Publication: Identificación de biomarcadores de úlcera gástrica en caballos
Authors
Matas Quintanilla, Marta
item.page.secondaryauthor
Escuela Internacional de Doctorado
item.page.director
Gutiérrez Montes, Ana María ; Rodolfo Ipharraguerre, Ignacio
Publisher
Universidad de Murcia
publication.page.editor
publication.page.department
DOI
item.page.type
info:eu-repo/semantics/doctoralThesis
Description
Abstract
Dada la elevada prevalencia del síndrome de úlcera gástrica equina (EGUS), los múltiples factores de riesgo implicados, la inespecificidad de sus signos clínicos y la complejidad diagnóstica de la gastroscopia, que constituye el único método diagnóstico antemortem aceptado y fiable, en esta tesis doctoral se planteó la identificación y validación de biomarcadores en fluidos biológicos mínimamente invasivos, que pudieran contribuir a la detección, cribado y monitorización de la enfermedad.En esta tesis doctoral se llevaron a cabo tres experimentos: 1) Identificación y validación de biomarcadores de úlceras gástricas en muestras de suero y saliva de caballos mediante técnicas proteómicas en gel; 2) Obtención de métodos cuantitativos apropiados para medir biomarcadores que faciliten la detección de úlceras gástricas en caballos; y 3) Evaluación del comportamiento de los biomarcadores de úlceras gástricas en caballos en condiciones clínicas. En dichos experimentos se trabajó con tres grupos de caballos. El primero estuvo constituido por ocho caballos sanos a los que se les indujeron úlceras gástricas experimentales. El segundo grupo incluyó veinte caballos de alta competición clínicamente sanos y veintiocho caballos con signos clínicos compatibles con úlceras gástricas y lesiones confirmadas mediante gastroscopia. El tercer grupo estuvo formado por caballos con lesiones ulcerativas en la mucosa gástrica, tanto con signos clínicos (n=28) como sin ellos (n=28), junto con un grupo control adicional de veintidós caballos clínicamente sanos y sin lesiones en la gastroscopia.En el Experimento 1, de carácter experimental, se trabajó con los ocho caballos a los que se les indujeron las úlceras gástricas mediante privación de alimento. Usando técnicas proteómicas (2DE y LC-MS/MS), se identificaron quince proteínas en saliva y cinco en suero con diferencias significativas entre los animales antes y después de la inducción de las úlceras. De esas proteínas, se validaron nueve en saliva (IL1-F5, IL1-F6, PIP, FABP5, CA-VI, albúmina, Apo A-4, ###1-antitripsina y serotransferrina), y las tres últimas también en suero, mediante SDS-PAGE y Western blotting.Posteriormente, en el Experimento 2, cinco de estos biomarcadores fueron cuantificados en saliva (IL1-F5, PIP, CA-VI, albúmina y serotransferrina), junto con dos marcadores de inflamación (ADA y S100A12). Los ensayos desarrollados presentaron precisión, exactitud y sensibilidad adecuadas, de acuerdo con las guías generales. En el Experimento 3, se evaluó el comportamiento de los siete biomarcadores bajo distintos escenarios clínicos y se estudió su valor diagnóstico. Los animales con úlceras mostraron concentraciones más altas que los animales sanos, con variaciones relacionadas con el tipo de lesión y la presencia de signos clínicos. El análisis de sus curvas ROC arrojó valores de AUC entre 0,80 y 0,84, para seis de los biomarcadores evaluados (IL1-F5, CA-VI, serotransferrina, albúmina, ADA y S100A12), lo que sugiere tener potencial clínico real como herramientas complementarias a la gastroscopia para la identificación temprana de EGUS. Únicamente PIP presentó un rendimiento limitado como marcador individual.Los resultados confirman que la saliva es un fluido biológico idóneo para la evaluación de biomarcadores asociados al EGUS, ya que permite una recogida rápida, sencilla y no invasiva. Se aporta, por primera vez, un conjunto validado de biomarcadores y métodos cuantitativos aplicables en la práctica clínica, lo que sienta las bases para el desarrollo de paneles multibiomarcadores que complementen la gastroscopia, actualmente el único método diagnóstico antemortem aceptado. Los biomarcadores evaluados abarcan diversas vías fisiopatológicas implicadas en el EGUS, incluyendo la respuesta inmune, la inflamación sistémica, la protección de la mucosa gástrica y el daño tisular, lo que refuerza su relevancia biológica. En conjunto, estos hallazgos respaldan su potencial para mejorar la detección temprana, la monitorización evolutiva y la toma de decisiones clínicas, contribuyendo a una gestión más eficaz y al bienestar de los caballos afectados.
Equine gastric ulcer syndrome (EGUS) has a high prevalence, involves multiple risk factors, presents non-specific clinical signs, and its diagnosis is complex, with gastroscopy being the only accepted and reliable antemortem diagnostic method. In this doctoral thesis, the identification and validation of biomarkers in minimally invasive biological fluids was proposed, with the aim of contributing to the detection, screening and monitoring of the disease.To carry out this doctoral thesis, three experiments were conducted: 1) Identification and validation of gastric ulcer biomarkers in serum and saliva samples from horses using gel-based proteomic techniques;2) Development of appropriate quantitative methods to measure biomarkers that facilitate the detection of gastric ulcers in horses; and 3) Evaluation of the behaviour of gastric ulcer biomarkers in horses under clinical conditions.Three groups of horses were used in the experiments. The first consisted of eight healthy horses in which gastric ulcers were experimentally induced. The second consisted of twenty clinically healthy high-performance horses and twenty-eight horses with clinical signs of gastric ulcers and evident lesions in the gastric mucosa, confirmed by gastroscopy. The third group consisted of horses with ulcerative lesions in the gastric mucosa and clinical signs, together with another twenty-eight horses with lesions in the gastric mucosa but without clinical signs, as well as twenty-two clinically healthy horses without lesions in the gastric mucosa on gastroscopy.In Experiment 1, which was experimental, eight horses were subjected to feed deprivation to induce gastric ulcers. Using proteomic techniques (2DE and LC-MS/MS), fifteen proteins in saliva and five in serum were identified as showing significant differences before and after ulcer induction. Of these proteins, nine were validated in saliva (IL1-F5, IL1-F6, PIP, FABP5, CA-VI, albumin, Apo A-4, ###1-antitrypsin and serotransferrin), and the last three were also validated in serum, using SDS-PAGE and Western blotting.Subsequently, in Experiment 2, five of these biomarkers were quantified in saliva (IL1-F5, PIP, CA-VI, albumin and serotransferrin), together with two inflammatory markers (ADA and S100A12). The assays developed showedadequate precision, accuracy and sensitivity in accordance with FDA (2018) guidelines.In Experiment 3, the behaviour of the seven biomarkers under different clinical scenarios was evaluated, and their diagnostic value was studied. Horses with ulcers showed higher concentrations than healthy animals, with variations related to lesion type and the presence of clinical signs. ROC curve analysis yielded AUC values between 0.80 and 0.84 for six of the biomarkers evaluated (IL1-F5, CA-VI, serotransferrin, albumin, ADA and S100A12), suggesting real clinical potential as complementary tools to gastroscopy for the early identification of EGUS. Only PIP showed limited diagnostic performance as an individual marker.The results of this thesis confirm that saliva is an ideal biological fluid for the evaluation of biomarkers associated with EGUS, as it allows rapid, simple and non-invasive sampling. For the first time, a validated set of biomarkers and quantitative methods applicable in clinical practice is provided, laying the groundwork for the development of multibiomarker panels that complement gastroscopy, currently the only accepted antemortem diagnostic method. The biomarkers evaluated encompass various pathophysiological pathways involved in EGUS, including immune response, systemic inflammation, gastric mucosal protection and tissue damage, reinforcing their biological relevance. Altogether, these findings support their potential to improve early detection, longitudinal monitoring and clinical decision-making, contributing to more effective management and improved welfare of affected horses.
Equine gastric ulcer syndrome (EGUS) has a high prevalence, involves multiple risk factors, presents non-specific clinical signs, and its diagnosis is complex, with gastroscopy being the only accepted and reliable antemortem diagnostic method. In this doctoral thesis, the identification and validation of biomarkers in minimally invasive biological fluids was proposed, with the aim of contributing to the detection, screening and monitoring of the disease.To carry out this doctoral thesis, three experiments were conducted: 1) Identification and validation of gastric ulcer biomarkers in serum and saliva samples from horses using gel-based proteomic techniques;2) Development of appropriate quantitative methods to measure biomarkers that facilitate the detection of gastric ulcers in horses; and 3) Evaluation of the behaviour of gastric ulcer biomarkers in horses under clinical conditions.Three groups of horses were used in the experiments. The first consisted of eight healthy horses in which gastric ulcers were experimentally induced. The second consisted of twenty clinically healthy high-performance horses and twenty-eight horses with clinical signs of gastric ulcers and evident lesions in the gastric mucosa, confirmed by gastroscopy. The third group consisted of horses with ulcerative lesions in the gastric mucosa and clinical signs, together with another twenty-eight horses with lesions in the gastric mucosa but without clinical signs, as well as twenty-two clinically healthy horses without lesions in the gastric mucosa on gastroscopy.In Experiment 1, which was experimental, eight horses were subjected to feed deprivation to induce gastric ulcers. Using proteomic techniques (2DE and LC-MS/MS), fifteen proteins in saliva and five in serum were identified as showing significant differences before and after ulcer induction. Of these proteins, nine were validated in saliva (IL1-F5, IL1-F6, PIP, FABP5, CA-VI, albumin, Apo A-4, ###1-antitrypsin and serotransferrin), and the last three were also validated in serum, using SDS-PAGE and Western blotting.Subsequently, in Experiment 2, five of these biomarkers were quantified in saliva (IL1-F5, PIP, CA-VI, albumin and serotransferrin), together with two inflammatory markers (ADA and S100A12). The assays developed showedadequate precision, accuracy and sensitivity in accordance with FDA (2018) guidelines.In Experiment 3, the behaviour of the seven biomarkers under different clinical scenarios was evaluated, and their diagnostic value was studied. Horses with ulcers showed higher concentrations than healthy animals, with variations related to lesion type and the presence of clinical signs. ROC curve analysis yielded AUC values between 0.80 and 0.84 for six of the biomarkers evaluated (IL1-F5, CA-VI, serotransferrin, albumin, ADA and S100A12), suggesting real clinical potential as complementary tools to gastroscopy for the early identification of EGUS. Only PIP showed limited diagnostic performance as an individual marker.The results of this thesis confirm that saliva is an ideal biological fluid for the evaluation of biomarkers associated with EGUS, as it allows rapid, simple and non-invasive sampling. For the first time, a validated set of biomarkers and quantitative methods applicable in clinical practice is provided, laying the groundwork for the development of multibiomarker panels that complement gastroscopy, currently the only accepted antemortem diagnostic method. The biomarkers evaluated encompass various pathophysiological pathways involved in EGUS, including immune response, systemic inflammation, gastric mucosal protection and tissue damage, reinforcing their biological relevance. Altogether, these findings support their potential to improve early detection, longitudinal monitoring and clinical decision-making, contributing to more effective management and improved welfare of affected horses.
publication.page.subject
Citation
item.page.embargo
Collections
Ir a Estadísticas
Este ítem está sujeto a una licencia Creative Commons. http://creativecommons.org/licenses/by-nc-nd/4.0/