Expansión de la distribución de escorpiones del género Tityus C. L. Koch 1836 en Argentina: Implicancias sanitarias / Expansion of the distribution of scorpions of the genus Tityus C. L. Koch 1836 in Argentina: Implications in public health

Se ha observado la presencia de especies de Tityus en diferentes regiones del país, en las cuales su presencia no había sido comunicada previamente: 1- Tityus bahiensis en las provincias de Entre Ríos y Buenos Aires, en esta última en la localidad de Lanús y en San Clemente del Tuyú, y 2- Tityus confluens en la Ciudad Autónoma de Buenos Aires (CABA) y en la provincia de Buenos Aires en las localidades de Pilar, La Plata, Mar del Plata y Bahía Blanca. Estos hallazgos modifican el mapa de la distribución de escorpiones de importancia sanitaria en Argentina por lo que ante la picadura de escorpiones deben considerarse estos nuevos hallazgos. Esto es especialmente importante en el ámbito de la CABA y la provincia de Buenos Aires, en donde la enorme mayoría de los accidentes por escorpiones han sido causados por T. trivittatus y en donde ahora, al menos en algunas de sus regiones se pueden encontrar T. confluens y T. bahiensis. Se discuten posibles razones de esta nueva distribución así como la ocurrencia de accidentes graves en zonas donde no ocurrían históricamente y de sus posibles causas. En base a los casos graves producidos en los últimos tiempos y a este nuevo mapa de distribución, se hace énfasis en la necesidad de capacitación al personal de salud en general y de los médicos de guardia y terapistas en particular, para tratar adecuadamente los accidentes por escorpiones.

Several species of Tityus have been described in regions of Argentina where their presence had not been previously described. These are: 1- Tityus bahiensis in the provinces of Entre Ríos and Buenos Aires (in the localities of Lanús and San Clemente del Tuyú), and 2- Tityus confluens in the city of Buenos Aires and in the province of Buenos Aires in the localities of Pilar, La Plata, Mar del Plata and Bahía Blanca. These findings modify the distribution map of scorpions of sanitary importance in Argentina, reason for which this new distribution must be considered when facing a scorpion sting. This is especially important in the city of Buenos Aires and the province of Buenos Aires, where most of the accidents by scorpions are caused by Tityus trivittatus, and where at least in some of their regions, T. confluens or T. bahiensis can be found at present. The possible reasons of this new distribution, as well as the possible causes for the occurrence of severe envenomations in regions where these were not observed historically, are discussed. Based on the severe envenomations observed and on this new distribution map, emphasis is placed on the need to capacitate health personnel in general and intensivists or critical care physicians in particular to adequately treat scorpion accidents.

cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom.

A mRNA transcript that codes for a phospholipase (PLA2) was isolated from a single venom gland of the Bothrops ammodytoides viper. The PLA2 transcript was cloned onto a pCR®2.1-TOPO vector and subsequently expressed heterologously in the E. coli strain M15, using the pQE30 vector. The recombinant phospholipase was named rBamPLA2_1, and is composed of an N-terminal fusion protein of 16 residues, along with 122 residues from the mature protein that includes 14 cysteines that form 7 disulfide bonds. Following bacterial expression, rBamPLA2_1 was obtained from inclusion bodies and extracted using a chaotropic agent. rBamPLA2_1 had an experimental molecular mass of 15,692.5 Da that concurred with its theoretical molecular mass. rBamPLA2_1 was refolded in in vitro conditions and after refolding, three main protein fractions with similar molecular masses, were identified. Although, the three fractions were considered to represent different oxidized cystine isoforms, their secondary structures were comparable. All three recombinant isoforms were active on egg-yolk phospholipid and recognized similar cell membrane phospholipids to be native PLA2s, isolated from B. ammodytoides venom. A mixture of the three rBamPLA2_1 cystine isoforms was used to immunize a horse in order to produce serum antibodies (anti-rBamPLA2_1), which partially inhibited the indirect hemolytic activity of B. ammodytoides venom. Although, anti-rBamPLA2_1 antibodies were not able to recognize crotoxin, a PLA2 from the venom of a related but different viper genus, Crotalus durissus terrificus, they recognized PLA2s in other venoms from regional species of Bothrops.

Isolation, characterization, cloning and expression of an alpha-neurotoxin from the venom of the Mexican coral snake Micrurus laticollaris (Squamata: Elapidae).

A new member of short chain α-neurotoxic protein family from venom of the Mexican coral snake, Micrurus laticollaris, was characterized. This protein, named MlatA1, possesses 61 amino acids with 8 conserved cysteine residues, sharing 30-91% sequence identity with other fully sequenced Micrurus toxins. MlatA1 (LD50i.v. = 0.064 mg/kg) antagonizes with both fetal and adult nicotinic acetylcholine receptor (nAChR) as well as α-7 neuronal nAChR in a dose-dependent way. Specific rabbit anti-Mlat serum (titer higher than 18,000) does not show any protective ability against this toxin, nevertheless it was able to recognize protein bands in six out of twelve Micrurus venoms showing the existence of two distinct antigenic groups for α-neurotoxins in North American coral snakes species. The MlatA1 gene was cloned and used to produce recombinant toxin (rMlatA1) that was recognized by rabbit anti-native toxin but was depleted of toxic activity.

Caracterización tóxica del veneno de Bothrops (Rhinocerophis) alternatus de diferentes regiones de la provincia de Córdoba (Argentina) / [The toxicity of venom of Bothrops (Rhinocerophris) alternatus in different areas of Cordoba State in Argentina].

Snake venoms can show biochemical and toxicological variability even in specimens from the same specie. The geographical localization of the snakes is one of the factors that can influence those variations. By these reasons the venom from specimens of Bothrops (Rhinocerophis) alternatus ("crucera", "yararágrande"), one of the snakes of highest medical importance in Argentina, from three different regions of Córdoba was studied. Lehtal potency, hemorrhagic, coagulant on plasma and thrombin like activities as well as the electrophoretic patterns of venom from snakes of Calamuchita, Traslasierras and the East of the province were determined. The venom from the snakes of the three regions showed the characteristic activities of the venom of the majority of Bothrops, causing hemorrhage, hemostatic disturbances acting on plasma or directly on fibrinogen with a "thrombin like activity". The different samples were very similar regarding their biochemical characteristics and toxic potencies at difference of previous observations on venoms from the same specie in different regions of other provinces fro Argentina. Bivalent antivenom, the one used by the Provincial Ministry of Health to treat the bothropic accidents, neutralized in all the cases the toxic activities of the venom in very similar range of neutralizing potency.

Envenenamiento de Chelydra serpentina (Reptilia: Testudines) por Tityus trivittatus (Scorpionida: Buthidae) / Envenomation of Chelydra serpentina (Reptilia: Testudines) by Tityus trivittatus (Scorpionida: Buthidae)

Se describe el caso de un ejemplar de tortuga mordedora (Chelydra serpentina) que fue hallada con los miembros tetanizados en extensión, midriasis y poca respuesta a estímulos externos, en cuyo recinto se encontró un ejemplar de escorpión Tityus trivittatus. Ante el claro cuadro de envenenamiento, se trató al quelonio con antiveneno escorpiónico específico retornando a un estado de relajación muscular a las seis horas y encontrándoselo totalmente normal a las 24 horas sin mostrar secuelas posteriores. Este es el primer comunicado sobre el envenenamiento de quelonios por escorpiones. Se discuten algunos aspectos de este envenenamiento escorpiónico y su tratamiento con antiveneno específico.

We report the case of a snapping turtle (Chelydra serpentina) found tetanized, with the limbs in extension, mydriasis and poor response to external stimuli, in whose terrarium was found a Tityus trivittatus scorpion. Based on the clear clinical picture of envenoming, the turtle was treated with a specific scorpion antivenin, returning to a state of muscle relaxation after six hours of treatment and it was found totally normal at 24 hours, without envenoming sequelae. This is the first report on turtle envenomation by scorpion. The scorpion envenomation in reptiles and the treatment with specific antivenom is discussed.

A novel toxin from the venom of the scorpion Tityus trivittatus, is the first member of a new alpha-KTX subfamily.

The first example of a new sub-family of toxins (alpha-KTx20.1) from the scorpion Tityus trivittatus was purified, sequenced and characterized physiologically. It has 29 amino acid residues, three disulfide bridges assumed to adopt the cysteine-stabilized alpha/beta scaffold with a pI value of 8.98. The sequence identities with all the other known alpha-KTx are less than 40%. Its effects were verified using seven different cloned K(+) channels (vertebrate Kv1.1-1.5, Shaker IR and hERG) expressed in Xenopus leavis oocytes. The toxin-induced effects show large differences among the different K(+) channels and a preference towards Kv1.3 (EC50=7.9+/-1.4 nM).

Envenenamiento por picaduras de abeja / Poisoning by bee sting

Dentro de las patologías humanas producidas por animales con la capacidad de inocular veneno, las picaduras de abeja producen el mayor número de accidentes por animales en muchos países, superando a menudo en mortalidad a los producidos por serpientes, escorpiones y arañas. El cuadro clínico por la picadura de estos himenópteros puede consistir en fenómenos alérgicos o en cuadros de envenenamiento. Estos últimos se producen por el ataque de enjambres constituyendo un hecho grave que puede comprometer la vida. En el sujeto envenenado pueden observarse hemólisis, rabdomiólisis e insuficiencia renal, que junto a otras alteraciones sistémicas pueden conducir a la muerte. El conocimiento de los acontecimientos fisiopatológicos que se producen ante los ataques masivos de abejas es de suma importancia para el personal de salud dado que hasta la fecha no existen antivenenos que hayan demostrado tener eficacia clínica comprobada. En esta revisión se consideran los aspectos biológicos de las abejas y de la composición de su veneno relacionado con la ocurrencia y severidad de los accidentes, así como datos epidemiológicos de utilidad para enfrentarse a este tipo de cuadro.

Among the human pathologies produced by venomous animals, bee stings constitute the largest number of accidents in several countries, exceeding the mortality rate caused by other venomous animals such as snakes, spiders or scorpions. The clinical picture after the bee sting may include anaphylaxis or poisoning. The latter is produced by massive attacks and is a serious problem that may put the patient's life at risk. People that are poisoned display hemolysis, rhabdomiolysis and acute renal failure that together with other systemic failures can bring about death. The knowledge of the physiopathological mechanisms involved in the massive attack of bees is crucial for health care professionals as to date we do not ha ve antivenoms with proven clinical efficacy. In this review we include the bee's biological aspects, venom composition and its relation with the occurrence and severity of accidents as well as epidemiological data that can be useful for this type of accidents.

Toxicidad de venenos de serpientes de importancia médica en México / Toxicity of venoms from snakes of medical importance in Mexico

La caracterización de las actividades tóxicas de los venenos de serpientes es necesaria para el cabal entendimiento de los procesos fisiopatológicos que se producen ante su mordedura, como también para evaluar la potencia neutralizante de los antivenenos utilizados para tratar estos envenenamientos. A causa de los pocos datos disponibles sobre la toxicidad del veneno de serpientes con importancia sanitaria en México, estudiamos las actividades tóxicas de los venenos de Bothrops asper, Athropoides nummifer, Agkistrodon billineatus> Crotalus durissus durissus, Crotalus basiliscus, Crotalus scutulatus, Crotalus atrox y Micrurus nigrocinctus. A los venenos se les realizaron los siguientes estudios: SDS-PAUE, determinación de la potencia letal, y de las actividades hemorrágica, necrotizante, coagulante en plasma y fibrinógeno, fosfolipásica y fibrinogenolítica. Se estudió además la capacidad neutralizante de un antiveneno de uso corriente para la terapéutica de las mordeduras de serpientes venenosas en México, sobre varias de estas actividades. Los venenos de vipéridos mostraron actividades hemorrágicas, necrotizante, coagulante sobre plasma, protrombínica, fibrinogenolítica y fosfolipásica importantes. Los venenos de mayor potencia letal fueron los de Micrurus nigrocinctus y Crotalus scutulatus, sin embargo el veneno que presentó en general potencias tóxicas mayores fue el de Bothrops asper. Las diferentes potencias tóxicas halladas se encontraron dentro de los márgenes descritos para especies de vipéridos y elápidos de Sudamérica. La actividad sobre el plasma y el fibrinógeno fue muy diferente en los diferentes venenos viperinos, sin embargo todos mostraron ser capaces de afectar componentes del sistema de la coagulación. El antiveneno probado no sólo neutralizó la letalidad del veneno sino también sus actividades tóxicas.

The characterization of the toxic activities of snake venoms is necessary to understand the physiopathology of the envenomation and to test the potency of the antivenoms used to treat this pathology. Because of the lack of data on the toxic activities of venoms from Mexican snakes of medical importance, we studied the venoms from Bothrops asper, Athropoides nummifer, Agkistrodon billineatus, Crotalus durissus durissus, Crotalus basiliscus, Crotalus scutulatus, Crotalus atrox and Micrurus nigrocinctus. The studies performed were : SDS-PAOE, determination of lethal potency, hemorrhagic, necrotizing, coagulation on plasma and fibrinogen, phospholipasic and fibrinogenolytic activities. In addition we studied the neutralizing capacity of the toxic activities of an antivenom currently used for the treatment of snakebites in Mexico. The venom from viperids showed important hemorrhagic, necrotizing, coagulative on plasma, prothrombinic, fibrinogenolytic and phospholipase activities. The venoms with the highest lethal potency were those of Micrurus nigrocinctus and Crotalus scutulatus; however, the viperine venom that globally displayed the most potent toxic activities was from Bothrops asper. All the venoms showed toxic activities of similar range to those described for other American venomous snakes. The activity on plasma or fibrinogen varied widely among the different venoms but all displayed capacity to act on the coagulation system. The antivenom tested not only neutralized the lethalityB. asper venom but also its other toxic activities.

Recombinant GRA4 or ROP2 protein combined with alum or the gra4 gene provides partial protection in chronic murine models of toxoplasmosis.

The efficacy of vaccination with Toxoplasma gondii recombinant GRA4 (rGRA4) and ROP2 (rRPO2) proteins and a mix of both combined with alum were evaluated in C57BL/6 and C3H mice. In C57BL/6 mice, rGRA4 and rGRA4-rROP2 immunizations generated similar levels of immunoglobulin G1 (IgG1) and IgG2a isotypes against GRA4, whereas immunizations with rROP2 and the mix induced a predominant IgG1 production against ROP2. All groups of C3H vaccinated mice exhibited higher levels of IgG1 than IgG2a. rGRA4-stimulated splenocytes from vaccinated mice produced primarily gamma interferon while those stimulated with rROP2 produced interleukin-4. Challenge of rGRA4- or rGRA4-rROP2-vaccinated mice from both strains with ME49 cysts resulted in fewer brain cysts than the controls, whereas vaccination with rROP2 alone only conferred protection to C3H mice. Immunization with a plasmid carrying the entire open reading frame of GRA4 showed a protective level similar to that of rGRA4 combined with alum. These results suggest that GRA4 can be a good candidate for a multiantigen anti-T. gondii vaccine based on the use of alum as an adjuvant.

Disulfide bridges and blockage of Shaker B K(+)-channels by another butantoxin peptide purified from the Argentinean scorpion Tityus trivittatus.

A peptide was isolated from the venom of the scorpion Tityus trivittatus. It is an isoform of the toxin TsTX-IV earlier described [Toxicon 37 (1999) 651] and identical to butantoxin [Arch. Biochem. Biophys. 379 (2000) 18], both isolated from the Brazilian scorpion Tityus serrulatus. This newly characterized peptide contains 40 amino acid residues with a molecular mass of [M+H(+)] 4507.0, cross-linked by four disulfide bridges, made between the cysteine pairs: Cys2-Cys5, Cys10-Cys31, Cys16-Cys36 and Cys20-Cys38. It blocks in a completely reversible manner the Shaker B K(+)-channels, with a K(d) around 660nM. It belongs to the sub-family 12 and it is now being classified as alpha-KTx 12.2.

Phase I and pharmacokinetics study of crotoxin (cytotoxic PLA(2), NSC-624244) in patients with advanced cancer.

A Phase I clinical trial was performed on patients with solid tumors refractory to conventional therapy. Crotoxin was administered i.m. for 30 consecutive days at doses ranging from 0.03 to 0.22 mg/m(2). Patients entered the study after providing a written informed consent. Although 26 patients were entered only 23 were evaluated. Reversible, nonlimiting neuromuscular toxicity evidenced as diplopia because of pareses of the external ocular muscles was present in 13 patients. It started at doses of 0.18 mg/m(2) and lasted from 2 to 6 h. These episodes did not require dose adjustment and disappeared in 1-3 weeks of treatment. Three patients experienced palpebral ptosis, nystagmus (grade 2), and anxiety (grade 2-3) at the dose-limiting toxicity of 0.22 mg/m(2). Also at dose-limiting toxicity, 1 patient showed nystagmus (grade 2) and anxiety (grade 3) without evidence of palpebral ptosis. Transient increases (grades 1-3) in the levels of creatinine kinase, aspartate aminotransferase, and alanine transaminase attributed to crotoxin myotoxicity were observed but returned to normal by the last week of treatment. At 0.21 mg/m(2) there was a case of grade-3 anaphylactic reaction on day 31, which required treatment. Hypersensitivity was regarded as an adverse drug-related reaction, and the patient was removed from the protocol. Two patients at different doses (0.12 mg/m(2) and 0.22 mg/m(2)) had sialorrhea. Four patients had asymptomatic transient increase in blood pressure (up to 20 mm Hg) 12 h after the first injection, which lasted 24 h. No treatment was required and toxicity did not reappear. Six patients experienced slight eosinophilia during the first 2 weeks. The maximum tolerated dose was set at 0.21 mg/m(2). Objective measurable partial responses (>50% reduction of tumor mass) were noted in 2 patients treated at 0.21 mg/m(2) and 1 at 0.12 mg/m(2). One patient (at 0.21 mg/m(2)) presented a complete response on day 110. Crotoxin pharmacokinetics showed rapid absorption from the injection site to blood (t(1/2 A) = 5.2 +/- 0.6 min). Plasma concentration reached a peak (C(max) = 0.79 +/- 0.1 ng/ml) at tau(max) = 19 +/- 3 min. The half-life of the distribution (alpha) phase is 22 +/- 2 min. Starting at 1.5 h after injection, the decrease in plasma concentration becomes slower, reaching 14 +/- 3 pg/ml 24 h after injection. The profile is dominated by the elimination (beta) phase with a half-life of 5.2 +/- 0.6 h. Consequently, 24 h after the injection ( approximately 5 half-life) 97% of the product was eliminated. The area under plasma concentration versus time curve was 0.19 +/- 0.05 microg/min/ml. Assuming availability (F) approximately 1, the clearance is C(L) = 26.3 +/- 7 ml/min, and the apparent volume of distribution is V(d) = 12 +/- 3 liter/kg. The recommended dose for a Phase II study is 0.18 mg/m(2).

Accidentes por lepidópteros con especial referencia a Lonomia sp / Accidents due to lepidoptera with special reference to Lonomia sp

Human poisoning by Lepidoptera (butterflies, caterpillars and moths) has been known to physicians since old times. In the last few years, several factors have been causative of a great number of contacts between different stages of these insects and humans, occasionally with epidemic features. Species of medical interest in Argentina are those related to Megalopygidae, Saturniidae, Hemileucidae, Arctiidae, Notodentidae and Eucliedae families. Among all the species involved, it is important to describe the pathology produced by contact with Lonomia obliqua caterpillar (Saturniidae). The caterpillar Lonomia has several enzymes with procoagulant and fibrinolytic activities, acting on various components of the hemostatic system inducing hemorrhage that can lead to death. In the South of Brazil during the last years caterpillars of this butterfly caused a great number of cases of erucism including some deaths. In the past two years, there have been some cases of poisoning by Lonomia obliqua in Misiones, in the North East of Argentina on the border with Southern Brazil. This presents a potential risk, taking into account the poor knowledge regarding this insect and its venom in Argentina. This brief actualization gives some biochemical, clinical and epidemiologic tools towards understanding human Lepidopterism, an uncommon medical problem in this country.