrygon gr. Orbignyi, and Cathorops spixii and was effective in pre-incubation trials. In an independent test, the antiserum applied immediately to the topical application of T. nattereri, P. gr orbygnyi, and C. spixii venoms completely abolished the toxic effects on the microcirculation, preventing alterations such as arteriolar contraction, slowing of blood flow in postcapillary venules, venular stasis, myofibrillar hypercontraction, and increased leukocyte rolling and adherence. The edematogenic and nociceptive activities induced by these venoms were also neutralized by the immediate application of the antiserum. Importantly, the antiserum prevented the acute inflammatory response in the lungs induced by the S. plumieri venom. The success of antiserum containing neutralizing polyclonal antibodies in controlling the toxic effects induced by different venoms offers a new strategy for the treatment of fish envenomation in Brazil.

Chemical composition, antioxidant activity and development of a facial serum formulation from the extract of Hancornia speciosa.

The goal of this work was to chemically characterise Hancornia speciosa extracts to develop an antioxidant serum formulation. Stem and bark extracts were prepared using 70% hydroethanol solution by Sohxlet and ultrasound assisted extraction. The content of total phenols, flavonoids, and antioxidant activity were evaluated, and chemical characterization was performed by HPLC -detector UV-VIS (SPD - 10 A). The formulation was developed with stem extract (0.250 mg/g) in hydroxyethylcellulose fluid gel. Stem extracts had higher total phenols and flavonoids, and higher antioxidant activity than bark extracts. The formulation presented low viscosity, a yellowish colour, 81.28% ± 0.14 of antioxidant activity. In the stability test, the physicochemical characteristics showed small variations, remaining more stable at a temperature of 5 °C, with an antioxidant activity of 64.81% ± 0.75. Therefore, the stem of H. speciosa has the potential to be used in antioxidant formulations.

Paratrygon aiereba irradiated anti-mucus serum reduce edematogenic activity induced in experimental model.

Accidents by freshwater stingrays are common in northern Brazil, there is no specific therapy for high morbidity and local tissue destruction. The irradiation of venoms and toxins by ionizing radiation has been used to produce appropriate immunogens for the production of antisera. We planned to study the efficacy of stinging mucus irradiation in the production of antisera, with serum neutralization assays of edematogenic activity and quantification of cytokines performed in animal models of immunization with native and irradiated mucus of Paratrygon aiereba, a large freshwater stingray. Antiserum potency and its cross-reactivity with mucus from other freshwater stingrays were detected by ELISA. Immunization models demonstrated the ability to stimulate a strong humoral response with elevated levels of serum IgG detectable by ELISA, and both native and irradiated mucus were immunogenic and capable of recognizing mucus proteins from other freshwater neotropical stingrays. Mucus P. aiereba causes cellular and humoral adaptive immune responses in cells of immunized mice producing antibodies and cytokines such as TNF-α, IL-6 and IL-17. Rabbit antisera immunized with mucus from P. aiereba irradiated at 2 kGy showed a significant reduction of mucus-induced edematogenic activity in mice. Our data suggest that the use of antisera against freshwater stingray mucus show the possibility of specific therapy for these accidents.

Stingray (Potamotrygon rex) maturity is associated with inflammatory capacity of the venom.

Several studies have been carried out with venom from sting and mucus of stingrays of marine or fluvial environments to compare the toxicity of their venom. However, studies demonstrating the existence of the influence of both sex and the maturation stage of stingrays on the variability of the toxic effects of venom are still scarce. Here, we investigated whether the sex and/or the stage maturation of the Potamotrygon rex stingray influence the toxic capacity of the venom to develop acute inflammation in mice. We carried out the main toxic activities in mice using venom from female or male of young and adult stingrays. Our results described here show that the nociception is mainly induced by venom from young female stingrays. In contrast, we observed the action of venom from both sex of adult stingrays in the induction of exudative phase of inflammatory process, including vascular leakage and neutrophil infiltration. Our data illustrate that the composition of the venom of P. rex is influenced by the stage of maturity of the stingray, modulating the production of peptides and proteins capable of acting on leukocytes-endothelial interactions and favoring neutrophil infiltration to the damage tissue.

Stingray (Potamotrygon rex) maturity is associated with inflammatory capacity of the venom

Several studies have been carried out with venom from sting and mucus of stingrays of marine or fluvial environments to compare the toxicity of their venom. However, studies demonstrating the existence of the influence of both sex and the maturation stage of stingrays on the variability of the toxic effects of venom are still scarce. Here, we investigated whether the sex and/or the stage maturation of the Potamotrygon rex stingray influence the toxic capacity of the venom to develop acute inflammation in mice. We carried out the main toxic activities in mice using venom from female or male of young and adult stingrays. Our results described here show that the nociception is mainly induced by venom from young female stingrays. In contrast, we observed the action of venom from both sex of adult stingrays in the induction of exudative phase of inflammatory process, including vascular leakage and neutrophil infiltration. Our data illustrate that the composition of the venom of P. rex is influenced by the stage of maturity of the stingray, modulating the production of peptides and proteins capable of acting on leukocytes-endothelial interactions and favoring neutrophil infiltration to the damage tissue.

Stingray (Potamotrygon rex) maturity is associated with inflammatory capacity of the venom

Several studies have been carried out with venom from sting and mucus of stingrays of marine or fluvial environments to compare the toxicity of their venom. However, studies demonstrating the existence of the influence of both sex and the maturation stage of stingrays on the variability of the toxic effects of venom are still scarce. Here, we investigated whether the sex and/or the stage maturation of the Potamotrygon rex stingray influence the toxic capacity of the venom to develop acute inflammation in mice. We carried out the main toxic activities in mice using venom from female or male of young and adult stingrays. Our results described here show that the nociception is mainly induced by venom from young female stingrays. In contrast, we observed the action of venom from both sex of adult stingrays in the induction of exudative phase of inflammatory process, including vascular leakage and neutrophil infiltration. Our data illustrate that the composition of the venom of P. rex is influenced by the stage of maturity of the stingray, modulating the production of peptides and proteins capable of acting on leukocytes-endothelial interactions and favoring neutrophil infiltration to the damage tissue.

Stingray venom activates IL-33 producing cardiomyocytes, but not mast cell, to promote acute neutrophil-mediated injury.

One of the hallmarks of acute inflammation is neutrophil infiltration of tissues. We investigated molecular mechanisms implicated in acute neutrophilic inflammation induced by the venom of a freshwater stingray (Potamotrygon cf. henlei) in mice. Ray venom induced early mobilization of neutrophil in the microvasculature of cremaster mice and infiltration of the peritoneal cavity 2 hours after injury, in a dose-response manner. IL-1ß, IL-6, TNF-α, and KC were produced. The neutrophilic infiltration did not occur in mice with ST2 receptor and MyD88 adapters neutralized, or in those with PI3K and p38 MAPK signaling blocked. Drastic reduction of neutrophil infiltration to peritoneal cavities was observed in ST2-/-, TLR2/TLR4-/-, MyD88-/-, TRIF-/- and IL-17A-/- mice, and a partial reduction was observed in IL-18R-/- mice. Mast cell Kit W(sh)/W(sh)-, AHR-, NLRP3-, ICE-, IL-1ß-, P2RX7-, CD39-, IL-17RA-, and TBX21 KO mice retain the ability to induce neutrophilia in peritoneal cavity after ray venom injection. IL-6 and TNF-α alone were insufficient for promote neutrophilia in the absence of ST2 signaling. Finally, abundant production of IL-33 by cardiomyocytes was observed. These results refine our understanding of the importance of the IL-33/ST2 axis and IL-33-producing cardiomyocytes in the early acute neutrophilia induced by freshwater stingray venoms.

Stingray venom activates IL-33 producing cardiomyocytes, but not mast cell, to promote acute neutrophil-mediated injury

One of the hallmarks of acute inflammation is neutrophil infiltration of tissues. We investigated molecular mechanisms implicated in acute neutrophilic inflammation induced by the venom of a freshwater stingray (Potamotrygon cf. henlei) in mice. Ray venom induced early mobilization of neutrophil in the microvasculature of cremaster mice and infiltration of the peritoneal cavity 2 hours after injury, in a dose-response manner. IL-1 beta, IL-6, TNF-alpha, and KC were produced. The neutrophilic infiltration did not occur in mice with ST2 receptor and MyD88 adapters neutralized, or in those with PI3K and p38 MAPK signaling blocked. Drastic reduction of neutrophil infiltration to peritoneal cavities was observed in ST2(-/-), TLR2/TLR4(-/-), MyD88(-/-), TRIF-/- and IL-17A(-/-) mice, and a partial reduction was observed in IL-18R(-/-) mice. Mast cell Kit W(sh)/W(sh)-, AHR-, NLRP3-, ICE-, IL-1 beta-, P2RX7-, CD39-, IL-17RA-, and TBX21 KO mice retain the ability to induce neutrophilia in peritoneal cavity after ray venom injection. IL- 6 and TNF-alpha alone were insufficient for promote neutrophilia in the absence of ST2 signaling. Finally, abundant production of IL-33 by cardiomyocytes was observed. These results refine our understanding of the importance of the IL-33/ST2 axis and IL-33-producing cardiomyocytes in the early acute neutrophilia induced by freshwater stingray venoms.

Triagem das hemoglobinas S e C e a influência das condições sociais na sua distribuição: um estudo em quatro comunidades quilombolas do Estado do Tocantins / Screening of hemoglobins S and C and the influence of social conditions in their distribution: a study in four quilombola communities, Tocantins state

Neste trabalho foi realizada a triagem das hemoglobinas S e C e o estudo do perfil social de quatro comunidades quilombolas. O estudo foi desenvolvido nas comunidades quilombolas de Malhadinha, Córrego Fundo, Curralinho do Pontal e Manoel João, município de Brejinho de Nazaré (TO). O perfil das hemoglobinas foi obtido com a triagem de 167 amostras de sangue, utilizando o teste de eletroforese em pH alcalino (pH 8,6). Para confirmar o diagnóstico, as amostras alteradas foram submetidas à eletroforese em pH ácido (pH 6,2). Foram identificadas 11 amostras (6,6 por cento) com hemoglobinas variantes: oito (4,8 por cento) com traço falciforme e três com hemoglobina C (1,8 por cento). Os dados sociais foram obtidos através de entrevista realizada com representantes de 48 famílias, levantando informações sobre a renda familiar, idade, sexo e atividade ocupacional de cada indivíduo. O estudo mostrou que essas comunidades são formadas predominantemente por adultos e idosos, com renda mensal maior que um salário mínimo, para 50 por cento das famílias entrevistadas. Apesar do percentual das hemoglobinas S e C encontradas nas comunidades estarem dentro do observado para várias regiões do Brasil, a ausência de informação sobre a doença e os aspectos sociais podem aumentar o número de indivíduos com doença falciforme no município ou áreas vizinhas.

This research performed the screening of S and C hemoglobins and a social profile study of four Quilombola communities. The study was performed at the communities of Malhadinha, Córrego Fundo, Curralinho do Pontal and Manoel João Quilombola, located in the Brejinho de Nazaré municipality, Tocantins state, Brazil. The hemoglobin profile was obtained by screening on 167 blood samples using the electrophoresis in alkaline pH (pH 8.6) test. Chemically altered hemoglobin was subject to electrophoresis in acid pH (pH 6.2) to confirm the diagnosis. We identified 11 samples (6.6%) with hemoglobin changes, eight (4.8%) had sickle cell trait and three were C hemoglobin (1.8%). Social data was obtained by interviews conducted with members of 48families, raising information such as family income, age, gender and occupational activity. Our results showed that these communities are formed predominantly by adults and elderly people with monthly revenue higher than the minimum wage, accounting for 50% of the interviewed families. Although the percentage of hemoglobin S and C found in all the communities are within the values observed within the various regions of Brazil, the absence of information about the disease and social aspects may increase the number of people with sickle cell disease in the municipality and surrounding areas.

Potamotrygon cf. henlei stingray mucus: biochemical features of a novel antimicrobial protein.

Antimicrobial molecules are important components of the innate immune system in vertebrates. They have been studied widely in several fishes, but little is known about these defence factors in stingrays, which are thought to have less sophisticated adaptive immune systems when compared to other teleosts. Stingrays from the specie Potamotrygon cf. henlei are distributed throughout the rivers of central-west Brazil, being the cause of numerous envenomations occurring in the dry seasons. In a previous study, we reported that the mucus of the stingray P. cf. henlei shows antimicrobial effects. Here, to analyze the antimicrobial compounds from the mucus of P. cf. henlei, we employed solid-phase extraction, chromatographic separation followed by ESI-MS, and Edman degradation. A protein similar to the ß-chain of hemoglobin was identified, isolated and partially sequenced by Edman degradation. This protein has a molecular weight of 16072.8 Da, and was shown to be active against bacteria (Micrococcus luteus and Escherichiacoli) and yeast (Candida tropicalis) without hemolytic activity. Effects of this new protein in the microcirculation environment were also evaluated. The results obtained provide fundamental information for future basic research, clinical diagnosis and development of new therapies to accident treatment. To the best of our knowledge, this is the first description of a bioactive polypeptide from the mucus of a stingray.

Studies on pharmacological properties of mucus and sting venom of Potamotrygon cf. henlei.

Stingrays from the Potamotrygon cf. henlei species are widely distributed in high numbers throughout the rivers of central-west Brazil, being the source of numerous envenomations occurring in the dry season, posing a serious public health problem even if not properly reported. The accidents usually involve fishermen and bathers, and to date there is no effective treatment for the injured. Considering these facts and limitations of studies aiming at understanding the effects induced by P. cf. henlei envenoming, this study aimed to describe the principal pharmacological and certain biochemical properties of the mucus and sting venom. We found that mucus and sting venom is toxic to mice having nociceptive, edematogenic and proteolysis activities. Our results also indicate that the inflammatory cellular influx observed could be triggered by the venom and mucus. Furthermore the venom and mucus were partially purified by solid-phase extraction tested for antimicrobial activity in which only the mucus presented activity. It could be inferred from the present study that P. cf. henlei venom possesses a diverse mixture of peptides, enzymes and pharmacologically active components.

Studies on pharmacological properties of mucus and sting venom ofPotamotrygon cf. henlei

Stingrays from the Potamotrygon cf. henlei species are widely distributed in high numbers throughout therivers of central-west Brazil, being the source of numerous envenomations occurring in the dry season, posinga serious public health problem even if not properly reported. The accidents usually involve fishermen andbathers, and to date there is no effective treatment for the injured. Considering these facts and limitations ofstudies aiming at understanding the effects induced by P. cf. henlei envenoming, this study aimed to describethe principal pharmacological and certain biochemical properties of the mucus and sting venom. We foundthat mucus and sting venom is toxic to mice having nociceptive, edematogenic and proteolysis activities. Ourresults also indicate that the inflammatory cellular influx observed could be triggered by the venom andmucus. Furthermore the venom and mucus were partially purified by solid-phase extraction tested forantimicrobial activity in which only the mucus presented activity. It could be inferred from the present studythat P. cf. henlei venom possesses a diverse mixture of peptides, enzymes and pharmacologically activecomponents.

Lonomia obliqua (Lepidoptera, Saturniidae) caterpillar bristle extract induces direct lysis by cleaving erythrocyte membrane glycoproteins.

Lonomia obliqua caterpillar bristle extract induces hemolysis in vitro on washed human and rat erythrocytes, in either the absence or presence of exogenous lecithin. In the former condition, phospholipases A(2) are key enzymes involved in hemolysis. However, the mechanism whereby this extract causes direct hemolysis is not known. Thus, the aim of this study was to investigate the hemolytic mechanism of the crude extract of the caterpillar L. obliqua on human erythrocytes in the absence of lecithin. The extract significantly increased the erythrocyte osmotic fragility and promoted the removal of glycophorins A and C, and band 3 from the erythrocyte membrane. The use of Ca2+ and Mg2+ ions significantly potentiated glycoprotein removal, remarkably of erythrocyte band 3. The composition of fatty acids was analyzed by HPLC in both L. obliqua caterpillar bristle extract and human erythrocyte membranes incubated with the extract. The levels of unsaturated fatty acids were remarkably augmented in erythrocytes incubated with the extract than in control erythrocytes, modifying thereby the saturated/unsaturated fatty acid ratio. Altogether, evidence is provided here that the interplay of at least three mechanisms of action accounts for the direct activity of the bristle extract on erythrocyte membrane, leading to hemolysis: the removal of glycoproteins and band 3; the insertion of fatty acids; and the action of phospholipases. Such mechanisms might affect erythrocyte flexibility and deformability, which may induce hemolysis by increasing erythrocyte fragility. However, whether the direct hemolytic activity of L. obliqua caterpillar is the major cause of intravascular hemolysis during envenomation still needs further investigation.

Purification of a phospholipase A2 from Lonomia obliqua caterpillar bristle extract.

Lonomia obliqua caterpillar bristle extract induces both direct and indirect hemolytic activity on human and rat washed erythrocytes, and provokes intravascular hemolysis in Wistar rats. Indirect hemolytic activity is assumed to be caused by a phospholipase A(2) (PLA(2)) present in this extract, and this investigation was initiated in order to characterize this enzyme. Phospholipase A(2) activity of crude extract was inhibited by both a PLA(2)-specific inhibitor (pBpb) and the metal ion chelator EDTA. L. obliqua PLA(2) was purified by liquid chromatography from the crude bristle extract and had a molecular mass of 15kDa and a pI of 5.9; its N-terminal sequence showed high homology to a sequence of a putative PLA(2) obtained from a cDNA library of L. obliqua bristles, and it is tentatively placed among Group III phospholipases A(2). This enzyme was stable at 4 degrees C, sensitive to higher temperatures, and its maximum catalytic activity was at pH 8.0. L. obliqua PLA(2) induced hemolysis only when incubated with exogenous lecithin. Thus, the PLA(2) purified herein appears to be responsible for the indirect hemolytic activity of the crude bristle extract.

Intravascular hemolysis induced by Lonomia obliqua caterpillar bristle extract: an experimental model of envenomation in rats.

Hemostatic disturbances are frequent findings in human accidents caused by Lonomia obliqua caterpillars in the southern region of Brazil. In severe envenomation, patients may present life-threatening bleedings. Such disturbances may be mimicked in rats, which also develop intravascular hemolysis. The scope of this study was to investigate the time-course and intensity of intravascular hemolysis induced by i.d. injection of 750 microg/kg crude L. obliqua bristle extract in rats. Total blood cell count, reticulocyte count, plasma hemoglobin and haptoglobin assays were performed in control and envenomed rats at 1, 6, 24 and 48 h after envenomation. Rats presented a drastic drop of haptoglobin levels at 1 and 6h with increased plasma hemoglobin levels, a decrease in packed cell volume values at 6, 24 and 48 h, and increased reticulocyte counts throughout after envenomation. Such observations indicated that intravascular hemolysis occurred as early as 1h following envenomation, and lasted for more than 6h. Intravascular hemolysis is probably induced by phospholipase A(2) and other proteins with direct hemolytic activity present in crude caterpillar bristle extract.

In vitro hemolytic activity of Lonomia obliqua caterpillar bristle extract on human and Wistar rat erythrocytes.

Human accidental envenomation caused by skin contact with the bristles of Lonomia obliqua caterpillar causes coagulation and fibrinolysis disorders. Alterations of hematologic parameters are observed only in severe cases of envenomation, but with no clinical evidence of intravascular hemolysis. However, since we have observed intravascular hemolysis in preliminary studies using Wistar rats as an experimental model for investigating L. obliqua envenomation, the objective of the present study was to investigate the in vitro hemolytic activity of the bristle extract of L. obliqua caterpillars on human and rat erythrocytes. Our results showed that the bristle extract has indirect and direct hemolytic activity on human and rat erythrocytes, although direct hemolytic activity was only observed at higher bristle extract concentrations. We also observed that the bristle extract has a proteolytic activity on band 3 of human and rat erythrocyte membranes. Thus, crude L. obliqua bristle extract was found to contain at least two components with hemolytic activity on erythrocytes, a phospholipase enzyme and another protein with a direct activity on the erythrocyte membrane.