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At least 469 records · Page 26Linked to original sources
PubMed2000

Targeted antigen delivery to antigen-presenting cells including dendritic cells by engineered Fas-mediated apoptosis.

Immunity to tumors as well as to viral and bacterial pathogens is often mediated by cytotoxic T lymphocytes (CTLs). Thus, the ability to induce a strong cell-mediated immune response is an important requirement of novel immunotherapies. Antigen-presenting cells (APCs), including dendritic cells (DCs), are specialized in initiating T-cell immunity. Harnessing this innate ability of these cells to acquire and present antigens, we sought to improve antigen presentation by targeting antigens directly to DCs in vivo through apoptosis. We engineered Fas-mediated apoptotic death of antigen-bearing cells in vivo by co-expressing the immunogen and Fas in the same cell. We then observed that the death of antigen-bearing cells results in increased antigen acquisition by APCs including DCs. This in vivo strategy led to enhanced antigen-specific CTLs, and the elaboration of T helper-1 (Th1) type cytokines and chemokines. This adjuvant approach has important implications for viral and nonviral delivery strategies for vaccines or gene therapies.

Animals↗
PubMed1993

Mammalian PC-12 cell genetically engineered for human cytochrome P450 2E1 expression.

The stable expression of the human cytochrome CYP2E1 (P450 alcohol) was performed in the mammalian cell line PC-12. This cell line expressed cytochrome b5 (58 +/- 12 pmol/mg microsomal protein vs 528 +/- 80 pmol/mg in microsomal human liver) and a high level of NADPH: cytochrome P450 reductase (140 +/- 20 nmol.min-1.mg microsomal protein-1 vs 68 +/- 48 nmol.min-1.mg-1 in microsomal human liver). An expression plasmid was constructed using the cDNA for the human CYP2E1 mRNA and the Rous sarcoma virus (RSV) promoter. This plasmid was co-transfected with the plasmid RSVneo into PC-12 cells. Clones were selected for resistance to the neomycin analog, G418, and then screened for expression of the CYP2E1 isozyme by testing for 6-hydroxylation of chlorzoxazone, a specific substrate for CYP2E1. Expression of CYP2E1 was confirmed in one clone, DB-7, by Western blot analysis and by measurement of monooxygenase activities which were not detectable in PC-12 cells. Chlorzoxazone 6-hydroxylation, n-butanol oxidation and dimethylnitrosamine N-demethylation were localized in microsomes (62, 60 and 63 pmol.min-1.mg microsomal protein-1, respectively) and were inhibited by carbon monoxide and diethyldithiocarbamate, both inhibitors of P450 enzymes. Although the level of the enzyme activities was about a tenth of that measured in human liver microsomes, CYP2E1 expressed in DB-7 cells has catalytic competence similar to human liver CYP2E1. DB-7 cells metabolized acetaminophen and this metabolic activation was shown to be toxic to these cells by release of lactate dehydrogenase. Construction of recombinant cell lines expressing CYP2E1 provides a useful tool for studying the catalytic properties of this enzyme and the consequent cytotoxic effects of substrates metabolized by this enzyme.

1-Butanol↗
PubMed1993

Transplantation of a polymer-encapsulated cell line genetically engineered to release NGF.

The delivery of nerve growth factor (NGF) to the lateral ventricle of a fimbria-fornix-lesioned rat prevents the lesion-induced reduction in choline acetyltransferase (ChAT) expression by medial septal cells. Although delivery has been achieved through neural grafting of genetically engineered cell lines which release NGF, transplanted cells have grown beyond the implantation site and formed tumors. The encapsulation of cells within a permselective polymer capsule prior to transplantation allows cell growth only within the capsule space, while allowing molecular exchange between the host tissue and enclosed cells. Rat fibroblasts from the parent cell line (R208F) or fibroblasts genetically modified to produce NGF (R208N.8) were loaded within a thermoplastic hollow fiber-based capsule. Only the capsules loaded with the genetically engineered cells released measurable amounts of NGF in culture. Adult rats received unilateral aspirative fimbria-fornix lesions, followed by intraventricular implantation of a R208F capsule (n = 6) or a R208N.8 capsule (n = 6). After 2 weeks, rats receiving encapsulated cells showed no undue reaction to the implants. With both cell types, the cells remained viable and confined to the capsule space. R208N.8 capsules released sufficient NGF to prevent the lesion-induced loss of septal ChAT expression, whereas R208F capsules did not. This study suggests that encapsulated genetically engineered cells can provide an efficient means for future applications involving delivery of neurotrophic factors.

Acetylcholinesterase↗
PubMed1996

Gene therapy for amyotrophic lateral sclerosis (ALS) using a polymer encapsulated xenogenic cell line engineered to secrete hCNTF.

The gene therapy approach presented in this protocol employs a polymer encapsulated, xenogenic, transfected cell line to release human ciliary neurotrophic factor (hCNTF) for the treatment of Amyotrophic Lateral Sclerosis (ALS). A tethered device, containing around 10(6) genetically modified cells surrounded by a semipermeable membrane, is implanted intrathecally; it provides for slow continuous release of hCNTF at a rate of 0.25 to 1.0 micrograms/24 hours. The semipermeable membrane prevents immunologic rejection of the cells and interposes a physical, virally impermeable barrier between cells and host. Moreover, the device and the cells it contains may be retrieved in the event of side effects. A vector containing the human CNTF gene was transfected into a line of baby hamster kidney cells (BHK) with calcium phosphate using a dihydrofolate reductase-based selection vector with a SV40 promoter and contains a HSV-tk killer gene. hCNTF is a potent neurotrophic factor which may have utility for the treatment of ALS. Systemic delivery of hCNTF in humans has been frustrated by peripheral side effects, the molecule's short half life, and its inability to cross the blood-brain barrier. The gene therapy approach described in this protocol is expected to mitigate such difficulties by local intrathecal delivery of a known quantity of continuously-synthesized hCNTF from a retrievable implant.

Amyotrophic Lateral Sclerosis↗
PubMed2004

Converse relationship between in vitro osteogenic differentiation and in vivo bone healing elicited by different populations of muscle-derived cells genetically engineered to express BMP4.

UNLABELLED: In this study, we compared the use of primary muscle-derived osteoprogenitor cells (PP6 cells) for the delivery of BMP4 to improve bone healing to that of muscle-derived non-osteoprogenitor cells (PP1 cells). Surprisingly, the use of PP1 cells resulted in an improved outcome because of the lack of adverse responses to BMP4 involving cell differentiation, proliferation, and apoptosis. INTRODUCTION: Although researchers frequently opt to use osteogenic cells for osteogenic bone morphogenetic protein (BMP)-based ex vivo gene therapy to improve bone healing, it remains unclear whether the osteogenic potential of a cellular vehicle affects the outcome of bone healing applications. Here we compared the use of muscle-derived non-osteoprogenitor cells (PP1 cells) to that of primary muscle-derived osteoprogenitor cells (PP6 cells) for the delivery of BMP4 to improve the healing of bone defects. MATERIALS AND METHODS: Two distinct populations of primary rat muscle-derived cells--PP1 and PP6--were selected, transduced with retroviral vectors to express BMP4 or a marker gene (LacZ), and implanted into critical-sized calvarial defects created in syngeneic rats. The bone healing was monitored radiographically and histologically at 7 and 14 weeks after implantation. Cellular responses to BMP4 were evaluated by alkaline phosphatase histochemical staining and RT-PCR of another osteogenic marker to indicate osteogenic differentiation, a cell proliferation assay and BrdU (bromodeoxyuridine) labeling to assess cell proliferation, and the TUNEL assay to determine apoptosis. RESULTS AND CONCLUSIONS: In all animals (nine rats per group), transduced PP1 cells expressing BMP4 demonstrated significantly advanced healing compared with PP6 cells expressing BMP4 and control cells expressing LacZ. We found that constitutive BMP4 expression negatively impacted the in vitro proliferation and in vivo survival rates of PP6 cells, but not PP1 cells. BMP4 exposure also directly inhibited the proliferation and induced the apoptosis of PP6 cells, but not PP1 cells. The impairment in PP6 cell proliferation was directly associated with the osteogenic differentiation of these cells. These results indicate that PP1 cells are better suited than osteoprogenitor cells for use as cellular vehicles to deliver osteogenic BMP4 to improve bone healing and that cellular behavior in response to a particular gene can be used to predict the cells' performance as delivery vehicles in ex vivo gene therapy.

Animals↗
PubMed1999

Development of systemic immunity to glioblastoma multiforme using tumor cells genetically engineered to express the membrane-associated isoform of macrophage colony-stimulating factor.

We investigated the ability of Fischer rat T9 glioblastoma cells transduced with cDNA genes for the secreted (s) or membrane-associated (m) isoform of M-CSF to elicit an antitumor response when implanted into syngeneic animals. Intracranial (i.c.) implantation of 1 x 10(5) T9 cells expressing mM-CSF (T9/mM-CSF) resulted in 80% tumor rejection. Electron microscopy of the T9/mM-CSF tumor site, 2-4 days postimplantation, showed marked infiltration by macrophages, many of which were in physical contact with the T9/mM-CSF cells. Animals that rejected T9/mM-CSF cells were resistant to i.c. rechallenge with T9 cells, but not syngeneic MadB106 breast adenocarcinoma cells, suggesting that T9-specific immunity can be generated within the brain via the endogenous APCs. Intracranial injection of parental T9, vector control (T9/LXSN), or T9 cells secreting M-CSF (T9/sM-CSF) was 100% fatal. Subcutaneous injection of 1 x 10(7) T9/sM-CSF, T9/LXSN, or parental T9 cells resulted in progressive tumors. In contrast, T9/mM-CSF cells injected s.c. were destroyed in 7-10 days and animals developed systemic immunity to parental T9 cells. Passive transfer of CD3+ T cells from the spleens of immune rats into naive recipients transferred T9 glioma-specific immunity. In vitro, splenocytes from T9/mM-CSF-immunized rats specifically proliferated in response to various syngeneic glioma stimulator cells. However, only marginal T cell-mediated cytotoxicity was observed by these splenocytes in a CTL assay against T9 target cells, regardless of restimulation with T9 cells. Subcutaneous immunization with viable T9/mM-CSF cells was effective in eradicating i.c. T9 tumors.

Animals↗
PubMed2006

Neotendon formation induced by manipulation of the Smad8 signalling pathway in mesenchymal stem cells.

Tissue regeneration requires the recruitment of adult stem cells and their differentiation into mature committed cells. In this study we describe what we believe to be a novel approach for tendon regeneration based on a specific signalling molecule, Smad8, which mediates the differentiation of mesenchymal stem cells (MSCs) into tendon-like cells. A biologically active Smad8 variant was transfected into an MSC line that coexpressed the osteogenic gene bone morphogenetic protein 2 (BMP2). The engineered cells demonstrated the morphological characteristics and gene expression profile of tendon cells both in vitro and in vivo. In addition, following implantation in an Achilles tendon partial defect, the engineered cells were capable of inducing tendon regeneration demonstrated by double quantum filtered MRI. The results indicate what we believe to be a novel mechanism in which Smad8 inhibits the osteogenic pathway in MSCs known to be induced by BMP2 while promoting tendon differentiation. These findings may have considerable importance for the therapeutic replacement of tendons or ligaments and for engineering other tissues in which BMP plays a pivotal developmental role.

Achilles Tendon↗
PubMed2003

Tissue engineering and cell therapy of cartilage and bone.

Trauma and disease of bones and joints, frequently involving structural damage to both the articular cartilage surface and the subchondral bone, result in severe pain and disability for millions of people worldwide and represent major challenges for the orthopedic surgeons. Therapeutic repair of skeletal tissues by tissue engineering has raised the interest of the scientific community, providing very promising results in preclinical animal models and clinical pilot studies. In this review, we discuss this approach. The choice of a proper cell type is addressed. The use of terminally differentiated cells, as in the case of autologous chondrocyte implantation, is compared with the advantages/disadvantages of using more undifferentiated cell types, such as stem cells or early mesenchymal progenitors that retain multi-lineage and self-renewal potentials. The need for proper scaffold matrices is also examined, and we provide a brief overview of their fundamental properties. A description of the natural and biosynthetic materials currently used for reconstruction purposes, either of cartilage or bone, is given. Finally, we highlight the positive aspects and the remaining problems that will drive future research in articular cartilage and bone repair.

Bone Diseases↗
PubMed1998

Molecular dissection of domains in mutant presenilin 2 that mediate overproduction of amyloidogenic forms of amyloid beta peptides. Inability of truncated forms of PS2 with familial Alzheimer's disease mutation to increase secretion of Abeta42.

Mutations in presenilin (PS) 1 or PS2 genes account for the majority of early-onset familial Alzheimer's disease, and these mutations have been shown to increase production of species of amyloid beta peptide (Abeta) ending at residue 42, i.e. the most amyloidogenic form of Abeta. To gain insight into the molecular mechanisms whereby mutant PS induces overproduction of Abeta42, we constructed cDNAs encoding mutant and/or truncated forms of PS2 and examined the secretion of Abeta42 from COS or neuro2a cells transfected with these genes. Cells expressing full-length PS2 harboring both N141I and M239V mutations in the same polypeptide induced overproduction of Abeta42, although the levels of Abeta42 were comparable with those in cells engineered to express PS2 with one or the other of these PS2 mutations. In contrast, cells engineered to express partially truncated PS2 (eliminating the COOH-terminal third of PS2 while retaining the endoproteolytic NH2-terminal fragment) and harboring a N141I mutation, as well as cells expressing COOH-terminal fragments of PS2, did not overproduce Abeta42, and the levels of Abeta42 were comparable with those in cells that expressed full-length, wild-type PS2 or fragments thereof. These data indicate that: (i) the Abeta42-promoting effects of mutant PS2 proteins reach the maximum level with a given single amino acid substitution (i.e. N141I or M239V); and (ii) the expression of full-length mutant PS2 is required for the overproduction of Abeta42. Hence, cooperative interactions of NH2- and COOH-terminal fragments generated from full-length mutant PS2 may be important for the overproduction of Abeta42 that may underlie familial Alzheimer's disease.

Alzheimer Disease↗
PubMed2005

eIF2alpha phosphorylation, stress perception, and the shutdown of global protein synthesis in cultured CHO cells.

The perception of environmental stress in animal cells engineered to produce heterologous protein leads to the induction of stress signaling pathways and ultimately apoptosis and cell death. Protein synthesis is regulated in response to various environmental stresses by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2). In this study we have utilized a model system of Chinese hamster ovary cells engineered to secrete recombinant TIMP-1 protein to investigate the relationship between the cellular rate of protein synthesis, eIF2alpha phosphorylation, cellular stress perception, and the rate of cell specific recombinant protein synthesis. The rate of total protein synthesis was maximal after 48 hours of culture, remaining relatively high until 96 hours of culture, after which a decline was observed. Towards the end of culture a marked increase in labeled secreted protein was observed. Total eIF2alpha expression levels were high during the exponential growth phase and decreased slightly towards the end of culture. On the other hand, the relative expression of phosphorylated eIF2alpha showed a bi-phasic response with a small increase in phosphorylated eIF2alpha observed at 48 hours of culture, and a significant increase at 120 hours post-inoculation. The large increase in phosphorylated eIF2alpha coincided with the observed increase in labeled secreted protein and the decline in total cellular protein synthesis. A marked increase in ubiquitination was also observed at 120 hours post-inoculation that coincided with reduced rates of cellular protein synthesis and mRNA translation attenuation. We suggest that eIF2alpha phosphorylation is an indicator of cellular stress perception, which could be exploited in recombinant protein manufacturing to commence feeding and engineering strategies.

Animals↗
PubMed2005

Inhibition of vascular smooth muscle cell proliferation in vitro by genetically engineered marrow stromal cells secreting calcitonin gene-related peptide.

Calcitonin gene-related peptide (CGRP) has a beneficial effect in pulmonary hypertension and is a target for cardiovascular gene therapy. Marrow stromal cells (MSCs), also known as mesenchymal stem cells, hold promise for use in adult stem cell-based ex vivo gene therapy. To test the hypothesis that genetically engineered MSCs secreting CGRP can inhibit vascular smooth muscle cell proliferation, rat MSCs were isolated, ex vivo expanded, and transduced with adenovirus containing CGRP. Immunocytochemical analysis demonstrated that wild type rat MSCs express markers specific for stem cells, endothelial cells, and smooth muscle cells including Thy-1, c-Kit, von Willebrand Factor and alpha-smooth muscle actin. Immunocytochemistry confirmed the expression of CGRP by the transduced rat MSCs. The transduced rat MSCs released 10.3+/-1.3 pmol CGRP/1 x 10(6) cells/48 h (mean+/-S.E.M., n=3) into culture medium at MOI 300 and the CGRP-containing culture supernatant from the transduced cells inhibited the proliferation of rat pulmonary artery smooth muscle cells (PASMCs) and rat aortic smooth muscle cells (ASMCs) in culture. Co-culture of the transduced rat MSCs with rat PASMCs or rat ASMCs also inhibited smooth muscle cell proliferation. These findings suggest that this novel adult stem cell-based CGRP gene therapy has potential for the treatment of cardiovascular diseases including pulmonary hypertension.

Actins↗
PubMed1994

Chronic exposure to benzodiazepine receptor ligands uncouples the gamma-aminobutyric acid type A receptor in WSS-1 cells.

Chronic exposure to benzodiazepines can result in an "uncoupling" of gamma-aminobutyric acid (GABA) receptors and benzodiazepine receptors (BzR) both in primary neuronal cell cultures and in vivo. The effect of chronic exposure to BzR ligands was examined in an engineered cell line (WSS-1) stably expressing "type I" GABAA receptors. Chronic exposure to flurazepam produced a concentration- (EC50, approximately 1.1 microM after a 48-hr exposure) and time-dependent (t1/2, approximately 3 hr at 100 microM) reduction in the efficacy (Emax) of GABA to enhance [3H]flunitrazepam binding to BzR, a characteristic of uncoupling in native GABAA receptor isoforms. Uncoupling of GABAA receptors and BzR without concomitant changes in BzR density was also produced by chronic exposure to other, structurally diverse, BzR ligands, including Ro 15-1788 and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, but was not manifested after exposure to the 5-hydroxytryptamine reuptake blocker fluoxetine. Chronic (12-48-hr) exposure to flurazepam did not remarkably alter levels of alpha 1 and gamma 2 mRNAs, which constitute GABAA receptors in this cell line. Based on these findings, it is hypothesized that uncoupling of GABAA receptors and BzR in this engineered cell line can proceed without the elaboration of additional novel subunits and could involve either post-translational modification of GABAA receptor proteins or changes in subunit stoichiometry.

Cell Line↗
PubMed2001

Lymphotactin expression by engineered myeloma cells drives tumor regression: mediation by CD4+ and CD8+ T cells and neutrophils expressing XCR1 receptor.

The C chemokine lymphotactin has been characterized as a T cell chemoattractant both in vitro and in vivo. To determine whether lymphotactin expression within tumors could influence tumor growth, we transfected an expression vector for lymphotactin into SP2/0 myeloma cells and tested their ability to form tumors in BALB/c and nude mice. Transfection did not alter cell growth in vitro. Whereas SP2/0 cells gave rise to a 100% tumor incidence, lymphotactin-expressing SP2/0-Lptn tumors invariably regressed in BALB/c mice and became infiltrated with CD4(+) and CD8(+) T cells and neutrophils. Regression of the SP2/0-Lptn tumors was associated with a type 1 cytokine response and dependent on both CD4(+) and CD8(+) T cells, but not NK cells. Both SP2/0 and SP2/0-Lptn tumors grew in nude mice, but growth of the latter tumors was retarded and associated with heavy neutrophil responses; this retardation of SP2/0-Lptn tumor growth was reversed by neutrophil depletion of the mice. Our data also indicate that mouse neutrophils express the lymphotactin receptor XCR1 and that lymphotactin specifically chemoattracts these cells in vitro. Thus, lymphotactin has natural adjuvant activities that may augment antitumor responses via effects on both T cells and neutrophils and thereby could be important in gene transfer immunotherapies for some cancers.

Animals↗
PubMed2005

Cytokine production by dendritic cells genetically engineered to express IL-4: induction of Th2 responses and differential regulation of IL-12 and IL-23 synthesis.

Dendritic cells (DCs) retrovirally transduced with IL-4 have recently been shown to inhibit murine collagen-induced arthritis and associated Th1 immune responses in vivo, but the mechanisms that underly these effects are not yet understood. In this report we demonstrate that IL-4-transduced DCs loaded with antigen led to lower T cell production of IFN-gamma, increased production of IL-4, and an attenuated, delayed type hypersensitivity response. We hypothesized that the ability of such DCs to regulate the Th1 immune response in vivo depends in part on their capacity to produce IL-12 and IL-23. Quantitative mRNA analysis revealed that IL-4-transduced DCs stimulated with CD40 ligand expressed higher levels of IL-12p35 mRNA, but lower levels of mRNA for IL-23p19 and the common subunit p40 found in both IL-12 and IL-23, compared with control DCs. These results, which indicate that expression of the IL-12 and IL-23 subunits is differentially regulated in IL-4-transduced DCs, were confirmed by ELISA of the IL-12 and IL-23 heterodimers. Thus, therapeutic suppression of Th1 -mediated autoimmunity (as recently shown in murine collagen-induced arthritis) and induction of Th2 responses in vivo by IL-4-transduced DCs occurs despite their potential to produce increased levels of IL-12, but could reflect, in part, decreased production of IL-23.

Animals↗
PubMed1992

Genetically engineered endothelial cells remain adherent and viable after stent deployment and exposure to flow in vitro.

Intravascular stents, currently in experimental human use for recurrent arterial stenosis, are plagued by subacute thrombosis. As a therapeutic approach to stent-related thrombosis, we and others have suggested coating stents with endothelial cells before implantation. In a previous study we demonstrated the feasibility of coating stents with endothelial cells that were genetically modified to secrete large amounts of human tissue plasminogen activator. In the present study we attempted both to develop a clinically applicable protocol for stent seeding and to test whether seeded cells would remain adherent to stents after exposure to pulsatile flow. Endothelial cells were harvested from the saphenous veins of sheep with survival of the donor animals. Harvested cells were transduced with a retroviral vector containing a marker gene and seeded onto catheter-mounted stents under sterile conditions. Scanning electron microscopy revealed complete coverage of the stent surfaces by seeded cells. Stents were expanded and exposed to pulsatile flow in vitro. Substantial cell retention was observed on the lateral stent surfaces by light microscopy and scanning electron microscopy; fewer cells were seen on the luminal and abluminal surfaces. Removal of seeded cells from flow-exposed stents by trypsin digestion resulted in the recovery of approximately 70% of the seeded cells. These cells were viable and healthy as judged by their ability to proliferate to confluence with the same kinetics as control (non-flow-exposed) cells. Autologous genetically modified endothelial cells can be seeded onto catheter-mounted stents in a sterile manner, and stent deployment under flow conditions results in substantial retention of viable cells.

Animals↗