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At least 577 records · Page 32Linked to original sources
PubMed1991

Production of immunogenic HIV-1 viruslike particles in stably engineered monkey cell lines.

A proviral fragment from human immunodeficiency virus type 1 (HIV-1) (LAV-1BRU) containing only protein-coding information, was expressed in COS cells using constitutive promoters in transient and stable transfection experiments. The presence of viruslike particles in cell supernatants was verified by Western blot analysis, density gradient centrifugation, and electron microscopy. Transfection of Vero cells with a similar construct employing the human metallothionein promoter led to the isolation of stable cell lines exhibiting inducible viruslike particle expression in response to cadmium chloride treatment. Induction ratios for viruslike particle expression were in excess of 1000-fold with production levels of p24 core antigen as high as 0.6 mg/L per 24 h. HIV-1 viruslike particles were immunogenic in mice, leading to strong envelope and core-specific humoral responses after two immunizations. The development of stable cell lines expressing significant quantities of HIV-1 viruslike particles offers an alternative to the use of live virus vectors for the production and evaluation of particle-based AIDS vaccines.

Animals↗
PubMed1998

Bystander effect-mediated therapy of experimental brain tumor by genetically engineered tumor cells.

Transfer of the herpes simplex virus-thymidine kinase (HSV-tk) gene, followed by administration of ganciclovir (GCV), generates the "bystander effect," in which HSV-tk-negative wild-type cells, as well as HSV-tk-expressing cells, are killed by GCV. To eradicate an intracranial tumor by this bystander effect, we injected the tumor cells transduced with the HSV-tk gene (TK cells) in the vicinity of the preimplanted wild-type tumor and then administered GCV. Wild-type 9L-gliosarcoma cells (1 x 10[5]) were implanted into the brain of syngeneic Fisher rats. On the next day, rats were injected with TK cells (1 x 10(5) or 3 x 10[5]) or medium alone at the same brain coordinate and then treated with GCV or saline. Administration of GCV significantly prolonged the survival of the rats injected with TK cells compared with that injected with medium alone (p < 0.01). Reduction in tumor size and retardation of tumor growth were observed by serial magnetic resonance imaging in the rats that received the combination of TK cells and GCV. The results show that the bystander effect is also achieved in vivo even when TK cells and wild-type cells are not simultaneously implanted. This treatment modality circumvents potential risks accompanied with in vivo gene transfer. Because there remained substantially no HSV-tk-positive cells in the recurrent tumors, this modality offers a "safe" therapeutic strategy against human malignant gliomas.

Animals↗
PubMed2006

Differential growth of IFN-beta-engineered tumor cells in nude and IFN receptor-null mice.

The purpose of this study was to investigate the therapeutic potential of interferon-beta (IFN-beta) against tumors that resist its antiproliferative effects. Mouse fibrosarcoma cells (UV-2237m-P) and their counterparts, transfected with either IFN-beta cDNA (UV-2237m-IFN-beta) or its control vector (UV-2237m-neo), were used in the study. UV-2237m-IFN-beta cells, still expressing functional IFN receptors, were resistant to the antiproliferative effects of IFN-beta. UV-2237m-P and UV-2237m-neo cells produced progressive tumors in both nude and IFN receptor-null nude (IFNAR-/-nude) mice. In contrast, growth of UV-2237m-IFN-beta cells was significantly delayed in nude mice. UV-2237m-IFN-beta tumors from nude mice contained fewer microvessels, fewer proliferating cells, and more apoptotic cells than did UV-2237m-P and UV-2237m-neo tumors. They expressed high levels of inducible nitric oxide synthase (iNOS) and were densely infiltrated by macrophages. Incubation with macrophages from nude mice, but not those from IFNAR-/- nude mice or iNOS-null/nude mice, led to more significant killing of UV-2237m-IFN-beta cells than that of control cells, which was blocked by iNOS inhibitor N-methylarginine. Similarly, more UV-2237m-IFN-beta cells were killed when they were incubated with spleen lymphocytes from nude mice. These data indicate that IFN-beta can inhibit growth of IFN-beta-resistant tumors by T cell-independent host-mediated mechanisms, including the role of macrophages, natural killer (NK) cells, and iNOS activity.

Animals↗
PubMed2005

In vitro assays for anticancer drug discovery--a novel approach based on engineered mammalian cell lines.

Despite decisive progress in understanding the molecular biology of cancer development, cytotoxic anticancer drugs continue to be the cornerstone of modern antitumor therapies. The developmental therapeutics program, initiated by the US National Institutes of Health's National Cancer Institute in the early 1990s, pioneered massive-scale screening for agents able to phenotypically interfere with the growth and viability of neoplastic cell lines derived from a representative panel of human carcinogenic tissues. Capitalizing on advanced knowledge of molecular processes particular for neoplastic cell characteristics, target-specific screening scenarios became since increasingly popular. With drug targets defined, natural and synthetic (combinatorial) compound/peptide/nucleic acid libraries available and the high-throughput screening technology of the systems' biology era in place, the quo vadis of anticancer drug discovery seems to be well determined. We review recent advances in cytotoxic anticancer drug assay design with emphasis on a novel mammalian cell-based anticancer drug finder technology for the discovery of cytotoxic drugs with fewer side-effects on non-dividing cells.

Animals↗
PubMed2005

Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome.

The minimization of a genome is necessary to identify experimentally the minimal gene set that contains only those genes that are essential and sufficient to sustain a functioning cell. Recent developments in genetic techniques have made it possible to generate bacteria with a markedly reduced genome. We developed a simple system for formation of markerless chromosomal deletions, and constructed and characterized a series of large-scale chromosomal deletion mutants of Escherichia coli that lack between 2.4 and 29.7% of the parental chromosome. Combining deletion mutations changes cell length and width, and the mutant cells with larger deletions were even longer and wider than the parental cells. The nucleoid organization of the mutants is also changed: the nucleoids occur as multiple small nucleoids and are localized peripherally near the envelope. Inhibition of translation causes them to condense into one or two packed nucleoids, suggesting that the coupling of transcription and translation of membrane proteins peripherally localizes chromosomes. Because these phenotypes are similar to those of spherical cells, those may be a consequence of the morphological change. Based on the nucleoid localization observed with these mutants, we discuss the cellular nucleoid dynamics.

Cell Nucleus↗
PubMed1993

Engineered yeast cells as model to study coupling between human xenobiotic metabolizing enzymes. Simulation of the two first steps of benzo[a]pyrene activation.

Human microsomal epoxide hydrolase and cytochrome P450 (P450) 1A1 were coexpressed in Saccharomyces cerevisiae from expression cassettes integrated respectively into the host chromosomal DNA and on a multicopy plasmid in a strain already overexpressing yeast NADPH-cytochrome P450 reductase (P450 reductase). A styrene-oxide-hydrolase activity (2 nmol.min-1.mg microsomal protein-1) and a 7-ethoxyresorufin-O-deethylase activity (320 pmol.min-1.mg microsomal protein-1) characteristic respectively of microsomal epoxide hydrolase and P450 1A1 were detected. The conversion of benzo[a]pyrene (B[a]P) to B[a]P-7,8-dihydrodiol both in microsomal preparations and in growing yeast cells was observed, demonstrating an efficient coupling between the two human enzymes. Kinetic analysis indicated that the B[a]P-7,8-oxide produced by the P450-1A1-dependent reaction does not accumulate before hydrolysis by microsomal epoxide hydrolase. This system was also used as a control to evaluate the coupling efficiency of a mixture of microsomes or of yeast cells containing separately the individual enzymes (i.e., human P450 1A1 and microsomal epoxide hydrolase). B[a]P-7,8-oxide was well converted to the corresponding dihydrodiol with a mixture of microsomes. In contrast, when the same experiment was repeated with a mixture of cells expressing independently the two activities, dihydrodiol formation was not observed. Coexpression of human phase I and phase II enzymes in a single yeast cell and microsome mixture thus appear to be complementary tools for the simulation of human-drug-metabolism or carcinogen-metabolism pathways.

Base Sequence↗
PubMed2003

Virus replication in engineered human cells that do not respond to interferons.

The V protein of the paramyxovirus simian virus 5 blocks interferon (IFN) signaling by targeting STAT1 for proteasome-mediated degradation. Here we report on the isolation of human cell lines that express the V protein and can no longer respond to IFN. A variety of viruses, particularly slow-growing wild-type viruses and vaccine candidate viruses (which are attenuated due to mutations that affect virus replication, virus spread, or ability to circumvent the IFN response), form bigger plaques and grow to titers that are increased as much as 10- to 4,000-fold in these IFN-nonresponsive cells. We discuss the practical applications of using such cells in vaccine development and manufacture, virus diagnostics and isolation of newly emerging viruses, and studies on host cell tropism and pathogenesis.

Animals↗
PubMed1997

Heparin/heparan sulfate chelation inhibits control of vascular repair by tissue-engineered endothelial cells.

The relative importance of heparin-like compounds in mediating vascular repair is unclear. We investigated how protamine, a chelator of heparin, affected endothelial cell inhibition of vascular smooth muscle cell growth and intimal hyperplasia. The 52% (P < 0.001) reduction in smooth muscle cell proliferation produced by postconfluent endothelial cell-conditioned medium was entirely reversed by pretreatment of medium with heparinase and heparitinase and was inhibited in a dose-dependent fashion by the coadministration of protamine. Pretreatment of conditioned medium with heparinase and heparitinase largely prevented protamine's mitogenic activity, suggesting that protamine affects growth by interacting with heparin-like compounds. Perivascular implantation of polymerengrafted endothelial cells reduced neointima formation in denuded rat carotid arteries by 92% (P < 0.001) and cell proliferation by 81% (P < 0.001). Coadministration of protamine abolished the inhibitory potential of the cell implants, resulting in a nearly twofold exacerbation of intimal hyperplasia compared with controls (P < 0.001). Thus heparin-like molecules are essential to the biochemical regulation of vascular repair provided by endothelial cells, and the continued routine clinical use of heparin chelators, like protamine, may be questionable.

Animals↗
PubMed1997

Creation of an In vivo cytosensor using engineered mesangial cells. Automatic sensing of glomerular inflammation controls transgene activity.

Automatic control over exogenous gene expression in response to the activity of disease is a crucial hurdle for gene transfer-based therapies. Towards achieving this goal, we created a "cytosensor" that perceives local inflammatory states and subsequently regulates foreign gene expression. alpha-Smooth muscle actin is known to be expressed in glomerular mesangial cells exclusively in pathologic situations. CArG box element, the crucial regulatory sequence of the alpha-smooth muscle actin promoter, was used as a sensor for glomerular inflammation. Rat mesangial cells were stably transfected with an expression plasmid that introduces a beta-galactosidase gene under the control of CArG box elements. In vitro, the established cells expressed beta-galactosidase exclusively after stimulation with serum. To examine whether the cells are able to automatically control transgene activity in vivo, serum-stimulated or unstimulated cells were transferred into normal rat glomeruli or glomeruli subjected to anti-Thy 1 glomerulonephritis. When stimulated cells were transferred into the normal glomeruli, beta-galactosidase expression was switched off in vivo within 3 d. In contrast, when unstimulated cells were transferred into the nephritic glomeruli, transgene expression was substantially induced. These data indicate the feasibility of using the CArG box element as a molecular sensor for glomerular injury. In the context of advanced forms of gene therapy, this approach provides a novel concept for automatic regulation of local transgene expression where the transgene is required to be activated during inflammation and deactivated when the inflammation has subsided.

Actins↗
PubMed2006

Engineered T cell receptors and their potential in molecular medicine.

T cell receptors are among the most specific biological structures found in nature and are therefore excellent candidates for the molecular targeting of antigen. It is becoming increasingly apparent that common sets of T cell receptors are frequently used in humans to combat pathogen and cancer derived threats. Given that many of these conserved T cell receptors have high affinity for their target ligands, there is potential to amass virtual banks of "off-the-shelf" receptors for use in a wide range of immunotherapeutic strategies. Additionally, such T cell receptors could become basic blueprints for artificial enhancement through mutagenesis, thereby creating an even better 3-dimensional fit for their cognate targets. Indeed, preliminary approaches using both "natural" and "supernatural" T cell receptors have shown promise in treating autoimmunity and malignancy. This review will discuss these studies and other approaches through which T cell receptors can be exploited in immunodiagnostics, pathogen control and gene therapy.

Animals↗
PubMed2001

Engineering dendritic cell grafts for clinical trials in cellular immunotherapy of cancer: example of chronic myelogenous leukemia.

Dendritic cells are pivotal regulators of immune reactivity and immune tolerance. The observation that dendritic cells can recruit naive T-cells has invigorated cancer immunology and stimulated clinical trials of dendritic cells in immunotherapy. However, variables inherent in preparation and use of dendritic cell grafts remain to be tested. Here we discuss the role of ex vivo dendritic cell processing for in vivo antigen presentation in clinical trials. As an example of the complexity in a clinical trial of dendritic cell vaccines, we present our ongoing trial in immunotherapy of chronic myelogenous leukemia.

Antigen Presentation↗
PubMed2002

In vitro study of encapsulation therapy for Fabry disease using genetically engineered CHO cell line.

Fabry disease is an X-linked recessive disorder caused by a deficiency of the lysosomal hydrolase alpha-galactosidase A (alpha-gal). The deficiency of this enzyme leads to the systemic deposition of ceramide trihexoside (CTH) in various tissues and organs. Enzyme replacement using IV doses of recombinant human alpha-gal produced in CHO cells or in human fibroblasts is currently being evaluated in clinical trials as a potential therapy for this disease. However, it requires lifelong therapy involving a large amount of purified alpha-gal. As a novel approach for treatment of Fabry disease we used polymer encapsulated Chinese hamster ovary (CHO) cells genetically modified to express alpha-gal. The secreted high levels of alpha-gal passed through the semipermeable polymeric membrane. Using coculture system with Fabry fibroblasts, the secreted enzyme was taken up in cells, resulting in reduced accumulation of CTH in Fabry fibroblasts. This in vitro study demonstrated that an encapsulated alpha-gal-secreting cell line can be used to treat Fabry mice by transplantation in vivo. Judging from the protection against immune rejection by a semipermeable synthetic membrane, this novel approach may be applied to treat patients with Fabry disease and other lysosomal storage diseases.

Animals↗
PubMedDate not supplied

Inhibition or promotion of tumor growth by granulocyte-macrophage colony stimulating factor derived from engineered tumor cells is dose-dependent.

BACKGROUND: Granulocyte-macrophage colony stimulating factor (GM-CSF) has been widely investigated as an adjuvant factor for tumor immunotherapy. However, the results are controversial with antitumor effects in some studies and a tumor growth promotion effect in others. MATERIALS AND METHODS: In order to determine whether there is a dose-dependent effect of GM-CSF on tumor growth, murine GM-CSF-expressing vector was constructed and transfected into TC-1 tumor cells and various clones stably expressing different levels of GM-CSF were obtained. The growth of these clones in vivo was studied. RESULTS: Although these clones grow at a similar rate in vitro, their growth in vivo is dramatically different. Clones expressing high levels (>10,000 pg/ml) of GM-CSF grow significantly faster than the control (p <0.001); clones expressing low levels (<100 pg/ml) of GM-CSF grow significantly slower than the control (p<0.001); while clones expressing intermediate levels (1000-2000 pg/ml) of GM-SCF grow at a similar rate as the control (p >0. 05). The high levels of GM-CSF secreted by tumor cells induced granulocytosis and lymphopenia. The antitumor growth effect induced by low levels of GM-CSF is not due to the function of lymphocytes. CONCLUSION: The inhibition or promotion of tumor growth by GM-CSF secreted from tumor cells is dose-dependent.

Animals↗
PubMed1991

Functional aspects of mammalian neural transplantation.

Although initially perceived as a method to study neural development and regeneration, neural transplantation has recently become a very promising approach in its own right as a therapeutic tool to treat neurodegenerative disorders. The development of several animal models which mimic aspects of clinical disorders such as Parkinson's disease, Alzheimer's disease, and Huntington's chorea, provides systems in which to study the potential benefits of grafts derived from different sources. Both fetal and adult donor tissues presently are under investigation. Additionally, cell lines and genetically engineered cells also are being developed as suitable graft material. Important aspects of graft-host interaction, including cell survival, host regeneration, immune interactions, improved behavior, and blood-brain barrier phenomena, may be studied in these transplant models. Advances in this field of biomedical research have led to clinical trials in patients afflicted with Parkinson's disease who now are undergoing transplantation therapy. Thus far, the results have been equivocal, raising important ethical questions about continued clinical studies until more is understood about how neural transplants function and interact with the host. Nevertheless, neural transplantation holds tremendous promise as a future therapeutic tool to treat progressive and irreversible neural disorders.

Animals↗
PubMed2001

[Human GDNF cDNA-engineered SH-SY5Y cells' neurotrophic and protective effect on primary dopaminergic neurons of rat].

OBJECTIVE: To construct a kind of engineered cell secreting human GDNF and study its possible effects on gene therapy of Parkinson's disease. METHOD: Human GDNF cDNA with Kozak sequence was cloned by RT-PCR, and then was transfected into SH-SY5Y cell line of human neuroblastoma. These engineered cells were co-cultured with primary mesencephalic cells of rats. Dopaminergic neurons were examined by immunohistochemistry. RESULTS: The number of dopaminergic neurons protected by engineered cells increased at least by 95.4% in comparison with the control cells (P < 0.01). The number of dopaminergic neurons protected by engineered cells against MPP+ toxicity increased 9.5-10.8 times (P < 0.01). CONCLUSION: A kind of engineered SH-SY5Y cells secreting human GDNF has been constructed successfully. These cells obviously protect dopaminergic neurons against degeneration and MPP+ toxication and may play an important role in gene therapy of Parkinson's disease.

1-Methyl-4-phenylpyridinium↗
PubMed1995

Tissue engineered perivascular endothelial cell implants regulate vascular injury.

Molecular biomaterial engineering permits in vivo transplantation of cells and tissues, offering the promise of restoration of physiologic control rather than pharmacologic dosing with isolated compounds. We engrafted endothelial cells on Gelfoam biopolymeric matrices with retention of viability, normal growth kinetics, immunoreactivity, and biochemical activity. The production of heparan sulfate proteoglycan and inhibition of basic fibroblast growth factor binding and activity by engrafted cells were indistinguishable from endothelial cells grown in culture. Perivascular implantation of Gelfoam-endothelial cell scaffolds around balloon-denuded rat carotid arteries reduced intimal hyperplasia 88.1%, far better than the isolated administration of heparin, the most effective endothelial mimic compound. In concert with a reduction in intimal area, cell proliferation was reduced by > 90%. To our knowledge, there have been no previous reports of extravascular cell implants controlling vasculoproliferative disease. Tissue engineered cells offer the potential for potent methods of vascular growth regulation and insight into the complex autocrine-paracrine control mechanisms within the blood vessel wall.

Animals↗
PubMed2006

Polymeric gene transfection on insulin-secreting cells: sulfonylurea receptor-mediation and transfection medium effect.

PURPOSE: In vitro transfection of secreting cells is regarded as one strategy for improved cell engineering/ transplantation. Insulin-secreting insulinoma cell lines or pancreatic beta-cells could be genetically engineered using designed polymeric vectors which are safer than viral vectors. This study investigates the effects of the constituents in transfection media on polymeric transfection. METHODS: Polyplexes conjugated with sulfonylurea (SU) were evaluated under different transfection conditions for gene transfection and their effects on cytotoxicity and insulin secretion. Several components in transfection media specifically associated with the insulin secretion pathway were amino acids, vitamins, Ca2+ and K+. The interactions of the polyplexes with insulin were monitored by surface charge and particle size to monitor how insulin as a protein influences transfection. RESULTS: For an insulin-secreting cell line (RINm5F), polyplexes in Ca2+--containing KRH medium (Ca2+(+)KRH) enhanced transfection and did not cause damage to biological functions. When adding amino acids, vitamins, or K+ or depleting Ca2+ from Ca2+(+)KRH, poly(L-lysine)/DNA complexes showed a greater reduction in transfection than SU receptor (SUR)-targeting polyplexes (SU-polyplex). Positively charged polyplexes interacted with insulin, developing a negative surface charge, and these interactions may cause a decrease in transfection. CONCLUSION: The findings suggest that in vitro and ex vivo polymeric transfection of insulin-secreting cells can be modulated and enhanced by adjusting the transfection conditions.

ATP-Binding Cassette Transporters↗