RESUMEN
The necessity of animal-free performance tests for novel ophthalmic formulation screening is challenging. For this, we developed and validated a new device to simulate the dynamics and physical-chemical barriers of the eye for in vitro performance tests of topic ophthalmic formulations. The OphthalMimic is a 3D-printed device with an artificial lacrimal flow, a cul-de-sac area, a support base, and a simulated cornea comprised of a polymeric membrane containing poly-vinyl alcohol 10 % (w/v), gelatin 2.5 % (w/v), and different proportions of mucin and poloxamer, i.e., 1:1 (M1), 1:2 (M2), and 2:1 (M3) w/v, respectively. The support base is designed to move between 0° and 50° to replicate the movement of an eyelid. We challenged the model by testing the residence performance of poloxamer®407 16 % and poloxamer®407 16 % + chitosan 1 % (PLX16CS10) gels containing fluconazole. The test was conducted with a simulated tear flow of 1.0 mL.min-1 for 5 min. The OphthalMimic successfully distinguished PLX16 and PLX16C10 formulations based on their fluconazole drainage (M1: 65 ± 14 % and 27 ± 10 %; M2: 58 ± 6 % and 38 ± 9 %; M3: 56 ± 5 % and 38 ± 18 %). In conclusion, the OphthalMimic is a promising tool for comparing the animal-free performance of ophthalmic formulations.
Asunto(s)
Soluciones Oftálmicas , Poloxámero , Poloxámero/química , Soluciones Oftálmicas/química , Administración Oftálmica , Fluconazol/administración & dosificación , Impresión Tridimensional , Córnea/efectos de los fármacos , Córnea/metabolismo , Animales , Quitosano/química , Alternativas a las Pruebas en Animales/métodos , Lágrimas/química , Humanos , Gelatina/químicaRESUMEN
The ocular irritation potential of products that may come into contact with the eyes should be assessed by the combination of different in vitro alternative methods to determine different mechanisms of toxicity previously evaluated by the Draize in vivo assay. Thus, this study proposed to apply two strategies for the prediction of the eye irritation potential of different concentrations of surfactants and silicones, the first one involving evaluation Hen's Egg Test - Chorioallantoic Membrane (HET-CAM), and the other one using Bovine Corneal Opacity and Permeability (BCOP) followed by histopathological. HET-CAM was considered important in assessing the ocular irritation potential and, despite classifying almost all surfactants as "severe irritants", it could discriminate moderate and slight irritant SLES concentrations as well as Cocoamidopropyl Betaine as a severe irritant, when the coagulation score was taken into consideration. The BCOP assay alone also did not offer a good prediction of the irritant potential of surfactants, since almost all of them were classified as "no prediction can be made". However, the histopathological evaluation of the BCOP corneas was very important for establishing the degree and depth of damage related to reversibility. The present study also showed those strategies are sensitive to small variations in the studied anionic, cationic amphoteric surfactant concentrations and can be used for predicting their toxicity in the final product and can be used depending on the focus of the analysis.
Asunto(s)
Opacidad de la Córnea , Cosméticos , Alternativas a las Pruebas en Animales/métodos , Animales , Bovinos , Pollos , Opacidad de la Córnea/patología , Cosméticos/toxicidad , Ojo , Femenino , Irritantes/toxicidad , Siliconas/farmacología , Tensoactivos/toxicidadRESUMEN
Therapeutic options for the treatment of leishmaniasis are insufficient and need improvements owing to their low efficiency and high toxicity as well as the emergence of resistant strains. The limited number of new drugs for neglected diseases and lack of innovation in your development are still challenges. In this context, the process of discovery and development of biological assays play a pivotal role for the identification of bioactive compounds. The assays currently used for screening of drugs with cytotoxic activity against Leishmania parasites, include different processes that utilize intact parasite (free or intracellular) or specific enzymes of metabolism as a target cell. These assays allow the screening of large numbers of samples followed by more detailed secondary confirmatory assays to confirm the observed activity and assess their toxicity. In the present study, we described the development of a new functional and more complete assay that enables simultaneous assessment of potential anti-Leishmania compounds through evaluation of internalization of fluorescein-labeled L. braziliensis promastigotes by human peripheral blood monocytes and their cytotoxicity by flow cytometry. We standardized the conditions for parasite labeling to achieve better phagocytosis analysis by setting the ratio of number of parasites per cell as 1 to 2, at incubation time of 6h. The cytotoxicity assessment was performed by the quantification of cells undergoing early/late apoptosis and necrosis using a double labelling platform employing 7AAD for late apoptosis and necrosis analysis and Annexin-V for early apoptosis evaluation. Hemolysis analysis was an additional parameter to test cytotoxicity. Two drugs used on clinic (Amphotericin B and Glucantime®) were used to validate the proposed methodology, and the assay was able to detect their known leishmanicidal activity and immunotoxicity properties. This new predictive assay will contribute to the development of translational medicine strategies in drug discovery for neglected diseases such as leishmaniasis.
Asunto(s)
Alternativas a las Pruebas en Animales/métodos , Antiprotozoarios/toxicidad , Citometría de Flujo/métodos , Leishmania/efectos de los fármacos , Enfermedades Desatendidas/tratamiento farmacológico , Adulto , Anfotericina B/farmacología , Anfotericina B/toxicidad , Animales , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Humanos , Leishmania braziliensis/efectos de los fármacos , Leishmaniasis/tratamiento farmacológico , Leucocitos/efectos de los fármacos , Leucocitos/parasitología , Antimoniato de Meglumina/farmacología , Antimoniato de Meglumina/uso terapéutico , Antimoniato de Meglumina/toxicidad , Microscopía Confocal , Persona de Mediana Edad , Monocitos/efectos de los fármacos , Monocitos/parasitología , Factores de Tiempo , Adulto JovenRESUMEN
The development of alternative approaches for safety and efficacy testing that avoid the use of animals is a worldwide trend, which relies on the improvement of current models and tools so that they better reproduce human biology. Human skin from elective plastic surgery is a promising experimental model to test the effects of topically applied products. As the structure of native skin is maintained, including cell population (keratinocytes, melanocytes, Langerhans cells and fibroblasts) and dermal matrix (containing collagen, elastin, glycosaminoglycans, etc.), it most closely matches the effects of substances on in vivo human skin. In this review, we present a collection of results that our group has generated over the last years, involving the use of human skin and scalp explants, demonstrating the feasibility of this model. The development of a test system with ex vivo skin explants, of standard size and thickness, and cultured at the air-liquid interface, can provide an important tool for understanding the mechanisms involved in several cutaneous disorders.
Asunto(s)
Alternativas a las Pruebas en Animales , Técnicas de Cultivo de Célula , Piel , Alternativas a las Pruebas en Animales/métodos , Alternativas a las Pruebas en Animales/normas , Animales , Técnicas de Cultivo de Célula/normas , Células Cultivadas , Humanos , Piel/citología , Cirugía PlásticaRESUMEN
The Bovine Corneal Opacity and Permeability (BCOP) assay is an alternative method used to ocular toxicity potential assessment of chemicals and mixtures. The standard BCOP test provides information about permeability and opacity, however, corneal histopathological analysis has been recommended as an additional parameter to better categorize eye irritants. Moreover, such analysis associated with depth of substance-induced corneal injury analysis may provide additional scientific measurement for the refinement of BCOP test. The aim of this study was to measure the depth of injury into the bovine cornea induced by eye irritants and associate it with the damage severity. For this purpose, BCOP assay was performed for 12 substances from different Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) categories and, additionally, corneal sections of 5⯵m thickness were obtained and analyzed by fluorescence microscopy. The results showed that the fluorescein permeation depth was directly proportional to the substances irritation degree. Severe irritants promoted highest rates of permeation followed by moderate and mild irritants, while non-irritants showed similar permeation indexes to the negative control. The refinement of BCOP by the depth of injury analysis through epithelial permeation of fluorescein can be considered a useful quantitative parameter to better categorize eye irritants.
Asunto(s)
Córnea/efectos de los fármacos , Irritantes/toxicidad , Alternativas a las Pruebas en Animales/métodos , Animales , Bioensayo/métodos , Bovinos , Córnea/metabolismo , Córnea/patología , Opacidad de la Córnea/inducido químicamente , Opacidad de la Córnea/metabolismo , Opacidad de la Córnea/patología , Fluoresceína/metabolismo , Permeabilidad , Pruebas de Toxicidad/métodosRESUMEN
In the present work, we established an adipogenesis inhibition assay as an adequate and sensitive in vitro model for reducing animal use by estimating the starting dose for the acute toxic class (ATC) method. First, human adipose-derived stem cells (ADSCs) underwent adipogenic differentiation induction for 14 days. Then, by high-content imaging analysis, we determined the percentage and area of cell differentiation that we considered suitable for negative and positive internal control according to the quality control criteria strictly standardized mean difference (SSMD) and robust SSMD. Moreover, we established sodium dodecyl sulfate (SDS) as an external positive control in this assay. To measure reduction in animal use to estimate the starting dose for the ATC method, we evaluated 10 chemicals representing Globally Harmonized System of Classification and Labeling of Chemicals (GHS) toxicity categories 1-5 and unclassified toxicity and determined the dose-response curves for percentage and area of cell differentiation by using the Hill function with an R2 ≥ 0.85. The resulting IC50 values were used for LD50 prediction and for estimating the starting dose for the ATC method. Our results indicated that use of the inhibition of adipogenesis assay to estimate the starting dose for the ATC method would decrease animal use for 7 out of 10 tested substances, possibly all substances if we consider the more toxic test substances in GHS categories 1, 2, and 3. We can conclude that the present assay is a suitable alternative to reduce animal testing in the first steps of predicting highly toxic substances. Moreover, this method also presents internal and external controls as differentials, which guarantee the quality of the assay as well as the results. These features are important for suggesting a methodology for regulatory purposes.
Asunto(s)
Adipogénesis/efectos de los fármacos , Tejido Adiposo/efectos de los fármacos , Alternativas a las Pruebas en Animales/métodos , Bioensayo/métodos , Células Madre/efectos de los fármacos , Pruebas de Toxicidad Aguda/métodos , Tejido Adiposo/citología , Tejido Adiposo/inmunología , Tejido Adiposo/metabolismo , Alternativas a las Pruebas en Animales/normas , Bioensayo/normas , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ensayos Analíticos de Alto Rendimiento , Humanos , Concentración 50 Inhibidora , Dosificación Letal Mediana , Fenotipo , Reproducibilidad de los Resultados , Células Madre/inmunología , Células Madre/metabolismo , Células Madre/patología , Factores de Tiempo , Pruebas de Toxicidad Aguda/normasAsunto(s)
Dermatitis Fototóxica , Queratinocitos/efectos de la radiación , Pruebas de Toxicidad/métodos , Alternativas a las Pruebas en Animales/métodos , Animales , Células 3T3 BALB , Técnicas de Cultivo de Célula/métodos , Línea Celular , Supervivencia Celular/efectos de la radiación , Humanos , Ratones , Reproducibilidad de los Resultados , Factores de TiempoAsunto(s)
Humanos , Animales , Ratones , Queratinocitos/efectos de la radiación , Dermatitis Fototóxica , Pruebas de Toxicidad/métodos , Factores de Tiempo , Línea Celular , Supervivencia Celular/efectos de la radiación , Reproducibilidad de los Resultados , Técnicas de Cultivo de Célula/métodos , Células 3T3 BALB , Alternativas a las Pruebas en Animales/métodosRESUMEN
Eye toxicity is a mandatory parameter in human risk and safety evaluation for products including chemicals, pesticides, medicines and cosmetics. Historically, this endpoint has been evaluated using the Draize rabbit eye test, an in vivo model that was never formally validated. Due to advances in scientific knowledge, economic and ethical issues, non-animal methods based on mechanisms of toxicity are being developed and validated for increasing the capability of these models to predict eye toxicity. In this study, the Cytometric Bead Array (CBA) and ELISA assays were used to evaluate the inflammatory cytokine profile produced by HaCaT human keratinocytes after exposure to chemicals with different UN GHS eye toxicity classifications, aiming to stablish a correlation between inflammatory endpoints and eye toxicity (damage/irritation) potential. As a first step, cytotoxic profile of the chemicals, including 3 non-irritants and 10 eye toxicants (GHS Category 1, 2A and 2B), was evaluated after 24â¯h exposure using MTT assay and Inhibitory Concentration of 20% of cell viability (IC20) was calculated for each chemical. Then, the cells were exposed to these chemicals at IC20 for 24â¯h and supernatants and cell lysates were analyzed by CBA assay for quantification of the following cytokines: IL-6, IL-8, IL-10, IL-1ß, TNF and IL-12p70. Regarding cytotoxicity evaluation, chemicals showed different cytotoxicity profiles and data demonstrated no correlation with their UN GHS classification. Among the cytokines evaluated, IL-1ß production has changed after exposure and such alterations were confirmed by quantification employing ELISA method. The higher intracellular levels of IL-1ß were found in GHS Category 1 chemicals, followed by Category 2A and 2B, while non irritants did not induce such increase. Thus, these findings show that IL-1ß measurement, using HaCaT model, can be a considerable biomarker to identify chemicals according to their potential in promote eye toxicity, differentiating damage from irritation potential.
Asunto(s)
Alternativas a las Pruebas en Animales/métodos , Alternativas a las Pruebas en Animales/normas , Irritantes/toxicidad , Queratinocitos/efectos de los fármacos , Pruebas de Toxicidad Aguda/métodos , Pruebas de Toxicidad Aguda/normas , Bioensayo/normas , Línea Celular , Citocinas/análisis , Ensayo de Inmunoadsorción Enzimática , Humanos , Inflamación/inducido químicamente , Concentración 50 Inhibidora , Queratinocitos/química , Modelos BiológicosRESUMEN
Mammalian models have served as a basis for R&D over the past decades. Nevertheless, these models are expensive, laborious, may yield results that cannot always be translated into the human in vivo situation and, more recently, have reverberated great social and ethical dilemmas. Hence, the prospect of changes in the global scientific scenario and the Three Rs principle (Reduction, Replacement and Refinement) have encouraged the development of alternative methods to the use of mammals. Despite the efforts, suitable alternative tests are not available in all areas of biomedical research, as regulatory acceptance requires time, prior validation and robust financial and scientific investment. In this perspective, we aim to shed light on the concepts, challenges and perspectives for implementation of innovative alternative animal and non-animal methods in scientific research. The applicability and meaningfulness of invertebrate animal models, in silico analysis and reverse pharmacology are discussed, among other aspects of relevance in today's scenario. Overall, the use of alternative models, including Artemia salina (brine shrimp), Caenorhabditis elegans (roundworm), Danio rerio (zebra fish), Drosophila melanogaster (fruit fly), Galleria mellonella (greater waxmoth) and in silico modelling, increased 909% from 1990 to 2015, as compared to 154% of conventional mammals in the same period. Thus, technological and scientific advancements in the fields of toxicology and drug development seem to have diminished the need for mammalian models. Today, however, mammals still remain critically indispensable to provide - in most cases -reliable data subsidizing and validating translation into the clinical setting.
Asunto(s)
Alternativas a las Pruebas en Animales/métodos , Modelos Animales de Enfermedad , Descubrimiento de Drogas/métodos , Animales , Investigación Biomédica/métodos , Simulación por Computador , Humanos , Farmacología/métodos , Toxicología/métodosRESUMEN
Substantial progress has been made in the development of alternative methods for skin sensitization in the last decade in several countries around the world. Brazil is experiencing an increasing concern about using animals for product development, since the publication of the Law 9605/1998, which prohibits the use of animals when an alternative method is available. In this way, an in vitro test to evaluate allergenic potential is a pressing need.This preliminary study started setting the use of myelomonocytic THP-1 cell line, according to the human cell line activation test (h-CLAT), already under validation process. We found that 48-h chemical exposure was necessary to identify 22 out of 23 sensitizers by the analyses of CD86 expression. In addition, the CD54 expression analyses presented a poor efficiency to discriminate sensitizers from non-sensitizers in our conditions. In view of these results, we looked for changes of pro-inflammatory interleukin profile. The IL-8 secretion analyses after 24-h chemical incubation seemed to be an alternative for CD54 expression assessing.Altogether, our findings showed that the combination of the analyses of CD86 expression and IL-8 secretion allowed predicting allergenicity.
Asunto(s)
Alérgenos/inmunología , Antígeno B7-2/análisis , Interleucina-8/metabolismo , Piel/inmunología , Alternativas a las Pruebas en Animales/métodos , Línea Celular Tumoral , Humanos , Molécula 1 de Adhesión Intercelular/análisis , Interleucina-8/genética , ARN Mensajero/análisis , Piel/metabolismoRESUMEN
Introducción: la perspectiva histórica demuestra lo eficaz y esencial que ha sido la utilización de animales como sujetos de prueba. Se ha logrado salvar vidas y responder a diferentes preguntas biomédicas, a partir de esta práctica.Objetivo: realizar una revisión bibliográfica sobre la investigación preclínica en las ciencias biomédicas.Métodos: se realizó una revisión sobre la investigación preclínica en las ciencias biomédicas en Internet. La búsqueda abarcó artículos publicados fundamentalmente en los últimos 5 años. Se evaluaron revistas internacionales de impacto de la Web of Sciencies relacionadas con el tema (32 revistas) y 2 cubanas. Se consultaron las bases de datos de sistemas referativos, como MEDLINE, PubMed y SciELO con la utilización de descriptores como animal research, preclinical investigation, biomodels, laboratory animals, translational medicine y sus equivalentes en español. Se incluyeron artículos en idioma inglés, portugués y español. Se obtuvieron 136 artículos, se circunscribió el estudio a 53 que enfocaron esta temática de manera más integral. Se revisó 1 libro.Análisis e integración de la información: al analizar el comportamiento de los artículos sobre la temática de estudio respecto a su representatividad en las diferentes revistas científicas donde fueron publicados, 9,4 por ciento de ellos correspondieron a la revista Journal of clinical periodontology e igual porciento a la PloS biology. Los restantes artículos estuvieron distribuidos de manera uniforme entre las otras revistas. La mayoría de las investigaciones sugieren que el empleo de biomodelos constituye una forma eficiente de desarrollar investigaciones, pero que estas deben estar bien diseñadas, pues frecuentemente se introducen sesgos en la validez interna y externa de la investigación, que conllevan a errores en la publicación científica...(AU)
Introduction: a historical analysis would reveal that the use of animals as test subjects has been both effective and essential. Such a practice has served both to save lives and to answer a variety of biomedical questions.Objective: carry out a bibliographic review about preclinical biomedical research.Methods: abibliographic review was conducted about preclinical biomedical research on the Internet. Most of the papers included in the search have been published in the last five years. An evaluation was performed of international high impact journals from the Web of Sciences dealing with the subject (32 journals), as well as two Cuban journals. Databases from reference systems such as MEDLINE, PubMed and SciELO were consulted with the aid of search terms like animal research, preclinical investigation, biomodels, laboratory animals, translational medicine and their Spanish counterparts. The papers included were in English, Portuguese or Spanish. Of the 136 papers obtained, the reviewers selected the 53 which approached the study subject in a more comprehensive manner. One book was also reviewed.Data analysis and integration: an analysis of the representativeness of papers in the scientific journals where they were published showed that 9.4 percent corresponded to the Journal of Clinical Periodontology, and the same percentage to PloS Biology. The remaining papers were evenly distributed among the other journals. Most studies suggest that biomodels are an effective tool in scientific research, but they should be correctly designed, for it is common to find bias in the internal and external validity of the research, leading to errors in scientific publications...(AU)
Asunto(s)
Animales , Diseño de Investigaciones Epidemiológicas , Alternativas a las Pruebas en Animales/métodos , Ingeniería BiomédicaRESUMEN
Introducción: la perspectiva histórica demuestra lo eficaz y esencial que ha sido la utilización de animales como sujetos de prueba. Se ha logrado salvar vidas y responder a diferentes preguntas biomédicas, a partir de esta práctica. Objetivo: realizar una revisión bibliográfica sobre la investigación preclínica en las ciencias biomédicas. Métodos: se realizó una revisión sobre la investigación preclínica en las ciencias biomédicas en Internet. La búsqueda abarcó artículos publicados fundamentalmente en los últimos 5 años. Se evaluaron revistas internacionales de impacto de la Web of Sciencies relacionadas con el tema (32 revistas) y 2 cubanas. Se consultaron las bases de datos de sistemas referativos, como MEDLINE, PubMed y SciELO con la utilización de descriptores como animal research, preclinical investigation, biomodels, laboratory animals, translational medicine y sus equivalentes en español. Se incluyeron artículos en idioma inglés, portugués y español. Se obtuvieron 136 artículos, se circunscribió el estudio a 53 que enfocaron esta temática de manera más integral. Se revisó 1 libro. Análisis e integración de la información: al analizar el comportamiento de los artículos sobre la temática de estudio respecto a su representatividad en las diferentes revistas científicas donde fueron publicados, 9,4 por ciento de ellos correspondieron a la revista Journal of clinical periodontology e igual porciento a la PloS biology. Los restantes artículos estuvieron distribuidos de manera uniforme entre las otras revistas. La mayoría de las investigaciones sugieren que el empleo de biomodelos constituye una forma eficiente de desarrollar investigaciones, pero que estas deben estar bien diseñadas, pues frecuentemente se introducen sesgos en la validez interna y externa de la investigación, que conllevan a errores en la publicación científica. Conclusiones: el desconocimiento de las características inherentes a los biomodelos, de aspectos propios de la investigación preclínica, los concernientes al diseño metodológico y el mismo desarrollo de la investigación pueden introducir errores en el análisis, y publicación de los resultados; consecuentemente, se afecta la calidad de la investigación, y se contribuye a la frecuente falta de estudios animales confiables(AU)
Introduction: a historical analysis would reveal that the use of animals as test subjects has been both effective and essential. Such a practice has served both to save lives and to answer a variety of biomedical questions. Objective: carry out a bibliographic review about preclinical biomedical research. Methods: abibliographic review was conducted about preclinical biomedical research on the Internet. Most of the papers included in the search have been published in the last five years. An evaluation was performed of international high impact journals from the Web of Sciences dealing with the subject (32 journals), as well as two Cuban journals. Databases from reference systems such as MEDLINE, PubMed and SciELO were consulted with the aid of search terms like animal research, preclinical investigation, biomodels, laboratory animals, translational medicine and their Spanish counterparts. The papers included were in English, Portuguese or Spanish. Of the 136 papers obtained, the reviewers selected the 53 which approached the study subject in a more comprehensive manner. One book was also reviewed. Data analysis and integration: an analysis of the representativeness of papers in the scientific journals where they were published showed that 9.4 percent corresponded to the Journal of Clinical Periodontology, and the same percentage to PloS Biology. The remaining papers were evenly distributed among the other journals. Most studies suggest that biomodels are an effective tool in scientific research, but they should be correctly designed, for it is common to find bias in the internal and external validity of the research, leading to errors in scientific publications. Conclusions: lack of knowledge about characteristics inherent to biomodels, preclinical research, methodological design and the very development of the research may lead to errors in the analysis and publication of results, affecting the quality of the research and contributing to the frequent scarcity of reliable animal studies(AU)
Asunto(s)
Animales , Diseño de Investigaciones Epidemiológicas/veterinaria , Experimentación Animal , Investigación Biomédica/métodos , Alternativas a las Pruebas en Animales/métodos , Literatura de Revisión como Asunto , Interpretación Estadística de Datos , Bases de Datos Bibliográficas/estadística & datos numéricosRESUMEN
Ehrlichiosis is a worldwide illness, endemic in tropical and subtropical countries where seroprevalence can reach up to 33% as it is the case in México and Israel. However, the low sensitivity of the serological tests used for diagnosis, 67% in tests with IFI, has led to a required searching for new alternatives to diagnose the disease quickly and effectively. PCR is one of these techniques that could provide high sensitivity and specificity. The target of this study was to implement the PCR test for the diagnosis of Ehrlichia spp., on blood samples from animals with suspected illness from veterinary clinics in the city of Medellin. 90 samples were taken, 33 samples from animals suspected of having symptoms and 57 from healthy animals as probable negatives. DNA from the Madrid strain was used as a positive control. The PCR was performed using as an example the protocol suggested by Aguirre et al (2004). EEC and ECB primers reported by Dawson et al (2004) were used (aquí el propio texto en español no está claro). In this study the 500pb band was amplified, corresponding to the 16s rRNA of Ehrlichia spp., in 11 samples of the animals with suspected illness, obtaining a presentation rate of 33.3%, confirming the presence of bacteria in the animals of the environment, and achieving the implementation of PCR for Ehrlichia spp. as a diagnostic tool in the city.
La Ehrlichiosis es una enfermedad de distribución mundial, es endémica en los países tropicales y subtropicales en donde la seroprevalencia puede llegar a ser hasta del 33%, como en México e Israel. La baja sensibilidad de las pruebas utilizadas para el diagnóstico, 67% en las pruebas con IFI, han llevado a la necesidad de buscar nuevas alternativas que permitan diagnosticar la enfermedad de manera rápida y eficaz. La PCR es una de estas técnicas que podría ofrecer una alta sensibilidad y especificidad. El objetivo de este trabajo fue implementar la prueba de PCR, para el diagnóstico de Ehrlichia spp., en muestras de sangre de caninos sospechosos provenientes de consultorios veterinarios de la ciudad de Medellín. Se tomaron 90 muestras, 33 de animales sospechosos de ehrlichiosis por sintomatología, y 57 de animales sanos como probables negativos. Se utilizó como control positivo el ADN de la cepa Madrid. La PCR fue realizada utilizando como ejemplo el protocolo sugerido por Aguirre et al. (2004). Se utilizaron los primers EEC y ECB reportados por Dawson et al. (1994). En este estudio se logró amplificar la banda de 500 pb correspondientes al gen 16s ARNr de Ehrlichia spp., en 11 muestras de los animales sospechosos, obteniendo una frecuencia de presentación del 33,3%, confirmando la presencia de la bacteria en los animales de nuestro medio, y logrando la implementación de PCR para Ehrlichia spp. como herramienta diagnóstica en nuestra ciudad.
A ehrlichiose é uma doença em todo o mundo, é endêmica em países tropicais e subtropicais, onde a soroprevalência pode atingir até 33%, como no México e Israel. A baixa sensibilidade dos testes utilizados para o diagnóstico, 67% nos testes de IFI, levaram à necessidade de buscar novas formas de diagnosticar a doença de forma rápida e eficaz. PCR é uma técnica de tal forma que poderia fornecer alta sensibilidade e especificidade. O objetivo deste trabalho foi implementar o teste de PCR para o diagnóstico de Ehrlichia spp., Em amostras de sangue de cães suspeitos de clínicas veterinárias da cidade de Medellín. Demorou 90 amostras, 33 amostras de suspeitos que apresentavam sintomas de erliquiose e 57 animais saudáveis como negativo provável. Foi utilizado como controle positivo de ADN a partir da estirpe de Madrid. A PCR foi padronizada utilizando o exemplo do protocolo proposto por Aguiar et al. (2004). Utilizou-se primers BCE CES e relatado por Dawson et al. (1994). Neste estudo foram amplificados de banda de 500 pb para o gene 16S rRNA do gênero Ehrlichia em 11 amostras de animais suspeitos, a obtenção de uma taxa de apresentação de 33,3%, o que confirma a presença de bactérias em animais nosso meio ambiente e obter a implementação de PCR por Ehrlichia spp. como uma ferramenta de diagnóstico em nossa cidade.
Asunto(s)
Animales , Alternativas a las Pruebas en Animales/métodos , Diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Ciencia de los Animales de Laboratorio , Métodos , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa/veterinariaRESUMEN
Precision-cut liver slices (PCLS) are mainly used to evaluate hepatotoxicity and metabolism of chemicals, as well as to study mechanisms of liver damage and repair. However, recently they have been used as a system to study amoebic infections. The aim of this study was to validate this model as an alternative for experimental amoebic liver absess (ALA) in animals. To do this, the PCLS was analyzed for the expression of amoebapore and cysteine proteinases 1 and 5, three of the most studied virulence factors of Entamoeba histolytica, as well as the induction of apoptosis and cytokines production in response to the ex vivo infection. PCHLS were prepared with the Brendel-Vitron tissue slicer and then, infected with 200,000 trophozoites of E. histolytica. Samples were taken at 0, 6, 12, 18, and 24 h and compared to control non-infected slices. Morphological studies were performed in order to verify the infection; while apoptosis was studied by TUNEL and PAS techniques. The expression of cysteine proteinases (1 and 5), and amoebapore, was analyzed by real-time PCR. By using ELISA assays, the production of cytokines was also studied. PCHLS were found to be a reproducible infection system, and E. histolytica caused the expression of cysteine proteinases and amoebapore in infected slices. At the same time, trophozoites induce release of cytokines and apoptotic death of the hepatocytes close to them. PCHLS represent a new and suitable alternative model to study the pathogenesis of hepatic amoebiasis.
Asunto(s)
Apoptosis , Citocinas/metabolismo , Entamoeba histolytica/patogenicidad , Hígado/parasitología , Factores de Virulencia/metabolismo , Alternativas a las Pruebas en Animales/métodos , Animales , Cricetinae , Proteasas de Cisteína/genética , Proteasas de Cisteína/metabolismo , Modelos Animales de Enfermedad , Entamoeba histolytica/inmunología , Regulación Enzimológica de la Expresión Génica , Etiquetado Corte-Fin in Situ , Hígado/inmunología , Hígado/patología , Masculino , Mesocricetus , Reacción del Ácido Peryódico de Schiff , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Virulencia/análisis , Factores de Virulencia/genéticaRESUMEN
Preclinical investigations can start with preliminary in vitro studies before using animal models. Following this approach, the number of animals used in preclinical acute toxicity testing can be reduced. In this study, we employed an in-house validated in vitro cytotoxicity test based on the Spielmann approach for toxicity evaluation of the lignan grandisin, a candidate anticancer agent, and its major metabolite, the 4-O-demethylgrandisin, by neutral red uptake (NRU) assay, on mouse fibroblasts Balb/c 3T3 cell line. Using different concentrations of grandisin and its major metabolite (2.31; 1.16; 0.58; 0.29; 0.14; 0.07; 0.04; 0.002 µM) in Balb/c 3T3-A31 NRU cytotoxicity assay, after incubation for 48 h, we obtained IC(50) values for grandisin and its metabolite of 0.078 and 0.043 µM, respectively. The computed LD(50) of grandisin and 4-O-demethylgrandisin were 617.72 and 429.95 mg/kg, respectively. Both were classified under the Globally Harmonized System as category 4. Since pharmacological and toxicological data are crucial in the developmental stages of drug discovery, using an in vitro assay we demonstrated that grandisin and its metabolite exhibit distinct toxicity profiles. Furthermore, results presented in this work can contribute to reduce the number of animals required in subsequent pharmacological/toxicological studies.
Asunto(s)
Alternativas a las Pruebas en Animales/métodos , Furanos/toxicidad , Lignanos/toxicidad , Pruebas de Toxicidad Aguda/métodos , Animales , Células 3T3 BALB , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Furanos/química , Furanos/aislamiento & purificación , Furanos/metabolismo , Lignanos/química , Lignanos/aislamiento & purificación , Lignanos/metabolismo , Ratones , Estructura Molecular , Piper/química , Extractos Vegetales/química , Hojas de la Planta/químicaRESUMEN
The search for new antimalarials, which in the past relied on animal models, is now usually performed with cultures of Plasmodium falciparum (PF) blood parasites by evaluation of parasite growth inhibition. Field isolates of PF human malaria parasite, parasite strains and clones, well characterized for their susceptibility to chloroquine and other standard antimalarials are available for the in vitro tests. The simplest method to evaluate parasite growth is the determination of parasitemias in Giemsa stained blood smears through light microscopy. Other methodologies have proven to be more precise and allow mass screening of new compounds against PF blood stages, such as: (i) measuring the incorporation of radioactive hypoxanthine by the parasites; (ii) indirect colorimetric assays in which specific parasite enzyme activities, and histidine-rich protein II (HRP2) production are measured with the help of monoclonal antibodies; (iii) the beta-haematin formation, and; (iv) assays using green fluorescent protein (GFP) in gene-expressing parasites. The advantages and disadvantages of the different in vitro screening methods, as well as the different in vivo models for antimalarial tests, are described in this review. Such tests can be used for the evaluation of medicinal plants, synthetic and hybrid molecules or drug combinations.
Asunto(s)
Antimaláricos/farmacología , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Alternativas a las Pruebas en Animales/métodos , Animales , Productos Biológicos/farmacología , Modelos Animales de Enfermedad , Diseño de Fármacos , Resistencia a Medicamentos , Humanos , Malaria Falciparum/parasitología , Plantas Medicinales/químicaRESUMEN
Several initiatives have recently taken place in Brazil in order to foster the creation of centers dedicated to alternatives to animal testing. In 2008, Vanessa Sá-Rocha organized a meeting with Brazilian regulatory authorities and the major stakeholders in the field of testing to foster discussions on the process of funding, development, and validation of alternative methods in Brazil. Octavio Presgrave published a scientific article on "The Need for the Establishment of a Brazilian Centre for the Validation of Alternative Methods." Also in 2008, Jadir Nunes, together with Dermeval de Carvalho, prepared and presented a proposal to the Brazilian National Agency of Health Surveillance (ANVISA) for the creation of a Centre for the Validation of Alternative Methods. ECVAM and other European stakeholders have been involved in the initiatives. Furthermore, also in 2008, a new legislation has been adopted in Brazil regarding the use of animals for scientific purposes ("lei Arouca"). The legislation establishes, among other provisions, the task of monitoring and evaluating the introduction of alternative methods. However, the legislation does not provide for promotion of or information about, existing alternative methods to the larger Brazilian scientific community. In order to streamline the different activities, Chantra Eskes acted as a facilitator by establishing a new joint proposal with the current Brazilian stakeholders, aimed at setting up a Brazilian Center on Alternative Test Methods.
Asunto(s)
Alternativas a las Pruebas en Animales/organización & administración , Bienestar del Animal , Evaluación Preclínica de Medicamentos/métodos , Alternativas a las Pruebas en Animales/métodos , Animales , Brasil , Estudios de Validación como AsuntoRESUMEN
We examined the correlation between results obtained from the in vivo Draize test for ocular irritation and in vitro results obtained from the sheep red blood cell (RBC) haemolytic assay, which assesses haemolysis and protein denaturation in erythrocytes, induced by cosmetic products. We sought to validate the haemolytic assay as a preliminary test for identifying highly-irritative products, and also to evaluate the in vitro test as alternative assay for replacement of the in vivo test. In vitro and in vivo analyses were carried out on 19 cosmetic products, in order to correlate the lesions in the ocular structures with three in vitro parameters: (i) the extent of haemolysis (H50); (ii) the protein denaturation index (DI); and (iii) the H50/DI ratio, which reflects the irritation potential (IP). There was significant correlation between maximum average scores (MAS) and the parameters determined in vitro (r = 0.752-0.764). These results indicate that the RBC assay is a useful and rapid test for use as a screening method to assess the IP of cosmetic products, and for predicting the IP value with a high level of concordance (94.7%). The assay showed high sensitivity and specificity rates of 91.6% and 100%, respectively.