Rol del receptor GPBAR-1 en la gravedad de la infección causada por Clostridium difficile
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2017
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es
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Universidad Andrés Bello
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Resumen
Clostridium difficile es un bacilo Gram positivo, anaerobio estricto y formador de endoesporas, que a su vez son el morfotipo de persistencia, transmisión e infección. La infección causada por C. difficile (ICD) corresponde al 30% de las colitis asociadas a antibióticos en centros asistenciales de salud y tiene una tasa de mortalidad global que alcanza un 5%. El daño se produce principalmente por las toxinas TcdA y TcdB de C. difficile que actúan sobre las proteínas de las uniones estrechas del epitelio colónico formando poros, lo que se traduce en la disrupción de la barrera epitelial y la activación de la respuesta inmune del hospedero. Las manifestaciones clínicas pueden variar desde leves con síntomas como una diarrea acuosa, a moderadas y graves, presentando colitis pseudomembranosa o megacolon tóxico, respectivamente. Hasta el momento, la terapia más utilizada y efectiva es la administración de vancomicina y metronidazol, sin embargo aún no existe una terapia que tenga una eficiencia total. Atendido lo anterior, se hace necesario buscar nuevos objetivos terapéuticos para atenuar los daños causados durante la ICD.
Por otro lado, GPBAR-1 es un receptor que posee acción anti-inflamatoria y que modula la integridad de la barrera epitelial intestinal en colitis química en modelo murino y, se ha descrito que el uso de ácido oleanólico (AO), agonista del receptor, disminuye los niveles de inflamación y daño clínico. Se hipotetiza con esto que GPBAR-1 disminuye la gravedad en una colitis infecciosa con C. difficile.
El objetivo de esta tesis fue estudiar el efecto de GPBAR-1 en la gravedad de una colitis infecciosa como la ICD. Es por esto que se estudió en una primera instancia como afecta la ausencia del receptor en la gravedad de los síntomas. Luego se analizó la variación en la integridad de la barrera epitelial, el grado de permeabilidad intestinal y la inflamación por medio del reclutamiento de células mediadoras de la inflamación en el epitelio en ausencia de GPBAR-1 en la ICD. Por último, para estudiar su uso terapeútico se suplementaron ratones infectados con AO. No se obtuvo incremento de la gravedad de la ICD en ausencia de GPBAR-1. La ausencia del receptor no incrementó los síntomas, el daño del epitelio ni la inflamación en una ICD de 4 días. No obstante, los resultados sugieren que la suplementación con ácido oleanólico podría disminuir la inflamación causada por la ICD. Se hace necesario realizar nuevos ensayos de infección de mayor duración y mayor tamaño de muestra para concluir el potencial de GPBAR-1 como blanco terapeútico.
Clostridium difficile is a gram-positive, anaerobic and endospore-forming bacillus, which in turn is the morphotype of persistence, transmission and infection. Infection caused by C. difficile (CDI) accounts for 30% of antibiotic-associated colitis in health care settings and has a global mortality rate of 5%. The damage is mainly caused by C. difficile toxins TcdA and TcdB that act on the proteins of the tight junctions of the colonic epithelium forming pores, which results in the breakdown of the epithelial barrier and the activation of the host immune response. The clinical manifestations can vary from mild symptoms such as watery diarrhea, to moderate and severe symptoms, presenting pseudomembranous colitis or toxic megacolon, respectively. Until now, the most widely used and effective therapy is the administration of vancomycin and metronidazole, however, there is still no therapy with total efficiency. Given the above, it is necessary to seek new therapeutic targets to mitigate the damage caused during the CDI. On the other hand, GPBAR-1 is a receptor that possesses anti-inflammatory action and modulates the integrity of the intestinal epithelial barrier in chemical colitis in murine model and, it has been described that the use of oleanolic acid (OA), agonist of the receptor, decreases levels of inflammation and clinical damage. Whit this, it is hypothesized that GPBAR-1 decreases the severity in an infectious colitis with C. difficile. The aim of this thesis was to study the effect of GPBAR-1 on the severity of an infectious colitis such as CDI. This is why it was studied in the first instance as it affects the absence of the receptor in the severity of the symptoms. The variation in the integrity of the epithelial barrier, the degree of intestinal permeability and the inflammation by means of the recruitment of cells mediating the inflammation in the epithelium in the absence of GPBAR-1 in the CDI were analyzed. Finally, to study their therapeutic use, infected mice were supplemented with OA. There was no increase in the severity of CDI in the absence of GPBAR-1. Absence of the receptor did not increase symptoms, epithelial damage, or inflammation in a 4-day ICD. However, the results suggest that supplementation with OA may decrease the inflammation caused by CDI. It is necessary to perform new infection tests of longer duration and larger sample size to conclude the potential of GPBAR-1 as a therapeutic target.
Clostridium difficile is a gram-positive, anaerobic and endospore-forming bacillus, which in turn is the morphotype of persistence, transmission and infection. Infection caused by C. difficile (CDI) accounts for 30% of antibiotic-associated colitis in health care settings and has a global mortality rate of 5%. The damage is mainly caused by C. difficile toxins TcdA and TcdB that act on the proteins of the tight junctions of the colonic epithelium forming pores, which results in the breakdown of the epithelial barrier and the activation of the host immune response. The clinical manifestations can vary from mild symptoms such as watery diarrhea, to moderate and severe symptoms, presenting pseudomembranous colitis or toxic megacolon, respectively. Until now, the most widely used and effective therapy is the administration of vancomycin and metronidazole, however, there is still no therapy with total efficiency. Given the above, it is necessary to seek new therapeutic targets to mitigate the damage caused during the CDI. On the other hand, GPBAR-1 is a receptor that possesses anti-inflammatory action and modulates the integrity of the intestinal epithelial barrier in chemical colitis in murine model and, it has been described that the use of oleanolic acid (OA), agonist of the receptor, decreases levels of inflammation and clinical damage. Whit this, it is hypothesized that GPBAR-1 decreases the severity in an infectious colitis with C. difficile. The aim of this thesis was to study the effect of GPBAR-1 on the severity of an infectious colitis such as CDI. This is why it was studied in the first instance as it affects the absence of the receptor in the severity of the symptoms. The variation in the integrity of the epithelial barrier, the degree of intestinal permeability and the inflammation by means of the recruitment of cells mediating the inflammation in the epithelium in the absence of GPBAR-1 in the CDI were analyzed. Finally, to study their therapeutic use, infected mice were supplemented with OA. There was no increase in the severity of CDI in the absence of GPBAR-1. Absence of the receptor did not increase symptoms, epithelial damage, or inflammation in a 4-day ICD. However, the results suggest that supplementation with OA may decrease the inflammation caused by CDI. It is necessary to perform new infection tests of longer duration and larger sample size to conclude the potential of GPBAR-1 as a therapeutic target.
Notas
Tesis (Magíster en Biotecnología)
Esta tesis fue realizada en el Departamento de Gastroenterología de la Facultad de Medicina de la Pontificia Universidad Católica de Chile y en el Departamento de microbiología de la Facultad de Ciencias Biológicas de la Universidad Andrés Bello; financiada por el proyecto Fondecyt 11130502 del Dr. Cristian Hernández-Rocha, Fondecyt 1131012 del Dr. Manuel Álvarez-Lobos M.A-L y Fondecyt 1151025 del Dr. Daniel Paredes-Sabja
Esta tesis fue realizada en el Departamento de Gastroenterología de la Facultad de Medicina de la Pontificia Universidad Católica de Chile y en el Departamento de microbiología de la Facultad de Ciencias Biológicas de la Universidad Andrés Bello; financiada por el proyecto Fondecyt 11130502 del Dr. Cristian Hernández-Rocha, Fondecyt 1131012 del Dr. Manuel Álvarez-Lobos M.A-L y Fondecyt 1151025 del Dr. Daniel Paredes-Sabja
Palabras clave
Clostridium Difficile, Enfermedades Infecciosas, Receptor GPBAR-1