RESUMEN
RNA-based vaccines have recently emerged as a promising alternative to the use of DNA-based and viral vector vaccines, in part because of the potential to simplify how vaccines are made and facilitate a rapid response to newly emerging infections. SAM vaccines are based on engineered self-amplifying mRNA (SAM) replicons encoding an Ag, and formulated with a synthetic delivery system, and they induce broad-based immune responses in preclinical animal models. In our study, in vivo imaging shows that after the immunization, SAM Ag expression has an initial gradual increase. Gene expression profiling in injection-site tissues from mice immunized with SAM-based vaccine revealed an early and robust induction of type I IFN and IFN-stimulated responses at the site of injection, concurrent with the preliminary reduced SAM Ag expression. This SAM vaccine-induced type I IFN response has the potential to provide an adjuvant effect on vaccine potency, or, conversely, it might establish a temporary state that limits the initial SAM-encoded Ag expression. To determine the role of the early type I IFN response, SAM vaccines were evaluated in IFN receptor knockout mice. Our data indicate that minimizing the early type I IFN responses may be a useful strategy to increase primary SAM expression and the resulting vaccine potency. RNA sequence modification, delivery optimization, or concurrent use of appropriate compounds might be some of the strategies to finalize this aim.
Asunto(s)
Diseño de Fármacos , Interferón Tipo I/inmunología , ARN Mensajero/inmunología , Vacunas Virales/inmunología , Adyuvantes Inmunológicos , Animales , Anticuerpos Antivirales , Antígenos/inmunología , Imagenología Tridimensional/métodos , Interferón Tipo I/biosíntesis , Ratones , ARN Mensajero/administración & dosificación , ARN Mensajero/fisiología , ARN Viral/inmunología , Virus Sincitiales Respiratorios/química , Virus Sincitiales Respiratorios/inmunología , Vacunación , Potencia de la Vacuna , Vacunas Virales/genéticaRESUMEN
TLR7 is the mammalian receptor for ssRNA and some nucleotide-like small molecules. We have generated a mouse by N-nitrose-N'-ethyl urea mutagenesis in which threonine 68 of TLR7 was substituted with isoleucine. Cells bearing this mutant TLR7 lost the sensitivity to the small-molecule TLR7 agonist resiquimod, hence the name TLR7(rsq1). In this work, we report the characterization of this mutant protein. Similar to the wild-type counterpart, TLR7(rsq1) localizes to the endoplasmic reticulum and is expressed at normal levels in both primary cells and reconstituted 293T cells. In addition to small-molecule TLR7 agonists, TLR7(rsq1) fails to be activated by ssRNA. Whole-transcriptome analysis demonstrates that TLR7 is the exclusive and indispensable receptor for both classes of ligands, consistent with the fact that both ligands induce highly similar transcriptional signatures in TLR7(wt/wt) splenocytes. Thus, TLR7(rsq1) is a bona fide phenocopy of the TLR7 null mouse. Because TLR7(rsq1) binds to ssRNA, our studies imply that the N-terminal portion of TLR7 triggers a yet to be identified event on TLR7. TLR7(rsq1) mice might represent a valuable tool to help elucidate novel aspects of TLR7 biology.
Asunto(s)
Mutación Puntual/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/metabolismo , Animales , Línea Celular , Células Cultivadas , Células HEK293 , Humanos , Imidazoles/farmacología , Ligandos , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mutagénesis Sitio-Dirigida , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Unión Proteica/inmunología , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 7/deficienciaRESUMEN
Glioblastoma, the most malignant type of primary brain tumor, is one of the solid cancers where cancer stem cells have been isolated, and studies have suggested resistance of those cells to chemotherapy and radiotherapy. Here, we report the establishment of CSC-enriched cultures derived from human glioblastoma specimens. They grew as neurospheres in serum-free medium with epidermal growth factor and fibroblast growth factor 2, varied in the level of CD133 expression and very efficiently formed highly invasive and/or vascular tumors upon intracerebral implantation into immunodeficient mice. As a novel therapeutic strategy for glioblastoma-derived cancer stem-like cells (GBM-SC), we have tested oncolytic herpes simplex virus (oHSV) vectors. We show that although ICP6 (UL39)-deleted mutants kill GBM-SCs as efficiently as wild-type HSV, the deletion of gamma34.5 significantly attenuated the vectors due to poor replication. However, this was significantly reversed by the additional deletion of alpha47. Infection with oHSV G47Delta (ICP6(-), gamma34.5(-), alpha47(-)) not only killed GBM-SCs but also inhibited their self-renewal as evidenced by the inability of viable cells to form secondary tumor spheres. Importantly, despite the highly invasive nature of the intracerebral tumors generated by GBM-SCs, intratumoral injection of G47Delta significantly prolonged survival. These results for the first time show the efficacy of oHSV against human GBM-SCs, and correlate this cytotoxic property with specific oHSV mutations. This is important for designing new oHSV vectors and clinical trials. Moreover, the new glioma models described in this study provide powerful tools for testing experimental therapeutics and studying invasion and angiogenesis.
Asunto(s)
Neoplasias Encefálicas/patología , Neoplasias Encefálicas/terapia , Células Madre Embrionarias/patología , Glioblastoma/patología , Glioblastoma/terapia , Células Madre Neoplásicas/patología , Viroterapia Oncolítica/métodos , Simplexvirus/fisiología , Animales , Neoplasias Encefálicas/virología , Células Madre Embrionarias/virología , Glioblastoma/virología , Humanos , Ratones , Mutación , Células Madre Neoplásicas/virología , Simplexvirus/genética , Simplexvirus/crecimiento & desarrollo , Células Tumorales Cultivadas , Replicación Viral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We present a method to create multi-layered engineered tissue composites consisting of human skin fibroblasts and keratinocytes which mimic skin layers. Three-dimensional (3D) freeform fabrication (FF) technique, based on direct cell dispensing, was implemented using a robotic platform that prints collagen hydrogel precursor, fibroblasts and keratinocytes. A printed layer of cell-containing collagen was crosslinked by coating the layer with nebulized aqueous sodium bicarbonate. The process was repeated in layer-by-layer fashion on a planar tissue culture dish, resulting in two distinct cell layers of inner fibroblasts and outer keratinocytes. In order to demonstrate the ability to print and culture multi-layered cell-hydrogel composites on a non-planar surface for potential applications including skin wound repair, the technique was tested on a poly(dimethylsiloxane) (PDMS) mold with 3D surface contours as a target substrate. Highly viable proliferation of each cell layer was observed on both planar and non-planar surfaces. Our results suggest that organotypic skin tissue culture is feasible using on-demand cell printing technique with future potential application in creating skin grafts tailored for wound shape or artificial tissue assay for disease modeling and drug testing.