Validation of a zebrafish developmental defects assay as a qualified alternative test for its regulatory use following the ICH S5(R3) guideline.

Zebrafish is a popular toxicology model and provides an ethically acceptable small-scale analysis system with the complexity of a complete organism. Our goal is to further validate this model for its regulatory use for reproductive and developmental defects by testing the compounds indicated in the "Guideline on detection of reproductive and developmental toxicity for human pharmaceuticals" (ICH S5(R3) guideline.) To determine the embryotoxic and developmental risk of the 32 reference compounds listed in the ICH S5(R3) guideline, the presence of morphological alterations in zebrafish embryos was analyzed at two different stages to calculateLC50 and EC50 values for each stage. Teratogenic Indexes were established as the ratio between LC50 and EC50 critical for the proper compound classification as teratogenic when it is ≥ 2. A total of three biological replicates have been conducted to study the reproducibility of the assay. The chemicals' concentration in the medium and internally in the zebrafish embryos was evaluated. In this study, the 3 negative compounds were properly categorized while 23 compounds out of the 29 reference ones (sensitivity of 79.31%) were classified as teratogenic in zebrafish. The 6 that had false-negative results were classified 4 as inconclusive, 1 as not toxic, and 1 compound resulted toxic for zebrafish embryos under testing conditions. After the bioavailability experiments, some of the obtained inconclusive results were refined. The developmental defects assay in zebrafish gives an accuracy of 89.66%, sensitivity of 88.46%, specificity and repeatability of 100% compared to mammals; therefore, this is a well-integrated strategy using New Alternative Methods, to minimize the use of animals in developmental toxicity studies.

Screening for chemicals with thyroid hormone-disrupting effects using zebrafish embryo.

Thyroid disruption is an increasingly recognized issue in the use and development of chemicals and new drugs, especially to help toxicologist to complement the reproductive and developmental toxicology information of chemicals. Still, adequate assessment methods are scarce and often suffer a trade-off between physiological relevance and labor- and cost-intensive assays. Here, we present a tiered approach for a medium-throughput screening of chemicals to identify their thyroid disrupting potential in zebrafish embryos as a New Approach Methodology (NAM). After identifying the maximum tolerated concentrations, we exposed zebrafish larvae to sub-adverse effect levels of the reference compounds benzophenone-2, bisphenol A, phenylthiourea, potassium perchlorate, propylthiouracil, and phloroglucinol to exclude any systemic toxicity. Applying the transgenic zebrafish line that carries a gene for the red fluorescence protein (Tg(tg:mCherry)) under the thyroglobulin promoter, we could identify the thyroid disrupting effects of the chemicals by a time and cost-effective image analysis measuring the fluorescence levels in the thyroid glands. Our observations could be confirmed by altered expression patterns of genes involved in the hypothalamus-pituitary-thyroid (HPT) axis. Finally, to anchor the observed thyroid disruption, we determined some changes in the Thyroid hormone levels of triiodothyronine (T3) and Thyroxine (T4) using a newly developed liquid chromatography mass spectrometric (LCMS) method. The presented approach carries the potential to extend the toolbox for legislative authorities and chemical producers for the assessment of thyroid-specific endocrine disruption and to overcome current challenges in the evaluation of endocrine disruptors.

Comparación de compuestos metabólicos en muestras de sudor entre pacientes con enfermedad pulmonar obstructiva crónica y cáncer de pulmón / Sweat sample metabolic compound comparison between patients with chronic obstructive pulmonary disease and lung cancer

Objetivo: Evaluar en pacientes con enfermedad pulmonar obstructiva crónica respecto a pacientes con cáncer epidermoide de pulmón en estadio inicial (CP I-II) si en el metaboloma del sudor existen diferencias en los compuestos. Metodología: Se incluyeron 11 pacientes con EPOC y 9 pacientes con CP I-II. El sudor se recogió siguiendo una técnica estandarizada y la muestra fue congelada a -80ºC hasta el análisis metabolómico, para lo que se utilizó un cromatógrafo de líquidos acoplado a un espectrómetro de masas de alta resolución (LC-QTOF) con ionización por electrospray. Se realizó un análisis de cambio (AC) para detectar las diferencias de concentración relativa de metabolitos entre grupos. Resultados. Las características basales de los sujetos incluidos en los dos grupos fueron similares. En la clínica destaca que un 67% de los enfermos con CP I-II (67%) no manifestaron síntomas atribuibles al tumor. El análisis metabolómico mostró que en el análisis de cambio una tetrahexosa presentó diferencias entre el grupo de enfermos con EPOC y con CP I-II (AC: - 4,021), igual tendencia se observó en un trisacárido fosfato (AC: -1,741) y en un lípido sulfónico (AC: -1,920). Conclusión: En muestras de sudor, el análisis de cambio muestra metabolitos con potencialidad para diferenciar entre pacientes EPOC y con CP I-II. Este resultado puede tener aplicabilidad en el cribado del cáncer de pulmón

Objective: To evaluate whether there are differences in sweat metabolite compounds in patients with chronic obstructive pulmonary disease compared to patients with early-stage squamous cell lung cancer (LC I-II). Methods: 11 patients with COPD and 9 patients with LC I-II were included. Sweat was collected using a standardized technique and the sample was frozen at -80ºC until the metabolic analysis was performed, which used a liquid chromatograph coupled with a high-resolution mass spectrometer (LC-QTOF) with electrospray ionization. A change analysis (CA) was done to detect the differences in the relative concentrations of metabolites between groups. Results: The baseline characteristics of subjects included in the two groups were similar. In the clinical presentation, it is worth noting that 67% of patients with LC I-II (67%) did not show symptoms that could be attributed to the tumor. The metabolic analysis showed that in the change analysis, a tetra-hexose showed differences between the COPD group and LC I-II group (CA: -4.021), the same pattern observed in a phosphate trisaccharide (CA: -1.741) and in a sulphonic lipid (AC: -1.920). Conclusion: In sweat samples, the change analysis shows metabolites with the potential to differ between patients with COPD and those with LC I-II. This result can be applied in lung cancer screening

Long-term Prognosis After Lung Transplantation: A Monocentric Study in 510 Patients.

INTRODUCTION: Lung transplantation is the final treatment option in patients with respiratory failure. Morbidity and mortality rates associated with the management of complications is high despite advances. Postoperative complications include acute transplant rejection, bronchiolitis obliterans, and infections. Because of that, the success of this treatment option depends on the correct choice of donor and candidates to receive a transplant. OBJECTIVE: This study aims to perform a survival analysis of transplanted patients in our center and determine predictive variables of mortality. PATIENTS AND METHODS: This study is a retrospective assessment of data collected from 510 patients at the Hospital University Reina Sofía from October 1993 to December 31, 2016. Patients who were retransplanted were excluded. We collected data regarding basal characteristics of the donors and candidates to receive a transplant. We analyzed the impact in terms of future survival of basal variables from donor and donor recipients. RESULTS: Five hundred ten patients were included (average age 44 ± 17 years, 69% male), with a maximum follow-up period of 21.6 years (average follow-up 4.52 years, interquartile ratio: 0.13 to 6.97 years). Two hundred twenty-seven patients died (54.3% of the total amount). The influence of donor's basal characteristics on mortality was analyzed; moreover, the relationship between basal variables and survival were analyzed using univariate analysis. Available variables were analyzed through multivariate analysis. CONCLUSION: Lung transplantation is a treatment option with an acceptable risk of morbidity and mortality. Increased awareness of features of evolution could help to reduce postoperative complications.

Relación entre las variables observadas en una prueba de marcha de 6 minutos y la desaturación nocturna en pacientes con EPOC / Relationship regarding the variables observed in a 6-minute walking test and desaturation at night in COPD

OBJETIVO: Determinar, en pacientes con enfermedad pulmonar obstructiva crónica (EPOC) las variables que en la prueba de 6 minutos marcha (P6MM) aportan información sobre el grado de desaturación nocturna. METODOLOGÍA: Estudio prospectivo, transversal con muestreo consecutivo. Se incluyeron sujetos ambulatorios, en estabilidad clínica, con una saturación periférica de oxígeno (SapO2 ≥92%) y sin contraindicación para realizar una P6MM. A todos los sujetos se les realizaron dos determinaciones con el mismo pulsioxímetro (Pulsox-300i) durante la P6MM y sueño. Se realizó correlación entre las variables de la P6MM y el porcentaje del tiempo nocturno con SapO2 <90% (T90) y se construyó un modelo de regresión para evaluar la variabilidad del T90. RESULTADOS: Fueron incluidos 47 enfermos, 37 hombres (79%), edad = 61,6 ± 7,5 años, y un FEV1 post-BD = 50 ± 18,3 %. Respecto al T90 nocturno, las variables que mostraron correlación significativa en la P6MM fueron la SapO2 , basal, media y mínima, y el T90 y T88 (SapO2 <88%). El modelo de regresión lineal mostró una R2 ajustada de 0,644; (p <0,001) siendo las variables que se asociaron de forma independiente al T90 nocturno: el IMC (p = 0,049), SapO2 basal (p = 0,001), SapO2 media-P6MM (p = 0,006) y el T88-P6MM (p = 0,048). CONCLUSIONES: La P6MM aporta información relevante sobre el grado de desaturación nocturna. La SapO2 basal, y la SapO2 media y el valor del T88 fueron las variables que en la P6MM mostraron mayor influencia sobre la variabilidad del T90 nocturno

OBJECTIVE: Determine in patients with chronic obstructive pulmonary disease (COPD) those variables that provide information about degree of nocturnal desaturation in the 6-minute walk test (6MWT). METHODS: Prospective, transversal study with consecutive sampling. Outpatients were included in clinical stability, with peripheral oxygen saturation (SapO2 ≥92%) and without contraindication for a 6MWT. In all subjects were performed two determinations with the same pulse oximeter (Pulsox-300i) during the 6MWT and sleep. Was performed correlation between variables 6MWT and percentage nigh time with SapO2<90% (T90) and a regression model were constructed to evaluate the variability of the T90. RESULTS: We included 47 patients, 37 men (79%), age = 61.6 ± 7.5 years, and post-BD FEV1 = 50 ± 18.3%. Regard to T90 nocturnal, the variables that showed significant correlation in 6MWT were SapO2 basal, mean and minimum, and T90 and T88 (SapO2 <88%). The linear regression model showed an adjusted R2 of 0.644 (p< 0.001), and BMI (p = 0.049), baseline SapO2 6MWT (p = 0.001), mean SapO2 (p = 0.006) and T88-6MWT (p = 0.048) were the only variables independently associated to nocturnal T90. CONCLUSIONS: The 6MWT provides relevant information on the level of nocturnal desaturation. The basal SapO2 and mean and the value of T88 were the 6MWT variables that showed the greatest influence on variability nocturnal T90

Monocyte-mediated enhancement of endometrial cell proliferation in women with endometriosis.

OBJECTIVE: To investigate the capacity of monocytes from women with endometriosis to influence endometrial cell proliferation. DESIGN: Uterine endometrial cells were cultured in the presence and absence of autologous blood monocytes for 72 hours before assessment of endometrial cell proliferation by thymidine incorporation. SETTING: Patients were tested at initial presentation for evaluation of infertility and/or endometriosis. PATIENTS, PARTICIPANTS: Fertile controls, n = 17; infertile controls, n = 9; untreated endometriosis, n = 29. INTERVENTIONS: None. RESULTS: Endometrial cell proliferation was enhanced significantly by blood monocytes in patients with endometriosis but was suppressed significantly by blood monocytes in fertile controls. Endometrial cell proliferation was not affected significantly by blood monocytes in infertile controls analyzed as a group, but a subset of infertile patients also showed enhancement of endometrial cell proliferation by blood monocytes. CONCLUSIONS: Blood monocytes from patients with endometriosis and a subset of patients with unexplained infertility enhance autologous endometrial cell proliferation, whereas blood monocytes from fertile patients suppress endometrial cell proliferation. The capacity of monocytes to enhance endometrial cell proliferation appears to require both monocyte-derived factors that stimulate endometrial cell proliferation and endometrial cells capable of responding to those stimulatory factors. If either of these factors is absent, monocytes either suppress or have no effect on endometrial cell proliferation.