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PubMed2006

Autologous adult human Schwann cells genetically modified to provide alternative cellular transplants in peripheral nerve regeneration.

OBJECT: The purpose of this study was to provide genetically modified adult human Schwann cells as tools for cell transplantation in peripheral nerve repair. The application of transfected autologous Schwann cells overexpressing regeneration-promoting proteins, for example, neurotrophic or growth factors, is a promising approach in the aforementioned context. To achieve an optimal clinical outcome, it is highly important to perform enrichment, genetic modification, and retransplantation of cells in a short time. METHODS: To enable the development of these autologous cellular prostheses, the authors tested the properties of adult human Schwann cells obtained from differently treated human peripheral nerve biopsy samples. The use of "cold jet," a fast and effective enrichment procedure, as well as selective, serum-free culture conditions, resulted in very pure adult human Schwann cell cultures. Using an optimized electroporation protocol, as many as 48.4% of adult human Schwann cells were successfully transfected. CONCLUSIONS: The authors present a very fast protocol to establish adult human Schwann cell cultures that demonstrably express plasmid proteins after plasmid DNA insertion by electroporation. These autologous human Schwann cells transfected to enhance the endogenous production of regeneration-supporting proteins will likely constitute a major component of tissue-engineered peripheral nerve grafts.

Adolescent↗
PubMed2001

Subretinal transplantation of genetically modified human cell lines attenuates loss of visual function in dystrophic rats.

Royal College of Surgeons rats are genetically predisposed to undergo significant visual loss caused by a primary dysfunction of retinal pigment epithelial (RPE) cells. By using this model, we have examined the efficacy of subretinal transplantation of two independent human RPE cell lines each exhibiting genetic modifications that confer long-term stability in vitro. The two cell lines, a spontaneously derived cell line (ARPE19) and an extensively characterized genetically engineered human RPE cell line (h1RPE7), which expresses SV40 large T (tumor) antigen, were evaluated separately. Both lines result in a significant preservation of visual function as assessed by either behavioral or physiological techniques. This attenuation of visual loss correlates with photoreceptor survival and the presence of donor cells in the areas of rescued photoreceptors at 5 months postgrafting (6 months of age). These results demonstrate the potential of genetically modified human RPE cells for ultimate application in therapeutic transplantation strategies for retinal degenerative diseases caused by RPE dysfunction.

Animals↗
PubMed2002

Characterization of genetically modified human retinal pigment epithelial cells developed for in vitro and transplantation studies.

PURPOSE: To develop, by specific genetic modification, a differentiated human retinal pigment epithelial (RPE) cell line with an extended life span that can be used for investigating their function in vitro and for in vivo transplantation studies. METHODS: Primary human RPE cells were genetically modified by transfecting with a plasmid encoding the simian virus (SV)40 large T antigen. After characterization, two cell lines, designated h1RPE-7 and h1RPE-116, were chosen for further investigation, along with the spontaneously derived RPE cell line ARPE-19. Factors reported to be important in RPE and photoreceptor cell function and survival in vivo were examined. RESULTS: Both h1RPE-7 and h1RPE-116 cells exhibited epithelial morphology, expressed cytokeratins, and displayed junctional distribution of ZO-1, p100-p120 and beta-catenin. The cells expressed mRNA for RPE65 and cellular retinaldehyde-binding protein (CRALBP) and the trophic and growth factors brain-derived neurotropic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF), pigment epithelium-derived factor (PEDF), nerve growth factor (NGF), platelet-derived growth factor (PDGF)-alpha, insulin-like growth factor (IGF)-1, and vascular endothelial growth factor (VEGF). Secreted BDNF, bFGF, and VEGF, but not CNTF, were identified in cell supernatants. The cell lines constitutively expressed HLA-ABC, CD54, CD58, and CD59. After activation with IFN-gamma both HLA-ABC and CD54 were upregulated, and the expression of HLA-DR was induced. Both cell lines failed to express CD80, CD86, CD40, or CD48 in vitro and in a mixed lymphocyte reaction were unable to induce T-cell proliferation. Fas ligand (CD95L) was not detected in vitro by RT-PCR. Similar results were obtained with the ARPE-19 cell line. CONCLUSIONS: RPE lines h1RPE-7 and h1RPE-116 retain many of the morphologic and biochemical characteristics of RPE cells in vivo and may serve as a source of cells for in vitro analysis of RPE cell function, as well as for orthotopic transplantation studies.

Antigens, Polyomavirus Transforming↗
PubMed2005

Toxic secondary metabolite production in genetically modified potatoes in response to stress.

Potatoes produce a number of toxic secondary metabolites, which are divided into two groups: the sesquiterpenes and the glycoalkaloids (PGAs): whereas PGAs are largely preformed and present in toxic quantities in both the foliage and "green" potatoes, it is well documented that the levels of PGAs and sesquiterpenes are effected by many biotic an abiotic stresses. The development of genetically modified potato varieties has made it prudent to ascertain whether there may be changes in the amounts or types of these secondary metabolites either as a direct effect of the transgene or due to its interactions with environmental variables. Transgenic potato lines were exposed, along with nontransgenic lines, to a range of biotic and abiotic stresses and a range of environmental conditions in the field and store. Following stressing, a comparison was made of levels of potato glycoalkaloid and sesquiterpene levels between the two groups. Significant differences were observed in the levels of both glycoalkaloid and sesquiterpene levels between transgenic and control material and between infected and noninfected material.

Alkaloids↗
PubMedDate not supplied

Subtle effects of herbicide use in the context of genetically modified crops: a case study with glyphosate (Roundup).

Research on the effects of glyphosate and seed germination spans 30 years. Despite several studies reporting detrimental effects of the herbicide on seedling germination and growth, glyphosate is still being registered for use as a weed killer and preharvest desiccant. Its nonselective nature and low chance of species developing resistance has lead to the development of genetically modified crops tolerant to the herbicide which also raises concerns about increased reliance on herbicide use, and subtle ecological impact. This paper presents the result of a literature review on past studies mostly, on crop species, and the results of a new experiment performed with emphasis on noncrop species. The new experimental part attempted to determine whether glyphosate (Roundup) would have an effect on the germination and growth of the F1 generation of seeds produced by plants sprayed with the herbicide. It was designed to mirror spray drift which might affect noncrop plants in nontarget drift zones. Of the 11 species tested using treatments of 0% (control), 1%, 10% or 100% of a 890 g a.i./ha label rate solution sprayed near seed maturity, seven showed a significant effect of the glyphosate treatment on germination and/or growth characteristics. Results of this experiment together with several previous studies reviewed in this paper suggest that there are significant effects to keep in mind when using herbicides such as glyphosate as severe ecological changes could occur.

Germination↗
PubMed2001

Gene flow from a large scale release of genetically modified herbicide tolerant and hybrid winter oilseed rape (Brassica napus L.).

One of the major concerns regarding the cultivation of transgenic crops is the uncontrolled spread of transgenes among fields or to related wild species. To address this concern a long-term study has been set up to assess the amount of gene flow that is occurring from a large scale field trial of genetically modified herbicide tolerant and hybrid (GMHTH) oilseed rape. During the first year of the project, outcrossing of the herbicide tolerance gene from a large scale release of GMHTH winter oilseed rape to a non-transgenic oilseed rape crop in the vicinity and to wild relatives in the environment has been monitored. After harvesting seeds on oilseed rape plants or wild relatives, the seeds are sown in the greenhouse. Seedlings are sprayed with the herbicide on two different occasions. In this way escapes of the first treatment that are not transgenic will be eliminated. To confirm the transgene state of the surviving plants, leaf material is collected from resistant plants. DNA is extracted and the material is examined for the presence of the herbicide tolerance gene by means of a PCR assay. Results on the levels of cross pollination will be discussed.

Agriculture↗
PubMed2005

Development of a peptide nucleic acid array platform for the detection of genetically modified organisms in food.

Two previously developed platforms, a multiplex polymerase chain reaction (PCR) and a peptide nucleic acid (PNA) array, the former allowing for the simultaneous detection of five transgenes and two endogenous controls in food and feed matrices and the latter for the assessment of the identity of amplified PCR products, were combined in order to develop a PNA array device for the screening of genetically modified organisms (GMOs) in food. PNA probes were opportunely designed, synthesized, and deposited on commercial slides. The length of the probes as well as the distance of the probes from the surface were evaluated and found to be critical points. The most suitable probes were found to be 15-mer PNAs linked to the slide surface by means of two 2-(2-aminoethoxy)ethoxyacetic acids as spacers. The device was tested on a model system constituted by flour samples containing a mixture of standards at known concentrations of transgenic material, in particular Roundup Ready soybean and Bt11, Bt176, Mon810, and GA21 maize: The DNA was amplified using the specific multiplex PCR method and tested on the PNA array. The method proposed was found to be able to correctly identify every GMO present in the tested samples.

DNA, Plant↗
PubMed2005

Genetically modified dendritic cells for cancer immunotherapy.

The ability to grow and differentiate dendritic cells (DC) ex vivo has allowed their genetic manipulation to enhance immune activation against tumor antigens. Gene engineering of DC can be achieved with a variety of physical methods and using different viral vectors. RNA or DNA transfection, either alone (naked), coated with liposomes or using electroporation or gene guns leads to T cell activation while transgene expression is frequently undetectable. Adenoviral and retroviral vectors have proven to be highly efficient in DC genetic modification, and have been widely used in preclinical models. Other vectors like lentivirus, poxvirus, herpes virus and adeno-associated virus (AAV) can also lead to foreign transgene expression in DC leading to immune cell activation. DC have been genetically engineered to provide constitutive and high level of tumor antigen expression or to introduce genes that further enhance their immune stimulatory ability. The promising results from preclinical animal models and from in vitro human immune cell culture systems have provided a strong rationale to initiate pilot clinical trials. Recently published or communicated clinical experiences and ongoing trials have used defined tumor antigen RNA transfection for prostate carcinoma and melanoma, liposome-encoated DNA transfection for breast or pancreatic cancer, adenoviral vector tumor antigen gene modification for melanoma and small cell lung cancer, and poxvirus-mediated expression of costimulatory molecules for colon carcinoma. These preliminary experiences suggest that genetically modified DC can safely induce T cell responses but few clinical responses.

Animals↗
PubMed2001

Environmental risks of chemicals and genetically modified organisms: a comparison. Part I: Classification and characterisation of risks posed by chemicals and GMOs.

Risks can be characterised by several parameters. A risk is commonly defined to be the product of the extent of damage and the probability of its occurrence. But there are several other characteristics to be taken into account: degree of certainty in determining extent and probability, persistency, ubiquity, irreversibility, delay effect and mobilisation potential. As potential risks of genetically modified plants (GMPs), resistance to antibiotics, impact on non-target organisms, spread of genes and GMOs, and secondary consequences, e.g. on cultivation practice, are discussed in detail. Risks of GMPs are, in general, characterised by high uncertainty of the magnitude and probability of damage, a high mobilisation potential and a delay effect.

Environmental Pollutants↗
PubMed1998

Systemic treatment with a recombinant erbB-2 receptor-specific tumor toxin efficiently reduces pulmonary metastases in mice injected with genetically modified carcinoma cells.

Receptor-mediated targeted tumor therapy is an important applied consequence of the studies on the genetic causes of cancer. These therapy concepts have to be evaluated in novel animal models that reflect the molecular aberrations found in human tumors. Here we introduce an animal model that allows the evaluation of drugs directed against a surface receptor that is frequently altered in primary human adenocarcinomas. Tumor toxins are polypeptides in which a tumor cell-specific recognition domain and a toxic effector domain have been joined by DNA recombination in vitro. Tumor cell recognition is contributed by a single-chain antibody domain specific for the extracellular domain of the erbB-2 receptor [scFv(FRP5)] and cytotoxicity by the enzymatically active domain of a bacterial exotoxin (exotoxin A from Pseudomonas aeruginosa). The erbB-2 receptor is overexpressed in many primary human cancer cells and is a favorable target for directed tumor therapy. The fusion protein scFv(FRP5)-exotoxin A has previously been shown to be able to efficiently and specifically kill erbB-2 receptor-expressing tumor cells. We have investigated the potential of this tumor toxin to detect and eliminate metastasizing tumor cells upon systemic administration. Murine renal carcinoma cells genetically modified with human erbB-2 receptor and bacterial beta-galactosidase genes form large pulmonary metastases when injected into the tail vein of BALB/c mice. Administration of the tumor toxin over a 10-day time period starting 1 day after tumor cell transplantation totally suppressed the formation of metastases. The treatment of animals 11 days after tumor cell transplantation, allowing the establishment of many pulmonary metastases, led to a drastic reduction in their number and size.

ADP Ribose Transferases↗
PubMedDate not supplied

Current methods for assessing safety of genetically modified crops as exemplified by data on Roundup Ready soybeans.

Several laboratories have used recombinant DNA technology in plant breeding to improve compositional, processing, and agronomic characteristics of plants. These transformed plants have been extensively tested in field trials, have gained full regulatory approvals and are currently being marketed in a number of countries around the world. This paper briefly summarizes the approach used to assure the safety of foods and feeds derived from these genetically modified crops, as exemplified by data on Roundup Ready soybeans that has been developed by Monsanto Company using biotechnology in order to confer tolerance to glyphosate, the active ingredient in Roundup herbicide, by the production of the CP4 enolpyruvylshikimate-3-phosphate synthase protein. The results of the studies demonstrate that Roundup Ready soybeans are as safe as traditional soybeans with respect to food and feed safety.

Agriculture↗
PubMed1999

Genetically modified organisms in food-screening and specific detection by polymerase chain reaction.

PCR methods for the detection of genetically modified organisms (GMOs) were developed that can be used for screening purposes and for specific detection of glyphosate-tolerant soybean and insect-resistant maize in food. Primers were designed to amplify parts of the 35S promoter derived from Cauliflower Mosaic Virus, the NOS terminator derived from Agrobacterium tumefaciens and the antibiotic marker gene NPTII (neomycin-phosphotransferase II), to allow for general screening of foods. PCR/hybridization protocols were established for the detection of glyphosate-tolerant RoundUp Ready soybean and insect-resistant Bt-maize. Besides hybridization, confirmation of the results using restriction analysis was also possible. The described methods enabled a highly sensitive and specific detection of GMOs and thus provide a useful tool for routine analysis of raw and processed food products.

Blotting, Southern↗
PubMed2002

Human NK cell-mediated cytotoxicity triggered by CD86 and Gal alpha 1,3-Gal is inhibited in genetically modified porcine cells.

Delayed xenograft rejection is a major hurdle that needs to be addressed to prolong graft survival in pig-to-primate xenotransplantation. NK cell activation has been implicated in delayed xenograft rejection. Both Ab-dependent and independent mechanisms are responsible for the high susceptibility of porcine cells to human NK cell-mediated cytotoxicity. Previous reports demonstrated a role of Galalpha1,3-Gal Ag in triggering the Ab-independent responses. We hypothesize that expression of CD80 and/or CD86 on porcine cells may also play a role in NK cell activation as human NK cells express a variant of CD28. Our initial analysis showed that porcine endothelial cells and fibroblasts express CD86, but not CD80. Genetic engineering of these cells to express hCD152-hCD59, a chimeric molecule designed to block CD86 in cis, was accompanied by a reduction in susceptibility to human NK cell-mediated cytotoxicity. The use of a specific anti-porcine CD86-blocking Ab and the NK92 and YTS cell lines further confirmed the involvement of CD86 in triggering NK cell-mediated lysis of porcine cells. Maximal protection was achieved when hCD152-hCD59 was expressed in H transferase-transgenic cells, which show reduced Galalpha1,3-Gal expression. In this work, we describe two mechanisms of human NK cell-mediated rejection of porcine cells and demonstrate that genetically modified cells resist Ab-independent NK cell-mediated cytotoxicity.

Abatacept↗
PubMed2004

Detection of genetically modified maize MON810 and NK603 by multiplex and real-time polymerase chain reaction methods.

In this study, the event-specific primers for insecticide-resistant maize, MON810, and herbicide-tolerance maize, NK603, have been designed. Simplex PCR and multiplex PCR detection method have been developed. The detection limit of the multiplex PCR is 0.5% for MON810 and NK603 in 50 ng of the template for one reaction. Quantitative methods based on real-time quantitative PCR were developed for MON810 and NK603. Plasmid pMulM2 as reference molecules for the detection of MON810 and NK603 was constructed. Quantification range was from 0.5 to 100% in 100 ng of the DNA template for one reaction. The precision of real-time Q-PCR detection methods, expressed as coefficient of variation for MON810 and NK603 varied from 1.97 to 8.01% and from 3.45 to 10.94%, respectively. The range agreed with European interlaboratories test results (25%). According to the results, the methods for quantitative detection of genetically modified maize were acceptable.

DNA, Plant↗
PubMed2007

Interleukin-17 as a molecular target in immune-mediated arthritis: immunoregulatory properties of genetically modified murine dendritic cells that secrete interleukin-4.

OBJECTIVE: Our previous studies have shown that murine dendritic cells (DCs) genetically modified to express interleukin-4 (IL-4) reduce the incidence and severity of murine collagen-induced arthritis. The present studies were performed to assess the immunoregulatory mechanisms underlying this response, by assessing the effects of IL-4 DCs on cytokine production by subsets of T helper cells. METHODS: Male DBA mice ages 6-8 weeks old were immunized with type II collagen. Splenic T cells obtained during the initiation phase and the end stage of arthritis were cultured with IL-4 DCs or untransduced DCs in the presence of collagen rechallenge. Interferon-gamma (IFNgamma) and IL-17 responses were measured. Antibodies to IL-4, IL-12, and IL-23, and recombinant IL-4, IL-12, and IL-23 were used to further study the regulation of T cell cytokine production by IL-4 DCs. RESULTS: Splenic T cells obtained during the initiation phase of arthritis produced less IL-17 when cultured in the presence of IL-4 DCs, despite their production of increased quantities of other proinflammatory cytokines (IFNgamma and tumor necrosis factor). T cell IL-17 production after collagen rechallenge was not inhibited by a lack of IL-23, since IL-4-mediated suppression of IL-17 was not reconstituted by IL-23, an otherwise potent inducer of IL-17 production by T cells. Although IL-4 DCs can produce increased quantities of IL-12 and IFNgamma, suppression of IL-17 production by IL-4 DCs was independent of both. While IL-17 production by T cells obtained during the initiation phase of arthritis was regulated by IL-4 DCs, IL-17 production by T cells obtained during end-stage arthritis was not altered. CONCLUSION: Our data suggest that IL-4 DCs exert a therapeutic effect on collagen-induced arthritis by targeting IL-17. IL-17 suppression by IL-4 DCs is robust and is not reversed by IL-23. Timing might be important in IL-17-targeted therapy, since IL-17 production by T cells obtained during end-stage arthritis did not respond to suppression by IL-4 DCs.

Animals↗
PubMed2004

Synergistic interactions between the genetically modified bacterial polysaccharide P2 and carob or konjac mannan.

Rheological studies have confirmed that the bacterial polysaccharide P2, a genetically modified variant of the Acetobacter xylinum polysaccharide acetan, undergoes synergistic gelation with either of the plant polysaccharides carob or konjac mannan. X-ray fibre diffraction data shows that P2 can form a 5-fold helical structure of pitch 4.7nm and an axial rise per disaccharide repeat of 0.92nm. Optical rotation data demonstrate that P2 undergoes a coil-helix transition in solution and that deacylation enhances the stability of the helical structure in solution. Studies made on mixtures prepared at different temperatures and ionic strengths suggest that denaturation of the P2 helix favours interaction and gelation. Deacetylation of P2 enhances gelation. X-ray diffraction data for oriented fibres prepared from deacetylated P2-konjac mannan mixed films reveal a 6-fold helical structure of pitch 5.54nm with an axial rise per disaccharide repeat also of 0.92nm. This mixed helix provides direct evidence for binding between the two polysaccharides. P2 contains two sites of acetylation: one on the backbone and one on the sidechain. The former site of acetylation inhibits helix formation for P2. It is suggested that this site of acetylation also inhibits formation of the mixed helix, explaining the enhanced gelation of mixtures on deacetylation.

Amorphophallus↗
PubMed2006

The Ferrier Lecture 1998. The molecular biology of consciousness investigated with genetically modified mice.

The question is raised of the relevance of experimental work with the mouse and some of its genetically modified individuals in the study of consciousness. Even if this species does not go far beyond the level of 'minimal consciousness', it may be a useful animal model to examine the elementary building blocks of consciousness using the methods of molecular biology jointly with investigations at the physiological and behavioural levels. These building blocks which are anticipated to be universally shared by higher organisms (from birds to humans) may include: (i) the access to multiple states of vigilance, like wakefulness, sleep, general anaesthesia, etc.; (ii) the capacity for global integration of several sensory and cognitive functions, together with behavioural flexibility resulting in what is referred to as exploratory behaviour, and possibly a minimal form of intentionality. In addition, the contribution of defined neuronal nicotinic receptors species to some of these processes is demonstrated and the data discussed within the framework of recent neurocomputational models for access to consciousness.

Animals↗