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Previous issue date: 2008 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / Dentre os efeitos induzidos pela toxina pertussis (TP) em mamíferos, ocupa um lugar de destaque o fenômeno conhecido por sensibilização aos efeitos biológicos e letais da histamina, cuja intensidade e constância proporcionaram o estabelecimento de um ensaio in vivo de controle de qualidade para avaliação da segurança de vacinas contra a pertussis (coqueluche) e tríplice bacteriana contra a difteria, tétano e coqueluche (DTP). O ensaio de sensibilização à histamina (ESH) em camundongos NIH fêmeas mostrou-se altamente sensível à toxina pertussis de referência NIBSC 90/518 (TPR) detectando níveis tão baixos quanto 20 ng de TP/dose. Todas as 5 vacinas DTP de um produtor nacional noESH apresentaram níveis de TP ativa que variaram entre 84 a 147 ng/mL, valores inferiores ao valor limite de 1789 ng/mL obtido para a vacina pertussis de referência NIBSC 66/303 (VPR), logo, todas as vacinas foram aprovadas para uso humano. Embora o ESH tenha sido conclusivo quanto à alta especificidade à TP, o elevado número de animais, no mínimo, 40 por ensaio, acarretando alto custo e o sofrimento dos animais são fatores limitantes que dificultam o uso rotineiro como ensaio de controle de qualidade da vacina DTP. O objetivo do nosso estudo foi desenvolver uma metodologia in vitro em preparações de íleo isolado de cobaias Short Hair fêmeas (250 a 300 g) fornecidas pelo CECAL/FIOCRUZ /Rio de Janeiro para avaliação do fenômeno de sensibilização à histamina pela TPR. Todos os experimentos foram aprovados de acordo com as diretrizes estabelecidas pela CEUA/FIOCRUZ. Curvas concentração-efeito à histamina em íleos isolados de cobaias foram analisadas e os parâmetros de concentração efetiva média(CE50), concentração efetiva máxima (CEmax) e de constante de dissociação no equilíbrio do complexo droga-receptor (Kd) para histamina foram determinados [...]. / Among the effects induced by pertussis toxin (PT) in mammalian species, a prominence place is occupied by the phenomenon known as sensitization to the biological and lethal effects of the histamine, whose intensity and constancy promoted the establishment of an in vivo quality control assay to evaluate the safety of the pertussis vaccine (PV) against whooping cough and the triple bacterial vaccine, (DPT) against diphtheria, whooping cough and tetanus. The histamine sensitization assay (HSA) performed with NIH female mice was highly sensitive to reference pertussis toxin NIBSC 90/518 (RPT), detecting levels as low as 20 ng of administered RPT/dose, which caused 50% lethality. All five samples of DPT vaccines from one Brazilian producer presented active PT levels in the range of 84 and 147 ng/ml by the HSA, inferior to the limit value of 1789 ng/mL obtained for reference pertussis vaccine NIBSC 66/303 (RPV), thus all the vaccines were approved for use. Although the HSA has been conclusive in relation to its high specificity for RPT, the large number of mice used (at least 40 per assay) results in high costs and the suffering of the mice are limiting factors that make its routine use as a DPT vaccine quality control assay difficult. The aim of our study was to develop an in vitro methodology in ileum segments from female Short Hair guinea pigs (250-300 g) maintained in the animal facilities of the Oswaldo Cruz Foundation in Rio de Janeiro (FIOCRUZ), Brazil, to evaluate the histamine sensitization phenomenon by RPT. All experiments were approved in accordance with the guidelines of the Committee for Ethics in Animal Use of the FIOCRUZ. Concentration effects curves for histamine in guinea pig ileum were studied and the parameters of mean effective concentration (EC50), maximum effective concentration (ECmax) and dissociation constant of drug-receptor complex (Kd) were determined. No increase in ileum contractile response to histamine was detected in relation to control PBS 4 days after intraperitoneal treatment of guinea pigs with doses and dilutions corresponding to mean histamine sensitization dose (HSD50) obtained in NIH female mice of RPT (40 ng), RPV and of 5 DPT vaccines (0.26 IU). In all the ten assays performed on the experimental group, the data followed normal distribution, the variances were homogeneous and no significant differences occurred between assays. With doses 10 times higher than the HSD50 of RPT (400 ng) and of RPV (2.6 IU), analysis of the data showed the same behavior above. Contrary to the anticipated results, histamine EC50 and Kd values in ileum of guinea pigs treated in vivo with RPT were significantly higher than the control and RPV (p< 0.05) with no alteration in ECmax (p= 0.3672). In vitro 15 min treatment of guinea pig ileum with 30 ng/ml of RPT reduced the ECmax to about half in relation to control (p= 0.0028), with no significant reduction in the mean values of histamine EC50 and Kd (p= 0.09). In contrast, in vitro 15 min treatment of ileum with 40 ng/ml of RPT significantly reduced histamine ECmax (p< 0.0069), EC50 (p= 0.0261) and Kd (p= 0.0479) in relation to control ileum. In vitro 15 min treatment with PBS (390 and 520 µL in 13 mL of Tyrode) did not significantly alter the mean values of histamine EC50 (p=0.4043 and p= 0.1035), ECmax (p= 0.2366 and p= 0.2708) or KD (p= 0.4564 and p= 0.1158) in relation to control without treatment, demonstrating no effect of the control solvent (PBS) on ileum contractile response by histamine. In conclusion, increased histamine sensitization in female guinea pig ileum after in vitro treatment of 30 and 40 ng/ml of RPT was demonstrated.
| Identifer | oai:union.ndltd.org:IBICT/oai:www.arca.fiocruz.br:icict/9265 |
| Date | January 2008 |
| Creators | Miller, Reginaldo Assad |
| Contributors | Corrado, Alexandre Pinto, Almeida, Antonio Eugenio Castro Cardoso de, Silva, Marco Antonio Mota da, Zamith, Helena Pereira da Silva, Zamith, Helena Pereira da Silva |
| Source Sets | IBICT Brazilian ETDs |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Source | reponame:Repositório Institucional da FIOCRUZ, instname:Fundação Oswaldo Cruz, instacron:FIOCRUZ |
| Rights | info:eu-repo/semantics/openAccess |
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