RESUMO
Haematopoietic humanization of mice is used frequently to study the human immune system and its reaction upon experimental intervention. Immunocompromised non-obese diabetic (NOD)-Rag1(-/-) mice, additionally deficient for the common gamma chain of cytokine receptors (γc) (NOD-Rag1(-/-) γc(-/-) mice), lack B, T and natural killer (NK) cells and allow for efficient human peripheral mononuclear cell (PBMC) engraftment. However, a major experimental drawback for studies using these mice is the rapid onset of graft-versus-host disease (GVHD). In order to elucidate the contribution of the xenogenic murine major histocompatibility complex (MHC) class II in this context, we generated immunodeficient mice expressing human MHC class II [human leucocyte antigen (HLA)-DQ8] on a mouse class II-deficient background (Aß(-/-) ). We studied repopulation and onset of GVHD in these mouse strains following transplantation of DQ8 haplotype-matched human PBMCs. The presence of HLA class II promoted the repopulation rates significantly in these mice. Virtually all the engrafted cells were CD3(+) T cells. The presence of HLA class II did not advance B cell engraftment, such that humoral immune responses were undetectable. However, the overall survival of DQ8-expressing mice was prolonged significantly compared to mice expressing mouse MHC class II molecules, and correlated with an increased time span until onset of GVHD. Our data thus demonstrate that this new mouse strain is useful to study GVHD, and the prolonged animal survival and engraftment rates make it superior for experimental intervention following PBMC engraftment.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Leucócitos Mononucleares/citologia , Linfócitos T/imunologia , Transferência Adotiva , Animais , Linfócitos B/imunologia , Progressão da Doença , Antígenos HLA-DQ/genética , Antígenos de Histocompatibilidade Classe II/genética , Proteínas de Homeodomínio/genética , Humanos , Imunidade Humoral/genética , Hospedeiro Imunocomprometido , Subunidade gama Comum de Receptores de Interleucina/genética , Leucócitos Mononucleares/transplante , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos TransgênicosRESUMO
RNAi represents a powerful technology to specifically downregulate the expression of target genes. For cancer research and therapy, an efficient in vivo delivery system is supposed to distribute RNAi to all tumour cells upon systemic administration. We present replication-competent murine leukaemia virus (MLV) vectors, which deliver RNAi to tumour tissue upon tail vein injection. In HT1080 cells stably expressing GFP or luciferase, GFP expression was suppressed by more than 80% and luciferase (luc) activity by more than 90%, even when only 0.1% of the cells were initially infected with reporter gene specific vectors. To demonstrate its potential, PLK1- and MMP14-specific small hairpin RNA expression cassettes were applied in the system. Upon infection, PLK1 and MMP14 levels were reduced on mRNA and protein level. MLV-shPLK1-infected cells were arrested in the G2-phase and underwent apoptosis. MLV-shMMP14-infected cells showed reduced MMP2 activity, as well as substantially reduced invasion and tumour growth. In vivo, MLV-shLuc silenced luc expression in HT1080-luc tumour tissue by more than 80% and MLV-shPLK1 reduced tumour growth substantially, demonstrating the therapeutic relevance of this system. This RNAi vector system allows long-term downregulation of target gene expression as well as efficient delivery to and distribution throughout tumour tissue in vivo.
Assuntos
Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Vetores Genéticos/genética , Vetores Genéticos/uso terapêutico , Vírus da Leucemia Murina/genética , Neoplasias/metabolismo , Neoplasias/terapia , Interferência de RNA , Animais , Western Blotting , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Primers do DNA/genética , Feminino , Citometria de Fluxo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Luciferases/metabolismo , Metaloproteinase 14 da Matriz/genética , Camundongos , Camundongos SCID , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Reação em Cadeia da Polimerase em Tempo Real , Quinase 1 Polo-LikeRESUMO
Authoring functions--integrated in a Hospital Information System (HIS)--provide a means for physicians and nurses to adapt partly their man/machine interface. We successfully implemented task-specific authoring functions that enable end-users to comprehend and structure a large body of multimedia documents, to focus attention during medical decision making, to speed up medical tasks and to standardise the drafting of documents.
