Evaluation of miRNAs regulation of BDNF and IGF1 genes in T2DM insulin resistance in experimental models: bioinformatics based approach / Avaliação da regulação por miRNAs dos genes BDNF e IGF1 na resistência a insulina no diabetes do tipo 2 em modelos experimentais: in silico

microRNAs (miRNAs) are recognized as diabetes mellitus type 2 (T2DM) biomarkers useful for disease metabolism comprehension and have great potential as therapeutics targets. BDNF and IGF1 increased expression are highly involved in the benefits of insulin and glucose paths, however, they are down-regulated in insulin resistance conditions, while their expression increase is correlated to the improvement of glucose and insulin metabolism. Studies suggest the microRNA regulation of these genes in several different contexts, providing a novel investigation approach for comprehending T2DM metabolism and revealing potential therapeutic targets. In the present study, we investigate in different animal models (human, rat, and mouse) miRNAs that target BDNF and IGF1 in skeletal muscle tissue with T2DM physiological conditions. Bioinformatics tools and databases were used to miRNA prediction, molecular homology, experimental validation of interactions, expression in the studied physiological condition, and network interaction. The findings showed three miRNAs candidates for IGF1(miR-29a, miR-29b, and miR-29c) and one for BDNF (miR-206). The experimental evaluations and the search for the expression in skeletal muscle from T2DM subjects confirmed the predicted interaction between miRNA-mRNA for miR-29b and miR-206 through human, rat, and mouse models. This interaction was reaffirmed in multiple network analyses. In conclusion, our results show the regulation relationship between miR-29b and miR-206 with the investigated genes, in several tissues, suggesting an inhibition pattern. Nevertheless, these data show a large number of possible interaction physiological processes, for future biotechnological prospects.

Os microRNAs (miRNAs) são reconhecidos como biomarcadores do diabetes mellitus tipo 2 (DM2), úteis para a compreensão do metabolismo da doença, e possuem grande potencial como alvos terapêuticos. O aumento da expressão de BDNF e IGF1 está altamente envolvido nos benefícios as vias de insulina e glicose, porém, são regulados negativamente em condições de resistência à insulina, enquanto seu aumento de expressão está correlacionado com a melhora do metabolismo da glicose e da insulina. Estudos sugerem a regulação desses genes por microRNA em vários contextos diferentes, proporcionando uma nova abordagem de investigação para compreender o metabolismo do DM2 e revelar potenciais alvos terapêuticos. No presente estudo, investigamos em diferentes modelos animais (humanos, ratos e camundongos) miRNAs que têm como alvo BDNF e IGF1 em tecido muscular esquelético com condições fisiológicas de DM2. As análises foram realizadas utilizando ferramentas de bioinformática e bancos de dados para predição de miRNA, homologia molecular, validação experimental de interações, expressão na condição fisiológica estudada e interação em rede. Os resultados mostraram três candidatos a miRNAs para IGF1 (miR-29a, miR-29b e miR-29c) e um para BDNF (miR-206). As avaliações experimentais e a busca pela expressão no músculo esquelético de indivíduos com DM2 confirmaram a interação prevista entre miRNA-mRNA para miR-29b e miR-206 através de modelos humanos, ratos e camundongos. Essa interação foi reafirmada em múltiplas análises de rede. Em conclusão, nossos resultados mostram a relação de regulação entre miR-29b e miR-206 com os genes investigados, em diversos tecidos, sugerindo um padrão de inibição. Contudo, esses dados mostram um grande número de possíveis processos fisiológicos de interação para perspectivas biotecnológicas.

Role of cyclooxygenases 1 and 2 in the maintenance of colonic mucosal integrity in an experimental colitis model.

The role of cyclooxygenase (COXs) isoforms in maintaining colonic mucosal integrity is not fully understood. This study aimed to evaluate the role of COX-1 and -2 on colonic mucosal integrity in an experimental colitis model. Colitis was induced in Wistar rats by intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid (20 mg + 50% ethanol). The control group (sham group) received saline only. After 7, 14, or 28 days, colonic samples were removed, and macroscopic lesion scores, wet weight, myeloperoxidase activity, and transepithelial electrical resistance (TER) were determined. In other rat groups, colonic samples from the sham group and a 7th day post-colitis group were mounted in Üssing chambers with the luminal side exposed to a buffer solution (control), acetylsalicylic acid (ASA), SC-560 (COX-1 inhibitor), or celecoxib (COX-2 inhibitor). TER and epithelial permeability to fluorescein were measured. The 7th day colitis group had higher macroscopic damage scores, wet weight, and myeloperoxidase activity and lower basal TER than the sham, 14th day colitis, and 28th day colitis groups. Inhibition of COX-1 but not COX-2 significantly decreased TER and increased permeability to fluorescein in the 7th day post-colitis group compared to the sham group. Additionally, ASA decreased the colonic mucosal integrity on day seven post-colitis compared to the sham group. A decrease in the colonic mucosa integrity in the experimental colitis model can be aggravated only by the inhibition of COX-1, which demonstrated the importance of this enzyme in the maintenance of colonic mucosal integrity.

Role of cyclooxygenases 1 and 2 in the maintenance of colonic mucosal integrity in an experimental colitis model

The role of cyclooxygenase (COXs) isoforms in maintaining colonic mucosal integrity is not fully understood. This study aimed to evaluate the role of COX-1 and -2 on colonic mucosal integrity in an experimental colitis model. Colitis was induced in Wistar rats by intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid (20 mg + 50% ethanol). The control group (sham group) received saline only. After 7, 14, or 28 days, colonic samples were removed, and macroscopic lesion scores, wet weight, myeloperoxidase activity, and transepithelial electrical resistance (TER) were determined. In other rat groups, colonic samples from the sham group and a 7th day post-colitis group were mounted in Üssing chambers with the luminal side exposed to a buffer solution (control), acetylsalicylic acid (ASA), SC-560 (COX-1 inhibitor), or celecoxib (COX-2 inhibitor). TER and epithelial permeability to fluorescein were measured. The 7th day colitis group had higher macroscopic damage scores, wet weight, and myeloperoxidase activity and lower basal TER than the sham, 14th day colitis, and 28th day colitis groups. Inhibition of COX-1 but not COX-2 significantly decreased TER and increased permeability to fluorescein in the 7th day post-colitis group compared to the sham group. Additionally, ASA decreased the colonic mucosal integrity on day seven post-colitis compared to the sham group. A decrease in the colonic mucosa integrity in the experimental colitis model can be aggravated only by the inhibition of COX-1, which demonstrated the importance of this enzyme in the maintenance of colonic mucosal integrity.

Evaluation of miRNAs regulation of BDNF and IGF1 genes in T2DM insulin resistance in experimental models: bioinformatics based approach.

microRNAs (miRNAs) are recognized as diabetes mellitus type 2 (T2DM) biomarkers useful for disease metabolism comprehension and have great potential as therapeutics targets. BDNF and IGF1 increased expression are highly involved in the benefits of insulin and glucose paths, however, they are down-regulated in insulin resistance conditions, while their expression increase is correlated to the improvement of glucose and insulin metabolism. Studies suggest the microRNA regulation of these genes in several different contexts, providing a novel investigation approach for comprehending T2DM metabolism and revealing potential therapeutic targets. In the present study, we investigate in different animal models (human, rat, and mouse) miRNAs that target BDNF and IGF1 in skeletal muscle tissue with T2DM physiological conditions. Bioinformatics tools and databases were used to miRNA prediction, molecular homology, experimental validation of interactions, expression in the studied physiological condition, and network interaction. The findings showed three miRNAs candidates for IGF1(miR-29a, miR-29b, and miR-29c) and one for BDNF (miR-206). The experimental evaluations and the search for the expression in skeletal muscle from T2DM subjects confirmed the predicted interaction between miRNA-mRNA for miR-29b and miR-206 through human, rat, and mouse models. This interaction was reaffirmed in multiple network analyses. In conclusion, our results show the regulation relationship between miR-29b and miR-206 with the investigated genes, in several tissues, suggesting an inhibition pattern. Nevertheless, these data show a large number of possible interaction physiological processes, for future biotechnological prospects.

Role of TRPV1 receptor in inflammation and impairment of esophageal mucosal integrity in a murine model of nonerosive reflux disease.

BACKGROUND: Microscopic inflammation and impairment of the esophageal epithelial barrier are considered relevant for perception of symptoms in patients with nonerosive reflux disease (NERD). In these patients, the receptor transient receptor potential vanilloid 1 (TRPV1) is overexpressed in the esophageal mucosa, but its role is not yet fully understood. We evaluated the role of TRPV1 in esophageal inflammation and mucosal barrier impairment in a murine model of NERD. METHODS: Nonerosive reflux disease was surgically induced in Swiss mice by pyloric substenosis and ligature of the gastric fundus, and the mice were killed 7 days post surgery. The experimental groups were: I, sham surgery (negative control); II, NERD untreated; III and IV, NERD + SB366791 or capsazepine (TRPV1 antagonists); and V, NERD + resiniferatoxin (for long-term desensitization of TRPV1). The esophagus was collected for western blotting and histopathology and for evaluation of wet weight, myeloperoxidase (MPO), keratinocyte-derived chemokine (KC), transepithelial electrical resistance (TEER), and basal permeability to fluorescein. KEY RESULTS: Compared to sham, NERD mice had increased esophageal wet weight and MPO and KC levels. The mucosa had no ulcers but exhibited inflammation. NERD mice showed mucosal TRPV1 overexpression, a more pronounced decrease in TEER at pH 0.5 (containing pepsin and taurodeoxycholic acid), and increased basal permeability. Pharmacological modulation of TRPV1 prevented esophageal inflammation development, TEER changes by acidic exposure, and increase in esophageal permeability. CONCLUSIONS & INFERENCES: The TRPV1 receptor has a critical role in esophageal inflammation and mucosal barrier impairment in NERD mice, suggesting that TRPV1 might be a pharmacological target in patients with NERD.

Sensory and inflammatory colonic changes induced by vincristine in distinct rat models of colitis.

Preclinical and clinical studies show that gastrointestinal (GI) inflammation can evoke sensory changes occasionally far from the original inflammatory site. Animal models of colitis with either trinitrobenzenesulphonic acid (TNBS) or mustard oil (MO) produce distinct patterns of somatic and visceral sensory changes. We evaluated the effects of four doses of i.v. vincristine 150 µg kg(-1) (total of 600 µg kg(-1) ) treatment on the somatic (thermal nociceptive threshold) and colonic (morphological) changes induced by TNBS or MO in rats. TNBS and MO groups were further submitted to vincristine or saline pretreatments. TNBS induced somatic hypersensitivity, while MO induced somatic hyposensitivity (P < 0.05) when compared to the saline and ethanol control groups. Vincristine per se induced somatic hypersensitivity (P < 0.05). This effect was enhanced by TNBS and reversed by MO treatments. Although vincristine increased the colitis area (colonic weight length(-1) ratio) and the Morris' score in TNBS-treated rats, it did not alter the colitis area and even lowered the Morris' score in MO-treated rats. Compared to the saline (control) group, vincristine did not alter the colonic microscopic pattern. However, such lesions scores are higher (P < 0.05) in colitis groups induced by TNBS and MO, pretreated or not with vincristine. In conclusion, the somatic changes induced by different models of experimental colitis are diverse and modulated differently by vincristine.

INTERCORRÊNCIA ENTRE TUBERCULOSE E LEUCOSE ENZOÓTICA EM REBANHO BOVINO LEITEIRO

A Tuberculose (TB) e a Leucose Enzoótica dos Bovinos (LEB) são doenças infectocontagiosas, caracterizadas pela evolução crônica e pelos prejuízos gerados à pecuária bovina. Essas doenças comprometem o desempenho produtivo dos rebanhos, causando condenações de carcaças em frigoríficos e restringindo o comércio de animais, além do aumento dos custos com serviços veterinários. A TB, além da importância em saúde pública, causa reduções de até 25% na produtividade animal. O vírus da LEB está associado ao desencadeamento de bacterioses oportunistas. Admite-se, que o comprometimento da integridade do sistema imunitário orgânico pela ação imunodepressora do vírus, que penetra e incorpora-se no genoma linfócitário por tempo indeterminado aumenta a susceptibilidade do hospedeiro a outras infecções. Objetivou-se com este trabalho, avaliar a ocorrência da Tuberculose Bovina e Leucose Enzoótica dos Bovinos (LEB), em um rebanho bovino leiteiro. Foram examinados 316 bovinos de ambos os sexos, com idade entre 6 meses e 16 anos, pelo teste alérgico-cutâneo, o exame utilizado foi o Teste Cervical Comparativo (TCC). Para o diagnóstico da LEB, foram avaliados 85 animais, escolhidos aleatoriamente, empregou-se a técnica de Imunodifusão em Gel de Ágar (IDGA) segundo o protocolo do fabricante do antígeno TECPAR, por meio de um substrato de difusão gelatinoso, utilizando o antígeno glicoprotéi

Intercorrência entre tuberculose e leucose enzoótica em rebanho bovino leiteiro

A Tuberculose (TB) e a Leucose Enzoótica dos Bovinos (LEB) são doenças infectocontagiosas, caracterizadas pela evolução crônica e pelos prejuízos gerados à pecuária bovina. Essas doenças comprometem o desempenho produtivo dos rebanhos, causando condenações de carcaças em frigoríficos e restringindo o comércio de animais, além do aumento dos custos com serviços veterinários. A TB, além da importância em saúde pública, causa reduções de até 25% na produtividade animal. O vírus da LEB está associado ao desencadeamento de bacterioses oportunistas. Admite-se, que o comprometimento da integridade do sistema imunitário orgânico pela ação imunodepressora do vírus, que penetra e incorpora-se no genoma linfócitário por tempo indeterminado aumenta a susceptibilidade do hospedeiro a outras infecções. Objetivou-se com este trabalho, avaliar a ocorrência da Tuberculose Bovina e Leucose Enzoótica dos Bovinos (LEB), em um rebanho bovino leiteiro. Foram examinados 316 bovinos de ambos os sexos, com idade entre 6 meses e 16 anos, pelo teste alérgico-cutâneo, o exame utilizado foi o Teste Cervical Comparativo (TCC). Para o diagnóstico da LEB, foram avaliados 85 animais, escolhidos aleatoriamente, empregou-se a técnica de Imunodifusão em Gel de Ágar (IDGA) segundo o protocolo do fabricante do antígeno TECPAR, por meio de um substrato de difusão gelatinoso, utilizando o antígeno glicoprotéi

The hydrogen sulfide donor, Lawesson's reagent, prevents alendronate-induced gastric damage in rats.

Our objective was to investigate the protective effect of Lawesson's reagent, an H2S donor, against alendronate (ALD)-induced gastric damage in rats. Rats were pretreated with saline or Lawesson's reagent (3, 9, or 27 µmol/kg, po) once daily for 4 days. After 30 min, gastric damage was induced by ALD (30 mg/kg) administration by gavage. On the last day of treatment, the animals were killed 4 h after ALD administration. Gastric lesions were measured using a computer planimetry program, and gastric corpus pieces were assayed for malondialdehyde (MDA), glutathione (GSH), proinflammatory cytokines [tumor necrosis factor (TNF)-α and interleukin (IL)-1ß], and myeloperoxidase (MPO). Other groups were pretreated with glibenclamide (5 mg/kg, ip) or with glibenclamide (5 mg/kg, ip)+diazoxide (3 mg/kg, ip). After 1 h, 27 µmol/kg Lawesson's reagent was administered. After 30 min, 30 mg/kg ALD was administered. ALD caused gastric damage (63.35 ± 9.8 mm(2)); increased levels of TNF-α, IL-1ß, and MDA (2311 ± 302.3 pg/mL, 901.9 ± 106.2 pg/mL, 121.1 ± 4.3 nmol/g, respectively); increased MPO activity (26.1 ± 3.8 U/mg); and reduced GSH levels (180.3 ± 21.9 µg/g). ALD also increased cystathionine-γ-lyase immunoreactivity in the gastric mucosa. Pretreatment with Lawesson's reagent (27 µmol/kg) attenuated ALD-mediated gastric damage (15.77 ± 5.3 mm(2)); reduced TNF-α, IL-1ß, and MDA formation (1502 ± 150.2 pg/mL, 632.3 ± 43.4 pg/mL, 78.4 ± 7.6 nmol/g, respectively); lowered MPO activity (11.7 ± 2.8 U/mg); and increased the level of GSH in the gastric tissue (397.9 ± 40.2 µg/g). Glibenclamide alone reversed the gastric protective effect of Lawesson's reagent. However, glibenclamide plus diazoxide did not alter the effects of Lawesson's reagent. Our results suggest that Lawesson's reagent plays a protective role against ALD-induced gastric damage through mechanisms that depend at least in part on activation of ATP-sensitive potassium (KATP) channels.

The hydrogen sulfide donor, Lawesson's reagent, prevents alendronate-induced gastric damage in rats

Our objective was to investigate the protective effect of Lawesson's reagent, an H2S donor, against alendronate (ALD)-induced gastric damage in rats. Rats were pretreated with saline or Lawesson's reagent (3, 9, or 27 µmol/kg, po) once daily for 4 days. After 30 min, gastric damage was induced by ALD (30 mg/kg) administration by gavage. On the last day of treatment, the animals were killed 4 h after ALD administration. Gastric lesions were measured using a computer planimetry program, and gastric corpus pieces were assayed for malondialdehyde (MDA), glutathione (GSH), proinflammatory cytokines [tumor necrosis factor (TNF)-α and interleukin (IL)-1β], and myeloperoxidase (MPO). Other groups were pretreated with glibenclamide (5 mg/kg, ip) or with glibenclamide (5 mg/kg, ip)+diazoxide (3 mg/kg, ip). After 1 h, 27 µmol/kg Lawesson's reagent was administered. After 30 min, 30 mg/kg ALD was administered. ALD caused gastric damage (63.35±9.8 mm2); increased levels of TNF-α, IL-1β, and MDA (2311±302.3 pg/mL, 901.9±106.2 pg/mL, 121.1±4.3 nmol/g, respectively); increased MPO activity (26.1±3.8 U/mg); and reduced GSH levels (180.3±21.9 µg/g). ALD also increased cystathionine-γ-lyase immunoreactivity in the gastric mucosa. Pretreatment with Lawesson's reagent (27 µmol/kg) attenuated ALD-mediated gastric damage (15.77±5.3 mm2); reduced TNF-α, IL-1β, and MDA formation (1502±150.2 pg/mL, 632.3±43.4 pg/mL, 78.4±7.6 nmol/g, respectively); lowered MPO activity (11.7±2.8 U/mg); and increased the level of GSH in the gastric tissue (397.9±40.2 µg/g). Glibenclamide alone reversed the gastric protective effect of Lawesson's reagent. However, glibenclamide plus diazoxide did not alter the effects of Lawesson's reagent. Our results suggest that Lawesson's reagent plays a protective role against ALD-induced gastric damage through mechanisms that depend at least in part on activation of ATP-sensitive potassium (KATP) channels.

AVALIAÇÃO DA REATIVIDADE DE IMUNOGLOBULINAS Y DE GALINHAS IMUNIZADAS COM VACINA B19 E SUA APLICAÇÃO EM IMUNODIAGNÓSTICO / EVALUATION OF IMMUNOGLOBULIN Y REACTIVITY OF CHICKENS IMMUNIZED WITH B19 AND ITS APPLICATION IN IMMUNODIAGNOSTICS

A IgY  é assim classificada porque os anticorpos maternos são transferidos do soro sanguíneo para a gema do ovo, desta forma, para adquirir anticorpos reativos para determinado tipo de antígeno, o sacrifício de animais seria evitado, uma vez que os mesmos podem ser extraídos da gema em grande quantidade, o que não ocorre na utilização de mamíferos. Objetiva-se avaliar a reatividade de imunoglobulinas Y de galinhas imunizadas com vacina B19 através produção de anticorpos policlonais específicos, detectáveis em testes oficiais para brucelose, tais como, Antígeno Acidificado Tamponado (AAT) e 2-Mercaptoetanol (2-ME), e no ELISA indireto. Foram utilizadas quatro galinhas, divididas aleatoriamente em dois grupos experimentais, sendo um grupo controle (Grupo 1) e um grupo imunizado com vacina B19 de Brucela abortus (Grupo 2). No Grupo 1 as galinhas foram imunizadas com 250 µL de PBS, e no Grupo 2 com 250 µL da Vacina B19 diluída em PBS, ambos adicionados de adjuvante. Os dois grupos foram imunizados seis vezes durante 13 semanas, quinzenalmente. Para avaliar a produção e reatividade da IgY foram realizadas sete coletas de sangue quinzenais, sendo a p

Avaliação da reatividade de imunoglobulinas y de galinhas imunizadas com vacina b19 e sua aplicação em imunodiagnóstico / Evaluation of immunoglobulin y reactivity of chickens immunized with b19 and its application in immunodiagnostic

A IgY é assim classificada porque os anticorpos maternos são transferidos do soro sanguíneo para a gema do ovo, desta forma, para adquirir anticorpos reativos para determinado tipo de antígeno, o sacrifício de animais seria evitado, uma vez que os mesmos podem ser extraídos da gema em grande quantidade, o que não ocorre na utilização de mamíferos. Objetiva-se avaliar a reatividade de imunoglobulinas Y de galinhas imunizadas com vacina B19 através produção de anticorpos policlonais específicos, detectáveis em testes oficiais para brucelose, tais como, Antígeno Acidificado Tamponado (AAT) e 2-Mercaptoetanol (2-ME), e no ELISA indireto. Foram utilizadas quatro galinhas, divididas aleatoriamente em dois grupos experimentais, sendo um grupo controle (Grupo 1) e um grupo imunizado com vacina B19 de Brucela abortus (Grupo 2). No Grupo 1 as galinhas foram imunizadas com 250 µL de PBS, e no Grupo 2 com 250 µL da Vacina B19 diluída em PBS, ambos adicionados de adjuvante. Os dois grupos foram imunizados seis vezes durante 13 semanas, quinzenalmente. Para avaliar a produção e reatividade da IgY foram realizadas sete coletas de sangue quinzenais, sendo a primeira, uma semana antes da primeira imunização e as demais uma semana após cada imunização; e coletas diárias de ovos a partir de uma semana antes da primeira imunização, sendo estes separados por grupo e por semana. A IgY proveniente da gema do ovo foi purificada a partir de um pool semanal gema de ovos de cada grupo, utilizando-se os métodos de delipidação através da diluição em água ácida e a precipitação com sulfato de amônio. As galinhas do Grupo 1, não foram reagentes aos testes, enquanto do Grupo 2 produziram anticorpos reativos a este antígeno detectáveis em todos os testes realizados para o diagnóstico de brucelose bovina. Conclui-se que as galinhas produziram anticorpos IgY reagentes nos testes sorológicos realizados, sendo a imunoglobulina Y um potencial antígeno para produção de anticorpos específicos, a fim de serem utilizados em testes diagnósticos.(AU)

The IgY is classified as such because maternal antibodies are transferred from the serum to the egg yolk. Thereby to acquire antibodies reactive to a particular type of antigen, animal killing could be avoided since the antibodies can now be extracted from the yolk in large quantity, something that does not occur with mammals. This study aims to evaluate the reactivity of immunoglobulin Y of chickens immunized with B19 through the production of specific polyclonal antibodies, detectable in official tests for brucellosis such as Buffered Acidified Antigen (AAT) and 2-mercaptoethanol (2-ME), and ELISA. Four hens were randomly divided into two experimental groups, one of which the control group (Group 1) and the second, a group immunized with Brucella abortus B19 vaccine (Group 2). The chickens of Group 1 were immunized with 250 μL of PBS while in Group 2 with 250 μL of B19 vaccine diluted in PBS; adjuvant was added in both. The two groups were immunized six times during 13 weeks, fortnightly. To evaluate the production and reactivity of IgY seven blood samples were collected biweekly, the first, a week before the first immunization and the others a week after each immunization; and eggs were harvested daily starting from a week before the first immunization, and separated by group per week. The IgY from the egg yolk was purged from an egg yolk pool of each group prepared weekly, using the delipidation by dilution with water and acid precipitation with ammonium sulfate method. The chickens of Group 1 were not reactive to the tests, whereas the chickens in Group 2 produced antibodies reactive to this antigen detectable in all brucellosis diagnosis tests. It is concluded that the chickens produced IgY antibody reagents in serological tests performed, and the immunoglobulin Y is a potential antigen for production of specific antibodies that can be used in diagnostic tests.(AU)

Avaliação da reatividade de imunoglobulinas y de galinhas imunizadas com vacina b19 e sua aplicação em imunodiagnóstico / Evaluation of immunoglobulin y reactivity of chickens immunized with b19 and its application in immunodiagnostic

A IgY é assim classificada porque os anticorpos maternos são transferidos do soro sanguíneo para a gema do ovo, desta forma, para adquirir anticorpos reativos para determinado tipo de antígeno, o sacrifício de animais seria evitado, uma vez que os mesmos podem ser extraídos da gema em grande quantidade, o que não ocorre na utilização de mamíferos. Objetiva-se avaliar a reatividade de imunoglobulinas Y de galinhas imunizadas com vacina B19 através produção de anticorpos policlonais específicos, detectáveis em testes oficiais para brucelose, tais como, Antígeno Acidificado Tamponado (AAT) e 2-Mercaptoetanol (2-ME), e no ELISA indireto. Foram utilizadas quatro galinhas, divididas aleatoriamente em dois grupos experimentais, sendo um grupo controle (Grupo 1) e um grupo imunizado com vacina B19 de Brucela abortus (Grupo 2). No Grupo 1 as galinhas foram imunizadas com 250 µL de PBS, e no Grupo 2 com 250 µL da Vacina B19 diluída em PBS, ambos adicionados de adjuvante. Os dois grupos foram imunizados seis vezes durante 13 semanas, quinzenalmente. Para avaliar a produção e reatividade da IgY foram realizadas sete coletas de sangue quinzenais, sendo a primeira, uma semana antes da primeira imunização e as demais uma semana após cada imunização; e coletas diárias de ovos a partir de uma semana antes da primeira imunização, sendo estes separados por grupo e por semana. A IgY proveniente da gema do ovo foi purificada a partir de um pool semanal gema de ovos de cada grupo, utilizando-se os métodos de delipidação através da diluição em água ácida e a precipitação com sulfato de amônio. As galinhas do Grupo 1, não foram reagentes aos testes, enquanto do Grupo 2 produziram anticorpos reativos a este antígeno detectáveis em todos os testes realizados para o diagnóstico de brucelose bovina. Conclui-se que as galinhas produziram anticorpos IgY reagentes nos testes sorológicos realizados, sendo a imunoglobulina Y um potencial antígeno para produção de anticorpos específicos, a fim de serem utilizados em testes diagnósticos.

The IgY is classified as such because maternal antibodies are transferred from the serum to the egg yolk. Thereby to acquire antibodies reactive to a particular type of antigen, animal killing could be avoided since the antibodies can now be extracted from the yolk in large quantity, something that does not occur with mammals. This study aims to evaluate the reactivity of immunoglobulin Y of chickens immunized with B19 through the production of specific polyclonal antibodies, detectable in official tests for brucellosis such as Buffered Acidified Antigen (AAT) and 2-mercaptoethanol (2-ME), and ELISA. Four hens were randomly divided into two experimental groups, one of which the control group (Group 1) and the second, a group immunized with Brucella abortus B19 vaccine (Group 2). The chickens of Group 1 were immunized with 250 μL of PBS while in Group 2 with 250 μL of B19 vaccine diluted in PBS; adjuvant was added in both. The two groups were immunized six times during 13 weeks, fortnightly. To evaluate the production and reactivity of IgY seven blood samples were collected biweekly, the first, a week before the first immunization and the others a week after each immunization; and eggs were harvested daily starting from a week before the first immunization, and separated by group per week. The IgY from the egg yolk was purged from an egg yolk pool of each group prepared weekly, using the delipidation by dilution with water and acid precipitation with ammonium sulfate method. The chickens of Group 1 were not reactive to the tests, whereas the chickens in Group 2 produced antibodies reactive to this antigen detectable in all brucellosis diagnosis tests. It is concluded that the chickens produced IgY antibody reagents in serological tests performed, and the immunoglobulin Y is a potential antigen for production of specific antibodies that can be used in diagnostic tests.

Avaliação da reatividade de imunoglobulinas y de galinhas imunizadas com vacina b19 e sua aplicação em imunodiagnóstico / evaluation of immunoglobulin y reactivity of chickens immunized with b19 and its application in immunodiagnostics

A IgY  é assim classificada porque os anticorpos maternos são transferidos do soro sanguíneo para a gema do ovo, desta forma, para adquirir anticorpos reativos para determinado tipo de antígeno, o sacrifício de animais seria evitado, uma vez que os mesmos podem ser extraídos da gema em grande quantidade, o que não ocorre na utilização de mamíferos. Objetiva-se avaliar a reatividade de imunoglobulinas Y de galinhas imunizadas com vacina B19 através produção de anticorpos policlonais específicos, detectáveis em testes oficiais para brucelose, tais como, Antígeno Acidificado Tamponado (AAT) e 2-Mercaptoetanol (2-ME), e no ELISA indireto. Foram utilizadas quatro galinhas, divididas aleatoriamente em dois grupos experimentais, sendo um grupo controle (Grupo 1) e um grupo imunizado com vacina B19 de Brucela abortus (Grupo 2). No Grupo 1 as galinhas foram imunizadas com 250 µL de PBS, e no Grupo 2 com 250 µL da Vacina B19 diluída em PBS, ambos adicionados de adjuvante. Os dois grupos foram imunizados seis vezes durante 13 semanas, quinzenalmente. Para avaliar a produção e reatividade da IgY foram realizadas sete coletas de sangue quinzenais, sendo a p

Gastroprotective activity of Zanthoxylum rhoifolium Lam. in animal models.

ETHNOPHARMACOLOGICAL RELEVANCE: The stem barks of Zanthoxylum rhoifolium Lam. (Rutaceae), locally known as "mamica de cadela", are popularly used in dyspepsies, stomachic, tonic, antitumoral, antipyretic and are used in treating flatulence and colic. The objective of this study was to evaluate the gastroprotective effect of the ethanolic extract of Zanthoxylum rhoifolium (EEZR) stem barks in acute gastric lesion models, investigating their possible mechanisms. MATERIALS AND METHODS: Mice were used for the evaluation of the acute toxicity, and mice and rats to study the gastroprotective activity. The gastroprotective action of EEZR was analyzed in the absolute ethanol, HCl/ethanol and indomethacin-induced gastric lesion models in mice, hypothermic-restraint stress, and ischemia/reperfusion in rats. In the investigation of the gastroprotective mechanisms of EEZR, the participation of the NO-synthase pathway, ATP-sensitive potassium channels (K(ATP)), the levels of the non-protein sulfhydril groups (NP-SH) and the catalase activity using the ethanol-induced gastric mucosa lesion model and the quantification of the gastric mucus and the antisecretory activity through pylorus ligature model in rats were analyzed. RESULTS: The animals did not present any signs of acute toxicity for the EEZR (up to the 4 g/kg dose, po), and it was not possible to calculate the DL(50). EEZR (125-500 mg/kg) exhibited a significant gastroprotective effect in absolute ethanol, HCl/ethanol, hypothermic-restraint stress, and ischemia/reperfusion-induced gastric lesion models. EEZR (250 and 500 mg/kg) exhibited still a gastroprotective activity in the indomethacin-induced ulcer model. Gastroprotection of EEZR was significantly decreased in pre-treated mice with l-NAME or glibenclamide, the respective nitric oxide synthase and K(ATP) channels inhibitors. Our studies revealed that EEZR (500 mg/kg) prevented the decrease of the non-protein sulfhydril groups (NP-SH) and increased the catalase levels in ethanol-treated animals. Furthermore, the extract (500 mg/kg) significantly increased the mucus production, however, the gastric secretion parameters (volume, [H(+)], pH) did not show any alteration. CONCLUSIONS: Our results indicate that the ethanolic extract of Zanthoxylum rhoifolium exhibits a significant gastroprotection, because it inhibits the formation of gastric lesions using different models. The release of the nitric oxide, the opening of the K(ATP) channels, the participation of the non-protein sulfhydril groups (NP-SH), catalase and the increase of mucous secretion seem to be involved in the gastroprotection activity of the EEZR. Nevertheless, this activity does not seem to be related to antisecretory mechanisms.

Testicular Sertoli cell function in male systemic lupus erythematosus.

OBJECTIVE: To assess the testicular Sertoli cell function in male SLE patients. METHODS: Thirty-four consecutive patients were prospectively selected to evaluate serum inhibin B. Clinical features, treatment, semen analysis, urological evaluation, testicular ultrasound, hormones and anti-sperm antibodies were determined. RESULTS: Patients were subdivided into two groups: low serum inhibin B (Group 1, n = 8) and normal levels (Group 2, n = 26). The median sperm concentration (P = 0.024), total sperm count (P = 0.023) and total motile sperm count (P = 0.025) were lower in Group 1. Inhibin B levels were positively correlated with sperm concentration (r = 0.343), total motile sperm count (r = 0.357), and negatively correlated with follicule-stimulating hormone (FSH) (r = 0.699) and luteinizing hormone (r = 0.397). The median serum inhibin B was lower in SLE patients treated with intravenous cyclophosphamide (IVCYC) compared with those without this therapy (P = 0.031). Further evaluation of the 26 SLE patients with normal inhibin B and FSH levels revealed that medians of inhibin B/FSH ratio were lower in SLE patients with oligozoospermia compared with normozoospermia (P = 0.004). This ratio was also lower in SLE patients treated with IVCYC than those without this therapy (P = 0.04). In contrast, inhibin B serum level alone did not discriminate the later group of patients (P = 0.12). CONCLUSIONS: This is the first study to identify a high frequency of testicular Sertoli cell dysfunction in male SLE associated with semen abnormalities. Further prospective studies are necessary to determine if inhibin levels and inhibin B/FSH ratio will be an earlier and useful marker of IVCYC toxicity in these patients.

Role of the NO/cGMP/K(ATP) pathway in the protective effects of sildenafil against ethanol-induced gastric damage in rats.

BACKGROUND AND PURPOSE: Sildenafil is a selective inhibitor of cGMP-specific phosphodiesterase. Sildenafil, acting via NO-dependent mechanisms, prevents indomethacin-induced gastropathy. Activation of ATP-sensitive potassium channels (K(ATP)) is involved in gastric defence. Our objective was to evaluate the role of the NO/cGMP/K(ATP) pathway in the protective effects of sildenafil against ethanol-induced gastric damage. EXPERIMENTAL APPROACH: Rats were treated with L-NAME (1 or 3 mg kg(-1), i.p.) or with L-arginine (200 mg kg(-1), i.p.) + L-NAME (3 mg kg(-1), i.p.), the guanylate cyclase inhibitor, ODQ (10 mg kg(-1), i.p.), glibenclamide (0.1, 0.3, 1 or 3 mg kg(-1), i.p.) or with glibenclamide (1 mg kg(-1), i.p.) + diazoxide (3 mg kg(-1), i.p.). After thirty minutes, the rats received sildenafil (1 mg kg(-1), by gavage), followed by intragastric instillation of absolute ethanol (4 ml kg(-1)) to induce gastric damage. One hour later, gastric damage (haemorrhagic or ulcerative lesions) was measured with a planimetry programme. Samples of stomach were also taken for histopathological assessment and for assays of tissue glutathione and haemoglobin. KEY RESULTS: Sildenafil significantly reduced ethanol-induced gastric damage in rats. L-NAME alone, without L-arginine, significantly reversed the protection afforded by sildenafil. Inhibition of guanylate cyclase by ODQ completely abolished the gastric protective effect of sildenafil against ethanol-induced gastric damage. Glibenclamide alone reversed sildenafil's gastric protective effect. However, glibenclamide plus diazoxide did not alter the effects of sildenafil. CONCLUSIONS: Sildenafil had a protective effect against ethanol-induced gastric damage through the activation of the NO/cGMP/K(ATP) pathway.

Temporal variation of chemical composition and relaxant action of the essential oil of Ocimum gratissimum L. (Labiatae) on guinea-pig ileum.

The medicinal plant Ocimum gratissimum L. (Labiatae) is widely encountered in the Northeast of Brasil where it is used to treat digestive problems. Its leaves have an essential oil (EOOG) content whose chemical composition varies according to the time of plant collection. We have compared the effects of the EOOG, collected at 08:00 a.m. (EOOG8) and at 12:00 a.m. (EOOG12), on the relaxation of guinea-pig isolated ileum. Both EOOG8 and EOOG12 (30-300 microg/ml) reversibly relaxed the spontaneous tonus of the guinea-pig ileum in a concentration-dependent manner, with similar IC50 values (49.3 and 23.8 microg/ml, respectively). The magnitude of the decrease in resting tonus was similar to that of the recognised smooth muscle relaxant papaverine. EOOG8 and EOOG12 relaxed 60 mM KCl-precontracted preparations similarly (38.33 +/- 9.91 microg/ml and 35.53 +/- 6.70), whereas a significantly more potent relaxant effect of EOOG12 compared to EOOG8 was observed when tissues were contracted using 10 microM acetylcholine (IC50 values of 69.55 +/- 4.93 and 128.16 +/- 15.70 microg/ml, respectively; p < 0.05). The principal constituents of the essential oil, eugenol and cineole, also relaxed KCl-precontracted preparations, although they were less potent than EOOG, suggesting that they alone were not responsible for EOOG-induced relaxations. Our results show that the essential oil extracted from the leaves of O. gratissimum L., collected at different time periods, exerts significant relaxant effects on isolated guinea-pig ileum which may underlie the therapeutic action of the plant.