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1.
The versatility of stem cells has only recently been fully recognized. There is evidence that upon adoptive bone marrow (BM) transplantation (BMT), donor-derived cells can give rise to neuronal phenotypes in the brains of recipient mice. Yet only few cells with the characteristic shape of neurons were detected 1-6 mo post-BMT using transgenic or newborn mutant mice. To evaluate the potential of BM to generate mature neurons in adult C57BL/6 mice, we transferred the enhanced green fluorescent protein (GFP) gene into BM cells using a murine stem cell virus-based retroviral vector. Stable and high level long-term GFP expression was observed in mice transplanted with the transduced BM. Engraftment of GFP-expressing cells in the brain was monitored by intravital microscopy. In a long-term follow up of 15 mo post-BMT, fully developed Purkinje neurons were found to express GFP in both cerebellar hemispheres and in all chimeric mice. GFP-positive Purkinje cells were also detected in BM chimeras from transgenic mice that ubiquitously express GFP. Based on morphologic criteria and the expression of glutamic acid decarboxylase, the newly generated Purkinje cells were functional.  相似文献
2.
Gene therapy in the central nervous system (CNS) is hindered by the presence of the blood-brain barrier, which restricts access of serum constituents and peripheral cells to the brain parenchyma. Expression of exogenously administered genes in the CNS has been achieved in vivo using highly invasive routes, or ex vivo relying on the direct implantation of genetically modified cells into the brain. Here we provide evidence for a novel, noninvasive approach for targeting potential therapeutic factors to the CNS. Genetically-modified hematopoietic cells enter the CNS and differentiate into microglia after bone-marrow transplantation. Up to a quarter of the regional microglial population is donor-derived by four months after transplantation. Microglial engraftment is enhanced by neuropathology, and gene-modified myeloid cells are specifically attracted to the sites of neuronal damage. Thus, microglia may serve as vehicles for gene delivery to the nervous system.  相似文献
3.
DNA vaccination elicits humoral and cellular immune responses and has been shown to confer protection against several viral, bacterial, and parasitic pathogens. Here we report that optimized codon usage of an injected DNA sequence considerably increases both humoral and cellular immune responses. We recently generated a synthetic human immunodeficiency virus type 1 gp120 sequence in which most wild-type codons were replaced with codons from highly expressed human genes (syngp120). In vitro expression of syngp120 is considerably increased in comparison to that of the respective wild-type sequence. In BALB/c mice, DNA immunization with syngp120 resulted in significantly increased antibody titers and cytotoxic T-lymphocyte reactivity, suggesting a direct correlation between expression levels and the immune response. Moreover, syngp120 is characterized by rev-independent expression and a low risk of recombination with viral sequences. Thus, synthetic genes with optimized codon usage represent a novel strategy to increase the efficacy and safety of DNA vaccination.  相似文献
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Adoptive T cell therapy has been successfully used for treatment of viral and malignant diseases. However, little is known about the fate and trafficking of transferred Ag-specific T cells. Using the tetramer (TM) technology which allows for detection and quantification of Ag-specific CTL, we assessed the frequency of circulating Melan-A-specific CTL in advanced melanoma patients during adoptive T cell therapy. Melan-A-specific CTL were generated from HLA-A2.1(+) patients by in vitro stimulation of CD8(+) T cells with dendritic cells pulsed with a mutated HLA-A2-binding Melan-A (ELAGIGILTV) peptide. Eight patients received three infusions of 0.25-11 x 10(8) Melan-A-specific CTL i.v. at 2-wk intervals along with low-dose IL-2. The transferred T cell product contained a mean of 42.1% Melan-A-TM(+) CTL. Before therapy, the frequencies of Melan-A-specific CTL in patients' circulating CD8(+) T cells ranged from 0.01 to 0.07%. Characterization of the TM frequencies before and at different time points after transfer revealed an increase of circulating Melan-A-specific CTL up to 2%, correlating well with the number of transferred CTL. An elevated frequency of TM(+) T cells was demonstrated up to 14 days after transfer, suggesting long-term survival and/or proliferation of transferred CTL. Combining TM analysis with a flow cytometry-based cytokine secretion assay, unimpaired production of IFN-gamma was demonstrated in vivo for at least 24 h after transfer. Indium-111 labeling of Melan-A-specific CTL demonstrated localization of transferred CTL to metastatic sites as early as 48 h after injection. Overall, the results suggest that in vitro-generated Melan-A-specific CTL survive intact in vivo for several weeks and localize preferentially to tumor.  相似文献
6.
Adipophilin is a sensitive marker for lipid loading in human blood monocytes.   总被引:18,自引:0,他引:18  
Adipophilin, a marker of lipid accumulation initially described in adipocytes, was recently shown to be induced in macrophage foam cells. We found that even freshly isolated blood monocytes express adipophilin and that the amount of adipophilin protein is variable in monocytes from different healthy individuals. However, the physiological expression of adipophilin does not correlate with the levels of free fatty acids, cholesterylesters or free cholesterol. Enzymatically modified low-density lipoprotein (E-LDL) induces rapid foam cell formation in monocytes and upregulates adipophilin mRNA and protein within 2 h of incubation. This rapid induction of adipophilin is accompanied by a significant increase of free fatty acids in monocytes incubated with E-LDL. Adipophilin facilitates the uptake of free fatty acids, and here we demonstrate that free fatty acids increase is related to the early upregulation of adipophilin expression in blood monocytes. Fatty acids are ligands for peroxisome proliferator-activated receptor-gamma (PPARgamma), and the upregulation of adipophilin mRNA by PPARgamma agonists like 15d-PGJ(2) and ciglitazone indicates that PPARgamma may mediate the induction of adipophilin expression in human blood monocytes.  相似文献
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Several statistical tests based on population genetic theory are used to identify genes that have recently acquired a beneficial mutation. Here, I describe the extension of these tests to a multilocus approach for a genome-wide survey for genes that have been under recent positive selection. As this strategy could potentially identify genes with weak phenotypic effects, it will be very useful in population genetic approaches aimed at understanding adaptation processes in natural populations. Furthermore, this 'hitchhiking mapping' could also help in the functional characterization of genomes.  相似文献
9.
Angiogenesis in situ includes coordinated interactions of various microvascular cell types, i.e., endothelial cells, pericytes and perivascular fibroblasts. To study the cellular interactions of microvascular cells in vitro, we have developed a microcarrier-based cocultivation system. The technical details of this method include seeding of endothelial cells on unstained cytodex-3 microcarriers and seeding of pericytes, fibroblasts or vascular smooth muscle cells on microcarriers which have been labeled by trypan blue staining. A mixture of both unstained and trypan blue-stained microcarriers was subsequently embedded in a three-dimensional fibrin clot. The growth characteristics of each cell type could be conveniently observed since the majority of cells left their supporting microcarriers in a horizontal direction to migrate into the transparent fibrin matrix. As differently stained microcarriers were randomly arranged in the fibrin matrix, the characteristic patterns of the microcarriers allowed location of particular points of interest at different developmental stages, facilitating the observation of cellular growth over the course of time. One further advantage of this microcarrier-based system is the possibility of reliably quantifying capillary growth by determination of average numbers of capillary-like formations per microcarrier. Thus, this model allows convenient evaluation of the effects of non-endothelial cells on angiogenesis in vitro. By using this coculture system, we demonstrate that endothelial capillary-like structures in vitro do not become stabilized by contacting vascular smooth muscle cells or pericytes during the initial stages of capillary formation.  相似文献
10.
Because the neural differentiation capacity of bone marrow stromal cells (BMSCs) is still a matter of controversial debate, we performed a thorough investigation into the differentiation capacity of human BMSCs and examined their therapeutic potency. BMSCs were isolated from the femur and kept in cell cultures with various cultivation protocols being applied. In standard culture conditions using a fetal calf serum-enriched medium, while not exhibiting a neural phenotype, the majority of cells expressed a variety of neuronal marker proteins as well as the astrocyte marker GFAP. Only a minority of stem cells expressed nestin, a marker for neural precursor cells. Cultivation in serum-free medium supplemented with specific growth factors resulted in a markedly higher percentage of nestin-positive cells. To establish the therapeutic potency of bone marrow-derived cells, the synthesis of neurotrophic factors such as NGF, BDNF and GDNF was analyzed under non-stimulating standard culture conditions as well as after a neural selection procedure. The therapeutic potency of BMSCs was further examined with regard to their migratory potential in vitro and after transplantation in vivo. After stereotactic engraftment into the lateral ventricle of adult rats, mesenchymal stem cells were seen to adhere to the ependymocytes and cells of the choroids plexus. Afterwards grafted cells passed through the ependymal barrier, locating in the subventricular space. Their BMSCs took up a close host graft interaction without any degenerative influence on the host cells. Furthermore, there was morphological as well as immunohistochemical evidence for a transdifferentiation within the host tissue. In addition, BMSCs could be efficiently transduced using a third-generation adenoviral vector, indicating their potential feasibility for a gene-therapeutic option.  相似文献
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