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Results for “Cell engineering”

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At least 685 records · Page 38Linked to original sources

PubMed2000

Conditionally-immortalized astrocytic cell line expresses GAD and secretes GABA under tetracycline regulation.

We have engineered conditionally-immortalized mouse astrocytes to express beta-galactosidase or GAD(65) in a tetracycline-controlled fashion. The engineered cell lines, BASlinbetagal and BASlin65, divide at 33 degrees C but cease division at 39 degrees C. We carried out morphological and biochemical analyses to further understand GABA production and release, and to determine the suitability of these cells for transplantation. Using the BASlinbetagal cell line, we showed a dramatic regulation of beta-galactosidase expression by tetracycline. The BASlin65 cell line showed functional GAD(65) enzymatic activity and GABA production, both of which were suppressed by growth in the presence of tetracycline. When cultured in the absence of tetracycline, BASlin65 cells have a total GABA content equal to or greater than other GABA-ergic cell lines. Immunofluorescence microscopy revealed that GAD(65) had a distinct perinuclear localization and punctate staining pattern. GABA, on the other hand, showed diffuse staining throughout the cytoplasm. BASlin65 cells not only synthesize GABA, they also release it into the extracellular environment. Their ability to produce and release significant amounts of GABA in a tetracycline-regulated manner makes BASlin65 cells a useful cellular model for the study of GABA production and release. Furthermore, their non-tumorigenicity makes them excellent candidates for transplantation into specific regions of the brain to provide a localized and regulatable source of GABA to the local neuronal circuitry.

Animals↗

PubMed1997

Tumour cell killing using chemically engineered antibody constructs specific for tumour cells and the complement inhibitor CD59.

Immunotherapy using MoAbs is inefficient due to limited activation of human effectors by mouse antibodies and multiple protective mechanisms available to host cells against autologous complement. We have used chemically engineered antibody constructs and human complement in vitro to specifically target and kill neoplastic B lymphoid cells (Raji). Fab'gamma Fc gamma2 chimaeric antibody (specific for human CD37) was used to activate the classical pathway of human complement on Raji cells, whilst CD59 was neutralized using one of two different bispecific F(ab'gamma)2 antibody constructs which contained both cell-targeting (anti-CD19 or anti-CD38) and CD59-neutralizing moieties. When either bispecific construct was used to neutralize CD59, 15-25% of cells were lysed. If CD55 was also neutralized using specific antibody, Raji cells were efficiently killed (70% lysis). When added to a mixture of target (Raji) and bystander (K562) cells, one bispecific antibody (anti-CD38 x anti-CD59) could be specifically delivered to Raji, avoiding significant uptake on CD59-expressing bystander cells (K562). The second bispecific antibody (anti-CD19 x anti-CD59) bound equally well to either cell type. Cell-specific targeting was dependent upon combination of a low-affinity anti-CD59 Fab'gamma with a high-affinity anti-tumour cell Fab'gamma. When Raji and K562 cells were mixed and incubated with a combination of the engineered constructs and anti-CD55 antibodies, Raji cell lysis (30-40%) was observed in the absence of K562 killing. We propose that combinations of these constructs may be of use for treatments such as ex vivo purging of autologous bone marrow or in vivo targeting of tumour cells.

Animals↗

PubMed2006

Adipose tissue engineering by human adipose-derived stromal cells.

Tissue engineering has emerged as a promising alternative approach to current clinical treatments for restoration of soft tissue defects. The purpose of this study was to investigate adipose tissue formation in vitro and in vivo by using human adipose-derived stromal cells (ADSCs) utilizing a gelatin sponge (Gelform) as a scaffold. Adipogenic potentials of human ADSCs were demonstrated by Oil-O-red staining and cellular morphology. After seeding human ADSCs in a density of 3 x 10(6) cells/ml on three-dimensional gelatin sponges, tissue-engineered constructs were exposed to adipogenic differentiation medium for in vitro studies and implanted in the backs of severe combined immunodeficient (SCID) mice for in vivo adipose regeneration. Adipogenesis of ADSC-seeded gelatin sponges was confirmed by Oil-O-red staining after 4 weeks of in vitro incubation. The optical density of the elution from Oil-O-red staining of adipogenic constructs is significantly higher than that of the control group (p < 0.05, n = 4). With short-term in vitro differentiation, adipogenic constructs turned into fat tissue 4 weeks after in vivo implantation, confirmed by biochemical and immunohistochemical examination. No adipogenic-morphological change or fat formation was observed in in vitro or in vivo studies when ADSCs were exposed to a control medium without adipogenic stimulation. These results indicate that engineered adipose tissue can be achieved using human ADSCs and biocompatible and degradable gelatin sponges.

Adipose Tissue↗

PubMed2000

[Cultivating human Schwann cells for tissue engineering of peripheral nerves].

Cultivation of human cells is well established. The cultivation of human Schwann cells may offer a new therapeutic approach for treatment of degenerative and traumatic lesions of the peripheral nervous system. Currently, Schwann cells in combination with other biological matrices are used as tissue-engineered biological nerve grafts in animal models. Cultivation of human Schwann cells, however, is more difficult than cultivation of rodent cells. A high cell yield is only achieved by pharmacological stimulation, which should not be used in clinical therapy. Thus, we aimed to establish an easy method of cultivating human Schwann cells from peripheral nerve neuromas that have developed after a complete nerve lesion. As these neuromas have to be resected in any case to allow proper nerve reconstruction, their removal does not lead to additional neurological defects. Schwann cells were cultivated from the neuromas of eleven patients, aged 5 to 75 years. All patients suffered from a complete median nerve lesion at the level of the distal forearm, which could not be treated primarily. Two weeks after trauma, the patients underwent secondary reconstruction by autologous sural nerve grafts. During this operation, the neuromas were resected. Control cultures were established from remaining parts of the sural nerve. Cell yield was determined on the first, third and seventh day in vitro. Schwann cells were stained for S100. Viability was assessed with fluoresceine-fluorescence. The cell count was assessed with regard to the donor age. The growth rate of Schwann cells was found to be donor-age dependent. The highest cell yield was obtained from adult neuromas. By the third day in vitro, they showed a 1.5 fold increased cell count compared with juvenile nerves and neuromas. By the seventh day in vitro, Schwann cells from adult neuromas were increased 2.5 times compared with cells from juvenile nerves and 7 times compared to cells from adult sural nerves. Cultivation of Schwann cells taken from sural nerves of patients older than 65 years was not possible. The utilization of neuromas as a source for human Schwann cells allows an age-independent cultivation within a short time period without any pharmacological treatment. These neuromas are virtually predegenerated and show an activation of Schwann cells implying good adherence and high mitotic activity in culture. Normal nerve tissue as a source for Schwann cells for tissue-engineered nerves is only sufficient in young patients due to its greater proliferative potential. The age-dependent proliferation underlines the need for alternative sources for Schwann cells.

Adolescent↗

PubMed2003

Complications of a trophic xenotransplant approach in parkinsonian monkeys.

Various restorative cell transplantation strategies have been investigated to substitute for lost dopamine (DA) neurons or to enhance DA synthesis in Parkinson's disease. Intracerebral implantation of engineered cells encapsulated in a semipermeable polymer membrane constitutes one way to deliver bioactive substances unable to cross the blood-brain barrier while avoiding the need for long-term immunosuppression. Glial cell line-derived neurotrophic factor (GDNF) has shown trophic effects on DA neurons but effective and sustained delivery within the brain parenchyma remains problematic. The long-term efficacy and late complications of a xenotransplant approach utilizing GDNF-expressing encapsulated baby hamster kidney (BHK) cells were examined. Each of five MPTP-lesioned parkinsonian cynomolgus monkeys received five devices containing active or inert cells grafted bilaterally in the striatum in a two-stage procedure 9 months apart and animals were sacrificed 4 months later for analyses. No definite motor benefit was observed, DA levels were comparable between GDNF- and control cell-implanted striata, and tyrosine hydroxylase (TH) immunoreactivity in the substantia nigra showed no consistent recovery. Cell viability and GDNF synthesis in the explanted devices were negligible. The brain tissue surrounding all implants showed an intense immune reaction with prominent "foreign body" inflammatory infiltrates. Membrane biophysics, the cell type used, and the extended period of time the devices remained in situ may have contributed to the negative outcome and should be addressed in future investigations using this approach.

Animals↗

PubMed2002

Genetic modification of adenovirus 5 tropism by a novel class of ligands based on a three-helix bundle scaffold derived from staphylococcal protein A.

The use of adenovirus (Ad) as an efficient and versatile vector for in vivo tumor therapy requires the modulation of its cellular tropism. We previously developed a method to genetically alter the tropism of Ad5 fibers by replacing the fiber knob domain by an extrinsic trimerization motif and a new cellular ligand. However, fibers carrying complex ligands such as single-chain antibody fragments did not assemble into functional pentons in vitro in the presence of penton base, and failed to be rescued into infectious virions because of their inability to fold correctly within the cytoplasm of Ad-infected cells. Here we show that the coding sequence for a disulfide bond-independent three-helix bundle scaffold Z, derived from domain B of Staphylococcal protein A and capable of binding to the Fc portion of immunoglobulin (Ig) G1, could be incorporated into modified knobless Ad fiber gene constructs with seven shaft repeats. These fiber gene constructs could be rescued into viable virions that were demonstrated to enter 293 cells engineered for IgG Fc surface expression but not unmodified 293 cells, via a mechanism that could be specifically blocked with soluble Fc target protein. However, the tropism modified viruses showed a slightly impaired cellular entry and a lower infectivity than wildtype (WT) virus. In addition, we generated recombinant fibers containing an IgA binding Affibody ligand, derived from combinatorial specificity-engineering of the Z domain scaffold. Such fiber constructs also showed the expected target specific binding, indicating that the affibody protein class is ideally suited for genetic engineering of Ad tropism.

Adenoviridae↗

PubMed2006

Divided loyalties: transdetermination and the genetics of tissue regeneration.

Most tissues contain cells capable of the self-renewal and differentiation necessary to maintain tissue and organ integrity. These somatic stem cells are generally thought to have limited developmental potential. The mechanisms that restrict cell fate decisions in somatic stem cells are only now being understood. This understanding will be important in the clinical exploitation of adult stem cells in tissue repair and replacement. Experiments performed over fifty years ago in Drosophila showed that developmental restriction could be relaxed in the proliferating larval cells that are destined to form the adult fly integument. This phenomenon, called transdetermination, can serve as a model for mechanisms of stem-cell commitment. A recent publication (1) sheds new light on the mechanism of transdetermination by demonstrating that loss of homeotic gene silencing leads to increased frequency of transdetermination. In addition, the authors link a specific signaling pathway induced by tissue regeneration to the relaxation of homeotic gene silencing. The data identify key mechanisms that control developmental homeostasis and cell fate restriction that could be manipulated to make somatic stem-cell engineering possible.

Animals↗

PubMed2002

FasL-transfected endothelial cells decrease the proliferative response of allogeneic PBL.

Graft endothelium has a key role in organ transplantation because it regulates graft infiltration by allogeneic activated T cells. Overexpression of death molecules that could induce apoptosis of alloreactive T cells might be an alternative to the immunosuppressive treatment currently used in graft transplantation. Several studies have shown that immune-privileged sites express Fas ligand (FasL) and induce apoptosis of activated T-cells. We propose that endothelial cells engineered to express FasL could inhibit alloreactive T cell-proliferation by inducing apoptosis. An expression vector was constructed with human FasL cDNA and used to transfect an endothelial cell line (ECV304 cells). We demonstrated that FasL-transfected ECV304 cells were effective in inducing apoptosis of Jurkat T cell lymphoma as an agonist anti-Fas antibody. Using a mixed lymphocyte-endothelial cell culture model we observed that FasL-transfected ECV304 cells which conserved their two principal costimulatory pathways inhibited alloreactive T cell-proliferation by inducing activated T-cell apoptosis. These results suggest that endothelial cells could be interesting candidates to convey a death signal and induce hyporesponsiveness of alloreactive T cells during organ transplantation.

Apoptosis↗

PubMed2005

The NF1 tumor suppressor critically regulates TSC2 and mTOR.

Loss-of-function mutations in the NF1 tumor suppressor gene underlie the familial cancer syndrome neurofibromatosis type I (NF1). The NF1-encoded protein, neurofibromin, functions as a Ras-GTPase activating protein (RasGAP). Accordingly, deregulation of Ras is thought to contribute to NF1 development. However, the critical effector pathways involved in disease pathogenesis are still unknown. We show here that the mTOR pathway is tightly regulated by neurofibromin. mTOR is constitutively activated in both NF1-deficient primary cells and human tumors in the absence of growth factors. This aberrant activation depends on Ras and PI3 kinase, and is mediated by the phosphorylation and inactivation of the TSC2-encoded protein tuberin by AKT. Importantly, tumor cell lines derived from NF1 patients, and a genetically engineered cell system that requires Nf1-deficiency for transformation, are highly sensitive to the mTOR inhibitor rapamycin. Furthermore, while we show that the activation of endogenous Ras leads to constitutive mTOR signaling in this disease state, we also demonstrate that in normal cells Ras is differentially required for mTOR signaling in response to various growth factors. Thus, these findings identify the NF1 tumor suppressor as an indispensable regulator of TSC2 and mTOR. Furthermore, our results also demonstrate that Ras plays a critical role in the activation of mTOR in both normal and tumorigenic settings. Finally, these data suggest that rapamycin, or its derivatives, may represent a viable therapy for NF1.

Animals↗

PubMed2004

Mobilized blood CD34+ cells transduced and selected with a clinically applicable protocol reconstitute lymphopoiesis in SCID-Hu mice.

We developed a clinically applicable gene transfer procedure into mobilized peripheral blood (MPB) CD34(+) hematopoietic progenitor cells, based on single viral exposure and selection of engineered cells. CD34(+) cells were transduced with a retroviral vector carrying the truncated form of the nerve growth factor receptor (Delta NGFR) marker gene, and immunoselected for Delta NGFR expression. Optimal time and procedure for viral exposure, length of culture, and transgene expression of MPB CD34(+) cells were determined using in vitro assays. The multipotent capacity of MPB CD34(+)-transduced cells was demonstrated in the SCID-hu bone/liver/thymus mouse model. Transduced Delta NGFR(+) cells retained 50% of long-term culture-colony forming cells (LTC-CFC) compared to unmanipulated CD34(+) cells. In SCID-hu mice, 52% of CD45(+) cells, 27% of CD34(+) cells, 49% of B cells, and more than 50% of T cells were derived from transplanted CD34(+)/Delta NGFR(+) cells. Furthermore, transplantation of purified transduced cells greatly reduced the competition with untransduced progenitors occurring in unselected grafts. These data demonstrate that MPB CD34(+) cells, transduced with a single viral exposure and selected by transgene expression, retain multilineage reconstitution capacity and remarkable transgene expression.

Animals↗

PubMed2006

Self assembly of DNA nanoparticles with polycations for the delivery of genetic materials into cells.

Increasing attention has been paid to technology used for the delivery of genetic materials into cells for gene therapy and the generation of genetically engineered cells. So far, viral vectors have been mainly used because of their inherently high transfection efficiency of gene. However, there are some problems to be resolved for the clinical applications, such as the pathogenicity and immunogenicity of viral vectors themselves. Therefore, many research trials with non-viral vectors have been performed to enhance their efficiency to a level comparable to the viral vector. Two directions of these trials exist: Material improvement of non-viral vectors and their combination with various external physical stimuli. In this study gelatin was selected as a non-viral carrier for DNA. To give a positive charge to gelatin, different extents introduction of ethylenediamine (Ed), spermidine (Sd), and spermine (Sm) were reacted with gelatin in the presence of a water-soluble carbodiimide. When positively charged gelatin derivatives (Ed, Sd, and Sm) were mixed with negatively charged DNA, a self assembly of DNA nanoparticle (complex) was formed within few minutes through electrostatic interaction. Irrespective of the type of gelatin derivatives, the apparent molecular size of DNA was reduced by increasing the gelatin/DNA mixing ratio to attain a saturated value of about 150 nm. The condensed gelatin/DNA complexes showed the zeta potential of 10-15 mV. The amount of DNA internalized into the cells was significantly increased by the complexation with every gelatin derivative. The cells incubated with the gelatin/DNA complexes exhibited significantly stronger luciferase activities than naked plasmid DNA. This study clearly demonstrates and self-assembled DNA complexes has potential as a gene delivery vechile and are stable to transfer genetic materials to cells.

Animals↗

PubMed2003

Mutations in PTPN11 implicate the SHP-2 phosphatase in leukemogenesis.

The PTPN11 gene encodes SHP-2 (Src homology 2 domain-containing protein tyrosine Phosphatase), a nonreceptor tyrosine protein tyrosine phosphatase (PTPase) that relays signals from activated growth factor receptors to p21Ras (Ras) and other signaling molecules. Mutations in PTPN11 cause Noonan syndrome (NS), a developmental disorder characterized by cardiac and skeletal defects. NS is also associated with a spectrum of hematologic disorders, including juvenile myelomonocytic leukemia (JMML). To test the hypothesis that PTPN11 mutations might contribute to myeloid leukemogenesis, we screened the entire coding region for mutations in 51 JMML specimens and in selected exons from 60 patients with other myeloid malignancies. Missense mutations in PTPN11 were detected in 16 of 49 JMML specimens from patients without NS, but they were less common in other myeloid malignancies. RAS, NF1, and PTPN11 mutations are largely mutually exclusive in JMML, which suggests that mutant SHP-2 proteins deregulate myeloid growth through Ras. However, although Ba/F3 cells engineered to express leukemia-associated SHP-2 proteins cells showed enhanced growth factor-independent survival, biochemical analysis failed to demonstrate hyperactivation of the Ras effectors extracellular-regulated kinase (ERK) or Akt. We conclude that SHP-2 is an important cellular PTPase that is mutated in myeloid malignancies. Further investigation is required to clarify how these mutant proteins interact with Ras and other effectors to deregulate myeloid growth.

Amino Acid Sequence↗

PubMed2003

Ultrasound enhances the transfection of plasmid DNA by non-viral vectors.

Animals↗

PubMed2001

Soluble decoy receptor 3 is expressed by malignant gliomas and suppresses CD95 ligand-induced apoptosis and chemotaxis.

Decoy receptor 3 (DcR3) is a newly identified soluble protein that binds to CD95 ligand (CD95L) and inhibits its proapoptotic activity. Here we report that DcR3 is expressed by the majority of long-term and ex vivo malignant glioma cell lines as well as in human glioblastoma in vivo. Expression of DcR3 correlates with the grade of malignancy: 15 of 18 (83%) glioblastomas (WHO grade IV) but none of 11 diffuse astrocytomas (WHO grade II) exhibited DcR3 immunoreactivity. We also demonstrate that human malignant glioma cells engineered to release high amounts of DcR3 into the cell culture supernatant are protected from CD95L-induced apoptotic cell death. In contrast, DcR3 does not confer protection from the death ligand Apo2 ligand (TRAIL). Importantly, ectopic expression of DcR3 resulted in substantial differences in immune cell infiltration in the 9L rat gliosarcoma model. Thus, the infiltration of CD4+ and CD8+ T cells as well as microglia/macrophages into glioma was substantially decreased in DcR3-producing tumors compared with control tumors. Chemotaxis assays revealed that DcR3 counteracts the chemotactic activity of CD95L against microglial cells in vitro. These findings suggest that DcR3 may be involved in the progression and immune evasion of malignant gliomas.

Animals↗

PubMed2001

Erythropoietin mediates terminal granulocytic differentiation of committed myeloid cells with ectopic erythropoietin receptor expression.

OBJECTIVES: The precise role of hematopoietic cytokine/cytokine receptor interactions in lineage-restricted hematopoietic differentiation giving rise to mature blood cells of diverse function is incompletely defined. To study lineage-specific effects of cytokines during terminal hematopoietic differentiation, we examined the ability of erythropoietin (Epo) to mediate terminal granulocytic differentiation and induction of myeloid gene expression in committed myeloid cells, engineered to ectopically express Epo receptor (EpoR). METHODS: A cell culture model for granulocyte-macrophage colony stimulating factor (GM-CSF)-mediated granulocytic differentiation was used. EpoR was introduced by retrovirus-mediated gene transfer into multipotential, hematopoietic murine cell line EML, from which GM-CSF-responsive, promyelocytic EPRO cells were generated. In EPRO cells ectopically expressing EpoR, we examined the ability of Epo to mediate granulocytic differentiation and determined whether Epo-mediated neutrophil differentiation is associated with a pattern of myeloid gene expression comparable to that induced by GM-CSF. RESULTS: Studies of EpoR function in myeloid EPRO cells revealed that Epo/EpoR interaction can mediate terminal granulocytic differentiation of committed myeloid cells. In EPRO cells expressing EpoR, Epo-mediated neutrophil differentiation was associated with surface CD11b/CD18 (Mac-1) expression and induction of mRNA expression of specific myeloid genes including lactoferrin, gelatinase and C/EBPepsilon, in a manner similar to GM-CSF-mediated differentiation. CONCLUSIONS: These results indicate that Epo can deliver differentiative signals along a non-erythroid lineage, providing evidence for interchangeable cytokine receptor signals that mediate terminal differentiation of committed myeloid cells.

Animals↗

PubMed1997

Extracorporal Immunopharmacotherapy.

The methods of extracorporal immunopharmacotherapy may be attributed to cell engineering immunocorrection therapy and allows to apply regulatory immune cells and their short-distance mediators after pharmaceutical activation in vitro (extracorporally). The therapy implores: (1) in vitro drug-dependent activation of leukocytes obtained from patient's blood, and (2) withdrawal of the activatory drugs and reinfusion of activated leukocytes back to the patient. By doing this it is possible to perform drug treatment of the patient not in his body, but in vitro through stimulating only a part of his cells. This significantly abolishes endotoxication and prevents the endless part of immune cells from drug-dependent cytotoxicity. The methods of extracorporal immunopharmacotherapy gives reliable opportunities for managing altered parameters of the immune system towards norm in fast and fair efficient manner. Here we report some positive results of application of extracorporal immunopharmacotherapy in patients with atopic asthma, Layell and severe atopic syndromes.

Journal Article↗

PubMed1992

Antiviral activity of tumor necrosis factor is signaled through the 55-kDa type I TNF receptor [corrected].

Agonist antibodies (Ab) to the two TNF receptors, TNF-R1 (55 kDa) and TNF-R2 (75 kDa), have been shown to signal many of the distinct functions induced by TNF-alpha. We have found that anti-TNF-R1, but not anti-TNF-R2, Ab trigger antiviral activity in human hepatoma Hep-G2 cells and enhance the antiviral activity of IFN-gamma in human lung fibroblast A549 cells. Likewise, anti-human-TNF-R1 Ab had antiviral enhancing activity on murine L929 cells engineered to express human TNF-R1. However, L929 cells that express human TNF-R1 lacking most of the intracellular domain fail to respond to anti-human-TNF-R1 Ab. This demonstrates that the intracellular domain of TNF-R1 is necessary to generate antiviral activity. TNF-R1 but not TNF-R2 also signals killing of virus-infected cells by TNF-alpha. Thus, all the known antiviral activities of TNF-alpha are mediated through TNF-R1.

Animals↗

PubMedDate not supplied

Development of metabolically engineered Saccharomyces cerevisiae cells for the production of lactic acid.

Interesting challenges from metabolically engineered Saccharomyces cerevisiae cells arise from the opportunity to obtain yeast strains useful for the production of chemical(s). In this paper, we describe the accumulation of lactic acid in the culture medium of growing, engineered yeast cells expressing a mammalian lactate dehydrogenase gene (LDH-A). High and reproducible productions (20 g/L) and productivities (up to 11 g/L/h) of lactic acid have been obtained by modulating the physiological growth conditions. Since yeast cells are acid tolerant and survive at very low pH values, the production of lactate can be avoided. In perspective, the approaches described could be useful for the production of lactic acid, outflanking the problems related to the synthesis from bacteria cells. In fact, during industrial productions, there is an inhibitory effect on the metabolic activities of the growing bacteria (i.e., Lactobacillus spp.) caused by the acid produced and by the low pH value. Thus, strategies to prevent the lowering of pH are conventional operations. These processes allow the production of lactate(s) and require the purification of the acid from its salt. The biotechnological implications of this study are also discussed.

Animals↗