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At least 937 records · Page 52Linked to original sources

PubMed1994

Fibroblasts genetically engineered to secrete interleukin 12 can suppress tumor growth and induce antitumor immunity to a murine melanoma in vivo.

Interleukin 12 (IL-12), a disulfide-linked heterodimeric cytokine produced primarily by macrophages, is composed of light (p35) and heavy (p40) chains. It binds to a receptor on T-cells and natural killer cells, promoting the induction of primarily a TH1 response in vitro and in vivo. To determine whether paracrine IL-12 secretion can alter tumor cell growth or promote antitumor immunity, we have developed a delivery system using genetically engineered fibroblasts in murine tumor models. NIH3T3 cells were stably transfected to express 100-240 units/10(6) cells/48 h of IL-12 using expression plasmids carrying both the murine p35 and p40 genes of murine IL-12. The effects of paracrine secretion of IL-12 on tumor establishment and vaccination models were examined using the poorly immunogenic murine melanoma cell line (BL-6) in C57BL/6 mice. To determine the effects of IL-12 on tumor formation, nonirradiated BL-6 cells were inoculated s.c. into C57BL/6 mice admixed with NIH3T3 cells transfected with both subunits of mIL-12 (3T3-IL-12) or with cells transfected with only the neomycin phosphotransferase gene (3T3-Neo). Compared to mice given injections of BL-6 alone, the day of emergence of detectable tumors was significantly delayed in mice given injections of BL-6 admixed with 3T3-IL-12, but not in mice with BL-6 admixed with 3T3-Neo. Effectiveness in this system was related to the amount of IL-12 expressed by the 3T3-IL-12. To determine the ability of locally secreted IL-12 at the tumor site to induce antitumor immunity, 10(6) irradiated tumor cells mixed with 3T3-IL-12 or 3T3-Neo were injected as a vaccine, and the response to a tumor challenge was subsequently examined. With a tumor challenge of less than 1 x 10(5) nonirradiated BL-6 cells, significant delay of establishment of tumor was noted with a relatively small amount of IL-12 secretion (1.2 units/5 x 10(5) cells/48 h). Larger amounts of secreted IL-12 provided no additional therapeutic benefit. Histological examination of tumor inoculum with 3T3-IL-12 secreting a high level of IL-12 showed peritumoral accumulation of macrophages, a characteristic capsule around the tumor composed of palisades of fibroblasts, and decreased numbers of CD4+ cells in the tumor. These results suggest that local delivery of IL-12 inhibits tumor growth in a dose dependent manner but leads to the development of an antitumor immune response when IL-12 is expressed at the tumor site at the relatively small amount indicated above.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗

PubMed1999

Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells.

The analysis of many natural glycoproteins and their recombinant counterparts from mammalian hosts has revealed that the basic oligosaccharide structures and the site occupancy of glycosylated polypeptides are primarily dictated by the protein conformation. The equipment of many frequently used host cells (e.g. BHK-21 and CHO-cells) with glycosyltransferases, nucleotide-sugar synthases and transporters appears to be sufficient to guarantee complex-type glycosylation of recombinant proteins with a high degree of terminal alpha2-3 sialylation even under high expression conditions. Some human tissue-specific terminal carbohydrate motifs are not synthesized by these cells since they lack the proper sugar-transferring enzymes (e.g. alpha1-3/4 fucosyltransferases, alpha2-6 sialyltransferases). Glycosylation engineering of these hosts by stable transfection with genes encoding terminal human glycosyltransferases allows to obtain products with tailored (human tissue-specific) glycosylation in high yields. Using site-directed mutagenesis, unglycosylated polypeptides can be successfully converted in N- and/or O-glycoproteins by transferring glycosylation domains (consisting of 7-17 amino acids) from donor glycoproteins to different loop regions of acceptor proteins. The genetic engineering of glycoproteins and of host cell lines are considered to provide a versatile tool to obtain therapeutic glyco-products with novel/improved in-vivo properties, e.g. by introduction of specific tissue-targeting signals by a rational design of terminal glycosylation motifs.

Animals↗

PubMed1991

Experimental therapy of human glioma by means of a genetically engineered virus mutant.

Malignant gliomas are the most common malignant brain tumors and are almost always fatal. A thymidine kinase-negative mutant of herpes simplex virus-1 (dlsptk) that is attenuated for neurovirulence was tested as a possible treatment for gliomas. In cell culture, dlsptk killed two long-term human glioma lines and three short-term human glioma cell populations. In nude mice with implanted subcutaneous and subrenal U87 human gliomas, intraneoplastic inoculation of dlsptk caused growth inhibition. In nude mice with intracranial U87 gliomas, intraneoplastic inoculation of dlsptk prolonged survival. Genetically engineered viruses such as dlsptk merit further evaluation as novel antineoplastic agents.

Animals↗

PubMed2001

Delivery of FGF-2 but not VEGF by encapsulated genetically engineered myoblasts improves survival and vascularization in a model of acute skin flap ischemia.

Stimulating angiogenesis by gene transfer approaches offers the hope of treating tissue ischemia which is untreatable by currently practiced techniques of vessel grafting and bypass surgery. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2) are potent angiogenic molecules, making them ideal candidates for novel gene transfer protocols designed to promote new blood vessel growth. In this study, an ex vivo gene therapy approach utilizing cell encapsulation was employed to deliver VEGF and FGF-2 in a continuous and localized manner. C(2)C(12) myoblasts were genetically engineered to secrete VEGF(121), VEGF(165) and FGF-2. These cell lines were encapsulated in hollow microporous polymer membranes for transplantation in vivo. Therapeutic efficacy was evaluated in a model of acute skin flap ischemia. Capsules were positioned under the distal, ischemic region of the flap. Control flaps showed 50% necrosis at 1 week. Capsules releasing either form of VEGF had no effect on flap survival, but induced a modest increase in distal vascular supply. Delivery of FGF-2 significantly improved flap survival, reducing necrosis to 34.2% (P < 0.001). Flap vascularization was significantly increased by FGF-2 (P < 0.01), with numerous vessels, many of which had a large lumen diameter, growing in the proximity of the implanted capsules. These results demonstrate that FGF-2, delivered from encapsulated cells, is more efficacious than either VEGF(121) or VEGF(165) in treating acute skin ischemia and improving skin flap survival. Furthermore, these data attest to the applicability of cell encapsulation for the delivery of angiogenic factors for the treatment and prevention of tissue ischemia.

Acute Disease↗

PubMed2005

Cell-based vaccines for renal cell carcinoma: genetically-engineered tumor cells and monocyte-derived dendritic cells.

Initial vaccine developments for renal cell carcinoma (RCC) have concentrated on cell-based approaches in which tumor cells themselves provide mixtures of unknown tumor-associated antigens as immunizing agents. Antigens derived from autologous tumors can direct responses to molecular composites characteristic of individual tumors, whereas antigens derived from allogeneic tumor cells must be commonly shared by RCC. Three types of cell-based vaccine for RCC have been investigated: isolated tumor cell suspensions, gene modified tumor cells and dendritic cells (DCs) expressing RCC-associated antigens. Approaches using genetic modification of autologous RCC have included ex vivo modification of tumor cells or modification of tumors in vivo. We have used gene-modification of allogeneic tumor cell lines to create generic RCC vaccines. More recently, emphasis has shifted to the use of DCs as cell-based vaccines for RCC. DCs have moved to a position of central interest because of their excellent stimulatory capacity, combined with their ability to process and present antigens to both naive CD4 and CD8 cells. The long impasse in identifying molecular targets for specific immunotherapy of RCC is now rapidly being overcome through the use of tools and information emerging from human genome research. Identification of candidate molecules expressed by RCC using cDNA arrays, combined with protein arrays and identification of peptides presented by MHC molecules, allow specific vaccines to be tailored to the antigenic profile of individual tumors, providing the basis for development of patient-specific vaccines.

Cancer Vaccines↗

PubMed2004

Genetically engineered Sertoli cells are able to survive allogeneic transplantation.

The immunoprotective nature of the testis has led to numerous investigations for its ability to protect cellular grafts. Sertoli cells (SCs) are at least partially responsible for this immunoprotective environment and survive allogeneic and xenogeneic transplantation. The ability of SCs to survive transplantation leads to the possibility that they could be engineered to deliver therapeutic proteins. As a model to test this hypothesis, we examined the ability of SCs that produce green fluorescent protein (GFP) to survive transplantation and continue expressing GFP. SCs were isolated from transgenic mice engineered to express GFP and transplanted as aggregates under the kidney capsule of severe combined immunodeficient (SCID) and Balb/c mice. Using this paradigm, it was possible to compare the survival of transgenic SCs directly in both immunodeficient and immunocompetent recipients. Fluorescence microscopy of the kidney capsule and immunohistochemistry of the grafts for GFP and GATA-4 revealed the presence of GFP-expressing SCs under the kidney capsule of SCID and Balb/c mice at both 30 and 60 days post-transplantation. In contrast, islets transplanted to Balb/c mice were rejected. Thus, SCs survive transplantation and continue to express GFP raising the possibility that SCs can be engineered using transgenic technology to produce proteins, such as insulin, factor VIII, or dopamine for the treatment of diabetes, hemophilia or Parkinson's disease, respectively.

Animals↗

PubMed2005

Genetically engineered myeloma cell vaccine.

Tumor cells engineered to express immunogenes have been used for cancer vaccines to induce antitumor immunity and to study the antitumor immune mechanisms derived from immunogene expression. In this chapter, we describe the design and methods for cloning a cDNA gene coding for the mouse CD40L molecule and for construction of the expression vector pcDNA-CD40L, as well as the methods for generation of engineered myeloma cells J558/CD40L expressing CD40 ligand. We also demonstrate that the engineered J558/CD40L tumor cells lose their tumorigenicity in syngeneic mice, and that the inoculation of J558/CD40L tumor cells further leads to protective immunity against wild-type J558 tumors.

Animals↗

PubMed1994

A genetically engineered, protein-based optical biosensor of myosin II regulatory light chain phosphorylation.

Myosin II is an important motor in the contraction of smooth and striated muscle as well as in a variety of non-muscle cell motile events including cytokinesis, cortical contractions during migration of fibroblasts, and capping of receptors. Phosphorylation of the 20-kDa light chain by myosin light chain kinase is part of the regulation of smooth muscle and mammalian nonmuscle myosin II. We designed, characterized, and tested the use of a protein-based optical biosensor to monitor this phosphorylation "switch." A regulatory light chain was genetically engineered to contain a single cysteine at amino acid position 18. The mutant light chain (Cys18.LC20), reacted with the fluorophore acrylodan, responded to phosphorylation of serine 19 with a fluorescence emission quenching of 60% and a 28-nm red-shift. When the acrylodan-labeled mutant light chain (AC-Cys18.LC20) was exchanged into turkey gizzard myosin II, it exhibited a 25% fluorescence emission quenching and a 10-nm red-shift upon phosphorylation of serine 19. The myosin II optical biosensor exhibited nearly control levels of the rate of phosphorylation, K+ATPase activity, and in vitro motility. The acrylodan-labeled light chain was exchanged into the A-bands of chicken pectoralis myofibrils in situ to demonstrate the localization and activity of the biosensor in a highly ordered contractile system. Fluorometry and quantitative fluorescence microscopic imaging experiments demonstrated that AC-Cys18.LC20 exchanged myofibrils expressed a phosphorylation-dependent fluorescence change. Labeled light chains were also incorporated into stress fibers of living fibroblasts and smooth muscle cells. This general approach of combining molecular biology and fluorescence spectroscopy to create novel protein-based optical biosensors should provide valuable tools for investigations with model systems and solution studies and ultimately yield important information about temporal-spatial chemical and molecular changes in live cells.

2-Naphthylamine↗

PubMed2005

[Research progress in genetic engineering of plant secondary metabolism].

Secondary metabolism plays an important role in plant life as well as the interaction between plants and environmental factors. Many secondary metabolites derived from plants have been used for the production of medicines, dyes, insecticides, food flavors, fragrances and so on. With increasingly comprehensive understanding of the plant metabolic networks, great progress has been made in the genetic improvement of plant secondary metabolic pathways through gene engineering. Strategies for the genetic engineering of plant secondary metabolism include: (1) enabling the host plant to accumulate a novel desirable compound by transformation of single/multiple enzyme gene (s) or a whole metabolic pathway; (2) decreasing target gene expression or inhibiting competitive metabolic pathway to achieve metabolic flux towards higher production of particular molecules through antisense RNA and RNA interference technologies; (3) effectively manipulating the transcription factors responsible for the metabolic regulation at multiple steps in a given pathway so as to have a great synthesis of the target bio-chemicals. Basing on author's research work on flavonoid synthesis mechanism in soybean seed and its gene engineering, recent progress in the engineering of plant secondary metabolism involved in the synthesis of anthocyanins, flavonoids, alkaloids, terpenoids, benzoic acid derivatives etc are reviewed.

Gene Expression Regulation, Plant↗

PubMed1989