New procedures and parameters for better evaluation of Androctonus australis garzonii (Aag) and Buthus occitanus tunetanus (Bot) scorpion envenomations and specific serotherapy treatment.

International audienceNew procedures describing intoxication with variable amounts of scorpion venoms (from 1 to 5 LD50) allowed us to introduce new parameters to evaluate Aag and Bot envenomations. Significant differences between the fatal limit time (FLT) and the last mortality time (LMT) were observed when the amount of Aag and Bot venom injected was equal to 1 LD50 and equal to or higher than 2 LD50. For Aag and Bot, the percentage of the fast mortality (FM) and the delayed mortality (DM) varied conversely when the amount of injected venom increased from 1 to 5 LD50. The relationship between the venom LD50 (from 2 to 20), the median protective dose (PD50) and the neutralizing activity of specific antivenom have been established. PD50 increased in a parallel manner with LD50. The neutralizing titres (LD50/ml) of Aag antivenom decreased from 74 +/- 3 to 44 +/- 2 and that of Bot antivenom from 52 +/- 2 to 36 +/- 1 when the number of LD50 injected increased from 2 to 20. Antivenom potency was evaluated using different protocols based on the presence or the absence of preincubation of the venom with the antivenom. In experiments where venom and antivenom were simultaneously but immediately injected, PD50 were twice as high as those found when venom and antivenom were preincubated (30 min at 37 degrees C). On the contrary, the corresponding neutralizing titres were two times lower. In an attempt to simulate accidental envenomations and subsequent serotherapy, Aag and Bot venom (4 LD50) were subcutaneously injected and the appropriate PD50S of antivenom were intravenously administered at different time intervals after envenomation. When the time of antivenom administration was shorter than the FLT, all envenomed mice might be protected by increasing volume of antivenom. However, when the antivenom is injected closer to the FLT only 50 to 60% of mice envenomed, respectively, by Aag and Bot could be saved even when more than 5 PD50 were injected

Effect of Some Variables on theIn VivoDetermination of Scorpion and Viper Venom Toxicities

International audienceAbstractAn adequate assessment of scorpion and snake venom LD50 is an important step for accurate evaluation of antivenom sera potencies and the optimization of serotherapy. The LD50 variation of Tunisian scorpion (Androctonus australis garzonii: Aag and Buthus occitanus tunetanus: Bot) venoms with body weight, sex and strain (Swiss or C57BI/6) of mice used, the route of venom injection, the venom-milking procedures (manually or electrically) and the venom batches have been studied over a 7-year period (1990-1996). Aag venom is 3-4 times more toxic than Bot venom. However for both venoms, the LD50 determined in C57BI/6 mice, in small body weight animal or by intraperitoneal route were respectively significantly lower than those determined in Swiss mice, in high body weight or by subcutaneous route. Significant LD50 variations (25-50%) were also seen from one electrically prepared batch to another. A good correlation (r = 0.982) was observed between the concentrations of the crude venom toxic fraction determined by ELISA and LD50 values when assessed in vivo. The LD50 variation of Tunisian viper (Cerastes cerastes: Cc and Vipera lebetina: VI) venoms with the strain (Swiss or BALB/c), sex and body weight of mice used, the season and the year of venom milking were also investigated over a 3-year period (1990-1992). No significant LD50 variations were observed with the mouse strain, the sex or the season of venom milking. However, LD50 varies significantly with the year of the venom collection and the body weight of mice used. Furthermore, SDS-PAGE analysis shows annual variation for VI venom composition where no such variations were observed for Cc venom. These results stress the need either for the standardization of the venom LD50 evaluation or of the venom quality used for the development of an efficient antivenom

Evaluation of antivenom therapy in children severely envenomed by Androctonus australis garzonii (Aag) and Buthus occitanus tunetanus (Bot) scorpions.

International audienceOne hundred and forty-seven cases of envenomed children under 15 years old presenting local and general symptoms without failure in vital functions (clinical grade II) or presenting serious general symptoms with failure in vital functions (clinical grade III) were collected during the summer seasons of 1993-1997. They were classified in six groups according to the use or not of antivenom, the route and the frequency of antivenom administration. The determination, by a sensitive ELISA, of blood venom concentration before and until 6 h after antivenom therapy, allowed the establishment of the venom toxicokinetic curve for each group. The intramuscular administration of antivenom did not show significant effects on venom toxicokinetic curves and on patients recovery time. However, the same amount of antivenom administered by intravenous route clear rapidly the blood free venom toxins. Also, the patient recovery time was significantly shortened. These data are in favor of intravenous application of an adequate dose of an efficient antivenom in order to treat successfully severe scorpion envenoming cases

Effects of antivenom on Buthus occitanus tunetanus (Bot) scorpion venom pharmacokinetics: towards an optimization of antivenom immunotherapy in a rabbit model

International audienceThe pharmacokinetic parameters of Bot venom were determined in a rabbit model using a specific sandwich type ELISA. After intravenous injection, Bot venom seems to follow a three-compartment pharmacokinetic open model. However, after subcutaneous injection, the distribution and elimination kinetics of Bot venom are best characterized by a bi-compartment pharmacokinetic open model. Bot venom is completely absorbed from its SC injection site, since the absolute bioavailability is higher than 95%; the maximum plasma venom concentration is reached between 30 and 60 min after venom injection. Bot venom diffuses rapidly to tissues and is distributed in a high body volume. The total body clearance of Bot venom is relatively high in agreement with a low mean residence time. Antivenom immunotherapy experiments were carried out in the rabbit model, in order to select the most appropriate strategy for the adequate use of this treatment. The effects of the route, the dose and the delay of antivenom injection on Bot venom pharmacokinetic parameters and on the antivenom immunotherapy efficacy were then studied. These studies indicated in particular that: (1) the injection of a minimal neutralizing antivenom dose is required for a complete and permanent neutralization of circulating venom antigens; this dose is named minimal (threshold) efficacious antivenom dose; (2) the intramuscular route is not the most appropriate way for antivenom injection; and (3) a delayed antivenom immunotherapy remains efficacious especially on the neutralization of the remaining circulating venom. In short, these experimental studies show that early intravenous injection of an appropriate antivenom dose (at least the threshold efficacious dose) is the indicated way for a rapid and permanent neutralization of circulating scorpion venom toxins

Pharmacokinetic studies of scorpion venom before and after antivenom immunotherapy

International audienceThe improvement of the immunotherapeutic treatment of envenomations requires a better knowledge of the pharmacological actions of the scorpion venom and of the mechanism of its in vivo neutralization by antivenom. In the present work, we determined the toxicokinetic parameters of the toxic fraction of Androctonus australis garzonii venom in the absence and after antivenom immunotherapy, in experimentally envenomed rabbits. After subcutaneous injection of the scorpion venom, toxins showed a fast and complete resorption from the site of injection associated with a simultaneous distribution in a large extracellular compartment and with an important body clearance. The precocious intravenous injection of an appropriate antivenom dose was shown to induce an immediate, complete and durable neutralization of toxins, as well as their rapid redistribution from the peripheric compartment to the vascular one. On the contrary, the intramuscular injection of the same antivenom dose produced a slower and partial redistribution of toxins, leading to a delayed neutralization of the venom. The intravenous injection of smaller antivenom doses induced transient decreases of circulating toxins, indicating that a minimal antivenom dose has to be administered to allow an efficient and durable neutralization of the venom. We concluded also that this minimal effective dose of antivenom has to be injected precociously, by intravenous route, to achieve an efficient immunotherapy

Quantitative variability in the biodistribution and in toxinokinetic studies of the three main alpha toxins from the Androctonus australis hector scorpion venom

International audienceScorpion stings represent a medical problem in numerous countries. The scorpion Androctonus australis hector produces three alpha toxins (Aah I to III), which are responsible for most of the lethality in mammals. These toxins act on sodium channel and do not cross-react immunologically. We used RIA and ELISA to measure the concentrations of these three toxins in plasma, urine and different organs after i.v. and s.c. injections of water extracts of venoms in rabbits or mice. In both animals, the toxins rapidly appeared in plasma after s.c. injection as it was previously described for the whole venom. However, the toxins disappeared from the blood more quickly than did other main components of the venom. Thus, serotherapy must be initiated immediately to prevent the toxin from reaching its target. We also detected the toxins in urine, kidneys, heart and lungs, but not in the brain. However, the concentration of Aah II was always lower than that of Aah I. Analysis of five samples of venom collected in different areas of southern Tunisia showed that a large polymorphism exists for the three toxins. This is yet another difficulty for serotherapy as there is no cross-antigenicity between them

Development of an ELISA for the detection of scorpion venoms in sera of humans envenomed by Androctonus australis garzonii (Aag) and Buthus occitanus tunetanus (Bot): correlation with clinical severity of envenoming in Tunisia.

This work was codirected by M. El Ayeb and K. Dellagi.International audienceA sandwich ELISA was set up for measuring scorpion venom levels in sera of accidentally envenomed humans with the aim to establish a quantitative relationship between these levels, envenoming severity and clinical symptoms. This assay used equine polyclonal F(ab')2, specific to two North African scorpion (Androctonus australis garzonii: Aag and Buthus occitanus tunetanus: Bot) venoms. The test proved to be simple, reproducible, very sensitive (detection limit = 0.9 ng/ml) and linear between 0.5 and 15 ng/ml of venom concentrations. A large survey on scorpion sting envenomings was conducted from 1993 to 1996 in Tunisia to gather accurate epidemiological, clinical and biological data from victims as well as informations on the treatment that they had received. Victims were classified into three grades (GI, GII and GIII) of increasing severity according to clinical signs of envenoming. Blood samples were collected from victims and tested by ELISA for their content of Aag and Bot venoms. A strong correlation was found between clinical symptoms of envenoming and the level of scorpion venom antigens in serum (r = 0.980). Mean serum venom concentrations were: 2.65 +/- 0.81 ng/ml in GI envenoming, 9.79 +/- 4.08 ng/ml in GII and 21.7 +/- 6.51 ng/ml in GIII. The difference between each group was statistically significant (p < 0.01). This ELISA may prove to be helpful to establish a rationale approach of specific antivenom therapy

Anti-platelet activity of the peptides composing the lebetin 1 family, a new class of inhibitors of platelet aggregation.

International audienceWe have purified from Vipera lebetina venom a family of inhibitors of platelet aggregation, named Lebetins. They are composed of two peptide groups of short (Lebetin 1: L1alpha: GDNKPPKKGPPNG; L1beta: DNKPPKKGPPNG) and long (Lebetin 2: L2alpha: GDNKPPKKGPPNGCFGHKIDRIGSHSGLGCNKVDDNKG; L2beta: DNKPPKKGPPNGCFGHKIDRIGSHSGLGCNKVDDNKG) size. The sequence presenting anti-platelet activity is mainly present within the Lebetin 1 sequence [Barbouche, R. Marrakchi, N., Mansuelle, P., Krifi, M., Fenouillet, E., Rochat, H. and El Ayeb, M. (1996) Novel anti-platelet aggregation polypeptides from Vipera lebetina venom: isolation and characterization. FEBS Lett. 392, 6-10]. Here, the peptides that compose the Lebetin 1 family were synthesized. Their respective activity was determined. Synthetic L1alpha and L1beta inhibited collagen-induced platelet aggregation in the nanomolar range. A peptide corresponding to L1beta deleted by D at its N terminus (L1gamma) also inhibited platelet aggregation potently; further truncation of L1gamma impaired its activity. Because L1 peptides efficiently inhibited fibrinogen-induced alpha-chymotrypsin treated-platelet aggregation, we tested whether they act mainly through the inhibition of platelet binding to fibrinogen and showed that they failed to inhibit platelet binding to fibrinogen-coated wells. The activity of L1 peptides was also tested in vivo: their intravenous administration strongly inhibited collagen-induced thrombocytopenia in rats