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Ts6 and Ts2 from Tityus serrulatus venom induce inflammation by mechanisms dependent on lipid mediators and cytokine production
Abstract
Inflammatory mediators are thought to be involved in the systemic and local immune response induced by the Tityus serrulatus scorpion envenomation. New functional aspects of lipid mediators have recently been described. Here, we examine the unreported role of lipid mediators in cell recruitment to the peritoneal cavity after an injection with Ts2 or Ts6 toxins isolated from the T. serrulatus scorpion venom. In this report, we demonstrate that following a single intraperitoneal (i.p.) injection of Ts2 or Ts6 (250 μg/Kg) in mice, there was an induction of leukocytosis with a predominance of neutrophils observed at 4, 24, 48 and 96 hours. Moreover, total protein, leukotriene (LT)B(4), prostaglandin (PG)E(2) and pro-inflammatory cytokine levels were increased. We also observed an increase of regulatory cytokines, including interleukin (IL)-10, after the Ts2 injection. Finally, we observed that Ts2 or Ts6 injection in wild type (WT) 129sv mice and 5-lipoxygenase (LO) deficient mice pre-treated with LTs and PGs inhibitors (MK-886 and celecoxib, respectively) reduced the influx of leukocytes compared to WT. The recruitment of these cells demonstrated a phenotype characteristic of neutrophils, macrophages, CD4 and CD8 lymphocytes expressing GR1+, F4/80+, CD3+/CD4+ and CD3+/CD8+, respectively. In conclusion, our data demonstrate that Ts2 and Ts6 induce inflammation by mechanisms dependent on lipid mediators and cytokine production. Ts2 may play a regulatory role whereas Ts6 exhibits pro-inflammatory activity exclusively.
... Independent of the envenoming severity, intense pain is a hallmark of Ts stings, which is induced mainly by venom-derived neurotoxins [3,6] able to modulate sodium channels (NaTxs) or potassium channels (KTxs) [7][8][9][10]. Furthermore, these neurotoxins may also release histamine, 5-hydroxytryptamine, and other pro-inflammatory mediators (e.g., cytokines), amplifying the nociceptive response [11][12][13][14]. Ts venom (TsV) is composed by neurotoxins that bind specifically to voltage-gated sodium channels or Navs (e.g., Ts1, Ts2, Ts4, and Ts5) [15][16][17][18] as well as neurotoxins targeting voltage-gated potassium channels or Kvs (e.g., Ts6, Ts7, Ts8, Ts9, Ts15, and Ts19 fragments I and II) [7,[19][20][21]. ...
... Neurotoxins can act on sodium, potassium, calcium, and chloride channels being able to induce a high flux of neurotransmitters that is considered the major driver of the pathology following a scorpion sting [6]. Moreover, many of the toxins that affect ion channels are also able to activate the immune system inducing exacerbated inflammatory responses, which is a research area that has been recently explored [13,14,20,42]. ...
... Immediately after a sting caused by Ts scorpion, local pain is the primary manifestation and, although further studies are needed to lighten this event, cutaneous hyperalgesia and local swelling result in a quick local inflammation with the production of pro-inflammatory mediators, amplifying the pain intensity [14]. For instance, Ts5 toxin acts as a pro-inflammatory mediator through its ability to stimulate the release of macrophage TNF-α and IL-6 [16]. ...
Scorpion sting envenomations (SSE) are feared by the intense pain that they produce in victims. Pain from SSE is triggered mainly by the presence of neurotoxins in the scorpion venom that modulates voltage-gated ion channels. In Brazil, SSE is mostly caused by Tityus serrulatus, popularly known as yellow scorpion. Here, we evaluated experimental spontaneous nociception induced by T. serrulatus venom as well as its isolated neurotoxins Ts1, Ts5, Ts6, Ts8, and Ts19 frag II, evidencing different degrees of pain behavior in mice. In addition, we developed a mice-derived polyclonal antibody targeting Ts5 able to neutralize the effect of this neurotoxin, showing that Ts5 presents epitopes capable of activating the immune response, which decreased considerably the nociception produced by the whole venom. This is the pioneer study to explore nociception using different classes of T. serrulatus neurotoxins on nociception (α-NaTx, β-NaTx, α-KTx, and β-KTx), targeting potassium and sodium voltage-gated channels, besides demonstrating that Ts5 plays an important role in the scorpion sting induced-pain.
... Based on this, it is possible to reduce the IKur current through Kv1.5 using Osu1 and thereby influence the shape and duration of the AP in the atrium of the heart. Figure 11) [92][93][94]. Ts6 is primarily known as a peptide that inhibits Kv1.2 and Kv1.3 ion channels at nM concentrations (the IC50 values were 6.19 ± 0.35 nM for Kv1.2 and 0.55 ± 0.20 nM for Kv1.3) [92], but in the selectivity experiments, Ts6 also inhibited the current flowing through Kv1.5 at µM concentrations. Besides its effects on ion channels, Ts6 has also shown a pro-inflammatory effect by increasing the levels of cytokines, such as interleukin-6 (IL-6), both in in vivo and in vitro models [94,95]. ...
... Ts6 is primarily known as a peptide that inhibits Kv1.2 and Kv1.3 ion channels at nM concentrations (the IC50 values were 6.19 ± 0.35 nM for Kv1.2 and 0.55 ± 0.20 nM for Kv1.3) [92], but in the selectivity experiments, Ts6 also inhibited the current flowing through Kv1.5 at µM concentrations. Besides its effects on ion channels, Ts6 has also shown a pro-inflammatory effect by increasing the levels of cytokines, such as interleukin-6 (IL-6), both in in vivo and in vitro models [94,95]. IL-6 increase can cause cardiac or systemic inflammation, which in turn can rapidly lead to atrial electrical remodeling [96]. ...
... This peptide was isolated from the venom of the scorpion Tityus serrulatus. It consists of 40 amino acids with a molecular mass of 4506 Da and with 8 cysteine residues ( Figure 11) [92][93][94]. Ts6 is primarily known as a peptide that inhibits Kv1.2 and Kv1.3 ion channels at nM concentrations (the IC 50 values were 6.19 ± 0.35 nM for Kv1.2 and 0.55 ± 0.20 nM for Kv1.3) [92], but in the selectivity experiments, Ts6 also inhibited the current flowing through Kv1.5 at µM concentrations. Besides its effects on ion channels, Ts6 has also shown a pro-inflammatory effect by increasing the levels of cytokines, such as interleukin-6 (IL-6), both in in vivo and in vitro models [94,95]. ...
The human voltage gated potassium channel Kv1.5 that conducts the IKur current is a key determinant of the atrial action potential. Its mutations have been linked to hereditary forms of atrial fibrillation (AF), and the channel is an attractive target for the management of AF. The development of IKur blockers to treat AF resulted in small molecule Kv1.5 inhibitors. The selectivity of the blocker for the target channel plays an important role in the potential therapeutic application of the drug candidate: the higher the selectivity, the lower the risk of side effects. In this respect, small molecule inhibitors of Kv1.5 are compromised due to their limited selectivity. A wide range of peptide toxins from venomous animals are targeting ion channels, including mammalian channels. These peptides usually have a much larger interacting surface with the ion channel compared to small molecule inhibitors and thus, generally confer higher selectivity to the peptide blockers. We found two peptides in the literature, which inhibited IKur: Ts6 and Osu1. Their affinity and selectivity for Kv1.5 can be improved by rational drug design in which their amino acid sequences could be modified in a targeted way guided by in silico docking experiments.
... Additionally, over the years, several studies have demonstrated that venoms and toxins from different scorpions are potent activators of the immune system, changing the concept that perturbations of the autonomic nervous system are the sole drivers of pathology in scorpionism. In fact, many of the toxins that affect ion channels also induce exacerbated inflammatory responses, which is a research area that has been intensively explored by us and other researchers employing the venom of the Brazilian scorpion Tityus serrulatus (Fialho et al., 2011;Paneque Peres et al., 2009;Pessini et al., 2003;Pucca et al., 2015aPucca et al., , 2015bPucca et al., , 2015cVan Fraga et al., 2015;Zoccal et al., 2011Zoccal et al., , 2018Zoccal et al., , 2016Zoccal et al., , 2015Zoccal et al., , 2014Zoccal et al., , 2013. Other scorpion species also stimulate host immune responses; however, there are very few studies addressing the whole magnitude of immunopathological mechanisms during scorpion poisoning (Cupo, 2015;FUNASA, 2001). ...
... Zoccal et al. found that J774.1 murine macrophages responded to Ts1 and Ts6 by producing pro-inflammatory mediators, while Ts2 induced contrasting responses (Zoccal et al., 2011). Importantly, Ts2 and Ts6 induced the production of bioactive lipids involved in the inflammatory response, such as prostaglandin E 2 (PGE 2 ) and leukotriene B 4 (LTB 4 ) (Zoccal et al., 2013). ...
... Due to its pro-inflammatory properties, TsV is known to induce cellular recruitment to the site of injection, including an intense accumulation of neutrophils and macrophages early after inoculation and of lymphocytes at later time points (Zoccal et al., 2013). Interestingly, prostaglandins and leukotrienes promote the recruitment of CD4 and CD8 lymphocytes to the peritoneal cavity of mice injected with TsV, because reduced numbers of these cells were recovered after pharmacological inhibition of eicosanoids (Zoccal et al., 2013). ...
Scorpion envenomation results in a wide range of clinical manifestations that are mostly attributed to the activation of the autonomic nervous system by venom toxins. In fact, sympathetic and parasympathetic disturbances play important roles during poisoning. However, scorpion venom also induces a complex hyperinflammatory state that occurs parallel to systemic inflammatory response syndrome and acute sepsis. After a scorpion sting, innate immune cells are exposed to the venom molecules, which bind to pattern recognition receptors and activate pro-inflammatory pathways that contribute toward the promotion of severe symptoms, such as pulmonary edema, and eventually lead to death. In this review, we highlight studies that pointed out inflammation as a major pathological facet of scorpion envenomation, so as to provide novel targets to improve therapeutics for scorpionism.
... A Dunnett's multiple comparison tests were applied; the statistical tests show significant differences (**p < 0.001 ***p < 0.0001). purified toxins (Magalhaes et al., 1999;Fukuhara et al., 2003;Petricevich, 2006;Adi-Bessalem et al., 2008;Liu et al., 2008;Abdoon and Fatani, 2009;Zoccal et al., 2011Zoccal et al., , 2013Hadaddezfuli et al., 2015;Saadi et al., 2015;Zoccal et al., 2016). ...
... IL-6 and TNF-a) and the presence of anti-inflammatory cytokine IL-10, this is the most characteristic footprint of an organism or cell exposed to the whole venom or purified peptide toxins (Petricevich, 2006;Petricevich et al., 2007;Fialho et al., 2011;Zoccal et al., 2011Zoccal et al., , 2013. Recently Zoccal et al. (2016) brought a new molecular concept of envenomation, which implicate the role of Tityus serrulatus scorpion venom regulating the inflammasome (Zoccal et al., 2016). ...
... The results reported is this manuscript, in comparison to other reports is that cytokine production was detected only at incubation times larger than 5 h after in vitro exposure to purified toxins. Previous studies showing cytokine production at short times (2e5 h) used enriched cell populations or plasma samples while our studies used total PBMCs where responding cells may be a minor subset of this population (Magalhaes et al., 1999;D'Suze et al., 2003;Fukuhara et al., 2003;D'Suze et al., 2004;Petricevich, 2006;Adi-Bessalem et al., 2008;Liu et al., 2008;Abdoon and Fatani, 2009;Zoccal et al., 2011Zoccal et al., , 2013Ramirez-Bello et al., 2014;Hadaddezfuli et al., 2015;Saadi et al., 2015;Zoccal et al., 2016). ...
... Human envenomation caused by scorpion stings is a serious public health issue worldwide. The recognition of scorpion venom by mammalian cells triggers a strong inflammatory response that is characterized by increased levels of local and circulating leukocytes [1,2] and that, in severe cases, can cause pulmonary edema and death in children [3]. ...
... It has previously been reported that interleukin-1a (IL-1a), tumor necrosis factor-a (TNF-a,) interleukin-6 (IL-6), interferon-c (INF-c), granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-10 (IL-10), leukotriene B 4 (LTB 4 ), and prostaglandin E 2 (PGE 2 ) are the primary mediators induced by TsV and its purified toxins, Ts1, Ts2 and Ts6 [1,2,10,11,12,13]. Data from the literature have demonstrated that inflammatory mediators are also released during inflammatory responses that are induced as the result of the recognition of either pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) by pattern recognition receptors (PRRs) on innate immune cells [14,15,16,17,18]. ...
... Lipid mediators are involved in inflammation, the regulation of the immune response and several homeostatic biological functions [34,35,36,37,38]. Additionally, LTB 4 and PGE 2 are produced in response to Ts2 and Ts6, which are toxins that can be isolated from scorpion venom [2], and PGE 2 participates in TsV-induced edematous responses in mice [39]. To determine whether TsV and Ts1 also induce PGE 2 and LTB 4 production in peritoneal macrophages, we measured the concentration of these lipid mediators in the cell culture supernatants after 24 h of stimulation. ...
Scorpion sting-induced human envenomation provokes an intense inflammatory reaction. However, the mechanisms behind the recognition of scorpion venom and the induction of mediator release in mammalian cells are unknown. We demonstrated that TLR2, TLR4 and CD14 receptors sense Tityus serrulatus venom (TsV) and its major component, toxin 1 (Ts1), to mediate cytokine and lipid mediator production. Additionally, we demonstrated that TsV induces TLR2- and TLR4/MyD88-dependent NF-κB activation and TLR4-dependent and TLR2/MyD88-independent c-Jun activation. Similar to TsV, Ts1 induces MyD88-dependent NF-κB phosphorylation via TLR2 and TLR4 receptors, while c-Jun activation is dependent on neither TLR2 nor TLR4/MyD88. Therefore, we propose the term venom-associated molecular pattern (VAMP) to refer to molecules that are introduced into the host by stings and are recognized by PRRs, resulting in inflammation.
... We further confirmed that bvSOD3-mediated H 2 O 2 overproduction promoted the expression of proinflammatory mediators and cytokines, such as tumor necrosis factor (TNF)-α, cyclooxygenase (COX)-2, interleukin (IL)-1β, and IL-6, in mice ( Figure 2E), which demonstrated bvSOD3 s critical role in promoting the immediate inflammatory response. Previous studies have shown that venoms from venomous animals [33][34][35], including bees [16,17], induce an inflammatory response in a proinflammatory mediator-and cytokine-dependent manner. We investigated the induction of proinflammatory molecules, which was attributed to bvSOD3. ...
... Furthermore, bee venom upregulates the level of proinflammatory mediators and cytokines [16,17]. Venom from animals such as scorpions [33], scorpionfish [45], centipedes [34], and snakes [35,[46][47][48][49] induces a local inflammatory response in a proinflammatory mediator-and cytokine-dependent manner. Herein, we observed an elevation in the secretion of proinflammatory mediators and cytokine, including TNF-α, COX-2, IL-1β, and IL-6, which was attributed to bvSOD3. ...
Venoms from venomous arthropods, including bees, typically induce an immediate local inflammatory response; however, how venoms acutely elicit inflammatory response and which components induce an inflammatory response remain unknown. Moreover, the presence of superoxide dismutase (SOD3) in venom and its functional link to the acute inflammatory response has not been determined to date. Here, we confirmed that SOD3 in bee venom (bvSOD3) acts as an inducer of H2O2 production to promote acute inflammatory responses. In mouse models, exogenous bvSOD3 rapidly induced H2O2 overproduction through superoxides that are endogenously produced by melittin and phospholipase A2, which then upregulated caspase-1 activation and proinflammatory molecule secretion and promoted an acute inflammatory response. We also showed that the relatively severe noxious effect of bvSOD3 elevated a type 2 immune response and bvSOD3 immunization protected against venom-induced inflammation. Our findings provide a novel view of the mechanism underlying bee venom-induced acute inflammation and offer a new approach to therapeutic treatments for bee envenoming and bee venom preparations for venom therapy/immunotherapy.
... According to another study an immediate increase in serum cytokine concentrations of rats treated with Androctonus australis venom was observed during one hour of exposure (Adi-Bessalem et al., 2008). Elevated levels IL-4 were also observed previously in serum of humans, envenomed by Tityus serrulatus (Magalheaes et al., 1999) and in the mice which were experimentally injected with venom and Ts2 fraction from venom of T. serrulatu (Petricevich et al., 2007;Zoccal et al., 2013). IL-4 is secreted by T cells and found to suppress various pro-inflammatory cytokines produced by monocytes which may help to quell hyperactive inflammatory process (Heller et al., 2008;Barros et al., 2009). ...
... Another study suggested that venom of T. serrulatus and its different fractions (Ts2) enhance IL-6 and IL-10 production which may impose antiinflammatory effect by acting on macrophages in a dose dependent manner. According to them, same fraction was responsible for inhibition of pro-inflammatory cytokines in presence of inflammation inducer (Lipopolyscharide) (Zoccal et al., 2011(Zoccal et al., & 2013. The increase in IL-10 in response to TsV is much rapid that within 6 hours of venom treatment considerable elevation could be observed (Fialho et al., 2011). ...
Scorpion venom has great medicinal value as it contains biologically active compounds. Some of these compounds enhance production of anti-inflammatory cytokines; the mediators of hyperactive immune system. Present study is based on the fact that scorpion venom or its components have anti-inflammatory potentials by boosting anti-inflammatory mediators. Venom of Androctonus finitimus was extracted and its toxicity was determined by calculating LD50 using Probit analysis. Remedial activity of venom and its selected peptide fractions on carrageenan induced paw edema in Mus musculus (Swiss Webster albino mice) was estimated. Total leukocyte count (TLC), differential leukocyte count (DLC), and anti-inflammatory cytokines i.e., interleukin 4 (IL-4), IL-6, IL-10 and IL-13 were measured in blood of venom treated animals. Results of study illustrated that protein fractions of venom were more potent anti-inflammatory agents as compared to whole venom because animals treated with different protein fractions showed rapid recovery in paw edema as well as elevated TLC, DLC and concentrations of anti-inflammatory cytokines. As venom and protein fractions under study were found effective in healing the inflammation, further study on isolation and biochemical characterization of these fractions is recommended to use them for synthesis of new and cheap drugs for curing edema/inflammation related diseases. © 2019, Pakistan Agricultural Scientists Forum. All rights reserved.
... The neutrophils are usually the first cells type that reaches the site of injury in an immediate inflammatory response (Smith 1994). Scorpion envenoming induces also an activation of the cytokines (IL-6, IL-1, and TNFa), and lipid-derived mediators of inflammation including platelet activator factor (PAF), leukotrienes, and prostaglandins , Zoccal et al. 2013, Costal-Oliveira et al. 2015, Zoccal et al. 2015. Obtained results indicate that Aah venom-induced elevations of all tested cytokines in kidney homogenates with maximum production at 24 h after venom administration. ...
... Obtained results indicate that Aah venom-induced elevations of all tested cytokines in kidney homogenates with maximum production at 24 h after venom administration. Similar findings reported the involvement of cytokines in the systemic inflammatory response and multiple organ failures after scorpion envenoming (Petricevich 2010, Saidi et al. 2013, Zoccal et al. 2013. Increased levels of cytokines are responsible for the release of large amounts of the production of free radicals and may contribute to the induction of oxidative stress in the kidneys. ...
The effect of Androctonus australis hector (Aah) venom in comparison with nephrotoxic model was investigated in vivo. Aah venom induced a rapid inflammatory response characterized by recruitment of neutrophil and eosinophil cells, increased levels of cytokines, and lipid peroxidation. These results were in accordance with the histological findings showing structural damage in the nephrons, similar and faster to that induced by cisplatin model. The modifications in renal function parameters as an elevation of blood urea and creatinine levels with a significant increase in the serum aspartate aminotransferase and lactate dehydrogenase were indicative of tissue damages responsible for the renal failure.
... In patients with moderate to severe systemic manifestations, leukocytosis and high plasma levels of the inflammatory cytokines IL-6, TNF-α, IL-8, IL1-β, INF-γ, GM-CSF, and RANTES, as well as the anti-inflammatory cytokines IL-10 and IL-4, are observed [113][114][115][116][117]. A positive correlation between increased levels of cytokines and the severity of scorpion envenomation in humans was described [113]. In this context, studies with experimental models of severe scorpion envenomation have demonstrated that multiple organ dysfunction and death were associated with an increased production of inflammatory cytokines [110,[118][119][120][121][122][123]. The role of IL-6 and TNF-α in hyperglycemia and metabolic deregulation described in severe envenomation by scorpion stings was evidenced after injection of Androctonus australis hector venom in mice [124]. ...
... Participation of eicosanoids derived from the cyclooxygenase (COX) pathway in hyperthermia caused by the scorpion Leiurus quinquestriatus sting in children has been shown, since treatment of patients with nonsteroidal anti-inflammatory drugs ameliorated the venom-induced effect [125]. In line with this report, experimental studies have demonstrated that prostaglandins released by the scorpion venom also have a role in the cardiorespiratory, edematogenic, and nociceptive effects induced by the injection of this venom in experimental animals [126][127][128][129]. Release of PGE 2 and LTB 4 induced by neurotoxins Ts2 and Ts6 isolated from T. serrulatus venom, as well as participation of these mediators in the leukocyte infiltration into the site of injection of these toxins, has been described [123]. With regard to the molecular mechanisms underlying the biosynthesis of eicosanoids, the role of peroxisome proliferator-activated receptor gamma (PPAR-γ) was experimentally shown in macrophages stimulated by T. serrulatus venom [130]. ...
... IL-1Ra was kindly provided by Dr Stephen Poole, from the National Institute for Biological Standards and Control (South Mimms, Hertfordshire, UK). In a specific experiment, the mice were either treated or not treated with MK886 (5-LO inhibitor, 5 mg kg À 1 i.p., in 200 ml of 1% alcohol in water) 64 , indomethacin (COX1/2 inhibitor, 2 mg kg À 1 i.p. in 200 ml of Tris[hydroxymethyl]aminomethane-HCl; TRIS-HCl, pH 8.2) 65 , SC-560 (selective COX1 inhibitor, 3 mg kg À 1 i.p., in 200 ml of PBS, Sigma-Aldrich) 66 , celecoxib ( COX2 inhibitor, 5 mg kg À 1 i.p., in 200 ml of water) 64 or EP2 antagonist (AH6809, 5 mg kg À 1 i.p., in 200 ml of PBS, Cayman Chemical) 67 . The drugs (MK886 or indomethacin) or vehicles were administered four times, at 4 h and 0.5 h before and again 4 and 8 h after the lethal dose of TsV. ...
... IL-1Ra was kindly provided by Dr Stephen Poole, from the National Institute for Biological Standards and Control (South Mimms, Hertfordshire, UK). In a specific experiment, the mice were either treated or not treated with MK886 (5-LO inhibitor, 5 mg kg À 1 i.p., in 200 ml of 1% alcohol in water) 64 , indomethacin (COX1/2 inhibitor, 2 mg kg À 1 i.p. in 200 ml of Tris[hydroxymethyl]aminomethane-HCl; TRIS-HCl, pH 8.2) 65 , SC-560 (selective COX1 inhibitor, 3 mg kg À 1 i.p., in 200 ml of PBS, Sigma-Aldrich) 66 , celecoxib ( COX2 inhibitor, 5 mg kg À 1 i.p., in 200 ml of water) 64 or EP2 antagonist (AH6809, 5 mg kg À 1 i.p., in 200 ml of PBS, Cayman Chemical) 67 . The drugs (MK886 or indomethacin) or vehicles were administered four times, at 4 h and 0.5 h before and again 4 and 8 h after the lethal dose of TsV. ...
Tityus serrulatus sting causes thousands of deaths annually worldwide. T. serrulatus-envenomed victims exhibit local or systemic reaction that culminates in pulmonary oedema, potentially leading to death. However, the molecular mechanisms underlying T. serrulatus venom (TsV) activity remain unknown. Here we show that TsV triggers NLRP3 inflammasome activation via K+ efflux. Mechanistically, TsV triggers lung-resident cells to release PGE2, which induces IL-1β production via E prostanoid receptor 2/4-cAMP-PKA-NFκB-dependent mechanisms. IL-1β/IL-1R actions account for oedema and neutrophil recruitment to the lungs, leading to TsV-induced mortality. Inflammasome activation triggers LTB4 production and further PGE2 via IL-1β/IL-1R signalling. Activation of LTB4-BLT1/2 pathway decreases cAMP generation, controlling TsV-induced inflammation. Exogenous administration confirms LTB4 anti-inflammatory activity and abrogates TsV-induced mortality. These results suggest that the balance between LTB4 and PGE2 determines the amount of IL-1β inflammasome-dependent release and the outcome of envenomation. We suggest COX1/2 inhibition as an effective therapeutic intervention for scorpion envenomation.
... Previously studies have reported significant anti-tumor activity of scorpion venom (Jain and Kumar, 2012;Ortiz et al., 2015;Liu et al., 2003). A previous in vitro study reported that the toxic components from Tityus serrulatus scorpion (Ts1, Ts2 and Ts6) induce different types of immune response following macrophage activation (Zoccal et al., 2011(Zoccal et al., , 2013. While Ts1 and Ts6 stimulated the production of NO, interleukin (IL)-6 and tumor necrosis factor (TNF)-a, Ts2 stimulated the production of the anti-inflammatory cytokine IL-10. ...
... However, this hypothesis needs to be confirmed in further studies where lower non-toxic concentrations are tested. A previous study on the venom from T. serrulatus scorpion (Zoccal et al., 2011(Zoccal et al., , 2013, 100 mg/ml concentration of the raw venom which produced 12% cytotoxicity, measured by MTT assay, was among the employed concentrations for assessment of the immune stimulating or modulating efficacies, while the initial lowest concentration employed in our study, measured by LDH assay, produced 28% cytotoxicity. ...
... Previously studies have reported significant anti-tumor activity of scorpion venom (Jain and Kumar, 2012;Ortiz et al., 2015;Liu et al., 2003). A previous in vitro study reported that the toxic components from Tityus serrulatus scorpion (Ts1, Ts2 and Ts6) induce different types of immune response following macrophage activation (Zoccal et al., 2011(Zoccal et al., , 2013. While Ts1 and Ts6 stimulated the production of NO, interleukin (IL)-6 and tumor necrosis factor (TNF)-a, Ts2 stimulated the production of the anti-inflammatory cytokine IL-10. ...
... However, this hypothesis needs to be confirmed in further studies where lower non-toxic concentrations are tested. A previous study on the venom from T. serrulatus scorpion (Zoccal et al., 2011(Zoccal et al., , 2013, 100 mg/ml concentration of the raw venom which produced 12% cytotoxicity, measured by MTT assay, was among the employed concentrations for assessment of the immune stimulating or modulating efficacies, while the initial lowest concentration employed in our study, measured by LDH assay, produced 28% cytotoxicity. ...
Abstract Hemiscorpius lepturus (H. lepturus), one of the most venomous scorpions in tropical and sub-tropical areas, belongs to the Hemiscorpiidae family. Studies of antibodies in sera against the protein component of the venom from this organism can be of great use for the development of engineered variants of proteins for eventual use in the diagnosis/treatment of, and prevention of reactions to, stings. In the present in vitro study, the proteins of H. lepturus venom, which could specifically activate the production of immunoglobulin G (IgG) in victims accidently exposed to the venom from this scorpion, were evaluated and their cross-reactivity with venoms from two other important scorpion species including Androctonus crassicauda and Mesobuthus eupeus assessed. H. lepturus venom was analyzed with respect to its protein composition and its antigenic properties against antibodies found in sera collected from victims exposed to the venom of this scorpion within a previous 2-month period. The cross-reactivity of the H. lepturus venom with those from A. crassicauda and M. eupeus was assessed using ELISA and immunoblotting. Electrophoretic analysis of the venom of H. lepturus revealed several protein bands with weights of 8-116 KDa. The most frequent IgG-reactive bands in the test sera had weights of 34, 50, and 116 kDa. A weak cross-reactivity H. lepturus of venom with venoms from A. crassicauda and M. eupeus was detected. The results of immunoblotting and ELISA experiments revealed that H. lepturus venom activated the host immune response, leading to the production of a high titer of antibodies. Clearly, a determination of the major immunogenic components of H. lepturus venom could be valuable for future studies and ultimately of great importance for the potential production of recombinant or hypo-venom variants of these proteins.
... The presence of DNA damage induced by different toxins is also reported on literature (Kang et al., 2011;Marcussi et al., 2011;. Specifically, on T. serrulatus it is known that Ts2 and Ts6 toxins are capable of affecting the immune system and trigger inflammation in human cells (Zoccal et al., 2013), could result in the formation of ROS causing DNA damage. Cells in CD process, or that present inadequate transport of K + ions do not divide (Lebaudy;Véry;Sentenac, 2007). ...
The effects of Tityus serrulatus venom was accessed on the cell cycle and genetic material of the Lactuca sativa L. and compared with the damages to human leukocytes, in order to evaluate the genotoxic and mutagenic potential of toxins on cell types very different. The occurrence of cell cycle alterations and DNA fragmentation was evaluated in Lactuca sativa. Incubations of venom with human leukocytes were also held with subsequent evaluation of cell proliferation index, micronucleus and fragmented DNA. The T. serrulatus venom showed cytogenotoxic, reducing mitotic index and induced alterations in the cell cycle and micronuclei formation. The occurrence of cell death was evidenced by the detection of condensed nuclei, positive TUNEL signals, and presence of DNA fragmentation on lettuce cells. The scorpion venom induced DNA fragmentation and micronuclei in leukocytes. Further, the role of peptides and proteases from this venom in inducing the observed damage was discussed.
... Gamma irradiation of honey bee venom resulted in a decrease in IL-10 by reducing allergen compounds (histamine and PLA 2 ) [35,41]. IL-10 and the antiinflammatory compounds of the irradiated honey bee venom in part are involved in fundamental homeostatic mechanisms that control the degree and duration of the inflammatory response [34,47]. These results are in agreement with the histopathological and RT-PCR findings observed in our assays. ...
In this study, the effect of gamma-irradiated honey bee venom (doses of 0, 2, 4, 6, and 8 kGy, volume of 0.1 ml and concentration of 0.2 mg/ml) was evaluated on the reduction of allergen compounds and the gene expression of inflammatory and anti-inflammatory cytokines in mice. Hence, edema activity induced by the bee venom irradiated at 4, 6, and 8 kGy was reduced, compared with the control group and that irradiated at 2 kGy. In contrast, the paw edema induced by the bee venom irradiated at 8 kGy increased, compared with 4 and 6 kGy. At all the time periods, there was a significant decrease in the gene expression of interferon gamma (IFN-γ), interleukin 6 (IL-6), and interleukin 10 (IL-10) in the bee venoms irradiated at 4, 6, and 8 kGy, compared with the control group and that irradiated at 2 kGy. In contrast, there was an increase in the gene expression of IFN-γ and IL-6 in the bee venom irradiated at 8 kGy, compared with those irradiated at 4 and 6 kGy. Therefore, gamma irradiation at 4 and 6 kGy reduced the gene expression of cytokines at each time period by decreasing the allergen compounds of honey bee venom.
... This β-toxin binds to TLR2 and TLR4, inducing the production of cytokines and lipid mediators in a MyD88-dependent manner, leading to the activation of proinflammatory signaling, such as the NF-κB, AP-1 or MAPK pathways (ERK1/2 and MAPK p38) [19]. The greatest toxic effect of Ts1 occurs at the level of sodium channels [20][21][22][23]. MT-III is a group IIA secreted phospholipase A2 (sPLA2) present in Bothrops atrox and Bothrops asper snake venom and is also recognized by TLR2, leading to the production of eicosanoids such as prostaglandin E2 (PGE2) and leukotriene B4 (LTB4), which are potent neutrophil chemoattractants that favor the resolution of the acute response at the injury site [24,25]. ...
Venoms are products of specialized glands and serve many living organisms to immobilize and kill prey, start digestive processes and act as a defense mechanism. Venoms affect different cells, cellular structures and tissues, such as skin, nervous, hematological, digestive, excretory and immune systems, as well as the heart, among other structures. Components of both the innate and adaptive immune systems can be stimulated or suppressed. Studying the effects on the cells and molecules produced by the immune system has been useful in many biomedical fields. The effects of venoms can be the basis for research and development of therapeutic protocols useful in the modulation of the immunological system, including different autoimmune diseases. This review focuses on the understanding of biological effects of diverse venom on the human immune system and how some of their components can be useful for the study and development of immunomodulatory drugs.
... Eicosanoids are lipid signaling molecules derived from the enzymatic oxygenation of arachidonic acid (AA) which are generated by cycloxygenase (COXs) and lipoxygenase (LOXs) pathways. These enzymes are well known mediators and regulators of inflammation, which the important role is to control key cellular processes (Zoccal et al., 2013). Many reports reveal that the lipid mediators induced by animal's venom are involved in inflammation and homeostatic biological functions. ...
The ability of Russell's viper (Daboia siamensis) venom (total RVV) and phospholipase A2 (purified PLA2) to induce the local pathological effects were investigated by the local inflammatory events and the release of inflammatory mediators. Both 0.5 μg of total RVV/mouse and 0.15 μg of purified PLA2/mouse were administered via intra-peritoneal injection. After 30 min, 1 h, 2 h, and 4 h incubation time, the peritoneal cavity was flooded with normal saline and the total leukocytes were collected. The eicosanoids (lipid mediators) and the leukocyte expression of cyclooxygenase (COX-1 and COX-2) were investigated by ELISA assay and western blotting, respectively. The amounts of total leukocytes were increased from 30 min to 2 h, then decreased at 4 h, by both total RVV and purified PLA2. Both treatments also induced the expression of COX-2 which was increased at 2 h and then decreased at 4 h, whereas only purified PLA2 induced the expression level of a COX-1 protein which was increased at 30 min, then constantly expressed until 4 h. In addition, total RVV and purified PLA2 caused the release of the eicosanoids; PGE2, TXB2, and LTB4, which reached the peak after 2 h. The findings of this study indicate that purified PLA2 has the potential effects to induce the local inflammation relating the amounts of leukocytes cells, lipid mediators and COX-2 more than the total RVV.
... Moreover, 5-LO −/− mice showed a systemic increase of IL-6 and MCP-1. This genetically deficient lineage had been already used in a previous study with the Tityus serrulatus scorpion's venom [32], in which the animals developed attenuated inflammatory response. This validates the used experimental model, and shows the complexity of interactions between different toxins from animal venoms and the immune response. ...
Bitis arietans is a snake of medical importance, as it is responsible for more accidents in humans and domestic animals than all other African snakes put together. The accidents are characterized by local and systemic alterations, such as inflammation, cardiovascular and hemostatic disturbances, which can lead victims to death or permanent disability. However, little is known about the envenomation mechanism, especially regarding the inflammatory response, which is related to severe clinical conditions triggered by the venom. Therefore, the aim of the present study was to evaluate the inflammatory response related to the B. arietans envenomation using a peritonitis mice model. By pharmacological interventions and use of mice genetically deficient of the 5-lipoxygenase enzyme (5-LO-/-) or platelet-activating factor (PAF) receptor (PAFR-/- the participation of eicosanoids and PAF in this response was also investigated. The obtained results demonstrated that the venom induces an in vivo inflammatory response, characterized by an early increased vascular permeability, followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity, accompanied by the production of the eicosanoids LTB4, LTC4, TXB2 and PGE2, as well as the local and systemic production of IL-6 and MCP-1. These inflammatory events were attenuated by the pre-treatment with anti-inflammatory drugs that interfere in lipid mediators' functions. However, 5-LO-/- mice did not show a reduction of inflammatory response induced by the venom, while PAFR-/- mice showed a reduction in both the PMN leukocytes number and the local and systemic production of IL-6 and MCP-1. This study demonstrated that the Bitis arietans venom contains toxins that trigger an inflammatory process, which is partially dependent on lipid mediators, and may contribute to the envenomation pathology.
... Pattern recognition receptors (PRRs), such as Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), cluster of differentiation 14 (CD14), and cluster of differentiation 36 (CD36), recognize TsV toxins and activate a range of molecular pathways in innate immune cells [7][8][9][10]. These interactions activate transcriptional factors such as activator protein 1 (AP-1), nucelar factor kappa-light-chain-enhancer of activated B cells (NF-κB), and peroxisome proliferator-activated receptor gamma (PPAR-γ), which induce the formation of lipid bodies, as well as the production of eicosanoids and cytokines. ...
Tityus serrulatus causes numerous scorpion envenomation accidents and deaths worldwide. The symptoms vary from local to systemic manifestations, culminating in pulmonary edema and cardiogenic shock. Among these events, transitory hyperglycemia is a severe manifestation that influences pulmonary edema, hemodynamic alterations, and cardiac disturbances. However, the molecular mechanism that leads to increased glucose levels after T. serrulatus envenomation remains unknown. This study aimed to investigate our hypothesis that hyperglycemia due to scorpion envenomation involves inflammatory signaling in the pancreas. The present study showed that T. serrulatus venom induces the production of IL-1α and IL-1β in the pancreas, which signal via IL-1R and provoke nitric oxide (NO) production as well as edema in β-cells in islets. Il1r1-/-mice were protected from transitory hyperglycemia and did not present disturbances in insulin levels in the serum. These results suggest that the pathway driven by IL-1α/IL-1β-IL-1R-NO inhibits insulin release by β-cells, which increases systemic glucose concentration during severe scorpion envenomation. A supportive therapy that inhibits NO production, combined with antiserum, may help to prevent fatal outcomes of scorpion envenomation. Our findings provide novel insights into the design of supportive therapy with NO inhibitors combined with antiscorpion venom serum to overcome fatal outcomes of scorpion envenomation. Key Contribution: TsV induces the production of IL-1α/IL-1β in pancreas, which binds to IL-1R in β-cells of islets, inducing NO production and inhibiting insulin secretion. NO inhibition (pharmacological) rescues animals from transitory hyperglycemia during scorpion envenomation.
... AMPs can be found in venom glands of many animals such as scorpions [9,10]. The scorpion venom peptides can be classified in two groups: disulfide bridge peptides (DBPs) and non-disulfide bridge peptides (NDBPs). ...
Antimicrobial peptides (AMPs) are small molecules with microbicidal and immunoregulatory activities. In this study we evaluated the anti-inflammatory and antimicrobial activities of peptides ToAP3 and ToAP4, AMPs from the venom of the Brazilian scorpion Tityus obscurus. To test the peptides' activity, murine bone marrow-derived macrophages (BMDMs) or dendritic cells (BMDCs) were stimulated with peptides plus LPS to analyze their ability to modulate cytokine release as well as phenotypic markers. For antimicrobial analysis, we evaluated the indirect activity against macrophage-internalized Cryptococcus neoformans and direct activity against Mycobacterium massiliense. Our data demonstrate that they were able to reduce TNF-α and IL-1β transcript levels and protein levels for BMDM and BMDC. Furthermore, the reduction of TNF-α secretion, before LPS-in-flammatory stimuli, is associated with peptide interaction with TLR-4. ToAP4 increased MHC-II expression in BMDC, while ToAP3 decreased co-stimulatory molecules such as CD80 and CD86. Although these peptides were able to modulate the production of cytokines and molecules associated with antigen presentation, they did not increase the ability of clearance of C. neoformans by macrophages. In antimicrobial analysis, only ToAP3 showed potent action against bacteria. Altogether, these results demonstrate a promising target for the development of new immunomodulatory and anti-bacterial therapies.
... In recent years, it became clear that beyond the classical neuroexcitatory syndrome, inflammation also underlies the pathology and mortality caused by TsV. We and others have demonstrated that crude TsV or isolated toxins (Ts1, Ts2, Ts6) induce the production of inflammatory mediators by innate immune cells, including cytokines, nitric oxide (NO), and bioactive lipids such as prostaglandin E 2 (PGE 2 ) and leukotriene B 4 (LTB 4 ) (Petricevich, 2002;Petricevich and Lebrun, 2005;Petricevich et al., 2007;Zoccal et al., 2011Zoccal et al., , 2013. Macrophages sense TsV via TLR2, TLR4, or CD14, which control the release of PGE 2 and interleukin-1β (IL-1β) (Zoccal et al., 2014(Zoccal et al., , 2016(Zoccal et al., , 2018b. ...
Over 1 million cases of scorpion stings are estimated every year, whereas current treatment is limited to antivenom serum combined with supportive therapy. Tityus serrulatus scorpion venom (TsV) is composed of diverse molecules, including toxins that induce a catecholamine storm and mediate classical symptoms of scorpion envenomation. However, the same toxins promote an intense inflammatory response coordinated by innate immune cells, such as macrophages, contributing significantly to the lung edema and mortality caused by TsV injection. Macrophages sense TsV via innate immune receptors, including TLR2, TLR4, and CD14 that promote inflammation and mortality via PGE2/cAMP/PKA/NF-κB/IL-1β axis. The scavenger receptor CD36 also recognizes TsV, but in contrast to the other receptors, it drives the production of leukotriene B4 (LTB4). This lipid mediator operates via BLT1 receptor to reduce cAMP production and consequently IL-1β release, which results in resistance to fatal outcomes of experimental scorpion envenomation. EP80317 is an hexapeptide that serves as a ligand for CD36 and features protective effects under conditions such as atherosclerosis and vascular inflammation. In this study, we evaluated the effects of EP80317 treatment during experimental scorpion envenomation. EP80317 treatment suppressed mouse peritoneal macrophage production of IL-1β, IL-6, tumor necrosis factor (TNF-α), CCL3, and PGE2in vitro. EP80317 treatment also boosted the production of LTB4 and IL-10 in response to TsV. Importantly, EP80317 restrained lung inflammation and mortality caused by TsV in vivo. Taken together, these data indicate a strong therapeutic potential of EP80317 as a supportive treatment to control inflammation induced by scorpion envenomation.
... Androctonus australis hector venom increases pro-inflammatory (IL-1β, IL-6, TNF-α) and antiinflammatory cytokine levels (IL-4, IL-10) and activates the complement system, effects that are correlated with tissue damage [20,35]. Scorpion envenomation also induces other cytokines (IL-4, IL-5, and IFNϒ) as well as lipid-derived mediators of inflammation, including platelet activator factor (PAF), leukotrienes, and prostaglandins [20, [46][47][48] (Figure 1). Indeed, bioactive lipids play a critical role in initiating and resolving inflammation. ...
Because of their venom lethality towards mammals, scorpions of the Androctonus genus are considered a critical threat to human health in North Africa. Several decades of exploration have led to a comprehensive inventory of their venom components at chemical, pharmacological, and immunological levels. Typically, these venoms contain selective and high affinity ligands for the voltage-gated sodium (Nav) and potassium (Kv) channels that dictate cellular excitability. In the well-studied Androctonus australis and Androctonus mauretanicus venoms, almost all the lethality in mammals is due to the so-called α-toxins. These peptides commonly delay the fast inactivation process of Nav channels, which leads to increased sodium entry and a subsequent cell membrane depolarization. Markedly, their neutralization by specific antisera has been shown to completely inhibit the venom’s lethal activity, because they are not only the most abundant venom peptide but also the most fatal. However, the structural and antigenic polymorphisms in the α-toxin family pose challenges to the design of efficient serotherapies. In this review, we discuss past and present accomplishments to improve serotherapy against Androctonus scorpion stings.
... The venom-gland transcriptome of T. serrulatus also revealed the presence of NaTxs, KTxs, AMPs and MPs (Alvarenga et al., 2012), most of which were later proteomically confirmed in the venom along with CRISPs and HYALs (de Oliveira et al., 2018). Several isolated T. serrulatus toxins have also been functionally characterized (Zoccal et al., 2011(Zoccal et al., , 2013Guo et al., 2013;Pucca et al., 2015b,c;2016a, 2016bCerni et al., 2017). ...
Scorpions are an ancient and diverse venomous lineage, with over 2200 currently recognized species. Only a small fraction of scorpion species are considered harmful to humans, but the often life-threatening symptoms caused by a single sting are significant enough to recognize scorpionism as a global health problem. The continued discovery and classification of new species has led to a steady increase in the number of both harmful and harmless scorpion species. The purpose of this review is to update the global record of medically significant scorpion species, assigning each to a recognized sting class based on reported symptoms, and provide the major toxin classes identified in their venoms. We also aim to shed light on the harmless species that, although not a threat to human health, should still be considered medically relevant for their potential in therapeutic development. Included in our review is discussion of the many contributing factors that may cause error in epidemiological estimations and in the determination of medically significant scorpion species, and we provide suggestions for future scorpion research that will aid in overcoming these errors.
... Inset: Representative densitometry of electrophoretic scan of the negative control, Ts venom (TsV 20 mg, positive control) and Ts19 Frag-II (8 mg). , 2008Zoccal et al., 2011;Zoccal et al., 2013) and humans (Fukuhara et al., 2003). In our study, Ts venom was able to significantly increase IL-6 cytokine production with no changes in the TNF-a levels. ...
Tityus serrulatus (Ts) venom is composed of a mixture of toxins presenting diverse biological functions. However, although this venom has been studied over the past three decades, omics analysis revealed that most of its toxins are not identified or their biological activities are unknown. Ts19 Frag-II is included is this group, which function is still uncertain. This study aimed to expand the biological activities of Ts19 Frag-II through in vivo investigation. Our results demonstrates that mice challenged with Ts19 Frag-II presented biochemical alterations, increasing serum levels of urea, ALT and β-globulin, besides decreasing γ-globulins. Moreover, this toxin was also able to induce immunological alterations, increasing NO, IL-6, TNF-α and IL-17, being considered a proinflammatory toxin. The increase of IL-17 was unprecedented regarding Ts toxins and could be a result of the overall produced-effect of cells of innate immunity (neutrophils, monocytes, natural killers and lymphoid tissue inducers - LTis) cells) as well as of adaptive immunity (Th17 cells). This study expanded the biological activities of Ts19 Frag-II, suggesting that this toxin could be contributing to the Ts envenoming through alterations of biochemical parameters as well as triggering the inflammatory response.
... Here, we demonstrated that Bj-CRP should also contribute to the inflammatory responses of B. jararaca venom, inducing the migration of neutrophils and the production of the cytokine IL-6 in the first hours after inoculation into the peritoneal cavity of mice. These results corroborate with other toxins from Tityus serrulatus scorpion venom (Pessini et al., 2003;Zoccal et al., 2013), and are consistent with the literature, since IL-6 is associated with the initiation and maintenance of inflammation (Fielding et al., 2008), appearing in increasing levels at the beginning of this process. This cytokine is a proinflammatory mediator produced by various cells, such as B and T lymphocytes, monocytes, fibroblasts and endothelial cells (Ataie-Kachoie et al., 2013), being responsible for the migration of neutrophils to the tissues, the activation of their maturation and the regulation of adhesion molecules involved in leukocyte adhesion to the endothelium (Suwa et al., 2002;Teixeira et al., 2009;Kishimoto, 2006). ...
Cysteine-rich secretory proteins (CRISPs) are commonly described as part of the protein content of snake venoms, nevertheless, so far, little is known about their biological targets and functions. Our study describes the isolation and characterization of Bj-CRP, the first CRISP isolated from Bothrops jararaca snake venom, also aiming at the identification of possible targets for its actions. Bj-CRP was purified using three chromatographic steps (Sephacryl S-200, Source 15Q and C18) and showed to be an acidic protein of 24.6 kDa with high sequence identity to other snake venom CRISPs. This CRISP was devoid of proteolytic, hemorrhagic or coagulant activities, and it did not affect the currents from 13 voltage-gated potassium channel isoforms. Conversely, Bj-CRP induced inflammatory responses characterized by increase of leukocytes, mainly neutrophils, after 1 and 4 h of its injection in the peritoneal cavity of mice, also stimulating the production of IL-6. Bj-CRP also acted on the human complement system, modulating some of the activation pathways and acting directly on important components (C3 and C4), thus inducing the generation of anaphylatoxins (C3a, C4a and C5a). Therefore, our results for Bj-CRP open up prospects for better understanding this class of toxins and its biological actions.
... Moreover, Nav channels are expressed mainly in sensory neurons (Dib-Hajj et al., 2010), easily reached by subcutaneous or intraplantar injection of toxins. The redundancy of action of these toxins, their promiscuity to different channels, the induced-production of inflammatory mediators such as cytokines (IL-6, TNF-a, IL-1b) and PGE2 e LBT4 (Cerni et al., 2016;Pessini et al., 2008;Pucca et al., 2015c;Zoccal et al., 2011Zoccal et al., , 2013Zoccal et al., , 2014Zoccal et al., , 2015Zoccal et al., , 2016 along with Ts8 inhibiting effect on Kv4.2 could explain the painful sting triggered by Ts scorpion envenomation. Fig. 8 describes the possible mechanism Ts venom enhances pain sensitivity, based on our results and previous studies. ...
The venom from the scorpion Tityus serrulatus (Ts) has been extensively studied mainly because of its rich cocktail of neurotoxins. Neurotoxins are the major and the most known components based on their modulation of voltage-gated ion channels. Until now, electrophysiological studies demonstrated that the Ts venom comprises toxins that affect Nav and Kv channels. However, although many studies have been conducted in this field, many peptides from Ts venom await further studies, including Ts8 toxin. Here we report the isolation and electrophysiological study of Ts8. The toxin Ts19 Frag-II was used as negative control. Ts8 demonstrates, among 20 tested channels, to be a selective modulator of Kv4.2 channels. Based on studies investigating the involvement of Kv4.2 on controlling nociception, we further investigated the modulation of pain by Ts8. Using intraplantar injections, Ts8 induced overt nociception (licking and lifting behaviors) and decreased the mechanical nociceptive threshold (hyperalgesia). Furthermore, the hyperalgesia was prolonged when intrathecal injections were performed. Independent of the severity, most of the victims stung by Ts scorpions report an intense and persistent pain as the major manifestation. The new role of Ts8 on nociception could explain, at least partially, this phenomenon. Additionally, our study also stresses the involvement of toxins specific to Nav channels and inflammatory mediators on the Ts painful sting. This work provides useful insights for a better understanding of the prolonged and intense pain associated with Ts envenoming for the development of specific therapies.
... Scorpion venoms and their isolated toxins are responsible for several immunological properties (e.g., inflammation) observed after scorpion envenoming [9][10][11]. Neurotoxins specific for voltage-gated K + and Na + channels can affect many cells, such as macrophages, which participate in the inflammatory response of Ts envenoming [12,13]. ...
Background:
The yellow scorpion Tityus serrulatus (Ts) is responsible for the highest number of accidents and the most severe scorpion envenoming in Brazil. Although its venom has been studied since the 1950s, it presents a number of orphan peptides that have not been studied so far. The objective of our research was to isolate and identify the components present in the fractions VIIIA and VIIIB of Ts venom, in order to search for a novel toxin. The major isolated toxins were further investigated for macrophage modulation.
Methods:
The fractions VIIIA and VIIIB, obtained from Ts venom cation exchange chromatography, were rechromatographed on a C18 column (4.6 × 250 mm) followed by a reversed-phase chromatography using another C18 column (2.1 × 250 mm). The main eluted peaks were analyzed by MALDI-TOF and Edman's degradation and tested on macrophages.
Results:
The previously described toxins Ts2, Ts3-KS, Ts4, Ts8, Ts8 propeptide, Ts19 Frag-II and the novel peptide Ts19 Frag-I were isolated from the fractions VIIIA and VIIIB. Ts19 Frag-I, presenting 58 amino acid residues, a mass of 6,575 Da and a theoretical pI of 8.57, shares high sequence identity with potassium channel toxins (KTx). The toxins Ts4, Ts3-KS and the partially purified Ts19 Frag-I did not produce cytotoxic effects on macrophage murine cells line (J774.1). On the other hand, Ts19 Frag-I induced the release of nitric oxide (NO) by macrophages, while Ts4 and Ts3-KS did not affect the NO production at the tested concentration (50 μg/mL). At the same concentration, Ts19 Frag-I and Ts3-KS increased the production of interleukin-6 (IL-6). Ts19 Frag-I and Ts4 did not induce the release of IL-10, IL-1β or tumor necrosis factor-α by macrophage cells using the tested concentration (50 μg/mL).
Conclusions:
We partially purified and determined the complete sequence and chemical/physical parameters of a new β-KTx, denominated Ts19 Frag-I. The toxins Ts4, Ts3-KS and Ts19 Frag-I showed no cytotoxicity toward macrophages and induced IL-6 release. Ts19 Frag-I also induced the release of NO, suggesting a pro-inflammatory activity.
... In vitro, Ts venom induced an increase of IL-6 and IFN-g production in the supernatants of peritoneal macrophages (Petricevich, 2002). Moreover, isolated Ts toxins were extensively investigated in the macrophage immunomodulation: Ts1 was able to increase the production of IL-6 and TNF-a; Ts2 stimulated the production of TNF-a, IL-10 and inhibited NO release; and Ts6 induced the production of IL-6 and NO, and decreased the production of TNF-a (Zoccal et al., 2011(Zoccal et al., , 2013. Recently, Ts5 also showed a pro-inflammatory effect through its ability to stimulate the release of macrophage TNF-a and IL-6 and, based on the induced-cytokines produced by Ts toxins, a new pathway of macrophage activation was suggested (Pucca et al., 2015c). ...
Tityus serrulatus (Ts) is the main scorpion species of medical importance in Brazil. Ts venom is composed of several compounds such as mucus, inorganic salts, lipids, amines, nucleotides, enzymes, kallikrein inhibitor, natriuretic peptide, proteins with high molecular mass, peptides, free amino acids and neurotoxins. Neurotoxins are considered the most responsible for the envenoming syndrome due to their pharmacological action on ion channels such as voltage-gated sodium (Nav) and potassium (Kv) channels. The major goal of this review is to present important advances in Ts envenoming research, correlating both the crude Ts venom and isolated toxins with alterations observed in all human systems. The most remarkable event lies in the Ts induced massive releasing of neurotransmitters influencing, directly or indirectly, the entire body. Ts venom proved to extremely affect nervous and muscular systems, to modulate the immune system, to induce cardiac disorders, to cause pulmonary edema, to decrease urinary flow and to alter endocrine, exocrine, reproductive, integumentary, skeletal and digestive functions. Therefore, Ts venom possesses toxins affecting all anatomic systems, making it a lethal cocktail. However, its low lethality may be due to the low venom mass injected, to the different venom compositions, the body characteristics and health conditions of the victim and the local of Ts sting. Furthermore, we also described the different treatments employed during envenoming cases. In particular, throughout the review, an effort will be made to provide information from an extensive documented studies concerning Ts venom in vitro, in animals and in humans (a total of 151 references).
... Similarly, Fernandes et al. (2006) showed that the metalloproteinase BaP1 also induced a significant leukocyte infiltrate with neutrophils recruitment in the initial phases of the response, and inflammatory response formed mainly by mononuclear cells such as lymphocytes and monocytes after 24 h. The results of leukocyte recruitment combined with increased protein levels indicate a local inflammatory response (Petricevich, 2010;Zoccal et al., 2013). ...
In this study, we evaluated the edema and hyperalgesic response induced by BpirMP, a P-I class metalloproteinase isolated from Bothrops pirajai snake venom. The animals were injected with the metalloproteinase or sterile PBS (control group) and evaluated for 1, 2, 3, 4, 5, 6 and 24h. The intraplantar injection of BpirMP (5-50μg/paw) induced a dose- and time-dependent response. BpirMP (50μg) induced paw edema in rats rapidly, with peak response two hours after injection of the toxin. Also, BpirMP injection caused a significant reduction in the nociceptive threshold of the animals tested, with peak response three hours after injection of the toxin. The inflammatory mediators involved in these responses were assayed by pretreatment of animals with synthesis inhibitors or receptor antagonists. Peak responses were significantly reduced by pretreatment of animals with pyrilamine, a histamine receptor antagonist, sodium cromoglycate, a mast cell degranulation inhibitor and valeryl salicylate and meloxicam, cyclooxygenase inhibitors. The analysis of the peritoneal cavity exudate revealed an acute inflammatory response with recruitment of leukocytes, increased levels of total proteins, nitric oxide and the cytokines IL-6, TNF-α and IL-10. In conclusion, our results demonstrated that BpirMP induces inflammation mediated by mast cell degranulation, histamine, prostaglandins and cytokine production.
... The literature shows that venoms and toxins can activate the cells from the immune system. Further, several Ts toxins have been immunologically investigated [60][61][62][63][64][65]. However, most of these studies demonstrate the involvement of Ts toxins in the inflammatory response focusing mainly on macrophages. ...
The toxin, previously described as a "non-toxic" toxin, was isolated from the scorpion venom of Tityus serrulatus (Ts), responsible for the most severe and the highest number of accidents in Brazil. In this study, the subtype specificity and selectivity of Ts4 was investigated using six mammalian Nav channels (Nav1.2→Nav1.6 and Nav1.8) and two insect Nav channels (DmNav1 and BgNav). The electrophysiological assays showed that Ts4 specifically inhibited the fast inactivation of Nav1.6 channels, the most abundant sodium channel expressed in the adult central nervous system, and can no longer be classified as a "non-toxic peptide". Based on the results, we could classify the Ts4 as a classical α-toxin. The Ts4 3D-structural model was built based on the solved X-ray Ts1 3D-structure, the major toxin from Ts venom with which it shares high sequence identity (65.57%). The Ts4 model revealed a flattened triangular shape constituted by three-stranded antiparallel β-sheet and one α-helix stabilized by four disulfide bonds. The absence of a Lys in the first amino acid residue of the N-terminal of Ts4 is probably the main responsible for its low toxicity. Other key amino acid residues important to the toxicity of α- and β-toxins are discussed here.
... Indeed, our group has previously shown that Ts venom (TsV) and its toxins Ts1, Ts2, or Ts6 were able to induce the production of nitric oxide (NO), interleukin (IL)-6, and tumor necrosis factor (TNF)-a by macrophages in vitro (Zoccal et al., 2011). Besides cytokine production, we have also shown that intraperitoneal injection of Ts2 and Ts6 in mice induced the production of the arachidonic-acid derived inflammatory lipid mediators prostaglandin (PG)E 2 and leukotriene (LT)B 4 (Zoccal et al., 2013). The production of these pro-inflammatory mediators involved the activation of MAPKs and NF-kB dependent pathways through the recognition of Toll-like receptors 2 (TLR2) and 4 (TLR4), and of CD14 (Zoccal et al., 2014). ...
Tityus serrulatus venom (TsV) consists of numerous peptides with different physiological and pharmacological activities. Studies have shown that scorpion venom increases pro-inflammatory cytokine production, contributing to immunological imbalance, multiple organ dysfunction, and patient death. We have previously demonstrated that TsV is a venom-associated molecular pattern (VAMP) recognized by TLRs inducing intense inflammatory reaction through the production of pro-inflammatory cytokines and arachidonic acid-derived lipid mediators prostaglandin (PG)E2 and leukotriene (LT)B4. Lipid bodies (LBs) are potential sites for eicosanoid production by inflammatory cells. Moreover, recent studies have shown that the peroxisome proliferator-activated receptor gamma (PPAR-γ) is implicated in LB formation and acts as an important modulator of lipid metabolism during inflammation. In this study, we used murine macrophages to evaluate whether the LB formation induced by TsV after TLR recognition correlates with lipid mediator generation by macrophages and if it occurs through PPAR-γ activation. We demonstrate that TsV acts through TLR2 and TLR4 stimulation and PPAR-γ activation to induce LB formation and generation of PGE2 and LTB4. Our data also show that PPAR-γ negatively regulates the pro-inflammatory NF-κB transcription factor. Based on these results, we suggest that during envenomation, LBs constitute functional organelles for lipid mediator production through signaling pathways that depend on cell surface and nuclear receptors. These findings point to the inflammatory mechanisms that might also be triggered during human envenomation by TsV.
... Other studies in the literature have already demonstrated that PGE2 is involved in the inflammatory response and in the neutrophil recruitment [37] in mice inoculated with T. serrulatus scorpion venom [38]. As in certain sodium channel toxins and potassium (Ts2 and Ts6), T. serrulatus is involved in the release of cytokines and cell migration by inducing the production and release of PGE2 and LTB4 [39]. As already has been stated, the aqueous extract of M. tenuiflora is rich in saponins and tannins [18]. ...
Scorpion bite represents a significant and serious public health problem in certain regions of Brazil, as well as in other parts of the world. Inflammatory mediators are thought to be involved in the systemic and local immune response induced by Tityus serrulatus scorpion envenomation. The aim of this study was to evaluate the effect of extracts of Mimosa tenuiflora on model envenomation. In mice, the envenomation model is induced by Tityus serrulatus venom. Previous treatment of mice with fractions from M. tenuiflora was able to suppress the cell migration to the peritoneal cavity. The treatment of mice with M. tenuiflora extracts also decreased the levels of IL-6, IL-12, and IL-1β. We concluded that the administration of the extract and fractions resulted in a reduction in cell migration and showed a reduction in the level of proinflammatory cytokines. This study demonstrates, for the first time, the anti-inflammatory effect of aqueous extract from the Mimosa tenuiflora plant on T. serrulatus venom.
... (BuTX), since they derive from species from different geographic regions [18]. Recently, our group demonstrated that Ts6 stimulates the release of NO, IL-6 and TNF-α in J774.1 cells [19] and also presents a pro-inflammatory activity in mice [20]. ...
In Brazil, Tityus serrulatus (Ts) is the species responsible for most of the scorpion related accidents. Among the Ts toxins, the neurotoxins with action on potassium channels (α-KTx) present high interest, due to their effect in the envenoming process and the ion channel specificity they display. The α-KTx toxins family is the most relevant because its toxins can be used as therapeutic tools for specific target cells. The improved isolation method provided toxins with high resolution, obtaining pure Ts6 and Ts7 in two chromatographic steps. The effects of Ts6 and Ts7 toxins were evaluated in 14 different types of potassium channels using the voltage-clamp technique with two-microelectrodes. Ts6 toxin shows high affinity for Kv1.2, Kv1.3 and Shaker IR, blocking these channels in low concentrations. Moreover, Ts6 blocks the Kv1.3 channel in picomolar concentrations with an IC50 of 0.55 nM and therefore could be of valuable assistance to further designing immunosuppressive therapeutics. Ts7 toxin blocks multiple subtypes channels, showing low selectivity among the channels analyzed. This work also stands out in its attempt to elucidate the residues important for interacting with each channel and, in the near future, to model a desired drug.
Animal venoms are a rich and complex source of components, including peptides (such as neurotoxins, anionic peptides and hypotensins), lipids, proteins (such as proteases, hyaluronidases and phospholipases) and inorganic compounds, which affect all biological systems of the envenoming victim. Their action may result in a wide range of clinical manifestations, including tachy/bradycardia, hyper/hypotension, disorders in blood coagulation, pain, edema, inflammation, fever, muscle paralysis, coma and even death. Scorpions are one of the most studied venomous animals in the world and interesting bioactive molecules have been isolated and identified from their venoms over the years. Tityus spp. are among the scorpions with high number of accidents reported in the Americas, especially in Brazil. Their venoms have demonstrated interesting results in the search for novel agents with antimicrobial, anti-viral, anti-parasitic, hypotensive, immunomodulation, anti-insect, antitumor and/or antinociceptive activities. Furthermore, other recent activities still under investigation include drug delivery action, design of anti-epileptic drugs, investigation of sodium channel function, treatment of erectile disfunction and priapism, improvement of scorpion antivenom and chelating molecules activity. In this scenario, this paper focuses on reviewing advances on Tityus venom components mainly through the modern omics technologies as well as addressing potential therapeutic agents from their venoms and highlighting this abundant source of pharmacologically active molecules with biotechnological application.
Peptides are potential therapeutic alternatives against global diseases, such as antimicrobial-resistant infections and cancer. Venoms are a rich source of bioactive peptides that have evolved over time to act on specific targets of the prey. Peptides are one of the main components responsible for the biological activity and toxicity of venoms. South American organisms such as scorpions, snakes, and spiders are important producers of a myriad of peptides with different biological activities. In this review, we report the main venom-derived peptide families produced from South American organisms and their corresponding activities and biological targets.
Toxoplasmosis, caused by Toxoplasma gondii, is a major public concern owing to its neurotropic nature and high morbidity and mortality rates in immunocompromised patients and newborns. Current treatment for this disease is inefficient and produces side effects. Inflammatory mediators produced during T. gondii infection (e.g., cytokines and nitric oxide) are crucial in controlling parasite replication. In this context, Tityus serrulatus venom (TsV) induces the production of inflammatory mediators by immune cells. Thus, this study aimed to isolate and identify the components of TsV with potential anti-T. gondii activity. TsV was extracted from scorpions and lyophilized or loaded onto a column to obtain its fractions. TsV subfractions were obtained using chromatography, and its amino acid sequence was identified and applied to peptide design using bioinformatics tools. The C57BL/6 mice and their harvested macrophages were used to test the anti-Toxoplasma activity of TsV components and peptides. TsV and its fraction F6 attenuated the replication of tachyzoites in macrophages and induced nitric oxide and cytokine (IL-12, TNF, and IL-6) production by infected cells, without host cell toxicity. Moreover, Su6-B toxin, a subfraction of F6, demonstrated anti-T. gondii activity. The partially elucidated and characterized amino acid sequence of Sub6-B demonstrated 93% similarity with T. serrulatus 2 toxin (Ts2). Ts2 mimetic peptides (“Pep1,” “Pep2a,” and “Pep2b”) were designed and synthesized. Pep1 and Pep2a, but not Pep2b, reduced the replication of tachyzoites in macrophages. In vivo, treatment of T. gondii-infected mice with Pep1, Pep2a, or Pep2b decreased the number of cerebral cysts and did not induce hepatotoxicity in the animals. Taken together, our data show promising immunomodulatory and antiparasitic activity of TsV that could be explored and applied in future therapies for treating infectious parasitic diseases such as toxoplasmosis.
Venoms are complex mixtures of toxic compounds delivered by bite or sting. In humans, the consequences of envenomation range from self-limiting to lethal. Critical host defence against envenomation comprises innate and adaptive immune strategies targeted towards venom detection, neutralisation, detoxification, and symptom resolution. In some instances, venoms mediate immune dysregulation that contributes to symptom severity. This review details the involvement of immune cell subtypes and mediators, particularly of the dermis, in host resistance and venom-induced immunopathology. We further discuss established venom-associated immunopathology, including allergy and systemic inflammation, and investigate Irukandji syndrome as a potential systemic inflammatory response. Finally, this review characterises venom-derived compounds as a source of immune modulating drugs for treatment of disease.
Scorpionism is responsible for most accidents involving venomous animals in Brazil, which leads to severe symptoms that can evolve to death. Scorpion venoms consist of complexes cocktails, including peptides, proteins, and non-protein compounds, making separation and purification procedures extremely difficult and time-consuming. Scorpion toxins target different biological systems and can be used in basic science, for clinical, and biotechnological applications. This study is the first to explore the venom content of the unexplored scorpion species Rhopalurus crassicauda, which inhabits exclusively the northernmost state of Brazil, named Roraima, and southern region of Guyana. Here, we pioneer the fractionation of the R. crassicauda venom and isolated and characterized a novel scorpion beta-neurotoxin, designated Rc1, and a monomeric hyaluronidase. R. crassicauda venom and Rc1 (6,882 Da) demonstrated pro-inflammatory activities in vitro and a nociceptive response in vivo. Moreover, Rc1 toxin showed specificity for activating Nav1.4, Nav1.6, and BgNav1 voltage-gated ion channels. This study also represents a new perspective for the treatment of envenomings in Roraima, since the Brazilian scorpion and arachnid antivenoms were not able to recognize R. crassicauda venom and its fractions (with exception of hyaluronidase). Our work provides useful insights for the first understanding of the painful sting and pro-inflammatory effects associated with R. crassicauda envenomings.
In Brazil, the scorpion species responsible for most severe incidents belong to the Tityus genus and, among this group, T. serrulatus, T. bahiensis, T. stigmurus and T. obscurus are the most dangerous ones. Other species such as T. metuendus, T. silvestres, T. brazilae, T. confluens, T. costatus, T. fasciolatus and T. neglectus are also found in the country, but the incidence and severity of accidents caused by them are lower. The main effects caused by scorpion venoms – such as myocardial damage, cardiac arrhythmias, pulmonary edema and shock – are mainly due to the release of mediators from the autonomic nervous system. On the other hand, some evidence show the participation of the central nervous system and inflammatory response in the process. The participation of the central nervous system in envenoming has always been questioned. Some authors claim that the central effects would be a consequence of peripheral stimulation and would be the result, not the cause, of the envenoming process. Because, they say, at least in adult individuals, the venom would be unable to cross the blood-brain barrier. In contrast, there is some evidence showing the direct participation of the central nervous system in the envenoming process. This review summarizes the major findings on the effects of Brazilian scorpion venoms on the central nervous system, both clinically and experimentally. Most of the studies have been performed with T. serrulatus and T. bahiensis. Little information is available regarding the other Brazilian Tityus species.
The present study purifies two T. serrulatus non-disulfide-bridged peptides (NDBPs), named venom peptides 7.2 (RLRSKG) and 8 (KIWRS) and details their synthesis and biological activity, comparing to the synthetic venom peptide 7.1 (RLRSKGKK), previously identified. The synthetic replicate peptides were subjected to a range of biological assays: hemolytic, antifungal, antiviral, electrophysiological, immunological and angiotensin-converting enzyme (ACE) inhibition activities. All venom peptides neither showed to be cytolytic nor demonstrated significant antifungal or antiviral activities. Interestingly, peptides were able to modulate macrophages’ responses increasing IL-6 production. The three venom peptides also demonstrated potential to inhibit ACE in the following order: 7.2 > 7.1 > 8. The ACE inhibition activity was unexpected, since peptides that display this function are usually proline-rich peptides. In an attempt to understand the origin of such small peptides, we discovered that the isolated peptides 7.2 and 8 are fragments of the same molecule, named Pape peptide precursor. Furthermore, the study discusses that Pape fragments could be originated from a post-splitting mechanism resulting from metalloserrulases and other proteinases cleavage, which can be seen as a clever mechanism used by the scorpion to enlarge its repertoire of venom components. Scorpion venom remains as an interesting source of bioactive proteins and this study advances our knowledge about three NDBPs and their biological activities.
Background:
Tityus serrulatus scorpion venom (TsV) contains toxins that act on K(+) and Na(+) channels and account for the venom's toxic effects. TsV can activate murine peritoneal macrophages, but its effects on human lymphocytes have been poorly investigated. Considering that lymphocytes may play an important role in envenomation, we assessed whether TsV affects the expression of phenotypic (CD3, CD4, and CD8) and activation (CD69, CD25, and HLA-DR) markers, cell proliferation, and cytokine production in peripheral blood mononuclear cells.
Methods:
Cytotoxicity of TsV was evaluated via the MTT assay. Cell proliferation, expression of phenotypic and activation markers, and release of cytokines were assessed using flow cytometry, after treatment with non-cytotoxic concentrations of TsV. The combined use of carboxyfluorescein diacetate succinimidyl ester and monoclonal antibodies against phenotypic and activation markers enabled us to simultaneously assess cell proliferation extent and cell activation status, and to discriminate among cell subpopulations.
Results:
TsV at concentrations of 25 to 100 μg/mL were not cytotoxic towards peripheral blood mononuclear cells. TsV did not induce significant changes in lymphocyte subpopulations or in the expression of activation markers on CD4(+) and CD8(+) T cells. TsV inhibited the phytohemagglutinin-stimulated lymphocyte proliferation, particularly in the CD8(+) CD25(+) T lymphocyte subset. TsV alone, at 50 and 100 μg/mL, did not induce peripheral blood mononuclear cell proliferation, but elicited the production and release of IL-6, a proinflammatory cytokine that plays an important role in innate and adaptive immune responses.
Conclusions:
TsV is a potential source of molecules with immunomodulatory action on human T lymphocytes.
Scorpion envenomation (SE) is a common medical problem in many countries; it is an important cause of morbidity and mortality, especially among children. In certain cases scorpion stings lead to multiorgan failure that may be fatal; the manifestations include acute respiratory distress syndrome and systemic inflammatory response syndrome. Neurotoxins are the most active components of the scorpion venom responsible for the toxic effects induced after SE. They induce a massive release of neurotransmitters during stimulation of sympathetic and parasympathetic of the autonomic nervous system. The pathophysiological disturbances caused by scorpion venom are not exclusively assigned to the released neurotransmitters. The activation and release of inflammatory mediators (cytokines, kinins, eicosanoids, reactive oxygen species, and nitric oxide) may also play an important role in the pathophysiology of envenomation after stings and may be responsible for some of the inflammatory manifestations and organ failure. The massive release of these mediators from injured and activated cells promotes the inflammatory response and may be responsible for its exacerbation and its maintenance. The present chapter focuses on the role of inflammatory mediators and on elucidation of the potential mechanisms by which the immune system affects the pathophysiology following SE. Understanding of involved inflammatory cascade in scorpion envenoming syndromes may have future therapeutic and diagnostic benefits.
Scorpion venoms are known to cause different inflammatory disorders through complex mechanisms in various tissues. In the study here, the involvement of phospholipase A2 (PLA2) and cyclo-oxygenase (COX)-derived metabolites in hepatic and renal inflammation responses were examined. Mice were envenomed with Androctonus australis hector scorpion venom in the absence or presence of inhibitors that can interfere with lipid inflammatory mediator synthesis, i.e., dexamethasone (PLA2 inhibitor), indomethacin (non-selective COX-1/COX-2 inhibitor), or celecoxib (selective COX-2 inhibitor). The inflammatory response was assessed by evaluating vascular permeability changes, inflammatory cell infiltration, oxidative/nitrosative stress marker levels, and by histologic and functional analyses of the liver and kidney. Results revealed that the venom alone induced an inflammatory response in this tissues marked by increased microvascular permeability and inflammatory cell infiltration, increases in levels of nitric oxide and lipid peroxidation, and decreases in antioxidant defense. Moreover, significant alterations in the histological architecture of these organs were associated with increased serum levels of some metabolic enzymes, as well as urea and uric acid. Pre-treatment of mice with dexamethasone led to significant decreases of the inflammatory disorders in the hepatic parenchyma; celecoxib pre-treatment seemed to be more effective against renal inflammation. Indomethacin pre-treatment only slightly reduced the inflammatory disorders in the tissues. These results suggest that the induced inflammation response in liver was mediated mainly by PLA2 activation, while the renal inflammatory process was mediated by prostaglandin formation by COX-2. These findings provide additional insight toward the understanding of activated pathways and related mechanisms involved in scorpion envenoming syndrome.
Scorpion envenomation (SE) is a common medical problem in many countries; it is an important cause of morbidity and mortality, especially among children. In certain cases scorpion stings lead to multiorgan failure that may be fatal; the manifestations include acute respiratory distress syndrome and systemic inflammatory response syndrome. Neurotoxins are the most active components of the scorpion venom responsible for the toxic effects induced after SE. They induce a massive release of neurotransmitters during stimulation of sympathetic and parasympathetic of the autonomic nervous system. The pathophysiological disturbances caused by scorpion venom are not exclusively assigned to the released neurotransmitters. The activation and release of inflammatory mediators (cytokines, kinins, eicosanoids, reactive oxygen species, and nitric oxide) may also play an important role in the pathophysiology of envenomation after stings and may be responsible for some of the inflammatory manifestations and organ failure. The massive release of these mediators from injured and activated cells promotes the inflammatory response and may be responsible for its exacerbation and its maintenance. The present chapter focuses on the role of inflammatory mediators and on elucidation of the potential mechanisms by which the immune system affects the pathophysiology following SE. Understanding of involved inflammatory cascade in scorpion envenoming syndromes may have future therapeutic and diagnostic benefits.
Tityus serrulatus (Ts) venom is composed of mainly neurotoxins specific for voltage-gated K(+) and Na(+) channels, which are expressed in many cells such as macrophages. Macrophages are the first line of defense invasion and they participate in the inflammatory response of Ts envenoming. However, little is known about the effect of Ts toxins on macrophage activation. This study investigated the effect of Ts5 toxin on different sodium channels as well as its role on the macrophage immunomodulation. The electrophysiological assays showed that Ts5 inhibits the rapid inactivation of the mammalian sodium channels Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6 and Nav1.7. Interestingly, Ts5 also inhibits the inactivation of the insect Drosophila melanogaster sodium channel (DmNav1), and is therefore classified as the first Ts α-like toxin. The immunological experiments on macrophages reveal that Ts5 is a pro-inflammatory toxin inducing the cytokine production of tumor necrosis factor (TNF)-α and interleukin (IL)-6. On the basis of recent literature, our study also stresses a possible mechanism responsible for venom-associated molecular patterns (VAMPs) internalization and macrophage activation and moreover we suggest two main pathways of VAMPs signaling: direct and indirect. This work provides useful insights for a better understanding of the involvement of VAMPs in macrophage modulation.
Copyright © 2015. Published by Elsevier B.V.
Envenoming caused by scorpion sting is a serious public health problem. In Brazil, 13,038 accidents caused by venomous animals have been reported. Of this total, 53% of the cases and 14 deaths were caused by scorpions. Furthermore, Tityus serrulatus (Buthidae) is the most dangerous scorpion due to the high toxicity of its venom. The treatment is the common supportive therapy and the serum therapy, but some people do not have access to both therapies and seek healing through the use of medical plants.
This study evaluated the ability of the crude extract and fractions from the leaves of Ipomoea asarifolia in neutralizing the main biological effects caused by T. serrulatus envenoming in mice.
BALB/c mice were pretreated (i.v.) with 100μλ of aqueous extracts and fractions dichloromethane, ethyl acetate, and n-butanol (CH2Cl2, EtOAc, and n-BuOH, respectively) of I. asarifolia, rutin or saline. Then, the animals received 100μλ (i.p.) of venom of T. serrulatus (0.8mg/kg). After six hours, the peritoneal lavage was performed with PBS and the number cells were determined using a Neubauer chamber. The supernatants were collected for determination of cytokines, such as IL-6, IL-12, and IL-1β.
The aqueous extract, fractions and rutin, at all doses, significantly reduced cell migration, which was endorsed by the reduction of the levels of certain cytokines.
This is the first study that demonstrated the potential effect of I. asarifolia against inflammation caused by T. serrulatus venom, suggesting that these extracts and / or their bioactive molecules, especially the flavonoid rutin, have potential use in the therapy of this envenomation.
Abstract— The scope of this work was to investigate the nature, chronology and mechanisms of the cardiovascular disorders induced by scorpion envenoniation. Anaesthetized rats were instrumented for measurement of cardiac output (CO), renal (RBF) and muscular (HBF) blood flows (pulsed Doppler flowmetry), blood pressure, heart rate and dP/dt. Buthus occitanus venom (BO) was administered intravenously in the absence/presence of different pre-treatments. BO dose-dependently (150–300 μg/kg) increased blood pressure, dP/dt, total peripheral (TPR), renal (RVR) and muscular (HVR) vascular resistances, and decreased CO, RBF and HBF. Recovery occurred after 150 but not after 300 μg/kg. BO, 600 μg/kg, produced qualitatively similar effects but arrhythmias developed and mortality increased. Pre-treatment with phentolamine prevented the rises in TPR, RVR, HVR and blood pressure and the decreases in CO, RBF and HBF induced by BO, 300 μg/kg. Pre-treatment with propranolol prevented the rise in dP/dt and the occurrence of arrhythmias and limited the rise in RVR and the drop in RBF induced by BO, 300 μg/kg. Phentolamine, propranolol and their combination also prevented BO, 600 μg/kg-induced mortality. Other pre-treatments (bosentan, losartan, diltiazem, mepyramine) were almost ineffective vs. BO effects. Finally, BO, 300 μg/kg, induced a 30–40-fold increase in plasma epinephrine and norepinephrine levels, but no change in plasma endothelin-1 levels. Thus in anaesthetized rats, the pattern of the cardiac and systemic and regional haemodynamic effects of BO is typically that of and results from catecholamine outpouring-induced α- and β-adrenoceptor stimulation.
Scorpion venoms consist of a complex of several toxins that exhibit a wide range of biological properties and actions, as well as chemical compositions, toxicity, and pharmacokinetic and pharmacodynamic characteristics. These venoms are associated with high morbility and mortality, especially among children. Victims of envenoming by a scorpion suffer a variety of pathologies, involving mainly both sympathetic and parasympathetic stimulation as well as central manifestations such as irritability, hyperthermia, vomiting, profuse salivation, tremor, and convulsion. The clinical signs and symptoms observed in humans and experimental animals are related with an excessive systemic host inflammatory response to stings and stings, respectively. Although the pathophysiology of envenomation is complex and not yet fully understood, venom and immune responses are known to trigger the release of inflammatory mediators that are largely mediated by cytokines. In models of severe systemic inflammation produced by injection of high doses of venom or venoms products, the increase in production of proinflammatory cytokines significantly contributes to immunological imbalance, multiple organ dysfunction and death. The cytokines initiate a cascade of events that lead to illness behaviors such as fever, anorexia, and also physiological events in the host such as activation of vasodilatation, hypotension, and increased of vessel permeability.
Three toxic polypeptides were purified from the venom of the Brazilian scorpion Tityus serrulatus by means of gel filtration in Sephadex G-50 and ion-exchange chromatography in carboxymethylcellulose. The peptides are basic molecules with molecular weights in the range of 7000 for which the amino acid compositions and sequences were determined. The effect of the purified peptides on pancreatic exocrine secretion in the guinea pig was studied. Biochemical measurements show that the cells are stimulated by these peptides to discharge their zymogen granules. Light and electron microscopic images confirm the biochemical measurements. At the light microscope level, acinar cells show dramatically fewer zymogen granules than in control pancreas with the appearance of large vacuoles and some loss of morphological integrity. Electron micrographs display apical regions devoid of zymogen granules and condensing vacuoles whereas acinar lumina contain crystalline secretory material. The secretory effect observed in vitro is comparable to that of carbamylcholine and that of the peptidergic secretagogue cholecystokinin-pancreozymin.
The beneficial actions of nonsteroid anti-inflammatory drugs (NSAID) can be associated with inhibition of cyclo-oxygenase (COX)-2 whereas their harmful side effects are associated with inhibition of COX-1. Here we report data from two related assay systems, the human whole blood assay and a modified human whole blood assay (using human A549 cells as a source of COX-2). This assay we refer to as the William Harvey Modified Assay. Our aim was to make meaningful comparisons of both classical NSAIDs and newer COX-2-selective compounds. These comparisons of the actions of >40 NSAIDs and novel COX-2-selective agents, including celecoxib, rofecoxib and diisopropyl fluorophosphate, demonstrate a distribution of compound selectivities toward COX-1 that aligns with the risk of serious gastrointestinal complications. In conclusion, this full in vitro analysis of COX-1/2 selectivities in human tissues clearly supports the theory that inhibition of COX-1 underlies the gastrointestinal toxicity of NSAIDs in man.
After a variety of pathophysiologic stimuli, neutrophils accumulate in lung capillaries and contribute to the pathogenesis of acute lung injury. Lung neutrophil sequestration has previously been attributed to mechanical retention of stiffened neutrophils, but L-selectin-mediated leukocyte/endothelial interaction may be an essential step. We investigated the effect of the anti-L-selectin antibody HuDreg 200 on leukocyte sequestration and microhemodynamics in alveolar capillaries in a model of acute endotoxemia. We used in vivo fluorescence microscopy to analyze kinetics of fluorescently labeled red and white blood cells in alveolar capillary networks of the rabbit lung. Investigations were performed over 2 h after an intravenous infusion of 0.2 ml/kg body weight (bw) NaCl, 2 mg/kg bw HuDreg 200, 20 microg/kg bw lipopolysaccharide (LPS) of Escherichia coli 0111:B4, or the combination of HuDreg 200 and LPS, respectively. Infusion of LPS induced leukocyte sequestration in alveolar capillaries, which was accompanied by a reduction of alveolar capillary perfusion and functional capillary density. These effects could be completely blocked by pretreatment of animals with HuDreg 200. We conclude that L-selectin-mediated leukocyte/endothelial interaction is a necessary prerequisite for leukocyte sequestration in alveolar capillaries in this model. Impaired alveolar capillary perfusion appeared to result directly from capillary leukocyte sequestration.
Prostaglandins and leukotrienes are potent eicosanoid lipid mediators derived from phospholipase-released arachidonic acid that are involved in numerous homeostatic biological functions and inflammation. They are generated by cyclooxygenase isozymes and 5-lipoxygenase, respectively, and their biosynthesis and actions are blocked by clinically relevant nonsteroidal anti-inflammatory drugs, the newer generation coxibs (selective inhibitors of cyclooxygenase-2), and leukotriene modifiers. The prime mode of prostaglandin and leukotriene action is through specific G protein-coupled receptors, many of which have been cloned recently, thus enabling specific receptor agonist and antagonist development. Important insights into the mechanisms of inflammatory responses, pain, and fever have been gleaned from our current understanding of eicosanoid biology.
Unlabelled:
We performed two-dimensional echocardiograms and determined plasma norepinephrine levels on admission and at 24h after hospitalization, in 16 children with scorpion envenomation. All patients came from areas where scorpions have been identified as Tityus zulianus and received antivenin at the site of the accident or upon admission. Based on the presence or absence of cardiovascular manifestations, patients were divided into two groups.
Group a:
10 patients had cardiovascular manifestations of pulmonary edema. Four patients had mild pulmonary edema (Left ventricular ejection fraction: 0.43+/-0.19) and six had moderate to severe pulmonary edema (Ejection fraction: 0.31+/-0.09. p=NS, M+/-SD). Plasma norepinephrine was elevated on admission (1279+/-824) and decreased at 24h in seven of eight patients (474+/-140 pg/ml, p<0.03).
Group b:
Six patients had no cardiovascular manifestations. These patients had normal chest X-rays and normal echocardiograms. Plasma norepinephrine was not elevated (188+/-180 pg/ml). Time interval from the accident to antivenin administration was significantly longer in Group A compared to Group B (4.5+/-3.3 vs 1.2+/-0.4h, p<0.03) and correlated directly with the absolute change in plasma norepinephrine (r=0.76, p<001). Consequently, we strongly recommend very early administration of antivenin in the medical management of scorpion envenomation by T. zulianus.
Filarial infections evoke exuberant inflammatory responses in the peritoneal cavities of immunocompetent mice. Clearance of infection appears to be dependent on complex interactions between B1 and B2 B lymphocytes, T cells, eosinophils, macrophages, and the products of these cells. In an earlier communication, we described the course of infection in normal immunocompetent mice. In this study, we utilize mice with well-characterized mutations that disable one or more effector components of adaptive immunity in order to determine their roles in host protection. We characterize peritoneal exudate cells by flow cytometry and determine the kinetics of accumulation of each of the different cell types following infection with Brugia pahangi. We find that (i) four-color flow-cytometric analysis of peritoneal exudate cells using anti-CD3, -CD11b, -CD19, and -Gr1 can distinguish up to six different populations of cells; (ii) an initial influx of neutrophils occurs within 24 h of infection, independent of the adaptive immune status of mice, and these cells disappear by day 3; (iii) an early influx of eosinophils is seen at the site of infection in all strains studied, but a larger, second wave occurs only in mice with T cells; (iv) the presence of T cells and eosinophils is important in causing an increase in macrophage size during the course of infection; and (v) most unexpectedly, T-cell recruitment appears to be optimal only if B cells are present, since JHD mice recruit significantly fewer T cells to the site of infection.
Trauma is considered to be the leading cause of death during the first three decades of life in the USA (Shires 1985; Trunkey 1982,1983) ranking overall as the fourth leading cause of death. Accidental injury accounts for approximately 165 000 deaths each year (Trunkey 1982). Furthermore, sepsis is the major non-neurologic cause of death after trauma, thermal injury, and major surgery. It carries an overall mortality rate of 60% (Baker et al. 1980). Numerous studies have been carried out exploring the causal relationships between the traumatic insults to the organism and its predisposition to develop septic complications and generalized septicemia (Alexander et al. 1979; Baker et al. 1985; Bjornson et al. 1978; Christou et al. 1980). Earlier studies indicated that patients with significant thermal and soft tissue injuries showed a marked tendency for multiple organ failure usually observed in association with persistent septicemia (Baue and Chaudry 1980; Miller et al. 1982). Efforts were therefore directed towards the eradication of septic foci by surgical procedures, antibiotic coverage, and overzealous fluid and nutritional therapy. However, the prognosis of such complications in the setting of major traumatic injuries has remained grim, mostly because of uncontrollable sepsis leading to severe homeostatic derangements and cellular dysfunction.
In a study of the transformation of polyunsaturated fatty acids in rabbit polymorphonuclear leukocytes in 1976 it was discovered that arachidonic acid is oxygenated at C-5 (1). Subsequently a number of derivatives, including leukotriene B 4 (LTB 4 ), were identified (2-4). Extension of these studies led in 1979 to the discovery of the pivot epoxide intermediate, LTA 4 (5), and the elucidation of the structure of SRS-A (slow-reacting substance of anaphylaxis) as a group of cysteinyl-leukotrienes, namely LTC 4 , LTD 4 , and LTE 4 (6-10). Earlier work had shown that the prostaglandins are formed by oxygenation and further transformation of arachidonic acid and other polyunsaturated fatty acids (11). The first intermediate in the formation of prostaglandins, the endoperoxide PGG 2 , was isolated and identified in 1974 (12). At the same time we introduced the name cyclooxygenase for the enzyme catalyzing the transformation of arachidonic acid into PGG 2 (12). However, the endoperoxides, PGG 2 and PGH 2 , had some biological effects that could not be explained by their conversion to the known prostaglandins (12). This finding eventually led to the discovery of thromboxane A 2 , an unstable platelet-aggregating and vasoconstrictor substance (13). Subsequent work showed that the endoperoxide can also be converted into a derivative, prostacyclin (PGI 2 ), with opposite biological effects (14). Prostaglandins E 2 and I 2 have strong proinflammatory effects. Aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs) inhibit the enzyme (cyclooxygenase) responsible for conversion of arachidonic acid into prostaglandins and thromboxanes (15). An induced form of the cyclooxygenase (COX-2) seems to play an important role in inflammation, thus opening the possibility of developing antiinflammatory NSAIDs that lack the side effects of the previous generation of such drugs (16, 17). In 1975 antiinflammatory steroids were proposed to inhibit prostaglandin formation by blocking of the release of the precursor acid from phospholipid stores (18). Since steroids and NSAIDs have significantly different antiinflammatory effects it seemed conceivable that some of these differences might be explained by the formation of proinflammatory derivatives of arachidonic acid by cyclooxygenase-independent reactions. Our studies of the metabolism of arachidonic acid in leukocytes unraveled a new metabolic pathway and led to the discovery of the leukotrienes (8). These compounds play a role in allergy and asthma and have pronounced proinflammatory effects (19, 20).
The inflammatory cell influx towards the peritoneal cavity in mice inoculated i.p. with live or dead Histoplasma capsulatum or with its subcellular preparations was studied. We also evaluated the effects of dexamethasone (Dexa) or MK886, an inhibitor of leukotriene (LT) biosynthesis, on the recruitment of leukocytes.
Live yeast form of fungus (LYH) induced an increase in neutrophils (NE) which was highest 4 to 24 h after inoculation. Mononuclear cell (MN) migration beginning at 24 h with a gradual increase over 48 and 168 h, and an eosinophil (EO) recruitment occurs between 24 and 48 h.
NE and EO recruitment induced by dead mycelial form of fungus (DMH) was greater than that observed for dead yeast form of fungus (DYH). A similar leukocyte migration pattern was seen after i.p. injection of the alkali-insoluble fraction (F1) from DYH (F1Y) and F1 from DMH (F1M) this being more active than former. The difference in concentration of β-glucan in DYH and DMH could explain the different inflammatory capacity exhibited by the two forms of H. capsulatum.
LT seems to be the principal mediator of leukocyte migration in response to LYH, DYH or DMH or to β-glucan. However, other mediators appear to contribute to NE and EO migration since the treatment with Dexa was more effective in inhibiting cell migration than MK886. Complement dependent leukocyte migration may participate in this recruitment. Treatment with MK886 completely abolished MN cell migration, indicating its dependence on the presence of LT.
British Journal of Pharmacology (1999) 128, 1529–1537; doi:10.1038/sj.bjp.0702912
Tityus serrulatus is considered the most dangerous scorpion in South America and responsible for most of the fatal cases. This review will focus on Tityus serrulatus scorpion venom (Tsv), its long-chain Na+-channel toxins (NaTx), which include α- and β-neurotoxins, short-chain K+-channel toxins (KTx), hyaluronidase, proteases and other peptides hitherto identified.
Voltage-gated sodium channels are the molecular targets for a broad range of neurotoxins that act at six or more distinct receptor sites on the channel protein. These toxins fall into three groups. Both hydrophilic low molecular mass toxins and larger polypeptide toxins physically block the pore and prevent sodium conductance. Alkaloid toxins and related lipid-soluble toxins alter voltage-dependent gating of sodium channels via an allosteric mechanism through binding to intramembranous receptor sites. In contrast, polypeptide toxins alter channel gating by voltage sensor trapping through binding to extracellular receptor sites. The results of recent studies that define the receptor sites and mechanisms of action of these diverse toxins are reviewed here.
Scorpion envenomation is a common medical problem in many countries and an important cause of morbidity and mortality, especially among children. The plasma levels of pro-inflammatory (IL-1β, IL-6, IL-8 and TNF-α) and anti-inflammatory (IL-10) cytokines were measured in individuals stung by Tityus serrulatus (Ts) scorpions. According to clinical manifestations patients were classified, as defined by the Brazilian Ministry of Health, as having mild (n=15, mean age=42.2 years), moderate (n=8, mean age=26 years) or severe (n=4, mean age=14 years) envenomation. Blood samples were taken immediately (T1) and 6 h (T2) after admission to the hospital. Eighteen age-matched healthy volunteers were used as control. TNF-α, IL-1β, IL-6 and IL-8 levels were significantly increased in moderate and severe cases and the levels of these cytokines were positively correlated with the severity of envenomation, as evaluated by clinical profile and plasma venom concentration. IL-10 levels were increased in severe and moderate cases and reduced in mild cases. The results reported in the present study suggest that the physiopathological manifestation of Ts envenomation may be mediated, at least in part, by cytokines, and that the early treatment after scorpion sting with drugs that inhibit cytokine production, such as glucocorticoids, may have a potential beneficial effect, ameliorating the severity of the clinical manifestations observed, particularly in severe and moderate cases.
Scorpion toxins targeting voltage-gated sodium (NaV) channels are peptides that comprise 60–76 amino acid residues cross-linked by four disulfide bridges. These toxins can be divided in two groups (α and β toxins), according to their binding properties and mode of action. The scorpion α-toxin Ts2, previously described as a β-toxin, was purified from the venom of Tityus serrulatus, the most dangerous Brazilian scorpion. In this study, seven mammalian NaV channel isoforms (rNaV1.2, rNaV1.3, rNaV1.4, hNaV1.5, mNaV1.6, rNaV1.7 and rNaV1.8) and one insect NaV channel isoform (DmNaV1) were used to investigate the subtype specificity and selectivity of Ts2. The electrophysiology assays showed that Ts2 inhibits rapid inactivation of NaV1.2, NaV1.3, NaV1.5, NaV1.6 and NaV1.7, but does not affect NaV1.4, NaV1.8 or DmNaV1. Interestingly, Ts2 significantly shifts the voltage dependence of activation of NaV1.3 channels. The 3D structure of this toxin was modeled based on the high sequence identity (72%) shared with Ts1, another T. serrulatus toxin. The overall fold of the Ts2 model consists of three β-strands and one α-helix, and is arranged in a triangular shape forming a cysteine-stabilized α-helix/β-sheet (CSαβ) motif.
Database Model data are available in the PMDB under accession number PM0077533.
Inflammation underlies a wide variety of physiological and pathological processes. Although the pathological aspects of many types of inflammation are well appreciated, their physiological functions are mostly unknown. The classic instigators of inflammation - infection and tissue injury - are at one end of a large range of adverse conditions that induce inflammation, and they trigger the recruitment of leukocytes and plasma proteins to the affected tissue site. Tissue stress or malfunction similarly induces an adaptive response, which is referred to here as para-inflammation. This response relies mainly on tissue-resident macrophages and is intermediate between the basal homeostatic state and a classic inflammatory response. Para-inflammation is probably responsible for the chronic inflammatory conditions that are associated with modern human diseases.
In this study, the effects of sublethal dose of Androctonus australis hector (Aah) venom on the enzymatic activities (creatine phospho-kinase and lactate dehydrogenase) and histopathological changes of heart and lungs' organs were determined 24h following envenoming NMRI mice. The effects of Aah venom on the lytic activity of the complement system, plasma cytokine rates (IL1-beta, IL-6, TNF-alpha, IL-4 and IL-10) and the peripheral blood cell infiltration were also studied. Microscopically, treated animals showed severe myocardial edema, hemorrhages and necroses and severe acute bronchopneumonia with alveolar edema and hemorrhages. High serum levels of lactate dehydrogenase and creatine kinase correlate to the tissue lesions. The results showed fast kinetics of production of pro-inflammatory (IL1-beta, IL-6, TNF-alpha) and anti-inflammatory (IL-4 and IL-10) cytokines at 30min in blood sera. An increase in serum lytic activity of envenomed animals and leucocytosis in peripheral blood with predominance of mononuclear and neutrophil cells were also observed. In conclusion, the results reported in the present study suggest that pathophysiological manifestations of Aah envenomation may be mediated sequentially or simultaneously by cytokines and the complement system, which in turn activate leukocyte to produce tissue damage.
Voltage-gated potassium channel toxins (KTxs) are basic short chain peptides comprising 23-43 amino acid residues that can be cross-linked by 3 or 4 disulfide bridges. KTxs are classified into four large families: α-, β-, γ- and κ-KTx. These peptides display varying selectivity and affinity for K(v) channel subtypes. In this work, a novel toxin from the Tityus serrulatus venom was isolated, characterized and submitted to a wide electrophysiological screening on 5 different subtypes of Na(V) channels (Na(V)1.4; Na(V)1.5; Na(V)1.6; Na(V)1.8 and DmNa(V)1) and 12 different subtypes of K(V) channels (K(V)1.1 - K(V)1.6; K(V)2.1; K(V)3.1; K(V)4.2; K(V)4.3; Shaker IR and ERG). This novel peptide, named Ts15, has 36 amino acids, is cross-linked by 3 disulfide bridges, has a molecular mass of 3956 Da and pI around 9. Electrophysiological experiments using patch clamp and the two-electrode voltage clamp techniques show that Ts15 preferentially blocks K(V)1.2 and K(V)1.3 channels with an IC₅₀ value of 196 ± 25 and 508 ± 67 nM, respectively. No effect on Na(V) channels was observed, at all tested concentrations. Since Ts15 shows low amino acid identity with other known KTxs, it was considered a bona fide novel type of scorpion toxin. Ts15 is the unique member of the new α-Ktx21 subfamily and therefore was classified as α-Ktx21.1.
Scorpion envenomation induces a systemic immune response, and neurotoxins of venom act on specific ion channels, modulating neurotransmitter release or activity. However, little is known about the immunomodulatory effects of crude venom from scorpion Tityus serrulatus (TsV) or its toxins (Ts1, Ts2 and Ts6) in combination with lipopolysaccharide (LPS). To investigate the immunomodulatory effects of TsV and its toxins (Ts1, Ts2 and Ts6), J774.1 cells were stimulated with different concentrations (25, 50 and 100 μg/mL) of venom or toxins pre-stimulated or not with LPS (0.5 μg/mL). Macrophage cytotoxicity was assessed, and nitric oxide (NO) and cytokine production were analyzed utilizing the culture supernatants. TsV and its toxins did not produce cytotoxic effects. Depending on the concentrations used, TsV, Ts1 and Ts6 stimulated the production of NO, interleukin (IL)-6 and tumor necrosis factor (TNF)-α in J774.1 cells, which were enhanced under LPS co-stimulation. However, LPS + Ts2 inhibited NO, IL-6 and TNF-α production, and Ts2 alone stimulated the production of IL-10, suggesting an anti-inflammatory activity for this toxin. Our findings are important for the basic understanding of the mechanisms involved in macrophage activation following envenomation; additionally, these findings may contribute to the discovery of new therapeutic compounds to treat immune-mediated diseases.
In this study, the production of prostaglandin E₂ (PGE₂) and up-regulation in cyclooxygenase (COX) pathway induced by a phospholipase A₂ (PLA₂), myotoxin-III (MT-III), purified from Bothrops asper snake venom, in isolated neutrophils were investigated. The arachidonic acid (AA) production and the participation of intracellular PLA₂s (cytosolic PLA₂ and Ca(2+)-independent PLA₂) in these events were also evaluated. MT-III induced COX-2, but not COX-1 gene and protein expression in neutrophils and increased PGE₂ levels. Pretreatment of neutrophils with COX-2 and COX-1 inhibitors reduced PGE₂ production induced by MT-III. Arachidonyl trifluoromethyl ketone (AACOCF₃), an intracellular PLA₂ inhibitor, but not bromoenol lactone (BEL), an iPLA₂ inhibitor, suppressed the MT-III-induced AA and PGE₂ release. In conclusion, MT-III directly stimulates neutrophils inducing COX-2 mRNA and protein expression followed by production of PGE₂. COX-2 isoform is preeminent over COX-1 for production of PGE₂ stimulated by MT-III. PGE₂ and AA release by MT-III probably is related to cPLA₂ activation.
Analysis of the molecular mechanisms governing the ability of IL-10 to keep inflammation under control has highlighted the existence of a great degree of plasticity and specificity with regard to innate immune cells. In this respect, neutrophils represent a perfect example of innate immune cells conditioned by external signals (for instance, by LPS), as well as by intracellular regulatory pathways, that render them optimally responsive to IL-10 only when required. The focus of this review are the recent experimental findings that have uncovered the sophisticated and complex molecular mechanisms responsible for the modulation of pro- and anti-inflammatory cytokine production by IL-10 in neutrophils and other innate immune cells. Understanding how IL-10 exerts its anti-inflammatory response, particularly in the case of neutrophils, will provide novel clues leading, hopefully, to the therapeutic control of neutrophil-driven inflammatory reactions, such as septic infections, rheumatoid arthritis, osteoarthritis and chronic obstructive pulmonary disease.
Neutrophils are one of the first lines of defense against microbial pathogens and are rapidly recruited at the infection site upon inflammatory conditions. We show here that after bacterial stimulation, and in contrast to monocytes and macrophages, murine neutrophils contributed poorly to inflammatory responses; however, they secreted high amounts of the anti-inflammatory cytokine IL-10 in a DAP12 adaptor-Syk kinase and MyD88 adaptor-dependent manner. Cotriggering of TLR-MyD88- and C-type lectin receptor (CLR)-Syk-dependent pathways led to a quick and sustained phosphorylation of p38 MAP and Akt kinases in neutrophils. In vivo, both Gram-negative bacteria and mycobacteria induced the recruitment of neutrophils secreting IL-10. In acute mycobacterial infection, neutrophil-derived IL-10 controlled the inflammatory response of dendritic cells, monocytes and macrophages in the lung. During a chronic infection, neutrophil depletion promoted inflammation and decreased the mycobacterial burden. Therefore, neutrophils can have a previously unsuspected regulatory role during acute and chronic microbial infections.
Macrophages are critical components of diverse microenvironments (ME) in adulthood, as well as during embryogenesis. Their role in development precludes the use of gene-targeting and knockout approaches for studying their function. Hence, we proposed to create a macrophage-specific inducible transgenic mouse where genes can be turned on or off at will.
A transgenic mouse in which the reverse tetracycline activator (rtTA-M2) is expressed under the hCD68 promoter for macrophage-specific gene induction was developed and crossed with a second transgenic reporter mouse strain in which the gene for green fluorescent protein (GFP) is under the control of tetracycline responsive element promoter. After doxycycline induction of the double transgenic animals (designated CD68-rtTA-tet-GFP), inducible expression of GFP was characterized by multicolor flow cytometric analysis of blood, marrow, and spleen cells and by demonstration of GFP expression in fresh-frozen sections in diverse tissues.
In bone marrow, inducible GFP expression was not confined to, or inclusive of, all cells expressing the classical macrophage markers, such as F4/80. However, GFP-expressing cells in thioglycollate-elicited peritoneal macrophages were also positive for F4/80 and monocyte-macrophage-specific 2 antigen. Interestingly, flow analysis also indicated little overlap between the F4/80 and CSF-1R-positive populations. Fresh-frozen samples of tissues known to contain macrophages revealed GFP-expressing cells with variable morphologies.
Our results show that the hCD68 promoter directs gene expression in a macrophage population distinct from that defined by classical monocyte-macrophage markers or promoters. Whether this population is functionally distinct remains to be established.
Tityus serrulatus is considered the most dangerous scorpion in South America and responsible for most of the fatal cases. This review will focus on Tityus serrulatus scorpion venom (Tsv), its long-chain Na(+)-channel toxins (NaTx), which include alpha- and beta-neurotoxins, short-chain K(+)-channel toxins (KTx), hyaluronidase, proteases and other peptides hitherto identified.
Crotoxin (CTX), a neurotoxin isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, induces analgesia. In this study, we evaluated the antinociceptive effect of CTX in a model of neuropathic pain induced by rat sciatic nerve transection. Hyperalgesia was detected 2 h after nerve transection and persisted for 64 days. Immersion of proximal and distal nerve stumps in CTX solution (0.01 mM for 10 s), immediately after nerve transection, blocked hyperalgesia. The antinociceptive effect of CTX was long-lasting, since it was detected 2 h after treatment and persisted for 64 days. CTX also delayed, but did not block, neurectomy-induced neuroma formation. The effect of CTX was blocked by zileuton (100 mg/kg, p.o.) and atropine (10 mg/kg, i.p.), and reduced by yohimbine (2 mg/kg, i.p.) and methysergide (5 mg/kg, i.p.). On the other hand, indomethacin (4 mg/kg, i.v.), naloxone (1 mg/kg, i.p.), and N-methyl atropine (30 mg/kg, i.p.) did not interfere with the effect of CTX. These results indicate that CTX induces a long-lasting antinociceptive effect in neuropathic pain, which is mediated by activation of central muscarinic receptors and partially, by activation of alpha-adrenoceptors and 5-HT receptors. Eicosanoids derived from the lipoxygenase pathway modulate the action of crotoxin.
The toxin Ts II from the venom of the Brazilian scorpion Tityus serrulatus was purified in two successive chromatographic steps. The amino acid sequence was then determined by automated Edman degradation of the reduced and S-carboxymethylated protein and of proteolytic peptides derived from it. This sequence appears to differ from that of previously characterized toxins found in this venom. However, it is identical to the recently published sequence of protein III-8 from the same venom [Possani et al., J Biol Chem 266:3178-3185, 1991], except that the C-terminus was found to be amidated. Homologies were found between the sequence of Ts II and that of other toxins from Tityus; in particular, the amino acid sequence of Ts II displays 72% sequence identity with Ts VII (also called Titx gamma). Consistent with this structural similarity, some biological properties of Ts II were found to be similar to those of Ts VII: Ts II has an intracerebroventricular LD50 of 6 ng, as compared to 0.6 ng for Ts VII; in a receptor binding assay Ts II, like Ts VII, was found to behave as a beta-type toxin and to inhibit the binding of the reference labelled toxin with a K0.5 of 5 x 10(-9) M, as compared to 7 x 10(-11) M for Ts VII. Nevertheless, Ts II is unable to bind to anti-Ts VII antibodies in radioimmunoassay experiments, indicating the non-conservation between the two toxins of at least some antigenically important residues.(ABSTRACT TRUNCATED AT 250 WORDS)
Eosinophils are implicated in the pathogenesis of a variety of allergic inflammatory diseases such as asthma. Several substances have been shown to be chemotactic for eosinophils in vitro, but the inflammatory mediators involved in the accumulation of eosinophils in vivo are as yet unidentified. In this study we have developed a system to measure the accumulation of 111In-eosinophils in guinea-pig skin in vivo. Horse serum-induced guinea-pig peritoneal eosinophils were radiolabelled with 111In and injected intravenously into recipient animals. 125I-albumin was also injected intravenously in order to measure local oedema formation simultaneously. A range of putative mediators was injected intradermally and responses measured for up to 2 hr. Of the mediators tested, guinea-pig C5a des Arg in zymosan-activated plasma was the most active. Recombinant human C5a (rHC5a) was also highly active, but less than the guinea-pig material. C5a des Arg in maximally activated plasma induced a 1500% increase in eosinophil accumulation, while rHC5a (10(-10) mol dose) induced a 600% increase. Platelet-activating factor (PAF) and leukotriene B4 (LTB4) were also tested for comparison. With respect to 111In-eosinophil accumulation, the order of potency of the mediators tested was as follows: guinea-pig C5a des Arg greater than LTB4 greater than PAF. In contrast, the order of potency of the mediators with respect to oedema formation was: PAF greater than guinea-pig C5a des Arg greater than LTB4. The techniques described will facilitate analysis of the mechanisms involved in eosinophil accumulation in defined inflammatory reactions.
This article has no abstract; the first 100 words appear below.
THE slow-reacting substance of anaphylaxis was recognized as a discrete biologic entity ¹,² three to four decades before it was chemically defined as three specific cysteinyl leukotriene products of the 5-lipoxygenase pathway.³⁴⁵⁶⁷ In the interim, it was hypothesized that this moiety might function as a proinflammatory mediator of bronchoconstriction and vasopermeability in diseases of immediate hypersensitivity.⁸⁹¹⁰¹¹ Since the chemical characterization of the slow-reacting substance of anaphylaxis as several cysteinyl leukotrienes and the identification and biochemical assessment of the enzymes in the 5-lipoxygenase pathway (Fig. 1 and Glossary), the products of this pathway have been implicated in a much broader variety of functions . . .
Five toxins from the venom of the Brazilian scorpion Tityus serrulatus were purified to homogeneity by a combination of ion exchange chromatography with ammonium bicarbonate buffer (pH 7.8) on CM-cellulose-52 and rechromatography on the same resin equilibrated with ammonium acetate buffer (pH 4.7). Four of these proteins, obtained in one or two steps in high yield and lethality (named toxins IX3, IX5, and X4 and XIII) were shown to be identical with other toxins already described. A fifth one, TsTX-IV, is reported as a new toxin. Except for IX3, which showed Gly as the sole N-terminal residue, the other four toxins showed Lys. TsTX-IV has an approximate mol. wt of 6880, an i.v. LD50, in mice, of 826 +/- 156 micrograms/kg and an intracisternal LD50 of 11 +/- 9 micrograms/kg, compared to 375 +/- 45 and 4.9 +/- 0.8, respectively, for the whole venom extract. It has 61 amino acid residues and an amino acid composition different from that of any other toxin from Tityus serrulatus venom so far described. Toxins IX5, TsTX-IV and XIII induced a prejunctional type of supersensitivity on the guinea pig vas deferens, probably due to an increased release of noradrenaline.
Arachidonic acid is metabolised either by cyclooxygenases to produce prostaglandins and thromboxanes or by lipoxygenases to produce mono-, di- and trihydroxyeicosatetraenoic acids (HETEs). Polymorphonuclear leukocytes (PMNs) release HETEs, including mono- and dihydroxy fatty acids, when exposed to stimuli such as the calcium ionophore A23187 (refs 1, 2). The mono-HETEs are assumed to be of particular importance with respect to effects on leukocyte function because they have been shown to possess both chemotactic and chemokinetic activities towards PMNs and eosinophils. However, we have now shown that the chemokinetic and aggregating activities released from rat and human PMNs exposed to ionophore A23187 (ref. 5) are not due to the release of mono-HETEs but to that of 5, 12-di-HETE (leukotriene B). This compound is active over the concentration range 10 pg ml-1 to 5 ng ml-1.
Forty-two cases of serious scorpion envenomation, of which 4 had a fatal outcome, are presented. The clinical profile, differential diagnosis and management of scorpionism are discussed. Most envenomations occurred in the summer months, peaking in January and February. An immediate local burning pain was the most prominent symptom. Systemic symptoms and signs developed within 4 hours of the sting in most instances, characterised by general paraesthesia, hyperaesthesia, muscle pain and cramps. Other striking features included dysphagia, dysarthria and sialorrhoea with varying degrees of loss of pharyngeal reflexes. The blood pressure and the temperature were often raised and the tendon reflexes increased, while motor power was often impaired. In a considerable number of patients the course was complicated by varying degrees of respiratory dysfunction, which tended to be more serious in children. The oustanding feature in children was an extreme form of restlessness characterised by excessive neuromuscular activity. Victims of scorpion sting, particularly in high-risk localities, should be closely observed for 12-24 hours. Children and other high-risk patients should be hospitalised. All patients with symptoms and signs of systemic envenomation should receive antivenom. Parabuthus granulatus (Hemprich & Ehrenberg, 1828) has been identified as the most important venomous species in the western Cape. The antivenom is produced from the venom of the medically less important P. transvaalicus Purcell, 1899. A strong case can therefore be made for the inclusion of P. granulatus venom in the production of a polyvalent antivenom.
Interleukin-6 levels were measured in the serum of ten children following severe scorpion envenomation. Measurements were taken on arrival, at the emergency room, and 12 and 24 hr after arrival. Interleukin-6 was markedly elevated in the serum of eight out of ten children on arrival. Interleukin-6 levels gradually decreased toward normal values on 12 and 24 hr measurements, but remained above control levels on all measurements. These results imply that signs and symptoms following scorpion envenomation may in part be explained by release of cytokines. Human and experimental animal studies are required in order to verify the assumption that interleukin-6 and other cytokines are involved in the pathogenesis of scorpion envenomation.
Scorpion venoms contain a variety of low mol. wt peptides toxic to different organisms. These peptides have been intensively studied because they represent excellent models for investigating structure-function relationships and they are also fine probes for studying ionic channel functions. This review deals with the biological and chemical aspects of toxic peptides that affect Na+ or K+ channels and the cloning of the cDNAs and genes encoding the main alpha and beta neurotoxins present in the venom of the three most dangerous species of Brazilian scorpion, Tityus bahiensis, Tityus stigmurus and Tityus serrulatus, and the Venezuelan scorpion Tityus discrepans. At least 16 different peptides specific for Na+ channels and five affecting K+ channels were isolated and characterized from the venom of these scorpions. The isolation of cDNAs and genes encoding four distinct toxins has permitted the elucidation of their nucleotide sequences as well as their genomic organization. Venoms and isolated toxins from scorpions of the genus Tityus were shown to enhance the secretory activity of the pancreas. Antisera obtained against venom of T. serrulatus show cross-reactivity with other species of the Brazilian scorpions.
The primary structure of TsTX-IV, a neurotoxin isolated from Tityrus serrulatus scorpion venom, is reported. Its amino acid sequence was determined by automated Edman sequential degradation of the reduced and carboxymethylated toxin and of relevant peptides obtained by digestion with Staphylococcus aureus strain V8 protease or trypsin and cleavage by CNBr. The complete sequence showed 41 amino acid residues, which account for an estimated molecular weight of 4520, and eight half-cystine residues which cross-link the toxin molecule with four disulfide bonds. The molecular weight determined by mass spectrometry was 4518. Comparison of this sequence with those from other scorpion toxins showed a resemblance with toxins which act on different types of K+ channels. TsTx-IV was able to block Ca2+-activated K+ channels of high conductance. TsTX-IV is the first four-disulfide-bridged short toxin from T. serrulatus so far completely sequenced.
Seventeen patients stung by Tityus serrulatus scorpion were classified as mild (pain at the site of the sting, n = 6), moderate (local pain and one of the following manifestations: vomiting, psychomotor agitation, prostration, sweating, tachypnea, tachycardia and mild arterial hypertension, n = 10) and severe cases (equal moderate cases plus cardiac failure, pulmonary edema and shock, n = 1). Venous blood was sampled for biochemical and hematological analysis and for IL-1alpha, IL-6, IL-10, TNF-alpha, IFN-gamma and GM-CSF ELISAs at the time of hospital admission, 6 h (moderate and severe cases), and 12, 18, 36 and 72 h (severe case) later. Ten age-matched healthy volunteers were used as control. Increased serum levels of IL-1alpha was noticed in all patients, high levels of IL-6, IFN-gamma and GM-CSF were observed only in a patient with severe envenomation. Our data suggest that a systemic inflammatory response-like syndrome is triggered during severe envenomation caused by T. serrulatus sting and that release of cytokines may be involved in this response.
Leukotrienes (LTs) are potent bioactive lipids derived from the 5-lipoxygenase (5-LO) pathway of arachidonic acid (AA) metabolism. Although they have been conventionally viewed as paracrine mediators of inflammatory disease processes such as asthma (1), more recent information suggests that they are also important participants in disease processes characterized by cellular proliferation and fibrogenesis (2-4), and that they subserve a homeostatic role in antimicrobial host defense (5). In view of the actions and importance of LTs, substantial effort has been directed at increasing our understanding of the regulation of their synthesis. An extensive body of research, reviewed elsewhere in this symposium, has identified three key factors that regulate LT synthesis: (1) the steady state levels of key proteins necessary for AA release and 5-LO metabolism; (2) posttranslational modifications (such as phosphorylation) that alter the catalytic activities of these proteins; and (3) the concentrations of small molecules (e.g., Ca 2+ , ATP, and glutathione) that serve as cofactors for certain of these proteins. One additional determinant of LT biosynthesis that has been recognized is the intracellular compartmentalization of these LT-forming proteins. This topic has become a focus of investigation in our own and other laboratories, and this article reviews the current state of knowledge in this area. Indeed, a number of unexpected findings that have emerged from these studies underscore the appropriateness of a perspective on LT biology that transcends conventional notions.
Voltage-gated sodium channels (VGSCs) are responsible for the initial inwards current during the depolarisation phase of action potential in excitable cells. Therefore, VGSCs are crucial for cardiac and nerve function, since the action potential of nerves and muscle cannot occur without them. Their importance in generation and transmission of signals has been known for more than 40 years but the more recent introduction of new electrophysiological methods and application of molecular biology techniques has led to an explosion of research on many different ion channels, including VGSCs. Their extraordinary biological importance makes them logical and obvious targets for toxins produced by animals and plants for attack or defence. The action of these and similar substances modulating the function of the VGSCs is interesting with respect to their possible use in medicine or use as tools in the study of these molecules. This review summarises recent progress in this research field and, in particular, considers what is known about the relationship of the structure to function, including a current understanding of the pharmacological modulation of VGSCs.
The 1,4- and the 1,5-benzodiazepines (BDZ) are commonly used as anxiolytic and anticonvulsive drugs. It has been suggested that they influence, particularly through stimulation of peripheral BDZ receptors, some immune cell properties such as pro-inflammatory cytokine production. The availability of a new class of [1,2,4]triazolo[4,3-a][1,5]benzodiazepine derivatives (compounds IV), endowed with anti-inflammatory and/or analgesic properties but no anti-pentylenetetrazole activity, prompted us to investigate in more detail the anti-inflammatory properties of three selected compounds IV (N,N-dimethyl-1-phenyl-4H-[1,2,4]triazolo[4,3-a][1,5]benz- odiazepin-5-amine; N,N-dibutyl-4H-[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5-amine; 1-methyl-N,N-dimethyl-4H-[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5-amine) and one structurally related compound (1-phenyl-4H-[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5(6H)-one). These BDZ derivatives have lost their affinity for the central and peripheral BDZ receptors. The in vivo effect on leukocyte migration of these compounds was investigated by using the mouse air-pouch model of local inflammation. Compounds A and B, significantly inhibited the carrageenan-induced leukocyte recruitment in a dose-dependent manner starting from the dose of 50 mgkg(-1), whereas compound C was effective only at the higher dose of 100 mgkg(-1). Compound D did not exert such effects at any of the doses considered. The effect of compounds A, B and C on leukocyte recruitment was paralleled by a significant inhibition of interleukin-6 and prostaglandin E(2)production in the exudate, similarly to indomethacin, and by a partial reduction of vascular permeability. These features may be relevant for the design and development of innovative anti-inflammatory molecules among the 4H-[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5-amine derivatives.
1. The autonomic effects of venoms and toxins from several species of scorpions, including the Indian red scorpion Mesobuthus tamulus, the Chinese scorpion Buthus martensi Karsch and the Israeli scorpion Leiurus quinquestriatus quinquestriatus, all belonging to Buthidae, and the Asian black scorpions Heterometrus longimanus and Heterometrus spinifer, belonging to Scorpionidae, are reviewed.
2. The effects of the venoms of M. tamulus and L. q. quinquestriatus on noradrenergic and nitrergic transmission in the rat isolated anococcygeus muscle revealed that both venoms mediated their pharmacological effects via a prejunctional mechanism involving the activation of voltage-sensitive sodium channels with consequent release of neurotransmitters that mediate target organ responses, similar to the effects mediated by other α-scorpion toxins.
3. Two new toxins, Makatoxin I and Bukatoxin, were purified to homogeneity from the venom of B. martensi Karsch. Determination of their complete amino acid sequences confirmed that both toxins belonged to the class of α-scorpion toxins. The effects of both toxins on noradrenergic and nitrergic transmission in the rat anococcygeus muscle provided firm evidence that their pharmacological actions also closely resembled those mediated by other α-scorpion toxins on neuronal voltage-sensitive sodium channels.
4. The venoms of H. longimanus and H. spinifer were found to have high concentrations of noradrenaline (1.8 ± 0.3 mmol/L) and relatively high concentrations of acetylcholine (79.8 ± 1.7 µmol/L) together with noradrenaline (146.7 ± 19.8 µmol/L), respectively, which can account for their potent direct cholinergic and noradrenergic agonist actions in the rat anococcygeus muscle.
5. Our studies confirmed that the rat anococcygeus muscle is an excellent nerve–smooth muscle preparation for investigating the effects of bioactive agents on noradrenergic and nitrergic transmission, as well as the direct agonist actions of these agents on post-synaptic α-adrenoceptors and M3 muscarinic cholinoceptors. Although many studies, including our own, have documented that scorpion venoms and toxins mediate their primary effects via a prejunctional mechanism that leads to the marked release of various autonomic neurotransmitters, our studies have shown that there are exceptions to this generally accepted phenomenon. In particular, we have provided firm evidence to show that the venoms from H. longimanus and H. spinifer do not have such a prejunctional site of action but, instead, the venoms mediate their autonomic effects through direct agonist actions on post-junctional muscarinic M3 cholinoceptors and α-adrenoceptors.
Scorpion envenomation is a common medical problem in many countries and an important cause of morbidity and mortality, especially among children. The plasma levels of pro-inflammatory (IL-1beta, IL-6, IL-8 and TNF-alpha) and anti-inflammatory (IL-10) cytokines were measured in individuals stung by Tityus serrulatus (Ts) scorpions. According to clinical manifestations patients were classified, as defined by the Brazilian Ministry of Health, as having mild (n=15, mean age=42.2 years), moderate (n=8, mean age=26 years) or severe (n=4, mean age=14 years) envenomation. Blood samples were taken immediately (T1) and 6h (T2) after admission to the hospital. Eighteen age-matched healthy volunteers were used as control. TNF-alpha, IL-1beta, IL-6 and IL-8 levels were significantly increased in moderate and severe cases and the levels of these cytokines were positively correlated with the severity of envenomation, as evaluated by clinical profile and plasma venom concentration. IL-10 levels were increased in severe and moderate cases and reduced in mild cases. The results reported in the present study suggest that the physiopathological manifestation of Ts envenomation may be mediated, at least in part, by cytokines, and that the early treatment after scorpion sting with drugs that inhibit cytokine production, such as glucocorticoids, may have a potential beneficial effect, ameliorating the severity of the clinical manifestations observed, particularly in severe and moderate cases.
A peptide was isolated from the venom of the scorpion Tityus trivittatus. It is an isoform of the toxin TsTX-IV earlier described [Toxicon 37 (1999) 651] and identical to butantoxin [Arch. Biochem. Biophys. 379 (2000) 18], both isolated from the Brazilian scorpion Tityus serrulatus. This newly characterized peptide contains 40 amino acid residues with a molecular mass of [M+H(+)] 4507.0, cross-linked by four disulfide bridges, made between the cysteine pairs: Cys2-Cys5, Cys10-Cys31, Cys16-Cys36 and Cys20-Cys38. It blocks in a completely reversible manner the Shaker B K(+)-channels, with a K(d) around 660nM. It belongs to the sub-family 12 and it is now being classified as alpha-KTx 12.2.
Omega-6 (omega-6) polyunsaturated fatty acids (PUFA), abundant in the Western diet, are precursors for a number of key mediators of inflammation including the 2-series of prostaglandins (PG). PGE(2), a cyclooxygenase (COX) metabolite of arachidonic acid, a omega-6 PUFA, is a potent mediator of inflammation and cell proliferation. Dietary supplements rich in omega-3 PUFA reduce the concentrations of 2-series PG and increase the synthesis of 3-series PG (e.g., PGE(3)), which are believed to be less inflammatory. However, studies on cellular consequences of increases in 3-series PG in comparison to 2-series PG have not been reported. In this study, we compared the effects of PGE(2) and PGE(3) on (i) cell proliferation in NIH 3T3 fibroblasts, (ii) expression and transcriptional regulation of the COX-2 gene in NIH 3T3 fibroblasts, and (iii) the production of an inflammatory cytokine, IL-6, in RAW 264.7 macrophages. PGE(3), unlike PGE(2), is not mitogenic to NIH 3T3 fibroblasts. PGE(2) and PGE(3) both induce COX-2 mRNA via similar signaling mechanisms; however, compared with PGE(2), PGE(3) is significantly less efficient in inducing COX-2 gene expression. Furthermore, although both PGE(2) and PGE(3) induce IL-6 synthesis in RAW 264.7 macrophages, PGE(3) is substantially less efficient compared with PGE(2). We further show that increasing the omega-3 content of membrane phospholipid results in a decrease in mitogen-induced PGE(2) synthesis. Taken together, our data suggest that successful replacement of omega-6 PUFA with omega-3 PUFA in cell membranes can result in a decreased cellular response to mitogenic and inflammatory stimuli.
A toxin with four disulfide bridges from Tityus serrulatus venom was able to compete with 125I-kaliotoxin on rat brain synaptosomal preparations, with an IC50 of 46 nM. The obtained amino acid sequence and molecular mass are identical to the previously described butantoxin. Enzymatic cleavages in the native peptide followed by mass spectrometry peptide mapping analysis were used to determine the disulfide bridge pattern of alpha-KTx12-1. Also, after the cleavage of the first six N-terminal residues, including the unusual disulfide bridge which forms an N-terminus ring, the potency of the cleaved peptide was found to decrease about 100 fold compared with the native protein.
Fatty acids have diverse roles in all cells. They are important as a source of energy, as structural components of cell membranes, as signalling molecules and as precursors for the synthesis of eicosanoids. Recent research has suggested that the organization of fatty acids into distinct cellular pools has a particularly important role in cells of the immune system and that forms of lipid trafficking exist, which are as yet poorly understood. This Review examines the nature and regulation of cellular lipid pools in the immune system, their delivery of fatty acids or fatty acid derivatives to specific locations and their potential role in health and disease.
Potassium channels are widespread in living cells and are involved in many diseases. The scorpion toxin alpha-KTx(12.1) interacts with various K(+) channels, suggesting its capacity to match diverse channel pores. It is recognized that tissue injuries may affect the pH at toxins site of action, thereby modulating both protein conformation and activity. To better understand its molecular mechanism of action, we studied alpha-KTx(12.1) using pH as a tool to explore its plasticity and NMR in combination with MD calculations to detect it. The toxin solution structure consists of an alpha-helix and a triple-stranded beta-sheet stabilized by four disulfide bridges. The NMR results show, in addition, that His28 possesses an unusually low pK(a) of 5.2. The best set of protein conformers is obtained at pH 4.5, while at pH 7.0, the reduced number of NOEs resulting from a faster hydrogen exchange does not allow to reach a good structural convergence. Nonetheless, MD calculations show that the toxin structure does not vary significantly in that pH range, while conformational changes and modifications of the surface charge distribution occur when His28 is fully protonated. Moreover, essential dynamics analysis reveals variations in the toxin's coherent motions. In conclusion, His28, with its low pK(a) value, provides alpha-KTx(12.1) with the ability to preserve its active conformation over a wide pH interval, thus expanding the range of cellular conditions where the toxin can fully exhibit its activity. Overall, the results further underline the role of histidine as a natural controller of proteins' functionality.
In this study we characterized the nociceptive response and edema induced by the venom of the scorpion Tityus serrulatus in rats and mice and carried out a preliminary pharmacological investigation of the mechanisms involved in these responses. Intraplantar injection of the venom (1 or 10mug) induced edema and a marked ipsilateral nociceptive response, characterized by thermal and mechanical allodynia and paw licking behaviour. The nociceptive response was inhibited by previous intraperitoneal administration of indomethacin (4mg/kg), dipyrone (200mg/kg), cyproheptadine (10mg/kg) or morphine (5 or 10mg/kg), but not by dexamethasone (1 or 4mg/kg) or promethazine (1 or 5mg/kg). The edema was inhibited by previous treatment with promethazine (5 or 10mg/kg) or cyproheptadine (5 or 10mg/kg), but not by indomethacin (2 or 4mg/kg), dexamethasone (1 or 4mg/kg) or cromolyn (40 or 80mg/kg). Some bioactive amines, including histamine and 5-hydroxytryptamine, were found in the venom in low concentrations. In conclusion, the nociceptive response and edema induced by the venom of T. serrulatus may result from the action of multiple mediators including eicosanoids, histamine and 5-hydroxytryptamine. These results may lead to a better understanding of the host response to potent animal toxins and also give insights into a more rational pharmacological approach to alleviate the intense pain associated with the scorpion envenomation.
Animal venom can induce systemic alterations similar to those observed in acute-phase inflammatory response. In the present study, we report the systemic (circulatory) and local (peritoneal cavity) effects induced by Tityus serrulatus venom and its major toxin TsTX-I (Ts1) in mice over various time periods. Both the venom and TsTX-I elicited quite similar responses in most assays. Responses included reduction of albumin, increased C-reactive protein, IL-6, IL-1alpha and TNF-alpha. Local and systemic leucocytosis, with a predominance of polymorphonuclear cells, was also observed. These effects show that a systemic inflammation-like syndrome is triggered during the severe envenomation caused by the T. serrulatus sting. The initial increases of albumin and total protein were probably consequences of the dehydration that occurs at the beginning of envenomation. Time-course analysis of these effects shows that responses are most pronounced on the first day after poisoning. However, leucocytosis and changes in acute-phase protein concentrations can be observed up to 7 days after envenomation.