Immunomodulatory and protective properties of tacrolimus in experimental scorpion envenomation.

Involvement of imbalance between pro- and anti-inflammatory events has been reported in the developed pathogenesis after scorpion envenomation. The immunosuppressive and anti-inflammatory properties of tacrolimus (FK-506) have been investigated: i) to better understand evolution of signaling pathways which are involved in the immune system ii) to reduce observed clinical signs while keeping a balance between pro- and anti-inflammatory cytokines. Naval Medical Research Institute (NMRI) mice received tacrolimus (1 mg/kg every 12 hours per os) for 21 days before envenomation with a sublethal dose (10 microg/20 g body weight) of Androctonus australis hector venom (Aah). Cell migration, pulmonary edema, exudation, Myeloperoxydase (MPO), Eosinophil peroxydase (EPO), C-reactive protein (CRP), C3, Creatine phosphokinase (CPK), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), and hyperglycemia were analyzed 30 min, 3 and 24 hours after injection of Aah venom. Histological analysis of lung parenchyma was undertaken 24 hours after envenomation. Aah lethality was evaluated on mice with or without pretreatment with tacrolimus. (Fab’)2 fragments (40 mg/kg) were also used as specific treatment in all protocols. Tacrolimus significantly inhibited cell migration, pulmonary edema, exudation, CRP and hyperglycemia. It also decreased MPO and EPO activities and prevented tissue damage in lung tissue, balancing seric parameter levels (CPK, ASAT and ALAT). The pretreated animals seemed to be protected by this macrolide against the venom lethality. These findings suggest that the overactivation of the immune system is one of the causes involved in the aggravation of the pathophysiological effects induced after envenomation. The obtained results showed that the use of F(ab’)2 fragments as specific treatment cannot reduce the induced inflammatory response.

Incidence and severity of scorpion stings in Algeria

Scorpion stings are a public health problem in the Maghreb region. In Algeria, epidemiological data were collected over the past twenty years by the Algerian health authorities. This study is an analysis of morbidity and mortality data collected from 2001 to 2010. Annual incidence and mortality due to scorpion envenoming were 152 ± 3.6 stings and 0.236 ± 0.041 deaths per 100,000 people (95% CI), respectively. The risk of being stung by a scorpion was dramatically higher in southern areas and central highlands due to environmental conditions. Incidence of envenoming was especially higher in the adult population, and among young males. In contrast, mortality was significantly higher among children under 15 years, particularly ages 1-4. Upper limbs were more often affected than lower limbs. Most stings occurred at night, indoors and during the summer. Data collected since 2001 showed a reduction of mortality by nearly 50%, suggesting that the medical care defined by the national anti-scorpion project is bearing fruit.(AU)

Toxicokinetic and toxicodynamic analyses of Androctonus australis hector venom in rats: optimization of antivenom therapy.

This paper reports the simultaneous determination of toxicokinetic and toxicodynamic properties of Androctonus australis hector venom, in the absence and presence of antivenom (F(ab')(2) and Fab), in envenomed rats. After subcutaneous injection of the venom, toxins showed a complete absorption phase from the site of injection associated with a distribution into a large extravascular compartment. The injection of Fab and F(ab')(2) induced the neutralization of venom antigens in the blood compartment, as well as the redistribution of venom components from the extravascular compartment to the blood compartment. Interestingly, F(ab')(2) and Fab showed distinct efficiencies depending on their route of injection. F(ab')(2) induced a faster venom neutralization and redistribution than Fab when injected intravenously. Fab was more effective than F(ab')(2) by the intramuscular route. The hemodynamic effects of Aah venom were further investigated. Changes in mean arterial pressure and heart rate were observed in parallel with an upper airway obstruction. Fab was more effective than F(ab')(2) for preventing early symptoms of envenomation, whatever their route of administration. Intraperitoneal injection of F(ab')(2) and Fab was similar for the prevention of the delayed symptoms, even after a late administration. Fab was more effective than F(ab')(2) in the inhibition of airway resistance, independent of the route and time of administration. These results show that the treatment for scorpion stings might be improved by the intravascular injection of a mixture of Fab and F(ab')(2). If antivenom cannot be administered intravenously, Fab might be an alternative as they are more effective than F(ab')(2) when injected intramuscularly.

[Effect of immunotherapy on metabolic and histopathological modifications after experimental scorpion envenomation]. / Effet de l'immunothérapie sur les modifications métaboliques et histopathologiques après envenimation scorpionique expérimentale.

Scorpion envenoming is a serious public health problem in many areas in the world. The most dangerous scorpion species in Algeria are Androctonus australis hector (Aah). Little is known about biochemical and histopathological effects of Androctonus australis hector venom after experimental envenomation. In this study, the effects of sublethal dose and lethal dose 50 (LD50) of Aah venom on the enzymatic activities (transaminases, alkaline phosphatase, creatine kinase and lactate dehydrogenase) and histopathological changes from organs (liver, heart, kidneys and lungs) were determined 24 hr following envenoming (s.c) of the mice. The effect of F(ab')2 fragments anti-FToxG-50-Aah was also tested on the same organs. The histopathological studies following Aah envenoming showed degenerative changes in the liver where most hepatocytes were enlarged and necrotic. Nuclei were irregular in size. After envenoming with Aah venom the myocardium showed myocytolysis with interstitial edema and hemorrhage, in the lungs, hemorrhage and thickening of inter-alveolar septa. The glomeruli and the tubules structure of kidneys were disorganized. Results indicate also that the enzyme activities were significantly increased in the serum and decreased in the organs after envenomation by Aah venom. This increase may be due to the enzyme release from organs into the circulation. Neutralization by anti-FToxG-50-Aah F(ab')2 fragments leads to a normalization of the enzyme rate and a partial restoration of the tissues structure after envenomation with Aah venom.

[Application of ELISA for the quantification of Androctonus australis hector venom in the envenomed serum of people and rats before and after immunotherapy]. / Application du test ELISA pour la quantification du venin d'Androctonus australis hector dans les sérums de personnes et de rats envenimés avant et après immunothérapie.

Scorpion envenomation remains an important health problem in many countries in the world, especially in North Africa, Asia and America. In Algeria, the most dangerous species for humans are Androctonus australis hector (Aah) and Buthus occitanus tunetanus (Bot). These scorpions are responsible for the apparition of various symptoms in envenomed patients such as: pain, hypertention, hypotension, sudation and fever. An aggravation of clinical conditions of envenomed patients is characterized by a pulmonary cedema and myocardial damage. The evaluation of the severity of scorpion envenoming by immuno-enzymatic assay requires firstly, the preparation of a specific anti-horse F(ab')2 peroxydase conjugate not yet commercialized. The restatement of conditions of ELISA sandwich test allowed its utilization in determination of the venom concentration in envenomed patients and rats sera after envenoming by scorpion venom. Standardization of this test, its reproductibility, linearity and its detection limit were defined. The venom concentrations are directly deducted from standard curves prepared by the dilution of Androctonus australis hector in human serum collected from healthy donors and in non envenomed rats serum. The present study showed that ELISA test has a good linear response in a range of concentrations of venom antigen. Its detection limit was 0.5 ng/ml of Aah venom in serum. This specific test of scorpion envenoming aims in one side at establishing a correlation between venom levels and the clinical observations and at evaluating the severity of the envenoming before and after immunotherapy.

[Fifteen years' experience in scorpion envenomation control in Algeria]. / Expérience de quinze années de lutte contre l'envenimation scorpionique en Algérie.

In Algeria, scorpion envenomation is real public health problem. Since the creation of the National Committee of Control of Scorpion envenomations (CNLES), several steps have been taken to deal with this problem. After a brief historical introduction, we present the main elements of the action carried out both in terms of treatment and of prevention of scorpion proliferation. The epidemiological situation is presented by stressing the difficulties involved in collecting reliable data. We also address the question of citizen and stakeholder awareness since public participation is crucial in all prevention programmes. Training for healthcare providers is also one of the principal axes of the Committee's programme which includes national, regional, and even local seminars. We describe the improvement of production and research on venoms carried out by the Institute Pasteur of Algeria. We conclude by discussing the action plan for 2001 and prospects for an enhanced strategy in the fight against the scorpion envenomation.

KTX3, the kaliotoxin from Buthus occitanus tunetanus scorpion venom: one of an extensive family of peptidyl ligands of potassium channels.

A new ligand of the K+ channels sensitive to KTX was purified from the venom of Buthus occitanus tunetanus, using two steps of high-performance-liquid-chromatography and by following its ability to compete with [125I]-KTX for binding to the KTX receptor on rat brain synaptosomes. Amino-acid analysis, amino acid sequencing and mass spectroscopy defined this new ligand. KTX3, as a 37-amino acid peptide, with three disulfide bridges. Its sequence shares 76% identity with KTX. The main differences between the two peptides are in the N-terminal region and the residue position 34 located in the region involved in channel recognition. These differences may explain the 5-fold lower binding affinity of KTX3, IC50=50 pM, than KTX to rat brain synaptosomes. Specific antibodies raised against KTX (1-37) were not able to recognize KTX3.

[Use of toxic fraction isolated from Algerian Androctonus australis hector scorpion venom for the assessment of anti-venom serum]. / Utilisation de la fraction toxique majoritaire isolée à partir du venin de scorpion Androctonus australis hector d'Algérie dans la valorisation du sérum anti-scorpionique.

Today, serotherapy is the only specific treatment used against the scorpionique envenomation. This treatment was associated with other drugs as a symptomatic and supportive care. The anti-venom against scorpion stings is still prepared using the conventional manufacturing production. To evaluate the efficacy of the anti-venom, different protocols are used: The immunization of animals (rabbit and horse) with a whole toxic fraction prepared by molecular filtration was the first step to improve the anti-venom. Some observations also show the need for the quality control of this preparation of anti-venom. In this study, we describe the use the toxic fraction isolated from Androctonus australis Hector in the new protocol of anti-venom production. The quality control of the product is evaluated by using a combination of experimental immunological assay systems (ELISA, Hemaglutination, in vivo assessment of the neutralization of the lethal effect of the venom).

Characterization of PO1, a new peptide ligand of the apamin-sensitive Ca2+ activated K+ channel.

A new peptide ligand of the small conductance Ca2+ activated K+ channels has been purified from the venom (obtained by manual rather than electrical stimulation of the scorpion Androctonus mauretanicus mauretanicus), by following the inhibition of the 125I-apamin binding to its receptor on rat brain synaptosomes. Only one step on a C18 reversed-phase high-performance liquid chromatography column was necessary to obtain PO1. Its K0.5 for the apamin binding site was 100 nM. The amino acid sequence of PO1 is different from those of leiurotoxin and PO5. For the first time the same peptide was also purified from the venoms of two other species of North African scorpions, Androctonus australis and Buthus occitanus tunetanus. PO1 was chemically synthesized by the solid-phase technique and fully characterized. A model of PO1 was constructed by amino acid replacement using PO5 nuclear magnetic resonance studies as the starting model. Structure-activity relationships between these toxins and their receptor are discussed.

Afaâcytin, an alpha beta-fibrinogenase from Cerastes cerastes (horned viper) venom, activates purified factor X and induces serotonin release from human blood platelets.

Afaâcytin, a proteinase with caseinolytic, arginine-esterase and amidase activities, was purified from the venom of Cerastes cerastes (horned viper) in two steps by gel filtration through Sephadex G75, then HPLC on carboxymethyl-cellulose. Afaâcytin has an isoelectric point of 6.25, and consists of two subunits, alpha and beta, which have the same apparent molecular mass (40,000) and are indistinguishable in the absence of reduction or/and deglycosylation. Subunit beta is constituted of two disulfide-linked polypeptidic chains, beta and beta'. The respective apparent molecular mass of the chains are 43,000 (alpha), 35,500 (beta) and 10,200 (beta') as determined by SDS/PAGE under reducing conditions. Both chains alpha and beta are N-glycosylated. The two chains have the same N-terminal sequence (20 residues) which is similar to those of other proteinases from snake venom. Susceptibility of afaâcytin to diisopropyl fluorophosphate and benzamidine indicates the presence of a serine and an aspartic (or glutamic) acid residues in the catalytic site. Ca2+ appears to be required for structural cohesion of the afaâcytin molecule. Afaâcytin exhibits alpha beta-fibrinogenase and alpha-fibrinase properties. It replaces missing factors VIII and IX in deficient plasmas, and activates purified human factor X into factor Xa. It releases serotonin from platelets and directly aggregates human (but not rabbit) blood platelets. Despite its thrombin-like characteristics, however, afaâcytin is not inhibited by plasmatic thrombin inhibitors. The procoagulant properties of afaâcytin therefore have potential clinical applications.

The kaliotoxin family enlarged. Purification, characterization, and precursor nucleotide sequence of KTX2 from Androctonus australis venom.

Kaliotoxin (KTX) has been originally described as an inhibitor of the intermediate conductance Ca(2+)-activated K+ channel (Crest, M., Jacquet, G., Gola, M., Zerrouk, H., Benslimane, A., Rochat, H., Mansuelle, P., and Martin-Eauclaire, M.-F. (1992) J. Biol. Chem. 267, 1640-1647). However, the radioiodinated 125I-KTX-(1-37) was also able to bind to the dendrotoxin sensitive voltage-dependent K+ channel (Romi, R., Crest, M., Gola, M., Sampieri, F., Jacquet, G., Zerrouk, H., Mansuelle, P., Sorokine, O., Van Dorsselaer, A., Rochat, H., Martin-Eauclaire, M.-F., and Van Rietschoten, J. (1993) J. Biol. Chem. 268, 26302-26309). By following the ability to compete with 125I-KTX-(1-37) for binding to its receptor on rat brain synaptosomes, a new kaliotoxin-like peptide, KTX2, was isolated from Androctonus australis scorpion venom. It is a 37-amino acid residue peptide, and its sequence shares 76% identity with KTX. The differences between the two peptides concern the NH2-terminal region and the residues 31 and 34 located in the region involved in the channel recognition. These differences may explain the 5-fold decrease of the molluscan Ca(2+)-activated K+ channel blockage by KTX2 (kd = 135 nM) as well as of its binding affinity to rat brain synaptosomes (IC50 = 50 pM), compared with KTX. Specific antibodies raised against KTX-(1-37) were not able to recognize KTX2. Using degenerate primers, a 370-base pair cDNA encoding the KTX2 precursor was amplified by polymerase chain reaction from a cDNA library of A. australis venom glands. It encoded a presumed signal peptide of 22 residues followed by the sequence of the mature peptide.

A fibrinogen-clotting serine proteinase from Cerastes cerastes (horned viper) venom with arginine-esterase and amidase activities. Purification, characterization and kinetic parameter determination.

An enzyme displaying proteolytic activity toward the natural substrate casein as well as clotting activity on fibrinogen was purified to homogeneity from Cerastes cerastes (horned viper) venom and characterized. The enzyme is constituted of two identical subunits of mol. wt 48,500 as determined by SDS-polyacrylamide gel electrophoresis, and has an isoelectric point of 3.75. N-terminal sequencing up to the 33rd residue evidenced a high homology with other snake venom proteinases. The proteinase is of serine-type as indicated by high sensitivity to DFP and shows both arginine-ester hydrolase and amidase activities on synthetic substrates. Both specific activities were 30-fold higher than the respective activities found in the crude venom. The Km value determined for arginine-containing substrate BAEE was 3.0 x 10(-4) M and the Km for chromogenic substrate CBS 34-47 0.65 x 10(-4) M. The Vm/Km ratio, however, was two-fold higher for BAEE than for CBS 34-47; the arginine-esterase activity of this enzyme is thus slightly higher than its amidase activity.