Abstract New drug discovery involves preclinical studies to demonstrate its effectivity and safety for further tests in humans. This leads to the need to develop technologies that take advantage of the new analytical tools available within a context where the results of the tests carried out are fully documented, under auditable systems of good laboratory practice. This review updates and describes some of the tests carried out in the preclinical stage of the development of a new drug and the current state of the analytical technology used to measure different blood biomarkers of interest. Biomarker parameters were analyzed at the physiological level, considering both the validation regulations of the analytical techniques used for their determination as the problems that arise when trying to apply them, since many of these biomarkers are endogenous compounds in the used matrices.

Resumen El descubrimiento de un nuevo principio activo farmacéutico implica estudios preclínicos, que tienen como objetivo demostrar que es eficaz y seguro para un posterior ensayo en seres humanos. Esto conduce a la necesidad de desarrollar tecnologías que aprovechen las nuevas herramientas analíticas disponi bles dentro de un contexto donde los resultados de las pruebas realizadas, estén plenamente documentados, bajo sistemas de buenas prácticas de laboratorio auditables. En esta revisión se actualizan y describen algunos de los ensayos realizados en la etapa preclínica del desarrollo de un nuevo fármaco y el estado actual de la tecnología analítica empleada para el dosaje de diferentes biomarcadores sanguíneos de interés. Se analizaron los biomarcadores más relevantes, las normativas de validación de las técnicas analíticas empleadas para su determinación y los problemas que se presentan al tratar de aplicarlas.

Abstract It is well known that several of the swainsonine-containing plant species found widespread around the world have a negative economic impact in each country. In Argentina, most of the information on the poisonous plant species that produce α-mannosidosis is published in Spanish and thus not available to most English-speaking researchers interested in toxic plants. Therefore, the aim of this review is to summarize the information about swainsonine-containing plants in Argentina, which are extensively distributed throughout different ecoregions of the country. To date, five species from three genera have been shown to induce α-mannosidosis in livestock in Argentina: Ipomoea carnea subsp. fistulosa, Ipomoea hieronymi subsp. calchaquina (Convolvulaceae), Astragalus garbancillo, Astragalus pehuenches (Fabaceae), and Sida rodrigoi (Malvaceae). These species contain the indolizidine alkaloid swainsonine, which inhibits the lysosomal enzyme α-mannosidase and consequently affects glycoprotein metabolism, resulting in partially metabolized sugars. The prolonged consumption of these poisonous plants produces progressive weight loss and clinical signs related to a nervous disorder, characterized by tremors of head and neck, abnormalities of gait, difficulty in standing, ataxia and wide-based stance. Histological lesions are mainly characterized by vacuolation of different cells, especially neurons of the central nervous system. The main animal model used to study α-mannosidosis is the guinea pig because, when experimentally poisoned, it exhibits many of the characteristics of naturally intoxicated livestock.

A expressão de proteínas do citoesqueleto foi avaliada por imuno-histoquímica em ovários normais e císticos de porcas matrizes. Amostras de 36 ovários normais (18 porcas) e de 44 císticos (22 porcas) foram avaliadas. Todas as matrizes apresentaram histórico de problemas reprodutivos, como infertilidade ou subfertilidade. As áreas coradas por imuno-histoquímica (IHCSA) foram quantificadas por avaliação de imagens avaliando a expressão dessas proteínas na parede folicular de folículos secundários, terciários e císticos. A imuno-reatividade para citoqueratina (CK) foi forte na camada de células da granulosa (GC) e discreta nas tecas interna (TI) e externa (TE) dos folículos normais. Houve redução acentuada da reação de CK na CG dos folículos císticos, principalmente nos cistos luteinizados. A reação para vimentina foi mais intensa na CG dos folículos normais e císticos em comparação com outras estruturas foliculares. Nos cistos luteinizados, a IHCSA para vimentina foi significativamente maior na TI e, em ambos os cistos observados, a marcação foi mais acentuada na TE. A marcação de desmina e actina alfa de músculo liso foi restrita a TE, sem diferenças entre os folículos normais e císticos. Os resultados do presente estudo mostram que o desenvolvimento de cistos ovarianos em porcas matrizes está associado a alterações na expressão das proteínas do citoesqueleto CK e vimentina.

The expression of cytoskeletal proteins was evaluated immunohistochemically in 36 normal ovaries sampled from 18 sows and 44 cystic ovaries sampled from of 22 sows, was evaluated. All sows had history of reproductive problems, such as infertility or subfertility. The immunohistochemically stained area (IHCSA) was quantified through image analysis to evaluate the expression of these proteins in the follicular wall of secondary, tertiary, and cystic follicles. Cytokeratins (CK) immunoreactivity was strong in the granulosa cell layer (GC) and mild in the theca interna (TI) and externa (TE) of the normal follicles. There was severe reduction of the reaction to CK in the GC in the cystic follicles, mainly in the luteinized cysts. The immunoreactivity for vimentin was higher in the GC from normal and cystic follicles in contrast with the other follicular structures. In the luteinized cysts, the IHCSA for vimentin was significantly higher in TI and in both observed cysts, the labeling was more accentuated in TE. Immunohistochemical detection of desmin and α-SMA was restricted to the TE, without differences between the normal and cystic follicles. The results of the current study show that the development of ovarian cysts in sows is associated to changes in the expression of the cytoskeletal proteins CK and vimentin.

Numerosos modelos experimentais têm sido desenvolvimos para o estudo da síndrome do ovário policístico em ratos. No presente estudo, a síndrome foi inducida por exposição à luz constante. O processo foi avaliado durante sua indução e inclusive durante sua reversão. O ciclo estral foi analisado através de citologia vaginal; parámetros reprodutivos foram avaliados por acasalamento, bem como a morfologia ovariana. Todos animais desenvolveram a síndrome depois de 13 semanas de luz permanente. As características histológicas dos ovários, na semana 15, foram similares àquelas observadas na síndrome do ovário policístico em humanos e outras espécies. Após a regressão da síndrome, não houve diferenta em nenhum dos parámetros reprodutivos avaliados, quando comparados com o grupo controle.

Numerous experimental models have been developed for the study of the polycystic ovarian syndrome in the rat. In the present study, the syndrome was induced by exposure to constant light. The process was evaluated during its induction and also during its reversion. The estral cycle was analyzed through the vaginal cytology; reproductive parameters were evaluated by mating, as well as the ovarian morphology. All the animals developed the syndrome after 13 weeks of permanent light. The histologic characteristics of the ovaries, at week 15, were similar to those observed in the polycystic ovarian syndrome in human and other species. After regression of the syndrome, there was not difference in any of the evaluated reproductive parameters, when compared with the control group.

Oligosacarids expression in llama (Lama glama) mammary glands was evaluated during lactation period by means of lectins. The lactocite nuclear diameter, alveoli mean diameter and the percentage of the area occupiedby alveolar lumen were compared by morphometric analysis. Fifteen females were biopsed on days 5, 15, 30, 60 y 120 after freshening. Seven biotynilated lectines (Con-A, WGA, DBA, SBA, PNA, RCA-I, UEA-I) were used, following the pre-established protocols (ABC Method). Apical domain cytoplasm of lactocites reacted intensely with lectins WGA and RCA; and moderately with Con-A during the whole lactation period. Lactocites glycocalyx reacted intensely with SBA y PNA in every studied period; fromday 5 to day 15 the reaction was remarkable for RCA, and from day 15 to 120 days of lactation, it was so for DBA. There was not marking of the parenchyma with UEA-I during the whole assay length. The results showed that carbohydrates express themselves in different ways as the lactation progresses, which indicates the occurrence of bio-molecular changes due to intense functional activity. Morphometrical analysis revealed that there is not variation in the relative area occupied by alveoli, but it does exist a significative increase (p<0,05) in the alveolus size since day 15, together with a decrease in the lactocites nuclear size beginning on day 60

Se evaluó la expresión de oligosacáridos en la glándula mamaria de llamas (Lama glama) durante la lactogénesis utilizando lectinas. Mediante análisis morfométrico se evaluó el diámetro nuclear de los lactocitos, diámetro promedio de los alvéolos y el porcentaje del área ocupada por la luz alveolar. Se realizaron biopsias a 15 hembras, a los 5, 15, 30, 60 y 120 días postparto. Se utilizaron siete lectinas biotiniladas (Con-A, WGA, DBA, SBA, PNA, RCA-I, UEA-I), siguiendo protocolos preestablecidos (Método ABC). El citoplasma del dominio apical de los lactocitos reaccionó intensamente con las lectinas WGA y RCA; y en forma moderada a Con-A, durante toda la lactogénesis. El glucocálix de los lactocitos reaccionó intensamente con SBA y PNA, en todos los períodos estudiados; desde el día 5 hasta el día 15 la reacción fue marcada para la RCA, y desde el día 15 hasta los 120 días de lactogénesis con la DBA. No hubo marcación del parénquima con UEA-I en ninguno de los períodos estudiados. Los resultados mostraron que los hidratos de carbono se expresan de diferente manera a medida que progresa la producción láctea, lo que indica la ocurrencia de cambios biomoleculares debidos a una intensa actividad funcional. El análisis morfométrico reveló que no se producen variaciones en el área relativa ocupada por los alvéolos, pero sí un aumento significativo (p< 0,05) en el tamaño de los alvéolos a partir del día 15 acompañado por una disminución en el tamaño de los núcleos de lactocitos, a partir de los 60 días