Articular inflammation induced by an enzymatically-inactive Lys49 phospholipase A2: activation of endogenous phospholipases contributes to the pronociceptive effect

Background Arthritis is a set of inflammatory conditions that induce aching, stiffness, swelling, pain and may cause functional disability with severe consequences to the patient's lives. These are multi-mediated pathologies that cannot be effectively protected and/or treated. Therefore, the aim of this study was to establish a new model of acute arthritis, using a Lys49-PLA2 (Bothrops asper myotoxin II; MT-II) to induce articular inflammation. Methods The articular inflammation was induced by MT-II (10 μg/joint) injection into the left tibio-tarsal or femoral-tibial-patellar joints. Cellular influx was evaluated counting total and differential cells that migrated to the joint. The plasma extravasation was determined using Evans blue dye. The edematogenic response was evaluated measuring the joint thickness using a caliper. The articular hypernociception was determined by a dorsal flexion of the tibio-tarsal joint using an electronic pressure-meter test. The mediators involved in the articular hypernociception were evaluated using receptor antagonists and enzymatic inhibitors. Results Plasma extravasation in the knee joints was observed 5 and 15 min after MT-II (10 μg/joint) injection. MT-II also induced a polymorphonuclear cell influx into the femoral-tibial-patellar joints observed 8 h after its injection, a period that coincided with the peak of the hyperalgesic effect. Hyperalgesia was inhibited by the pretreatment of the animals with cyclooxygenase inhibitor indomethacin, with type-2 cyclooxygenase inhibitor celecoxib, with AACOCF3 and PACOCF3, inhibitors of cytosolic and Ca2+-independent PLA2s, respectively, with bradykinin B2 receptor antagonist HOE 140, with antibodies against TNFα, IL-1β, IL-6 and CINC-1 and with selective ET-A (BQ-123) and ET-B (BQ-788) endothelin receptors antagonists. The MT-II-induced hyperalgesia was not altered by the lipoxygenase inhibitor zileuton, by the bradykinin B1 receptor antagonist Lys-(Des-Arg9,Leu8)-bradykinin, by the histamine and serotonin antagonists promethazine and methysergide, respectively, by the nitric oxide inhibitor LNMMA and by the inhibitor of matrix 1-, 2-, 3-, 8- and 9- metalloproteinases GM6001 (Ilomastat). Conclusion These results demonstrated the multi-mediated characteristic of the articular inflammation induced by MT-II, which demonstrates its relevance as a model for arthritis mechanisms and treatment evaluation.(AU)

Articular inflammation induced by an enzymatically-inactive Lys49 phospholipase A2: activation of endogenous phospholipases contributes to the pronociceptive effect

Background Arthritis is a set of inflammatory conditions that induce aching, stiffness, swelling, pain and may cause functional disability with severe consequences to the patient's lives. These are multi-mediated pathologies that cannot be effectively protected and/or treated. Therefore, the aim of this study was to establish a new model of acute arthritis, using a Lys49-PLA2 (Bothrops asper myotoxin II; MT-II) to induce articular inflammation. Methods The articular inflammation was induced by MT-II (10 μg/joint) injection into the left tibio-tarsal or femoral-tibial-patellar joints. Cellular influx was evaluated counting total and differential cells that migrated to the joint. The plasma extravasation was determined using Evans blue dye. The edematogenic response was evaluated measuring the joint thickness using a caliper. The articular hypernociception was determined by a dorsal flexion of the tibio-tarsal joint using an electronic pressure-meter test. The mediators involved in the articular hypernociception were evaluated using receptor antagonists and enzymatic inhibitors. Results Plasma extravasation in the knee joints was observed 5 and 15 min after MT-II (10 μg/joint) injection. MT-II also induced a polymorphonuclear cell influx into the femoral-tibial-patellar joints observed 8 h after its injection, a period that coincided with the peak of the hyperalgesic effect. Hyperalgesia was inhibited by the pretreatment of the animals with cyclooxygenase inhibitor indomethacin, with type-2 cyclooxygenase inhibitor celecoxib, with AACOCF3 and PACOCF3, inhibitors of cytosolic and Ca2+-independent PLA2s, respectively, with bradykinin B2 receptor antagonist HOE 140, with antibodies against TNFα, IL-1β, IL-6 and CINC-1 and with selective ET-A (BQ-123) and ET-B (BQ-788) endothelin receptors antagonists. The MT-II-induced hyperalgesia was not altered by the lipoxygenase inhibitor zileuton, by the bradykinin B1 receptor antagonist Lys-(Des-Arg9,Leu8)-bradykinin, by the histamine and serotonin antagonists promethazine and methysergide, respectively, by the nitric oxide inhibitor LNMMA and by the inhibitor of matrix 1-, 2-, 3-, 8- and 9- metalloproteinases GM6001 (Ilomastat). Conclusion These results demonstrated the multi-mediated characteristic of the articular inflammation induced by MT-II, which demonstrates its relevance as a model for arthritis mechanisms and treatment evaluation.(AU)

Articular inflammation induced by an enzymatically-inactive Lys49 phospholipase A2: activation of endogenous phospholipases contributes to the pronociceptive effect

Abstract Background Arthritis is a set of inflammatory conditions that induce aching, stiffness, swelling, pain and may cause functional disability with severe consequences to the patients lives. These are multi-mediated pathologies that cannot be effectively protected and/or treated. Therefore, the aim of this study was to establish a new model of acute arthritis, using a Lys49-PLA2 (Bothrops asper myotoxin II; MT-II) to induce articular inflammation. Methods The articular inflammation was induced by MT-II (10 g/joint) injection into the left tibio-tarsal or femoral-tibial-patellar joints. Cellular influx was evaluated counting total and differential cells that migrated to the joint. The plasma extravasation was determined using Evans blue dye. The edematogenic response was evaluated measuring the joint thickness using a caliper. The articular hypernociception was determined by a dorsal flexion of the tibio-tarsal joint using an electronic pressure-meter test. The mediators involved in the articular hypernociception were evaluated using receptor antagonists and enzymatic inhibitors. Results Plasma extravasation in the knee joints was observed 5 and 15 min after MT-II (10 g/joint) injection. MT-II also induced a polymorphonuclear cell influx into the femoral-tibial-patellar joints observed 8 h after its injection, a period that coincided with the peak of the hyperalgesic effect. Hyperalgesia was inhibited by the pretreatment of the animals with cyclooxygenase inhibitor indomethacin, with type-2 cyclooxygenase inhibitor celecoxib, with AACOCF3 and PACOCF3, inhibitors of cytosolic and Ca2+-independent PLA2s, respectively, with bradykinin B2 receptor antagonist HOE 140, with antibodies against TNF, IL-1, IL-6 and CINC-1 and with selective ET-A (BQ-123) and ET-B (BQ-788) endothelin receptors antagonists. The MT-II-induced hyperalgesia was not altered by the lipoxygenase inhibitor zileuton, by the bradykinin B1 receptor antagonist Lys-(Des-Arg9,Leu8)-bradykinin, by the histamine and serotonin antagonists promethazine and methysergide, respectively, by the nitric oxide inhibitor LNMMA and by the inhibitor of matrix 1-, 2-, 3-, 8- and 9- metalloproteinases GM6001 (Ilomastat). Conclusion These results demonstrated the multi-mediated characteristic of the articular inflammation induced by MT-II, which demonstrates its relevance as a model for arthritis mechanisms and treatment evaluation.

First record of Porocephalus cf. clavatus (Pentastomida: Porocephalida) as a parasite on Bothrops asper (Squamata: Viperidae) in Costa Rica / Primeiro registro de Porocephalus cf. clavatus (Pentastomida: Porocephalida) como um parasita em Bothrops asper (Squamata: Viperidae) na Costa Rica

Pentastomids are parasites that infect respiratory cavities of vertebrates, they are pretty common but poorly known in wildlife veterinary. A Bothrops asper snake (Garman, 1884) was captured in the Caribbean region of Costa Rica and had its lung infested with pentastomids, identified as ca Porocephalus clavatus (Wyman, 1845). This represents the first record of Porocephalus (Humboldt, 1812) on B. asper as well as P. cf. clavatus in Costa Rica. Further studies are needed to clarify their taxonomic position, images and scanning electron microscopy photographs (SEM) of the specimens are given.(AU)

Pentastomídeos sâo parasitas que infectam as cavidades respiratórias dos vertebrados, eles são bastante comuns, mas pouco conhecido nos animais silvestres. Uma Bothrops asper (Garman, 1884) foi capturada na região do Caribe da Costa Rica e teve seu pulmão infestado de pentastomídeos, identificados como ca Porocephalus clavatus (Wyman, 1845). Isto representa o primeiro registro de Porocephalus (Humboldt, 1812) em B. asper, assim como P. cf. clavatus na Costa Rica. Mais estudos detalhados são necessários para esclarecer sua posição taxonómica. Imagens e fotografias de microscopia electrónica de varredura (MEV) dos espécimes são dadas.(AU)

Biologia reprodutiva de serpentes neotropicais dos grupos Bothrops atrox e Bothrops jararacussu (Viperidae: Crotalinae)

A extensa distribuição geográfica apresentada pelos grupos de espécies Bothrops asper, B. atrox e B. jararacussu sugere que as diferentes estratégias reprodutivas encontradas nestas espécies poderiam estar sendo moduladas pelos ritmos ambientais, principalmente temperatura e pluviosidade. As espécies de Crotalinae que habitam regiões frias ou temperadas possuem ciclos reprodutivos estritamente sazonais, os ciclos começam nos períodos mais quentes do ano. Embora vários estudos já tenham descrito as estratégias reprodutivas de diferentes espécies de Bothrops, nenhum deles teve como objetivo avaliar em conjunto os representantes do clado composto pelos grupos de espécies B. asper, B. atrox e B. jararacussu. Neste estudo, descrevemos aspectos da biologia reprodutiva, como a vitelogênese, a espermatogênese, a hipertrofia do segmento sexual renal (SSR) e o dimorfismo sexual das espécies seguintes: B. asper, B. atrox, B jararacussu, B. brazili e B. marajoenesis. Nossas análises indicam que machos de espécies distribuídas em regiões de clima equatorial, como B. atrox, B. asper, B. brazili e B. marajoenesis, tendem a apresentar o pico de espermatogênese principalmente na estação chuvosa. Por outro lado, B. jararacussu (espécie distribuída na região tropical e subtropical) apresenta alta freqüência de espermatogênese na estação seca e assincronia entre a espermiação e o nível máximo da hipertrofia do SSR (aumento de grânulos ricos em carboidratos neutros e ácidos). O crescimento dos folículos ovarianos em B. jararacussu e B. atrox ocorre no final da estação chuvosa ou no início da estação seca. Apenas B. asper apresenta vitelogênese ocorrendo principalmente durante a estação chuvosa. Considerando o período de vitelogênese secundária, as fêmeas de B. asper, B. atrox e B. jararacussu apresentam hipertrofia das glândulas do infundíbulo posterior e do útero, a qual regride durante a vitelogênese primária e prenhez. Essas variações morfológicas do oviduto podem estar diretamente associadas a estocagem e sobrevivência dos espermatozoides. Dentre as espécies do gênero Bothrops, B. jararacussu apresenta o maior índice de dimorfismo sexual (0,6), com fêmeas significativamente maiores que os machos.

The extensive geographic distribution presented by the species groups Bothrops asper, B. atrox and B. jararacussu, may suggest that different environmental rhythms could be responsible for shaping different reproductive strategies for the species within these groups. Species of Crotalinae living in cold or temperate regions have strictly seasonal reproductive cycles, with cycles beginning in the hottest periods of the year. Although several studies have already described the reproductive strategies of different species of Bothrops, none of them was focused on the clade composed by the B. asper, B. atrox and B. jararacussu species groups. Here we describe aspects of the reproductive biology, such as vitellogenesis, spermatogenesis, the hypertrophy of the sexual segment of the kidney (SSK) and the presence and degree of sexual dimorphism for the following five species: B. atrox, B. asper, B. jararacussu, B. brazili and B. marajoenesis. Our analyses indicate that males of species distributed on regions with an equatorial climate, as B. atrox, B. asper, B. brazili and B. marajoenesis, tend to present the peak of spermatogenesis mainly in the rainy season. On the other hand, B. jararacussua species distributed on tropical and subtropical regionshows a high frequency of spermatogenesis in the dry season, and an asynchrony between spermiation and the maximum level of the hypertrophy of the SSK (increase of granules rich in neutral and acidic carbohydrates). The growth of the ovarian follicles in B. jararacussu and B. atrox occurs at the end of the rainy season or early in the dry season. Only B. asper presents vitellogenesis mainly occurring during the rainy season. Considering the period of secondary vitellogenesis, females of B. asper, B. atrox, and B. jararacussu present hypertrophy of the posterior infundibulum glands and the uterus glands, which regress during primary vitellogenesis and pregnancy. These morphological variations of the oviduct are supposed to be directly associated with the storage and survival of spermatozoa. Among all species of Bothrops, B. jararacussu presents the highest sexual dimorphism index (0.6), with females significantly larger than males.

Current challenges for confronting the public health problem of snakebite envenoming in Central America

Snakebite envenoming is a serious public health problem in Central America, where approximately 5,500 casesoccur every year. Panama has the highest incidence and El Salvador the lowest. The majority, and most severe,cases are inflicted by the pit viper Bothrops asper (family Viperidae), locally known as ‘terciopelo, ‘barba amarilla or‘equis. About 1% of the bites are caused by coral snakes of the genus Micrurus (family Elapidae). Despite significantand successful efforts in Central America regarding snakebite envenomings in the areas of research, antivenommanufacture and quality control, training of health professionals in the diagnosis and clinical management of bites,and prevention of snakebites, much remains to be done in order to further reduce the impact of this medicalcondition. This essay presents seven challenges for improving the confrontation of snakebite envenoming inCentral America. Overcoming these challenges demands a coordinated partnership of highly diverse stakeholdersthough inter-sectorial and inter-programmatic interventions.(AU)

Current challenges for confronting the public health problem of snakebite envenoming in Central America

Snakebite envenoming is a serious public health problem in Central America, where approximately 5,500 casesoccur every year. Panama has the highest incidence and El Salvador the lowest. The majority, and most severe,cases are inflicted by the pit viper Bothrops asper (family Viperidae), locally known as ‘terciopelo’, ‘barba amarilla’ or‘equis’. About 1% of the bites are caused by coral snakes of the genus Micrurus (family Elapidae). Despite significantand successful efforts in Central America regarding snakebite envenomings in the areas of research, antivenommanufacture and quality control, training of health professionals in the diagnosis and clinical management of bites,and prevention of snakebites, much remains to be done in order to further reduce the impact of this medicalcondition. This essay presents seven challenges for improving the confrontation of snakebite envenoming inCentral America. Overcoming these challenges demands a coordinated partnership of highly diverse stakeholdersthough inter-sectorial and inter-programmatic interventions.

Development of a sensitive enzyme immunoassay (ELISA) for specific identification of Lachesis acrochorda venom

The snake genus Lachesis provokes 2 to 3% of snakebites in Colombia every year. Two Lachesis species, L. acrochorda and L. muta, share habitats with snakes from another genus, namely Bothrops asper and B. atrox. Lachesis venom causes systemic and local effects such as swelling, hemorrhaging, myonecrosis, hemostatic disorders and nephrotoxic symptoms similar to those induced by Bothrops, Portidium and Bothriechis bites. Bothrops antivenoms neutralize a variety of Lachesis venom toxins. However, these products are unable to avoid coagulation problems provoked by Lachesis snakebites. Thus, it is important to ascertain whether the envenomation was caused by a Bothrops or Lachesis snake. The present study found enzyme linked immunosorbent assay (ELISA) efficient for detecting Lachesis acrochorda venom in a concentration range of 3.9 to 1000 ng/mL, which did not show a cross-reaction with Bothrops, Portidium, Botriechis and Crotalus venoms. Furthermore, one fraction of L. acrochorda venom that did not show crossreactivity with B. asper venom was isolated using the same ELISA antibodies; some of its proteins were identified including one Gal-specific lectin and one metalloproteinase. This test may be useful to physicians, since it could be applicable for tracking the kinetic distribution of antigens in patients or experimentally envenomed animals.(AU)

Development of a sensitive enzyme immunoassay (ELISA) for specific identification of Lachesis acrochorda venom

The snake genus Lachesis provokes 2 to 3% of snakebites in Colombia every year. Two Lachesis species, L. acrochorda and L. muta, share habitats with snakes from another genus, namely Bothrops asper and B. atrox. Lachesis venom causes systemic and local effects such as swelling, hemorrhaging, myonecrosis, hemostatic disorders and nephrotoxic symptoms similar to those induced by Bothrops, Portidium and Bothriechis bites. Bothrops antivenoms neutralize a variety of Lachesis venom toxins. However, these products are unable to avoid coagulation problems provoked by Lachesis snakebites. Thus, it is important to ascertain whether the envenomation was caused by a Bothrops or Lachesis snake. The present study found enzyme linked immunosorbent assay (ELISA) efficient for detecting Lachesis acrochorda venom in a concentration range of 3.9 to 1000 ng/mL, which did not show a cross-reaction with Bothrops, Portidium, Botriechis and Crotalus venoms. Furthermore, one fraction of L. acrochorda venom that did not show crossreactivity with B. asper venom was isolated using the same ELISA antibodies; some of its proteins were identified including one Gal-specific lectin and one metalloproteinase. This test may be useful to physicians, since it could be applicable for tracking the kinetic distribution of antigens in patients or experimentally envenomed animals.(AU)

Ethnobotanic study of Randia aculeata (Rubiaceae) in Jamapa, Veracruz, Mexico, and its anti-snake venom effects on mouse tissue

In Mexico, medicinal plants are widely used. The use of Randia aculeata by healers against snakebites has never been scientifically tested in relation to possible effects on blood parameters and muscle tissue damage. Interviews were carried out in Jamapa, Veracuz, Mexico, with local residents to collect information about the traditional use of Randia aculeata. In this locality, seven pieces of fruit from the plant are mixed in a liter of alcohol, and then administered orally against snakebites. By using histological techniques and a murine model, we explored its cytoprotective properties against the effects of Crotalus simus and Bothrops asper venoms. Possible protections provided by the plant against tissue damage to skeletal and cardiac muscles and against the typical loss of red blood cells were analyzed. Randia aculeata caused an increase in microhematocrit and total hemoglobin, parameters that are often decremented in association with the loss of red blood cells, which is a characteristic effect of animal venom. Randia aculeata was also shown to protect against the lowering of platelet levels caused by Bothrops asper venom. Finally, Randia aculeata produced a partial inhibition of necrosis following administration of snake venom in skeletal and myocardial muscles. The present results provide solid evidence for the traditional use of Randia aculeata against snakebites, as demonstrated by protection against muscular tissue damage and the diminution of red blood cells.(AU)

Functional and structural characterization of phospholipases A2 isolated from Bothrops asper snake venom in Panamá

Envenoming by Bothrops snakes is the most serious type of envenoming from the medical and economic point of view in Central America. Bothrops asper is responsible for 90% of the snakebites registered in Panamá every year. Despite its medical and economic relevance, only the venom of Costa Rican and Guatemalan populations of this species has been studied to some detail, and there is very little information on intraspecies variability in venom composition and toxicity. In this study the crude venom of B. asper from Panamá was characterized and its pharmacological and biochemistry activities were investigated with standard laboratory assays. Furthermore, we described the isolation, functional and structural characterization of four basic phospholipases A2, namely MTX-I, MTX-II, MTX-III, MTX-IV, and a new acid phospholipase A2 called Basp-I-PLA2. The proteins were isolated from the crude venom by a combination of two chromatographic steps, using ion-exchange chromatography on CM-Sepharose (0.05 M NH4HCO3 pH 8.1 buffer), and hydrophobic chromatography on Phenyl-Sepharose (0.05 M Tris-HCl pH 7.4), followed by concentration gradient from 4 to 0 M NaCl at 25°C in the same buffer. Analyses of phospholipids hydrolyzed by these enzymes have shown that all phospholipases belong to type A2. The acidic isoform demonstrated more catalytic activity than basic PLA2s. This enzyme was more active on substrates such as phosphotidylcholine and phosphatidylglycerol. The isoelectric focusing evidenced pIs between 8.1 to 8.3 for MTXs and 4.6 for the isoform Basp-I-PLA2. The molecular weight was estimated by mass spectrometry to be: MTX-1 = 14,156.5; MTX-2 = 14,249.5 and MTX-3 = 14,253.0 and Basp-I-PLA2 = 14,246.0.8 Da.(AU)

Functional and structural characterization of phospholipases A² isolated from Bothrops asper snake venom in Panamá

Envenoming by Bothrops snakes is the most serious type of envenoming from the medical and economic point of view in Central America. Bothrops asper is responsible for 90% of the snakebites registered in Panamá every year. Despite its medical and economic relevance, only the venom of Costa Rican and Guatemalan populations of this species has been studied to some detail, and there is very little information on intraspecies variability in venom composition and toxicity. In this study the crude venom of B. asper from Panamá was characterized and its pharmacological and biochemistry activities were investigated with standard laboratory assays. Furthermore, we described the isolation, functional and structural characterization of four basic phospholipases A2, namely MTX-I, MTX-II, MTX-III, MTX-IV, and a new acid phospholipase A2 called Basp-I-PLA2. The proteins were isolated from the crude venom by a combination of two chromatographic steps, using ion-exchange chromatography on CM-Sepharose (0.05 M NH4HCO3 pH 8.1 buffer), and hydrophobic chromatography on Phenyl-Sepharose (0.05 M Tris-HCl pH 7.4), followed by concentration gradient from 4 to 0 M NaCl at 25°C in the same buffer. Analyses of phospholipids hydrolyzed by these enzymes have shown that all phospholipases belong to type A2. The acidic isoform demonstrated more catalytic activity than basic PLA2s. This enzyme was more active on substrates such as phosphotidylcholine and phosphatidylglycerol.(AU)

Purification and partial characterization of phospholipases A2 from Bothrops asper (barba amarilla) snake venom from Chiriguaná (Cesar, Colombia)

Components with phospholipase A2 activity were isolated by gel filtration and cationic exchange chromatography from the venom of Bothrops asper snakes from Chiriguaná, Colombia (9°22´N; 73°37´W). Five fractions were obtained by the gel filtration, and PLA2 activity was found in fraction 3 (F3). In the cationic exchange chromatography, F3 showed eight components with PLA2 activity. Six of these components appeared as one band in polyacrylamide gel electrophoresis (SDS-PAGE). Fractions II and VII exhibited an optimal activity at pH 9 and 52ºC. The optimum calcium concentration for fraction II was 48 mM and for fraction VII, 384 mM. Both fractions showed thermal stability. Fraction II was stable at pH values between 2.5 and 9, and fraction VII, between 2.5 and 8. The Michaelis Menten constant (K M) was 3.5x10-3 M for fraction II and 1.6x10-3 M for fraction VII. The molecular weight was 16,000 Dalton for fraction II and 17,000 Dalton for fraction VII. Both isoenzymes did not show any toxic activity (DL50) at 5.3 and 4 µg/g. The two fractions showed different kinetic constant (K M), calcium requirement, and substrate specificity for haemolytic activity.