Effect of crotapotin and heparin on the rat paw oedema induced by different secretory phospholipases A2.

The effects of crotapotin (a non-toxic and non-enzymatic acid polypeptide naturally complexed with phospholipase A2) and heparin on rat paw edema induced by different secretory phospholipases A2 (sPLA2) have been investigated. The ability of crotapotin to affect the enzymatic activity of the sPLA2(s) have also been evaluated. Secretory PLA2(s) obtained from both snake (Naja naja, Naja mocambique mocambique, Crotalus adamanteus and Crotalus durissus terrificus) and bee (Apis mellifera) venoms as well as that from bovine pancreas were used in this study. Rat paw oedema was induced by a single subplantar injection of the sPLA2s (5-30 microg/paw) in absence and presence of either crotapotin (10-100 microg/paw) or heparin (50 U/paw). Paw volume was measured using a hydroplethysmometer. Phospholipase A2 from Naja naja, Naja mocambique mocambique, Apis mellifera venoms and the basic component of Crotalus durissus terrificus venom all induced dose-dependent rat paw oedema whereas those from Crotalus adamanteus venom and bovine pancreas were ineffective. Paw oedema induced by PLA2(s) from both Naja naja and Apis mellifera venoms was significantly (P < 0.05) inhibited by crotapotin (0.1-100 microg/site) whereas the Naja mocambique mocambique venom PLA2-induced oedema was significantly potentiated (P < 0.05) by this polypeptide (40 microg/site). On the other hand, heparin (50 U/paw) had no effect on the paw oedema induced by PLA2 from Naja naja and Apis mellifera venoms but significantly inhibited the Naja mocambique mocambique venom PLA2-induced oedema. The measurement of the in vitro phospholipasic activity revealed that crotapotin inhibited by 60-70% the enzymatic activities of PLA2(s) from Crotalus adamanteus, Naja mocambique mocambique, Apis mellifera venoms and bovine pancreas. Our results suggest that despite the great homology between the various types of sPLA2 they interact with crotapotin on cell surfaces in different ways leading to either inhibition or potentiation of the paw oedema by a mechanism unrelated to their enzymatic activities. Since heparin reduced paw oedema induced by PLA2 from Naja mocambique mocambique venom it is likely that this sPLA2 is similar to the novel heparin-sensitive PLA2 found in mast cells.     

A comparative study of biological activities of crotoxin and CB fraction of venoms from Crotalus durissus terrificus, Crotalus durissus cascavella and Crotalus durissus collilineatus.

In Brazil, the Crotalus durissus terrificus subspecie is the most studied, particularly concerning its crotoxin. Crotoxin is the major toxic component of the South American rattlesnake Crotalus durissus venom. It is composed of two different subunits, CA called crotapotin and CB weakly toxic phospholipase A2 with high enzymatic activity. In this paper, we decided to make a study of the main toxic characteristics of crotoxin (CTX) and CB fraction from the other subspecies, Crotalus durissus cascavella and of Crotalus durissus collilineatus, in comparison with those of C. d. terrificus. Ours results have shown that the venoms presented similar chromatographic profiles and the purified fractions were free of contaminants. Regarding the toxic activities, the DL50 of the crotoxins showed no significant differences between the subspecies. The smaller toxicity of CB indicated that the toxicity of the crotoxin complex depends on the interaction between CA and CB. CTX and fraction CB of the three species of Crotalus showed negligible proteolytic activity. C. d. terrificus CTX presented higher PLA2 activity when compared with the others two subspecies. The oedema induced by CB developed later than the CTX and reached its peak 3 h after the injection. The myotoxic activity was determined by assaying serum CK levels. Mice injected with CTX of C. d. terrificus presented greater myotoxic activity compared to the others. The myotoxic activity of CB from the three subspecies was lower than the activity of the crotoxin, reinforcing the idea that the fraction CA increases the toxicity of CB.     

Cross-neutralization of the neurotoxicity of Crotalus durissus terrificus and Bothrops jararacussu venoms by antisera against crotoxin and phospholipase A2 from Crotalus durissus cascavella venom.

We have previously demonstrated that rabbit antisera raised against crotoxin from Crotalus durissus cascavella venom (cdc-crotoxin) and its PLA2 (cdc-PLA2) neutralized the neurotoxicity of this venom and its crotoxin. In this study, we examined the ability of these antisera to neutralize the neurotoxicity of Crotalus durissus terrificus and Bothrops jararacussu venoms and their major toxins, cdt-crotoxin and bothropstoxin-I (BthTX-I), respectively, in mouse isolated phrenic nerve-diaphragm preparations. Immunoblotting showed that antiserum to cdc-crotoxin recognized cdt-crotoxin and BthTX-I, while antiserum to cdc-PLA2 recognized cdt-PLA2 and BthTX-I. ELISA corroborated this cross-reactivity. Antiserum to cdc-crotoxin prevented the neuromuscular blockade caused by C. d. terrificus venom and its crotoxin at a venom/crotoxin:antiserum ratio of 1:3. Antiserum to cdc-PLA2 also neutralized the neuromuscular blockade caused by C. d. terrificus venom or its crotoxin at venom or toxin:antiserum ratios of 1:3 and 1:1, respectively. The neuromuscular blockade caused by B. jararacussu venom and BthTX-I was also neutralized by the antisera to cdc-crotoxin and cdc-PLA2 at a venom/toxin:antiserum ratio of 1:10 for both. Commercial equine antivenom raised against C. d. terrificus venom was effective in preventing the neuromuscular blockade typical of B. jararacussu venom (venom:antivenom ratio of 1:2), whereas for BthTX-I the ratio was 1:10. These results show that antiserum produced against PLA2, the major toxin in C. durissus cascavella venom, efficiently neutralized the neurotoxicity of C. d. terrificus and B. jararacussu venoms and their PLA2 toxins.     

Anti-sera raised in rabbits against crotoxin and phospholipase A2 from Crotalus durissus cascavella venom neutralize the neurotoxicity of the venom and crotoxin.

Crotoxin, the principal neurotoxin in venom of the South American rattlesnakes Crotalus durissus terrificus and Crotalus durissus cascavella, contains a basic phospholipase A2 (PLA2) and an acidic protein, crotapotin. In this work, we examined the ability of rabbit anti-sera against crotoxin and its PLA2 subunit to neutralize the neurotoxicity of venom and crotoxin from C. d. cascavella in mouse phrenic nerve-diaphragm and chick biventer cervicis preparations. Immunoblotting showed that the anti-sera recognized C. d. cascavella crotoxin and PLA2. This was confirmed by ELISA, with both anti-sera having end-point dilutions of 3 x 10(-6). Anti-crotoxin serum neutralized the neuromuscular blockade in phrenic nerve-diaphragm muscle preparations at venom or crotoxin:anti-serum ratios of 1:2 and 1:3, respectively. Anti-PLA2 serum also neutralized this neuromuscular activity at a venom or crotoxin:anti-serum ratio of 1:1. In biventer cervicis preparations, the corresponding ratio for anti-crotoxin serum was 1:3 for venom and crotoxin, and 1:1 and 1:2 for anti-PLA2 serum. The neutralizing capacity of the sera in mouse preparations was comparable to that of commercial anti-serum raised against C. d. terrificus venom. These results show that anti-sera against crotoxin and PLA2 from C. d. cascavella venom neutralized the neuromuscular blockade induced by venom and crotoxin in both nerve-muscle preparations, with the anti-serum against crotoxin being slightly less potent than that against crotoxin.     

Ability of rabbit antiserum against crotapotin to neutralize the neurotoxic, myotoxic and phospholipase A2 activities of crotoxin from Crotalus durissus cascavella snake venom.

The toxicity of crotoxin, the major toxin of Crotalus durissus terrificus (South American rattlesnake) venom, is mediated by its basic phospholipase A(2) (PLA(2)) subunit. This PLA(2) is non-covalently associated with crotapotin, an acidic, enzymatically inactive subunit of the crotoxin complex. In this work, rabbit antiserum raised against crotapotin purified from Crotalus durissus cascavella venom was tested for its ability to neutralize the neurotoxicity of this venom and its crotoxin in vitro. The ability of this antiserum to inhibit the enzymatic activity of the crotoxin complex and PLA(2) alone was also assessed, and its potency in preventing myotoxicity was compared with that of antisera raised against crotoxin and PLA(2). Antiserum to crotapotin partially neutralized the neuromuscular blockade caused by venom and crotoxin in electrically stimulated mouse phrenic nerve-hemidiaphragm preparations and prevented the venom-induced myotoxicity, but did not inhibit the enzymatic activity of crotoxin and purified PLA(2). In contrast, previous findings showed that antisera against crotoxin and PLA(2) from C. d. cascavella effectively neutralized the neuromuscular blockade and PLA(2) activity of this venom and its crotoxin. The partial neutralization of crotoxin-mediated neurotoxicity by antiserum to crotapotin probably reduced the binding of crotoxin to its receptor following interaction of the antiserum with the crotapotin moiety of the complex.     

Structural and biological characterization of a crotapotin isoform isolated from Crotalus durissus cascavella venom.

Envenoming by Crotalus durissus subspecies leads to coagulation disorders, myotoxicity, neurotoxicity and acute renal failure. The most serious systemic alteration and primary cause of death after snakebite is acute renal failure. In this work, we isolated crotapotin, an acid component (Crtp) of crotoxin from Crotalus durissus cascavella venom and we investigated its bactericidal and pro-inflammatory activities as well as its renal effects in rat isolated perfused kidneys. Crtp was bactericidal to the Gram-negative species Xanthomonas axonopodis pv. passiflorae, but was less effective against the Gram-positive Claribacteri ssp, probably because of differences in the cell wall composition. Crtp showed a high amino acid sequence homology with other Crtps described in the literature (around of 90%) and its A and B chains had high conserved regions corresponding to the calcium-binding loop, catalytic site and helix 3 of PLA2. The Crtp showed moderate pro-inflammatory activity and increased significantly the inflammation evoked by PLA2 when co-injected or co-incubated with PLA2. The renal parameters evaluated included the perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR) and percent of sodium tubular transport (%TNa+). Crotapotin (5 microg/ml) significantly increased the PP and RVR, whereas the GFR, UF and %TNa+ were unaffected. These results suggest that crotoxin is the main venom component responsible for nephrotoxicity and crotapotin contributes little to this phenomenom. The biological and bactericidal actions of Crtp also suggest that this protein may have functions other than simply acting as a chaperone for PLA2.     

Characterization of the insulinotropic action of a phospholipase A2 isolated from Crotalus durissus collilineatus rattlesnake venom on rat pancreatic islets.

The ability of PLA2 and crotapotin, isolated from Crotalus durissus collilineatus rattlesnake venom, to stimulate insulin secretion from isolated rat islets was examined. PLA2 and crotapotin stimulated insulin secretion at 2.8 mmol/L glucose, whereas at a high glucose concentrations (16.7 mmol/L) only PLA2 stimulated secretion. Nifedipine (10 micromol/L) did not alter the ability of PLA2 to increase insulin secretion stimulated by a depolarizing concentration of K+ (30 mmol/L). PLA2 did not affect 14CO2 production but significantly increased the efflux of arachidonic acid from isolated islets. These results indicate that PLA2-stimulated secretion is not dependent on an additional influx of Ca2+ through L-type Ca(2+)-channels but rather is associated with arachidonic acid formation in pancreatic islets.     

Mitochondrial dysfunction induced by pancreatic and crotalic (Crotalus durissus terrificus) phospholipases A2 on rabbit proximal tubules suspensions

In the present study we show that phospholipases A2 isolated from porcine pancreas (PP-PLA2) and Crotalus durissus terrificus snake venom (SV-PLA2) induced dose-dependent increases of LDH release from rabbit proximal tubules in suspension. Both porcine and crotalic PLA(2)s induced increases in non-esterified fatty acid (NEFA) levels (microg of NEFA/mg of tubule protein). It was observed that the NEFA levels in the pellets were higher than in the supernatant for both PLA2, and were dose-dependent for the crotalic PLA2 group. Furthermore, snake venom PLA2 induced a decrease in mitochondrial membrane potential (DeltaPsi(m)) assessed by both JC-1 uptake and safranin O uptake. Porcine PLA2 produced no effects on JC-1 uptake with the highest concentrations and an unexpected increase in the group treated with the lowest concentration. In contrast, the safranin O method revealed decreases of energization with both phospholipases, so it had higher sensitivity to the presence of the increased NEFA levels. Addition of delipidated bovine serum albumin (dBSA) completely reversed the effects induced by phospholipases on DeltaPsi(m) measured with safranin O. Incubation with pancreatic and crotalic phospholipases A2 produced no changes on cell ATP levels. We conclude that the treatment of proximal tubule suspensions with porcine or crotalic phospholipases disturbed membrane integrity as well as mitochondrial function. Specific early NEFA-mediated mitochondrial effects of the phospholipases used in the present study are indicated by the benefit provided by dBSA.     

Pharmacological evidence for a presynaptic action of venoms from Bothrops insularis (jararaca ilhoa) and Bothrops neuwiedi (jararaca pintada).

Whereas the presynaptic action of Crotalus durissus terrificus venom is well-established, Bothrops venoms have historically been considered to have only postsynaptic and muscular effects. However, some studies have also suggested a presynaptic action for these venoms. In this work, we used chick biventer cervicis preparations to compare the presynaptic actions of two Bothrops venoms (B. insularis and B. neuwiedi) with that of C. d. terrificus venom. At 10 microg/ml, all venoms produced irreversible blockade of the twitch tension responses, with no reduction in acetylcholine (ACh)-induced contractures and only a slight decrease in potassium induced-contractures. The times (in min) required to produce 50% neuromuscular blockade (C. d. terrificus: 16.3+/-0.7, n = 8; B. insularis: 30.0+/-1.9, n = 5; B. neuwiedi: 42.0+/-2.0, n = 8; mean +/- SEM) were significantly different among the venoms (p < 0.01). Lowering the temperature at which the experiments were done (from 37 to 24 degrees C) prevented neuromuscular blockade by the three venoms, indicating that enzyme activity may be involved in this response. At concentrations capable of causing complete neuromuscular blockade, creatine kinase release remained close to levels seen in control preparations incubated with Krebs solution alone (500-1200 IU/l). Commercial crotalic antivenom, but not bothropic antivenom, protected against the neuromuscular blockade caused by B. insularis and B. neuwiedi venoms. These observations indicate that bothropic venoms may contain components which act presynaptically in a manner similar to C. d. terrificus venom, and that at low venom concentrations a direct action on skeletal muscle does not contribute to this presynaptic neurotoxicity.     

Biological activities of a lectin from Bothrops jararacussu snake venom.

Snake venoms contain saccharide-binding lectins. In this work, we examined the biological activities of a lectin (BjcuL) purified from Bothrops jararacussu snake venom by chromatography on non-derivatized Sepharose 4B and Sephacryl S-200 HR. The protein, a homodimer with subunits of 14.5 kDa, gave a single immunoprecipitin line in immunoelectrophoresis and cross-reacted in ELISA with antivenoms raised against Bothrops spp. (lanceheads), Micrurus spp. (coral snakes), Crotalus durissus terrificus (South American rattlesnake), and arthropod (Loxosceles gaucho, Phoneutria nigriventer and Tityus serrulatus) venoms. BjcuL agglutinated human formaldehyde-fixed erythrocytes at > or = 100 ng/ml and was inhibited by lactose and EDTA (> or = 2 mM) and high concentrations (> 100 mM) of glucose and sucrose, but not by N-acetylglucosamine. BjcuL had no direct hemolytic activity and was devoid of esterase, PLA2 and proteolytic activities. The lectin (up to 200 microg/ml) did not aggregate human platelet-rich plasma (PRP) or washed platelets (WP), nor did it alter the aggregation induced by ADP in PRP or by thrombin in WP. When injected into mouse hind paws, BjcuL (10-100 microg/paw) caused edema and increased vascular permeability, with a maximum effect after 1h that persisted for up to 6 h (edema) or gradually decreased after the peak interval (vascular permeability). No hemorrhage was observed in BjcuL-injected paws. In anesthetized rats, B. jararacussu venom (200 microg/kg, i.v.) produced sustained hypotension (maximum decrease of approximately 60%) whereas a similar dose of BjcuL decreased the blood pressure by approximately 15%, with a rapid return to the resting level.     

Determination of Crotalus durissus cascavella venom components that induce renal toxicity in isolated rat kidneys.

Envenomation by Crotalus durissus terrificus leads to coagulation disorders, myotoxicity, neurotoxicity and acute renal failure (ARF). The most serious systemic change and primary cause of death is ARF. In this work, we used RP-HPLC to isolate crotoxin, convulxin and gyroxin from venom of the related subspecies Crotalus durissus cascavella and investigated the effects of these toxins on renal function in the isolated rat kidneys perfused with Krebs-Henseleit solution containing 6% of bovine serum albumin. The parameters studied included perfusion pressure (PP), renal vascular resistance (RVR), glomerular filtration rate (GFR), urinary flow (UF), percent of sodium tubular transport (%TNa(+)), percent of potassium tubular transport (%TK(+)) and percent of chloride tubular transport (%TCl(-)). Crotoxin (5 microg/ml) increased the PP, RVR, GFR, UF and decreased %TNa(+), %TK(+) and %TCl(-), with gyroxin (5 micro g/ml) the GFR remained stable during the 120 min of perfusion, whereas PP and RVR increased significantly and the %TNa(+), %TK(+) and %TCl(-) decreased significantly. Convulxin (5 micro g/ml) had no effect on renal function. Crotoxin caused alterations in all renal parameters. Gyroxin produced a minor effect compared to crotoxin. These results indicated that crotoxin is the main componenet responsible for acute nephrotoxicity caused by C. d. cascavella venom.     

Effects of convulxin,a toxin from rattlesnake venom,on platelets and leukocytes of anesthetized rabbits

Convulxin is a high molecular weight toxin isolated from rattlesnake (Crotalus durissus terrificus, Crotalus durissus collilineatus, Crotalus durissus cascavella) venom. When administered i.v. it reduces the number of circulating platelets and leukocytes of anesthetized rabbits in a dose dependent manner. Small doses induce reversible platelet aggregation, and larger doses cause lysis. The doses of convulxin that cause platelet lysis also evoke cardiovascular and respiratory disturbances in anesthetized animals. These effects were also observed when platelets which were preincubated with convulxin in vitro were administered to animals which had received antivenom serum. This indicates that convulxin may act by releasing substances contained in the platelets.     

Crotapotin induced modification of T lymphocyte proliferative response through interference with PGE2 synthesis.

The immunosuppressive property has been demonstrated for the venom of the Crotalus durissus terrificus. Using a simple, novel method for obtaining crotapotin and phospholipase A2 isoforms from venom, it was possible to demonstrate that the addition of crotapotin to cultures of isolated lymphocytes resulted in a significant inhibition of the cellular proliferative response to Concanavalin A. This reduction in blastogenic response of lymphocytes is accompanied by a significant increase in the production of PGE2 by macrophages. This effect on the innate immune response suggests that this compound may modify the subsequent adaptative immune response.     

Several isoforms of crotoxin are present in individual venoms from the South American rattlesnake Crotalus durissus terrificus

Crotalus durissus terrificus venoms collected either from individual snakes or from a large number of animals (more than 30) have been fractionated by high performance liquid chromatography on gel-filtration and ion exchange columns. The chromatographic patterns obtained with individual venom samples indicated that each Crotalus durissus terrificus snake synthesizes five to ten different crotoxin isoforms in widely variable relative proportions. Furthermore, the heterogeneity of venom samples collected from a large number of snakes did not appear significantly larger than that observed with venoms obtained from individual snakes. The comparison of the chromatographic patterns that we obtained with the various (individual and pooled) venoms allowed us to identify about 15 crotoxin isoforms, which may result from the expression of isogenes, since two amino acid variants have been reported to occur at several positions in the sequence of crotoxin component B. These observations confirm the existence of numerous molecular isoforms of crotoxin and suggest that an individual Crotalus durissus terrificus snake possesses several genes coding for the various crotoxin isoforms. The heterogeneity of venom samples collected from a large number of animals is explained, in a large measure, by the complexity of the venom obtained from the individual snakes.     

Effects of morin on snake venom phospholipase A2 (PLA2).

Flavonoids are potent anti-inflammatory compounds isolated from several plant extracts, and have been used experimentally against inflammatory processes. In this work, a PLA2 isolated from the Crotalus durissus cascavella venom and rat paw oedema were used as a model to study the effect of flavonoids on PLA2. We observed that a treatment of PLA2 with morin induces several modifications in the aromatic amino acids, with accompanying changes in its amino acid composition. In addition, results from circular dichroism spectroscopy and UV scanning revealed important structural modifications. Concomitantly, a considerable decrease in the enzymatic and antibacterial activities was observed, even though anti-inflammatory and neurotoxic activities were not affected. These apparent controversial results may be an indication that PLA2 possess a second pharmacological site which does not affect or depend on the enzymatic activity.     

Neurotoxic and myotoxic actions of crotoxin-like and Crotalus durissus cascavella whole venom in the chick biventer cervicis preparation.

Crotoxin from Crotalus durissus cascavella venom was purified by a combination of molecular exclusion chromatography (Superdex 75 column) and HPLC molecular exclusion (Protein Pack 300SW column). Neurotoxic and myotoxic effects from C. durissus cascavella whole venom and its main fraction, the crotoxin-like, were studied in the chick biventer cervicis (CBC) nerve-muscle preparation. Both venom and its crotoxin showed significant (p < 0.05) blockade of neuromuscular transmission at concentrations as low as 0.2-1, 5 and 25 microg/ml, but no significant effect has been shown with a concentration of 0.04 microg/ml (n = 5 each). The time required to produce 50% neuromuscular blockade with the venom and its crotoxin was 53.6+/-8.2 and 65.9+/-4.9 min (0.2 microg/ml), 29.7+/-1.9 and 34.3+/-1.9 min (1 microg/ml), 24.8+/-1.6 and 21.1+/-1.5 min (5 microg/ml), 20.9+/-3.7 and 20.1+/-1.4 min (25 microg/ml), respectively. The addition to the incubation bath of acetylcholine (55 and 110 microM) or KCl (20.1 mM), either before or after the venom or the crotoxin induced contracture in the presence of a total blockade, in all the concentrations used. Morphological analysis showed that the damage caused by C. durissus cascavella venom is stronger than that caused by crotoxin. The myonecrotic picture was more marked at higher venom and crotoxin doses (1, 5 or 25 microg/ml). Only at 25 microg/ml concentrations of the venom and crotoxin, marked muscle fiber changes were detected. We concluded that the crotoxin-like and the whole venom from C. durissus cascavella possess a preponderant and quite potent neurotoxic action in this preparation, and a myotoxic action which is observed only at higher doses.     

Renal effects of supernatant from macrophages activated by Crotalus durissus cascavella venom: the role of phospholipase A2 and cyclooxygenase

In Brazil, the genus Crotalus is responsible for approximately 1500 cases of snakebite annually. The most common complication in the lethal cases is acute renal failure, although the mechanisms of the damaging effects are not totally understood. In this work, we have examined the renal effects caused by a supernatant of macrophages stimulated by Crotalus durissus cascavella venom as well the potential role of phospholipase A2 and cyclo-oxygenase. Rat peritoneal macrophages were collected and placed in a RPMI medium and stimulated by crude Crotalus durissus cascavella venom (1, 3 or 10 microg/ml) for 1 hr. They were then washed and kept in a culture for 2 hr. The supernatant (1 ml) was tested in an isolated perfused rat kidney. The first 30 min. of each experiment were used as an internal control, and the supernatant was added to the system after this period. All experiments lasted 120 min. A study of toxic effect on perfusion pressure, glomerular filtration rate, urinary flow percent of sodium tubular transport and percent of proximal tubular sodium transport was made. The lowest concentration of venom (1 microg/ml) was not statistically different from the control values. The most intense effects were seen at 10 microg/ml for all renal parameters. The infusion of the supernatant of macrophages stimulated with crude venom (3 or 10 microg/ml) increased the perfusion pressure, glomerular filtration rate and urinary flow, decreased the percent of sodium tubular transport and percent of proximal tubular sodium transport. Dexamethasone (10 microM) and quinacrine (10 microM) provided protection against the effect of the venom on glomerular filtration rate, urinary flow, percent of sodium tubular transport, percent of proximal tubular sodium transport and perfusion pressure. Indomethacin (10 microM) and nordiidroguaretic acid (1 microM) reversed almost all functional changes, except those of the perfusion pressure. These results suggest that macrophages stimulated with Crotalus durissus cascavella venom release mediators capable of promoting nephrotoxicity in vitro. Moreover, phospholipase A2 and cyclooxygenase products are involved in these biologic effects.     

Enzyme-linked immunosorbant assay (ELISA) of size-selected crotalid venom antigens by Wyeth's polyvalent antivenom

The binding of Antivenom (Crotalidae) Polyvalent to fractions from crude venoms of eight crotalid and one viperid snake, obtained by high performance size-exclusion chromatography, was determined with an indirect enzyme-linked immunosorbent assay (ELISA). Most of the large (greater than 30,000 mol. wt) molecular mass crotalid venom fractions were associated with high (greater than 0.7 absorbance units) ELISA values. Similarly, the medium (13,000-30,000 mol. wt) and small (less than 14,000 mol. wt) molecular mass crotalid venom fractions were coincident with moderate (0.3-0.7 absorbance units) and low (less than 0.3 absorbance units) ELISA levels. Some variability in this pattern was seen with individual venom fractions. A distinctly different pattern of ELISA values were observed with two rattlesnake venoms: the South American (Crotalus durissus terrificus) and Mojave desert (Crotalus scutulatus scutulatus) rattlesnakes. The elution profile from these venoms showed a progression of low to moderate ELISA values within the large molecular mass fractions. This pattern was followed by a decline to low ELISA values throughout the remainder of the elution profile. When saw scaled viper (Echis carinatus leucogaster) venom fractions were tested, only background ELISA values were detected with antivenom. Similarly, background ELISA values were associated with the small molecular mass fractions of all venoms tested. In addition, the elution position for the basic peptides of southern Pacific (Crotalus viridis helleri) and timber (Crotalus h. horridus) rattlesnake venoms showed minimal ELISA values. These data support the view that except for the venom of C. durissus terrificus and C. s. scutulatus, most antivenom antibodies bind large (greater than 30,000 mol. wt) venom fractions. Thus, antivenom contains minimal levels of antibodies to the basic peptides in these venoms.     

Serum kinetics of crotoxin from Crotalus durissus terrificus venom in mice: evidence for a rapid clearance.

We report on an ELISA for the detection of crotoxin with a detection limit of 1-3 pg/ml of sample. Cross-reactivity with other animal venoms occurred only at concentrations above 1 microgram/ml. Serum kinetics of crotoxin were investigated in BALB/c mice after a single 10 micrograms s.c. dose of venom obtained from Crotalus durissus terrificus. Crotoxin levels were 254 +/- 141 ng/ml serum (X +/- S.E.) 15 min after venom injection, 3.9 +/- 0.5 ng/ml serum at 30 min and undetectable thereafter. The rapid clearance of crotoxin from the serum suggests that the test may be unsuitable for the clinical management of envenomation victims.     

Crotoxin alters lymphocyte distribution in rats: Involvement of adhesion molecules and lipoxygenase-derived mediators

Crotoxin is the main neurotoxic component of Crotalus durissus terrificus snake venom and modulates immune and inflammatory responses, interfering with the activity of leukocytes. In the present work, the effects of crotoxin on the number of blood and lymphatic leukocytes and on lymph nodes and spleen lymphocytes population were investigated. The toxin s.c. administered to male Wistar rats, decreases the number of lymphocytes in blood and lymph circulation and increases the content of B and T-lymphocytes in lymph nodes. These effects were detected 1-2h after treatment. The crotoxin molecule is composed of two subunits, an acidic non-toxic polypeptide, named crotapotin and a toxic basic phospholipase A(2) (PLA(2)). PLA(2), but not crotapotin, decreased the number of circulating blood and lymph lymphocytes. Crotoxin promotes leukocyte adherence to endothelial cells of blood microcirculation and to lymph node high endothelial venules, which might contribute to the drop in the number of circulating lymphocytes. Crotoxin increases expression of the adhesion molecule LFA-1 in lymphocytes. The changes in the expression of the adhesion molecule might contribute, at least in part, for the increased leukocyte adhesion to endothelium. Zileuton, a 5-lipoxygenase inhibitor, blocked the decrease in the number of circulating leukocytes induced by crotoxin and also abolished the changes observed in leukocyte-endothelial interactions, suggesting the involvement of lipoxygenase-derived mediators in the effects of the toxin.