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PubMedDate not supplied

Cell cycle and cancer.

PURPOSE: To evaluate the link between cell cycle dysfunctions and tumor formation. DESIGN: A review of the cell cycle mechanism and its regulatory factors which are involved in carcinogenesis. RESULT: Cell duplication is directed by a precise cellular machine. The engine of this machine is composed of the cyclin-dependent kinases (Cdks). Their function mainly consists of phosphorylating the pRb family of proteins to conduct the cell towards a series of events that end in generating two sister cells from one mother cell. The regulation of Cdk activity depends on several cellular proteins that are part of a major system that is able to sense extracellular factors and intracellular signals. Abnormalities in cell cycle regulation and in its checkpoints lead to development of malignant cells. Various components of the cell cycle machinery are mutated, overexpressed or eliminated in several human cancers. Some of them can be even classified as oncogenes or tumor suppressor genes. CONCLUSIONS: It is necessary to design new antitumoral strategies able to target cells harboring such alterations, in order to understand the events that regulate the cell cycle and its disruption during oncogenesis.

Cell Cycle↗

PubMed2000

The stress kit: a new method based on competitive reverse transcriptase-polymerase chain reaction to quantify the expression of human alphaB-crystallin, Hsp27, and Hsp60.

We describe a reverse transcriptase-polymerase chain reaction method for the semiquantitative detection of mRNAs encoding the human heat shock proteins alphaB-crystallin, Hsp27, and Hsp60. The method involves the coamplification of cellular mRNA-derived cDNA with a dilution series of a competitor fragment (internal standard), using 1 primer pair common to both templates. Internal standards were based on cellular-derived cDNA engineered to be slightly smaller to differentiate between the target and the standard on electrophoretic separation. Initial cDNA quantitations can be corrected for possible variations during cDNA synthesis by standardizing to the levels of beta-actin-encoding cDNA. We show that the coamplified templates accumulate in a parallel manner with the cellular-derived cDNA throughout both the exponential and the nonexponential phase of amplification. Furthermore, we illustrate the utility of this technique by quantifying increased expression of alphaB-crystallin, Hsp27, and Hsp60 mRNA in astroglioma cells on heat shock.

Astrocytoma↗

PubMed2005

Tissue regeneration. The past, the present and the future.

Tissue engineering has been a topic of extensive research over the last years. The ability of human body to regenerate tissue loss such as bone, cartilage, nerves, skin and muscle is limited leading often to amputations of limbs or functional disability. The isolation of mesenchymal stem cells (MSCs) and later the embryonic stem cells in conjunction with the advances made in cellular biology, tissue engineering, genetics and recombinant technology has initiated the development of new techniques and new therapeutic strategies allowing treatment of many pathological conditions providing restoration of tissue continuity and function.

Bone Transplantation↗

PubMed2006

Commercial considerations in tissue engineering.

Tissue engineering is a field with immense promise. Using the example of an early tissue-engineered skin implant, Dermagraft, factors involved in the successful commercial development of devices of this type are explored. Tissue engineering has to strike a balance between tissue culture, which is a resource-intensive activity, and business considerations that are concerned with minimizing cost and maximizing customer convenience. Bioreactor design takes place in a highly regulated environment, so factors to be incorporated into the concept include not only tissue culture considerations but also matters related to asepsis, scaleup, automation and ease of use by the final customer. Dermagraft is an allogeneic tissue. Stasis preservation, in this case cryopreservation, is essential in allogeneic tissue engineering, allowing sterility testing, inventory control and, in the case of Dermagraft, a cellular stress that may be important for hormesis following implantation. Although the use of allogeneic cells provides advantages in manufacturing under suitable conditions, it raises the spectre of immunological rejection. Such rejection has not been experienced with Dermagraft. Possible reasons for this and the vision of further application of allogeneic tissues are important considerations in future tissue-engineered cellular devices. This review illustrates approaches that indicate some of the criteria that may provide a basis for further developments. Marketing is a further requirement for success, which entails understanding of the mechanism of action of the procedure, and is illustrated for Dermagraft. The success of a tissue-engineered product is dependent on many interacting operations, some discussed here, each of which must be performed simultaneously and well.

Bioreactors↗

PubMed2006

Local myocardial insulin-like growth factor 1 (IGF-1) delivery with biotinylated peptide nanofibers improves cell therapy for myocardial infarction.

Strategies for cardiac repair include injection of cells, but these approaches have been hampered by poor cell engraftment, survival, and differentiation. To address these shortcomings for the purpose of improving cardiac function after injury, we designed self-assembling peptide nanofibers for prolonged delivery of insulin-like growth factor 1 (IGF-1), a cardiomyocyte growth and differentiation factor, to the myocardium, using a "biotin sandwich" approach. Biotinylated IGF-1 was complexed with tetravalent streptavidin and then bound to biotinylated self-assembling peptides. This biotin sandwich strategy allowed binding of IGF-1 but did not prevent self-assembly of the peptides into nanofibers within the myocardium. IGF-1 that was bound to peptide nanofibers activated Akt, decreased activation of caspase-3, and increased expression of cardiac troponin I in cardiomyocytes. After injection into rat myocardium, biotinylated nanofibers provided sustained IGF-1 delivery for 28 days, and targeted delivery of IGF-1 in vivo increased activation of Akt in the myocardium. When combined with transplanted cardiomyocytes, IGF-1 delivery by biotinylated nanofibers decreased caspase-3 cleavage by 28% and increased the myocyte cross-sectional area by 25% compared with cells embedded within nanofibers alone or with untethered IGF-1. Finally, cell therapy with IGF-1 delivery by biotinylated nanofibers improved systolic function after experimental myocardial infarction, demonstrating how engineering the local cellular microenvironment can improve cell therapy.

Animals↗

PubMed2006

Update on tissue-engineered biological dressings.

Tissue-engineered biological dressings offer promise in the treatment of burns, chronic ulcers, donor site and other surgical wounds, and a variety of blistering and desquamating dermatologic conditions. For example, the prevalence of diabetic foot ulcers ranges from 4.4% to 10.5% of diabetics, resulting in 82,000 lower extremity amputations annually; venous leg ulcers affect 0.18% to 1.35% of the population; and pressure ulcers are found in 5.0% to 8.8% of institutionalized patients and 14.8% of patients in acute care facilities. Despite the large number of potential beneficiaries, cellular tissue-engineered products have suffered setbacks in recent years and have garnered considerably lower market share than commercial promoters anticipated. The mechanism of action of these products is not universally agreed upon, but delivery of growth factors and extracellular matrix components to the wound is thought to be important; graft "take" is not usually considered to occur. These "engineered" products do not specifically match a treatment modality to an underlying pathology. Clinical effect is often modest, and sometimes not justi- fiable from a cost-benefit perspective. Nevertheless, clinical reports in the literature of uses of tissueengineered biological dressings continue to mount, indicating that these products are finding niche applications where clinical utility is high and the cost can be defended. Despite commercial setbacks, the first-approved products, Dermagraft, Apligraf, and Cultured Epidermal Autograft (Epicel) are still being marketed, and new ones, such as OrCel, continue to be developed. The major indications for these products are summarized and a brief review of the available clinical literature is offered.

Biological Dressings↗

PubMed1999

Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity.

The glycosylation pattern of chCE7, an antineuroblastoma chimeric IgG1, was engineered in Chinese hamster ovary cells with tetracycline-regulated expression of beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase catalyzing formation of bisected oligosaccharides that have been implicated in antibody-dependent cellular cytotoxicity (ADCC). Measurement of the ADCC activity of chCE7 produced at different tetracycline levels showed an optimal range of GnTIII expression for maximal chCE7 in vitro ADCC activity, and this activity correlated with the level of constant region-associated, bisected complex oligosaccharides determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The new optimized variants of chCE7 exhibit substantial ADCC activity and, hence, may be useful for treatment of neuroblastoma. The strategy presented here should be applicable to optimize the ADCC activity of other therapeutic IgGs.

Animals↗

PubMed2003

An engineered bifunctional recombinant molecule that regulates humoral and cellular effector functions of the immune system.

BACKGROUND: Humoral and cellular defense mechanisms mediate the rejection of transplanted cells, tissues, and organs after allogeneic or xenogeneic transplantation. Inhibition of complement and T-cell costimulation are strategies aimed at increasing transplant survival. METHODS: Engineered novel fusion proteins that contain the functional domains of human CD152 (hCTLA4) or porcine CD152 (pCD152) and human CD59 (hCD152-hCD59, pCD152-hCD59) were developed to form bifunctional chimeric proteins that retain the effector functions of both moieties. Porcine aortic endothelial cells and murine Balb/3T3 cells were transduced or transfected to express the novel fusion proteins. RESULTS: Fluorescence-activated cell sorter analysis of hCD152-hCD59 transduced primary porcine aortic endothelial cells or hCD152-hCD59 and pCD152-hCD59 transfected Balb/3T3 cells determined that the molecules were expressed on the cell surface, and that they retained conformational epitopes. We demonstrate that hCD152-hCD59 and pCD152-hCD59 chimeric proteins inhibit complement-mediated cell lysis. In addition, hCD152-hCD59 or pCD152-hCD59 expression resulted in a significant reduction in T-cell activation as the result of CD152 engagement of porcine CD86 or murine CD80 in when Jurkat cells were cocultured with the hCD152-hCD59 or pCD152-hCD59 expressing cells. Antibody-blocking experiments or phosphatidylinositol phospholipase C removal of the glycosyl-phosphatidylinositol-linked molecules resulted in increased serum-mediated cytolysis and eliminated the costimulatory blockade. CONCLUSIONS: These data illustrate that a single molecule can confer resistance to humoral and cellular immune attack.

3T3 Cells↗

PubMed2005

Nanotechnology approaches for the regeneration and neuroprotection of the central nervous system.

Nanotechnology is the science and engineering concerned with the design, synthesis, and characterization of materials and devices that have a functional organization in at least 1 dimension on the nanometer (ie, one-billionth of a meter) scale. The ability to manipulate and control engineered self-assembling (ie, self-organizing) substrates at these scales produces macroscopic physical and/or chemical properties in the bulk material not possessed by the constituent building block molecules alone. This in turn results in a degree of functional integration between the engineered substrates and cellular or physiological systems not previously attainable. Applied nanotechnology aimed at the regeneration and neuroprotection of the central nervous system (CNS) will significantly benefit from basic nanotechnology research conducted in parallel with advances in cell biology, neurophysiology, and neuropathology. Ultimately the goal is to develop novel technologies that directly or indirectly aid in providing neuroprotection and/or a permissive environment and active signaling cues for guided axon growth. In some cases, it is expected that the neurosurgeon will be required to administer these substrates to the patient. As such, in order for nanotechnology applications directed toward neurological disorders to develop to their fullest potential, it will be important for neuroscientists, neurosurgeons, and neurologists to participate and contribute to the scientific process alongside physical science and engineering colleagues. This review will focus on emerging clinical applications aimed at the regeneration and neuroprotection of the injured CNS, and discuss other platform technologies that have a significant potential for being adapted for clinical neuroscience applications.

Biomedical Engineering↗

PubMed2004

Comparative study of cellular and extracellular matrix composition of native and tissue engineered heart valves.

Tissue engineering of heart valves utilizes biodegradable or metabolizable scaffolds for remodeling by seeded autologous cells. The aim of this study was to determine and compare extracellular matrix (ECM) formations, cellular phenotypes and cell location of native and tissue engineered (TE) valve leaflets. Ovine carotid arteries, ovine and porcine hearts were obtained from slaughterhouses. Cells were isolated from carotid arteries and dissected ovine, porcine and TE leaflets. TE constructs were fabricated from decellularized porcine pulmonary valves, seeded ovine arterial cells and subsequent 16 days dynamic in vitro culture using a pulsatile bioreactor. Native and TE valves were studied by histology (hematoxylin-eosin, resorcin-fuchsin, Movat pentachrome), NIR femtosecond multiphoton laser scanning microscopy and scanning electron microscopy (SEM). Cells of native and TE tissues were identified and localized by immunohistochemistry. Arterial, valvular and re-isolated TE-construct cells were processed for immunocytochemistry and Western blotting. ECM analysis and SEM revealed characteristical and comparable structures in native and TE leaflets. Most cells in native leaflets stained strongly positive for vimentin. Cells positive to alpha-smooth muscle actin (alpha-SMA), myosin and calponin were only found at the ventricular (inflow) side of ovine aortic and porcine pulmonary valve leaflets. Cells from TE constructs had a strong expression of vimentin, alpha-SMA, myosin, calponin and h-caldesmon throughout the entire leaflet. Comparable ECM formation and endothelial cell lining of native and TE leaflets could be demonstrated. However, immunostaining revealed significant differences between valvular cell phenotypes of native and TE leaflets. These results may be essential for further cardiovascular tissue engineering efforts.

Animals↗

PubMedDate not supplied

Regulation of cell proliferation and apoptosis in CHO-K1 cells by the coexpression of c-Myc and Bcl-2.

Proliferation and cell death are regarded as key targets for the optimization of animal cell culture processes and for the maximization of product yield. Although chemical and physical factors are vitally important, of primary interest is the utilization of genetic engineering to regulate cellular processes. CHO cells were first genetically modified to enhance proliferation rate in both suspension and monolayer cultures. Under the constitutive control of c-myc overexpression the CHO cultures showed an increase in growth rate and maximum cell number accompanied by a similar decrease in specific glucose consumption rate. Although the c-myc transfected cell line exhibited apoptosis at much lower rates than is widely reported and associated with the overexpression of c-Myc, it was nevertheless apparent that c-Myc was responsible for the induction of higher apoptotic rates when compared with the control cell line. Hence, the anti-apoptotic gene bcl-2 was also used to transfect the c-Myc CHO cell line, to reduce cell death. Overexpression of both oncoproteins resulted in a cell line that exhibited higher proliferation rates and maximum cell numbers, with a decrease in apoptosis when compared to the parental cell line. In conclusion, it was shown that Bcl-2 protein overexpression specifically abrogates c-Myc-induced apoptosis without affecting the c-Myc mitogenic function.

Animals↗

PubMed1997

Hematopoietic transplantation: state of the art.

Bone marrow transplantation has developed from an experimental therapy for a small group of patients to a well-established form of treatment with well-defined indications for a large group of patients with hematological and non-hematological neoplasia. The availability of suitable donors has more than doubled due to large registries of persons volunteering for marrow donation. With improved techniques for histocompatibility typing, it has become possible to study the role of specific histoincompatibilities for graft-versus-host disease and graft-versus-leukemia reactions. The source of hematopoietic stem cells now comprises not only bone marrow, but also stem cells mobilized into the peripheral blood and stem cells from cord blood. Hematopoietic growth factors have found a large distribution in the mobilization of stem cells and support for hematological reconstitution. They are studied for the expansion of pools of stem cells. Reconstitution of antileukemia and antiviral activity has been achieved by adoptive immunotherapy using lymphocytes and dendritic cells cultured in vitro. The way from transplantation of bone marrow to cellular and genetic engineering leads to new indications such as treatment of severe autoimmune disease. Hematopoietic transplantation has come a long way and it still has possible new areas of application.

Graft vs Host Disease↗

PubMed2006

Study on the effects of nylon-chitosan-blended membranes on the spheroid-forming activity of human melanocytes.

Though reported limitedly in tissue engineering, modification of cellular functions can be achieved by culturing them into multicellular spheroids. We have shown melanocytes form spheroids on chitosan surface. However, how biomaterials promote spheroid formation has never been systemically investigated. In this work, nylon, which inhibits melanocyte spheroid formation, and chitosan, which promotes melanocyte spheroid formation, are used to prepare nylon/chitosan-blended membranes. Membranes composed of pure nylon, pure chitosan and various ratios of nylon and chitosan are employed to examine their effects on spheroid formation. Melanocytes show better adhesion to nylon membranes than that to chitosan membranes. In blended membranes, as more nylon is incorporated, cell adhesion increases and the trend for spheroid formation decreases. Melanocytes can only form spheroids on membranes with poorer cell adhesion. Examining the surface of the blended membranes shows phase separation of nylon and chitosan. As nylon content increases, the nylon phase on the membrane surface increases and thereby enhances cell adhesion. The opposite trend for cell adhesion and spheroid formation substantiates our hypothesis of spheroid formation on biomaterials: a balance between cell-substrate interaction and cell-cell interaction. The decrease in cell-substrate interaction tilts the balance to a state more favorable for spheroid formation. Our work can serve as a model to investigate the relative strengths of cell-cell and cell-substrate interactions and also pave way to design blended membranes with desired physical properties while preserving the spheroid-forming activity.

Biocompatible Materials↗

PubMed2005

Preparative expression of secreted proteins in bacteria: status report and future prospects.

The expression of heterologous secreted proteins in Escherichia coli is widely employed for laboratory and preparative purposes. Thanks to advances in expression technologies over the past 25 years, many mammalian proteins can now be produced routinely in secreted form with yields in the gram/litre scale. Nonetheless, ensuring efficient secretion across the inner membrane, and preventing proteolytic degradation, incorrect disulfide-bond formation and aggregation into periplasmic inclusion bodies, frequently presents significant challenges. Recent advances in the understanding of the periplasmic folding quality control system are leading to new strategies to facilitate the expression of heterologous secreted proteins. In parallel, protein design and directed evolution approaches are beginning to be exploited for engineering of the cellular protein folding machinery to achieve further improvements in protein expression.

Adenosine Triphosphatases↗

PubMed2025

The role of iPSC research for insight into inherited arrhythmia conditions.

Human induced pluripotent stem cells (iPSCs) have emerged as a transformative platform for modeling inherited cardiac arrhythmia syndromes and uncovering human-specific disease mechanisms. However, the promise of iPSC-derived cardiomyocytes lies beyond the recapitulation of arrhythmogenic phenotypes and channelopathies. In this review, we explore recent works which have enabled mechanistic interrogation and therapeutic insight for inherited arrhythmia syndromes, beyond the capabilities of traditional animal models. Such studies have leveraged iPSCs to elucidate the role of splice variants, transcriptional regulation, and mitochondrial stress in arrhythmogenesis. Further, iPSC systems have proven important for reclassifying variants of uncertain significance and in modeling idiopathic arrhythmias where genotype-phenotype links are elusive. Advances in directed differentiation now permit chamber-specific cardiac cell generation, allowing for atrial and ventricular disease modeling and revealing critical cell-cell interactions. iPSCs also serve as high-fidelity precursor platforms for drug testing, offering predictive insight into mutation-specific responses to pharmacologic and genetic therapies. Though limitations in maturation and scalability persist, ongoing efforts for integration with tissue engineering, multi-cellular models, and computational frameworks are evolving to improve model reliability. iPSC-based systems now occupy a critical role in arrhythmia research, bridging basic discovery with translational applications, thereby contributing to personalizing care and advancing therapeutics in inherited and idiopathic arrhythmic syndromes.

Humans↗

PubMed2003

Nucleus alignment and cell signaling in fibroblasts: response to a micro-grooved topography.

Cellular response to scaffold materials is of great importance in cellular and tissue engineering, and it is perhaps the initial cell contact with the scaffold that determines development of new tissue. Material surface morphology has strong effects on cell cytoskeleton and morphology, and it is thought that cells may react to the topography of collagen and surrounding cells during tissue embryology. A poorly understood area is, however, gene-level responses to topography. Thus, this paper used microarray to probe for consistent gene changes in response to lithographically produced topography (12.5 x 2-microm grooves) with time. The results showed many initial gene changes and also down-regulation of gene response with time. Cell and nucleus morphology were also considered, with nuclear deformation linked to cell signaling.

Cell Adhesion↗

PubMed2003

Coding of sweet, bitter, and umami tastes: different receptor cells sharing similar signaling pathways.

Mammals can taste a wide repertoire of chemosensory stimuli. Two unrelated families of receptors (T1Rs and T2Rs) mediate responses to sweet, amino acids, and bitter compounds. Here, we demonstrate that knockouts of TRPM5, a taste TRP ion channel, or PLCbeta2, a phospholipase C selectively expressed in taste tissue, abolish sweet, amino acid, and bitter taste reception, but do not impact sour or salty tastes. Therefore, despite relying on different receptors, sweet, amino acid, and bitter transduction converge on common signaling molecules. Using PLCbeta2 taste-blind animals, we then examined a fundamental question in taste perception: how taste modalities are encoded at the cellular level. Mice engineered to rescue PLCbeta2 function exclusively in bitter-receptor expressing cells respond normally to bitter tastants but do not taste sweet or amino acid stimuli. Thus, bitter is encoded independently of sweet and amino acids, and taste receptor cells are not broadly tuned across these modalities.

Action Potentials↗

PubMed1981

Liposome-mediated delivery of deoxyribonucleic acid to cells: enhanced efficiency of delivery related to lipid composition and incubation conditions.

Delivery of liposome-encapsulated simian virus 40 (SV40) DNA to African green monkey Related to been used as a probe to study liposome--cell interactions and to determine conditions which favor the intracellular delivery of liposome contents to cells. The efficiency of DNA delivery by various liposome preparations (monitored by infectivity assays) was found to be dependent both on the magnitude of vesicle binding to cells and on the resistance of liposomes to cell-induced leakage of contents. Acidic phospholipids were much more effective in both binding and delivery, and phosphatidylserine (PS) was the best in both aspects. The inclusion of 50 mol % cholesterol in liposomes reduces the cell-induced leakage of vesicle contents (2--5-fold) and substantially enhances the delivery of DNA to cells (2--10-fold). Following incubation of cells with negatively charged liposomes containing SV40 DNA, infectivity can be enhanced greatly by brief exposure of the cells to glycerol solutions. In contrast, only slight enhancement by glycerol was observed for SV40 DNA encapsulated in neutral or positively charged liposomes. The results of competition experiments between empty phosphatidylcholine liposomes and DNA-containing PS liposomes also suggest possible differences in the interaction of neutral and negatively charged liposome preparations with cells. Morphological studies indicate that the glycerol treatment stimulates membrane ruffling and vacuolization and suggest that the enhanced uptake of liposomes occurs by an endocytosis-like process. Results obtained with metabolic inhibitors are also consistent with the interpretation that the enhancement of liposome delivery in glycerol-treated cells occurs via an energy-dependent endocytotic pathway. Pretreatment of cells with chloroquine, a drug which alters lysosomal activity, further enhanced infectivity in glycerol-treated cells (4-fold). This observation suggests the involvement of a lysosomal processing step at some point in the expression of liposome-encapsulated DNA and, more importantly, illustrates the possibility of altering cellular mechanism to engineer more efficient delivery by liposomes. Under optimal conditions determined in this study, the efficiency of liposome-mediated SV40 DNA delivery was increased more than 1000-fold over that obtained by simply incubating cells with liposomes. It is also demonstrated that these conditions enhance delivery of other molecules, besides DNA, which are encapsulated in liposomes.

Animals↗