RESUMEN
Antisense oligonucleotides, short interfering RNAs (siRNAs) and aptamers are oligonucleotide-based pharmaceuticals with a promising role in targeted therapies. Currently, five oligonucleotide-based pharmaceuticals have achieved marketing authorization in Europe or USA and many more are undergoing clinical testing. However, several safety concerns have been raised in non-clinical and clinical studies. Oligonucleotides share properties with both chemical and biological pharmaceuticals and therefore they pose challenges also from the regulatory point of view. We have analyzed the safety data of oligonucleotides and evaluated the applicability of current non-clinical toxicological guidelines for assessing the safety of oligonucleotide-based pharmaceuticals. Oligonucleotide-based pharmaceuticals display a similar toxicological profile, exerting adverse effects on liver and kidney, evoking hematological alterations, as well as causing immunostimulation and prolonging the coagulation time. It is possible to extrapolate some of these effects from non-clinical studies to humans. However, evaluation strategies for genotoxicity testing of "non-natural" oligonucleotides should be revised. Additionally, the selective use of surrogates and prediction of clinical endpoints for non-clinically observed immunostimulation is complicated by its multiple potential manifestations, demanding improvements in the testing strategies. Utilizing more relevant and mechanistic-based approaches and taking better account of species differences, could possibly improve the prediction of relevant immunological/proinflammatory effects in humans.
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Sistema Inmunológico/efectos de los fármacos , Oligonucleótidos/efectos adversos , ARN Interferente Pequeño/efectos adversos , Animales , Guías como Asunto , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Pruebas de Mutagenicidad/métodos , Pruebas de Mutagenicidad/normas , Oligonucleótidos/farmacocinética , ARN Interferente Pequeño/farmacocinética , Especificidad de la Especie , Distribución TisularRESUMEN
In the past decade, the therapeutic value of mesenchymal stromal cells (MSCs) has been studied in various indications, thereby taking advantage of their immunosuppressive properties. Easy procurement from bone marrow, adipose tissue or other sources and conventional in vitro expansion culture have made their clinical use attractive. Bridging the gap between current scientific knowledge and regulatory prospects on the transformation potential and possible tumorigenicity of MSCs, the Cell Products Working Party and the Committee for Advanced Therapies organized a meeting with leading European experts in the field of MSCs. This meeting elucidated the risk of potential tumorigenicity related to MSC-based therapies from two angles: the scientific perspective and the regulatory point of view. The conclusions of this meeting, including the current regulatory thinking on quality, nonclinical and clinical aspects for MSCs, are presented in this review, leading to a clearer way forward for the development of such products.
Asunto(s)
Carcinogénesis , Proliferación Celular , Trasplante de Células Madre Mesenquimatosas/efectos adversos , Células Madre Mesenquimatosas/citología , Tejido Adiposo/citología , Células de la Médula Ósea/citología , Técnicas de Cultivo de Célula , Diferenciación Celular/genética , Humanos , Células Madre Mesenquimatosas/metabolismoRESUMEN
Genomic parasites have evolved distinctive lifestyles to optimize replication in the context of the genomes they inhabit. Here, we introduced new DNA into eukaryotic cells using bacteriophage Mu DNA transposition complexes, termed 'transpososomes'. Following electroporation of transpososomes and selection for marker gene expression, efficient integration was verified in yeast, mouse and human genomes. Although Mu has evolved in prokaryotes, strong biases were seen in the target site distributions in eukaryotic genomes, and these biases differed between yeast and mammals. In Saccharomyces cerevisiae transposons accumulated outside of genes, consistent with selection against gene disruption. In mouse and human cells, transposons accumulated within genes, which previous work suggests is a favorable location for efficient expression of selectable markers. Naturally occurring transposons and viruses in yeast and mammals show related, but more extreme, targeting biases, suggesting that they are responding to the same pressures. These data help clarify the constraints exerted by genome structure on genomic parasites, and illustrate the wide utility of the Mu transpososome technology for gene transfer in eukaryotic cells.
Asunto(s)
Bacteriófago mu/genética , Elementos Transponibles de ADN , Técnicas de Transferencia de Gen , Animales , Línea Celular , Mapeo Cromosómico , Electroporación , Células Madre Embrionarias/metabolismo , Marcadores Genéticos , Genoma Fúngico , Genoma Humano , Genómica , Células HeLa , Humanos , Ratones , Saccharomyces cerevisiae/genéticaRESUMEN
BACKGROUND: Individual differences between human embryonic stem cell (hESC) lines are poorly understood. Here, we describe the derivation of five hESC lines (called FES 21, 22, 29, 30 and 61) from frozen-thawed human embryos and compare their individual differentiation characteristic. RESULTS: The cell lines were cultured either on human or mouse feeder cells. The cells grew significantly faster and could be passaged enzymatically only on mouse feeders. However, this was found to lead to chromosomal instability after prolonged culture. All hESC lines expressed the established markers of pluripotent cells as well as several primordial germ cell (PGC) marker genes in a uniform manner. However, the cell lines showed distinct features in their spontaneous differentiation patterns. The embryoid body (EB) formation frequency of FES 30 cell line was significantly lower than that of other lines and cells within the EBs differentiated less readily. Likewise, teratomas derived from FES 30 cells were constantly cystic and showed only minor solid tissue formation with a monotonous differentiation pattern as compared with the other lines. CONCLUSION: hESC lines may differ substantially in their differentiation properties although they appear similar in the undifferentiated state.
Asunto(s)
Diferenciación Celular , Línea Celular , Embrión de Mamíferos/citología , Inducción Embrionaria/fisiología , Células Madre/metabolismo , Animales , Biomarcadores/análisis , Técnicas de Cultivo de Célula/métodos , Células Germinativas/metabolismo , Células Germinativas/ultraestructura , Humanos , Cariotipificación , Masculino , Ratones , Células Madre/ultraestructura , Teratoma/patologíaRESUMEN
Efficient response to environmental cues is crucial to successful infection by plant-pathogenic bacteria such as Erwinia carotovora ssp. carotovora. The expression of the main virulence genes of this pathogen, encoding extracellular enzymes that degrade the plant-cell wall, is subject to complex regulatory machinery where two-component systems play an important role. In this paper, we describe for the first time the involvement of the PmrA-PmrB two-component system in regulation of virulence in a plant-pathogenic bacterium. Disruption of pmrB resulted in reduced virulence both in potato and in Arabidopsis. This is apparently due to reduced production of the extracellular enzymes. In contrast, a pmrA mutant exhibited increased levels of these enzymes implying negative regulation of the corresponding genes by PmrA. Furthermore, the pmrB but not pmrA mutant exhibited highly increased resistance to the cationic antimicrobial peptide polymyxin B suggesting alterations in cell surface properties of the mutant. A similar increase of polymyxin resistance was detected in the wild type at mildly acidic pH with low Mg2+. Functional pmrA is essential for bacterial survival on excess iron at acidic pH, regardless of the Mg2+ concentration. We propose that PmrA-PmrB TCS is involved in controlling of bacterial response to external pH and iron and is crucial for bacterial virulence and survival in planta.
Asunto(s)
Hierro/farmacología , Pectobacterium carotovorum/fisiología , Pectobacterium carotovorum/patogenicidad , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana/genética , Regulación Bacteriana de la Expresión Génica , Homeostasis , Concentración de Iones de Hidrógeno , Magnesio/metabolismo , Magnesio/farmacología , Datos de Secuencia Molecular , Mutación , Operón , Pectobacterium carotovorum/efectos de los fármacos , Enfermedades de las Plantas/microbiología , Polimixina B/farmacología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Homología de Secuencia de Aminoácido , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/genética , Virulencia/genéticaRESUMEN
We have used an hrp-positive strain of the soft rot pathogen Erwinia carotovora subsp. carotovora to elucidate plant responses to this bacterial necrotroph. Purified virulence determinants, harpin (HrpN) and polygalacturonase (PehA), were used as tools to facilitate this analysis. We show that HrpN elicits lesion formation in Arabidopsis and tobacco and triggers systemic resistance in Arabidopsis. Establishment of resistance is accompanied by the expression of salicylic acid (SA)-dependent, but also jasmonate/ethylene (JA/ET)-dependent, marker genes PR1 and PDF1.2, respectively, suggesting that both SA-dependent and JA/ET-dependent defense pathways are activated. Use of pathway-specific mutants and transgenic NahG plants show that both pathways are required for the induction of resistance. Arabidopsis plants treated simultaneously with both elictors PehA, known to trigger only JA/ET-dependent defense signaling, and HrpN react with accelerated and enhanced induction of the marker genes PR1 and PDF1.2 both locally and systemically. This mutual amplification of defense gene expression involves both SA-dependent and JA/ET-dependent defense signaling. The two elicitors produced by E. carotovora subsp. carotovora also cooperate in triggering increased production of superoxide and lesion formation.
Asunto(s)
Apoptosis/efectos de los fármacos , Arabidopsis/efectos de los fármacos , Proteínas de la Membrana Bacteriana Externa/farmacología , Defensinas , Pectobacterium carotovorum/crecimiento & desarrollo , Poligalacturonasa/farmacología , Apoptosis/fisiología , Arabidopsis/genética , Arabidopsis/microbiología , Ciclopentanos/farmacología , Sinergismo Farmacológico , Etilenos/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/genética , Inmunidad Innata/fisiología , Mutación , Oxilipinas , Enfermedades de las Plantas/microbiología , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/microbiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Ácido Salicílico/farmacología , Transducción de Señal/fisiología , Superóxidos/metabolismo , Nicotiana/efectos de los fármacos , Nicotiana/microbiología , Nicotiana/fisiologíaRESUMEN
Intramolecular information specifying protein secretion through the type II (GSP) pathway of Gram-negative bacteria was investigated. Two regions of the polygalacturonase (PehA) of Erwinia carotovora containing residues proposed to be included in a targeting motif were located, one close to the C-terminus between residues 342 and 369 and another between residues 84 and 135 in the large central loops. The regions were required together to promote secretion. Further residues in the middle of the protein were required for proper positioning of the regions, suggesting that they were both involved in interaction with the GSP. To our knowledge, this is the first time that a possible three-dimensional targeting motif has been defined. At least one of the motifs comprises a cluster on the surface of the protein. The two motifs are structurally dissimilar, suggesting that there are two distinct recognition regions in the GSP apparatus. Finally, we propose that the targeting motifs are of a complex conformational nature with some variability accommodated, as illustrated by the observation that many mutations exhibited no clear phenotype individually but, in combination, severely compromised secretion.