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Results for “cellular engineering”

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At least 1,531 records · Page 85Linked to original sources

PubMed2000

Wnt1 and MEK1 cooperate to promote cyclin D1 accumulation and cellular transformation.

Members of the Wnt family of signal transducers regulate cellular differentiation/reorganization and cellular proliferation. However, few pro-proliferative targets of Wnt have been identified. We now show that cyclin D1, a critical mediator of cell cycle progression, is a downstream target of Wnt-dependent signaling. NIH-3T3 cell lines engineered to overexpress Wnt1 displayed reduced glycogen synthase kinase-3beta activity. Wnt1-dependent glycogen synthase kinase-3beta inhibition corresponded with decreased cyclin D1 proteolysis and, thus, hyperaccumulation of active cyclin D1.CDK4 (cyclin-dependent kinase 4) kinase. However, in the absence of serum-derived growth factors, Wnt-1 was not sufficient to drive cyclin D1 accumulation or S-phase entry. In contrast, cells engineered to co-express Wnt1 and activated MEK1 accumulated high levels of cyclin D1 and entered the DNA synthetic phase in the absence of serum-derived growth factors, a characteristic of neoplastic transformation. The ability of a dominant-negative cyclin D1 mutant, D1-T156A, to inhibit Wnt1/MEK1-dependent S-phase entry suggests that cyclin D1 is a critical downstream target for Wnt1- and MEK1-dependent cellular proliferation.

3T3 Cells↗

PubMed2004

Tissue engineering of ovine aortic blood vessel substitutes using applied shear stress and enzymatically derived vascular smooth muscle cells.

Compared to native blood vessels, all clinically available blood vessel substitutes perform suboptimally. Numerous approaches to tissue engineer (TE) blood vessels have been pursued using different scaffold materials, cell types, and culture conditions. Several limitations however remain to be overcome prior to the potential application in the arterial system. This study aimed at tissue engineering viable ovine blood vessels suitable for implantation into the systemic circulation of sheep. In recent studies vascular smooth muscle cells (vSMC) were derived by an explant technique. However, in this study we show that homogenous populations of differentiated vSMC were only obtained by enzymatic dispersion as characterized by immunostaining for specific vSMC marker proteins. In contrast the explant method yielded predominantly less differentiated myofibroblast-like cells. Enzymatically derived vSMC were seeded onto P-4-HB scaffolds and incubated either in a pulsatile flow bioreactor or under static conditions. Dynamically cultured TE blood vessel substitutes showed confluent layered tissue formation and were completely water resistant. They displayed significantly increased ECM synthesis, DNA, and protein content as well as vSMC marker expression. Mechanical properties of bioreactor cultured TE blood vessels approached those of native aorta. In conclusion ovine, aortic blood vessel substitutes were successfully created using enzymatically derived vSMC, bioabsorbable scaffolds, and applied shear stress.

Animals↗

PubMedDate not supplied

Tissue culture studies on Amaranthus viridis.

Amaranthus viridis is a plant that is very widely distributed in our country, grows easily, and is eaten by low-income groups. It has been found that the proteins of this plant, like most other plants, are deficient in some essential amino acids. We were able to develop cultures from the cut-end parts of this plant tissue. The commonly used media were modified to achieve good and fast growth. Results have been obtained on the techniques, histology, chromosomal, and biochemical studies which indicate that the plant could be useful as a research tool to study problems in crop improvement at the cellular level.

Chromosomes↗

PubMed1986

Scanning fluorometer for the rapid assessment of pyridine nucleotide and flavoprotein fluorescence changes in tissues in vivo.

This paper describes a scanning fluorometer which produces images in real time of the distribution of pyridine nucleotide or flavoprotein fluorescence at the surface of tissues in vivo. The basic difference between this device and others reported in the literature is that fluorescence changes at any selected point within the image can be quantified as they occur. We suggest that the apparatus has potential application in those areas of surgery where vascular replacement or repair is required and where it would be advantageous to have an immediate measure of the cellular response to a return of blood flow.

Animals↗

PubMed1992

Prospects for cellular immunotherapy for metastatic melanoma.

Systemic therapy for metastatic malignant melanoma has been disappointing. The search for alternate therapeutic methods includes investigation of the interaction between melanoma and the human immune system. A cellular immune response to melanoma has been documented in vitro and in vivo. In most patients with disseminated disease, however, immune T cells fail to eradicate the tumor. While this phenomenon is poorly understood, the occasional occurrence of spontaneous regression provides some indication that the immune response may, in fact, be capable of eradicating established tumor in vivo. Current efforts to augment and to direct the immune response to melanoma include investigation of specific and nonspecific adoptive immunotherapy. Specific therapy includes the generation of tumor-activated specific killer cells from peripheral blood, draining nodes, or metastatic tumor deposits. An increasing understanding of antigen recognition and improved methodology for T-cell culture are contributing toward the application of cellular immunotherapy to patients with melanoma.

Animals↗

PubMed2000

In vitro cell shearing device to investigate the dynamic response of cells in a controlled hydrodynamic environment.

Mechanical stresses and strains play important roles in the normal growth and development of biological tissues, yet the cellular mechanisms of mechanotransduction have not been identified. A variety of in vitro systems for applying mechanical loads to cell populations have been developed to gain insight into these mechanisms. However, limitations in the ability to control precisely relevant aspects of the mechanical stimuli have obscured the physical relationships between mechanical loading and the biochemical signals that mediate the cellular response. We present a novel in vitro cell shearing device based on the principles of a cone and plate viscometer that utilizes microstepper motor technology to control independently the dynamic and steady components of a hydrodynamic shear-stress environment. Physical measurements of the cone velocity demonstrated faithful reproduction of user-defined input wave forms. Computational modeling of the fluid environment for the unsteady startup confirmed small inertial contributions and negligible secondary flows. Finally, we present experimental results demonstrating the onset rate dependence of functional and structural responses of endothelial cell cultures to dynamically applied shear stress. The controlled cell shearing device is a novel tool for elucidating mechanisms by which mechanical forces give rise to the biological signals that modulate cellular morphology and metabolism.

Animals↗

PubMed1998

DNA vaccines: vector design, delivery, and antigen presentation.

Inoculations with antigen-expressing plasmid DNAs (DNA vaccines) in the production of protective immune responses. Since the initial development of DNA vaccines more than 5 years ago, major strides have been made in the design of efficient vaccine vectors and in the process of vaccine delivery. However, many questions remain regarding the mechanism of cellular transfection and in the development of immune responses. This review addresses functional aspects of DNA vaccines, including vector design and delivery, as well as cellular transfection and antigen presentation.

Animals↗

PubMed2002

Context-dependent functions of the PII and GlnK signal transduction proteins in Escherichia coli.

Two closely related signal transduction proteins, PII and GlnK, have distinct physiological roles in the regulation of nitrogen assimilation. Here, we examined the physiological roles of PII and GlnK when these proteins were expressed from various regulated or constitutive promoters. The results indicate that the distinct functions of PII and GlnK were correlated with the timing of expression and levels of accumulation of the two proteins. GlnK was functionally converted into PII when its expression was rendered constitutive and at the appropriate level, while PII was functionally converted into GlnK by engineering its expression from the nitrogen-regulated glnK promoter. Also, the physiological roles of both proteins were altered by engineering their expression from the nitrogen-regulated glnA promoter. We hypothesize that the use of two functionally identical PII-like proteins, which have distinct patterns of expression, may allow fine control of Ntr genes over a wide range of environmental conditions. In addition, we describe results suggesting that an additional, unknown mechanism may control the cellular level of GlnK.

Amino Acid Sequence↗

PubMed2002

Engineered expression of the Coxsackie B and adenovirus receptor (CAR) in human dendritic cells enhances recombinant adenovirus-mediated gene transfer.

Dendritic cells (DCs) are key antigen-presenting cells (APCs) that act as central modulators of cellular immune responses. Genetic modification of DCs has considerable therapeutic potential in the treatment of a wide spectrum of diseases, including cancer and persistent viral infection. In this report, we show that pre-treatment of DCs with a recombinant adenovirus encoding the major adenovirus receptor, Coxsackie B and adenovirus receptor (CAR), significantly increased the uptake of recombinant adenoviruses (Ads) by primary immature monocyte-derived DCs. This could be correlated with CAR mRNA and surface protein expression. Transduction of DCs by recombinant adenoviruses did not significantly alter cellular viability. Therefore, we propose that pre-treatment of DCs with Ad5-CAR is one strategy to increase the susceptibility of DCs to transduction by recombinant Ads.

Antigen Presentation↗

PubMed1995

Engineering liposomes for drug delivery: progress and problems.

Liposomes were first proposed and tested as a drug delivery system 25 years ago. Since then, advances in our understanding of the fate and behaviour of liposomes at the cellular and subcellular level in vivo have allowed the rational design of constructs for use in the treatment and prevention of disease, both in experimental animals and clinically. The involvement of several liposome-based biotechnology companies in the early 1980s, paralleled by great leaps in liposome technology, has culminated in the design and licencing of formulations for the treatment of certain microbial infections and cancers, and the first liposome-based vaccine for use in humans.

Drug Delivery Systems↗

PubMed2001

Tissue-engineered product: allogeneic cultured dermal substitute composed of spongy collagen with fibroblasts.

Recently, various types of allogeneic skin substitutes including cultured epidermal substitute (CES), cultured dermal substitute (CDS), and cultured skin substitute (CSS), which are composed of keratinocytes and/or fibroblasts as the cellular component(s), have been used as biological wound dressings. In our study, the allogeneic CDS was prepared by plating fibroblasts on a spongy collagen. The clinical evaluation was conducted using fresh or cryopreserved allogeneic CDS. In 145 of our clinical cases, 95% (138/145) of various wounds were evaluated as achieving good or excellent results, including 96% (22/23) of deep dermal burns (DDB) and dermal burns (DB), 100% (53/53) of partial-thickness donor wounds, 91% (21/23) of traumatic skin defects, 100% (5/5) of pressure ulcers, 82% (9/11) of chronic skin ulcers, 100% (6/6) of coverage for debrided DB, and 92% (22/24) of coverage for autologous meshed graft. The results obtained in our study suggest that the allogeneic CDS is able to provide an effective therapy for patients with partial and/or full-thickness skin defects.

Aged↗

PubMed2005

T-cell reconstitution and expansion after hematopoietic stem cell transplantation: 'T' it up!

Adoptive immunotherapy is the isolation and infusion of antigen-specific or nonspecific lymphocytes. Adoptive therapy with T cells may have a role in replacing, repairing, or enhancing immune function damaged by cytotoxic therapies, and rapid lymphocyte recovery may improve outcome after autologous and allogeneic stem cell transplantation (SCT). Recently, a plethora of information on the basic mechanisms of T-cell biology and regulation of cellular immune responses has emerged, permitting the development of new forms of adoptive cell therapy. Efficient ex vivo culture method for T-cell subsets affords the possibility of adoptive transfer of T cells engineered with enhanced capacity for central memory, effector cytotoxicity, Th1, Th2, veto cell, and T regulatory functions. Studies show that homeostatic T-cell proliferation is important for effective adoptive immunotherapy and pretreatment with chemotherapy may enhance the effects of infused T cells. Replicative senescence, in part due to telomere erosion, likely limits successful adoptive immunotherapy, though it may be possible to maintain T-cell pools by enforced expression of telomerase. Clinical trials now demonstrate that it is possible to enhance immune reconstitution after SCT with cytokines or infusions of ex vivo costimulated expanded T cells. These data all support the premise that adoptive therapy can accelerate reconstitution of cellular immunity with enhanced antitumor effects following SCT.

Adoptive Transfer↗

PubMed1999

Involvement of fibronectin in the regulation of urokinase production and binding in murine mammary tumor cells.

Tumor invasion and metastasis development is a multistep process involving adhesion molecules as well as tumor proteases. It has been reported that tumor cells lacking fibronectin (FN) expression and engineered to re-express FN showed a marked reduction in metastatic ability. Besides its effects on cell adhesion and migration, FN could be modulating other cellular events associated with the metastatic cascade. To test this hypothesis, we analyzed the production of urokinase-type plasminogen activator (uPA), and its receptor (uPAR), 2 molecules involved in the invasive phenotype, in cells over-expressing RGD wild-type FN (FNwt clones) or RGD-mutated FN (FN RGD-minus clones). Secreted uPA activity and antigen were significantly up-regulated in FN-expressing clones, although RGD-minus cells secreted approximately 50% less uPA than the FNwt ones. Interestingly, while control and FN RGD-minus clones were able to readily bind uPA to their surface, FNwt clones exhibited impaired uPA binding. Furthermore, treatment of the parental cell line as well as the control and FN-expressing clones with exogenous purified FN or RGD peptides induced up-regulation of uPA production and the reduction of uPA membrane binding, which was associated with lower expression of uPAR. This modulation by FN was found to be dependent on RGD sequence and beta1 integrin. These results strongly suggest a novel activity for the multifunctional glycoprotein FN regarding the regulation of uPA production as well as the capacity of tumor cells to bind uPA.

Animals↗

PubMed1994

Murine and humanized constructs of monoclonal antibody M195 (anti-CD33) for the therapy of acute myelogenous leukemia.

Long-term survival rates of patients with acute myelogenous leukemia treated with intensive chemotherapy are 15-20%, despite efforts to develop new treatment strategies. Murine M195 (131I-M195), an anti-CD33, immunoglobulin (Ig) G2a monoclonal antibody has reactivity restricted to early myeloid cells and myeloid leukemic blasts but not hematopoietic progenitors. Previous trials in patients with relapsed or refractory myeloid leukemia showed that 131I-M195 rapidly targeted to the bone marrow and internalized into target cells. This article describes a therapeutic dose escalation study in which 24 patients received from 50 mCi/m2 to 210 mCi/m2 of 131I-M195 in divided doses. Cytoreduction of peripheral cell counts and bone marrow blasts occurred without nonhematopoietic toxicity. Doses of 131I-M195 greater than 135 mCi/m2 were associated with marrow cytoreduction sufficient to necessitate bone marrow transplant. However, 37% of the patients developed human anti-mouse antibody, preventing retreatment. To decrease immunogenicity and improve effector function, chimeric IgG1 and IgG3, and complementarity-determining region-grafted, humanized IgG1 and IgG3 versions of mouse M195 were developed by genetic engineering techniques. The new versions maintained specificity and biologic function, and they were superior to the mouse M195 in their ability to perform antibody-dependent cellular cytotoxicity against leukemia cells. Humanized M195, but not chimeric M195, showed a 4-8.6 times higher avidity than its mouse counterpart. Because effector function of IgG depends to a large extent on Fc clustering, a homodimeric HuG1 also was developed. Homodimeric HuG1 showed an ability to cause additional dramatic improvements in effector functions, as well as an ability to internalize and retain radioisotope in target leukemia cells. Monomeric and dimeric forms of humanized M195 may be advantageous in the therapy of acute myelogenous leukemia.

Adult↗

PubMed1991

Mature mRNA 3' end formation stimulates RNA export from the nucleus.

We have analysed nucleocytoplasmic export of mRNAs in primate cells with the aim of identifying signals promoting RNA transport. Our results demonstrate that sequences directing either histone mRNA 3' processing or cleavage/polyadenylation of mRNA stimulate nucleocytoplasmic RNA transport. To elucidate the nature of this stimulation, we engineered test gene transcripts which could obtain a mature histone 3' end by the RNA cleaving activity of a cis-acting ribozyme, thus circumventing the cellular 3' end processing machinery. However, such transcripts were found to be transport deficient and accumulated in the nuclear compartment. Our experiments provide genetic evidence that there is a linkage between 3' end formation and the export of RNA transcripts from the nucleus. Analysis of a similar series of histone mRNAs in which the mature 3' end was generated by means of ribozyme cleavage led to the discovery of a second export mechanism which relies on features specific for mature histone RNA and which can be uncoupled from the cellular processing machinery. The presence of histone mRNA sequences and of the highly conserved histone hairpin structure, positioned close to the 3' terminus, are critical determinants for this export mechanism.

Animals↗

PubMed1999

Single-cell models: promise and limitations.

This article describes the development of single-cell models, their uses and accomplishments, the barriers to the greater adoption, and a perspective on challenges to the biochemical engineering community where the single-cell model approach may be used advantageously. In particular, it may become an important tool in relating genomic information to cellular regulation and dynamics.

Animals↗

PubMed2001

Topology of a human equilibrative, nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporter (hENT1) implicated in the cellular uptake of adenosine and anti-cancer drugs.

The human equilibrative nucleoside transporter hENT1, the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for the cellular uptake of physiologic nucleosides, including adenosine, and many anti-cancer nucleoside drugs. We have produced recombinant hENT1 in Xenopus oocytes and used native and engineered N-glycosylation sites in combination with immunological approaches to experimentally define the membrane architecture of this prototypic nucleoside transporter. hENT1 (456 amino acid residues) is shown to contain 11 transmembrane helical segments with an amino terminus that is intracellular and a carboxyl terminus that is extracellular. Transmembrane helices are linked by short hydrophilic regions, except for a large glycosylated extracellular loop between transmembrane helices 1 and 2 and a large central cytoplasmic loop between transmembrane helices 6 and 7. Sequence analyses suggest that this membrane topology is common to all mammalian, insect, nematode, protozoan, yeast, and plant members of the ENT protein family.

Adenosine↗

PubMed2003

Replicative oncolytic adenoviruses in multimodal cancer regimens.

The use of replication-competent viruses that have a cytolytic cycle has emerged as a viable strategy (oncolytic virotherapy) to specifically kill tumor cells and the field has advanced to the point of clinical trials. A theoretical advantage of replicative oncolytic viruses is that their numbers should increase via viral replication within infected tumor cells and resulting viral progeny can then infect additional cells within the tumor mass. The life cycle of a virus involves multiple interactions between viral and cellular proteins/genes, which maximize the ability of the virus to infect and replicate within cells. Understanding such interactions has led to the design of numerous genetically engineered adenovirus (Ad) vectors that selectively kill tumor cells while sparing normal cells. These viruses have also been modified to function as therapeutic gene delivery vehicles, thus augmenting their anticancer capacity. In addition, the oncolytic mode of tumor killing differs from that of standard anticancer therapies, providing the possibility for synergistic interactions with other therapies in a multimodal antitumor approach. In this review, we describe the oncolytic Ad vectors tested in preclinical and clinical models and their use in combination with chemo-, radio-, and gene therapies.

Adenoviridae↗