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At least 937 records · Page 52Linked to original sources
PubMed2002

High-throughput measurement of the Tp53 response to anticancer drugs and random compounds using a stably integrated Tp53-responsive luciferase reporter.

Human Tp53 is normally a short-lived protein. Tp53 protein is stabilized and levels are increased in response to a variety of cellular stresses, including those induced by genotoxic anticancer drugs and environmental exposures. To engineer an efficient assay based on this property, we constructed and integrated a Tp53-specific reporter system into human cancer cells, termed p53R cells. We tested a range of conventional chemotherapeutic agents as well as over 16 000 diverse small compounds. Ionizing radiation and two-thirds of conventional chemotherapeutic agents, but only 0.2% of diverse compounds activated Tp53 activity by two-fold or greater, consistent with the presumptive genotoxic activation of Tp53 function. Cytotoxicity was independent of TP53 genetic status when paired, syngeneic wild-type TP53 and TP53-null cells in culture were treated with compounds that activated Tp53. From the unbiased survey of random compounds, Tp53 activation was strongly induced by an analog of AMSA, an investigational anti-cancer agent. Tp53 was also strongly induced by an N-oxide of quinoline and by dabequine, an experimental antimalarial evaluated in humans; dabequine was reported to be negative in other screens of mutagenicity and clastogenicity but carcinogenic in animal studies. Further exploration of antimalarial compounds identified the common medicinals chloroquine, quinacrine, and amodiaquine as Tp53-inducers. Flavonoids are known to have DNA topoisomerase activity, a Tp53-inducing activity that is confirmed in the assay. A reported clinical association of Tp53 immunopositive colorectal cancers with use of the antihypertensive agents was extended by the demonstration of hydralazine and nifedipine as Tp53-inducers. p53R cells represent an efficient Tp53 functional assay to identify chemicals and other agents with interesting biologic properties, including genotoxicity. This assay may have utility in the identification of novel chemotherapeutic agents, as an adjunct in the pharmaceutical optimization of lead compounds, in the exploration of environmental exposures, and in chemical probing of the Tp53 pathway.

Amsacrine↗
PubMed2004

Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol.

BACKGROUND: The aim of this study was to isolate mesenchymal stem cells (MSCs) from amniotic fluid obtained by second-trimester amniocentesis. METHODS: A novel two-stage culture protocol for culturing MSCs was developed. Flow cytometry, RT-PCR and immunocytochemistry were used to analyse the phenotypic characteristics of the cultured MSCs. Von Kossa, Oil Red O and TuJ-1 stainings were used to assess the differentiation potentials of MSCs. RESULTS: Amniotic fluid-derived MSCs (AFMSCs) were successfully isolated, cultured and enriched without interfering with the routine process of fetal karyotyping. Flow cytometry analyses showed that they were positive for SH2, SH3, SH4, CD29, CD44 and HLA-ABC (MHC class I), low positive for CD90 and CD105, but negative for CD10, CD11b, CD14, CD34, CD117, HLA-DR, DP, DQ (MHC class II) and EMA. Importantly, a subpopulation of Oct-4-positive cells was detectable in our cultured AFMSCs. Under specific culture conditions, AFMSCs could be induced to differentiate into adipocytes, osteocytes and neuronal cells. CONCLUSIONS: We demonstrate that human multipotent MSCs are present in second-trimester amniotic fluid. Considering the great potential of cellular therapy using fetal stem cells and the feasibility of intrauterine fetal tissue engineering, amniotic fluid may provide an excellent alternative source for investigation of human MSCs.

Amniotic Fluid↗
PubMed1997

Can enhancement be distinguished from prevention in genetic medicine?

In discussions of the ethics of human gene therapy, it has become standard to draw a distinction between the use of human gene transfer techniques to treat health problems and their use to enhance or improve normal human traits. Some dispute the normative force of this distinction by arguing that it is undercut by the legitimate medical use of human gene transfer techniques to prevent disease-such as genetic engineering to bolster immune function, improve the efficiency of DNA repair, or add cellular receptors to capture and process cholesterol. If disease prevention is a proper goal of medicine, these critics argue, and the use of gene transfer techniques to enhance human health maintenance capacities will help achieve that goal, then the "treatment/enhancement" distinction cannot define the limits of legitimate gene therapy. In this paper, I argue that a line can be drawn between prevention and enhancement for gene therapy (and thus between properly medical and nonmedical uses of gene therapy), but only if one is willing to accept two rather old-fashioned claims: 1) Some health problems are best understood as if they were entities in their own right, reifiable as processes or parts in a biological system, with at least as much ontological objectivity and theoretical significance as the functions that they inhibit. 2) Legitimate preventive genetic health care should be limited to efforts to defend people from attack by these more robust pathological entities, rather than changing their bodies to evade social injustices.

Gene Transfer Techniques↗
PubMed2001

Comparison of activation requirements and activation phenotype of allogeneic and xenogeneic rodent responses in vivo and in vitro.

BACKGROUND: Both discordant and concordant xenogeneic responses are dominated by humoral immunity. Recent advances in molecular engineering approaches may largely prevent rejection by means of this pathway, leaving the cellular arm of the immune response as the principal remaining barrier to successful engraftment. METHODS: To characterize further the cellular response to xenogeneic tissues, we used the intracellular fluorescent marker CFSE (5-(and-6)-carboxyfluorescein diacetate succinimidyl ester) to track the mitotic record of T cells (and T cell subsets) after either xenogeneic or allogeneic activation in vitro or in vivo. Activation marker expression was monitored by simultaneous labeling with antibodies for either CD25 or CD134. RESULTS: The in vitro and in vivo responses of Lewis lymphocytes were generally similar in magnitude and timing comparing activation with allogeneic or xenogeneic stimulators. However, the xenogeneic T cell precursor frequency was found to be markedly higher than that previously reported and were comparable to that seen in allogeneic responses. Xenogeneic responses were unique in the continued expression of activation markers in later division cycles. In addition, CD4 and CD8 T-cell proliferation was highly dependent on stimulator class II expression, highlighting the importance of CD4 T cells and the indirect pathway in the xenogeneic response. CONCLUSIONS: An unexpectedly high precursor frequency was detected for xenogeneic cellular responses in the rat anti-mouse combination and was comparable to that seen in allogeneic responses. Differences in xenogeneic versus allogeneic activation profiles exist that may result from the cellular pathways used for activation.

Animals↗
PubMed2001

Distinct and overlapping functions of insulin and IGF-I receptors.

Targeted gene mutations have established distinct, yet overlapping, developmental roles for receptors of the insulin/IGF family. IGF-I receptor mediates IGF-I and IGF-II action on prenatal growth and IGF-I action on postnatal growth. Insulin receptor mediates prenatal growth in response to IGF-II and postnatal metabolism in response to insulin. In rodents, unlike humans, insulin does not participate in embryonic growth until late gestation. The ability of the insulin receptor to act as a bona fide IGF-II-dependent growth promoter is underscored by its rescue of double knockout Igf1r/Igf2r mice. Thus, IGF-II is a true bifunctional ligand that is able to stimulate both insulin and IGF-I receptor signaling, although with different potencies. In contrast, the IGF-II/cation-independent mannose-6-phosphate receptor regulates IGF-II clearance. The growth retardation of mice lacking IGF-I and/or insulin receptors is due to reduced cell number, resulting from decreased proliferation. Evidence from genetically engineered mice does not support the view that insulin and IGF receptors promote cellular differentiation in vivo or that they are required for early embryonic development. The phenotypes of insulin receptor gene mutations in humans and in mice indicate important differences between the developmental roles of insulin and its receptor in the two species.

Alternative Splicing↗
PubMed2005

Inactivation of NF1 in CNS causes increased glial progenitor proliferation and optic glioma formation.

The gene responsible for neurofibromatosis type 1 (NF1) encodes a tumor suppressor that functions as a negative regulator of the Ras proto-oncogene. Individuals with germline mutations in NF1 are predisposed to the development of benign and malignant tumors of the peripheral and central nervous system (CNS). Children with this disease suffer a high incidence of optic gliomas, a benign but potentially debilitating tumor of the optic nerve; and an increased incidence of malignant astrocytoma, reactive astrogliosis and intellectual deficits. In the present study, we have sought insight into the molecular and cellular basis of NF1-associated CNS pathologies. We show that mice genetically engineered to lack NF1 in CNS exhibit a variety of defects in glial cells. Primary among these is a developmental defect resulting in global reactive astrogliosis in the adult brain and increased proliferation of glial progenitor cells leading to enlarged optic nerves. As a consequence, all of the mutant optic nerves develop hyperplastic lesions, some of which progress to optic pathway gliomas. These data point to hyperproliferative glial progenitors as the source of the optic tumors and provide a genetic model for NF1-associated astrogliosis and optic glioma.

Animals↗
PubMed1999

New approaches for estimating risk from exposure to diethylstilbestrol.

A subgroup from a National Institute of Environmental Health Sciences, workshop concerned with characterizing the effects of endocrine disruptors on human health at environmental exposure levels considered the question, If diethylstilbestrol (DES) were introduced into the market for human use today and likely to result in low-dose exposure of the human fetus, what would be required to assess risk? On the basis of an analysis of the quality of data on human DES exposure, the critical times and doses for inducing genital tract malformations and cancer must be determined. This would be facilitated through analysis of the ontogeny of estrogen receptor expression in the developing human genital tract. Models of low-dose estrogenic effects will have to be developed for human and rodent genital tract development. Mouse models offer many advantages over other potential animal models because of the wealth of the earlier literature, the availability of sensitive end points, the availability of mutant lines, and the possibility of generating genetically engineered model systems. Through multidisciplinary approaches, it should be possible to elucidate the cellular and molecular mechanisms of endocrine disruption elicited by estrogens during development and facilitate an assessment of risk to humans.

Animals↗
PubMed2006

Stem cells--potential for repairing damaged lungs and growing human lungs for transplant.

Repair or regeneration of defective lung epithelium would be of great therapeutic potential. It is estimated by the British Lung Foundation that 1 in 7 people in the UK is affected by a lung disease and that 1 in 4 admissions to children's wards are as a result of respiratory problems. Potential cellular sources for the regeneration of lung tissue in vivo or lung tissue engineering in vitro include endogenous pulmonary epithelial stem cells, extrapulmonary circulating stem cells and embryonic stem cells. This article discusses the potential role of each of these stem cell types in future approaches to the treatment of lung injury and disease.

Animals↗
PubMed1997

Deletion of the S component inverted repeat sequence c' and the nonessential genes U(S)1 through U(S)5 from the herpes simplex virus type 1 genome substantially impairs productive viral infection in cell culture and pathogenesis in the rat central nervous system.

A distinctive feature of the genetic make-up of herpes simplex virus type 1 (HSV-1), a human neurotropic virus, is that approximately half of the 81 known viral genes are not absolutely required for productive infection in Vero cells, and most can be individually deleted without substantially impairing viral replication in cell culture. If large blocks of contiguous viral genes could be replaced with foreign DNA sequences, it would be possible to engineer highly attenuated recombinant HSV-1 gene transfer vectors capable of carrying large cellular genes or multiple genes having related functions. We report the isolation and characterization of an HSV-1 mutant, designated d311, containing a 12 kb deletion of viral DNA located between the L-S Junction a sequence and the U(S)6 gene, spanning the S component inverted repeat sequence c' and the nonessential genes U(S)1 through U(S)5. Replication of d311 was totally inhibited in rat B103 and mouse Neuro-2A neuroblastoma cell lines, and was reduced by over three orders of magnitude in human SK-N-SH neuroblastoma cells compared to wild-type (wt) HSV-1 KOS. This suggested that the deleted genes, while nonessential for replication in Vero cells, play an important role in HSV replication in neuronal cells, particularly those of rodent origin. Unlike wt KOS which replicated locally and spread to other regions of brain following stereotactic inoculation into rat hippocampus, d311 was unable to replicate and spread within the brain, and did not cause any apparent local neuronal cell damage. These results demonstrate that d311 is highly attenuated for the rat central nervous system. d311 and other mutants of HSV containing major deletions of the nonessential genes within U(S) have the potential to serve as useful tools for gene transfer applications to brain.

Animals↗
PubMed2005

[Synthesis of a bio-active bone-matrix material and study of the cellular biocompatibility].

To prepare poly(lactic acid/glycolic acid/ asparagic acid-co- polyethylene glycol) (PLGA-[ASP-PEG]) and examine the cellular biocompatibility. PLGA-[ASP-PEG] was obtained by bulk ring-opening copolymerization method, examined by infrared spectrometry (IR) and 1H nuclear magnetic resonance spectroscopy (1H NMR). Bone marrow stromal cells(BMSCs) were cultured with PLGA-[ASP-PEG] (experiment gruop) and PLGA (control group) in vitro respectively, and were observed by phase-contrast microscopy and scanning electron microscopy. The resuls showed that PLGA-[ASP-PEG] was obtained and proved by IR and 1H NMR. The BMSCs of the experiment group could well attach to and extend on the surface of the PLGA-[ASP-PEG], and could proliferate and secrete better extracellular matrix, compared with control. The PLGA-[ASP-PEG] has good cellular a biocompatibility. It can be used as a biomaterial for bone tissue engineering.

Aspartic Acid↗
PubMedDate not supplied

Novel approaches to tumor detection and therapy using a combination of monoclonal antibody and cytokine.

Cytokine-based tumor antigen augmentation is one of the approaches researchers and clinicians are using to improve the effectiveness of MAb-directed tumor diagnosis and therapy. Other efforts encompass the use of dose-fractionation for multiple administrations of radioimmunoconjugates, exploitation of genetic engineering to construct antibody molecules with specific biological properties (i.e., altered pharmacokinetics, activation of cellular immune responses, etc.) and use of MAb-directed conjugates that can enhance tumor MAb uptake by altering tumor perfusion. The studies summarized here as well as those from other laboratories have served as the framework for clinical investigations designed to determine the effectiveness of the interferons and other differentiation-inducing agents to alter the tumor antigen phenotype in patients. In an earlier study, patients given IFN-alpha had improved tumor uptake of an antimelanoma MAb. Subsequently, we reported that i.p. IFN-gamma administration substantially upregulated TAG-72 and CEA on the surface of human tumor cells isolated from malignant ascites. A seminal investigation showed significant increase of TAG-72 and CEA levels in tumor biopsies from patients diagnosed with colorectal carcinoma and given systemic IFN-alpha. Those studies led to a clinical trial in which late stage breast cancer patients were administered interferon in combination with therapeutic doses of CC49. Some clinical responses were observed, however, the cytokine and MAb combination may have also enhanced marrow toxicity. Future studies will continue to evaluate the ability to enhance tumor antigen expression in the context of genetically engineered MAbs designed to minimize normal organ toxicity.

Antibodies, Monoclonal↗
PubMedDate not supplied

Animal disease models generated by genetic engineering of polyamine metabolism.

The polyamines putrescine, spermidine and spermine are natural components of all living cells. Although their exact cellular functions are still largely unknown, a constant supply of these compounds is required for mammalian cell proliferation to occur. Studies with animals displaying genetically altered polyamine metabolism have shown that polyamines are intimately involved in the development of diverse tumors, putrescine apparently has specific role in skin physiology and neuroprotection and the higher polyamines spermidine and spermine are required for the maintenance of pancreatic integrity and liver regeneration. In the absence of ongoing polyamine biosynthesis, murine embryogenesis does not proceed beyond the blastocyst stage. The last years have also witnessed the appearance of the first reports linking genetically altered polyamine metabolism to human diseases.

Animals↗
PubMed2002

In vitro engineering of heart muscle: artificial myocardial tissue.

INTRODUCTION: Myocardial infarction followed by heart failure represents one of the major causes of morbidity and mortality, particularly in industrialized countries. Engineering and subsequent transplantation of contractile artificial myocardial tissue and, consequently, the replacement of ischemic and infarcted areas of the heart provides a potential therapeutic alternative to whole organ transplantation. METHODS: Artificial myocardial tissue samples were engineered by seeding neonatal rat cardiomyocytes with a commercially available 3-dimensional collagen matrix. The cellular engraftment within the artificial myocardial tissues was examined microscopically. Force development was analyzed in spontaneously beating artificial myocardial tissues, after stretching, and after pharmacologic stimulation. Moreover, electrocardiograms were recorded. RESULTS: Artificial myocardial tissues showed continuous, rhythmic, and synchronized contractions for up to 13 weeks. Embedded cardiomyocytes were distributed equally within the 3-dimensional matrix. Application of Ca(2+) and epinephrine, as well as electrical stimulation or stretching, resulted in enhanced force development. Electrocardiographic recording was possible on spontaneously beating artificial myocardial tissue samples and revealed physiologic patterns. CONCLUSIONS: Using a clinically well-established collagen matrix, contractile myocardial tissue can be engineered in vitro successfully. Mechanical and biologic properties of artificial myocardial tissue resemble native cardiac tissue. Use of artificial myocardial tissues might be a promising approach to reconstitute degenerated or failing cardiac tissue in many disease states and therefore provide a reasonable alternative to whole organ transplantation.

Animals↗
PubMed2002

Promoter competition assay for analyzing gene regulation in joint tissue engineering.

We describe a new biochemical technique, "promoter competition assay," for examining the role of cis-acting DNA elements in tissue cultures. Recent advances in tissue engineering permit the culture of a variety of cells. Many tissues are engineered, however, without an appropriate understanding of molecular machinery that regulates gene expression and cellular growth. For elucidating the role of cis-acting regulatory elements in cellular differentiation and growth, we developed the promoter competition assay. This assay uses a transient transfer into cells of double-stranded DNA fragments consisting of cis-acting regulatory elements. The transferred DNA fragments act as a competitor and titrate the function of their genomic counterparts. Using synovial cells derived from a rheumatoid arthritis patient, we examined a role of NF-kappa B binding sites in the regulation of the expression of matrix metalloproteinase (MMP) genes. The results support a stimulatory role of NF-kappa B in transcriptional regulation of MMP-1 and MMP-13.

Arthritis, Rheumatoid↗
PubMed1998

Protein-like molecular architecture: biomaterial applications for inducing cellular receptor binding and signal transduction.

The development of biomaterials with desirable biocompatibility has presented a difficult challenge for tissue engineering researchers. First and foremost, materials themselves tend to be hydrophobic and/or thrombogenic in nature, and face compatibility problems upon implantation. To mediate this problem, researchers have attempted to graft protein fragments onto biomaterial surfaces to promote endothelial cell attachment and minimize thrombosis. We envisioned a novel approach, based on the capability of biomolecules to self-assemble into well-defined and intricate structures, for creating biomimetic biomaterials that promote cell adhesion and proliferation. One of the most intriguing self-assembly processes is the folding of peptide chains into native protein structures. We have developed a method for building protein-like structural motifs that incorporate sequences of biological interest. A lipophilic moiety is attached onto a N alpha-amino group of peptide chain, resulting in a "peptide-amphiphile." The alignment of amphiphilic compounds at the lipid-solvent interface is used to facilitate peptide alignment and structure initiation and propagation, while the lipophilic region absorbs to hydrophobic surfaces. Peptide-amphiphiles containing potentially triple-helical or alpha-helical structural motifs have been synthesized. The resultant head group structures have been characterized by CD spectroscopy and found to be thermally stable over physiological temperature ranges. Triple-helical peptide-amphiphiles have been applied to studies of surface modification and cell receptor binding. Cell adhesion and spreading was promoted by triple-helical peptide-amphiphiles. Cellular interaction with the type IV collagen sequence alpha 1(IV) 1263-1277 increased signal transduction, with both the time and level of induction dependent upon triple-helical conformation. Collectively, these results suggest that peptide-amphiphiles may be used to form stable molecular structure on biomaterial surfaces that promote cellular activities and improve biocompatibility.

Amino Acid Sequence↗
PubMed2001

Poly(lactide-co-glycolide)/hydroxyapatite delivery of BMP-2-producing cells: a regional gene therapy approach to bone regeneration.

Currently, functional treatment of fracture non-unions and bone loss remains a significant challenge in the field of orthopaedic surgery. Tissue engineering of bone has emerged as a new treatment alternative in bone repair and regeneration. Our approach is to combine a polymeric matrix with a cellular vehicle for delivery of bone morphogenetic protein-2 (BMP-2), constructed through retroviral gene transfer. The objective of this study is to develop an osteoinductive, tissue-engineered bone replacement system by culturing BMP-2-producing cells on an osteoconductive, biodegradable, polymeric-ceramic matrix. The hypothesis is that retroviral gene transfer can be used effectively in combination with a biodegradable matrix to promote bone formation. First, we examined the in vitro attachment and growth of transfected BMP-producing cells on a PLAGA-HA scaffold. Second, the bioactivity of the produced BMP in vitro was evaluated using a mouse model. It was found that the polymer-ceramic scaffold supported BMP-2 production, allowing the attachment and growth of retroviral transfected, BMP-2-producing cells. In vivo, the scaffold successfully functioned as a delivery vehicle for bioactive BMP-2, as it induced heterotopic bone formation in a SCID mouse model.

Animals↗
PubMed2006

Engineering of therapeutic antibodies to minimize immunogenicity and optimize function.

One of the first difficulties in developing monoclonal antibody therapeutics was the recognition that human anti-mouse antibody (HAMA) response limited the administration of murine antibodies. Creative science has lead to a number of ways to counter the immunogenicity of non-human antibodies, primarily through chimeric, humanized, de-immunized, and most recently, human-sequence therapeutic antibodies. Once therapeutic antibodies of low or no immunogenicity were available, the creativity then turned to engineering both the antigen-binding domains (e.g., affinity maturation, stability) and altering the effector functions (e.g. antibody-dependent cellular cytotoxicity, complement-dependent cellular cytotoxicity, and clearance rate).

Animals↗