Desarrollo y validación de métodos cromatográficos ajustados a propósito para la monitorización de antimicrobianos de interés clínico. Aplicación a estudios farmacocinéticos

La infección en el entorno hospitalario es a día de hoy uno de los mayores desafíos en el ámbito sanitario. La elevada incidencia a nivel mundial en la aparición de estas infecciones incrementa la tasa de morbimortalidad y el gasto sanitario, y es considerado un problema de salud pública. Las infecciones tanto en pacientes críticos como oncohematológicos trasplantados presentan una incidencia elevada y se asocian a un incremento en la tasa de mortalidad hospitalaria. El tratamiento de estas infecciones se basa en el uso racional de antimicrobianos como antibacterianos y antifúngicos capaces de eliminar al patógeno. Las pautas empleadas para la dosificación de antimicrobianos en este tipo de pacientes pueden no ser adecuadas, ya que éstas derivan de estudios realizados con sujetos sanos con características diferentes a las de los pacientes a tratar. Esto puede dar lugar a concentraciones inadecuadas de fármaco en el organismo, con consecuencias no deseadas. La optimización de estas terapias mediante la individualización posológica es un objetivo en el tratamiento de estas infecciones. Así, la implementación de programas para la monitorización de antimicrobianos y su estudio farmacocinético ha demostrado ser efectiva en este tipo de pacientes. Para conseguir este objetivo, existen guías publicadas por parte de diferentes sociedades científicas que recomiendan el empleo de métodos validados basados en cromatografía de líquidos de alta resolución que sean capaces de dar un resultado analítico con rapidez para ser útiles en el ámbito asistencial (~24 h desde la extracción de la muestra hasta la notificación de los resultados analíticos). El objetivo general de esta Tesis Doctoral es el desarrollo, puesta a punto y validación de métodos cromatográficos ajustados a propósito para la monitorización de antimicrobianos de interés en muestras de plasma de pacientes tratados en el Hospital Clínico Universitario de Valencia. Los antimicrobianos monitorizados incluyen antibacterianos (meropenem, piperacilina, ceftolozano combinado con tazobactam y amikacina) y antifúngicos (voriconazol, caspofungina y anidulafungina). Se realizó una validación completa de las características analíticas de cada uno de los métodos desarrollados siguiendo las recomendaciones descritas en la guía publicada por la Agencia Europea del Medicamento. Los métodos cromatográficos desarrollados y validados en la presente Tesis Doctoral se emplearon en la monitorización clínica de pacientes bajo tratamiento antimicrobiano, así como en el estudio del perfil farmacocinético de ciertos antimicrobianos en pacientes sometidos a técnicas de depuración extracorpórea

Comparacion entre Valores de Vtec Predichos por el Modelo Nequick2 y los Obtenidos a Partir de Gps sobre Boulder durante muy baja actividad Solar

Una especificación confiable del estado de la ionósfera dada por modelos empíricos es importante para mitigar los efectos que esta zona atmosférica produce sobre las operaciones de los sistemas de posicionamiento y navegación basadas en satélites. Este estudio evalúa la capacidad del modelo NeQuick2 para predecir el contenido electrónico vertical total (VTEC), hasta la altura de los satélites GPS, sobre Boulder (40,00°N, 254,75°E), durante una época de muy baja actividad solar. Para ello se comparan las predicciones del modelo con valores de VTEC obtenidos a partir de señales de satélites GPS, publicados por el National Geophysical Data Center (NGDC) de EEUU. Teniendo en cuenta las incertezas de los valores de GPS VTEC publicados por el NGDC, los resultados muestran que el modelo estudiado, en general, se comporta como un buen predictor del contenido electrónico total en las condiciones solares consideradas

SPINK7 expression changes accompanied by HER2, P53 and RB1 can be relevant in predicting oral squamous cell carcinoma at a molecular level

The oral squamous cell carcinoma (OSCC), which has a high morbidity rate, affects patients worldwide. Changes in SPINK7 in precancerous lesions could promote oncogenesis. Our aim was to evaluate SPINK7 as a potential molecular biomarker which predicts OSCC stages, compared to: HER2, TP53, RB1, NFKB and CYP4B1. This study used oral biopsies from three patient groups: dysplasia (n = 33), less invasive (n = 28) and highly invasive OSCC (n = 18). The control group consisted of clinically suspicious cases later to be confirmed as normal mucosa (n = 20). Gene levels of SPINK7, P53, RB, NFKB and CYP4B1 were quantified by qPCR. SPINK7 levels were correlated with a cohort of 330 patients from the TCGA. Also, SPINK7, HER2, TP53, and RB1, were evaluated by immunohistofluorescence. One-way Kruskal–Wallis test and Dunn's post-hoc with a p < 0.05 significance was used to analyze data. In OSCC, the SPINK7 expression had down regulated while P53, RB, NFKB and CYP4B1 had up regulated (p < 0.001). SPINK7 had also diminished in TCGA patients (p = 2.10e-6). In less invasive OSCC, SPINK7 and HER2 proteins had decreased while TP53 and RB1 had increased with respect to the other groups (p < 0.05). The changes of SPINK7 accompanied by HER2, P53 and RB1 can be used to classify the molecular stage of OSCC lesions allowing a diagnosis at molecular and histopathological levels.Fil: Pennacchiotti, Graciela Laura. Universidad de Chile; ChileFil: Valdes Garrido, Fabio. Instituto Nacional del Cáncer; ChileFil: González Arriaga, Wilfredo Alejandro. Universidad de Valparaíso; ChileFil: Montes, Héctor Federico. Universidad Nacional de Cuyo. Facultad de Odontologia; ArgentinaFil: Parra, Judith Maria Roxana. Universidad Nacional de Cuyo. Facultad de Odontologia; ArgentinaFil: Guida, Valeria Andrea. Universidad Nacional de Cuyo. Facultad de Odontologia; ArgentinaFil: Gomez, Silvina Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Guerrero Gimenez, Martin Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Fernandez Muñoz, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Zoppino, Felipe Carlos Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Caron, Ruben Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Ezquer, Marcelo Eduardo. Universidad del Desarrollo; ChileFil: Ramires Fernández, Ricardo. Universidad Mayor; ChileFil: Bruna, Flavia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad Nacional de Cuyo. Facultad de Odontologia; Argentin

Post-entry blockade of small ruminant lentiviruses by wild ruminants

Small ruminant lentivirus (SRLV) infection causes losses in the small ruminant industry due to reduced animal production and increased replacement rates. Infection of wild ruminants in close contact with infected domestic animals has been proposed to play a role in SRLV epidemiology, but studies are limited and mostly involve hybrids between wild and domestic animals. In this study, SRLV seropositive red deer, roe deer and mouflon were detected through modified ELISA tests, but virus was not successfully amplified using a set of different PCRs. Apparent restriction of SRLV infection in cervids was not related to the presence of neutralizing antibodies. In vitro cultured skin fibroblastic cells from red deer and fallow deer were permissive to the SRLV entry and integration, but produced low quantities of virus. SRLV got rapidly adapted in vitro to blood-derived macrophages and skin fibroblastic cells from red deer but not from fallow deer. Thus, although direct detection of virus was not successfully achieved in vivo, these findings show the potential susceptibility of wild ruminants to SRLV infection in the case of red deer and, on the other hand, an in vivo SRLV restriction in fallow deer. Altogether these results may highlight the importance of surveilling and controlling SRLV infection in domestic as well as in wild ruminants sharing pasture areas, and may provide new natural tools to control SRLV spread in sheep and goats.Funded by CICYT (AGL2010-22341-C04-01 and AGL2013-49137-C3-1-R) and Navarra’s Government (IIQ010449.RI1 and IIQ14064.RI1). L. Sanjosé was a FPI fellow of the Spanish MINECO and R. Reina had contracts from the Public University of Navarra and CSIC. The auhors acknowledge support in the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)

High Fat Diet-Induced Skeletal Muscle Wasting Is Decreased by Mesenchymal Stem Cells Administration: Implications on Oxidative Stress, Ubiquitin Proteasome Pathway Activation, and Myonuclear Apoptosis

Obesity can lead to skeletal muscle atrophy, a pathological condition characterized by the loss of strength and muscle mass. A feature of muscle atrophy is a decrease of myofibrillar proteins as a result of ubiquitin proteasome pathway overactivation, as evidenced by increased expression of the muscle-specific ubiquitin ligases atrogin-1 and MuRF-1. Additionally, other mechanisms are related to muscle wasting, including oxidative stress, myonuclear apoptosis, and autophagy. Stem cells are an emerging therapy in the treatment of chronic diseases such as high fat diet-induced obesity. Mesenchymal stem cells (MSCs) are a population of self-renewable and undifferentiated cells present in the bone marrow and other mesenchymal tissues of adult individuals. The present study is the first to analyze the effects of systemic MSC administration on high fat diet-induced skeletal muscle atrophy in the tibialis anterior of mice. Treatment with MSCs reduced losses of muscle strength and mass, decreases of fiber diameter and myosin heavy chain protein levels, and fiber type transitions. Underlying these antiatrophic effects, MSC administration also decreased ubiquitin proteasome pathway activation, oxidative stress, and myonuclear apoptosis. These results are the first to indicate that systemically administered MSCs could prevent muscle wasting associated with high fat diet-induced obesity and diabetes

Pituitary changes involved in prolactin secretion induced by mifepristone and naloxone during late pregnancy

The antiprogesterone mifepristone facilitates prolactin release, an effect enhanced by administration of the opioid antagonist naloxone. The present study explores ultrastructural changes in lactotropes after mifepristone and naloxone administration, correlating them with the expression of pituitary prolactin. Methods/Results: Rats were sacrificed at 18:00 h on day 19 of pregnancy. Prolactin immunoelectron microscopy of lactotropes from control rats showed characteristics of quiescent cells with numerous small and spherical secretory granules. Naloxone administration did not modify lactotrope morphology or prolactin expression in terms of mRNA or protein abundances. Mifepristone treatment induced lactotrope activation with development of the rough endoplasmic reticulum and Golgi complex with prolactin immunoreactive small newly formed and large mature secretory granules. Mifepristone increased prolactin mRNA and protein expression. Naloxone administration to mifepristone-treated rats potentiated lactotrope activation compared with mifepristone alone showing exocytotic images of prolactin granules and some cells with evident signs of involution. Conclusions: (1) Blockade of progesterone action by mifepristone activated the lactotrope, increased significantly prolactin mRNA and protein expression and prepared the pituitary for naloxone action. (2) The high serum prolactin levels induced by mifepristone and naloxone may regulate negatively lactotrope activity as suggested by the presence of regressing cells neighboring the actively secreting cells.Fil: Villegas Gabutti, Carlos Mauricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Ezquer, Eduardo Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Deis, Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Maldonado, Cristina Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Soaje, Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentin

Hipothyroidism produces change in the uterine vasculature during the implantation process

Hypothyroidism is one of the most common endocrine abnormalities implicated in the recurrent loss of pregnancy. Our laboratory, have shown that hypothyroidism in the rat is associated with a lower number of pups per litter due to a lower number of implantation sites and a decrease in the proliferation of the endothelial and decidual cells during the process of implantation of the embryo. On the other hand, is known that angiogenesis is a critical process in the uterine endometrium for embryo implantation, maintenance of early pregnancy, and development of the placenta. During this period, steroid hormones (E2 and P4) stimulate the synthesis of vascular endothelial growth factor (VEGF-A), the main modulator of angiogenesis during peri-implantation period. Therefore we hypothesize that hypothyroidism affects the normal vascularization of endometrium during implantation. The aim of this work was to study the effect of hypothyroidism on the degree of vascularization of the uterine decidua during the implantation process. Hypothyroidism was induced in female Wistar rats by daily administration of 6-propyl-2-thiouracil (PTU) 0,1 g/L in drinking water. In addition, hormone replacement therapy with T3 was administered simultaneously to the treatment with PTU (PTU+T3), in daily physiological doses of 0.6ug/100g. Both groups were compared to rats that only drink tap water (Control),on day five (G5) and seven (G7) of gestation. Uterine vascularization was evaluated by immunofluorescence. Besides,mRNA expression of PECAM (Platelet endothelial cell adhesion molecule, an indicator of the presence of endothelial cells) and VEGF-A were evaluated during the same peri-implantation periods (G5 y G7)by RTqPCR. Our results demonstrate that hypothyroidism decreases vascularization density of the uterine tissue during the process of implantation of the embryo (p<0.05). On the other hand, our results demonstrated a significant increase of expression of VEGF mRNA when hypothyroid rats were treated with T3 before implantation, in comparison to the control group and hypothyroid group (p< 0.05). However, no changes were found in the levels of PECAM expression among the different groups. In conclusion, the failure of implantation due to hypothyroidism may be directly linked to expression of VEGF-A, and consequently to vascularization of the endometrium before implantation in early gestation. Although are necessary further studies that corroborate the exact mechanism, our results identify molecular targets regulated by thyroid hormones that may link hypothyroidism to implantation failure and recurrent miscarriage.Fil: Troncoso, Mariana Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Rinaldini, Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Ezquer, M. E.. Universidad del Desarrollo; ChileFil: Gamarra Luques, Carlos Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Hapon, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaXXXVI Reunión Científica Anual de la Sociedad Cuyo de BiologíaMendozaArgentinaSociedad de Biología de Cuy

Effect of Thyromimetic GC-1 Selective Signaling on Reproductive and Lactational Performance in the Hypothyroid Rat

Introduction: The potential of the thyroid hormone receptor β (TRβ1) selective analog GC-1 has been widely proven in animal models and humans. However, its effect on the reproductive stage of the female rat has not been evaluated. Methods: The effect of the administration of GC-1 or equimolar doses of triiodothyronine (T3) was evaluated on the reproductive performance of the hypothyroid female rat and the indirect effect on pup thyroid status, weight, and survival. Results: Hypothyroidism reduced the number of embryos implanted in the uterus, whereas T3 and GC-1 treatment in hypothyroid females reestablished the number of implanted embryos to normal. Initiation of labor was delayed by hypothyroidism, and T3 replacement treatment reinstated the normal timing of parturition. The administration of GC-1 alone to the lactating mother did not affect pup survival, weight, or thyroidal status. Conclusions: Our findings show the differential effect of thyroid hormone selective signaling during gestation and the indirect exposure of the pups; we also emphasize the plausible use of GC-1 for treatment of hypothyroid mothers during the lactation period.Fil: Rinaldini, Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Campo Verde Arbocco, Fiorella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Ezquer, Eduardo Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad del Desarrollo. Facultad de Medicina Clínica Alemana; ChileFil: Gamarra Luques, Carlos Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Hapon, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentin