Browsing by Author "López Lastra, Marcelo Andrés"

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    A cis-Acting Element Present within the gag Open Reading Frame Negatively Impacts on the Activity of the HIV-1 IRES
    (2013) Valiente Echeverria, Fernando; Vallejos, Maricarmen; Monette, Anne; Pino, Karla; Letelier, Alejandro; Huidobro Toro, J. Pablo; Mouland, Andrew J.; López Lastra, Marcelo Andrés
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    Correlation between female sex, IL28B genotype, and the clinical severity of bronchiolitis in pediatric patients
    (2020) Astudillo, P.; Angulo, J.; Pino, K.; de Carvalho, J. B.; de Morais, G. L.; Perez, S.; de Vasconcelos, A. T. R.; Ferrés, Marcela; López Lastra, Marcelo Andrés
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    Detection of high biliary and fecal viral loads in patients with chronic hepatitis C virus infection
    (2017) Monrroy Bravo, Hugo Alfonso; Angulo, J.; Pino, Karla; Labbé, P.; Miquel P., Juan Francisco; López Lastra, Marcelo Andrés; Soza, Alejandro
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    Dicistronic MLV-retroviral vectors transduce neural precursors in vivoand co-express two genes in their differentiated neuronal progeny
    (2005) López Lastra, Marcelo Andrés; Derrington, Edmund A.; Darlix, Jean-Luc
    Abstract Dicistronic MLV-based retroviral vectors, in which two IRESes independently initiate the translation of two proteins from a single RNA, have been shown to direct co-expression of proteins in several cell culture systems. Here we report that these dicistronic retroviral vectors can drive co-expression of two gene products in brain cells in vivo. Injection of retroviral vector producer cells leads to the transduction of proliferating precursors in the external granular layer of the cerebellum and throughout the ventricular regions. Differentiated neurons co-expressing both transgenes were observed in the cerebellum and in lower numbers in distant brain regions such as the cortex. Thus, we describe an eukaryotic dicistronic vector system that is capable of transducing mouse neural precursors in vivo and maintaining the expression of genes after cell differentiation.Abstract Dicistronic MLV-based retroviral vectors, in which two IRESes independently initiate the translation of two proteins from a single RNA, have been shown to direct co-expression of proteins in several cell culture systems. Here we report that these dicistronic retroviral vectors can drive co-expression of two gene products in brain cells in vivo. Injection of retroviral vector producer cells leads to the transduction of proliferating precursors in the external granular layer of the cerebellum and throughout the ventricular regions. Differentiated neurons co-expressing both transgenes were observed in the cerebellum and in lower numbers in distant brain regions such as the cortex. Thus, we describe an eukaryotic dicistronic vector system that is capable of transducing mouse neural precursors in vivo and maintaining the expression of genes after cell differentiation.Abstract Dicistronic MLV-based retroviral vectors, in which two IRESes independently initiate the translation of two proteins from a single RNA, have been shown to direct co-expression of proteins in several cell culture systems. Here we report that these dicistronic retroviral vectors can drive co-expression of two gene products in brain cells in vivo. Injection of retroviral vector producer cells leads to the transduction of proliferating precursors in the external granular layer of the cerebellum and throughout the ventricular regions. Differentiated neurons co-expressing both transgenes were observed in the cerebellum and in lower numbers in distant brain regions such as the cortex. Thus, we describe an eukaryotic dicistronic vector system that is capable of transducing mouse neural precursors in vivo and maintaining the expression of genes after cell differentiation.Abstract Dicistronic MLV-based retroviral vectors, in which two IRESes independently initiate the translation of two proteins from a single RNA, have been shown to direct co-expression of proteins in several cell culture systems. Here we report that these dicistronic retroviral vectors can drive co-expression of two gene products in brain cells in vivo. Injection of retroviral vector producer cells leads to the transduction of proliferating precursors in the external granular layer of the cerebellum and throughout the ventricular regions. Differentiated neurons co-expressing both transgenes were observed in the cerebellum and in lower numbers in distant brain regions such as the cortex. Thus, we describe an eukaryotic dicistronic vector system that is capable of transducing mouse neural precursors in vivo and maintaining the expression of genes after cell differentiation.
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    Differential expression profile of CXCR3 splicing variants is associated with thyroid neoplasia. Potential role in papillary thyroid carcinoma oncogenesis?
    (2018) Urra, Soledad; Fischer, Martin C.; Martinez, Jose R.; Veliz, Loreto; Orellana, Paulina; Solar González, Antonieta Alejandra; Bohmwald Prieto, Karen; Kalergis Parra, Alexis Mikes; Riedel, Claudia; Corvalán R., Alejandro; Roa Strauch, Juan Carlos Enrique; Fuentealba, Rodrigo; Cáceres, C. Joaquín; López Lastra, Marcelo Andrés; León Ramírez, Augusto; Droppelmann, Nicolás; Gonzalez, Hernan E.
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    Dual Mechanisms of Translation Initiation of the Full-Length HIV-1 mRNA Contribute to Gag Synthesis
    (2013) Monette, Anne; Valiente Echeverría, Fernando; Rivero, Matías; Cohen,Éric A.; López Lastra, Marcelo Andrés; Mouland, Andrew J.
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    Effect of ezetimibe in HCV viral load after liver transplantation
    (2016) Monrroy Bravo, Hugo Alfonso; Angulo, J.; Pino, Karla; Labbé, P.; López Lastra, Marcelo Andrés; Soza, Alejandro
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    Estructuras de RNA y su impacto sobre el inicio de la traducción del mRNA genómico de HIV-1 y del SmRNA de ANDV
    (2019) Carvajal Díaz, Felipe Alonso; López Lastra, Marcelo Andrés; Pontificia Universidad Católica de Chile. Facultad de Ciencias Biológicas
    La síntesis de proteínas es el proceso por el cual la información contenida en un RNA mensajero (mRNA) es decodificada para sintetizar la correspondiente cadena polipeptídica. El proceso de síntesis de proteínas se divide en 4 etapas principales: iniciación, elongación, terminación y reciclaje de ribosomas. El inicio de la síntesis de proteínas, la cual es la etapa más regulada de la traducción, comprende el reclutamiento de los factores de inicio de la traducción y de la subunidad 40S ribosomal sobre el mRNA a ser traducido, el proceso de búsqueda del codón de inicio o scanning, y el ensamblaje y posicionamiento del ribosoma 80S sobre el codón de inicio. En el mRNA existen estructuras canónicas necesarias para un proceso eficiente de inicio de la traducción, tales como la estructura cap en el extremo 5’, y la cola poliadenilada (poly(A)) en el extremo 3’ de éste. Dichas estructuras permiten el reclutamiento de los factores de inicio, así como la circularización no covalente del mRNA. A su vez, las regiones 5’ y 3’ no traducidas (UTRs) del mRNA contribuyen en la regulación del proceso de inicio, modulando la eficiencia del reclutamiento de la maquinaria traduccional. En este respecto, las estructuras de RNA presentes en las regiones UTR son moduladores importantes del proceso de inicio de la síntesis de proteínas. Algunos mRNAs de origen viral utilizan mecanismos alternativos para reclutar los complejos de inicio de la traducción, otorgándoles una ventaja por sobre los demás mRNAs dentro de la célula. Parte de estos mecanismos no canónico se basan en la presencia de estructuras de RNA en las regiones UTR del mRNA, las cuales pueden mediar la síntesis de proteínas de forma independiente de la estructura cap o de la cola poly(A). Para ahondar en el conocimiento de los mecanismos de regulación del inicio de la traducción no canónico, en este trabajo de tesis, se eligieron dos modelos de mRNAs virales, los cuales permiten caracterizar por un lado el inicio de la síntesis de proteínas cap-independiente, y por otro, el inicio de la traducción poly(A) independiente. El primer modelo utilizado fue el mRNA completo del virus de la inmunodeficiencia humana de tipo 1 (HIV-1). Este mRNA viral posee en su región 5’UTR un sitio interno de entrada al ribosoma (IRES). Para este modelo, se determinó el funcionamiento del elemento HIV-1 IRES en diferentes tipos celulares. Se estableció que este IRES es de naturaleza modular, diferenciándose así de los IRES de origen viral. Se determinó además que el elemento HIV-1 IRES no requiere del proceso de scanning para iniciar la síntesis de proteínas, reclutando los complejos de inicio directamente sobre el codón de inicio. El segundo modelo estudiado correspondió al mRNA del segmento S (SmRNA) del Orthohantavirus Andes (ANDV). Este mRNA viral posee una estructura 5’-cap, pero carece de cola poly(A). A pesar de esto, el SmRNA es traducido, dando origen a las proteínas virales N y NSs. En este trabajo se caracterizaron las estructuras secundarias de RNA presentes en la región 5’ y 3’ UTR del SmRNA de ANDV mediante la estrategia de SHAPE. Se determinó la estructura de la región 5’UTR y se estableció que las estructuras de RNA presentes en ella regulan el inicio de la traducción del SmRNA. Para la región 3’UTR del SmRNA, se caracterizó las estructuras de RNA existentes, sin embargo, no fue posible asignan una estructura única a esta región puesto que los resultados sugieren que más bien se comportaría como un elemento dinámico. En conjunto, los resultados obtenidos en este trabajo de tesis permiten ahondar en el entendimiento de la regulación del proceso de inicio de la síntesis de proteínas no canónico en modelos de mRNAs virales.La síntesis de proteínas es el proceso por el cual la información contenida en un RNA mensajero (mRNA) es decodificada para sintetizar la correspondiente cadena polipeptídica. El proceso de síntesis de proteínas se divide en 4 etapas principales: iniciación, elongación, terminación y reciclaje de ribosomas. El inicio de la síntesis de proteínas, la cual es la etapa más regulada de la traducción, comprende el reclutamiento de los factores de inicio de la traducción y de la subunidad 40S ribosomal sobre el mRNA a ser traducido, el proceso de búsqueda del codón de inicio o scanning, y el ensamblaje y posicionamiento del ribosoma 80S sobre el codón de inicio. En el mRNA existen estructuras canónicas necesarias para un proceso eficiente de inicio de la traducción, tales como la estructura cap en el extremo 5’, y la cola poliadenilada (poly(A)) en el extremo 3’ de éste. Dichas estructuras permiten el reclutamiento de los factores de inicio, así como la circularización no covalente del mRNA. A su vez, las regiones 5’ y 3’ no traducidas (UTRs) del mRNA contribuyen en la regulación del proceso de inicio, modulando la eficiencia del reclutamiento de la maquinaria traduccional. En este respecto, las estructuras de RNA presentes en las regiones UTR son moduladores importantes del proceso de inicio de la síntesis de proteínas. Algunos mRNAs de origen viral utilizan mecanismos alternativos para reclutar los complejos de inicio de la traducción, otorgándoles una ventaja por sobre los demás mRNAs dentro de la célula. Parte de estos mecanismos no canónico se basan en la presencia de estructuras de RNA en las regiones UTR del mRNA, las cuales pueden mediar la síntesis de proteínas de forma independiente de la estructura cap o de la cola poly(A). Para ahondar en el conocimiento de los mecanismos de regulación del inicio de la traducción no canónico, en este trabajo de tesis, se eligieron dos modelos de mRNAs virales, los cuales permiten caracterizar por un lado el inicio de la síntesis de proteínas cap-independiente, y por otro, el inicio de la traducción poly(A) independiente. El primer modelo utilizado fue el mRNA completo del virus de la inmunodeficiencia humana de tipo 1 (HIV-1). Este mRNA viral posee en su región 5’UTR un sitio interno de entrada al ribosoma (IRES). Para este modelo, se determinó el funcionamiento del elemento HIV-1 IRES en diferentes tipos celulares. Se estableció que este IRES es de naturaleza modular, diferenciándose así de los IRES de origen viral. Se determinó además que el elemento HIV-1 IRES no requiere del proceso de scanning para iniciar la síntesis de proteínas, reclutando los complejos de inicio directamente sobre el codón de inicio. El segundo modelo estudiado correspondió al mRNA del segmento S (SmRNA) del Orthohantavirus Andes (ANDV). Este mRNA viral posee una estructura 5’-cap, pero carece de cola poly(A). A pesar de esto, el SmRNA es traducido, dando origen a las proteínas virales N y NSs. En este trabajo se caracterizaron las estructuras secundarias de RNA presentes en la región 5’ y 3’ UTR del SmRNA de ANDV mediante la estrategia de SHAPE. Se determinó la estructura de la región 5’UTR y se estableció que las estructuras de RNA presentes en ella regulan el inicio de la traducción del SmRNA. Para la región 3’UTR del SmRNA, se caracterizó las estructuras de RNA existentes, sin embargo, no fue posible asignan una estructura única a esta región puesto que los resultados sugieren que más bien se comportaría como un elemento dinámico. En conjunto, los resultados obtenidos en este trabajo de tesis permiten ahondar en el entendimiento de la regulación del proceso de inicio de la síntesis de proteínas no canónico en modelos de mRNAs virales.
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    Genetic variations in host IL28B links to the detection of peripheral blood mononuclear cells-associated hepatitis C virus RNA in chronically infected patients
    (2013) Angulo, Jenniffer; Pino, Karla; Pavez, C.; Biel, F.; Labbé, Pilar; Miquel P., Juan Francisco; Soza, Alejandro; López Lastra, Marcelo Andrés
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    Hepatitis C virus may have an entero-hepatic cycle which could be blocked with ezetimibe
    (2017) Monrroy Bravo, Hugo Alfonso; López Lastra, Marcelo Andrés; Soza, Alejandro
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    HnRNPK and HuR are regulators of ires-mediated translation initiation of the mouse mammary tumot virus and the human tcell leukemia virus type-1 mRNAs
    (2025) Mendonça, Dafne Silva Furtado de; López Lastra, Marcelo Andrés; Pontificia Universidad Católica de Chile. Facultad de Medicina
    Riesgos multifactoriales, incluyendo factores genéticos e infecciones virales, pueden conducir al crecimiento celular incontrolable y atípico observado en cáncer. La desregulación de las proteínas de unión al RNA (RBPs) pueden causar una expresión proteica anormal y por modificaciones postraduccionales de éstas pueden contribuir a la tumorigénesis y a la adquisición de resistencia a fármacos quimioterapéuticos, aumentando la expresión de oncogenes o reduciendo la expresión de genes supresores de tumores. El microambiente tumoral caracterizado por múltiples condiciones de estrés obliga a las células a adaptarse y conservar energía, disminuyendo la traducción global del ARN mensajero (ARNm) al inhibir el inicio de la traducción cap-dependiente. No obstante, la síntesis de proteínas involucradas en la respuesta al estrés se mantiene mediante un mecanismo de iniciación de la traducción cap-independiente, utilizando un sitio interno de entrada al ribosoma (IRES). Se han descrito elementos IRES en ARNm oncogénicos, como c-myc, RBPs como el antígeno R humano (HuR) y miembros de la familia de la ribonucleoproteína nucleares heterogéneas (hnRNP), como hnRNPK, han sido identificados como reguladores clave de la iniciación de la traducción mediada por IRES de ARNm oncogénicos, lo que contribuye a la progresión tumoral o a la resistencia a la quimio/radioterapia. Las RBPs que regulan la iniciación de la traducción mediada por IRES se denominan factores que actúan en trans sobre el IRES (ITAFs). Los agentes infecciosos, como el virus linfotrópico de células T humanas tipo 1 (HTLV-1), agente causal de a leucemia/linfoma de células T en adultos, y el virus del tumor mamario murino (MMTV), asociado con el cáncer de mama en humanos, son responsables del 18 % de todos los casos de cáncer en el mundo. Los ARN de HTLV-1 y MMTV posee una actividad IRES regulada por ITAFs. Esta observación nos llevó a cuestionarnos si RBPs, como HuR y hnRNPK, asociadas con el cáncer, actuarían como ITAFs para los IRES de MMTV-1 y HTLV-1. Para responder a esta duda, se transfectaron células HEK293T y HeLa con un vector bicistrónico (dl MMTV IRES o dl HTLV-1 IRES) junto con un plásmido que codifica para la proteína de interés (hnRNPK o HuR). La distribución de las proteínas sobreexpresadas se visualizó mediante inmunofluorescencia. Se midió la actividad de la luciferasa para determinar la función de IRES. Se realizaron ensayos de Western blot para monitorear la sobreexpresión de proteínas y se realizó RISH-PLA para determinar la proximidad proteína-ARN. Este estudio establece que tanto las proteínas hnRNPK como HuR se distribuyen en el núcleo y el citoplasma. En células, la sobreexpresión de hnRNPK incrementó la actividad del IRES de MMTV, y la dimetilación asimétrica de hnRNPK mediada por la proteína arginina metiltransferasa 1 indujo significativamente la capacidad de esta proteína de promover la actividad del IRES de MMTV. Por otro lado, HuR disminuyó la actividad del IRES de HTLV-1 mientras aumentó la del IRES de MMTV. Además, se estableció que hnRNPK se encontraba en estrecha proximidad con el IRES del dl HTLV-1, lo que sugiere una interacción proteína-mRNA. Este trabajo proporciona evidencia de RBPs relacionadas con el cáncer, HuR y hnRNPK, actúan como ITAFs y regulan el inicio de la traducción mediada por IRES de los ARN de los oncoretrovirus MMTV y HTLV1. Además, las modificaciones postraduccionales de hnRNPK pueden afectar su efecto sobre la actividad de IRES. Comprender este mecanismo puede revelar posibles dianas terapéuticas para las infecciones retrovirales asociadas con el cáncer.
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    Identificación de las proteínas hnRNP A1, hnRNP K y HUR como moduladores de la traducción cap-independiente de los mRNAS de HTLV-1 y sHBZ
    (2023) López Ulloa, Brenda Patricia; López Lastra, Marcelo Andrés; Pontificia Universidad Católica de Chile. Facultad de Ciencias Biológicas
    HTLV-1 es un retrovirus clasificado en el género Deltaretrovirus, perteneciente a la subfamilia Orthoretrovirinae. La traducción del mRNA genómico de HTLV-1 puede iniciar siguiendo una vía cap-dependiente o bien ser mediada por un sitio interno de entrada a ribosoma (IRES), el HTLV-1 IRES. Durante su replicación HTLV-1 expresa dos mRNAs anti-sentido que codifican para el factor de cremallera de leucina de carácter básico (HBZ), generando dos isoformas diferentes de la proteína HBZ, sHBZ y usHBZ. El inicio de la traducción de la forma usHBZ es cap-dependiente, mientras que el mRNA de sHBZ inicia de manera dependiente de un IRES, el sHBZ IRES. Basado en evidencia previa que establece que los IRESs de origen retroviral son regulados por proteínas celulares clasificadas como factores trans-activadores del IRES (ITAF), en este trabajo de tesis se evaluó el potencial rol de ITAF de las proteínas: nuclear heterogénea (hnRNP) A1, hnRNP K y el antígeno humano R (HuR). Los resultados muestran que hnRNP A1 actua como ITAF que estimula la actividad del HTLV-1 IRES y sHBZ IRES. Sin embargo, la magnitud de la estimulación ejercida por hnRNP A1 sobre el sHBZ IRES es menor que la obtenida para el HTLV-1 IRES. La sobreexpresión de hnRNP K estimula la actividad del HTLV-1 IRES sin afectar la traducción mediada por el sHBZ IRES. Indicando que hnRNP K es un ITAF para el IRES de HTLV-1 pero no para el IRES de sHBZ. Finalmente, la proteína HuR inhibe al HTLV-1 IRES y sHBZ IRES, sugiriendo que HuR es un ITAF para ambos IRES. Posteriormente, se evaluó el rol de las modificaciones post-traduccionales (PTMs), fosforilaciones y metilaciones, en la actividad de ITAF de hnRNP A1, hnRNP K y HuR para el HTLV-1 IRES y sHBZ IRES. Los resultados muestran que a excepción de la mutante hnRNP A1 R218/225K, todas las mutantes evaluadas para hnRNP A1 se comportaban como la proteína wild type (WT) estimulando a ambos IRESs. Con respecto a las mutantes evaluadas de hnRNP K se encontró que su actividad sobre los IRES de HTLV-1 y sHBZ es similar a la proteína WT. Las mutantes evaluadas para HuR, se comportaron como la proteína WT para ambos IRES. Como conclusión de este trabajo de tesis se logró identificar que las proteínas hnRNP A1, hnRNP K y HuR que impactan de manera diferencial al HTLV-1 IRES y al sHBZ IRES.
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    IL28B gene polymorphism rs12979860, but not rs8099917, contributes to the occurrence of chronic HCV infection in Uruguayan patients
    (2018) Echeverría, Natalia; Angulo, Jenniffer; López Lastra, Marcelo Andrés; Chiodi, Daniela; López, Pablo; Sanchez Ciceron, Adriana; Silvera, Paola; Canavesi, Adrian; Bianchi, Carla; Colistro, Valentina
    Abstract Background Host single-nucleotide polymorphisms (SNPs) near the interleukin 28B (IL28B) locus are associated with sustained virological response to antiviral therapy and with spontaneous Hepatitis C Virus (HCV) clearance. Prevalence of these SNPs varies depending on ethnicity. The impact of IL28B SNPs in HCV-infected patients is currently unknown in Uruguay. Therefore, the aim of this study was to evaluate and compare the distribution of polymorphisms in the IL28B gene (rs12979860 and rs8099917) among HCV-infected patients and healthy individuals in Uruguay and thus assess their possible association with the establishment of HCV infection. Methods DNA was recovered from 92 non-infected individuals and 78 HCV-infected patients and SNPs were determined by RFLP and allelic discrimination by real-time PCR. Results The distribution of rs12979860 genotypes for the infected population was 29.5%-CC, 47.4%-CT and 23.1%-TT and for the control group 45.7%, 42.4% and 11.9%, respectively. Prevalence in both infected and uninfected individuals is similar to that reported in other countries with admixed populations. The distribution of rs8099917 genotypes for the infected population was 57.7%-TT, 27.2%-TG and 14.1%-GG and for the control group 60.9%, 33.7% and 5.4%, respectively. The comparison of rs12979860 genotype distribution between the two populations evidenced a higher prevalence of the favourable genotype (CC) in the uninfected control group (p < 0.05). Additionally, results generated using logistic regression analysis show that individuals carrying rs12979860-TT or CT genotypes have a higher likelihood of developing chronic hepatitis upon infection with HCV, when compared to CC carriers, considering rs8099917 genotype as constant. Conclusion Patients with HCV infection have a statistically significant lower prevalence of the favourable rs12979860 genotype when compared to uninfected individuals; therefore we can establish that only IL28B rs12979860-CT and TT genotypes seem to contribute to the occurrence of chronic HCV infection in the cohort of Uruguayan population studied. Considering that a trend towards a higher frequency of “good” response genotypes was observed in responder patients, we believe that IL28B rs12979860 genotyping could be a useful tool for predicting different therapies outcome, including in the DAA era.Abstract Background Host single-nucleotide polymorphisms (SNPs) near the interleukin 28B (IL28B) locus are associated with sustained virological response to antiviral therapy and with spontaneous Hepatitis C Virus (HCV) clearance. Prevalence of these SNPs varies depending on ethnicity. The impact of IL28B SNPs in HCV-infected patients is currently unknown in Uruguay. Therefore, the aim of this study was to evaluate and compare the distribution of polymorphisms in the IL28B gene (rs12979860 and rs8099917) among HCV-infected patients and healthy individuals in Uruguay and thus assess their possible association with the establishment of HCV infection. Methods DNA was recovered from 92 non-infected individuals and 78 HCV-infected patients and SNPs were determined by RFLP and allelic discrimination by real-time PCR. Results The distribution of rs12979860 genotypes for the infected population was 29.5%-CC, 47.4%-CT and 23.1%-TT and for the control group 45.7%, 42.4% and 11.9%, respectively. Prevalence in both infected and uninfected individuals is similar to that reported in other countries with admixed populations. The distribution of rs8099917 genotypes for the infected population was 57.7%-TT, 27.2%-TG and 14.1%-GG and for the control group 60.9%, 33.7% and 5.4%, respectively. The comparison of rs12979860 genotype distribution between the two populations evidenced a higher prevalence of the favourable genotype (CC) in the uninfected control group (p < 0.05). Additionally, results generated using logistic regression analysis show that individuals carrying rs12979860-TT or CT genotypes have a higher likelihood of developing chronic hepatitis upon infection with HCV, when compared to CC carriers, considering rs8099917 genotype as constant. Conclusion Patients with HCV infection have a statistically significant lower prevalence of the favourable rs12979860 genotype when compared to uninfected individuals; therefore we can establish that only IL28B rs12979860-CT and TT genotypes seem to contribute to the occurrence of chronic HCV infection in the cohort of Uruguayan population studied. Considering that a trend towards a higher frequency of “good” response genotypes was observed in responder patients, we believe that IL28B rs12979860 genotyping could be a useful tool for predicting different therapies outcome, including in the DAA era.Abstract Background Host single-nucleotide polymorphisms (SNPs) near the interleukin 28B (IL28B) locus are associated with sustained virological response to antiviral therapy and with spontaneous Hepatitis C Virus (HCV) clearance. Prevalence of these SNPs varies depending on ethnicity. The impact of IL28B SNPs in HCV-infected patients is currently unknown in Uruguay. Therefore, the aim of this study was to evaluate and compare the distribution of polymorphisms in the IL28B gene (rs12979860 and rs8099917) among HCV-infected patients and healthy individuals in Uruguay and thus assess their possible association with the establishment of HCV infection. Methods DNA was recovered from 92 non-infected individuals and 78 HCV-infected patients and SNPs were determined by RFLP and allelic discrimination by real-time PCR. Results The distribution of rs12979860 genotypes for the infected population was 29.5%-CC, 47.4%-CT and 23.1%-TT and for the control group 45.7%, 42.4% and 11.9%, respectively. Prevalence in both infected and uninfected individuals is similar to that reported in other countries with admixed populations. The distribution of rs8099917 genotypes for the infected population was 57.7%-TT, 27.2%-TG and 14.1%-GG and for the control group 60.9%, 33.7% and 5.4%, respectively. The comparison of rs12979860 genotype distribution between the two populations evidenced a higher prevalence of the favourable genotype (CC) in the uninfected control group (p < 0.05). Additionally, results generated using logistic regression analysis show that individuals carrying rs12979860-TT or CT genotypes have a higher likelihood of developing chronic hepatitis upon infection with HCV, when compared to CC carriers, considering rs8099917 genotype as constant. Conclusion Patients with HCV infection have a statistically significant lower prevalence of the favourable rs12979860 genotype when compared to uninfected individuals; therefore we can establish that only IL28B rs12979860-CT and TT genotypes seem to contribute to the occurrence of chronic HCV infection in the cohort of Uruguayan population studied. Considering that a trend towards a higher frequency of “good” response genotypes was observed in responder patients, we believe that IL28B rs12979860 genotyping could be a useful tool for predicting different therapies outcome, including in the DAA era.Abstract Background Host single-nucleotide polymorphisms (SNPs) near the interleukin 28B (IL28B) locus are associated with sustained virological response to antiviral therapy and with spontaneous Hepatitis C Virus (HCV) clearance. Prevalence of these SNPs varies depending on ethnicity. The impact of IL28B SNPs in HCV-infected patients is currently unknown in Uruguay. Therefore, the aim of this study was to evaluate and compare the distribution of polymorphisms in the IL28B gene (rs12979860 and rs8099917) among HCV-infected patients and healthy individuals in Uruguay and thus assess their possible association with the establishment of HCV infection. Methods DNA was recovered from 92 non-infected individuals and 78 HCV-infected patients and SNPs were determined by RFLP and allelic discrimination by real-time PCR. Results The distribution of rs12979860 genotypes for the infected population was 29.5%-CC, 47.4%-CT and 23.1%-TT and for the control group 45.7%, 42.4% and 11.9%, respectively. Prevalence in both infected and uninfected individuals is similar to that reported in other countries with admixed populations. The distribution of rs8099917 genotypes for the infected population was 57.7%-TT, 27.2%-TG and 14.1%-GG and for the control group 60.9%, 33.7% and 5.4%, respectively. The comparison of rs12979860 genotype distribution between the two populations evidenced a higher prevalence of the favourable genotype (CC) in the uninfected control group (p < 0.05). Additionally, results generated using logistic regression analysis show that individuals carrying rs12979860-TT or CT genotypes have a higher likelihood of developing chronic hepatitis upon infection with HCV, when compared to CC carriers, considering rs8099917 genotype as constant. Conclusion Patients with HCV infection have a statistically significant lower prevalence of the favourable rs12979860 genotype when compared to uninfected individuals; therefore we can establish that only IL28B rs12979860-CT and TT genotypes seem to contribute to the occurrence of chronic HCV infection in the cohort of Uruguayan population studied. Considering that a trend towards a higher frequency of “good” response genotypes was observed in responder patients, we believe that IL28B rs12979860 genotyping could be a useful tool for predicting different therapies outcome, including in the DAA era.Abstract Background Host single-nucleotide polymorphisms (SNPs) near the interleukin 28B (IL28B) locus are associated with sustained virological response to antiviral therapy and with spontaneous Hepatitis C Virus (HCV) clearance. Prevalence of these SNPs varies depending on ethnicity. The impact of IL28B SNPs in HCV-infected patients is currently unknown in Uruguay. Therefore, the aim of this study was to evaluate and compare the distribution of polymorphisms in the IL28B gene (rs12979860 and rs8099917) among HCV-infected patients and healthy individuals in Uruguay and thus assess their possible association with the establishment of HCV infection. Methods DNA was recovered from 92 non-infected individuals and 78 HCV-infected patients and SNPs were determined by RFLP and allelic discrimination by real-time PCR. Results The distribution of rs12979860 genotypes for the infected population was 29.5%-CC, 47.4%-CT and 23.1%-TT and for the control group 45.7%, 42.4% and 11.9%, respectively. Prevalence in both infected and uninfected individuals is similar to that reported in other countries with admixed populations. The distribution of rs8099917 genotypes for the infected population was 57.7%-TT, 27.2%-TG and 14.1%-GG and for the control group 60.9%, 33.7% and 5.4%, respectively. The comparison of rs12979860 genotype distribution between the two populations evidenced a higher prevalence of the favourable genotype (CC) in the uninfected control group (p < 0.05). Additionally, results generated using logistic regression analysis show that individuals carrying rs12979860-TT or CT genotypes have a higher likelihood of developing chronic hepatitis upon infection with HCV, when compared to CC carriers, considering rs8099917 genotype as constant. Conclusion Patients with HCV infection have a statistically significant lower prevalence of the favourable rs12979860 genotype when compared to uninfected individuals; therefore we can establish that only IL28B rs12979860-CT and TT genotypes seem to contribute to the occurrence of chronic HCV infection in the cohort of Uruguayan population studied. Considering that a trend towards a higher frequency of “good” response genotypes was observed in responder patients, we believe that IL28B rs12979860 genotyping could be a useful tool for predicting different therapies outcome, including in the DAA era.
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    IL28B Polymorphisms Among Latin American HCV Patients
    (2013) Soza, Alejandro; López Lastra, Marcelo Andrés
    Latin America is a diverse region with more than 500 million people and approximately 8 million HCV infected individuals. The recent description of genetic polymorphisms associated to the interleukin 28B gene that predicts the response to treatment in these patients has impacted the clinical algorithms of management. In this review we examine the studies of prevalence of the IL28B genotypes in the general population and in HCV infected patients in the region and its relationship with treatment response to peginterferon and ribavirin. We show that the prevalence in the different studies in the region show a CC genotype of the rs12979860 between 18 to 35 %. This particular SNP is more consistently associated with treatment response than rs8099917 in Latin America, as well as around the world.
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    Infection of human monocyte-derived dendritic cells by ANDES Hantavirus enhances pro-inflammatory state, the secretion of active MMP-9 and indirectly enhances endothelial permeability
    (2011) Marsac, Delphine; García, Stephanie; Pino, Karla; Ferrés Garrido, Marcela Viviana; López Lastra, Marcelo Andrés; Kalergis Parra, Alexis Mikes; Fournet, Alexandra; Aguirre, Adam; Veas, Francisco
    Abstract Background Andes virus (ANDV), a rodent-borne Hantavirus, is the major etiological agent of Hantavirus cardiopulmonary syndrome (HCPS) in South America, which is mainly characterized by a vascular leakage with high rate of fatal outcomes for infected patients. Currently, neither specific therapy nor vaccines are available against this pathogen. ANDV infects both dendritic and epithelial cells, but in despite that the severity of the disease directly correlates with the viral RNA load, considerable evidence suggests that immune mechanisms rather than direct viral cytopathology are responsible for plasma leakage in HCPS. Here, we assessed the possible effect of soluble factors, induced in viral-activated DCs, on endothelial permeability. Activated immune cells, including DC, secrete gelatinolytic matrix metalloproteases (gMMP-2 and -9) that modulate the vascular permeability for their trafficking. Methods A clinical ANDES isolate was used to infect DC derived from primary PBMC. Maturation and pro-inflammatory phenotypes of ANDES-infected DC were assessed by studying the expression of receptors, cytokines and active gMMP-9, as well as some of their functional status. The ANDES-infected DC supernatants were assessed for their capacity to enhance a monolayer endothelial permeability using primary human vascular endothelial cells (HUVEC). Results Here, we show that in vitro primary DCs infected by a clinical isolate of ANDV shed virus RNA and proteins, suggesting a competent viral replication in these cells. Moreover, this infection induces an enhanced expression of soluble pro-inflammatory factors, including TNF-α and the active gMMP-9, as well as a decreased expression of anti-inflammatory cytokines, such as IL-10 and TGF-β. These viral activated cells are less sensitive to apoptosis. Moreover, supernatants from ANDV-infected DCs were able to indirectly enhance the permeability of a monolayer of primary HUVEC. Conclusions Primary human DCs, that are primarily targeted by hantaviruses can productively be infected by ANDV and subsequently induce direct effects favoring a proinflammatory phenotype of infected DCs. Finally, based on our observations, we hypothesize that soluble factors secreted in ANDV-infected DC supernatants, importantly contribute to the endothelial permeability enhancement that characterize the HCPS.
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    Infectious and non-infectious diseases burden among Haitian immigrants in Chile : a cross-sectional study
    (2020) Fuster F.; Peirano F.; Vargas Domínguez, José Ignacio; Cox, Valentina; López Lastra, Marcelo Andrés; Zamora, F. X.; Núñez, R.; Soza, J.; González, K.; Estay, D.; Barchiesi, B.; Fuster, A.; López, I.; Utrera, N.; Landeros, J.; Chandía, J.; Paredes, A.; Reyes, D.; Arias, R.; Padilla, L.; Suárez, H.; Farcas, K.; Cannistra, M.; Muñoz, G.; Rodríguez, I.; Ormazábal, I.; Cortés, J.; Cornejo, B.; Manzur, F.; Reyes, A.; Leiva, Vicente; Raimann, M. V.; Arrau, C.; Soza, Alejandro
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    Is Single-Strand Conformation Polymorphism Analysis of the Full 5 ' Untranslated Region an Adequate Approach To Study Hepatitis C Virus Quasispecies Distribution?
    (2009) Vera-Otarola, J.; León, Úrsula; Carvallo de Saint Quentin, Pilar; Soza, Alejandro; López Lastra, Marcelo Andrés
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    LOOP IIId of the HCV IRES is essential for the structural rearrangement of the 40S-HCV IRES complex
    (2016) Angulo, J.; Ulryck, N.; Deforges, J.; Chamond, N.; López Lastra, Marcelo Andrés; Masquida, B.; Sargueil, B.
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    Mechanism of HIV-1 Tat RNA translation and its activation by the Tat protein
    (2009) Charnay, Nicolas; López Lastra, Marcelo Andrés; Ivanyi-Nagy, Roland; Soto Rifo, Ricardo; Ohlmann, Théophile; Darlix, Jean-Luc
    Abstract Background The human immunodeficiency virus type 1 (HIV-1) Tat protein is a major viral transactivator required for HIV-1 replication. In the nucleus Tat greatly stimulates the synthesis of full-length transcripts from the HIV-1 promoter by causing efficient transcriptional elongation. Tat induces elongation by directly interacting with the bulge of the transactivation response (TAR) RNA, a hairpin-loop located at the 5'-end of all nascent viral transcripts, and by recruiting cellular transcriptional co-activators. In the cytoplasm, Tat is thought to act as a translational activator of HIV-1 mRNAs. Thus, Tat plays a central role in the regulation of HIV-1 gene expression both at the level of mRNA and protein synthesis. The requirement of Tat in these processes poses an essential question on how sufficient amounts of Tat can be made early on in HIV-1 infected cells to sustain its own synthesis. To address this issue we studied translation of the Tat mRNA in vitro and in human cells using recombinant monocistronic and dicistronic RNAs containing the 5' untranslated region (5'-UTR) of Tat RNA. Results This study shows that the Tat mRNA can be efficiently translated both in vitro and in cells. Furthermore, our data suggest that translation initiation from the Tat mRNA probably occurs by a internal ribosome entry site (IRES) mechanism. Finally, we show that Tat protein can strongly stimulate translation from its cognate mRNA in a TAR dependent fashion. Conclusion These results indicate that Tat mRNA translation is efficient and benefits from a feedback stimulation by the Tat protein. This translational control mechanism would ensure that minute amounts of Tat mRNA are sufficient to generate enough Tat protein required to stimulate HIV-1 replication.
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    Non-canonical translation initiation of the spliced mRNA encoding the human T-cell leukemia virus type 1 basic leucine zipper protein
    (2018) Cáceres, C. Joaquín; Angulo, Jenniffer; Lowy de la Torre, Fernando; Contreras, Nataly; Walters, Beth; Olivares, Eduardo; Allouche, Delphine; Merviel, Anne; Pino, Karla; Sargueil, Bruno; Thompson, Sunnie R.; López Lastra, Marcelo Andrés