Production of a monoclonal antibody against hemorrhagic activity of Crotalus atrox (western diamondback rattlesnake) venom

J. C. Perez, V. E. Garcia and S. Y. Huang. Production of a monoclonal antibody against hemorrhagic activity of Crotalus atrox (western diamondback rattlesnake) venom. Toxicon22, 967 – 973, 1984. — Crotalid venoms have cytotoxic properties which could be useful in medical research. Crotalus atrox venom-hyperimmunized mouse spleen cells were fused with SP2/0 myeloma cells. Forty-one wells containing the hybridoma cells were positive for C. atrox venom, as determined by the enzyme linked immunosorbent assay (ELISA). Cell line 1-e12 was cloned and used to produce ascites tumors in BALB/c mice. The monoclonal antibody produced by cloned and subcultured 1-e12 cells reacted with both C. atrox venom and six other venoms in the ELISA and neutralized the hemorrhagic activity of crude C. atrox venom. A series of monoclonal antibodies could be used in studying the nature of snake venoms.     

In vivo diminution by chelators of snake venom-provoked hemorrhage and in vitro inhibition of proteolytic activity

Topical application of alcohol-soluble metal-complexing agents, particularly α-mercapto-β-(2-furyl)acrylic acid (MFA)1, diminished the severity of hemorrhages caused by s.c. injection to mice of thirteen hemorrhagic pit viper venoms and two viper venoms. Metal dependency of the alkaline protease activity ranged from 0 to 100% for these venoms, plus one non-hemorrhagic pit viper venom; MFA completely inhibited this activity in six pit viper and two viper venoms. The presence of zinc-dependent proteases in venoms of Crotalus atrox and Bitis arietans was indicated. Less compelling evidence suggested the presence of zinc metalloproteases in at least 11 of 14 other venoms. The potencies of hemorrhagic and alkaline protease activities were not related in the venoms and we did not investigate if metalloproteases were the targets of chelator antagonism of hemorrhage development. Topically active chelators may be useful as adjunctive therapy in some snake envenomations.     

The Direct Actinc α-Fibrin(Ogen)Olytic Enzymes from Snake Venoms

Abstract

A variety of α-fibrin(ogen)olytic enzymes have been found in snake venoms. More than 15 α-fibrin(ogen)ases have been isolated and characterized. Most work has been done with the venom of snakes belonging to a few species from the Agkistrodon, Crotalus, Trimeresurus, Bothrops, and Vipera. Only one α-fibrin(ogen)olytic enzyme is characterized from Elapidae snake venoms. The enzymatic properties of these proteinases are described in relation to action on fibrinogen, fibrin, and casein. The fibrinolytic enzymes act directly on fibrin and do not activate plasminogen. The proteolytic activity of these metalloproteinases is inhibited by EDTA. Most thoroughly investigated snake venom fibrinolytic enzymes are fibrolase from Agkistrodon contortrix contortrix venom, atroxase from Crotalus atrox venom and cerastase from Cerastes cerastes venom. Antibodies to fibrolase were prepared and their cross-reactions with other fibrinolytic components from several snake venoms have been detected. These antibodies were successfully used for purification of fibrolase from crude southern copperhead venom. Fibrolase and atroxase have no hemorrhagic activity, and they effectively solubilize artificial thrombi. Research in this area has a chance to provide new therapeutic agents for dissolving thrombi.     

Cloning of cDNAs encoding C-type lectins from Elapidae snakes Bungarus fasciatus and Bungarus multicinctus

A number of C-type lectins with various biological activities have been purified and characterized from Viperidae snake venoms. In contrast, only a few reports could be found in literature concerning the C-type lectins in Elapidae snake venoms. Based on the published cDNA sequences of C-type lectins from Viperidae snake venoms, oligonucleotide primers were designed and used to screen the cDNA libraries made from the venom glands of Bungarus fasciatus and Bungarus multicinctus. This allowed the cloning of three full length cDNAs encoding C-type lectins. The encoded proteins, named BFL-1, BFL-2 and BML, exhibit high degrees of sequence identities with Viperidae snake venom saccharide-binding lectins (around 60% with Trimeresurus stejnegeri venom lectin, Crotalus atrox venom lectin and Agkistrodon piscivorus venom lectin). They show much less identities with other venom C-type lectin-like proteins (around 30% with the platelet glycoprotein Ib-binding protein from Agkistrodon blomhoffi venom and the factor IX/X-binding protein from Trimeresurus flavoviridis venom). The cDNAs revealed that the precursors contain potential signal peptides characterized by a hydrophobic core. To our knowledge, these are the first cDNA cloning of group VII C-type lectins (Drickamer K. 1993. Prog. Nucleic Acid Res. Mol. Biol. 45, 207–232) from Elapidae snake venom glands.     

Toxoids and Antivenoms of Venomous Snakes in Taiwan

Abstract

There are in Taiwan six major venomous snakes which can inflict severe bite on human victims. They are three hemorrhagic species i.e. the Taiwan habu (Trimeresurus mucrosquamatus), the green tree viper (Trimeresurus stejnegeri), and the hundred-pace snake (Deinagkistrodon acutus); two neurotoxic species, i.e. the cobra (Naja naja atra) and the krait (Bungarus multicinctus); and the Russell's viper (Daboia r. formosensis) whose venom is both coagulopathy and neurotoxic. Our aim has been the production of highly potent antivenoms for snake-bite treatment in this country. Among individual antivenoms for Taiwan venomous snakes, only those from the pitvipers show partial cross-neutralizing capacity with venoms of other pit vipers.

As all snake venoms are quite lethal to animals, it is important to tame or detoxify the crude venom before using it on the animal to obtain antivenoms. We have demonstrated that glutaraldehyde can be used successfully not only in the detoxification of snake venoms but also improving their immunogenecity. Protocols for toxoid preparation from the crude venoms in the process of manufacturing highly potent antisera have been improved in our institute. Two bivalent equine antivenoms specific for either the combined glutaraldehyde-treated venoms of N. n. atra and B. multicinctus or those of T. mucrosquamatus and T. stejnegeri were successfully produced and proven to be effective and useful. A tetravalent equine antivenom has been prepared likewise against the four major viperid venoms in Taiwan. Recently, we also developed a process to prepare an efficient hexavalent antivenom against all the six major venomous snakes.     

Immunological studies on polyvalent and monovalent snake antivenins.

Polyvalent antivenin prepared against the Egyptian snake venoms, Naja haje, N. nigricollis, Cerastes vipera, C. cerastes, Echis carinatus and Walterinnesia aegyptia; against the African snake venoms, Dendroaspis polylepis, Bitis gabonica and B. arietans as well as the Asian snake venoms, Trimeresurus flavoviridis. was tested by neutralization and immunodiffusion experiments. The antivenin was of high potency against Egyptian venoms especially C. vipera and C. cerastes, followed by Walterinnesia aegyptia, N. haje, Echis carinatus, N. nigricollis, T. flavoviridis, B. arietans, and B. gabonica.Comparison of the polyvalent antivenin and monovalent B. gabonica antivenin revealed that while the polyvalent serum was of better effectiveness against the Egyptian viper venoms, especially Cerastes venoms, more protection was provided by the monovalent serum against B. gabonica venom.     

Rock squirrel (Spermophilus variegatus) blood sera affects proteolytic and hemolytic activities of rattlesnake venoms

Rock squirrels (Spermophilus variegatus) from two sites in south central New Mexico, where prairie (Crotalus viridis viridis) and western diamondback (Crotalus atrox) rattlesnakes are common predators, were assayed for inhibition of rattlesnake venom digestive and hemostatic activities. At statistically significant levels rock squirrel blood sera reduced the metalloprotease and hemolytic activity of venoms from C. v. viridis and C. atrox more than venom from an allopatric snake species, the northern Pacific rattlesnake (Crotalus oreganus). In contrast, general proteolytic activity of venom from C. oreganus was inhibited more by S. variegatus serum defenses than activity of venom from sympatric snakes. For all three venoms, incubation with squirrel sera increased the level of fibrinolysis over venom-only treatments. These results suggest that rock squirrels (S. variegatus) can defend against metalloproteases and other proteases after envenomation from at least two of five rattlesnake predators they might encounter. However, there were statistically significant differences between general proteolytic activity and fibrinolytic activity of C. v. viridis and C. atrox venom, suggesting that rock squirrels might be differentially vulnerable to these two predators. The hypothesis that prey resistance influences snake venom evolution in a predator-prey arms race is given further support by the previously cryptic variation in venoms detected when assayed against prey defenses.     

Preclinical assessment of the efficacy of a new antivenom (EchiTAb-Plus-ICP®) for the treatment of viper envenoming in sub-Saharan Africa

A preclinical assessment was performed on the neutralizing efficacy of a whole IgG polyspecific antivenom (EchiTAb-Plus-ICP^®), designed for the treatment of snakebite envenomings in Nigeria. It was generated by immunizing horses with the venoms of Echis ocellatus, Bitis arietans and Naja nigricollis, the most medically important species in Nigeria. Antivenom was tested against the venoms of E. ocellatus, Echis leucogaster, Echis pyramidum leakeyi, B. arietans, Bitis gabonica, Bitis rhinoceros and Bitis nasicornis. The neutralization of the venom toxins responsible for the lethal, hemorrhagic, coagulant and local necrotizing activities was assessed, since these are the most significant effects that characterize envenoming by these species. Echis sp venoms exerted lethal, hemorrhagic, coagulant and necrotizing effects, whereas the Bitis sp venoms tested induced lethality, hemorrhage and necrosis, but were devoid of coagulant activity. The antivenom was effective in the neutralization of all effects tested in all venoms. Highest neutralization was achieved against the venoms of E. ocellatus and B. arietans, and the lowest neutralizing potency was against the venom of B. nasicornis, a species that has a low clinical relevance. It is concluded that EchiTAb-Plus-ICP^®, whilst specifically designed for Nigeria, has a good preclinical neutralizing profile against homologous and heterologous viperid venoms from other sub-Saharan African locations. It therefore constitutes a promising therapeutic option for the treatment of snakebite envenoming in this region.     

Pharmacological activity of Costus spicatus in experimental Bothrops atrox envenomation

Context: Medicinal plants encompass a rich source of active compounds that can neutralize snake venoms or toxins. Costus spicatus (Jacq.) Sw. (Costaceae) is used by the Amazonian population to treat inflammation, pain and other pathological manifestations.

Objective: To evaluate the influence of C. spicatus aqueous extract on edema, peritonitis, nociception, coagulation, haemorrhage and indirect haemolytic activity induced by Bothrops atrox venom (BAV).

Materials and methods: Dried and pulverized leaves were extracted with distilled water. Envenoming was induced by administration of B. atrox snake venom in Swiss Webster mice. The experimental groups consisted of BAV (at the minimum dose to induce measurable biological responses) and C. spicatus extract (CSE, 1.25, 2.5, 5.0, 7.5 and 10?mg/kg/25?μl phosphate-buffered saline) administered individually and in combination (BAVCSE). PBS was used as a control. In vitro assays were also conducted in order to evaluate phospholipase A2 coagulant activities (indirect haemolytic method).

Results: CSE significantly reduced the venom-induced edema and nociception at all concentrations tested and inhibited migration of inflammatory cells at the three least concentrations (5.0, 7.5 and 10?mg/kg/25?μl PBS). CSE was not effective in inhibiting coagulant, haemorrhagic and indirect haemolytic activities of the venom.

Discussion and conclusion: The data suggest that CSE could exhibit a central mechanism for pain inhibition, and may also inhibit prostaglandin synthesis. These findings corroborate the traditional administration of C. spicatus decoction to treat inflammatory disorders, including those caused by B. atrox envenomation.     

Resistance of warm-blooded animals to snake venoms

Crotalus atrox venom resistance in 40 species of animal sera was studied by means of antihemorrhagic assay, ring precipitation test and serum protection test. The antihemorrhagic titers ranged from 0 to 256 with 16 animal sera having a titer greater than two. Five animal sera precipitated with C. atrox venom and the titers ranged from 0 to 64. Procyon lotor (racoon), Liomys irroratus (Mexican spiny pocket mouse) and two species of Peromyscus sp. (white footed deer mouse) were the only unimmunized animals with both a positive antihemorrhagic and ring precipitation titer. One of the resistant sera, Neotoma micropus (gray woodrat), was further studied to determine effects of five other snake venoms. Neotoma micropus serum neutralized hemorrhagic factors in all hemorrhagic venoms tested but would not neutralize the lethal factors of elapid venoms in the serum protection test. It appears that both reptiles and warm-blooded animals have antihemorrhagic factors in their sera.     

Neutralization of proteases from Bothrops snake venoms by the aqueous extract from Casearia sylvestris (Flacourtiaceae)

Aqueous extract from Casearia sylvestris leaves, a typical plant from Brazilian open pastures, was able to neutralize the hemorrhagic activity caused by Bothrops asper, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi and Bothrops pirajai venoms. It also neutralized two hemorrhagic metalloproteinases from Bothrops asper venom. Proteolytic activity on casein induced by bothropic venoms and by isolated proteases, including Bn2 metalloproteinase from B. neuwiedi venom, was also inhibited by the C. sylvestris extract in different levels. The α-fibrinogen chain was partially protected against degradation caused by B. jararacussu venom, when this venom was incubated with C. sylvestris extract. We also observed that this extract partially increased the time of plasma coagulation caused by B. jararacussu, B. moojeni and B. neuwiedi venoms. C. sylvestris extract did not induce proteolysis in any substrate assayed.     

Immunological studies on Naja nigricollis antivenin

Monovalent Naja nigricollis antivenin was prepared in horses using venom adsorbed to bentonite (first three injections) followed by increasing doses (up to 50 mg) of unmodified venom. The serum (1 ml) neutralized 51 ld₅₀'s of its homologous venom. Good neutralizing capacity (30–50 ld₅₀'s per ml) was also shown against venoms of N. haje, N. nivea, N. naja, Walterinnesia aegyptia, Cerastes cerastes and Cerastes vipera as well as Echis carinatus venom. For all these venoms, identity patterns, complete or partial, were established using Ouchterlony's technique. Moderate neutralization (22 ld₅₀'s per ml) was shown against Echis coloratus venom which gave a nonidentity pattern. Low neutralization (10 ld₅₀'s per ml) was observed with venoms of Bitis arietans, B. gabonica and Trimeresurus flavoviridis; all these venoms showing very weak or no precipitation lines with Naja nigricollis antivenin.     

Hemostatic properties of Venezuelan Bothrops snake venoms with special reference to Bothrops isabelae venom

In Venezuela, Bothrops snakes are responsible for more than 80% of all recorded snakebites. This study focuses on the biological and hemostatic characteristics of Bothrops isabelae venom along with its comparative characteristics with two other closely related Bothrops venoms, Bothrops atrox and Bothrops colombiensis. Electrophoretic profiles of crude B. isabelae venom showed protein bands between 14 and 100 kDa with the majority in the range of 14-31 kDa. The molecular exclusion chromatographic profile of this venom contains five fractions (F1-F5). Amidolytic activity evaluation evidenced strong thrombin-like followed by kallikrein-like activities in crude venom and in fractions F1 and F2. The fibrinogenolytic activity of B. isabelae venom at a ratio of 100:1 (fibrinogen/venom) induced a degradation of Aα and Bβ chains at 15 min and 2 h, respectively. At a ratio of 100:10, a total degradation of Aα and Bβ chains at 5 min and of γ chains at 24 h was apparent. This current study evidences one of rarely reported for Bothrops venoms, which resembles the physiologic effect of plasmin. B. isabelae venom as well as F2 and F3 fractions, contain fibrinolytic activity on fibrin plate of 36, 23.5 and 9.45 mm²/μg, respectively using 25 μg of protein. Crude venom and F1 fraction showed gelatinolytic activity. Comparative analysis amongst Venezuelan bothropoid venoms, evidenced that the LD₅₀ of B. isabelae (5.9 mg/kg) was similar to B. atrox-Puerto Ayacucho 1 (6.1 mg/kg) and B. colombiensis-Caucagua (5.8 mg/kg). B. isabelae venom showed minor hemorrhagic activity, whereas B. atrox-Parguasa (Bolivar state) was the most hemorrhagic. In this study, a relative high thrombin-like activity was observed in B. colombiensis venoms (502-568 mUA/min/mg), and a relative high factor Xa-like activity was found in B. atrox venoms (126-294 mUA/min/mg). Fibrinolytic activity evaluated with 10 μg protein, showed that B. isabelae venom contained higher specific activity (50 mm²/μg) than B. colombiensis and B. atrox venoms, which should encourage the isolation of these fibrinolytic molecules to improve the quality of immunotherapy.     

Comparative study on hemorrhagic and proteolytic activities of snake venoms

The hemorrhagic and proteolytic titers of nine snake venoms were measured. Suitable venom dilutions were selected to study the antihemorrhagic and antiproteolytic activities of three warm-blooded animal sera — gray woodrat (Neotoma micropus), hispidus cottonrat (Sigmodon hispidus) and Virginia opossum (Didelphis virginiana). It was demonstrated that all hemorrhagic venoms are proteolytic. The results show that the hemorrhagic activity of the venoms is readily neutralized by the sera; however, the proteolytic activity is not neutralized. We suggest that the mechanisms involved in neutralizing the two activities are not closely related.     

Cytotoxic germacranolide sesquiterpenes from the bark of <Emphasis Type="Italic">Magnolia kobus</Emphasis>

A new (3) and three known germacrane-type sesquiterpenoids were isolated from the chloroform-soluble fraction of the methanolic extract of the bark of Magnolia kobus (Magnoliaceae) through repeated silica gel and Sephadex LH-20 column chromatography. Their chemical structures were elucidated as costunolide (1), parthenolide (2), isobisparthenolidine (3), and bisparthenolidine (4) by spectroscopic analysis. Compounds 1–4 exhibited cytotoxicity against human A549, SK-OV-3, SK-MEL-2, and HCT15 tumor cells.     

Analysis of camelid antibodies for antivenom development: Neutralisation of venom-induced pathology

Camelid IgG has been reported to be less immunogenic, less able to activate the complement cascade and more thermostable than IgG from other mammals, and has the ability to bind antigens that are unreactive with other mammalian IgGs. We are investigating whether these attributes of camelid IgG translate into antivenom with immunological and venom-neutralising efficacy advantages over conventional equine and ovine antivenoms. The objective of this study was to determine the preclinical venom-neutralising effectiveness of IgG from camels immunised with venoms, individually or in combination, of the saw-scaled viper, Echis ocellatus, the puff adder, Bitis arietans and the spitting cobra, Naja nigricollis - the most medically-important snake species in West Africa. Neutralisation of the pathological effects of venoms from E. ocellatus, B. arietans and N. nigricollis by IgG from the venom-immunised camels, or commercial antivenom, was compared using assays of venom lethality (ED₅₀), haemorrhage (MHD) and coagulopathy (MCD). The E. ocellatus venom ED₅₀, MHD and MCD results of the E. ocellatus monospecific camel IgG antivenom were broadly equivalent to comparable ovine (EchiTAbG^®, MicroPharm Ltd, Wales) and equine (SAIMR Echis, South African Vaccine Producer, South Africa) antivenoms, although the equine antivenom required half the amount of IgG. The B. arietans monospecific camel IgG neutralised the lethal effects of B. arietans venom at one fourth the concentration of the SAIMR polyspecific antivenom (a monospecific B. arietans antivenom is not available). The N. nigricollis camel IgG antivenom was ineffective (at the maximum permitted dose, 100 μl) against the lethal effects of N. nigricollis venom. All the equine polyspecific antivenoms required more than 100 μl to be effective against this venom. The polyspecific camel IgG antivenom, prepared from five camels, was effective against the venom-induced effects of E. ocellatus but not against that of B. arietans and N. nigricollis venoms. No direct correlation was evident between either camel IgG relative avidity or titre and the effectiveness of venom neutralisation in preclinical assays.     

Determination of sphingomyelinase-D activity of Loxosceles venoms in sphingomyelin/cholesterol liposomes containing horseradish peroxidase

Based on degradation of sphingomyelin/cholesterol liposomes containing entrapped horseradish peroxidase, we evaluated the Sphingomyelinase-D (SMase-D) activity of scorpion, spider and snake venoms by monitoring spectrophotometrically the product of oxidation of HRP released. The results indicate that Loxosceles crude venoms (Loxosceles intermedia, Loxosceles laeta, Loxosceles gaucho and Loxosceles similis) displayed SMase-D activity in a concentration-dependent manner. Furthermore, this activity was blocked by the anti-loxoscelic antivenom. However, Tityus serrulatus scorpion venom, Phoneutria nigriventer spider venom and Bothrops jararaca, Crotalus durissus, Lachesis muta and Micrurus frontalis snake venoms did not show measurable SMase-D activity.     

Evaluation of the Lethal Potency of Scorpion and Snake Venoms and Comparison between Intraperitoneal and Intravenous Injection Routes

Scorpion stings and snake bites are major health hazards that lead to suffering of victims and high mortality. Thousands of injuries associated with such stings and bites of venomous animals occur every year worldwide. In North Africa, more than 100,000 scorpion stings and snake bites are reported annually. An appropriate determination of the 50% lethal doses (LD₅₀) of scorpion and snake venoms appears to be an important step to assess (and compare) venom toxic activity. Such LD₅₀ values are also commonly used to evaluate the neutralizing capacity of specific anti-venom batches. In the present work, we determined experimentally the LD₅₀ values of reference scorpion and snake venoms in Swiss mice, and evaluated the influence of two main venom injection routes (i.e., intraperitoneal (IP) versus intravenous (IV)). The analysis of experimental LD₅₀ values obtained with three collected scorpion venoms indicates that Androctonus mauretanicus (Am) is intrinsically more toxic than Androctonus australis hector (Aah) species, whereas the latter is more toxic than Buthus occitanus (Bo). Similar analysis of three representative snake venoms of the Viperidae family shows that Cerastes cerastes (Cc) is more toxic than either Bitis arietans (Ba) or Macrovipera lebetina (Ml) species. Interestingly, the venom of Elapidae cobra snake Naja haje (Nh) is far more toxic than viper venoms Cc, Ml and Ba, in agreement with the known severity of cobra-related envenomation. Also, our data showed that viper venoms are about three-times less toxic when injected IP as compared to IV, distinct from cobra venom Nh which exhibited a similar toxicity when injected IP or IV. Overall, this study clearly highlights the usefulness of procedure standardization, especially regarding the administration route, for evaluating the relative toxicity of individual animal venoms. It also evidenced a marked difference in lethal activity between venoms of cobra and vipers, which, apart from the nature of toxins, might be attributed to the rich composition of high molecular weight enzymes in the case of viper venoms.     

Comparative study of anticoagulant and procoagulant properties of 28 snake venoms from families Elapidae, Viperidae, and purified Russell’s viper venom-factor X activator (RVV-X)

Snake venoms consist of numerous molecules with diverse biological functions used for capturing prey. Each component of venom has a specific target, and alters the biological function of its target. Once these molecules are identified, characterized, and cloned; they could have medical applications. The activated clotting time (ACT) and clot rate were used for screening procoagulant and anticoagulant properties of 28 snake venoms. Crude venoms from Daboia russellii siamensis, Bothrops asper, Bothrops moojeni, and one Crotalus oreganus helleri from Wrightwood, CA, had procoagulant activity. These venoms induced a significant shortening of the ACT and showed a significant increase in the clot rate when compared to the negative control. Factor X activator activity was also measured in 28 venoms, and D. r. siamensis venom was 5-6 times higher than those of B. asper, B. moojeni, and C. o. helleri from Wrightwood County. Russell’s viper venom-factor X activator (RVV-X) was purified from D. r. siamensis venom, and then procoagulant activity was evaluated by the ACT and clot rate. Other venoms, Crotalus atrox and two Naja pallida, had anticoagulant activity. A significant increase in the ACT and a significant decrease in the clot rate were observed after the addition of these venoms; therefore, the venoms were considered to have anticoagulant activity. Venoms from the same species did not always have the same ACT and clot rate profiles, but the profiles were an excellent way to identify procoagulant and anticoagulant activities in snake venoms.