The edematogenic effect of Micrurus lemniscatus venom is dependent on venom phospholipase A2 activity and modulated by non-neurogenic factors.

Coral snakes mainly cause neurotoxic symptoms in human envenomation, but experimental studies have already demonstrated several pharmacological activities in addition to these effects. This investigation was carried out with the aim of evaluating (1) non-neurogenic mechanisms involved in the inflammatory response induced by Micrurus lemniscatus venom (MLV) in rat hind paws, (2) participation of PLA2 in this response, and (3) neutralizing efficiency of commercial anti-elapid antivenom on edema. MLV promoted a rapid, significant increase in vascular permeability, influx of leukocytes, and disorganization of collagen bundles, as demonstrated by histological analysis. Several pretreatments were applied to establish the involvement of inflammatory mediators in MLV-induced edema (5 µg/paw). Treatment of animals with chlorpromazine reduced MLV-induced edema, indicating participation of TNF-α. However, the inefficiency of other pharmacological treatments suggests that eicosanoids, leukotrienes, and nitric oxide have no role in this type of edema formation. In contrast, PAF negatively modulates this venom-induced effect. MLV was recognized by anti-elapid serum, but this antivenom did not neutralize edema formation. Chemical modification of MLV with p-bromophenacyl bromide abrogated the phospholipase activity and markedly reduced edema, demonstrating PLA2 participation in MLV-induced edema. In conclusion, the non-neurogenic inflammatory profile of MLV is characterized by TNF-α-mediated edema, participation of PLA2 activity, and down-regulation by PAF. MLV induces an influx of leukocytes and destruction of collagen fibers at the site of its injection.

Instituto Butantan and Instituto Clodomiro Picado: A long-standing partnership in science, technology, and public health.

Instituto Butantan (São Paulo, Brazil) and Instituto Clodomiro Picado (San José, Costa Rica) are public institutions devoted to scientific and technological research, production of antivenoms and other immunobiologicals, and a variety of public health interventions aimed at confronting the problem of snakebite envenoming in their countries and elsewhere. In the context of the 120th anniversary of Instituto Butantan, this work describes the historical developments in the relationship between these institutions, which has evolved into a solid cooperation platform in science, technology, and public health. The relationship between Instituto Butantan and Costa Rica started early in the 20th century, with the provision of Brazilian antivenoms to Costa Rica through the coordination of Instituto Butantan and the health system of Costa Rica, with the leadership of Clodomiro Picado Twight. After the decade of 1980, a prolific collaborative network has been established between Instituto Butantan and Instituto Clodomiro Picado (founded in 1970) in the areas of scientific and technological research in pharmacology, biochemistry, experimental pathology, immunology, and public health, as well as in antivenom development, production, preclinical evaluation, and quality control. In addition, both institutions have played a key role in the integration of regional efforts in Latin America to create a network of public institutions devoted to antivenom production and quality control, in close coordination with the Pan American Health Organization (PAHO). This long-standing partnership is an example of a highly productive south-south cooperation under a frame of solidarity and public well-being.

Lemnitoxin, the major component of Micrurus lemniscatus coral snake venom, is a myotoxic and pro-inflammatory phospholipase A2.

The venom of Micrurus lemniscatus, a coral snake of wide geographical distribution in South America, was fractionated by reverse-phase HPLC and the fractions screened for phospholipase A2 (PLA2) activity. The major component of the venom, a PLA2, here referred to as 'Lemnitoxin', was isolated and characterized biochemically and toxicologically. It induces myotoxicity upon intramuscular or intravenous injection into mice. The amino acid residues Arg15, Ala100, Asn108, and a hydrophobic residue at position 109, which are characteristic of myotoxic class I phospholipases A2, are present in Lemnitoxin. This PLA2 is antigenically related to M. nigrocinctus nigroxin, Notechis scutatus notexin, Pseudechis australis mulgotoxin, and Pseudonaja textilis textilotoxin, as demonstrated with monoclonal and polyclonal antibodies. Lemnitoxin is highly selective in its targeting of cells, being cytotoxic for differentiated myotubes in vitro and muscle fibers in vivo, but not for undifferentiated myoblasts or endothelial cells. Lemnitoxin is not lethal after intravenous injection at doses up to 2µg/g in mice, evidencing its lack of significant neurotoxicity. Lemnitoxin displays anticoagulant effect on human plasma and proinflammatory activity also, as it induces paw edema and mast cell degranulation. Thus, the results of this work demonstrate that Lemnitoxin is a potent myotoxic and proinflammatory class I PLA2.

Mast cells and histamine play an important role in edema and leukocyte recruitment induced by Potamotrygon motoro stingray venom in mice.

This work aimed to investigate mechanisms underlying the inflammatory response caused by Potamotrygon motoro stingray venom (PmV) in mouse paws. Pre-treatment of animals with a mast cell degranulation inhibitor (cromolyn) diminished edema (62% of inhibition) and leukocyte influx into the site of PmV injection. Promethazine (histamine type 1 receptor antagonist) or thioperamide (histamine type 3 and 4 receptor antagonist) also decreased edema (up to 30%) and leukocyte numbers, mainly neutrophils (40-50 %). Cimetidine (histamine type 2 receptor antagonist) had no effect on PmV-induced inflammation. In the RBL-2H3 lineage of mast cells, PmV caused proper cell activation, in a dose-dependent manner, with release of PGD2 and PGE2. In addition, the role of COXs products on PmV inflammatory response was evaluated. Indomethacin (COX-1/COX-2 inhibitor) or etoricoxib (COX-2 inhibitor) partially diminished edema (around 20%) in PmV-injected mice. Indomethacin, but not etoricoxib, modulated neutrophil influx into the site of venom injection. In conclusion, mast cell degranulation and histamine, besides COXs products, play an important role in PmV-induced reaction. Since PmV mechanism of action remains unknown, hindering accurate treatment, clinical studies can be performed to validate the prescription of antihistaminic drugs, besides NSAIDs, to patients injured by freshwater stingrays.

Role of cyclooxygenases in oedema-forming activity of bothropic venoms.

The venoms of Bothrops asper (BaV) and Bothrops jararaca (BjV), two of the most medically important poisonous snakes of Latin America, cause pronounced oedema in the victims through poorly understood mechanisms. In the present study, we examined the possible role of cyclooxygenases (COX) in the genesis of mouse paw oedema caused by BaV and BjV injections. BaV at 2.5 microg/paw and BjV at 0.75 microg/paw induced significant oedema that persisted for up to 6h following subplantar injection. Treatment with indomethacin (2 mg/kg), rofecoxib, (10 mg/kg), or dexamethasone (2 mg/kg) significantly reduced the BaV- and BjV-induced oedema formation. Treatment with SC-560 (30 mg/kg) significantly reduced the oedema formation induced by BjV but had no effect on that induced by BaV. Both venoms induced significant increases in the levels of prostaglandin E(2) (PGE(2)) and the expression of COX-1 and COX-2 in paw tissue. The peak of oedema formation and PGE(2) release correlated with marked expression of COX-2 in the paw tissue. These results demonstrate that injection of BaV and BjV results in a rapid increase in oedema formation that is, at least partially, mediated by arachidonic acid metabolites formed by COX-2. In the case of BjV, COX-1-derived prostanoids also appear to contribute significantly to the inflammatory changes.

Endothelins mediate neutrophil activation, ProMMP-9 release and endothelial cell detachment.

Neutrophils isolated from human peripheral blood added to a monolayer of human endothelial cells (ECV-304 cell line) stimulated with LPS (100 ng ml(-1)) resulted in: (a) neutrophil activation, measured by spreading and release of leukotriene B(4) (LTB(4)); (b) neutrophil degranulation, measured by release of matrix pro-metalloproteinase-9 (proMMP-9) and (c) loss of the monolayer integrity due to detachment of the endothelial cells. Stimulation of endothelial cells with tumor necrosis factor-alpha (TNF-alpha 10 ng ml(-1)) or interleukin-1 (IL-1; 10 ng ml(-1)) induced a similar dose-dependent increase in the neutrophil activation and endothelial cell detachment. Pre-treatment of LPS-activated ECV-304 cells with [Phe22]BigET-1(19-37) (10(-9) M; an inhibitor of endothelin converting enzyme (ECE)) or addition of BQ-123 (10(-6) M; a selective endothelin A (ET(A)) receptor antagonist) to the co-cultures, significantly reduced neutrophil spreading (50-70% inhibition) as well as the levels of LTB(4) (70-100% inhibition) and proMMP-9 (40-50% inhibition) in the co-culture supernatants. In addition, the detachment of endothelial cells was also reduced (60-75% inhibition). Moreover, the exogenous addition of ET-1 (10(-9) M) to neutrophil suspensions induced neutrophil spreading and release of LTB(4) and proMMP-9. Taken together, these findings indicate that neutrophils added to stimulated endothelial cells in the co-culture system employed in this study, get activated by products of these cells and degranulate. In parallel, the detachment of endothelial cell monolayer from the culture plates, possibly by the action of neutrophil granule-derived gelatinases, is observed. Endothelins (ETs) produced by the endothelial cells are suggested to play an essential role in these phenomena.

Effects of neutrophil depletion in the local pathological alterations and muscle regeneration in mice injected with Bothrops jararaca snake venom.

In order to study the role of neutrophils in the acute local pathological alterations induced by Bothrops jararaca snake venom, and in the process of skeletal muscle regeneration that follows, an experimental model was developed in mice pretreated with either an anti-mouse granulocyte rat monoclonal immunoglobulin G, which induces a profound neutropenia, or an isotype-matched control antibody. B. jararaca venom induced prominent haemorrhage and oedema, but only a moderate myonecrosis. No significant differences were observed in the extent of local haemorrhage, oedema and myonecrosis between neutropenic and control mice, suggesting that neutrophils do not play a determinant role in the acute pathological alterations induced by B. jararaca venom in this experimental model. Moreover, no differences were observed in skeletal muscle regeneration between these two experimental groups. In both the cases, limited areas of myonecrosis were associated with a drastic damage to the microvasculature and a scarce inflammatory infiltrate, with the consequent lack of removal of necrotic debris during the first week, resulting in a poor regenerative response at this time interval. Subsequently, a similar regenerative process occurred in both groups, and by 30 days, necrotic areas were substituted by groups of small regenerating muscle fibres. It is suggested that the drastic effect exerted by B. jararaca venom in the microvasculature precludes an effective access of inflammatory cells to necrotic areas, thereby compromising an effective removal of necrotic debris; this explains the poor regenerative response observed during the first week and the fact that there were no differences between neutropenic and control mice. As neutropenia in this model lasted only 7 days, the successful regenerative process observed at 30 days is associated with revascularization of necrotic regions and with a successful removal by phagocytes of necrotic debris in both groups.

Inflammatory events induced by Lys-49 and Asp-49 phospholipases A2 isolated from Bothrops asper snake venom: role of catalytic activity.

The inflammatory events induced in the peritoneal cavity of mice by two PLA2s isolated from Bothrops asper snake venom were investigated. MT-III, an Asp-49 catalytically active enzyme and MT-II, a catalytically inactive Lys-49 variant induced increase in vascular permeability. Inhibition of enzymatic activity of MT-III with p-bromophenacyl bromide reduced this effect. MT-III induced a larger neutrophil infiltrate than MT-II. This activity was significantly reduced after inhibition of catalytic activity. Reduction in the number of neutrophils was observed when antibodies against L-selectin, CD18 or LFA-1 were used, suggesting the involvement of these adhesion molecules in the effects of both PLA2s. There was no effect with antibodies against ICAM-1 and PECAM-1. Increase in the levels of LTB4 and TXA2, as well as of IL-1, IL-6 and TNF-alpha, were observed in the peritoneal exudates induced by MT-III. MT-II did not enhance levels of eicosanoids but increased those of cytokines. It is concluded that both PLA2s induce inflammatory events in this model. Since MT-III exerts a stronger proinflammatory effect, the enzymatic phospholipid hydrolysis may be relevant for these phenomena. However, the fact that MT-II induced inflammation suggests that molecular regions distinct from the catalytic site elicit inflammatory events perhaps by interacting with specific cell membrane acceptors.

Role of TNF-alpha, IL-1beta and IL-6 in the local tissue damage induced by Bothrops asper snake venom: an experimental assessment in mice.

The role of the cytokines TNF-alpha, IL-1beta and IL-6 in the acute local pathological effects induced by Bothrops asper snake venom was assessed in mice. Intramuscular injection of this venom induced increments in IL-1beta and IL-6 in muscle, but no elevations of TNF-alpha were detected. Pentoxifylline (PTX), a methylxanthine derivative that inhibits the synthesis of TNF-alpha, and antibodies against these three cytokines were used to assess the role of these cytokines in venom-induced effects. As a control, PTX pretreatment was effective at abrogating lethality and serum TNF-alpha increments in mice subjected to endotoxemia induced by injection of Escherichia coli lipopolysaccharide, although it did not affect the increment in IL-1beta and IL-6 in such endotoxic model. PTX failed to reduce lethality, hemorrhage, myonecrosis, dermonecrosis and edema induced by B. asper venom. Moreover, pretreatment with anti-cytokine antibodies was also ineffective at reducing venom-induced myonecrosis and hemorrhage. It is concluded that TNF-alpha, IL-1beta and IL-6 do not have a significant role in the pathogenesis of the acute local pathological effects induced by B. asper venom in mice, although this does not exclude the possibility that these cytokines play a role in other aspects of venom-induced local pathology, as well as in the reparative and regenerative responses that take place after the onset of tissue damage.

Activation of alpha(M)beta(2)-mediated phagocytosis by HF3, a P-III class metalloproteinase isolated from the venom of Bothrops jararaca.

The integrin alpha(M)beta(2) regulates important cell functions in inflammation being the primary phagocytic receptor on macrophages. HF3, a metalloproteinase isolated from Bothrops jararaca venom, is a potent hemorrhagic toxin. A cDNA encoding HF3 indicated that it is a multidomain molecule composed of a pro-domain, a catalytic domain with a zinc binding sequence, followed by disintegrin-like and cysteine-rich domains. It is known that metalloproteinases play a relevant role in the pathogenesis of venom-induced local tissue damage including inflammation. In this study we evaluated the effects of native HF3 and its recombinant disintegrin-like/cysteine-rich domains (DC-HF3) on alpha(M)beta(2)-mediated phagocytosis of opsonized-zymosan particles by macrophages. HF3 and DC-HF3 significantly increased phagocytosis and this activity was inhibited by anti-alpha(M) and anti-beta(2) antibodies. The data show the ability of P-III metalloproteinases to activate macrophages for phagocytosis through integrin alpha(M)beta(2) and suggest that the disintegrin-like/cysteine-rich domains are important for this effect. This is the first report on the activation of phagocytosis via alpha(M)beta(2) integrin by a metalloproteinase containing disintegrin-like/cysteine-rich domains.

Pharmacological characterization of mouse hind paw oedema induced by Bothrops insularis (jararaca ilhoa) snake venom.

Bothrops snake venoms produce marked local effects, including oedema, haemorrhage and necrosis. The ability of Bothrops insularis venom to induce oedema in mice was investigated. Venom was injected into hind paws and the change in volume over time was measured by plethysmometry. B. insularis venom (0.01-2.5 microg/paw) induced paw oedema which, at high doses (>/=0.5 microg/paw), was accompanied by haemorrhage. The peak oedematogenic response occurred 3 h after venom injection with all doses and decreased gradually thereafter, but was still elevated with high doses after 24 h. Pretreating the mice with cyproheptadine (histamine H(1) and serotonin 5-HT(2) receptor antagonist), mepyramine (histamine H(1) receptor antagonist), L-NAME (inhibitor of nitric oxide synthase), indomethacin and rofecoxib (inhibitors of cyclooxygenases), and dexamethasone (indirect inhibitor of PLA(2)) significantly attenuated venom-induced oedema, whereas methysergide, a serotonin 5-HT(1)/5-HT(2) receptor antagonist, had no effect. The administration of antivenom 30 min before or immediately after venom injection also significantly inhibited venom-induced oedema. These results show that B. insularis venom causes oedema in the mouse hind paw and that this response is mediated by histamine, nitric oxide, and arachidonic acid metabolites formed by cyclooxygenases 1 and 2. The neutralization by commercial antivenom indicates that the venom components responsible for oedema are recognized by the antivenom and share immunological identity with their counterparts in the venoms of mainland Bothrops species.

Increments in cytokines and matrix metalloproteinases in skeletal muscle after injection of tissue-damaging toxins from the venom of the snake Bothrops asper.

Envenomations by the snake Bothrops asper are characterized by prominent local tissue damage (i.e. myonecrosis), blistering, hemorrhage and edema. Various phospholipases A2 and metalloproteinases that induce local pathological alterations have been purified from this venom. Since these toxins induce a conspicuous inflammatory response, it has been hypothesized that inflammatory mediators may contribute to the local pathological alterations described. This study evaluated the local production of cytokines and matrix metalloproteinases (MMPs) as a consequence of intramuscular injections of an Asp-49 myotoxic phospholipase A2 (myotoxin III (MT-III)) and a P-I type hemorrhagic metalloproteinase (BaP1) isolated from B. asper venom. Both enzymes induced prominent tissue alterations and conspicuous increments in interleukin (IL)-1beta, IL-6 and a number of MMPs, especially gelatinase MMP-9, rapidly after injection. In contrast, no increments in tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma were detected. In agreement, MT-III and BaP1 did not induce the synthesis of TNF-alpha by resident peritoneal macrophages in vitro. Despite the conspicuous expression of latent forms of MMPs in muscle, evidenced by zymography, there were no increments in activated MMP-2 and only a small increase in activated MMP-9, as detected by a functional enzymatic assay. This suggests that MMP activity was regulated by a highly controlled activation of latent forms and, probably, by a concomitant synthesis of MMP inhibitors. Since no hemorrhage nor dermonecrosis were observed after injection of MT-III, despite a prominent increase in MMP expression, and since inflammatory exudate did not enhance hemorrhage induced by BaP1, it is suggested that endogenous MMPs released in the tissue are not responsible for the dermonecrosis and hemorrhage characteristic of B. asper envenomation. Moreover, pretreatment of mice with the peptidomimetic MMP inhibitor batimastat did not reduce myotoxic nor edema-forming activities of MT-III, suggesting that MMPs do not play a prominent role in the pathogenesis of these effects in this experimental model. It is concluded that MT-III and BaP1 induce a local inflammatory response associated with the synthesis of IL-1beta, IL-6 and MMPs. MMPs do not seem to play a prominent role in the acute local pathological alterations induced by these toxins in this experimental model.

Cell adhesion molecules involved in the leukocyte recruitment induced by venom of the snake Bothrops jararaca.

It has been shown that Bothrops jararaca venom (BjV) induces a significant leukocyte accumulation, mainly neutrophils, at the local of tissue damage. Therefore, the role of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), LECAM-1, CD18, leukocyte function-associated antigen-1 (LFA-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1) on the BjV-induced neutrophil accumulation and the correlation with release of LTB4, TXA2, tumor necrosis factor-alpha, interleukin (IL)-1 and IL-6 have been investigated. Anti-mouse LECAM-1, LFA-1, ICAM-1 and PECAM-1 monoclonal antibody injection resulted in a reduction of 42%, 80%, 66% and 67%, respectively, of neutrophil accumulation induced by BjV (250 microg/kg, intraperitoneal) injection in male mice compared with isotype-matched control injected animals. The anti-mouse CD18 monoclonal antibody had no significant effect on venom-induced neutrophil accumulation. Concentrations of LTB(4), TXA(2), IL-6 and TNF-alpha were significant increased in the peritoneal exudates of animals injected with venom, whereas no increment in IL-1 was detected. This results suggest that ICAM-1, LECAM-1, LFA-1 and PECAM-1, but not CD18, adhesion molecules are involved in the recruitment of neutrophils into the inflammatory site induced by BjV. This is the first in vivo evidence that snake venom is able to up-regulate the expression of adhesion molecules by both leukocytes and endothelial cells. This venom effect may be indirect, probably through the release of the inflammatory mediators evidenced in the present study.

Lipid mediators, tumor necrosis factor and nitric oxide and their interactions in immune-complex-induced lung injury

We investigated the contribution of eicosanoids, platelet-activating factor, tumor necrosis factor and nitric oxide to the neutrophil influx and development of pulmonary haemorrhagic lesions following immune-complex-induced pneumonitis in rats and possible interactions between these mediators. Increased levels of leukotriene B4 and tumor necrosis factor, measured by enzyme immunoassay and L-929 cytotoxicity assay, were found in the bronchoalveolar lavage 1 and 4 h after induction of the reaction, respectively, and their release was dependent on the previous generation of platelet activating factor. Antagonism of leukotriene B4 receptors by RO-0254094 (2-[(5-carboxypentyl])oxy]-6-[6-[3,4-dihydro-4-oxo-8-propyl-2H-1-benzopyran-7-yl)oxy]hexyl] benzenepropanoic acid), inhibition of nitric oxide synthesis by l-NAME (NW-nitro-l-arginine methyl ester) and antagonism of PAF-receptors by WEB-2170 (5-(2-chlorphenyl)-3-4-dihydro-10-methyl-3-((4-morpholinyl)carbonyl)-2H,7H-cyclopenta (4,5)thieno(3,2-f)(1,2,4)-triazolo-4,3,a)91,4)diazepine), significantly inhibited the intensity of haemorrhage, evaluated by the increased levels of extravascular hemoglobin in homogenates of lung tissues. Little evidence support the role of tumor necrosis factor in these lesions. The infiltration of neutrophils, evaluated by measuring myeloperoxidase in homogenates of lungs, was reduced by compounds L-663,536 (3-[1-(4 chlorobenzyl)-3-t-butyl thio-5-isopropylindol-2-yl]-2-2-dimethylpropanoic acid), WEB-2170 and l-NAME. These results indicate that neutrophil infiltration and haemorrhagic lesions in immune-complex-induced lung inflammation are mediated by platelet activating factor, leukotriene B4 and nitric oxide and point out to interesting interactions between these mediators.