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At least 595 records · Page 33Linked to original sources
PubMed1994

Fibroblast growth factor-4 enhanced G2 arrest and cell survival following ionizing radiation.

Fibroblast growth factors (FGFs) bind to cell membrane receptors and activate signal transduction pathways related to cell growth, angiogenesis, and tumorigenesis. FGFs have been shown to be abundantly expressed in some of the human tumors, which are known to be poorly responsive to radiation therapy. Using adrenal cortical carcinoma cells genetically engineered to express FGF-4, we have tested cellular survival following exposure to ionizing radiation. We report here that FGF-4 enhances cellular capacity to survive ionizing radiation. Furthermore, cell cycle analysis shows a pronounced increase in the duration of G2 arrest, suggesting perturbation of a cell cycle checkpoint. These findings implicate fibroblast growth factor-mediated signal transduction in cellular resistance of human tumors to radiation therapy.

Cell Survival↗
PubMed1994

Recombinant Salmonella vectors in vaccine development.

A diversity of means are available for the attenuation of Salmonella which can be used to immunize animals and humans orally to elicit mucosal, humoral and cellular immune responses. Avirulent Salmonellae can be genetically engineered to express foreign antigens and the recombinant avirulent Salmonellae are capable of stable, high-level expression of the foreign antigen in the orally immunized animal or human host. The resulting vaccines are safe, efficacious, and are easy and economical to use.

Administration, Oral↗
PubMed1998

Efficient direct priming of tumor-specific cytotoxic T lymphocyte in vivo by an engineered APC.

Although numerous studies have documented a role for B7-1 (CD80) in the induction of antitumor CTL immunity, it is presently unclear to what extent expression of this costimulatory molecule truly endows tumors with significant in vivo APC (antigen-presenting cell) capacity. Recent studies have, in fact, demonstrated that cross-priming, rather than direct priming, may constitute the major mechanism of CTL induction by B7-1 expressing tumors. We have, therefore, investigated the requirements for antigen density and costimulatory molecules in direct CTL priming with a prototype cell-based vaccine that uses a signal sequence-containing minigene to direct expression of a tumor-specific CTL epitope to the endoplasmic reticulum. This design limits sources of antigen available to professional APC in the host and, thereby, the contribution of cross-priming. Induction of antitumor CTL immunity by our prototype APC was shown to solely involve direct priming, independent of host APC, NKI.1+ cells, and CD4+ T cell help. CTL induction through this mechanism required the engineered APC to express the B7-1 molecule as well as a sufficiently high density of peptide/MHC complexes at its surface. Our data, in contrast to previous studies using modified tumor cells, clearly define the antigenic and costimulatory requirements for a suitably engineered "artificial" APC to directly prime peptide-specific CTL in vivo, and demonstrate that the signal sequence minigene approach allows the engineering of highly effective and well-defined cellular vaccines for activation of CTL against epitopes of choice.

Adenovirus E1A Proteins↗
PubMed2006

Designing a nano-interface in a microfluidic chip to probe living cells: challenges and perspectives.

Nanotechnology-based materials are beginning to emerge as promising platforms for biomedical analysis, but measurement and control at the cell-chip interface remain challenging. This idea served as the basis for discussion in a focus group at the recent National Academies Keck Futures Initiative. In this Perspective, we first outline recent advances and limitations in measuring nanoscale mechanical, biochemical, and electrical interactions at the interface between biomaterials and living cells. Second, we present emerging experimental and conceptual platforms for probing living cells with nanotechnology-based tools in a microfluidic chip. Finally, we explore future directions and critical needs for engineering the cell-chip interface to create an integrated system capable of high-resolution analysis and control of cellular physiology.

Biomedical Engineering↗
PubMed2025

Cellular Inflammation-Induced Cleavage of Phosphorothioate DNA Locker Activates CRISPR/Cas9 Regulator for Gene Editing.

The CRISPR technology is a highly promising strategy for developing a versatile toolbox to engineer genetic circuits. However, achieving precise and specific control over the activity of the CRISPR/Cas9 system in response to intracellular processes remains a challenging endeavor. In this study, we present a cellular inflammation-induced activation of an engineered CRISPR/Cas9 regulator for gene regulation. A phosphorothioate (PS)-modified DNA sequence, referred as the "locker," is employed to deactivate single guide RNA (sgRNA), whose locker sequence complements the spacer region of sgRNA. In the presence of myeloperoxidase during cellular inflammation, a halogenation process is triggered, leading to the generation of HClO, specifically cleaving the PS site of locker and activating CRISPR/Cas9 for gene editing. The target GFP gene has been successfully edited, downregulating the GFP protein expression in HeLa cells. This study provides valuable insights into the CRISPR-based gene regulation through specific endogenous processes.

Humans↗
PubMed2001

Humoral and cellular immune responses: independent forces or collaborators in the fight against cancer?

Recent advances in our understanding of immune function with regard to the generation of a potent antitumor response have resulted in a renewed interest in cancer vaccines and point to a role for immunotherapy in the treatment of cancer. Currently, the majority of vaccine strategies for the treatment of solid malignancies focus on the generation of cytotoxic T lymphocytes (CTL) that destroy tumor cells that express a given target protein, or antigen. However, antibody therapies have already been successful against some cancers. Current humoral immunotherapy typically involves the passive infusion of monoclonal antibodies, which usually target a specific tumor-encoded antigen. However, vaccines can be engineered to induce humoral immunity. By focusing on the cellular arm of the immune response at the expense of humoral immunity (or the converse), we may have inadvertently limited the potential efficacy of our anticancer vaccines. This article seeks to explore the notion that a vaccine designed to optimally activate both arms of the immune system may well generate an antitumor immune response greater than the sum of the two individual effector mechanisms alone.

Antibodies, Monoclonal↗
PubMed2002

Matrix-mediated cellular rejuvenation.

Biomaterial surface morphology and chemistry influence cell responses mediated via signaling cascades that regulate a wide range of metabolic processes. These responses may range from changes in surface adhesion and remodeling of the extracellular matrix to activation of cytokine, cytoskeletal and other biochemical pathways regulating or modulating cellular morphology and function. The present study has focused on collagen Type I, a key extracellular matrix protein, and its potential impact on the process of cellular aging. This study was undertaken for several reasons. First, several investigators reported that growth of cells on a collagen matrix markedly enhanced the resistance of cells to stresses. Second, a large body of accumulated data strongly indicated a relationship between the potential to respond to stresses and cellular aging with the former strongly influencing the rate of the latter. Finally, it has been recently demonstrated that in aged cells one of the key aging-related processes previously considered irreversible, attenuation of the expression of a major stress response protein, Hsp70, can be reversed. This fact together with a probable regulatory role of the stress response potential in cellular aging suggested a possibility that the cellular aging process as a whole can be altered. Indeed, in the present study, growth on a denatured collagen matrices reversed in aged cells not only the attenuation of Hsp70 expression but also other aging-related processes, such as beta-galactosidase expression, increase in protein oxidation and changes in cell morphology. Moreover, it appeared to reduce the rate of aging in young cells. Understanding the nature of collagen matrix-mediated cellular rejuvenation might suggest approaches for interfering with organismic aging. Some immediate applications include cell rejuvenation for purposes of cloning and reduction of the rate of aging during expansion of stem cells for purposes of tissue engineering.

Cell Line↗
PubMedDate not supplied

Hyaluronic acid-poly-D-lysine-based three-dimensional hydrogel for traumatic brain injury.

Brain tissue engineering in the postinjury brain represents a promising option for cellular replacement and rescue, providing a cell scaffold for either transplanted or resident cells. In this article, a hyaluronic acid (HA)-poly-D-lysine (PDL) copolymer hydrogel with an open porous structure and viscoelastic properties similar to neural tissue has been developed for brain tissue engineering. The chemicophysical properties of the hydrogel with HA:PDL ratios of 10:1, 5:1, and 4:1 were investigated by scanning electron microscopy (SEM) and X-ray photoelectron spectrometry. Neural cells cultured in the hydrogel were studied by phase-contrast microscope and SEM. The incorporation of PDL peptides into the HA-PDL hydrogel allowed for the modulation of neuronal cell adhesion and neural network formation. Macrophages and multinucleated foreign body giant cells found at the site of implantation of the hydrogel in the rat brain within the first weeks postimplantation decreased in numbers after 6 weeks, consistent with the host response to inert implants in numerous tissues. Of importance was the infiltration of the hydrogel by glial fibrillary acidic protein-positive cells-reactive astrocytes-by immunohistochemistry and the contiguity between the hydrogel and the surrounding tissue demonstrated by SEM. These findings indicated the compatibility of this hydrogel with brain tissue. Collectively, the results demonstrate the promise of an HA-PDL hydrogel as a scaffold material for the repair of defects in the brain.

Animals↗
PubMedDate not supplied

Metabolic engineering of Escherichia coli to enhance recombinant protein production through acetate reduction.

Genetic and metabolic engineering provide powerful and effective tools for the systematic manipulation and fine tuning of cellular metabolic activities. In this study, successful application of such techniques to enhance recombinant protein production by reducing acetate accumulation in Escherichia coli is presented. The alsS gene from Bacillus subtilis encoding the enzyme acetolactate synthase was introduced into E. coli cells using a multicopy plasmid. This newly introduced heterologous enzyme modifies the glycolytic fluxes by redirecting excess pyruvate away from acetate to acetolactate. Acetolactate is then converted to a nonacidic and less harmful byproduct acetoin, which appears in the broth. Furthermore, comparative fermentation studies show that the reduction in acetate accumulation leads to a significant improvement of recombinant protein production. The expression of a model recombinant CadA/beta-galactosidase fusion protein, under the control of a strong pH-regulated promoter, was found to increase by about 60% for the specific protein activity (to a level of 30% of total cellular protein) and 50% in terms of the volumetric activity in a batch fermenter. In fed-batch cultivation, the engineered strain achieved a volumetric recombinant protein yield of 1.6 million units/mL (about 1.1 g/L of beta-galactosidase), which represented a 220% enhancement over the control strain. In the meantime, acetate excretion was maintained below 20 mM compared with 80 mM for the control, and the final cell density was improved by 35%.

Acetates↗
PubMed2002

Electrospun nanofibrous structure: a novel scaffold for tissue engineering.

The architecture of an engineered tissue substitute plays an important role in modulating tissue growth. A novel poly(D,L-lactide-co-glycolide) (PLGA) structure with a unique architecture produced by an electrospinning process has been developed for tissue-engineering applications. Electrospinning is a process whereby ultra-fine fibers are formed in a high-voltage electrostatic field. The electrospun structure, composed of PLGA fibers ranging from 500 to 800 nm in diameter, features a morphologic similarity to the extracellular matrix (ECM) of natural tissue, which is characterized by a wide range of pore diameter distribution, high porosity, and effective mechanical properties. Such a structure meets the essential design criteria of an ideal engineered scaffold. The favorable cell-matrix interaction within the cellular construct supports the active biocompatibility of the structure. The electrospun nanofibrous structure is capable of supporting cell attachment and proliferation. Cells seeded on this structure tend to maintain phenotypic shape and guided growth according to nanofiber orientation. This novel biodegradable scaffold has potential applications for tissue engineering based upon its unique architecture, which acts to support and guide cell growth.

Animals↗
PubMed1998

Engineering Src family protein kinases with unnatural nucleotide specificity.

BACKGROUND: Protein kinases play a central role in controlling diverse signal transduction pathways in all cells. The identification of the direct cellular substrates of individual protein kinases remains the key challenge in the field. RESULTS: We describe the protein engineering of v-Src to produce a kinase which preferentially uses an ATP analog, N6-(benzyl) ATP, as a substrate, rather than the natural v-Src substrate, ATP. The sidechain of a single residue (Ile338) controls specificity for N6-substituted ATP analogs in the binding pocket of v-Src. Elimination of this sidechain by mutation to glycine produces a v-Src kinase which preferentially utilizes N6-(benzyl) ATP as a phosphodonor substrate. Our engineering strategy is generally applicable to the Src family kinases: mutation of the corresponding residue (Thr339 to glycine) in the Fyn kinase confers specificity for N6-(benzyl) ATP on Fyn. CONCLUSIONS: The v-Src tyrosine kinase has been engineered to exhibit specificity for an unnatural ATP analog, N6-(benzyl) ATP, even in a cellular context where high concentrations of natural ATP are present (1-5 mM), where preferential use of the ATP analog by the mutant kinase is essential. The mutant v-Src transfers phosphate more efficiently with the designed unnatural analog than with ATP. As the identical mutation in the Src-family kinase Fyn confers on Fyn the ability to recognize the same unnatural ATP analog, our strategy is likely to be generally applicable to other protein kinases and may help to identify the direct targets of specific kinases.

Adenosine Triphosphate↗
PubMed2001

[[Surgical intra-articular interventions in arthrosis].

The reconstruction of weight-bearing joint surfaces has long been a challenge for the orthopedic surgeon. The statistical rise in life expectancy, the spread of high-risk sports and leisure-time activities combined with high demands for painfree joint function even in older age as a factor for contemporary quality of life are the demographic factors of the bone and joint decade announced by the WHO. Osteoarthritis represents a multifactorial, irreversible, often heterogeneous joint disease with joint destruction as the inevitable consequence. Conventional and innovative intra-articular operative methods for the treatment of osteoarthritis are presented and discussed. Tissue engineering stands for vitalizing tissue modification by modification of cellular and humoral mechanisms. Thus, new therapy options for curing degenerative joint diseases will arise in the future. Autologous chondrocyte transplantation (ACT) represents one of the first clinically established methods. The paradigm of treating osteoarthritis by the orthopedic surgeon will be multimodal in the future. Innovative techniques in the biotechnological sector provide a promising horizon.

Arthroscopy↗
PubMed1998

Relative importance of surface wettability and charged functional groups on NIH 3T3 fibroblast attachment, spreading, and cytoskeletal organization.

Understanding the relationships between material surface properties, adsorbed proteins, and cellular responses is essential to designing optimal material surfaces for implantation and tissue engineering. In this study, we have prepared model surfaces with different functional groups to provide a range of surface wettability and charge. The cellular responses of attachment, spreading, and cytoskeletal organization have been studied following preadsorption of these surfaces with dilute serum, specific serum proteins, and individual components of the extracellular matrix. When preadsorbed with dilute serum, cell attachment, spreading, and cytoskeletal organization were significantly greater on hydrophilic surfaces relative to hydrophobic surfaces. Among the hydrophilic surfaces, differences in charge and wettability influenced cell attachment but not cell area, shape, or cytoskeletal organization. Moderately hydrophilic surfaces (20-40 degree water contact angle) promoted the highest levels of cell attachment. Preadsorption of the model surfaces with bovine serum albumin (BSA) resulted in a pattern of cell attachment very similar to that observed following preadsorption with dilute serum, suggesting an important role for BSA in regulating cell attachment to biomaterials exposed to complex biological media.

3T3 Cells↗
PubMedDate not supplied

Molecular/cell engineering approach to autocrine ligand control of cell function.

Tissue engineering, along with other modern cell- and tissue-based health care technologies, depends on successful regulation of cell function by molecular means, including pharmacological agents, materials, and genetics. This regulation is generally mediated by cell receptor/ligand interactions providing primary targets for molecular intervention. While regulatory ligands may often be exogenous in nature, in the categories of endocrine and paracrine hormone systems, they are being increasingly appreciated as crucial in local control of cell and tissue function. Improvements in design of health care technologies involving autocrine ligand interactions with cell receptors should benefit from increased qualitative and quantitative understanding of the kinetic and transport processes governing these interactions. In this symposium paper we offer a concise overview of our recent efforts combining molecular cell biology and engineering approaches to increase the understanding of how molecular and cellular parameters may be manipulated for improved control of cell and tissue function regulated by autocrine ligands.

Animals↗
PubMed2005

Electrospun chitosan-based nanofibers and their cellular compatibility.

Chitosan-based nanofibers with an average fiber diameter controllable from a few microns down to approximately 40 nm and a narrow size distribution were fabricated by electrospinning solutions containing chitosan, polyethylene oxide (PEO), and Triton X-100. Rheological study showed a strong dependence of spinnability and fiber morphology on solution viscosity and thus on chitosan-to-PEO ratio. The nanofibers can be deposited either as a nonwoven mat or as a highly aligned bundle of controllable size. Potential use of this nanofibrous matrix for tissue engineering was studied by examining its integrity in water and cellular compatibility. It was found that the matrix with a chitosan/PEO ratio of 90/10 retained excellent integrity of the fibrous structure in water. Experimental results from cell stain assay and SEM imaging showed that the nanofibrous structure promoted the attachment of human osteoblasts and chondrocytes and maintained characteristic cell morphology and viability throughout the period of study. This nanofibrous matrix is of particular interest in tissue engineering for controlled drug release and tissue remodeling.

Biocompatible Materials↗
PubMed2006

Repair of tendon defect with dermal fibroblast engineered tendon in a porcine model.

Harvesting autologous tenocytes for tendon engineering may cause secondary tendon defect at the donor site. Dermal fibroblasts are an easily accessible cell source and do not cause major donor site defect. This study aims to explore the possibility of tendon engineering using dermal fibroblasts. A total of 45 hybrid pigs were randomly divided into three groups: experimental group (n = 15)--repair of tendon defect with a dermal fibroblast engineered tendon; control group 1 (n = 15)--repair of defect with a tenocyte engineered tendon; and control group 2 (n = 15)-repair of defect with a scaffold alone. Both autologous dermal fibroblasts and tenocytes were seeded on polyglycolic acid (PGA) unwoven fibers to form a cell-scaffold construct and cultured in vitro for 7 days before in vivo implantation to repair a defect of flexor digital superficial tendon. Specimens were harvested at weeks 6, 14, and 26 for gross, histological, and mechanical analyses. Microscopy revealed good attachment of both dermal fibroblasts and tenocytes on PGA fibers and matrix production. In vivo results showed that fibroblast and tenocyte engineered tendons were similar to each other in their gross view, histology, and tensile strength. At 6 weeks, parallel collagen alignment was observed at both ends, but not in the middle in histology, with more cellular components than natural tendons. At weeks 14 and 26, both engineered tendons exhibited histology similar to that of natural tendon. Collagens became parallel throughout the tendon structure, and PGA fibers were completely degraded. Interestingly, dermal fibroblast and tenocyte engineered tendons did not express type III collagen at 26 weeks, which remained observable in normal pig skin and control group 2 tissue using polarized microscopy, suggesting a possible phenotype change of implanted dermal fibroblasts. Furthermore, both fibroblast and tenocyte engineered tendons shared similar tensile strength, about 75% of natural tendon strength. At 6 weeks in control group 2, neo-tissue was formed only at the peripheral area by host cells. A cord-like tissue was formed at weeks 14 and 26. However, the formed tissue was histologically disorganized and mechanically weaker than both cell-engineered tendons (p < 0.05). These results suggest that dermal fibroblasts may have the potential as seed cells for tendon engineering.

Absorbable Implants↗
PubMed2005

Cellular response to gelatin- and fibronectin-coated multilayer polyelectrolyte nanofilms.

Surface engineering is a critical effort in defining substrates for cell culture and tissue engineering. In this context, multilayer self-assembly is an attractive method for creating novel composites with specialized chemical and physical properties that is currently drawing attention for potential application in this area. In this work, effects of thickness, surface roughness, and surface material of multilayer polymer nanofilms on the growth of rat aortic smooth muscle cells were studied. Polyelectrolyte multilayers (PEMs) electrostatically constructed from poly(allylamine hydrochloride) and poly(sodium 4-styrenesulfonate) (PSS) with gelatin, fibronectin, and PSS surface coatings were evaluated for interactions with smooth muscle cells (SMCs) in an in vitro environment. The results prove that PEMs terminated with cell-adhesive proteins promote the attachment and further growth of SMCs, and that this property is dependent upon the number of layers in the underlying multilayer film architecture. Cell roundness and number of pseudopodia were also influenced by the number of layers in the nanofilms. These findings are significant in that they demonstrate that both surface coatings and underlying architecture of nanofilms affect the morphology and growth of SMCs, which means additional degrees of freedom are available for design of biomaterials. This work supports the excellent potential of nanoassembled ultrathin films for biosurface engineering, and points to a novel perspective for controlling cell-material interaction that can lead to an elegant system for defining the extracellular in vitro environment.

Animals↗