Effects of Crotalus durissus collilineatus venom in the isolated rat kidney.

Ophidian accidents caused by the subspecies Crotalus durissus are responsible for high morbity and mortality rates. Acute renal failure is a common complication observed in these accidents. The aim of the present study was to investigate the renal effects promoted by the venom of C. d. collilineatus and its fractions, crotoxin and phospholipase A2. C. d. collilineatus (Cdc; 30 microg mL(-1)), crotoxin (CTX; 10 microg mL(-1)) and phospholipase A2 (PLA2; 10 microg mL(-1)) were tested in isolated rat kidney. The first 30 min of each experiment were used as an internal control and Cdc or its fractions, CTX and PLA2 were added to the system after this period. All experiments lasted 120 min. The venom of Cdc decreased perfusion pressure (PP; control120 = 110.3 +/- 3.69 mmHg; Cdc120 = 96.7+/-8.1 mmHg), renal vascular resistance (RVR; control120 = 6.42+/-0.78 mmHg mL g(-1) min(-1); Cdc120 = 4.8+/-0.56 mmHg/mL g(-1) min(-1)), urinary flow (UF; control120 = 0.19+/-0.03 mL g(-1) min(-1); Cdc120 = 0.12 +/- 0.01 mL g(-1) min(-1)), and glomerular filtration rate (GFR; control120 = 0.79 +/- 0.07 mL g(-1) min(-1); Cdc120 = 0.53 +/- 0.09 mL g(-1) min(-1)), but had no effect on the percent of sodium tubular transport (%TNa+), percent of chloride tubular transport (%TK+) and percent of potassium tubular transport (%TCl-). CTX and PLA2 reduced the GFR, while UF, PP and RVR remained stable during the full 120 min of perfusion. Crotoxin administration also diminished the %TK+ (control120 = 69.94 +/- 6.49; CTX120 = 33.28 +/- 4.78) and %TCl- (control120 = 79.53 +/- 2.67; CTX120 = 64.62 +/- 6.93). PLA2 reduced the %TK+, but exerted no effect on the %TNa+ or on that of TCl-. In conclusion, the C. d. collilineatus venom altered the renal functional parameters evaluated. We suggest that crotoxin and phospholipase A2 were involved in this process, since the renal effects observed would be due to the synergistic action of the components of the venom.

Neutrophils do not contribute to local tissue damage, but play a key role in skeletal muscle regeneration, in mice injected with Bothrops asper snake venom.

Local tissue damage induced by crotaline snake venoms includes edema, myonecrosis, hemorrhage, and an inflammatory response associated with a prominent cellular infiltrate. The role of neutrophils in the local tissue damage induced by Bothrops asper snake venom and by myotoxin I, a phospholipase A2 isolated from this venom, was investigated. Male Swiss mice were pretreated with either an antimouse granulocyte rat monoclonal immunoglobulin G (IgG) antibody or with isotype-matched control antibody. No significant differences in these local effects were observed between mice pretreated with antigranulocyte antibodies and those receiving control IgG. Moreover, myotoxicity induced by B. asper myotoxin I was similar in neutrophil-depleted and control mice. The role of neutrophils in the process of skeletal muscle regeneration was also assessed. Muscle regeneration was assessed by quantifying the muscle levels of creatine kinase and by morphometric histological analysis of the area comprised by regenerating cells in damaged regions of skeletal muscle. Mice depleted of neutrophils and then injected with B. asper venom showed a more deficient regenerative response than mice pretreated with control IgG. Moreover, a drastic difference in the regenerative response was observed in mice injected with myotoxin I, because animals pretreated with control IgG showed a successful regeneration, whereas those depleted of neutrophils had abundant areas of necrotic tissue that had not been removed 7 days after injection, associated with reduced contents of creatine kinase. It is concluded that (1) neutrophils do not play a significant role in the acute local pathological alterations induced by the venom of B. asper, and (2) neutrophils play a prominent role in the process of skeletal muscle regeneration after injection of B. asper venom and myotoxin I, probably related to the phagocytosis of necrotic material and the recruitment of other inflammatory cells, two events directly associated with a successful muscle regenerative response.

Inflammatory oedema induced by phospholipases A2 isolated from Crotalus durissus sp. in the rat dorsal skin: a role for mast cells and sensory C-fibers.

The ability of the phospholipases A(2) (PLA(2)s) from Crotalus durissus cascavella, Crotalus durissus collilineatus and Crotalus durissus terrificus venoms and crotapotin to increase the vascular permeability in the rat skin as well as the contribution of both mast cells and sensory C-fibers have been investigated in this study. Vascular permeability was measured as the plasma extravascular accumulation at skin sites of intravenously injected 125I-human serum albumin. Intradermal injection of crotalic PLA(2)s (0.05-0.5 microg/site) in the rat skin resulted in dose-dependent increase in plasma extravascular whereas crotapotin (1 microg/site) failed to affect this response. Co-injection of crotapotin (1 microg/site) did not modify the increased vascular permeability induced by the PLA(2)s (0.05-0.5 microg/site). Previous treatment (30 min) of the animals with cyproheptadine (2 mg/kg, i.p.) markedly reduced PLA(2) (0.5 microg/site)-induced oedema. In rats treated neonatally with capsaicin to deplete neuropeptides, the plasma extravasation induced by all PLA(2)s (0.5 microg/site) was also significantly reduced. Similarly, the tachykinin NK(1) receptor antagonist SR140333 (1nmol/site) significantly reduced the PLA(2)-induced oedema. In addition, the combination of SR140333 with cyproheptadine further reduced the increased plasma extravasation by PLA(2) from C. d. cascavella venom, but not by PLA(2) from C. d. terrificus and C. d. collilineatus venoms. Our results suggest that increase in skin vascular permeability by crotalic PLA(2)s is mediated by activation of sensory C-fibers culminating in the release of substance P, as well as by activation of mast cells which in turn release amines such as histamine and serotonin.

In vitro hemolytic activity of Lonomia obliqua caterpillar bristle extract on human and Wistar rat erythrocytes.

Human accidental envenomation caused by skin contact with the bristles of Lonomia obliqua caterpillar causes coagulation and fibrinolysis disorders. Alterations of hematologic parameters are observed only in severe cases of envenomation, but with no clinical evidence of intravascular hemolysis. However, since we have observed intravascular hemolysis in preliminary studies using Wistar rats as an experimental model for investigating L. obliqua envenomation, the objective of the present study was to investigate the in vitro hemolytic activity of the bristle extract of L. obliqua caterpillars on human and rat erythrocytes. Our results showed that the bristle extract has indirect and direct hemolytic activity on human and rat erythrocytes, although direct hemolytic activity was only observed at higher bristle extract concentrations. We also observed that the bristle extract has a proteolytic activity on band 3 of human and rat erythrocyte membranes. Thus, crude L. obliqua bristle extract was found to contain at least two components with hemolytic activity on erythrocytes, a phospholipase enzyme and another protein with a direct activity on the erythrocyte membrane.

Role of crotoxin, a phospholipase A2 isolated from Crotalus durissus terrificus snake venom, on inflammatory and immune reactions.

BACKGROUND: Crotoxin (CTX) is a potent neurotoxin from Crotalus durissus terrificus snake venom (CdtV) composed of two subunits: one without catalytic activity (crotapotin), and a basic phospolipase A2. Recent data have demonstrated that CdtV or CTX inhibit some immune and inflammatory reactions. AIM: The aim of this paper was to investigate the mechanisms involved in these impaired responses. MATERIALS AND METHODS: Male Swiss mice were bled before and at different intervals of time after subcutaneous injection of CTX or bovine serum albumin (BSA) (control animals). The effect of treatments on circulating leukocyte mobilisation and on serum levels of interleukin (IL)-6, IL-10, interferon (IFN)-gamma and corticosterone were investigated. Spleen cells from treated animals were also stimulated in vitro with concanavalin A to evaluate the profile of IL-4, IL-6, IL-10 or IFN-gamma secretion. Cytokine levels were determined by immunoenzymatic assay and corticosterone levels by radioimmunoassay. To investigate the participation of endogenous corticosteroid on the effects evoked by CTX, animals were treated with metyrapone, an inhibitor of glucocorticoid synthesis, previous to CTX treatment. RESULTS: Marked alterations on peripheral leukocyte distribution, characterised by a drop in the number of lymphocytes and monocytes and an increase in the number of neutrophils, were observed after CTX injection. No such alteration was observed in BSA-treated animals. Increased levels of IL-6, IL-10 and corticosterone were also detected in CTX-injected animals. IFN-gamma levels were not modified after treatments. In contrast, spleen cells obtained from CTX-treated animals and stimulated with concanavalin A secreted less IL-10 and IL-4 in comparison with cells obtained from control animals. Metyrapone pretreatment was effective only to reverse the neutrophilia observed after CTX administration. CONCLUSIONS: Our results suggest that CTX may contribute to the deficient inflammatory and immune responses induced by crude CdtV. CTX induces endogenous mechanisms that are responsible, at least in part, for these impaired responses.

Histopathological changes in avian kidney caused by Bothrops insularis (jararaca ilh a) venom and a phospholipase A2-containing fraction.

The histopathological changes induced in avian kidney by the intramuscular injection of Bothrops insularis (jararaca ilh a) venom and its phospholipase A2 (PLA2)-containing fraction were examined. Acute experiments (3 h and 24 h) with B. insularis crude venom (20 microg and 80 microg) or its PLA2-contaning fraction (10 microg and 40 microg) resulted in significant structural damage to the kidneys of 5-12-day-old chicks. Histopathological analysis indicated that the venom and its fraction acted on the renal tubules and glomeruli. The morphological changes, although widespread, varied in intensity from cell to cell, and from tubule to tubule in venom-injected chicks. The tubular and glomerular changes produced by the venom and its PLA2-containing fraction may be the result of a direct cytotoxic effect potentiated by ischemia-related disturbances in the regional hemodynamics. The venom and its fraction affected more segments along reptilian-type nephrons than along mammalian ones. This divergent sensitivity to the venom and its fraction may reflect the species-specific characteristics of B. insularis snake, an example of geographical isolation influencing its diet which is almost exclusively avian.

In vivo effect of snake phospholipase A2 (crotoxin+cardiotoxin) on serum IL-1alpha, TNF-alpha and IL-1ra level in humans.

VRCTC-310-Onco (crotoxin, a secretory phospholipase A2+cardiotoxin) is under development as an anti-neoplastic agent. Pro-inflammatory cytokines TNF-alpha and interleukin 1 alpha (IL-1alpha) and anti-inflammatory cytokine IL-1 receptor antagonist (IL-1ra) were measured with commercial ELISA kits in sera corresponding to 23 cycles with doses between 0.0025 and 0.023 microg/kg body weight, obtained during the phase I trial of VRCTC-310-Onco. Neither serum TNF-alpha nor IL-1alpha did change significantly after VRCTC-310-Onco. Basal IL-1ra was 794 +/- 97 pg/ml, by 3 h it was similar, 651 +/- 99 pg/ml and at 24 h p.i. it increased to 1197 +/- 122 pg/ml (P<0.001). The increase was dose-dependent. The addition of dexamethasone (required to reduce pain with the highest doses) inhibited IL-1alpha and enhanced the induction of IL-1ra by VRCTC-310-Onco. Summing up, in vivo, in humans, in the dose range tested, VRCTC-310-Onco induces IL-1ra, and does not consistently modify IL-1alpha or TNF-alpha serum levels.

Evidence of a role for galectin-1 in acute inflammation.

Galectin-1 (Gal-1), a member of a family of beta-galactoside-binding proteins, has been suggested to play key roles in immunological and inflammatory processes. The present study deals with the concept of an in vivo role for Gal-1 in acute inflammation by using the rat hind paw edema test. Local administration of Gal-1 (0.5, 2, 4 and 8 microg/ml) inhibited acute inflammation induced by bee venom phospholipase A(2) (PLA(2)) when it was injected 30 min before the enzyme or co-injected together with PLA(2). The anti-inflammatory effect was prevented by a specific antibody, but independent of its carbohydrate-binding properties. In contrast, Gal-1 failed to inhibit histamine-induced edema. Histopathological studies showed a clear reduction of the inflammatory process when Gal-1 was injected before PLA(2), evidenced by a diminished number of infiltrated polymorphonuclear neutrophils and scarce degranulated mast cells. The anti-inflammatory effect was also assessed in vitro, showing that Gal-1 treatment reduced prostaglandin E(2) secretion and arachidonic acid release from stimulated peritoneal macrophages. Results presented here provide the first evidence for a role of Gal-1 in acute inflammation and suggest that the anti-inflammatory effect involves the inhibition of both soluble and cellular mediators of the inflammatory response.

Immunoenzymatic quantitation of antibodies to Bothrops asper myotoxins after polyvalent antivenom administration in mice.

1. Two quantitative enzyme-immunoassays (EIA) for Bothrops asper myotoxin and anti-myotoxin antibodies, respectively, were utilized to study their in vivo distribution in mice (Swiss, 18 to 20 g). 2. After polyvalent antivenom (0.4 ml) administration by the iv route, there was an immediate peak in plasma anti-myotoxin antibodies which declined rapidly during the first hour, and then decreased more gradually. Anti-myotoxin antibodies were detected in muscular tissue (gastrocnemius) following iv injection of antivenom. After im injection of antivenom (0.4 ml), a slow and steady increase in plasma anti-myotoxin levels was observed, with a peak at 24 h. 3. Mice that received antivenom (0.4 ml) by the iv or im route 15 min after im injection of B. asper venom (100 micrograms) had lower levels of plasma anti-myotoxin antibodies than controls injected with antivenom only, suggesting that at least a fraction of the antibodies combines with myotoxins in vivo. 4. Myotoxin was not detected in plasma at any time after venom injection by the im (100 micrograms) or ip (40 micrograms) route. Following iv injection of 50 micrograms of purified myotoxin II, all plasma samples were also negative, at a detection limit of 10 ng/ml. 5. It was demonstrated that myotoxin II binds to mouse erythrocytes in vitro, a fact that could partially explain its rapid in vivo disappearance from plasma. 6. The present results on the distribution of anti-myotoxin antibodies in vivo are in agreement with previous experimental studies reporting the poor neutralization of myotoxicity induced by B. asper venom when antivenom is injected im, in comparison to i.v. injection.

Increased phagocytic activity of peripheral blood monocytes after intravenous injection of phospholipase A2 to monkeys.

One of the expressions of the activity of phagocytic cells such as monocytes or macrophages is a burst of increased oxidative activity on stimulation. The free oxygen radicals liberated, mainly O2- and H2O2, lead to chemoluminescence, which is thus a measure of activation. Chemoluminescence also depends on arachidonic acid metabolism, and this depends on phospholipase A2 (PLA2). We modified monocyte activity in monkeys by injecting them i.v. with this enzyme and observed that 30 min after injection, the phagocytic activity of peripheral blood monocytes and the chemoluminescence they emitted was greater than that of controls. We suggest that PLA2 may act as an in vivo immunomodulator in mammals.

Is direct cardiotoxicity the primary cause of death following i.v. injection of the basic phospholipase A2 from Naja nigricollis venom?

The primary cause of death following i.v. injection of the basic phospholipase A2 (PLA2) from Naja nigricollis venom has been attributed to its direct cardiotoxicity. In view of our recent findings that cardiac failure caused by the basic PLA2 from Naja m. mossambica is primarily due to hyperkalemia resulting from cellular damage and possibly also from hemolysis, the cause of death due to the basic PLA2 from Naja nigricollis was re-investigated. In the anesthetized mice and rats, the PLA2 (0.3 micrograms/g, i.v.) produced a transient hypotension followed by recovery and subsequently by cardiac failure with ECG changes suggestive of hyperkalemia, such as P-R prolongation, tall T-wave, biphasic QRS-T complex, low voltage of QRS, A-V block, etc. Analysis of blood chemistry revealed marked increases in the plasma levels of K+, CPK, LDH, GOT, GPT, inorganic phosphate and hemoglobin (probably a mixture of hemoglobin and myoglobin). In the atrial preparation, however, no marked cardiotoxicity was observed except for a slight negative inotropic effect at 30 micrograms/ml. When 200 micrograms of the enzyme was injected into the coronary circulation in the Langendorff preparation, also no marked cardiotoxic effect was observed except for a decrease (about 40%) of coronary flow. From these results, it is concluded that the primary cause of death following i.v. injection of the basic PLA2 from Naja nigricollis is apparently cardiac failure due to hyperkalemia, resulting from cellular damage and possibly also from hemolysis, rather than direct cardiotoxicity.

Is direct cardiotoxicity the primary cause of death following i. v. injection of the basic phospholipase A2 from Naja Nigricollis venom?

Con objeto de elucidar la función del Ca intracelular en la transmisión neuromuscular investigamos en preparaciones de músculo de rana los efectos del ácido1,2-bis(o-aminofenoxi)etano-N,N,N',N'-tetraacético (BAPTA) sobre el aumento-potenciación por frecuencia (anteriormente llamado facilitación por frecuencia) el que ha sido de utilidad para identificar los sitios de acción de varios agentes colinérgicos. La disminución de los iones Ca del espacio intracelular por BAPTA sólo suprimió el componente dependiente de Ca del fenómeno (ma) sin modificar el factor de estimulación dependiente de frecuencia (K). La depresión causada por BAPTA en la facilitación de corto plazo del potencial de placa (EPP) fue la misma tanto en reposo como en la estimulación. El efecto del BAPTA fue parcialmente antagonizado, por el ionóforo de Ca A23187. Esto sugiere que la capacidad de "buffer" de Ca del BAPTA se mantiene durante la estimulación repetitiva de baja frecuencia. BAPTA no modificó la potenciación post-tetánica de los EPP miniatura en medio libre de Ca. Estos resultados indican que los iones Ca son esenciales para la liberación de transmisor y para la facilitación de corto plazo, pero no son responsables de todos los cambios en la liberación de transmisor

Is direct cardiotoxicity the primary cause of death following i. v. injection of the basic phospholipase A2 from Naja Nigricollis venom?

Con objeto de elucidar la función del Ca intracelular en la transmisión neuromuscular investigamos en preparaciones de músculo de rana los efectos del ácido1,2-bis(o-aminofenoxi)etano-N,N,N,N-tetraacético (BAPTA) sobre el aumento-potenciación por frecuencia (anteriormente llamado facilitación por frecuencia) el que ha sido de utilidad para identificar los sitios de acción de varios agentes colinérgicos. La disminución de los iones Ca del espacio intracelular por BAPTA sólo suprimió el componente dependiente de Ca del fenómeno (ma) sin modificar el factor de estimulación dependiente de frecuencia (K). La depresión causada por BAPTA en la facilitación de corto plazo del potencial de placa (EPP) fue la misma tanto en reposo como en la estimulación. El efecto del BAPTA fue parcialmente antagonizado, por el ionóforo de Ca A23187. Esto sugiere que la capacidad de "buffer" de Ca del BAPTA se mantiene durante la estimulación repetitiva de baja frecuencia. BAPTA no modificó la potenciación post-tetánica de los EPP miniatura en medio libre de Ca. Estos resultados indican que los iones Ca son esenciales para la liberación de transmisor y para la facilitación de corto plazo, pero no son responsables de todos los cambios en la liberación de transmisor (AU)

Anticoagulant effect of myotoxic phospholipase A2 isolated from the venom of the snake Bothrops asper (Viperidae)

La fosfolipasa A2 miotoxica del veneno de Bothrops asper (tercipelo) prolonga el tiempo de recalcificación de plasmas de cinco especies de mamíferos. La fosfolipasa A2 hidroliza los fosfolípidos del plasma, en tanto que su acción inhibitoria es revertida cuando se adicionan fosfolípidos plaquetarios. Estas dos observaciones sugieren que la acción anticoagulante se debe a una alteración de los fosfolípidos plasmáticos necesarios para la coagulación. Tanto la actividad enzimática como la anticoagulante se mantuvieron aún después de calentamiento a 95è-C durante 10 min. Pese a su acción anticoagulante in vitro, la fosfolipasa A2 no prolonga el tiempo de coagulación luego de inoculación intravenosa en ratones