Protocol for Efficient Generation of Chimeric Antigen Receptor T Cells with Multiplexed Gene Silencing by Epigenome Editing.

Multiplex gene regulation enables the controlled and simultaneous alteration of the expression levels of multiple genes and is generally pursued to precisely alter complex cellular pathways with a single intervention. Thus far, this has been typically exploited in combination with genome editing tools (i.e., base-/prime-editing, designer nucleases) to enable simultaneous genetic alterations and modulate complex physiologic cellular pathways. In the field of cancer immunotherapy, multiplex genome editing has been used to simultaneously inactivate three genes (i.e., TRAC, B2M, and PDCD1) and generate universal chimeric antigen receptor (CAR) T cells resistant to the inhibitory activity of the PD-1 ligand. However, the intrinsic risk of genomic aberrations driven by such tools poses concerns because of the generation of multiple single-strand or double-strand DNA breaks followed by DNA repair. Modulating gene expression without DNA damage using epigenome editing promises a safer and efficient approach to alter gene expression. This method enables for simultaneous activation and/or repression of target genes, offering superior fine-tuning capabilities with reduced off-targeting effects and potential reversibility as compared to genome editing. Here we describe a detailed protocol for achieving multiplexed and sustainable gene silencing in CAR T cells. In an exemplary approach, we use designer epigenome modifiers (DEMs) for the simultaneous inactivation of two T cell inhibitory genes, PDCD1 and LAG3 to generate CAR T cells with increased resistance to tumor-induced exhaustion.

Connective tissue growth factor autocriny in human hepatocellular carcinoma: oncogenic role and regulation by epidermal growth factor receptor/yes-associated protein-mediated activation.

UNLABELLED: The identification of molecular mechanisms involved in the maintenance of the transformed phenotype of hepatocellular carcinoma (HCC) cells is essential for the elucidation of therapeutic strategies. Here, we show that human HCC cells display an autocrine loop mediated by connective tissue growth factor (CTGF) that promotes DNA synthesis and cell survival. Expression of CTGF was stimulated by epidermal growth factor receptor (EGFR) ligands and was dependent on the expression of the transcriptional coactivator, Yes-associated protein (YAP). We identified elements in the CTGF gene proximal promoter that bound YAP-enclosing complexes and were responsible for basal and EGFR-stimulated CTGF expression. We also demonstrate that YAP expression can be up-regulated through EGFR activation not only in HCC cells, but also in primary human hepatocytes. CTGF contributed to HCC cell dedifferentiation, expression of inflammation-related genes involved in carcinogenesis, resistance toward doxorubicin, and in vivo HCC cell growth. Importantly, CTGF down-regulated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2 expression and was involved in the reduced sensitivity of these cells toward TRAIL-mediated apoptosis. CONCLUSION: We have identified autocrine CTGF as a novel determinant of HCC cells' neoplastic behavior. Expression of CTGF can be stimulated through the EGFR-signaling system in HCC cells in a novel cross-talk with the oncoprotein YAP. Moreover, to our knowledge, this is the first study that identifies a signaling mechanism triggering YAP gene expression in healthy and transformed liver parenchymal cells.

NADPH Oxidase 5 (NOX5) Overexpression Promotes Endothelial Dysfunction via Cell Apoptosis, Migration, and Metabolic Alterations in Human Brain Microvascular Endothelial Cells (hCMEC/D3).

NADPH oxidases (NOX) constitute the main reactive oxygen species (ROS) source in blood vessels. An oxidative stress situation due to ROS overproduction can lead into endothelial dysfunction, a molecular mechanism that precedes cardiovascular diseases (CVDs) such as atherosclerosis, myocardial infarction, and stroke. NOX5 is the last discovered member of the NOX family, studied in a lesser extent due to its absence in the rodent genome. Our objective was to describe the phenotypic alterations produced by an oxidative stress situation derived from NOX5 overexpression in an endothelial in vitro model. The in vitro model consists of the hCMEC/D3 cell line, derived from brain microvascular endothelium, infected with a recombinant NOX5-ß adenovirus. After an initial proteomic analysis, three phenotypic alterations detected in silico were studied: cell proliferation and apoptosis, general and mitochondrial metabolism, and migration capacity. NOX5 infection of hCMEC/D3 generates a functional protein and an increase in ROS production. This model produced changes in the whole cell proteome. The in silico analysis together with in vitro validations demonstrated that NOX5 overexpression inhibits proliferation and promotes apoptosis, metabolic alterations and cell migration in hCMEC/D3 cells. NOX5 overexpression in endothelial cells leads to phenotypic changes that can lead to endothelial dysfunction, the onset of atherosclerosis, myocardial infarction, and stroke.

A versatile reporter system for multiplexed screening of effective epigenome editors.

The formation and function of highly specialized cells and tissues in a multicellular organism from a single genome are enabled through differential spatiotemporal access to the information contained in the genomic DNA. The epigenome plays an essential role in how DNA information can be accessed, and in the last decade the link between epigenetic aberrations and pathologies has become increasingly clear. Methods to precisely modify the epigenome are hence attracting interest as potential novel therapeutics. We recently described a platform, designer epigenome modifier (DEM), capable of precisely editing the epigenome of a cell to control the expression of selected genes. Here, we provide a detailed protocol to streamline the process of identifying DEMs that efficiently and selectively bind to their intended target site and inactivate expression of the target gene. Further, we describe the procedure to simultaneously regulate the expression of up to three genes in a multiplexed fashion. The protocol is divided into four stages that guide the user through the generation of the multicolor reporter cell line and its use for selecting functional DEMs. The duration of the whole procedure described varies from ~6 weeks when using a single reporter up to 13 weeks for fine-tuning the multiplex epigenome editing abilities of selected DEMs using three reporters. Given the great interest in epigenome editing in various fields of biomedical research, this protocol will help scientists to explore these novel technologies for their research.

Characterization of cisplatin cytotoxicity delivered from PLGA-systems.

Biodegradable lactic acid-glycolic acid copolymer (PLGA) formulations incorporating cisplatin have been developed to evaluate the cytotoxicity of this agent in cultured cells. Two different W/O/W protocols were used to formulate micro- (MP) and nanoparticles (NP) under the solvent evaporation method. Although the amount of cisplatin encapsulated was higher in the MP, the efficiency of encapsulation was similar: 10.33% vs. 11.23%, for both MP and NP, respectively. The "in-vitro" release profiles displayed a significant difference in the initial burst effect, which had a significant impact in the antiproliferative effect of cisplatin. In addition, a duality in the cell cycle distribution was found for both formulations and low doses of free cisplatin (2.5, 10 microM) in comparison with the high doses of free cisplatin. The 50 microM caused a rapid inhibition of cells growth followed by a significant loss of cells in phases G0/G1 and G2/M which correlated with an increase in the number of cells in sub-G1. However, cisplatin released from controlled formulations induced an accumulation of cells in the phase G2/M. These results led to enhance the caspase-3 activity for MP and NP. These findings indicate that controlled release formulations of cisplatin are able to induce a more effective apoptosis than free cisplatin.

Prevalence of celiac disease: multicentric trial among pediatric population from five urban districts in Argentina.

INTRODUCTION: No published material on the prevalence of celiac disease (CD) in the pediatric population of Argentina has been found up to date. Objective. To estimate the prevalence of CD in a pediatric population (hospital-based sample) from 5 urban districts of Argentina. METHODS: In a cross-sectional descriptive study, we analyzed serum samples from 2219 children, aged 3-16 years old, which had been requested for pre-surgical tests and for physical aptitude certificates for sports in the province of Buenos Aires, and cities of Buenos Aires, Córdoba, Santa Fe and Salta. Children with a previous and accurate diagnosis of CD were also included. IgA class tissue transglutaminase antibodies were determined using serum samples, and those samples which turned out positive were also tested for IgA class endomysium antibodies. A small intestine biopsy was proposed for those who had a positive serology. RESULTS: Between May 2008 and August 2009, 29 positive serologies were found. A total of 22 duodenum biopsies were performed, and 21 turned out compatible with CD. Out of 2219 children, 7 had a previous diagnosis. A prevalence of 1.26% (1:79 children), with female gender predominance (p < 0.023) was found. Ninety percent of the celiac children were over 6 years old (p < 0.021). Silent celiac disease predominated but there was a 33% of symptomatic cases. CONCLUSIONS: The results of the trial show a higher prevalence of CD than expected. The finding of symptomatic patients (33%) suggests the undertaking of different activities to spread the knowledge on this disease and promote the indication for serology test, to avoid complications by means of an early diagnosis.

Epidermal Growth Factor Receptor (EGFR) Crosstalks in Liver Cancer.

Hepatocarcinogenesis is a complex multistep process in which many different molecular pathways have been implicated. Hepatocellular carcinoma (HCC) is refractory to conventional chemotherapeutic agents, and the new targeted therapies are meeting with limited success. Interreceptor crosstalk and the positive feedback between different signaling systems are emerging as mechanisms of targeted therapy resistance. The identification of such interactions is therefore of particular relevance to improve therapeutic efficacy. Among the different signaling pathways activated in hepatocarcinogenesis the epidermal growth factor receptor (EGFR) system plays a prominent role, being recognized as a "signaling hub" where different extracellular growth and survival signals converge. EGFR can be transactivated in response to multiple heterologous ligands through the physical interaction with multiple receptors, the activity of intracellular kinases or the shedding of EGFR-ligands. In this article we review the crosstalk between the EGFR and other signaling pathways that could be relevant to liver cancer development and treatment.

Prevalence of celiac disease: multicentric trial among pediatric population from five urban districts in Argentina / Prevalencia de enfermedad celíaca: estudio multicéntrico en población pediátrica de cinco distritos urbanos de la Argentina

Introduction. No published material on the preva lence of celiac disease (CD) in the pediatric population of Argentina has been found up to date. Objective. To estimate the prevalence of CD in a pediatric population (hospital-based sample) from 5 urban districts of Argentina. Methods. In a cross-sectional descriptive study, we analyzed serum samples from 2219 children, aged 3-16 years old, which had been requested for pre-surgical tests and for physical aptitude certifcates for sports in the province of Buenos Aires, and cities of Buenos Aires, Córdoba, Santa Fe and Salta. Children with a previous and accurate diagnosis of CD were also included. IgA class tissue transglu taminase antibodies were determined using serum samples, and those samples which turned out positive were also tested for IgA class endomysium an tibodies. A small intestine biopsy was proposed for those who had a positive serology. Results. Between May 2008 and August 2009, 29 positive serologies were found. A total of 22 duo denum biopsies were performed, and 21 turned out compatible with CD. Out of 2219 children, 7 had a previous diagnosis. A prevalence of 1.26% (1:79 children), with female gender predominance (p < 0.023) was found. Ninety percent of the celiac children were over 6 years old (p < 0.021). Silent celiac disease predominated but there was a 33% of symptomatic cases. Conclusions. The results of the trial show a higher prevalence of CD than expected. The finding of symptomatic patients (33%) suggests the undertak ing of different activities to spread the knowledge on this disease and promote the indication for serology test, to avoid complications by means of an early diagnosis.

Introducción. Hasta la fecha del estudio no se hallaron estudios poblacionales publicados sobre prevalencia de enfermedad celíaca en la población pediátrica argentina. Objetivo. Estimar la prevalencia de la enfermedad celíaca en población pediátrica a partir de una muestra de base hospitalaria de cinco distritos urbanos. Método. Diseño descriptivo de corte transversal. Bajo consentimiento informado, participaron 2219 niños, de 3 a 16 años, que realizaban estudios de laboratorio para exámenes prequirúrgicos o certificados de aptitud física deportiva del Conurbano bonaerense, y ciudades de Buenos Aires, Santa Fe, Córdoba y Salta. Se incluyeron niños con diagnóstico previo y certero de enfermedad celíaca dentro de esa población. Se determinaron anticuerpos antitransglutaminasa y, en las muestras positivas, anticuerpo antiendomisio. Se propuso biopsia de intestino delgado a quienes presentaron ambas serologías positivas. Resultados: 29 serologías fueron positivas. Se realizaron 22 biopsias de duodeno, 21 fueron compatibles con enfermedad celíaca y 7 presentaron diagnóstico previo. La prevalencia fue de 1,26% (1:79) IC 95% 0,84-1,81, con predominio del sexo femenino (p <0,039). El 90% de los niños celíacos hallados fueron mayores de 6 años. Las formas clínicas silentes predominaron, pero hubo un 33% de casos sintomáticos. Conclusión. Los resultados en la población estudiada muestran una prevalencia mayor que la esperada. El hallazgo de formas sintomáticas (33%) sugiere emprender acciones de difusión del conocimiento de la enfermedad y ampliar la indicación de serología para obtener diagnóstico precoz.

Prevalencia de enfermedad celíaca: estudio multicéntrico en población pediátrica en cinco distritos urbanos de Argentina / Prevalence of celiac disease: multicentric trial among pediatric population in five urban districts of Argentina

INTRODUCCIÓN: Hasta la fecha del estudio no se hallaron estudios poblacionales publicados sobre prevalencia de EC en población pediátrica argentina. OBJETIVO: estimar la prevalencia de la EC en población pediátrica de cinco distritos urbanos. MÉTODOS: diseño descriptivo de corte transversal. Se invitó a participar a 2.230 niños, de 3 a 16 años, que realizaban estudios de laboratorio para exámenes prequirúrgicos o certificados de aptitud física deportiva. Se determinaron Anticuerpos Antitransglutaminasa, y en las muestras positivas Anticuerpo Antiendomisio. Se propuso biopsia de intestino delgado a quienes presentaron ambas serologías positivas. Se incluyeron niños con diagnóstico previo de EC que cumplían los criterios de inclusión. La prevalencia se expresó mediante el porcentajey su IC exacto. Las comparaciones entre grupos se efectuaron mediante la prueba exacta de Fisher. RESULTADOS: se testearon los sueros de 2.219 niños, 29 serologías fueron positivas. Se realizaron 22 biopsias de duodeno, 21 fueron compatibles con EC y 7 presentaron diagnóstico previo. La prevalencia fue de 1,26% (1:79)IC 95% 0,84-1.81, con predominio del sexo femenino (p<0,039) e importantes diferencias regionales. El 90% de los niños celíacos hallados fueron > 6 años. Las formas clínicas silentes predominaron pero hubo un 33% de casos sintomáticos. CONCLUSIÓN: los resultados en la población estudiada muestran una prevalencia mayor que estudios previos en adultos. El hallazgo de formas sintomáticas (33%) sugiere emprender acciones de difusión del conocimientode la enfermedad y ampliar la indicación de serología para obtener diagnóstico precoz.

INTRODUCTION: up the date of this study, published material about the prevalence of CD in pediatric population in Argentina has not been found. OBJECTIVE: to estimate CD prevalencein a pediatric population of 5 urban districts. METHODS: descriptive and cross sectional cut design. Were invited 2.230 children, between 3 and 16 years, which had been requested forpre-surgical studies and physical aptitude certificates for sports. IgA class tissue transglutaminase antibodies were determined and to positive samples IgA class endomysium antibodies. A small intestine biopsy was proposed for those who had both positive serology. Children with a previous diagnosis of CD who met the inclusion criteria were included. The prevalence was expressed by means of the percentage and its exact confidence interval and the comparisons between groups were performed using Fisher´s exact test. RESULTS: 2.219 children´s sera were studied. 29 were positive serologies. 22 duodenum biopsies were performed, 21 turned out compatible with CD. 7 children presented a previous diagnosis. A prevalence of 1.26% (1:79) CI 95% (0,84-1,81) was found, with female sex predominance ( p<0.039) and important regional differences as well. Ninety percent of the celiac children found were> 6 years Silent clinical manifestations predominated but there were 33% of symptomatic cases. CONCLUSIONS: the results in the study population showed a higher prevalence than previous studies in adults. The finding of symptomatic manifestations (33%)suggests actions to spread the knowledge of this disease promotingthe indication of serology for early diagnosis.