RESUMO
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.
Assuntos
Venenos de Escorpião , Toxoplasmose , Animais , Técnicas de Química Sintética , Citocinas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Venenos de Escorpião/uso terapêutico , Escorpiões , Toxoplasma , Toxoplasmose/tratamento farmacológicoRESUMO
Scorpion envenomation is a public health problem in tropical and subtropical areas. In Brazil, Tityus serrulatus is the biggest cause of accidents with venomous animals. Tityus serrulatus venom causes symptoms related to a great activation of the autonomic system attributed to a massive release of sympathetic and parasympathetic mediators. This effect is attributed to the presence of toxins acting in Na+ and K+ ion channels, leading to an increase in cell excitability. Although gastrointestinal symptoms, like diarrhoea and sialorrhea, is observed in moderate to severe cases, little attention is given in clinical reports. Gastrointestinal motility is controlled by the enteric nervous system which is composed of a wide variety of interconnected neurons that are influenced by the sympathetic and parasympathetic nervous systems. Thus, this work aimed to characterize the effects of Tityus serrulatus venom on sympathetic and parasympathetic neurotransmission of rat jejunum, as well as to investigate possibles effects on other neurons of the enteric nervous system. To this, we verify the effects of Tityus serrulatus venom on the contractility of isolated rat jejunum through organ-bath experiments. We observed that venom can induce both contraction and relaxation. The contraction was partially inhibited by atropine (1 µM) and by suramin (0.1 mM) through tetrodotoxin-resistant and sensitive mechanisms. The relaxation was completely inhibited by 3 µM propranolol and partially inhibited by 1 µM phentolamine. Suramin induced a slowing of relaxation curve. Tetrodotoxin completely inhibits the relaxation induced by Tityus serrulatus venom, but the contraction curves were only partially reduced in their initial portion. The final part of the curve was largely enhanced by Tetrodotoxin. Atropine blocks almost completely the contraction curve in the presence of Tetrodotoxin. These results indicate that Tityus serrulatus venom induces the release of both excitatory (predominantly acetylcholine) and inhibitory (mainly noradrenaline) neurotransmitters. The effects of Tityus serrulatus venom on organ contractility was quite complex and seem to derive from a diffuse and nonspecific release of mediators from autonomic and enteric nervous systems. Further investigation of venom action and its isolated toxins can reveal important aspects to deepen our knowledge about the enteric nervous system transmission and the interaction between excitatory and inhibitory mediators as well as the physiological role of Na+ and K+ ion channels in gut motility.
Assuntos
Sistema Nervoso Entérico/efeitos dos fármacos , Jejuno/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Neurotoxinas/farmacologia , Sistema Nervoso Parassimpático/efeitos dos fármacos , Venenos de Escorpião/farmacologia , Sistema Nervoso Simpático/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Masculino , Ratos , Ratos WistarRESUMO
Scorpion envenomation is a public health problem in tropical and subtropical areas. In Brazil, Tityus serrulatus is the biggest cause of accidents with venomous animals. Tityus serrulatus venom causes symptoms related to a great activation of the autonomic system attributed to a massive release of sympathetic and parasympathetic mediators. This effect is attributed to the presence of toxins acting in Na+ and K+ ion channels, leading to an increase in cell excitability. Although gastrointestinal symptoms, like diarrhoea and sialorrhea, is observed in moderate to severe cases, little attention is given in clinical reports. Gastrointestinal motility is controlled by the enteric nervous system which is composed of a wide variety of interconnected neurons that are influenced by the sympathetic and parasympathetic nervous systems. Thus, this work aimed to characterize the effects of Tityus serrulatus venom on sympathetic and parasympathetic neurotransmission of rat jejunum, as well as to investigate possibles effects on other neurons of the enteric nervous system. To this, we verify the effects of Tityus serrulatus venom on the contractility of isolated rat jejunum through organ-bath experiments. We observed that venom can induce both contraction and relaxation. The contraction was partially inhibited by atropine (1 µM) and by suramin (0.1 mM) through tetrodotoxin-resistant and sensitive mechanisms. The relaxation was completely inhibited by 3 µM propranolol and partially inhibited by 1 µM phentolamine. Suramin induced a slowing of relaxation curve. Tetrodotoxin completely inhibits the relaxation induced by Tityus serrulatus venom, but the contraction curves were only partially reduced in their initial portion. The final part of the curve was largely enhanced by Tetrodotoxin. Atropine blocks almost completely the contraction curve in the presence of Tetrodotoxin. These results indicate that Tityus serrulatus venom induces the release of both excitatory (predominantly acetylcholine) and inhibitory (mainly noradrenaline) neurotransmitters. The effects of Tityus serrulatus venom on organ contractility was quite complex and seem to derive from a diffuse and nonspecific release of mediators from autonomic and enteric nervous systems. Further investigation of venom action and its isolated toxins can reveal important aspects to deepen our knowledge about the enteric nervous system transmission and the interaction between excitatory and inhibitory mediators as well as the physiological role of Na+ and K+ ion channels in gut motility.
RESUMO
The number of cases of envenomation by scorpions has grown significantly in Brazil since 2007, with the most severe cases being caused by the Tityus serrulatus scorpion. Although envenomed patients mostly suffer neurotoxic manifestations, other symptoms, such as hypertension, cannot be exclusively attributed to neurotoxins. Omics analyses have detected plentiful amounts of metalloproteases in T. serrulatus venom. However, the roles played by these enzymes in envenomation are still unclear. Endeavoring to investigate the functions of scorpion venom proteases, we describe here for the first time an Angiotensin I-Converting Enzyme-like peptidase (ACE-like) purified from T. serrulatus venom. The crude venom cleaved natural and fluorescent substrates and these activities were inhibited by captopril. Regarding the serum neutralization, the scorpion antivenom was more effective at blocking the ACE-like activity than arachnid antivenom, although neither completely inhibited the venom cleavage action, even at higher doses. ACE-like was purified from the venom after three chromatographic steps and its identity was confirmed by mass spectrometric and transcriptomic analyses. Bioinformatics analysis showed homology between the ACE-like transcript sequences from Tityus spp. and human testis ACE. These findings advance our understanding of T. serrulatus venom components and may improve treatment of envenomation victims, as ACE-like may contribute to envenomation symptoms, especially the resulting hypertension.
Assuntos
Peptídeo Hidrolases/metabolismo , Peptidil Dipeptidase A/metabolismo , Venenos de Escorpião/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Antivenenos/farmacologia , Captopril/farmacologia , Cloretos/farmacologia , Hipertensão/metabolismo , EscorpiõesRESUMO
Tityus serrulatus is the scorpion specie responsible for the majority of scorpion sting accidents in Brazil. Symptoms of envenomation by Tityus serrulatus range from local pain to severe systemic reactions such as cardiac dysfunction and pulmonary edema. Thus, this study has evaluated the participation of bronchial epithelial cells in the pulmonary effects of Tityus serrulatus scorpion venom (Tsv). Human bronchial epithelial cell line BEAS-2B were utilized as a model target and were incubated with Tsv (10 or 50 µg/mL) for 1, 3, 6 and 24 h. Effects on cellular response of venom-induce cytotoxicity were examined including cell viability, cell integrity, cell morphology, apoptosis/necrosis as well as cell activation through the release of pro-inflammatory cytokines IL-1ß, IL-6 and IL-8. Tsv caused a decrease in cell viability at 10 and 50 µg/mL, which was confirmed by lactate dehydrogenase (LDH) measurement. Flow cytometry analyses revealed necrosis as the main cell death pathway caused by Tsv. Furthermore, Tsv induced the release of IL-1ß, IL-6 and IL-8. Altogether, these results demonstrate that Tsv induces cytotoxic effects on bronchial epithelial cells, involving necrosis and release of pro-inflammatory cytokines, suggesting that bronchial epithelial cells may play a role in the pulmonary injury caused by Tsv.
Assuntos
Brônquios/efeitos dos fármacos , Citocinas/biossíntese , Venenos de Escorpião/toxicidade , Animais , Apoptose/efeitos dos fármacos , Brônquios/citologia , Brônquios/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Técnicas In Vitro , Necrose , EscorpiõesRESUMO
Background Tityus serrulatus scorpion venom (TsV) contains toxins that act on K + and Na + channels and account for the venoms 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 g/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 g/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.(AU)
Assuntos
Animais , Venenos de Escorpião , Animais Peçonhentos , Imunomodulação/efeitos dos fármacos , Linfócitos T/efeitos dos fármacosRESUMO
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.
RESUMO
Background Tityus serrulatus scorpion venom (TsV) contains toxins that act on K + and Na + channels and account for the venoms 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 g/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 g/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.
Assuntos
Animais , Animais Peçonhentos , Imunomodulação/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Venenos de EscorpiãoRESUMO
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.(AU)
Assuntos
Animais , Venenos de Escorpião , Linfócitos T , Proliferação de Células , Citometria de Fluxo , ToxicidadeRESUMO
BACKGROUND AND PURPOSE: The antipyretic and hypothermic prodrug dipyrone prevents PGE2 -dependent and -independent fever induced by LPS from Escherichia coli and Tityus serrulatus venom (Tsv) respectively. We aimed to identify the dipyrone metabolites responsible for the antipyretic and hypothermic effects. EXPERIMENTAL APPROACH: Male Wistar rats were treated i.p. with indomethacin (2 mg·kg(-1) ), dipyrone, 4-methylaminoantipyrine (4-MAA), 4-aminoantipyrine (4-AA) (60-360 mg·kg(-1) ), 4-formylaminoantipyrine, 4-acethylaminoantipyrine (120-360 mg·kg(-1) ) or vehicle 30 min before i.p. injection of LPS (50 µg·kg(-1) ), Tsv (150 µg·kg(-1) ) or saline. Rectal temperatures were measured by tele-thermometry and dipyrone metabolite concentrations determined in the plasma, CSF and hypothalamus by LC-MS/MS. PGE2 concentrations were determined in the CSF and hypothalamus by elisa. KEY RESULTS: In contrast to LPS, Tsv-induced fever was not followed by increased PGE2 in the CSF or hypothalamus. The antipyretic time-course of 4-MAA and 4-AA on LPS-induced fever overlapped with the period of the highest concentrations of 4-MAA and 4-AA in the hypothalamus, CSF and plasma. These metabolites reduced LPS-induced fever and the PGE2 increase in the plasma, CSF and hypothalamus. Only 4-MAA inhibited Tsv-induced fever. The higher doses of dipyrone and 4-MAA also induced hypothermia. CONCLUSIONS AND IMPLICATIONS: The presence of 4-MAA and 4-AA in the CSF and hypothalamus was associated with PGE2 synthesis inhibition and a decrease in LPS-induced fever. 4-MAA was also shown to be an antipyretic metabolite for PGE2 -independent fever induced by Tsv suggesting that it is responsible for the additional antipyretic mechanism of dipyrone. Moreover, 4-MAA is the hypothermic metabolite of dipyrone.