In vivo phagocytosis and hematology in Astyanax altiparanae, a potential model for surrogate technology / Fagocitose in vivo e hematologia em Astyanax altiparanae, um potencial modelo para tecnologia de propagação mediada

Abstract Although the potential of surrogate propagation technology for aquaculture and conservation of Neotropical fish, the poor understanding of the host immune system may results in rejection and destruction of the donor material. Thus, it is necessary to study and to develop methods to evaluate the effects of immunosuppressive drugs employment and to evaluate the immunocompatibility between donor and receptor. Thus, the present study aimed to optimize a methodology to assess in vivo phagocytosis in Astyanax altiparanae using Saccharomyces cerevisiae and to evaluate their hematological response resultant from the inflammatory induction. To this, S. cerevisiae were labeled with Congo red and injected in the coelomic cavity of A. altiparanae at the concentration of 2.5 x 106 cells mL-1. A PBS solution and a non-injected group were kept as control. Fish blood was sampled and the phagocytic capacity and index were determined at 1, 2, 3 and 6 h post-injection (hpi). The yeast injection successfully stimulated phagocytosis, with the best result for phagocytosis assessment after 2 hpi. Moreover, it was achieved a high traceability of phagocytized and non-phagocytized yeast under optic microscopy analysis due to the Congo red labeling. The hematological profile was similar to usually observed in early infections, indicating lymphocyte migration to inflammatory site and increase in number of circulating phagocytes due to natural response to inflammatory stimulus. In conclusion, our method was efficient to assess in vivo phagocytosis in A. altiparanae and will be an important tool to evaluate the efficacy of immunosuppressive drugs in this species. Additionally, these results may serve as support for further studies in fish immunocompetence, both in laboratory and in field conditions.

Resumo Apesar do potencial apresentado pela tecnologia de propagação mediada para a aquicultura e conservação de peixes Neotropicais, o pobre entendimento do sistema imune do hospedeiro pode resultar na rejeição e destruição do material do doador. Com isso, se fazem necessários o estudo e o desenvolvimento de métodos para análise tanto dos efeitos de drogas imunossupressoras quanto para a avaliação da imunocompatibilidade entre doadores e receptores. Logo, o presente estudo teve como objetivo aperfeiçoar um método para analisar a fagocitose in vivo em Astyanax altiparanae usando Saccharomyces cerevisiae marcado e avaliar seu perfil hematológico resultante da indução inflamatória. Para isso, S. cerevisiae foram marcados com vermelho Congo e injetados na cavidade celomática dos A. altiparanae na concentração de 2,5 x 106 células.mL-1. Peixes injetados com PBS e peixes não injetados foram mantidos como controle. Sangue foi colhido e a capacidade fagocítica e o índice fagocítico foram determinados após 1, 2, 3 e 6 horas após à injeção (hpi). A injeção de levedura estimulou a fagocitose com sucesso, com o melhor resultado atingido após 2 hpi. Ainda, foi observada uma alta rastreabilidade das leveduras fagocitadas e não fagocitadas sob microscopia óptica devido à marcação com vermelho Congo. O perfil hematológico foi similar ao observado usualmente em infecções recém-induzidas, indicando migração de linfócitos ao sítio inflamatório e aumento no número de fagócitos circulantes devido à resposta natural ao estímulo inflamatório. Como conclusão, nosso método foi eficiente para analisar a fagocitose in vivo em A. altiparanae e será uma ferramenta importante para a avaliação de eficácia de drogas imunossopressoras para esta espécie. Em adição, estes resultados podem contribuir para futuros estudos em imunocompetência em peixes, tanto em âmbito laboratorial quanto a campo.

Expression of myo-inositol cotransporters in the sciatic nerve and dorsal root ganglia in experimental diabetes

The transport of myo-inositol is the main mechanism for the maintenance of its high intracellular levels. We aimed to measure the mRNA and protein levels of myo-inositol cotransporters in the sciatic nerve (SN) and dorsal root ganglia (DRG) during experimental diabetes. Streptozotocin-induced (STZ; 4, 8, and 12 weeks; 65 mg/kg; ip) diabetic rats (DB) and age-matched euglycemic (E) rats were used for the analysis of mRNA and protein levels of sodium myo-inositol cotransporters 1, 2 (SMIT1, SMIT2) or H+/myo-inositol cotransporter (HMIT). There was a significant reduction in the mRNA levels for SMIT1 in the SN and DRG (by 36.9 and 31.0%) in the 4-week DB (DB4) group compared to the E group. SMIT2 was not expressed in SN. The mRNA level for SMIT2 was up-regulated only in the DRG in the DB4 group. On the other hand, the protein level of SMIT1 decreased by 42.5, 41.3, and 44.8% in the SN after 4, 8, and 12 weeks of diabetes, respectively. In addition, there was a decrease of 64.3 and 58.0% of HMIT in membrane and cytosolic fractions, respectively, in the SN of the DB4 group. In the DRG, there was an increase of 230 and 86.3% for SMIT1 and HMIT, respectively, in the DB12 group. The levels of the main inositol transporters, SMIT1 and HMIT, were greatly reduced in the SN but not in the DRG. SMIT-1 was selectively reduced in the sciatic nerve during experimental STZ-induced diabetes.

In vivo phagocytosis and hematology in Astyanax altiparanae, a potential model for surrogate technology

Abstract Although the potential of surrogate propagation technology for aquaculture and conservation of Neotropical fish, the poor understanding of the host immune system may results in rejection and destruction of the donor material. Thus, it is necessary to study and to develop methods to evaluate the effects of immunosuppressive drugs employment and to evaluate the immunocompatibility between donor and receptor. Thus, the present study aimed to optimize a methodology to assess in vivo phagocytosis in Astyanax altiparanae using Saccharomyces cerevisiae and to evaluate their hematological response resultant from the inflammatory induction. To this, S. cerevisiae were labeled with Congo red and injected in the coelomic cavity of A. altiparanae at the concentration of 2.5 x 106 cells mL-1. A PBS solution and a non-injected group were kept as control. Fish blood was sampled and the phagocytic capacity and index were determined at 1, 2, 3 and 6 h post-injection (hpi). The yeast injection successfully stimulated phagocytosis, with the best result for phagocytosis assessment after 2 hpi. Moreover, it was achieved a high traceability of phagocytized and non-phagocytized yeast under optic microscopy analysis due to the Congo red labeling. The hematological profile was similar to usually observed in early infections, indicating lymphocyte migration to inflammatory site and increase in number of circulating phagocytes due to natural response to inflammatory stimulus. In conclusion, our method was efficient to assess in vivo phagocytosis in A. altiparanae and will be an important tool to evaluate the efficacy of immunosuppressive drugs in this species. Additionally, these results may serve as support for further studies in fish immunocompetence, both in laboratory and in field conditions.

Resumo Apesar do potencial apresentado pela tecnologia de propagação mediada para a aquicultura e conservação de peixes Neotropicais, o pobre entendimento do sistema imune do hospedeiro pode resultar na rejeição e destruição do material do doador. Com isso, se fazem necessários o estudo e o desenvolvimento de métodos para análise tanto dos efeitos de drogas imunossupressoras quanto para a avaliação da imunocompatibilidade entre doadores e receptores. Logo, o presente estudo teve como objetivo aperfeiçoar um método para analisar a fagocitose in vivo em Astyanax altiparanae usando Saccharomyces cerevisiae marcado e avaliar seu perfil hematológico resultante da indução inflamatória. Para isso, S. cerevisiae foram marcados com vermelho Congo e injetados na cavidade celomática dos A. altiparanae na concentração de 2,5 x 106 células.mL-1. Peixes injetados com PBS e peixes não injetados foram mantidos como controle. Sangue foi colhido e a capacidade fagocítica e o índice fagocítico foram determinados após 1, 2, 3 e 6 horas após à injeção (hpi). A injeção de levedura estimulou a fagocitose com sucesso, com o melhor resultado atingido após 2 hpi. Ainda, foi observada uma alta rastreabilidade das leveduras fagocitadas e não fagocitadas sob microscopia óptica devido à marcação com vermelho Congo. O perfil hematológico foi similar ao observado usualmente em infecções recém-induzidas, indicando migração de linfócitos ao sítio inflamatório e aumento no número de fagócitos circulantes devido à resposta natural ao estímulo inflamatório. Como conclusão, nosso método foi eficiente para analisar a fagocitose in vivo em A. altiparanae e será uma ferramenta importante para a avaliação de eficácia de drogas imunossopressoras para esta espécie. Em adição, estes resultados podem contribuir para futuros estudos em imunocompetência em peixes, tanto em âmbito laboratorial quanto a campo.

Produção de embriões em ovinos Morada Nova e Somalis Brasileira / Embryo production in Morada Nova and Somalis Brasileira ewes

Superovulatory response and embryo yield in 19 Morada Nova and 20 Somalis Brasileira ewes was analyzed. All animals were synchronized with the insertion of an intravaginal device (CIDR®) on Day 0, replaced by a new device on Day 7, which remained in place until Day 14 and superovulated with 133mg of porcine FSH (pFSH) in decreasing doses at 12h intervals from Day 12 until Day 15 of the treatment, and a single dose of equine chorionic gonadotropin (eCG, 200UI) on Day 14 (i.e., administered in CIDR removal). Fifty hours after CIDR® removal, females were inseminated by laparoscopy. All embryos were recovered by laparotomy 5 days after insemination. Sheep which responded to the superovulation protocol (P>0.05) included 74% of the Morada Nova ewes and 50% of the Somalis Brasileira ewes. Morada Nova showed better results (P<0.05) than Somalis Brasileira in number of ovulations (15.38 ± 5.24 vs. 10.56 ± 2.83), total structures (11.00 ± 7.55 vs. 3.33 ± 1.94) and embryo yields (6.79 ± 5.35 vs. 2.90 ± 2.18). Despite the high fertilization rate, degenerate embryo rate was high for both breeds, with an overall rate of 39% (57/145). In conclusion, superovulatory response and embryo yields in Morada Nova ewes were considered sufficient to justify the use of this procedure in genetic resources conservation programs. However, improvements to embryo quality and control of precocious regression of corpus luteum are necessary to produce better results in the MOET program, with minimal variations and maximum embryo yield in Morada Nova and Somalis Brasileira ewes.(AU)

Plasmatic ADAMTS-13 metalloprotease and von Willebrand factor in children with cyanotic congenital heart disease

Changes in plasma von Willebrand factor concentration (VWF:Ag) and ADAMTS-13 activity (the metalloprotease that cleaves VWF physiologically) have been reported in several cardiovascular disorders with prognostic implications. We therefore determined the level of these proteins in the plasma of children with cyanotic congenital heart disease (CCHD) undergoing surgical treatment. Forty-eight children were enrolled (age 0.83 to 7.58 years). Measurements were performed at baseline and 48 h after surgery. ELISA, collagen-binding assays and Western blotting were used to estimate antigenic and biological activities, and proteolysis of VWF multimers. Preoperatively, VWF:Ag and ADAMTS-13 activity were decreased (65 and 71% of normal levels considered as 113 (105-129) U/dL and 91 ± 24% respectively, P < 0.003) and correlated (r = 0.39, P = 0.0064). High molecular weight VWF multimers were not related, suggesting an interaction of VWF with cell membranes, followed by proteolytic cleavage. A low preoperative ADAMTS-13 activity, a longer activated partial thromboplastin time and the need for cardiopulmonary bypass correlated with postoperative bleeding (P < 0.05). Postoperatively, ADAMTS-13 activity increased but less extensively than VWF:Ag (respectively, 2.23 and 2.83 times baseline, P < 0.0001), resulting in an increased VWF:Ag/ADAMTS-13 activity ratio (1.20 to 1.54, respectively, pre- and postoperative median values, P = 0.0029). ADAMTS-13 consumption was further confirmed by decreased ADAMTS-13 antigenic concentration (0.91 ± 0.30 to 0.70 ± 0.25 µg/mL, P < 0.0001) and persistent proteolysis of VWF multimers. We conclude that, in pediatric CCHD, changes in circulating ADAMTS-13 suggest enzyme consumption, associated with abnormal structure and function of VWF.

Study of irradiated bothropstoxin-1 with60Co gamma rays: immune system behavior

Ionizing radiation has been successfully employed to modify the immunological properties of biomolecules. Very promising results were obtained when crude animal venoms, as well as isolated toxins, were treated with 60Co gamma rays, yielding toxoids with good immunogenicity. The achievement of modified antigens with lower toxicity and preserved or improved immunogenicity can be very useful. Ionizing radiation has already been proven to be a powerful tool to attenuate snake venom toxicity without affecting, and even increasing, their immunogenic properties. However, little is known about the modifications that irradiated molecules undergo and even less about the immunological response that such antigens elicit. In the present work, we investigated the immunological behavior of bothropstoxin-1, a K49 phospholipase, before and after irradiation. Structural modifications of the toxin were analyzed by SDS-PAGE. Isogenic mice were immunized with either the native or the irradiated toxin. The circulating antibodies were isotyped and titrated by ELISA. According to our data, irradiation promoted structural modifications in the toxin characterized by higher molecular weight forms of proteins (aggregates and oligomers). The results also indicated that irradiated toxins were immunogenic and antibodies elicited by them were able to recognize the native toxin in ELISA. These findings suggest that irradiation of toxic proteins can promote significant modifications in their structures; however they still retain many of the original antigenic and immunological properties of native proteins. Also, our data indicate that irradiated proteins induce higher titers of IgG2a and IgG2b, suggesting that Th1 cells are predominantly involved in the immune response.

BTCI enhances guanylin-induced natriuresis and promotes renal glomerular and tubular effects

Guanylin and uroguanylin are small cysteine-rich peptides involved in the regulation of fluid and electrolyte homeostasis through binding and activation of guanylyl cyclases signaling molecules expressed in intestine and kidney. Guanylin is less potent than uroguanylin as a natriuretic agent and is degraded in vitro by chymotrypsin due to unique structural features in the bioactive moiety of the peptide. Thus, the aim of this study was to verify whether or not guanylin is degraded by chymotrypsin-like proteases present in the kidney brush-border membranes. The isolated perfused rat kidney assay was used in this regard. Guanylin (0.2 µM) induced no changes in kidney function. However, when pretreated by the black-eyed pea trypsin and chymotrypsin inhibitor (BTCI - 1.0 µM; guanylin - 0.2 µM) it promoted increases in urine flow (deltaUF of 0.25 ± 0.09 mL.g-1/min, P < 0.05) and Na+ excretion ( percent delta ENa+ of 18.20 ± 2.17, P < 0.05). BTCI (1.0 µM) also increased percentENa+ (from 22.8 ± 1.30 to 34.4 ± 3.48, P < 0.05, 90 minutes). Furthermore, BTCI (3.0 µM) induced increases in glomerular filtration rate (GFR; from 0.96 ± 0.02 to 1.28 0.02 mL.g-1/min, P < 0.05, 60 minutes). The present paper strongly suggests that chymotrypsin-like proteases play a role in renal metabolism of guanylin and describes for the first time renal effects induced by a member of the Bowman-Birk family of protease inhibitors.

Guanilina e uroguanilina são peptídeos pequenos, ricos em cisteína, envolvidos na regulação da homeostase de fluidos e eletrólitos através da ligação e ativação da guanilato ciclase expressa no intestino e nos rins. A guanilina é menos potente do que a uroguanilina como agente natriurético e é degradada in vitro pela quimiotripsina devido a características estruturais únicas no domínio bioativo do peptídeo. Portanto o objetivo deste trabalho foi verificar se a guanilina é degradada por proteases tipo quimiotripsina, presentes na membrana da borda em escova dos rins. Para esta investigação, foi usado o modelo do rim isolado de rato perfundido. A Guanilina (0,2 µM) não induziu mudanças na função renal. Entretanto, quando pré-tratada com inibidor de tripsina e de quimiotripsina de black-eyed pea (BTCI - 1,0 µM; guanilina - 0,2 µM) promoveu um aumento no fluxo urinário (deltaUF de 0,25 ± 0,09 mL.g-1/min, P < 0,05) e na excreção de Na+ ( por centoDENa+ de 18,20 ± 2,17, P < 0,05). BTCI (1,0 µM) também aumenta por centoENa+ (de 22,8 ± 1,30 a 34,4 ± 3,48, P < 0,0590 minutos). Além disto, BTCI (3,0 µM) induziu um aumento da taxa de filtração glomerular (GFR; de 0,96 ± 0,02 para 1,28 ± 0,02 mL.g-1/min, P < 0,05, 60 minutos). O presente trabalho sugere fortemente que proteases semelhantes à quimiotripsina desempenham um papel no metabolismo renal de guanilinas e descreve, pela primeira vez, os efeitos renais induzidos por um membro da família de inibidores de proteases do tipo Bowman-Birk.

Young ovine death during hyperimmunization: crotalic envenomation or copper toxicosis?

The unfavorable evolution of a young ovine during hyperimmunization process with Crotalus durissus terrificus venom was investigated in order to differentiate its origin between ophidic envenomation and copper toxicosis. Clinical, laboratory, necroscopic and histological exams as well as evaluation and measurement of heavy metals (copper) in the kidneys and in the liver were carried out. Blood counts revealed anemia and serological tests showed high levels of blood urea nitrogen, creatinine, aspartate aminotransferase, creatine phosphokinase, total bilirubin and indirect bilirubin; which indicates liver, kidney and skeletal muscle damages. At necropsy, the animal presented hepatopathy and nephropathy. Histological examination revealed renal and hepatic features that may imply copper intoxication. Copper levels were 237.8 µg/g in the liver and 51.2 µg/g in the kidneys. Although the amount of metal found in both organs was below the level that can cause death, according to the literature, anatomopathological signs were suggestive of copper intoxication. Therefore, the hypothesis of metal toxicosis during the hyperimmunization process became more consistent than the crotalic envenomation one.(AU)

Estudo de confiabilidade da aplicação da escala de Fugl-Meyer no Brasil / Reliability study on the application of the Fugl-Meyer scale in Brazil

OBJETIVOS: Os objetivos do estudo foram realizar uma versão brasileira da escala original de Fugl-Meyer e verificar a confiabilidade da aplicação inter e intra-observador desta versão em pacientes crônicos pós AVC. MÉTODO: Participaram do estudo 50 pacientes portadores de hemiparesia, os quais foram submetidos a duas avaliações (confiabilidade intra-observador), realizadas por três fisioterapeutas (confiabilidade interobservador), procedentes de três centros de reabilitação. RESULTADOS: Os resultados demonstraram alta confiabilidade inter e intra-observador da EFM total (IC = 0,99 e 0,98; respectivamente), assim como para todas as subescalas (interobservador IC = 0,99 a 0,94; intra-observador IC = 0,98 a 0,87). CONCLUSÃO: Conclui-se neste artigo que não foi verificado conflitos de interpretação na versão brasileira da escala de Fugl-Meyer. Obtivemos alto índice de confiabilidade, tanto intra como interobservador, permitindo assim seu uso como instrumento de avaliação clínica e de pesquisa no Brasil.

Laboratory evaluation of young ovines inoculated with natural or 60co-irradiated Crotalus durissus terrificus venom during hyperimmunization process

Laboratory profile of young ovines was studied in order to evaluate and compare their antiserum production from natural and Cobalt-60 irradiated Crotalus durissus terrificus (C.d.t.) venoms. The parameters analyzed included complete blood count, and urea, creatinine, aspartate aminotransferase, total proteins, albumin and globulin serum measurements. Three groups of six animals each were used. Group 1 (G1) received natural C.d.t. venom; Group 2 (G2) received irradiated C.d.t. venom; and Group 3 (G3) was used as control and did not receive venom, only adjuvants, using seven venom inoculations. During the experimental period, animals were fortnightly weighed. According to clinical and weight evaluation, sheep in post-weaning phase showed no changes in their physiological profiles but had excellent weight gain. The parameters analyzed were not statistically different (p<5 percent) among the groups tested. The hyperimmunization process was successfully accomplished with the production of specific antibodies against Crotalus durissus terrificus venom. Results bring a new possibility of utilizing ovines in the commercial production of anticrotalic serum, which may be used to treat human and animal envenomation. Its production cost may be reduced by subsequent use of hyperimmunized sheep for human consumption.

Avaliação toxicológica do extrato acetato de etila de Spigelia anthelmia Linn, em ratos e camundongos / Toxicological evaluation of an ethyl acetate extract of Spigelia anthelmia Linn in rats and mice

O extrato acetato de etila de Spigelia anthelmia (EASa) mostrou formalmente ser altamente eficaz contra o desenvolvimento larvar e a eclosão de ovos de Haemonchus contorlus, um importante parasito de ruminantes, in vitro. A OL, e a OL,o de EASa foram administradas subcrônica e cronicamente pela via oral em ratos wistar e o perfil bioquímico foi comparado antes e após cada tratamento e com veículo. Vários órgãos foram coletados e processados para análise histopatológica. Os parâmetros hematológicos foram avaliados antes e depois da administração de EASa durante 30 dias. E os efeitos do EASa administrado pela via oral durante o período embriogênico ou organogênico a camundongas gestantes foram estudados. Os efeitos diretos de EASa, in vivo, foram calculados na pressão sangüínea arterial média e no eletrocardiograma (ECG), e in vitro no coração isolado e no átrio isolado de ratos. A administração de EASa não afetou qualquer parâmetro bioquímico, hematológico ou reprodutivo estudado. EASa induziu um efeito hipotensivo de curto prazo em ratos normotensivos sem qualquer alteração concomitante nos parâmetros do ECG. As maiores doses de EASa induziram uma significante diminuição da amplitude de contração do coração e átrio direito. EASa é desprovido de toxicidade significante e tem leves efeitos no sistema cardiovascular(

Immunological assessment of mice hyperimmunized with native and cobalt-60-irradiated Bothrops venoms

ELISA was used to evaluate, accompany, and compare the humoral immune response of Swiss mice during hyperimmunization with native and Cobalt-60-irradiated (60Co) venoms of Bothrops jararaca, Bothrops jararacussu and Bothrops moojeni. Potency and neutralization were evaluated by in vitro challenges. After hyperimmunization, immunity was observed by in vivo challenge, and the side effects were assessed. The animals immunization with one LD50 of each venom occurred on days 1, 15, 21, 30, and 45, when blood samples were collected; challenges happened on the 60th day. Results showed that ELISA was efficient in evaluating, accompanying and comparing mouse immune response during hyperimmunization. Serum titers produced with natural venom were similar to those produced with irradiated venom. Immunogenic capacity was maintained after 60Co-irradiation. The sera produced with native venom showed neutralizing potency and capacity similar to those of the sera produced with irradiated venom. All antibodies were able to neutralize five LD50 from these venoms. Clinical alterations were minimum during hyperimmunization with irradiated venom, however, necrosis and death occurred in animals inoculated with native venom

Immunization with native and cobalt 60-irradiated Crotalus durissus terrificus venom in swiss mice: assessment of the neutralizing potency of antisera

ELISA was used to evaluate, follow, and compare the humoral immune response of Swiss mice during hyperimmunization with natural and Cobalt 60-irradiated (60Co) Crotalus durissus terrificus venom. Potency and neutralization were evaluated by in vitro challenges. After hyperimmunization, immunity was observed by “in vivo” challenge and the side effects were assessed. The animals immunization with one LD50 of the venom was on days one, 15, 21, 30, and 45, when blood samples were collected; the challenges occurred on the 60th day. Results showed that ELISA was efficient in evaluating, following, and comparing mouse immune response during hyperimmunization. Serum titers produced with natural venom were similar to those produced with irradiated venom. Immunogenic capacity was maintained after 60Co irradiation. Serum produced from Crotalus durissus terrificus irradiated venom showed higher potency and neutralization capacity than that from natural venom. All antibodies were able to neutralize five LD50 from these venoms. Clinical alterations were minimum during hyperimmunization with irradiated venom

Avaliação toxicológica do extrato acetato de etila de Spigelia anthelmia Linn, em ratos e camundongos

O extrato acetato de etila de Spigelia anthelmia (EASa) mostrou formalmente ser altamente eficaz contra o desenvolvimentolarvar e a eclosão de ovos de Haemonchus contortus, um importante parasito de ruminantes, in vitro. A DL1 e a DL10 de EASaforam administradas subcrônica e cronicamente pela via oral em ratos wistar e o perfil bioquímico foi comparado antes e apóscada tratamento e com veículo. Vários órgãos foram coletados e processados para análise histopatológica. Os parâmetroshematológicos foram avaliados antes e depois da administração de EASa durante 30 dias. E os efeitos do EASa administradopela via oral durante o período embriogênico ou organogênico a camundongas gestantes foram estudados. Os efeitos diretosde EASa, in vivo, foram calculados na pressão sangüínea arterial média e no eletrocardiograma (ECG), e in vitro no coraçãoisolado e no átrio isolado de ratos. A administração de EASa não afetou qualquer parâmetro bioquímico, hematológico oureprodutivo estudado. EASa induziu um efeito hipotensivo de curto prazo em ratos normotensivos sem qualquer alteraçãoconcomitante nos parâmetros do ECG. As maiores doses de EASa induziram uma significante diminuição da amplitude decontração do coração e átrio direito. EASa é desprovido de toxicidade significante e tem leves efeitos no sistema cardiovascular.

Hematological changes in sheep inoculated with natural and cobalt 60- irradiated Crotalus durissus terrificus venom (Laurenti, 1768)

Natural (NV)and cobalto 60-irradiated (IrV) Crotalus durissus terrificus venom were used to evaluate serum production capacity of sheep and possible hematological and biochemical effects. Freeze-dried venom aliquots were diluted in acidified saline solution (NaCl 150 mM, pH 3.0) and irradiated by a Cobalt 60 source at a dose of 5.54 x 102 Gy/h and a concentration of 2.000 Gy. Twelve sheep were divided into two groups of six animals. One group received irradiated venom (IrV) and the other natural venom (NV). Three antigen doses (venom) were administered at monthly intervals. Blood samples were collected weekly for analysis of serum neutralization potency and capacity, complete blood count (CBC), total plasma protein, fibrinogen, albumin, and globulin. At the end of the experiment, the animals were challenged with a LD50 for sheep and showed no signs of envenoming. The two groups did not present clinical alterations. Results of the total leukocyte count did not present interaction or time factor effect for both groups, but there was a different action between them, with the NV group presenting more cells than the IrV group. The leukocyte increase to 13,000/µl indicates that slight leukocytosis occurred in the week after the first inoculation in the NV group. There was no statistically significant difference between groups in the absolute count of segmented neutrophils, eosinophils, and lymphocytes but there were statistically significant oscillations in values ant the different collecting times. The NV group presented an increase in the absolute neutrophil count after the first inoculation that persisted for 5 weeks. In the IrV group, the increase in neutrophils occured only in the first week returning to normal in the following weeks. The alterations in the neutrophil count are indicative of systemic inflammatory response related to cytokine release; response was more marked in the NV group, showing its greater toxicity.

Avaliação toxicológica do extrato acetato de etila de Spigelia anthelmia Linn, em ratos e camundongos

O extrato acetato de etila de Spigelia anthelmia (EASa) mostrou formalmente ser altamente eficaz contra o desenvolvimentolarvar e a eclosão de ovos de Haemonchus contortus, um importante parasito de ruminantes, in vitro. A DL1 e a DL10 de EASaforam administradas subcrônica e cronicamente pela via oral em ratos wistar e o perfil bioquímico foi comparado antes e apóscada tratamento e com veículo. Vários órgãos foram coletados e processados para análise histopatológica. Os parâmetroshematológicos foram avaliados antes e depois da administração de EASa durante 30 dias. E os efeitos do EASa administradopela via oral durante o período embriogênico ou organogênico a camundongas gestantes foram estudados. Os efeitos diretosde EASa, in vivo, foram calculados na pressão sangüínea arterial média e no eletrocardiograma (ECG), e in vitro no coraçãoisolado e no átrio isolado de ratos. A administração de EASa não afetou qualquer parâmetro bioquímico, hematológico oureprodutivo estudado. EASa induziu um efeito hipotensivo de curto prazo em ratos normotensivos sem qualquer alteraçãoconcomitante nos parâmetros do ECG. As maiores doses de EASa induziram uma significante diminuição da amplitude decontração do coração e átrio direito. EASa é desprovido de toxicidade significante e tem leves efeitos no sistema cardiovascular.

Estimulacao eletrica neuromuscular e exercicios com movimentos na diagonal para ganho de forca em biceps e triceps braquial / Neuromuscular electrical stimulation and diagonal exercises to incremental of strength in biceps and triceps braquial

O proposito deste estudo foi avaliar os efeitos do exercicio contra-resistido em diagonal com halter e da estimulacao eletrica neuromuscular(EENM) combinada com exercicio isometrico ou combinada com exercicio contra-resistido em diagonal com halter no inremento de forca e no aumento de massa nos musculos biceps e triceps braquial. Para isso, 28 individuos de ambos os sexos foram divididos em quatro grupos (n=7 cada): grupo I - exercicios contra-resistidos em diagonal com halter; grupo II - EENM associado a isomtria; grupo III - exercicio contra-resistido em diagonal com halter associado a EENM; e o grupo IV - controle. Todos os voluntarios foram submetidos a um teste incremental de membros superiores (TIMS) com movimentos em diagonal, seguindo os principios do metodo de facilitacao neuromuscular proprioceptiva(FNP). Foram controladas, nesse teste, as seguintes variaveis: esforco de membros superiores(Escala de Borg), pressao arterial (PA), frequencia cardiaca (FC), perimetria e tempo em que o voluntario realizou o teste e a carga em que terminou. Apos 24 sessoes de treinamento, realizadas tres vezes por semana com a carga fixa de 50 por cento da carga maxima obtida no TMS, foi repetida a avaliacao. Diferencas significativas (p<0,05) foram encontradas na carga e no tempo pos-treinamento no grupo III e no aumento da perimetria no membro superior esquerdo do grupo I. Nao houve diferenca significativa para Escala de Borg entre os grupos, sugerindo a necessidade de mais pesuisas na area

Humoral response and neutralization capacity of sheep serum inoculated with natural and cobalt 60-irradiated Crotalus durissus terrificus venom (Laurenti, 1768)

The aim of this work was to investigate antigen irradiation on crotalic antivenom and the capacity of sheep as serum producers. Twelve sheep in two groups of six were inoculated with Crotalus durissus terrificus venom. One group was inoculated with natural venom (NV) and the other with Cobalt 60 gamma-irradiated venom (IrV). Three antigen doses were given to the animals at monthly intervals for immunization. The toxic activity of the venom was assessed by LD50 determination in mice. Blood samples were collected weekly analyses of serum neutralization capacity and potency. At the end of the experiment, the animals were challenged with a LD50 for sheep showed no signs of envenoming. These results showed that toxicity of the irradiated venom was 4.4 times less than the natural venom. The sera from the irradiated group neutralized LD50 14.6 times, and the sera from the natural group 4.4 times. Sera from the irradiated group were five times more potent. The two groups did not present clinical alterations. The results of this study show the potential for using sheep in crotalic antivenom production. The use of irradiated venom in sheep immunization induces a powerful and lasting humoral immune response shown by both the in vitro neutralization and potency tests and by the indirect ELISA antibody level detection technique.

A single-step purification of bothropstoxin-1

Bothrops venoms are complex mixtures of components with a wide range of biological activities. Among these substances, myotoxins have been investigated by several groups. Bothropstoxin-1 (Bthtx-1) is a phospholipase A2-like basic myotoxin from Bothrops jararacussu. The purification of this component involves two chromatographic steps. Although providing a pure material, the association of these two steps is time consuming and a single-step method using high performance chromatography media would be useful. In the present study, we describe a single-step purification method for Bthtx-1. Bothrops jararacussu venom was dissolved in 1 ml buffer. After centrifugation, the supernatant was injected into a Resource-S cation exchange column connected to an FPLC system and eluted with a linear salt gradient. The complete procedure took 20 min, representing a considerable time gain when compared to a previously described method (Homsi-Brandenburgo MI et al. (1988) Toxicon, 26: 615-627). Bthtx-1 purity and identity, assessed by SDS-PAGE and N-terminal sequencing, resulted in a single band with a molecular mass of about 14 kDa and the expected sequence of the first 5 residues, S-L-F-E-L. Although the amount of protein purified after each run is lower than in the previously described method, we believe that this method may be useful for small-scale purifications

Effect of gamma irradiation on the behavioral properties of crotoxin

Crotoxin has been detoxified with gamma radiation in order to improve crotalic antiserum production. Nevertheless, present knowledge of the biological characteristics of irradiated crotoxin is insufficient to propose it as an immunizing agent. Crotoxin is known to increase the emotional state of rats and to decrease their exploratory behavior (Moreira EG, Nascimento N, Rosa GJM, Rogero JR and Vassilieff VS (1996) Brazilian Journal of Medical and Biological Research, 29: 629-632). Therefore, we decided 1) to evaluate the effects of crotoxin in the social interaction test, which has been widely used for the evaluation of anxiogenic drugs, and 2) to determine if irradiated crotoxin induces behavioral alterations similar to those of crotoxin in the social interaction, open-field and hole-board tests. Male Wistar rats (l8O-220 g) were used. Crotoxin (100, 250, and 500 mug/kg) was injected intraperitoneally 2 h before the social interaction test. Similarly, irradiated crotoxin (2000 Gy gamma radiation from a 60Co source) was administered at the doses of 100, 250, and 500 mug/kg for the hole-board test, and at the doses of 1000 and 2500 mug/kg for the open-field and social interaction tests. ANOVA complemented with the Dunnett test was used for statistical analysis (P<0.05). Crotoxin decreased the social interaction time(s) at the doses of 1OO, 250 and 500 mug/kg (means + SEM) from 51.6 ñ 4.4 to 32.6 ñ 3.7,28.0 ñ 3.6 and 31.6 ñ 4.4, respectively. Irradiated crotoxin did not induce behavioral alterations. These results indicate that 1) crotoxin may be an anxiogenic compound, and 2) in contrast to crotoxin, irradiated crotoxin was unable to induce behavioral alterations, which makes it a promising compound for the production of crotalic antiserum.