Snake venom neutralization research -
An equal volume of saline was injected into the contralateral paw control. The volume increase edema of the paws was measured plethysmographically at 1 and 4 h or 2 and 4 h after BjV and BaV injection, respectively, according to the method of Van Arman et al.
The difference between the values obtained for both paws expressed as percent increase in paw volume was used as a measure of edema. Two kinds of experiments were performed: 1 injection of antivenom at different times before or after venom administration and 2 in vitro incubation of venom and antivenom before injection.
Assays with independent injection of venom and antivenom. Antivenoms 0. Hyperalgesia and edema measurements were carried out as described above.
Assays with preincubation of venom and antivenom. After incubation at 37ºC for 30 min, µl of each mixture containing the appropriate amounts of venoms 5 µg BjV or 15 µg BaV were injected by the intraplantar route, and hyperalgesia and edema were determined as described above.
Cross-neutralization studies. Cross-neutralization experiments were performed in order to determine if AVIB and AVCP antivenoms are able to neutralize the hyperalgesic and edematogenic effects induced by B.
jararaca venoms, respectively. The neutralization studies were carried out as described above for assays with independent injection of venom and antivenom or assays with preincubation of venom and antivenom.
The antivenoms did not alter, per se , the pain threshold of the animals, nor did they induce an edematogenic response data not shown. Neutralization of hyperalgesia and edema induced by Bothrops jararaca venom.
The peak of the hyperalgesic and edematogenic responses occurred 1 h after venom injection. After this time both phenomena started to decrease Figure 1 and completely disappeared at 24 h data not shown.
When venom and antivenom were injected separately, neutralization of BjV-induced hyperalgesia was observed only when AVIB was administered 15 min before venom injection Figure 1 A. Administration of AVIB 15 min or immediately before BjV induced a marked reduction of edema, although this effect was not completely neutralized Figure 1 B.
In experiments with incubation of venom and antivenom prior to injection, antivenom was effective in neutralizing the hyperalgesia and edema induced by BjV Figure 2.
Figure 1. Neutralization of the hyperalgesia and edema activity of Bothrops jararaca venom by bothropic antivenom in assays in which venom and antivenom were injected separately.
Antivenom AV, 0. The decrease in threshold response A and increase in paw volume B were determined in rat hind paws before and 1 and 4 h after injection of the venom. Sensitivity to pain was measured as the threshold response to pressure and is reported in grams.
Edema is reported as percent increase in relation to the initial volume of the paw. Each point indicates the mean ± SEM for 6 animals. Note that the abscissa scale is not linear. Figure 2.
Neutralization of the hyperalgesia and edema induced by Bothrops jararaca venom by bothropic antivenom in assays in which venom and antivenom were incubated together before injection into the rat paw.
Then, µl of the mixtures containing 5 µg of venom were injected by the intraplantar route. Neutralization of hyperalgesia and edema induced by Bothrops asper venom.
AVCP administered 15 min or immediately before or 15 min after B. asper venom injection did not modify the hyperalgesia or edema induced by the venom Figure 3.
Figure 3. Neutralization of the hyperalgesia and edema activity of Bothrops asper venom by polyvalent antivenom in assays in which venom and antivenom were injected separately. The decrease in threshold response A and increase in paw volume B were determined in rat hind paws before and 2 and 4 h after injection of the venom.
Figure 4. Neutralization of the hyperalgesia and edema induced by Bothrops asper venom by polyvalent antivenom in assays in which venom and antivenom were incubated together before injection into rats. A fixed amount of venom V was incubated with various dilutions of antivenom AV or with saline S, control for 30 min at 37ºC.
Then, µl of the mixtures containing 15 µg of venom were injected by the intraplantar route. AVIB or AVCP injected 15 min before BaV and BjV, respectively, did not modify the hyperalgesia or edema induced by both venoms data not shown.
In experiments involving incubation of venom and antivenom prior to injection, AVIB partially neutralized the hyperalgesia and edema induced by BaV Figure 5 A,B. On the other hand, AVCP did not modify either effect induced by B.
jararaca venom. One hour after intraplantar injection of neutralized venom, the pain threshold was 47 ± 1. Figure 5. Neutralizing activity of bothropic antivenom AVIB against Bothrops asper venom in assays involving incubation of the venom and the antivenom together before injection.
A fixed amount of venom was incubated with dilutions of antivenom or with saline S, control for 30 min at 37ºC. Then, µl of the mixture containing 15 µg of B. asper venom BaV was injected by the intraplantar route.
The decrease in threshold response A and increase in paw volume B were measured in rat hind paws before and 2 and 4 h after the injection of BaV. Bothropic and polyvalent antivenoms constitute the major therapeutic resource in snakebite envenomations in Brazil and Central America, respectively 3, Clinical investigations have demonstrated the efficacy of these products on the neutralization of life-threatening systemic effects associated with these envenomations 3,5, However, experimental and clinical evidence indicates that local effects in Bothrops sp envenomations are poorly neutralized by these and other antivenoms 3,8,10, The present results further demonstrate the complete ineffectiveness of these antivenoms in neutralizing the local edema and hyperalgesia induced by B.
jararaca venoms when antivenoms are administered to rats after envenomation. The ability of antivenoms to neutralize hyperalgesia has not been previously addressed in experimental studies, despite the fact that pain is a common manifestation of Bothrops sp envenomations 3,13, We selected venom doses which caused an increase in the sensitivity to pain hyperalgesia and local edema, without inducing macroscopically evident hemorrhage or tissue damage.
Such doses have been validated and used in previous pharmacological studies 15, Bothropic antivenom produced against B. jararaca at Instituto Butantan was ineffective in neutralizing edema or hyperalgesia when administered after envenomation.
Only when antivenom was injected before venom was neutralization achieved. In the case of B. asper venom, polyvalent antivenom produced in Costa Rica was ineffective in neutralizing edema or hyperalgesia when administered either before or after envenomation, in agreement with prior observations in mice 8, Moreover, Rucavado and Lomonte 24 demonstrated that the presence of antibodies in the circulation before injection of B.
asper venom in mice reduces, but does not abolish, local tissue damage. Such poor neutralizing efficacy cannot be explained on the basis of lack of neutralizing antibodies in these antivenoms, since both hyperalgesic and edematogenic responses were abolished if venom and antivenoms were incubated prior to injection.
A similar situation occurs regarding the neutralization of hemorrhage and myonecrosis induced by B. asper venom It has been suggested that the observed poor neutralization is based on the fact that hyperalgesia and edema develop at an extremely rapid rate after venom injection, associated with the release of endogenous mediators involved in these pharmacological effects 15,16,25, Thus, by the time antivenom antibodies reach the affected tissues, it is likely that the cascade of inflammatory events associated with hyperalgesia and edema has already started.
Moreover, since antivenoms are administered intravenously, the amount of antibodies diffusing to the affected tissues is probably insufficient and delayed.
Thus, an evident mismatch occurs between the pharmacokinetics and pharmacodynamics of venoms and antivenoms in this experimental model and, very likely, in the clinical situation as well. The present study also addressed the issue of cross-neutralization of these effects by antivenoms.
Previous investigations have documented cross-neutralization between bothropic and polyvalent antivenoms when assayed against a variety of venoms from Central and South America The present results demonstrate that bothropic antivenom partially neutralizes B.
asper venom, whereas polyvalent antivenom is largely ineffective in the neutralization of edema and hyperalgesia induced by B. It is likely that components immunologically related to hyperalgesic and edematogenic toxins in B. fasciatus victims, B.
multicinctus plus N. atra antivenin to treat king cobra patients [ 39 ]. Present investigation evaluated immunoreactivity of commercial B. multicinctus antivenin and prepared antisera with eight other dangerous Chinese terrestrial snake venoms. The immunoreactivity between venoms and clinic B.
Surprisingly, current used B. multicinctus antivenin showed very strong immunoreactivity with high MW of B. multicinctus venom under non-reducing condition but very weak immunoreactivity with these fractions under reducing conditions.
These results consistent with the conclusion that B. multicinctus antivenin well recognize crude venom but poorly recognize α-BGT, β-BGT and γ-BGT as depicted in Fig 1D.
Previously, Gao et al evaluated the immunoreactivity between four venoms and commercial antivenoms produced by Shanghai Serum Bio-technology Company [ 50 ]. Comparing their western-blotting results of B. multicinctus crude venom with present results, a big difference existed in the immunoreactivity of B.
multicinctus antivenin recognize B. multicinctus high MW fractions. multicinctus antivenin used by Gao et al reacted strongly with β-BGTs but not high MW fractions under non-reducing conditions, which was contrary with our present results. The most possible reason might be caused by the B.
multicinctus antivenin used by Gao et al was further purified by a HiTrap Protein G column or caused by the used crude venoms in producing of different batches of the antivenins. multicinctus antivenin could recognize high MW of B.
multicinctus antivenin showed very weak reaction with high MW of N. atra venom under non-reducing conditions but weak signals were seen under reducing conditions. However, B.
multicinctus antivenin showed strong immunoreactivity with high MW of O. hannah venom both under non-reducing and reducing conditions. Since no anti-king cobra venom antiserum available in China, using single N.
multicinctus antivenin or combining both antivenins to treat O. hannah envenomation was recommended by China Expert Consensus on the management of snake-bites and has practiced in clinic for many decades in China [ 39 ].
Thus, present results strongly indicated that high MW of O. hannah venom might contribute in the lethal activity of the venom. Furthermore, our previously reported L-amino acid oxidase [ 51 ] and blood coagulation factor X activator [ 52 ] represented the high MW of O.
hannah venom components that could be reacted with commercial B. multicinctus antivenin were determined. In accordance with previous results that β-BGTs are the most lethal and abundant components contained in the B.
The determined ED 50 values of prepared β-BGT antiserum and commercial B. multicinctus venom demonstrated that prepared β-BGT antiserum showed a better animal protection efficiency over commercial B. Our present results suggested that it might be feasible for commercial B.
multicinctus antivenin producer to add a single gel filtration step and us both β-BGT and α-BGT fractions to produce the B.
multicinctus antivenin to largely avoid unnecessary high MW reaction antibodies Figs 4B and S1. The LD 50 values of purified α-, β- and γ-BGTs via i. v injection routines were systematically determined in Kunming mice.
Administration routines did have effects on the lethal activity of different bungarotoxins were first revealed. Importantly, the recommended using B. multicinctus antivenin to treat king cobra victims was supported but using B. fasciatus envenomation in clinic practice in China was not supported by present evidences.
The purification process of β-bungarotoxin A-D. The purification process of α-bungarotoxin A, E, F. The purification process of γ-bungarotoxin A, E, G.
The buffer used in the experiments were PBS A , sodium acetate-acetate buffer solution 0. The bungarotoxins were digested by trypsin. SDS-PAGE under non-reducing conditions, 1. Western blot revealed by prepared anti-β-BGT antiserum under non-reducing conditions, 1.
M: Molecular marker. SDS-PAGE of O. Western-blot profile of commercial B. hannah venom B. We would like to thank Dr. Lin Zeng from the Public Technology Service Center, Kunming Institute of Zoology, Chinese Academy of Sciences for her help in mass spectrometry determination and analysis.
Article Authors Metrics Comments Media Coverage Reader Comments Figures. Abstract Bungarus multicinctus is the most venomous snake distributed in China and neighboring countries of Myanmar, Laos, north Vietnam and Thailand.
Author summary Snakebite envenoming is an important public health problem around the world. Calvete, Instituto de Biomedicina de Valencia, SPAIN Received: June 1, ; Accepted: October 12, ; Published: November 30, Copyright: © Lin et al.
Introduction The study of toxins and venomous animals has a long history, it affects human life and health [ 1 ]. Materials and methods Ethics statement All experiments on animals meet the requirements of National Institutes of Health guide for the care and use of Laboratory animals NIH Publications No.
Venoms and antivenin Pooled B. Purification of α-, β- and γ-BGTs Lyophilized B. Construction and expression of maltose binding protein fusion γ-BGT The amount of γ-BGT contained in the B. Mass spectrometry The mass spectrometry analysis was performed according to the reported methods [ 18 — 19 ].
SDS-PAGE and western blotting SDS-PAGE was performed according to the method of Laemmli [ 20 ]. Rabbit specific antiserum preparation The purified β-BGT, α-BGT and recombinant MBP-γ-BGT protein were separately used to immune rabbits according to common methods.
Immunoreactivity study of commercial antivenin and prepared antisera The immunoreactivity of antisera was defined as half of the maximum dilution factor at which the immunological binding can be observed [ 23 ].
Results Purification and characterization of α-, β- and γ-BGTs By a combination of gel filtration, ion exchange and HPLC procedures, high purity α-, β- and γ-BGTs were isolated from B. Download: PPT. Fig 1. Median lethal dose LD 50 determination The LD 50 values of α-BGT, β-BGT and γ-BGT were determined to be 0.
Table 1. Summary of the available LD 50 values of bungarotoxins via different administration routines. Immunoreactivity of bungarotoxin-specific antisera Both prepared anti-β-BGT and anti-α-BGT antisera could well recognize crude venom and corresponding purified β-BGT or α-BGT, respectively.
Fig 3. The immunoreactivity of antisera comparison by indirect ELISA. Immunoreactivity of the commercial B. multicinctus antivenin and prepared antisera To investigate whether commercial B. Fig 4. multicinctus antivenin and the prepared antiserum against top Chinese dangerous terrestrial snake venoms.
ED 50 values of the commercial used B. multicinctus antivenin and prepared antisera According to the determined LD 50 values of the B. Table 2. ED 50 determination of B.
multicinctus antivenin and the prepared bungarotoxin antiserum against B. multicinctus venom and purified bungarotoxins. Table 3. The protective effects of antiserum against different snake venoms. Conclusions In accordance with previous results that β-BGTs are the most lethal and abundant components contained in the B.
Supporting information. S1 Fig. Purification of bungarotoxins from B. s TIF. S2 Fig. S3 Fig. SDS-PAGE and Western blot of each fractions in S1B Fig. S4 Fig. Expression and purification of MBP-γ-BGT recombinant protein. S5 Fig. Acknowledgments We would like to thank Dr.
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htm accessed February 13, Explore More. Bacteria Can Live in Snake and Spider Venoms. May 23, Newly published research shows that, contrary to what is commonly believed, the venom of snakes and spiders is actually populated with microbes, including bacteria that could cause infection in Snake Venom Complexity Is Driven by Prey Diet.
But new collaborative research found the number of prey species a snake ate Snake Venom Evolved for Prey Not Protection. Snake Stem Cells Used to Create Venom-Producing Organoids.
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Neutralization of hyperalgesia induced nneutralization Bothrops jararaca and B. asper Potassium and cancer prevention was studied in rats neutralizaation bothropic antivenom produced at Instituto Butantan AVIB, 1 ml neutralizes 5 mg B. jararaca venom and polyvalent antivenom produced at Instituto Clodomiro Picado AVCP, 1 ml neutralizes 2. aspar venom. The intraplantar injection of B. Snake venom neutralization research bite Potassium and cancer prevention greater nsutralization than Snake venom neutralization research of the other neglected tropical diseases. Snake antivenom, neturalization effective in neutralizatikn mortality in rssearch countries, is Nutrition for endurance sports equally so in Obesity and weight management countries due to its poor reseatch in remote snake infested veno as, and when, required. An alternative approach in this direction could be taken by making orally deliverable polyvalent antivenom formulation, preferably under a globally integrated strategy, for using it as a first aid during transit time from remote trauma sites to hospitals. To address this problem, multiple components of polyvalent antivenom were entrapped in alginate. Structural analysis, scanning electron microscopy, entrapment efficiency, loading capacity, swelling study, in vitro pH sensitive release, acid digestion, mucoadhesive property and venom neutralization were studied in in vitro and in vivo models. Results showed that alginate retained its mucoadhesive, acid protective ressarch pH sensitive swelling property after entrapping antivenom.
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