RESUMEN

Coxsackievirus-B4 (CV-B4) E2 can persist in the pancreatic ductal-like cells (Panc-1 cell line), which results in an impaired differentiation of these cells into islet-like cell aggregates (ICA). In this study, primary pancreatic ductal cells obtained as a by-product of islet isolation from the pancreas of seven brain-dead adults were inoculated with CV-B4 E2, followed-up for 29 days, and the impact was investigated. Viral titers in culture supernatants were analyzed throughout the culture. Intracellular viral RNA was detected by RT-PCR. Levels of ductal cell marker CK19 mRNA and of insulin mRNA were evaluated by qRT-PCR. The concentration of c-peptide in supernatants was determined by ELISA. Ductal cells exposed to trypsin and serum-free medium formed ICA and resulted in an increased insulin secretion. Ductal cells from five brain-dead donors were severely damaged by CV-B4 E2, whereas the virus persisted in cultures of cells obtained from the other two. The ICAs whose formation was induced on day 14 post-inoculation were scarce and appeared tiny in infected cultures. Also, insulin mRNA expression and c-peptide levels were strongly reduced compared to the controls. In conclusion, CV-B4 E2 lysed human primary pancreatic ductal cells or persisted in these cells, which resulted in the impairment of differentiation into insulin-producing cells.

Asunto(s)

Infecciones por Coxsackievirus/virología , Enterovirus Humano B/fisiología , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Conductos Pancreáticos/virología , Diferenciación Celular , Células Cultivadas , Infecciones por Coxsackievirus/metabolismo , Infecciones por Coxsackievirus/fisiopatología , Enterovirus Humano B/genética , Células Epiteliales , Humanos , Células Secretoras de Insulina/citología

RESUMEN

INTRODUCTION: Although known as cytolytic viruses, group B coxackieviruses (CVB) are able to establish a persistent infection in vitro and in vivo. Viral persistence has been reported as a key mechanism in the pathogenesis of CVB-associated chronic diseases such as type 1 diabetes (T1D). The impact of CVB4 persistence on human pancreas ductal-like cells was investigated. METHODS: A persistent CVB4 infection was established in ductal-like cells. PDX-1 expression, resistance to CVB4-induced lysis and CAR expression were evaluated. The profile of cellular microRNAs (miRNAs) was investigated through miRNA-sequencing. Viral phenotypic changes were examined, and genomic modifications were assessed by sequencing of the viral genome. RESULTS: The CVB4 persistence in ductal-like cells was productive, with continuous release of infectious particles. Persistently infected cells displayed a resistance to CVB4-induced lysis upon superinfection and expression of PDX-1 and CAR was decreased. These changes were maintained even after virus clearance. The patterns of cellular miRNA expression in mock-infected and in CVB4-persistently infected ductal-like cells were clearly different. The persistent infection-derived virus (PIDV) was still able to induce cytopathic effect but its plaques were smaller than the parental virus. Several mutations appeared in various PIDV genome regions, but amino acid substitutions did not affect the predicted site of interaction with CAR. CONCLUSION: Cellular and viral changes occur during persistent infection of human pancreas ductal-like cells with CVB4. The persistence of cellular changes even after virus clearance supports the hypothesis of a long-lasting impact of persistent CVB infection on the cells.

Asunto(s)

Infecciones por Coxsackievirus/virología , Enterovirus Humano B/fisiología , Conductos Pancreáticos/citología , Conductos Pancreáticos/virología , Línea Celular Tumoral , Infecciones por Coxsackievirus/genética , Infecciones por Coxsackievirus/metabolismo , Enterovirus Humano B/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Páncreas/metabolismo , Páncreas/virología , Transactivadores/genética , Transactivadores/metabolismo , Replicación Viral

RESUMEN

Group B Coxsackieviruses (CVB) are involved in various acute clinical features and they can play a role in the development of chronic diseases like type 1 diabetes. The persistence of CVB has been described in vitro and in vivo in various models. Fluoxetine was reported to inhibit the replication of CVB1-3, which prompted us to study the in vitro antiviral activity of fluoxetine against CVB4 in models of acute infection. In addition we took advantage of a chronically CVB4-infected Panc-1 cell line to evaluate the antiviral effect of fluoxetine in a model of persistent CVB4 infection. An inhibition of the CVB4 replication was obtained when fluoxetine was added at 5.48µM to Hep-2 cell cultures. No inhibitory effect was observed when CVB4 was mixed with fluoxetine for 2h and filtered to eliminate fluoxetine before inoculation to cells, or when cells were treated up to 96h and washed before viral inoculation. Fluoxetine (5.48µM) reduced viral replication by more than 50% in acutely infected Panc-1 cell cultures. A dramatic decrease of infectious particles levels in supernatants of Panc-1 cells chronically infected with CVB4 was obtained a few days after treatment with fluoxetine and no infectious viral particle was found as soon as day 21 of treatment, and intracellular enteroviral RNA was undetectable by RT-PCR after three weeks of treatment. These data display that fluoxetine can inhibit the replication of CVB4 and can cure Panc-1 cells chronically infected with CVB4.

Asunto(s)

Antivirales/farmacología , Enterovirus Humano B/efectos de los fármacos , Fluoxetina/farmacología , Conductos Pancreáticos/virología , Replicación Viral/efectos de los fármacos , Línea Celular Tumoral , Enterovirus Humano B/fisiología , Humanos , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

The role of enteroviruses, especially Coxsackievirus B (CVB), in type 1 diabetes is suspected, but the mechanisms of the virus-induced or aggravated pathogenesis of the disease are unknown. The hypothesis of an enterovirus-induced disturbance of pancreatic ß-cells regeneration has been investigated in the human system. The infection of human pancreas ductal cells and pancreatic duct cell line, PANC-1, with CVB4E2 has been studied. Primary ductal cells and PANC-1 cells were infectable with CVB4E2 and a RT-PCR assay without extraction displayed that a larger proportion of cells harbored viral RNA than predicted by the detection of the viral capsid protein VP1 by indirect immunofluorescence. The detection of intracellular positive- and negative-strands of enterovirus genomes in cellular extracts by RT-PCR and the presence of infectious particles in supernatant fluids during the 37 weeks of monitoring demonstrated that CVB4E2 could persist in the pancreatic duct cell line. A persistent infection of these cells resulted in an impaired expression of Pdx1, a transcription factor required for the formation of endocrine pancreas, and a disturbed formation of islet-like cell aggregates of which the viability was decreased. These data support the hypothesis of an impact of enteroviruses onto pancreatic ductal cells which are involved in the renewal of pancreatic ß-cells.

Asunto(s)

Enterovirus Humano B , Proteínas de Homeodominio/metabolismo , Células Secretoras de Insulina/citología , Conductos Pancreáticos/citología , Conductos Pancreáticos/virología , Transactivadores/metabolismo , Diferenciación Celular , Línea Celular , Proliferación Celular , Cartilla de ADN , Diabetes Mellitus Tipo 2/metabolismo , Regulación Viral de la Expresión Génica , Humanos , Microscopía Fluorescente , Conductos Pancreáticos/metabolismo , ARN Viral/metabolismo , Factores de Tiempo

RESUMEN

CONTEXT: Pancreatic cancer is highly resistant to treatment. Previously, we showed that Newcastle disease virus (NDV) strain 73-T was highly cytotoxic to a range of tumor types in vitro and in vivo but the effects of NDV on pancreatic tumors are unknown. We determined the cytotoxicity of the lentogenic LaSota strain of NDV (NDV-LS) toward 7 different human pancreatic tumor cell lines and 4 normal human cell lines (keratinocytes, fibroblasts, pancreatic ductal cells, and vascular endothelial cells). METHODS: Cytotoxicity assays used serially diluted NDV incubated for 96 hours post-infection. Cells were fixed, stained, and minimum cytotoxic plaque forming unit (PFU) doses were determined (n = 10-24/cell line). RESULTS: Normal cells were killed only by high doses of NDV-LS. The cytotoxic doses for pancreatic ductal cells, fibroblasts, and vascular endothelial cells were 729, 626, and 1,217 plaque forming units, respectively. In contrast, most pancreatic cancer cells were killed by much lower doses. The doses for PL45, Panc 10.05, PANC-1, BxPC3, SU.86.86, Capan-1 and CFPAC-1 were 0.15, 0.41, 0.43, 0.55, 1.30, 17.1 and 153 plaque forming units, respectively. CONCLUSIONS: Most pancreatic tumor cells were more than 700 times more sensitive to NDV-LS killing than normal cells. Such avirulent, lentogenic NDV strains may have therapeutic potential in the treatment of pancreatic cancers.

Asunto(s)

Fibroblastos/virología , Células Endoteliales de la Vena Umbilical Humana/virología , Queratinocitos/virología , Virus de la Enfermedad de Newcastle/fisiología , Conductos Pancreáticos/virología , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/fisiología , Células Cultivadas , Fibroblastos/citología , Interacciones Huésped-Patógeno , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Queratinocitos/citología , Virus de la Enfermedad de Newcastle/clasificación , Virus Oncolíticos/fisiología , Conductos Pancreáticos/citología , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/virología

RESUMEN

Genetic analysis of pancreatic ductal adenocarcinomas and their putative precursor lesions, pancreatic intraepithelial neoplasias (PanIN), has shown a multistep molecular paradigm for duct cell carcinogenesis. Mutational activation or inactivation of the K-ras, p16(INK4A), Smad4, and p53 genes occur at progressive and high frequencies in these lesions. Oncogenic activation of the K-ras gene occurs in >90% of pancreatic ductal carcinoma and is found early in the PanIN-carcinoma sequence, but its functional roles remain poorly understood. We show here that the expression of K-ras(G12V) oncogene in a near diploid HPV16-E6E7 gene immortalized human pancreatic duct epithelial cell line originally derived from normal pancreas induced the formation of carcinoma in 50% of severe combined immunodeficient mice implanted with these cells. A tumor cell line established from one of these tumors formed ductal cancer when implanted orthotopically. These cells also showed increased activation of the mitogen-activated protein kinase, AKT, and nuclear factor-kappaB pathways. Microarray expression profiling studies identified 584 genes whose expression seemed specifically up-regulated by the K-ras oncogene expression. Forty-two of these genes have been reported previously as differentially overexpressed in pancreatic cancer cell lines or primary tumors. Real-time PCR confirmed the overexpression of a large number of these genes. Immunohistochemistry done on tissue microarrays constructed from PanIN and pancreatic cancer samples showed laminin beta3 overexpression starting in high-grade PanINs and occurring in >90% of pancreatic ductal carcinoma. The in vitro modeling of human pancreatic duct epithelial cell transformation may provide mechanistic insights on gene expression changes that occur during multistage pancreatic duct cell carcinogenesis.

Asunto(s)

Carcinoma Ductal Pancreático/genética , Transformación Celular Viral/genética , Regulación Neoplásica de la Expresión Génica , Genes ras/genética , Conductos Pancreáticos/fisiología , Neoplasias Pancreáticas/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/virología , Procesos de Crecimiento Celular/fisiología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Células Epiteliales/virología , Humanos , Inmunohistoquímica , Laminina/biosíntesis , Laminina/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Oncogénicas Virales/fisiología , Conductos Pancreáticos/citología , Conductos Pancreáticos/metabolismo , Conductos Pancreáticos/virología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/virología , Papillomaviridae/fisiología , Proteínas E7 de Papillomavirus , Proteínas Represoras/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta1 , Proteínas ras/biosíntesis , Proteínas ras/genética

RESUMEN

The effects of a diet enriched with 25% raw soya bean flour (RSF) on the pancreas and on the avian retrovirus Pts 56-induced pancreatic carcinogenesis in guinea fowl were studied. It has been shown that prolonged RSF feeding of new-hatched virus-infected and uninfected guinea fowl-poults induced enlargement of the pancreas, which was less pronounced when administration of the RSF supplemented diet started at the age of 75 days. Time-dependent multifocal inter- and intralobular hyperplasia of pleomorphic ducts lined by mucin-producing epithelium in the exocrine pancreas of virus-infected guinea fowls fed a RSF supplemented diet was regularly observed. Enlargement of virus-induced ductular neoplasms has been shown only after simultaneous RSF and virus administration.

Asunto(s)

Cocarcinogénesis , Harina/efectos adversos , Glycine max , Neoplasias Pancreáticas/etiología , Infecciones por Retroviridae/complicaciones , Retroviridae , Infecciones Tumorales por Virus/complicaciones , Animales , Animales Recién Nacidos , Peso Corporal , Pruebas de Carcinogenicidad , Femenino , Hiperplasia/etiología , Hiperplasia/virología , Masculino , Páncreas/patología , Conductos Pancreáticos/patología , Conductos Pancreáticos/virología , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/virología , Aves de Corral , Distribución Aleatoria

RESUMEN

OBJECTIVE: To determine if a viable cadaveric pancreas might be used to study viral transfection efficacy in a manner precisely mimicking in vivo human studies. DESIGN: Ex vivo gene transfer to an intact human pancreatic duct. SETTING: Molecular biology laboratory and organ procurement center. INTERVENTION: The recombinant adenoviral vector that contains the Escherichia coli beta-galactosidase (LacZ) gene driven by the human cytomegalovirus promoter, ie, AdCMVLacZ, was used to transfect the epithelial cells of the pancreatic ductal system. A human pancreas (150 g wt/wt) procured for transplantation, but subsequently found unsuitable, was used for the study. The splenic, superior mesenteric arteries and portal vein were cannulated and perfused in a heat-controlled organ procurement perfusion system. A segment of vascularized, perfused distal pancreatic duct was isolated with a balloon occlusion catheter. The recombinant adenoviral vector AdCMVLacZ was introduced into the lumen of the distal segment of the pancreatic duct and incubated for 6 hours at 25 degrees C. The proximal segment of the pancreatic duct was not exposed to the vector and served as control tissue. Tissue was harvested and processed for evaluation of beta-galactosidase activity. RESULTS: Adenoviral vector-infected pancreatic ducts exhibited intense blue staining, indicative of reporter gene expression in the epithelial cells of the pancreatic duct. The phenotype of these cells was confirmed by immunohistochemical studies using anti-annexin III polyclonal antibody. Control tissue not exposed to the adenoviral vector was subjected to an identical analysis and did not reveal evidence of expression of the reporter gene. CONCLUSIONS: This study demonstrates the first successful transfection of epithelial cells of the pancreatic duct from normal human pancreas with a recombinant adenovirus. This system will provide not only information on the efficacy of transfection but also a novel gene therapeutic approach to target pancreatic ductal adenocarcinoma.

Asunto(s)

Adenovirus Humanos/genética , Vectores Genéticos/genética , Conductos Pancreáticos/virología , Cadáver , Epitelio/virología , Escherichia coli/genética , Técnicas de Transferencia de Gen , Genes Bacterianos , Genes Reporteros/genética , Genes Virales/genética , Humanos , Operón Lac , Conductos Pancreáticos/citología , Perfusión/métodos , Coloración y Etiquetado/métodos , Transfección/genética , Transfección/métodos

RESUMEN

Pancreatic cancer is one of the most lethal cancers in humans. The majority of these cancers arise from the pancreatic duct epithelium. Research into the pathogenesis of pancreatic carcinoma has largely relied on animal models. In vitro models of pancreatic carcinogenesis using propagable cultured epithelial cells derived from the pancreatic ducts of rats and hamsters have been described. A human model, however, has been nonexistent due to the unavailability of propagable cultured duct epithelial cells derived from normal human pancreas. We report here a reproducible method for the long-term culture of pancreatic duct epithelial cells derived from normal and benign adult human pancreata by infection with a retrovirus containing the E6 and E7 genes of the human papilloma virus 16. One of these cell lines has become immortal and has propagated continuously for more than 20 passages. They remain anchorage dependent in their growth and nontumorigenic in nude mice. These cell lines and the methodology described here to establish them may provide new avenues for in vitro studies of the roles played by duct epithelium in human pancreatic diseases and cancers.

Asunto(s)

Línea Celular Transformada , Genes Virales , Proteínas Oncogénicas Virales/genética , Conductos Pancreáticos/citología , Papillomaviridae/genética , Proteínas Represoras , Anciano , Animales , Secuencia de Bases , División Celular , Células Epiteliales , Femenino , Genes ras/genética , Humanos , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Conductos Pancreáticos/virología , Proteínas E7 de Papillomavirus , Retroviridae/genética , Retroviridae/fisiología , Transfección