Pancreatic Cancer Heterogeneity Can Be Explained Beyond the Genome

Pancreatic ductal adenocarcinoma (PDAC) remains a major health problem because it induces almost systematic mortality. Carcinogenesis begins with genetic aberrations which trigger epigenetic modifications. While genetic mutations initiate tumorigenesis, they are unable to explain the vast heterogeneity observed among PDAC patients. Instead, epigenetic changes drive transcriptomic alterations that can regulate the malignant phenotype. The contribution of factors from the environment and tumor microenvironment defines different epigenetic landscapes that outline two clinical subtypes: basal, with the worst prognosis, and classical. The epigenetic nature of PDAC, as a reversible phenomenon, encouraged several studies to test epidrugs. However, these drugs lack specificity and although there are epigenetic patterns shared by all PDAC tumors, there are others that are specific to each subtype. Molecular characterization of the epigenetic mechanisms underlying PDAC heterogeneity could be an invaluable tool to predict personalized therapies, stratify patients and search for novel therapies with more specific phenotype-based targets. Novel therapeutic strategies using current anticancer compounds or existing drugs used in other pathologies, alone or in combination, could be used to kill tumor cells or convert aggressive tumors into a more benign phenotype

Chagas' disease in Aboriginal and Creole communities from the Gran Chaco Region of Argentina: Seroprevalence and molecular parasitological characterization

Most indigenous ethnias from Northern Argentina live in rural areas of "the Gran Chaco" region, where Trypanosoma cruzi is endemic. Serological and parasitological features have been poorly characterized in Aboriginal populations and scarce information exist regarding relevant T. cruzi discrete typing units (DTU) and parasitic loads. This study was focused to characterize T. cruzi infection in Qom, Mocoit, Pit'laxá and Wichi ethnias (N = 604) and Creole communities (N = 257) inhabiting rural villages from two highly endemic provinces of the Argentinean Gran Chaco.DNA extracted using Hexadecyltrimethyl Ammonium Bromide reagent from peripheral blood samples was used for conventional PCR targeted to parasite kinetoplastid DNA (kDNA) and identification of DTUs using nuclear genomic markers. In kDNA-PCR positive samples from three rural Aboriginal communities of "Monte Impenetrable Chaqueño", minicircle signatures were characterized by Low stringency single primer-PCR and parasitic loads calculated using Real-Time PCR.Seroprevalence was higher in Aboriginal (47.98%) than in Creole (27.23%) rural communities (Chi square, p = 4.e-8). A low seroprevalence (4.3%) was detected in a Qom settlement at the suburbs of Resistencia city (Fisher Exact test, p = 2.e-21).The kDNA-PCR positivity was 42.15% in Aboriginal communities and 65.71% in Creole populations (Chi square, p = 5.e-4). Among Aboriginal communities kDNA-PCR positivity was heterogeneous (Chi square, p = 1.e-4). Highest kDNA-PCR positivity (79%) was detected in the Qom community of Colonia Aborigen and the lowest PCR positivity in two different surveys at the Wichi community of Misión Nueva Pompeya (33.3% in 2010 and 20.8% in 2014).TcV (or TcII/V/VI) was predominant in both Aboriginal and Creole communities, in agreement with DTU distribution reported for the region. Besides, two subjects were infected with TcVI, one with TcI and four presented mixed infections of TcV plus TcII/VI. Most minicircle signatures clustered according to their original localities, but in a few cases, signatures from one locality clustered with signatures from other village, suggesting circulation of the same strains in the area. Parasitic loads ranged from undetectable to around 50 parasite equivalents/mL, showing higher values than those generally observed in chronic Chagas disease patients living in urban centers of Argentina. Our findings reveal the persistence of high levels of infection in these neglected populations.Fil: Lucero, R. H.. Universidad Nacional del Nordeste; ArgentinaFil: Brusés, B. L.. Universidad Nacional del Nordeste; ArgentinaFil: Cura, Carolina Inés. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Formichelli, L. B.. Universidad Nacional del Nordeste; ArgentinaFil: Juiz, Natalia Anahí. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Fernández, G. J.. Universidad Nacional del Nordeste; ArgentinaFil: Bisio, Margarita María Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires; ArgentinaFil: Deluca, Gerardo Daniel. Universidad Nacional del Nordeste; ArgentinaFil: Besuschio, Susana Alicia. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Hernández, D. O.. Universidad Nacional del Nordeste; ArgentinaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires; Argentin

Usefulness of Serial Blood Sampling and PCR Replicates for Treatment Monitoring of Patients with Chronic Chagas Disease

This work evaluated a serial blood sampling procedure to enhance the sensitivity of duplex real-time quantitative PCR (qPCR) for baseline detection and quantification of parasitic loads and posttreatment identification of failure in the context of clinical trials for treatment of chronic Chagas disease, namely, DNDi-CHE1224-001 (ClinicalTrials.gov registration no. NCT01489228) and the MSF-DNDi PCR Sampling Optimization Study (NCT01678599). Patients from Cochabamba (n 294), Tarija (n 257), and Aiquile (n 220) were enrolled. Three serial blood samples were collected at each time point, and qPCR triplicates were tested for each sample. The first two samples were collected during the same day and the third one 7 days later. A patient was considered PCR positive if at least one qPCR replicate was detectable. Cumulative results of multiple samples and qPCR replicates enhanced the proportion of pretreatment sample positivity from 54.8% to 76.2%, 59.5% to 77.8%, and 73.5% to 90.2% in Cochabamba, Tarija, and Aiquile cohorts, respectively. This strategy increased the detection of treatment failure from 72.9% to 91.7%, 77.8% to 88.9%, and 42.9% to 69.1% for E1224 low-, short-, and high-dosage regimens, respectively, and from 4.6% to 15.9% and 9.5% to 32.1% for the benznidazole arm in the DNDi-CH-E1224-001 and MSF-DNDi studies, respectively. The addition of the third blood sample and third qPCR replicate in patients with nondetectable PCR results in the first two samples gave a small, non-statistically significant improvement in qPCR positivity. No change in clinical sensitivity was seen with a blood volume increase from 5 to 10 ml. The monitoring of patients treated with placebo in the DNDi-CH-E1224-001 trial revealed fluctuations in parasitic loads and occasionally nondetectable results. In conclusion, a serial sampling strategy enhanced PCR sensitivity to detecting treatment failure during follow-up and has the potential for improving recruitment capacity in Chagas disease trials, which require an initial positive qPCR result for patient admission

Identification of Trypanosoma cruzi Discrete Typing Units (DTUs) in Latin-American migrants in Barcelona (Spain)

Trypanosoma cruzi, the causative agent of Chagas disease, is divided into six Discrete Typing Units (DTUs): TcI-TcVI. We aimed to identify T. cruzi DTUs in Latin-American migrants in the Barcelona area (Spain) and to assess different molecular typing approaches for the characterization of T. cruzi genotypes. Seventy-five peripheral blood samples were analyzed by two real-time PCR methods (qPCR) based on satellite DNA (SatDNA) and kinetoplastid DNA (kDNA). The 20 samples testing positive in both methods, all belonging to Bolivian individuals, were submitted to DTU characterization using two PCR-based flowcharts: multiplex qPCR using TaqMan probes (MTq-PCR), and conventional PCR. These samples were also studied by sequencing the SatDNA and classified as type I (TcI/III), type II (TclI/IV) and type I/II hybrid (TcV/VI). Ten out of the 20 samples gave positive results in the flowcharts: TcV (5 samples), TcII/V/VI (3) and mixed infections by TcV plus TclI (1) and TcV plus Tcll/VI (1). By SatDNA sequencing, we classified the 20 samples, 19 as type I/II and one as type I. The most frequent DTU identified by both flowcharts, and suggested by SatDNA sequencing in the remaining samples with low parasitic loads, TcV, is common in Bolivia and predominant in peripheral blood. The mixed infection by TcV-TclI was detected for the first time simultaneously in Bolivian migrants. PCR-based flowcharts are very useful to characterize DTUs during acute infection. SatDNA sequence analysis cannot discriminate T. cruzi populations at the level of a single DTU but it enabled us to increase the number of characterized cases in chronically infected patients. (C) 2016 Elsevier Ireland Ltd. All rights reserved

Identification of Trypanosoma cruzi Discrete Typing Units (DTUs) in Latin-American migrants in Barcelona (Spain)

Trypanosoma cruzi, the causative agent of Chagas disease, is divided into six Discrete Typing Units (DTUs): TcI-TcVI. We aimed to identify T. cruzi DTUs in Latin-American migrants in the Barcelona area (Spain) and to assess different molecular typing approaches for the characterization of T. cruzi genotypes. Seventy-five peripheral blood samples were analyzed by two real-time PCR methods (qPCR) based on satellite DNA (SatDNA) and kinetoplastid DNA (kDNA). The 20 samples testing positive in both methods, all belonging to Bolivian individuals, were submitted to DTU characterization using two PCR-based flowcharts: multiplex qPCR using TaqMan probes (MTq-PCR), and conventional PCR. These samples were also studied by sequencing the SatDNA and classified as type I (TcI/III), type II (TcII/IV) and type I/II hybrid (TcV/VI). Ten out of the 20 samples gave positive results in the flowcharts: TcV (5 samples), TcII/V/VI (3) and mixed infections by TcV plus TcII (1) and TcV plus TcII/VI (1). By SatDNA sequencing, we classified the 20 samples, 19 as type I/II and one as type I. The most frequent DTU identified by both flowcharts, and suggested by SatDNA sequencing in the remaining samples with low parasitic loads, TcV, is common in Bolivia and predominant in peripheral blood. The mixed infection by TcV-TcII was detected for the first time simultaneously in Bolivian migrants. PCR-based flowcharts are very useful to characterize DTUs during acute infection. SatDNA sequence analysis cannot discriminate T. cruzi populations at the level of a single DTU but it enabled us to increase the number of characterized cases in chronically infected patients

Analytical Performance of a Multiplex Real-Time PCR Assay Using TaqMan Probes for Quantification of Trypanosoma cruzi Satellite DNA in Blood Samples

Background: The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. Methods/Principal Findings: We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CLBrener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. Conclusions/Significance: The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment.Fil: Duffy, Tomas. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones En Ingeniería Genética y Biología Molecular; ArgentinaFil: Cura, Carolina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Ramírez, Juan C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Abate, Teresa. Universidad Central de Venezuela. Instituto de Medicina Tropical; VenezuelaFil: Cayo, Nelly M.. Universidad Nacional de Jujuy. Instituto de Biologia de la Altura; ArgentinaFil: Parrado, Rudy. Universidad San Simón; BoliviaFil: Diaz Bello, Zoraida. Universidad Central de Venezuela. Instituto de Medicina Tropical; VenezuelaFil: Velazquez, Elsa Beatriz. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; ArgentinaFil: Muñoz Calderón, Arturo. Universidad Central de Venezuela. Instituto de Medicina Tropical; VenezuelaFil: Juiz, Natalia Anahí. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Basile, Joaquín. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Garcia, Lineth. Universidad San Simón; BoliviaFil: Riarte, Adelina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; ArgentinaFil: Nasser, Julio Rubén. Universidad Nacional de Salta. Facultad de Ciencias Naturales; ArgentinaFil: Ocampo, Susana B.. Universidad Nacional de Jujuy. Instituto de Biologia de la Altura; ArgentinaFil: Yadon, Zaida E.. Pan-American Health Organization; Estados UnidosFil: Torrico, Faustino. Universidad San Simón; BoliviaFil: Alarcón de Noya, Belkisyole. Universidad Central de Venezuela. Instituto de Medicina Tropical; VenezuelaFil: Ribeiro, Isabela. Drugs and Neglected Diseases Initiative; SuizaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentin

Host genetic factors associated with Chagas disease: vertical transmission and chronic chagasic cardiopathy

La enfermedad de Chagas es producto de una compleja interacción entre factoresambientales, del parásito Trypanosoma cruzi y del hospedero. En el marco de esta Tesisnos hemos enfocado en describir el rol que estos últimos juegan en dos escenarios dealto impacto en salud pública como son la transmisión congénita y la cardiopatíachagásica crónica. La transmisión congénita es parcialmente responsable de la urbanización del Chagas, que puede perpetuarse en sucesivas generaciones. Aún no han sido esclarecidoslos mecanismos que la placenta ofrece en defensa contra la infección, así como tampocopor qué sólo una proporción de niños nacidos de madres infectadas adquieren Chagascongénito. Con el objetivo de describir la respuesta genética del microambiente placentarioproducido frente a la infección, hemos realizado un estudio de genómica funcional através de un microarreglo en modelo murino C57BL/6J, comparando placentas deanimales no infectados e infectados por dos cepas de T. cruzi: K98, no letal y miotrópica y VD, aislada de un caso humano de infección congénita. Nuestro análisis de redesbiológicas sugiere que el microambiente placentario ejerce una fuerte respuesta inmuneen detrimento del metabolismo celular modulada por la cepa parasitaria. Más aún, lasdiferencias encontradas entre ambas cepas podrían ser el resultado de un tropismoplacentario observado en VD. Por otra parte, en el marco de esta Tesis se han evaluado polimorfismos denucleótido simples (SNPs) en genes que codifican para enzimas de expresión placentariacomo marcadores genéticos de susceptibilidad a esta transmisión. Para analizar dichaasociación hemos realizado un estudio del tipo caso-control comparando dos grupos deniños con y sin infección congénita nacidos de madres seropositivas. Para lagenotipificación de las muestras de ADN humano, hemos diseñado técnicas de análisis decurvas de disociación de alta resolución para análisis de SNPs y de electroforesis capilarpara análisis de microsatélites. Los resultados obtenidos del estudio de 11 sitios sugierenun papel importante en la infección congénita por parte de los polimorfismos en los genes de las proteínas MMP2 y ADAM12, implicadas en procesos de remodelación de lamatriz extracelular y en la respuesta inmune. El último objetivo de este trabajo ha sido evaluar polimorfismos en los genes CCR2y CCR5 como posibles marcadores genéticos de susceptibilidad a desarrollar lacardiopatía chagásica crónica en poblaciones originarias de Argentina. En particular,hemos encontrado que individuos de la etnia wichi, con menor prevalencia de problemascardíacos, no presentan una de las mutaciones en CCR5, que es factor de riesgo enpoblaciones no-wichi.Chagas disease is caused by a complex interaction of environmental factors, Trypanosoma cruzi parasite and host. As part of this Thesis we have focused ondescribing the role that host factors play in two scenarios of high public health impact ascongenital transmission and chronic Chagas cardiomyopathy. Congenital transmission is partially responsible for the urbanization of Chagas,which can remain in the following generations. Until now, it has not been elucidated themechanisms that the placenta provides as defense against infection, nor why only aproportion of children born to infected mothers acquire congenital Chagas. In order to describe the genetic response of the placental environment producedduring infection, we performed a study of functional genomics using a microarrayapproach in a C57BL/6J murine model, comparing placentas from uninfected andinfected animals by two strains of T. cruzi: K98, a non-lethal myotropic strain and VD,isolated from a human case of congenital infection. The analysis of biological networkssuggests that the placenta environment exerts a strong immune response to thedetriment of cell metabolism modulated by the parasitic strain. Moreover, thedifferences between the two strains could be the result of a stronger placental tropism inthe VD strain. Moreover, in the framework of this Thesis we have evaluated single nucleotidepolymorphisms (SNPs) in genes coding for enzymes of placental expression as geneticmarkers of susceptibility to vertical transmission. To analyze the association weperformed a case-control study comparing two groups of children with and withoutcongenital infection born to seropositive mothers. In order to genotype human DNAsamples, we have designed techniques of high resolution melting analyses for SNPsdiscrimination and capillary electrophoresis for analysis of microsatellites. The results ofthe 11 loci studied suggest an important role in congenital infection by human polymorphisms in genes coding for MMP2 and ADAM12 proteins, involved in extracellularmatrix remodeling processes and in immune response. The last aim of this research was to evaluate polymorphisms in CCR2 and CCR5genes as potential genetic markers of susceptibility to develop chronic Chagascardiomyopathy in populations from Argentina. In particular, we have found thatindividuals belonging to the wichi ethnia, with lower prevalence of heart problems, donot exhibit one of the mutations in CCR5, a risk factor in non-wichi populations.Fil: Juiz, Natalia Anahí. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Host-parasite interaction: changes in human placental gene expression induced by Trypanosoma cruzi

Abstract Background Chagas disease is caused by Trypanosoma cruzi, a parasite endemic to Latin America. Most infections occur in children by vector or congenital transmission. Trypanosoma cruzi establishes a complexity of specific molecular parasite-host cell interactions to invade the host. However, most studies have been mainly focused on the interaction between the parasite and different cell types, but not on the infection and invasion on a tissue level. During congenital transmission, T. cruzi must cross the placental barrier, composed of epithelial and connective tissues, in order to infect the developing fetus. Here we aimed to study the global changes of transcriptome in the placental tissue after a T. cruzi challenge. Results Strong changes in gene expression profiling were found in the different experimental conditions, involving the reprogramming of gene expression in genes involved in the innate immune response. Conclusions Trypanosoma cruzi induces strong changes in genes involved in a wide range of pathways, especially those involved in immune response against infections

Validación analítica de métodos cuantitativos de PCR en tiempo real para la cuantificación del ADN de Trypanosoma cruzi en muestras de sangre de pacientes con enfermedad de Chagas

An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control.