- Email: [email protected]
168. Tityus serrulatus Venom Induces a Higher Lung Inflammation in Mice Selected for Maximal Inflammatory Response
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
eukaryotic cells and are involved in a variety of functions such as lipid metabolism, cell signaling and inflammation. However, nothing is known about the formation and function of LBs in alveolar macrophages stimulated with Ts1, Ts2 and Ts6 from the venom of T. serrulatus escorpion. Methodology and Results: Ts1, Ts2 and Ts6 were not cytotoxic in all concentrations used in MH-S cells. NO concentration was measured by Greiss method and interleukin (IL)-6, IL-10 and tumor necrosis factor (TNF)-a by ELISA. NO, IL-6 and TNF-a production by MH-S cells, stimulated with Ts1 or Ts6, were enhanced under LPS prestimulation. On the other hand, Ts2 inhibited the release of these inflammatory mediators and increased IL-10 production. LBs formation increased after toxin stimulation compared to non-stimulated cells. Discussion and Conclusion: Our results demonstrated that, in vitro, individual scorpion toxins possess different properties. Ts1 and Ts6 presented similar effects that were opposite to Ts2 regarding to NO, TNF-a, IL-6 and IL-10. Ts1, Ts2 and Ts6 induced the formation of LBs, which could be related with eicosanoids production. We might suggest that production of cytokine and lipid mediators by toxinstimulated macrophages is independent of toxin ion channel interactions, since that Ts1 and Ts2 act on Naþ ion channels, and Ts6 act on Kþ ion channels.
181
tested in electrophysiological assays on a panel of six Kþ channels (Kv1.1-1.6) using the two-electrode voltage clamp technique. A cDNA library from the telson was constructed and specific screening of genes was conducted. Different algorithms and bioinformatics tools were used for the sequences analysis. Results: In the present study, we report for the first time, the molecular, biochemical and electrophysiological characterization of the components present in the soluble venom from M. gibbosus. Three new alpha-KTx peptides were found and called MegKTx1, MegKTx2 and MegKTx3 (Mesobuthus gibbosus, Kþ channel toxin number 1 to 3). Biochemical and molecular characterization of MegKTx peptides and genes shows a relation with toxins of three different alpha-KTx subfamilies. Conclusions: The exploration of the components present in the venom from M. gibbosus and the identification of gene sequences are important to understand the biological role of venom components that interact with Kþ channels. Consequently, this information may help in the dissection and knowledge of the noxious effects produced by this scorpion sting. References
167. Novel Potassium Channel Blocker Venom Peptides from Mesobuthus gibbosus (Scorpiones: Buthidae)
Tytgat et al., 1999 Trends Pharmacol Sci. 11:444-7. Srinivasan et al., 2002 J. Biol. Chem. 277:30040-47. Rodríguez de la Vega and Possani, 2004 Toxicon 43:865-75. Ceraretli and Ozkan 2010 Rev. Inst. Med. Trop. Sao Paulo 52(4):215-20. Adiguzel, 2010 J. Venom Anim. Toxins Incl. Trop. Dis 16:198-211.
Elia Diego-García 1, Steve Peigneur 1, Sarah Debaveye 1, Eveline Gheldof 1, Jan Tytgat 1, Figen Caliskan 2
Keywords: scorpion, alpha-KTx, toxin, gene 10.1016/j.toxicon.2012.04.168
Keywords: MH-S, Tityus serrulatus, inflammatory mediators 10.1016/j.toxicon.2012.04.167
1
Laboratory of Toxicology, University of Leuven (KUL), Leuven, Belgium Department of Biology, Faculty of Science and Art, Eskisehir Osmangazi University, Campus Meselik, Eskisehir, Turkey E-mail address: [email protected] (E. Diego-García). 2
Background: Scorpion toxins specific for potassium channels (KTx) have been classified on the basis of the alignment of Cys and other conserved residues into four families, known as alpha-, beta-, gamma-KTx [1] and kappaKTx [2]. The alpha-KTx family is considered the largest [3]. Until now, twenty-two subfamilies have been reported and several new peptides are continuously being discovered. Mesobuthus gibbosus (Brullé, 1832) belongs to the Buthidae family. This species is widely distributed in the Eastern Mediterranean region; the geographical range includes the Balkan Peninsula (Albania, Montenegro, Macedonia and Greece) and Anatolia (Turkey, except for the coast of the Black Sea). According to the epidemiological and clinical situation of scorpion envenomations in Turkey, M. gibbosus is one of the most important health-threatening scorpion species [4]. Despite the medical importance reported for M. gibbosus [5], there is no additional information of toxin and venom components to clarify the toxic effect of a M. gibbosus sting. Methods: Biochemical characterization was performed using different protocols and techniques following a bioassay-guided strategy (HPLC, mass spectrometry and EDMAN degradation sequencing). Venom fractions were
168. Tityus serrulatus Venom Induces a Higher Lung Inflammation in Mice Selected for Maximal Inflammatory Response Priscila G. Lara 1, Thaís R. Narcizo 1, Fernanda C.V. Portaro 2, Nancy Starobinas 1, Vera Aiello V 3, Luiz A. Benvenuti 3, Osvaldo A. Sant'Anna 2, Orlando G. Ribeiro 1, Mônica Spadafora-Ferreira 1 1
Laboratório de Imunogenética, Instituto Butantan, São Paulo, Brazil Laboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil 3 Laboratório de Patologia, Instituto do Coração (InCor), São Paulo, Brazil E-mail address: [email protected] (M. Spadafora-Ferreira). 2
Background: Tityus serrulatus is the main cause of scorpion envenomations in Brazil. Cardiovascular failure complicated by pulmonary edema is the main cause of death after severe envenomation. T. serrulatus venom (TsV) induces a systemic inflammatory response with the release of inflammatory mediators and cytokines both in patients and animal models. Lung alterations have been reported with the presence of inflammatory infiltrating cells and edema. The amount of venom inoculated, age, physical condition and genetic factors of the victims directly influence the severity of symptoms reported by patients. This study aimed to evaluate the action of TsV in the lung inflammation in strains of mice
182
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
genetically selected for high (AIRmax) or low (AIRmin) acute inflammatory response and BALB/c and observe if genetic factors are involved in the response to the venom. Methods: AIRmax, AIRmin and BALB/c mice were inoculated (s.c.) with increasing doses for LD50 determination. A subletal dose of 0.75 mg/g of TsV was inoculated in mice and after different periods, the lungs were collected, for H&E histopathological analysis, myeloperoxidase (MPO) quantification, phenotype characterization of infiltrating cells and cytokine and chemokine analysis by ELISA or cytometry analysis. Results: Lung histology analysis of AIRmax and AIRmin TsV-treated mice showed an increased perivascular infiltrate and the presence of haemorrhage and alveolar edema 1 and 2h after venom inoculation, but no cardiac alterations were observed. MPO activity showed that the venom induced a significant migration of neutrophils in AIRmax when compared to AIRmin and BALB/c mice, 2h after venom inoculation. Phenotypic analysis of lung cell populations of TsV-treated mice showed that after 1 hour, AIRmax presented significantly more Ly6GþCD11bþ cells and after 2 hours an increase in F4/80þCD11bþ and CD3þ cells when compared to their controls and to AIRmin strain. In addition, AIRmin mice had an increased number of Ly6GþCD11bþ cells and F4/80þCD11bþ only after 2 hours of administration of the venom. Lungs of AIRmax TsV-treated mice presented higher levels of IL-6 and TNF-a and the chemokines MCP-1, MIP-1b and RANTES, compared to AIRmin and BALB/c. Discussion and Conclusions: Our results suggest that T. serrulatus scorpion venom is able to induce significant inflammatory response in the lung with presence of polymorphonuclear cells macrophages and lymphocytes, as well as pro-inflammatory cytokines. This inflammatory response is more pronounced in AIRmax mice, thus suggesting the importance of genetic factors in the inflammatory response to animal venoms. Financial support: FAPESP, INCTTOX Program – CNPq, Brazil. Keywords: Tityus serrulatus, scorpion venom, lung inflammation, inflammatory response 10.1016/j.toxicon.2012.04.169
169. Tityus serrulatus Scorpion Laboratory Breeding and Venom Collection for Antivenom Production and Research Claudio M.V. Souza 1, Jonathan R.L. Vieira 1, Jonathan R. Souza 1, Lana S. Sales 1, Luis E.R. Cunha 2 1
Laboratório de Artrópodos, Instituto Vital Brazil, Niterói, RJ, Brazil Diretoria Científica ,Instituto Vital Brazil, Niterói, RJ, Brazil E-mail address: [email protected] (J.R.L. Vieira).
capture for venom extraction and antigen and research application. In this study we present a successful laboratory breeding protocol for this parthenogenetic scorpion specie and venom obtaining. Methods: 372 young T. serrulatus scorpions were separated immediately after leaving their mothers’ back and accommodated in plastic black boxes (2508 cm2 and 1872 cm2) with cotton plugs for humidity and paperboard shelters. The animals were kept at 23o C to 25o C and 12 light/shadow period; live crickets (Gryllus sp.) were used as food. Young scorpions received 27 %; immature scorpions 38 % and adult scorpions 50 % body weight of food in a 7 days intervals scheme. The venom was milked by electrical stimulation (70 mV, DC) from the animals telsons. The compared toxicity of laboratory breeding animals venom and T. serrulatus venom captured in the field was determinated in a mice Lethal Dose 50% (LD50) model by intraperitoneal injection of lyophilized venoms (0.25; 0.5; 1.0 mg/kg, n¼8). Results: The monthly average scorpions weight gain was 37.6 % and after 11 months and 4.5 molts 275 adults (73.9 %) produced 1.7 mg liquid venom/scorpion by electric milking (not different from the field animals average: 2.0 mg liquid venom/scorpion). The DL 50% (48h) from laboratory scorpions, venom was 1.2 mg/kg i.p. and the venom from field animals, 1.6 mg/kg i.p. All mice showed signs of experimental scorpionism. Discussion: There is very little information on T. serrulatus’ field and laboratory biology. The widespread increase in scorpion stings in Brazil, mainly due to T. serrulatus’ urban colonization, requires basic biologic knowledge of this species. Conclusions: The present laboratory breeding protocol for this dangerous scorpion species is a very useful tool for standardizing bioterium conditions and venom collection for public health needs and research. Keywords: Tityus serrulatus; laboratory breeding; venom 10.1016/j.toxicon.2012.04.170
170. Identification of a Dynorphin-Degrading Metallopeptidase Releasing Leu-Enkephalin in Brazilian Tityus spp. Scorpion Venoms Emerson J. Venancio 1, 2, Fernanda C.V. Portaro 2, Alexandre K. Kuniyoshi 2, Daniela Cajado Carvalho 2, Giselle Pidde-Queiroz 2, Denise V. Tambourgi 2 1
Dept. of Pathological Science, State University of Londrina, Paraná, Brazil Immunochemistry Laboratory, Butantan Institute, São Paulo, Brazil E-mail address: [email protected] (D.V. Tambourgi). 2
2
Background: Tityus serrulatus is the most dangerous scorpion in Brazil. The number of scorpion stings due to this specie has sharply increased in the last years as documents from official information health systems. Antivenom therapy is mandatory on mild and severe cases of human scorpionism. Brazilian environmental legal system is very complex and creates serious difficulties to scorpions
Background: Accidents caused by scorpions from the genus Tityus are an important public health problem in Brazil, scorpion envenomings occur more frequently than those caused by other venomous animals, including snakes. In the present study, we have analysed some toxic characteristics of the venoms from three scorpion species of the genus Tityus. Methods: T. serrulatus, T. bahiensis and T. stigmurus venoms and antivenoms were provided by Butantan
eukaryotic cells and are involved in a variety of functions such as lipid metabolism, cell signaling and inflammation. However, nothing is known about the formation and function of LBs in alveolar macrophages stimulated with Ts1, Ts2 and Ts6 from the venom of T. serrulatus escorpion. Methodology and Results: Ts1, Ts2 and Ts6 were not cytotoxic in all concentrations used in MH-S cells. NO concentration was measured by Greiss method and interleukin (IL)-6, IL-10 and tumor necrosis factor (TNF)-a by ELISA. NO, IL-6 and TNF-a production by MH-S cells, stimulated with Ts1 or Ts6, were enhanced under LPS prestimulation. On the other hand, Ts2 inhibited the release of these inflammatory mediators and increased IL-10 production. LBs formation increased after toxin stimulation compared to non-stimulated cells. Discussion and Conclusion: Our results demonstrated that, in vitro, individual scorpion toxins possess different properties. Ts1 and Ts6 presented similar effects that were opposite to Ts2 regarding to NO, TNF-a, IL-6 and IL-10. Ts1, Ts2 and Ts6 induced the formation of LBs, which could be related with eicosanoids production. We might suggest that production of cytokine and lipid mediators by toxinstimulated macrophages is independent of toxin ion channel interactions, since that Ts1 and Ts2 act on Naþ ion channels, and Ts6 act on Kþ ion channels.
181
tested in electrophysiological assays on a panel of six Kþ channels (Kv1.1-1.6) using the two-electrode voltage clamp technique. A cDNA library from the telson was constructed and specific screening of genes was conducted. Different algorithms and bioinformatics tools were used for the sequences analysis. Results: In the present study, we report for the first time, the molecular, biochemical and electrophysiological characterization of the components present in the soluble venom from M. gibbosus. Three new alpha-KTx peptides were found and called MegKTx1, MegKTx2 and MegKTx3 (Mesobuthus gibbosus, Kþ channel toxin number 1 to 3). Biochemical and molecular characterization of MegKTx peptides and genes shows a relation with toxins of three different alpha-KTx subfamilies. Conclusions: The exploration of the components present in the venom from M. gibbosus and the identification of gene sequences are important to understand the biological role of venom components that interact with Kþ channels. Consequently, this information may help in the dissection and knowledge of the noxious effects produced by this scorpion sting. References
167. Novel Potassium Channel Blocker Venom Peptides from Mesobuthus gibbosus (Scorpiones: Buthidae)
Tytgat et al., 1999 Trends Pharmacol Sci. 11:444-7. Srinivasan et al., 2002 J. Biol. Chem. 277:30040-47. Rodríguez de la Vega and Possani, 2004 Toxicon 43:865-75. Ceraretli and Ozkan 2010 Rev. Inst. Med. Trop. Sao Paulo 52(4):215-20. Adiguzel, 2010 J. Venom Anim. Toxins Incl. Trop. Dis 16:198-211.
Elia Diego-García 1, Steve Peigneur 1, Sarah Debaveye 1, Eveline Gheldof 1, Jan Tytgat 1, Figen Caliskan 2
Keywords: scorpion, alpha-KTx, toxin, gene 10.1016/j.toxicon.2012.04.168
Keywords: MH-S, Tityus serrulatus, inflammatory mediators 10.1016/j.toxicon.2012.04.167
1
Laboratory of Toxicology, University of Leuven (KUL), Leuven, Belgium Department of Biology, Faculty of Science and Art, Eskisehir Osmangazi University, Campus Meselik, Eskisehir, Turkey E-mail address: [email protected] (E. Diego-García). 2
Background: Scorpion toxins specific for potassium channels (KTx) have been classified on the basis of the alignment of Cys and other conserved residues into four families, known as alpha-, beta-, gamma-KTx [1] and kappaKTx [2]. The alpha-KTx family is considered the largest [3]. Until now, twenty-two subfamilies have been reported and several new peptides are continuously being discovered. Mesobuthus gibbosus (Brullé, 1832) belongs to the Buthidae family. This species is widely distributed in the Eastern Mediterranean region; the geographical range includes the Balkan Peninsula (Albania, Montenegro, Macedonia and Greece) and Anatolia (Turkey, except for the coast of the Black Sea). According to the epidemiological and clinical situation of scorpion envenomations in Turkey, M. gibbosus is one of the most important health-threatening scorpion species [4]. Despite the medical importance reported for M. gibbosus [5], there is no additional information of toxin and venom components to clarify the toxic effect of a M. gibbosus sting. Methods: Biochemical characterization was performed using different protocols and techniques following a bioassay-guided strategy (HPLC, mass spectrometry and EDMAN degradation sequencing). Venom fractions were
168. Tityus serrulatus Venom Induces a Higher Lung Inflammation in Mice Selected for Maximal Inflammatory Response Priscila G. Lara 1, Thaís R. Narcizo 1, Fernanda C.V. Portaro 2, Nancy Starobinas 1, Vera Aiello V 3, Luiz A. Benvenuti 3, Osvaldo A. Sant'Anna 2, Orlando G. Ribeiro 1, Mônica Spadafora-Ferreira 1 1
Laboratório de Imunogenética, Instituto Butantan, São Paulo, Brazil Laboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil 3 Laboratório de Patologia, Instituto do Coração (InCor), São Paulo, Brazil E-mail address: [email protected] (M. Spadafora-Ferreira). 2
Background: Tityus serrulatus is the main cause of scorpion envenomations in Brazil. Cardiovascular failure complicated by pulmonary edema is the main cause of death after severe envenomation. T. serrulatus venom (TsV) induces a systemic inflammatory response with the release of inflammatory mediators and cytokines both in patients and animal models. Lung alterations have been reported with the presence of inflammatory infiltrating cells and edema. The amount of venom inoculated, age, physical condition and genetic factors of the victims directly influence the severity of symptoms reported by patients. This study aimed to evaluate the action of TsV in the lung inflammation in strains of mice
182
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
genetically selected for high (AIRmax) or low (AIRmin) acute inflammatory response and BALB/c and observe if genetic factors are involved in the response to the venom. Methods: AIRmax, AIRmin and BALB/c mice were inoculated (s.c.) with increasing doses for LD50 determination. A subletal dose of 0.75 mg/g of TsV was inoculated in mice and after different periods, the lungs were collected, for H&E histopathological analysis, myeloperoxidase (MPO) quantification, phenotype characterization of infiltrating cells and cytokine and chemokine analysis by ELISA or cytometry analysis. Results: Lung histology analysis of AIRmax and AIRmin TsV-treated mice showed an increased perivascular infiltrate and the presence of haemorrhage and alveolar edema 1 and 2h after venom inoculation, but no cardiac alterations were observed. MPO activity showed that the venom induced a significant migration of neutrophils in AIRmax when compared to AIRmin and BALB/c mice, 2h after venom inoculation. Phenotypic analysis of lung cell populations of TsV-treated mice showed that after 1 hour, AIRmax presented significantly more Ly6GþCD11bþ cells and after 2 hours an increase in F4/80þCD11bþ and CD3þ cells when compared to their controls and to AIRmin strain. In addition, AIRmin mice had an increased number of Ly6GþCD11bþ cells and F4/80þCD11bþ only after 2 hours of administration of the venom. Lungs of AIRmax TsV-treated mice presented higher levels of IL-6 and TNF-a and the chemokines MCP-1, MIP-1b and RANTES, compared to AIRmin and BALB/c. Discussion and Conclusions: Our results suggest that T. serrulatus scorpion venom is able to induce significant inflammatory response in the lung with presence of polymorphonuclear cells macrophages and lymphocytes, as well as pro-inflammatory cytokines. This inflammatory response is more pronounced in AIRmax mice, thus suggesting the importance of genetic factors in the inflammatory response to animal venoms. Financial support: FAPESP, INCTTOX Program – CNPq, Brazil. Keywords: Tityus serrulatus, scorpion venom, lung inflammation, inflammatory response 10.1016/j.toxicon.2012.04.169
169. Tityus serrulatus Scorpion Laboratory Breeding and Venom Collection for Antivenom Production and Research Claudio M.V. Souza 1, Jonathan R.L. Vieira 1, Jonathan R. Souza 1, Lana S. Sales 1, Luis E.R. Cunha 2 1
Laboratório de Artrópodos, Instituto Vital Brazil, Niterói, RJ, Brazil Diretoria Científica ,Instituto Vital Brazil, Niterói, RJ, Brazil E-mail address: [email protected] (J.R.L. Vieira).
capture for venom extraction and antigen and research application. In this study we present a successful laboratory breeding protocol for this parthenogenetic scorpion specie and venom obtaining. Methods: 372 young T. serrulatus scorpions were separated immediately after leaving their mothers’ back and accommodated in plastic black boxes (2508 cm2 and 1872 cm2) with cotton plugs for humidity and paperboard shelters. The animals were kept at 23o C to 25o C and 12 light/shadow period; live crickets (Gryllus sp.) were used as food. Young scorpions received 27 %; immature scorpions 38 % and adult scorpions 50 % body weight of food in a 7 days intervals scheme. The venom was milked by electrical stimulation (70 mV, DC) from the animals telsons. The compared toxicity of laboratory breeding animals venom and T. serrulatus venom captured in the field was determinated in a mice Lethal Dose 50% (LD50) model by intraperitoneal injection of lyophilized venoms (0.25; 0.5; 1.0 mg/kg, n¼8). Results: The monthly average scorpions weight gain was 37.6 % and after 11 months and 4.5 molts 275 adults (73.9 %) produced 1.7 mg liquid venom/scorpion by electric milking (not different from the field animals average: 2.0 mg liquid venom/scorpion). The DL 50% (48h) from laboratory scorpions, venom was 1.2 mg/kg i.p. and the venom from field animals, 1.6 mg/kg i.p. All mice showed signs of experimental scorpionism. Discussion: There is very little information on T. serrulatus’ field and laboratory biology. The widespread increase in scorpion stings in Brazil, mainly due to T. serrulatus’ urban colonization, requires basic biologic knowledge of this species. Conclusions: The present laboratory breeding protocol for this dangerous scorpion species is a very useful tool for standardizing bioterium conditions and venom collection for public health needs and research. Keywords: Tityus serrulatus; laboratory breeding; venom 10.1016/j.toxicon.2012.04.170
170. Identification of a Dynorphin-Degrading Metallopeptidase Releasing Leu-Enkephalin in Brazilian Tityus spp. Scorpion Venoms Emerson J. Venancio 1, 2, Fernanda C.V. Portaro 2, Alexandre K. Kuniyoshi 2, Daniela Cajado Carvalho 2, Giselle Pidde-Queiroz 2, Denise V. Tambourgi 2 1
Dept. of Pathological Science, State University of Londrina, Paraná, Brazil Immunochemistry Laboratory, Butantan Institute, São Paulo, Brazil E-mail address: [email protected] (D.V. Tambourgi). 2
2
Background: Tityus serrulatus is the most dangerous scorpion in Brazil. The number of scorpion stings due to this specie has sharply increased in the last years as documents from official information health systems. Antivenom therapy is mandatory on mild and severe cases of human scorpionism. Brazilian environmental legal system is very complex and creates serious difficulties to scorpions
Background: Accidents caused by scorpions from the genus Tityus are an important public health problem in Brazil, scorpion envenomings occur more frequently than those caused by other venomous animals, including snakes. In the present study, we have analysed some toxic characteristics of the venoms from three scorpion species of the genus Tityus. Methods: T. serrulatus, T. bahiensis and T. stigmurus venoms and antivenoms were provided by Butantan