Bothrops jararacussu snake venom lectin induces mast cell activation and vascular permeability enhance in an animal model.

Bothrops species trigger an acute inflammatory response in victims, with activated leukocytes releasing several mediators that may contribute to local and systemic effects. The effects of BjcuL, a lectin isolated from B. jararacussu snake venom, on mast cells and vasopermeability were investigated in this study. BjcuL activates mast cells and increases vasopermeability through the involvement of histamine and platelet activating factor, which may play a role in the victims' acute inflammatory reaction.

Peripheral mechanisms involved in Tityus bahiensis venom-induced pain.

Scorpionism is a public health burden in Brazil. Tityus bahiensis is responsible for most accidents in the Southeastern region of Brazil. Here, the hyperalgesic mechanisms of Tityus bahiensis venom were investigated, focusing on the role of pro-inflammatory cytokines (tumor necrosis factor alpha [TNF-α] and interleukin 1 beta [IL-1ß]) and activation of the transcription factor NFκB. Intraplantar (i.pl.) administration of Tityus bahiensis venom (0.2, 0.6, 1.2 and 2.4 µg/20 µL i.pl.) induced mechanical hyperalgesia and thermal hyperalgesia. The 2.4 µg dose of Tityus bahiensis venom induced overt pain-like behavior and increased myeloperoxidase (MPO) and N-acetyl-beta-D-glucosaminidase (NAG) activities, TNF-α and IL-1ß levels in the paw tissue. Systemic pre-treatment with etanercept (soluble TNF-α receptor; 10 mg/kg), IL-1ra (IL-1 receptor antagonist; 30 mg/kg) and pyrrolidine dithiocarbamate (PDTC, nuclear factor kappa B [NFκB] inhibitor; 100 mg/kg) inhibited Tityus bahiensis venom-induced mechanical and thermal hyperalgesia, MPO and NAG activity and overt pain-like behavior. These data demonstrate the involvement of TNF-α and IL-1ß signaling as well as NFκB activation in Tityus bahiensis venom-induced mechanical and thermal hyperalgesia, overt pain-like behavior, and MPO activity and NAG activity, indicating thus, that targeting these mechanisms might contribute to reducing the pain in this scorpionism.

A comparative study of pathophysiological alterations in scorpionism induced by Tityus serrulatus and Tityus bahiensis venoms.

Scorpionism is a relevant public health problem in several countries in tropical and subtropical regions. In Brazil, Tityus serrulatus sting can induce acute lung injury in part as a consequence of inflammation. Despite the occurrence of other scorpions of Tityus genus in Brazilian scorpiofauna, the knowledge regarding pulmonary alterations is related to T. serrulatus venom (Tsv). Here we studied, comparatively, the pathophysiological changes in the rat airways envenomed by Tsv or T. bahiensis venom (Tbv), since both scorpions are involved in human accidents but with severe envenomations occurring when victims are stung by T. serrulatus. After intravenous injection of the venoms (200 µg/kg), both were able to induce Evans blue extravasation (in 30 min) into airways and increased protein extravasation into lungs at 4 and 24 h, but the magnitude of such events was higher in Tsv group. Hemorrhage (in 60 min) in the lungs was higher in Tbv group, while IL-1ß (at 1 h) and IL-6 (at 1 and 4 h) in lung homogenates were detected only in Tsv group. Four and 24 h after envenomation, myeloperoxidase activity in lung was equally augmented in the envenomed groups, as well as an increased in polymorphonuclear cell numbers in bronchoalveolar lavage fluid. At 4 h blood leukogram showed increased leukocyte values with the highest neutrophilia in Tsv group. The numbers of leukocytes and neutrophils remained higher than control at 24 h in Tsv and Tbv groups, and it was accompanied by lympho (envenomed groups) and monocytosis (Tsv group). In conclusion, although Tbv was capable of inducing acute lung injury and blood leukocyte mobilization, most of the evaluated parameters were more affected by the Tsv. These results could help to explain the pathophysiology of the scorpionism and the clinical data arguing toward the greatest severity associated with T. serrulatus stings.

Human bronchial epithelial cells injury and cytokine production induced by Tityus serrulatus scorpion venom: An in vitro study.

Tityus serrulatus is the scorpion specie responsible for the majority of scorpion sting accidents in Brazil. Symptoms of envenomation by Tityus serrulatus range from local pain to severe systemic reactions such as cardiac dysfunction and pulmonary edema. Thus, this study has evaluated the participation of bronchial epithelial cells in the pulmonary effects of Tityus serrulatus scorpion venom (Tsv). Human bronchial epithelial cell line BEAS-2B were utilized as a model target and were incubated with Tsv (10 or 50 µg/mL) for 1, 3, 6 and 24 h. Effects on cellular response of venom-induce cytotoxicity were examined including cell viability, cell integrity, cell morphology, apoptosis/necrosis as well as cell activation through the release of pro-inflammatory cytokines IL-1ß, IL-6 and IL-8. Tsv caused a decrease in cell viability at 10 and 50 µg/mL, which was confirmed by lactate dehydrogenase (LDH) measurement. Flow cytometry analyses revealed necrosis as the main cell death pathway caused by Tsv. Furthermore, Tsv induced the release of IL-1ß, IL-6 and IL-8. Altogether, these results demonstrate that Tsv induces cytotoxic effects on bronchial epithelial cells, involving necrosis and release of pro-inflammatory cytokines, suggesting that bronchial epithelial cells may play a role in the pulmonary injury caused by Tsv.

Tityus serrulatus scorpion venom improves survival and lung inflammation in lethal sepsis induced by CLP in mice.

Tityus serrulatus venom (Tsv) modifies the behavior of immune cells and induces the production of inflammatory and anti-inflammatory cytokines; such action may interfere with physiological or pathological states. Because sepsis is characterized as an inflammatory disorder, the aim of present study was to investigate the effect of a non-lethal dose of Tsv in mice submitted to a polymicrobial infection by cecal ligation and puncture (CLP) model. The parameters evaluated were survival index, cellularity on lymphoid organs, peritoneal cavity and brochoalveolar space, production of IL-10, IL-12, IL-6, TNF-α, IFN-γ and MCP-1, pulmonary inflammation and oxidative burst. The results demonstrated that in sharp contrast to CLP group in which sepsis was lethal in a 24 h period all mice pretreated with Tsv survived even 60 h after CLP. Lung inflammation, another hallmark of CLP group, was also dramatically down regulated in Tsv/CLP group. Despite pretreatment with Tsv did not reduce the inflammatory serum cytokines when compared to CLP group; there was an increase in IL-10. In conclusion, subcutaneous Tsv administration 6 h before CLP was able to control the harmful effects of sepsis (lethality and lung inflammation). We suggest that both systemic IL-10 and oxidative burst are involved in this effect.

Tityus serrulatus venom increases vascular permeability in selected airway tissues in a mast cell-independent way.

Tityus serrulatus venom (Tsv)-induced pulmonary edema can occur in severe envenomation and the mechanisms involved are not completely understood. Therefore, we studied the effect of pharmacological modulation of the mast cell activation and the histamine antagonism on airways edema (investigated by Evans blue dye extravasation) and measured 5-hydroxytryptamine (5-HT) concentration in bronchoalveolar lavage fluid (BALF) in rats envenomed by Tsv. Additionally, the in vitro effect of Tsv on mast cells was studied using histological method and 5-HT release from mesenteric and peritoneal mast cells. We found that i.v. injection of Tsv increase vascular permeability in trachea, upper and lower bronchi and in lung parenchyma. This was not affected by ketotifen, a mast cell "stabilizer," or by pretreatment with pyrilamine (histamine H1 receptor antagonist). Moreover, 5-HT was not found in BALF of envenomed rats. In vitro experiments showed that Tsv did not induce mast cell degranulation nor release of 5-HT by mesenteric or peritoneal mast cells, in sharp contrast to preparations challenged by a mast cell activator, compound 48/80. In conclusion, our results show that Tsv causes strong edema in rat airways which is independent of mast cell activation and show that mast cells are not directly activated by Tsv.

Local and systemic biochemical alterations induced by Bothrops atrox snake venom in mice.

The local and systemic alterations induced by Bothrops atrox snake venom (BaV) injection in mice were studied. BaV induced superoxide production by migrated neutrophils, mast cell degranulation and phagocytosis by macrophages. Moreover, BaV caused hemorrhage in dorsum of mice after 2hr post- injection. Three hours post-injection in gastrocnemius muscle, we also observed myonecrosis, which was assessed by the determination of serum and tissue CK besides the release of urea, but not creatinine and uric acid, indicating kidney alterations. BaV also induced the release of LDH and transaminases (ALT and AST) indicating tissue and liver abnormalities. In conclusion, the data indicate that BaV induces events of local and systemic importance.

Immune cells recruitment and activation by Tityus serrulatus scorpion venom.

Despite several studies showed that the Tityus serrulatus scorpion venom (Tsv) induces an inflammatory response, just a few have investigated the effect of the venom on the immune response. Therefore, the aim of this study was to evaluate alterations of venom application on lymphoid organs and on the recruitment and activation of cells and also on the cytokine production. Swiss male mice (2-3 months, 20-25 g) received a non-lethal dose of crude Tsv (200 µg/kg), diluted in sterile PBS by subcutaneous route. Control animals received only sterile PBS. The animals were sacrificed after 30, 120 and 360 min. The inflammatory parameters studied were skin histology at the site of venom application, leukocyte count, and blood cytokine levels (IL-6, IL-10, and TNF-α). Inguinal lymph node, spleen and bone marrow cellularity was determined for evaluation of the Tsv effect on immune system organs. The results showed that Tsv caused no local inflammation, but it induced an increase of blood neutrophils and serum IL-6, TNF-α and IL-10. After 360 min of envenomation there was a reduction in the cells number from peritoneum and spleen, but there was an increase in the cell number from lymph nodes. In conclusion, the Tsv induces systemic alterations characterized by changes in the cell number in lymphoid organs, increase pro and anti-inflammatory cytokines.

Arthritis induced by adjuvant in spontaneously hypertensive and normotensive rats: endogenous glucocorticoid effects on inflammatory response.

The present study investigated arthritis induced by complete Freund adjuvant (AIA) in spontaneously hypertensive and normotensive rats (respectively, SHR and NTR rats). The inflammatory reaction was studied for 28 days by evaluating paw edema and secondary lesions found 10 days after complete Freund adjuvant (CFA) administration. The body weight of the animals and macroscopic alterations of several organs, including spleen, thymus, adrenal glands, and lymph nodes, were also analyzed. The results showed that the AIA manifestations were decreased in SHRs compared with NTRs. Moreover, this altered inflammatory response was not modified by surgical adrenalectomy.

Small bowel injury associated to allergy is triggered by platelet-activating factor, mast cells, neutrophils and protected by nitric oxide.

Allergy to components of the diet is followed by gut inflammation which in children, sometimes progress to mucosal lesions and anaphylaxis. In newborns suffering of cow's milk allergy, bloody stools, rectal bleeding and ulcerations are found. The rat systemic anaphylaxis is a suitable model to study the intestinal lesions associated to allergy. In the present study we used this model to investigate some mechanisms involved. We found that 15 min after antigen challenge of sensitized rats, hemorrhagic lesions develop in the small intestine. The lesions were more severe in jejunum and ileum compared to duodenum. Pretreatment of the rats with a platelet-activating factor-receptor antagonist (WEB-2170) reduced the lesions whereas inhibition of endogenous nitric oxide by l-NAME, greatly increased the hemorrhagic lesions and mortality. Both, lesions and mortality were reversed by l-arginine. The hemorrhagic lesions were also significantly reduced by the mast cell stabilizers, disodium cromoglycate and ketotifen as well as by neutrophils depletion (with anti-PMN antibodies) or inhibition of selectin binding (by treatment with fucoidan). Thus, the intestinal hemorrhagic lesions in this model are dependent on platelet-activating factor, mast cell granule-derived mediators and neutrophils. Endogenous nitric oxide and supplementation with l-arginine has a protective role, reducing the lesions and preventing mortality. These results contributed to elucidate mechanisms involved in intestinal lesions which could be of relevance to human small bowel injury associated to allergy.

Bronchoconstriction and endogenous nitric oxide in isolated lungs of spontaneously hypertensive rats.

Bronchoconstrictor responses were measured in lungs isolated from spontaneously hypertensive (SHR) and normotensive rats, perfused via the airways. Lungs from SHRs were more responsive than lungs from normotensive rats to methacholine, 5-hydroxytryptamine (5-HT), arachidonic acid or prostaglandin H(2). The responses of SHR airways to methacholine or 5-HT were unaffected by pretreatment in vivo with an inhibitor of nitric oxide (NO) synthase, N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME, 30 mg kg(-1)), although responses in normotensive airways to methacholine, but not to 5-HT, were enhanced. Antigen challenge of isolated lungs from actively sensitized rats elicited bronchoconstriction, not different between strains. Pretreatment with L-NAME increased the response to antigen challenge only in normotensive lungs. Compound 48/80 induced bronchoconstriction in lungs from either strain, equally. These responses to compound 48/80 were unaffected by L-NAME pretreatment. Thus, SHR airways lack relaxing factors and degranulation of mast cells in SHR lungs was not affected by endogenous NO.

Endogenous nitric oxide does not modulate mesenteric mast cell degranulation in rats.

The inhibitory effects of endogenous nitric oxide could explain the decreased mesenteric mast cell degranulation after anaphylaxis in genetically hypertensive rats (SHR). SHR and normotensive rats (NT) were sensitized to ovalbumin and challenged 14 days later. Degranulation of mast cells was assessed in duodenum, mesentery and skin by increased microvascular permeability using extravasation of Evans blue dye (20mg/kg, i.v.), and in the mesentery also by light microscopy after staining with toluidine blue. Pretreatment with an inhibitor of nitric oxide synthesis, L-NAME (30 mg/kg, i.v.) did not change dye extravasation after immunological challenge or after compound 48/80 in mesentery of either SHR or NT. PCA was also defective in SHR. Pretreatment with L-NAME did not affect either the defective PCA in SHR or the normal PCA reaction in NT. Our results show that inhibition by endogenous nitric oxide is not the cause of the defective mast cell degranulation in the SHR nor did it modulate degranulation of mesenteric or skin mast cells in NT.