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At least 721 records · Page 40Linked to original sources
PubMed2005

Low-density cultures of bovine chondrocytes: effects of scaffold material and culture system.

Chondrocytes were seeded on either agarose or polyglycolic acid (PGA) unwoven meshes at 10 million cells/ml of scaffold volume to evaluate the effect that these two biomaterials have on the low-density culture of chondrocytes in a rotating-wall bioreactor. For both static and bioreactor culture, agarose constructs contained more glycosaminoglycan than their PGA counterparts. However, the PGA constructs contained more collagen for both culture conditions when compared to agarose. For the low seeding density of this study, PGA constructs cultured in the bioreactor did not outperform static cultures when comparing collagen content after 8 weeks. The mechanical properties of the PGA constructs also did not improve with culture time. Similar results were observed with the agarose culture, though both static- and bioreactor-culture agarose constructs exhibited increases in aggregate modulus at the end of the culture period. As in PGA culture, chondrocytes cultured in agarose may require a higher density to reap the benefits of the bioreactor environment.

Animals↗
PubMed1998

Collagen--biomaterial for drug delivery.

The use of collagen as a biomaterial is currently undergoing a renaissance in the tissue engineering field. The biotechnological applications focus on the aspects of cellular growth or delivery of proteins capable of stimulating cellular response. However, basic knowledge about collagen biochemistry and the processing technology in combination with understanding of the physico-chemical properties is necessary for an adequate application of collagen for carrier systems. The purpose of this review article is to summarize information available on collagen dosage forms for drug delivery as well as to impart an overview of the chemical structures and the galenical properties including detailed description of the processing steps - extraction, purification, chemical crosslinking and sterilization. The most successful and stimulating applications are shields in ophthalmology, injectable dispersions for local tumor treatment, sponges carrying antibiotics and minipellets loaded with protein drugs. However, the scientific information about manipulating release properties or mechanistic studies is not as abundant as for some synthetic polymers.

Biocompatible Materials↗
PubMed2006

A bottom-up approach to gene regulation.

The ability to construct synthetic gene networks enables experimental investigations of deliberately simplified systems that can be compared to qualitative and quantitative models. If simple, well-characterized modules can be coupled together into more complex networks with behaviour that can be predicted from that of the individual components, we may begin to build an understanding of cellular regulatory processes from the 'bottom up'. Here we have engineered a promoter to allow simultaneous repression and activation of gene expression in Escherichia coli. We studied its behaviour in synthetic gene networks under increasingly complex conditions: unregulated, repressed, activated, and simultaneously repressed and activated. We develop a stochastic model that quantitatively captures the means and distributions of the expression from the engineered promoter of this modular system, and show that the model can be extended and used to accurately predict the in vivo behaviour of the network when it is expanded to include positive feedback. The model also reveals the counterintuitive prediction that noise in protein expression levels can increase upon arrest of cell growth and division, which we confirm experimentally. This work shows that the properties of regulatory subsystems can be used to predict the behaviour of larger, more complex regulatory networks, and that this bottom-up approach can provide insights into gene regulation.

Arabinose↗
PubMed1994

Production of human embryonic haemoglobin (Gower II) in a yeast expression system.

The cDNA coding for a human embryonic globin protein has been obtained from an erythroleukaemic cell line. A plasmid expression system for human embryonic haemoglobin Gower II containing cDNA copies of the appropriate pair of globin genes coupled to synthetic galactose-regulated hybrid promoters has been engineered. Transformation of Saccharomyces cerevisiae with this plasmid yields a cellular system capable of high-level production of fully functional tetrameric embryonic haemoglobin. We have developed a purification scheme which gives high yields of pure human embryonic haemoglobin suitable for structural and functional studies. Preliminary characterization studies are reported.

Amino Acid Sequence↗
PubMed1996

Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety.

Vaccinia virus, no longer required for immunization against smallpox, now serves as a unique vector for expressing genes within the cytoplasm of mammalian cells. As a research tool, recombinant vaccinia viruses are used to synthesize and analyze the structure-function relationships of proteins, determine the targets of humoral and cell-mediated immunity, and investigate the types of immune response needed for protection against specific infectious diseases and cancer. The vaccine potential of recombinant vaccinia virus has been realized in the form of an effective oral wild-life rabies vaccine, although no product for humans has been licensed. A genetically altered vaccinia virus that is unable to replicate in mammalian cells and produces diminished cytopathic effects retains the capacity for high-level gene expression and immunogenicity while promising exceptional safety for laboratory workers and potential vaccine recipients.

AIDS Vaccines↗
PubMed1998

Charge-dependent translocation of Bordetella pertussis adenylate cyclase toxin into eukaryotic cells: implication for the in vivo delivery of CD8(+) T cell epitopes into antigen-presenting cells.

Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain (400-aa residues) into the cytosol of eukaryotic target cells, directly through the cytoplasmic membrane. We have previously shown that CyaA can be used as a vehicle to deliver T cell epitopes, inserted within the catalytic domain of the toxin, into antigen-presenting cells and can trigger specific class I-restricted CD8(+) cytotoxic T cell responses in vivo. Here, we constructed a series of recombinant toxins harboring at the same insertion site various peptide sequences of 11-25 amino acids, corresponding to defined CD8(+) T cell epitopes and differing in the charge of the inserted sequence. We show that inserted peptide sequences containing net negative charges (-1 or -2) decreased or completely blocked (charge of -4) the internalization of the toxin into target cells in vitro and abolished the induction of cytotoxic T cell responses in vivo. The blocking of translocation due to the inserted acidic sequences can be relieved by appropriate mutations in the flanking region of CyaA that counterbalance the inserted charges. Our data indicate that (i) the electrostatic charge of the peptides inserted within the catalytic domain of CyaA is critical for its translocation into eukaryotic cells and (ii) the delivery of T cell epitopes into the cytosol of antigen-presenting cells by recombinant CyaA toxins is essential for the in vivo stimulation of specific cytotoxic T cells. These findings will help to engineer improved recombinant CyaA vectors able to stimulate more efficiently cellular immunity.

Adenylate Cyclase Toxin↗
PubMed1984

The impact of new technologies on vaccine development.

The ability to move genetic determinants between species using in vitro gene-manipulation techniques has opened up new approaches to vaccine development. This has rapidly grown into an exciting area of research in both academic and industrial laboratories. There are numerous scientific challenges which require multidisciplinary teams to solve problems in creating new immunogens. This has challenged our existing knowledge about protein structure and conformation, microbial pathogenicity and the immune system. Recombinant-DNA techniques are invaluable as tools of analysis and antigen production. The surface of micro-organisms can also be minutely explored with the use of synthetic peptides and monoclonal antibodies. Nevertheless, these new technologies do not allow us to circumvent the need for detailed understanding of pathogens and the disease process. What is apparent from the work carried out so far is that there are few easy answers to vaccine development and it is not realistic to expect rapid solutions to these problems. As there are many potential targets for constructing novel vaccines for both human and animal diseases, it is helpful to establish some priorities. There is a tendency to look at the existing effective vaccines and simply direct research at producing them more economically or with enhanced safety and stability. The advantage of this approach is that considerable background work will have already been carried out establishing the basis for the application of recombinant DNA techniques. However, this can also lead to conflicts (often within the same institute or company) between the new and old technologies. This could be to the detriment of the new technologies which are still only partly developed and may not be good enough yet to compete with existing vaccines in cost or efficacy. The more ambitious, and eventually more rewarding, approach is to attempt to develop new vaccines where none had existed before. There is a vast untapped market, especially in the parasitic diseases, but the scientific problems may be considerable and much more background work is likely to be necessary. Indeed, most of the work in this area is more accurately referred to as basic research rather than vaccine development as totally new, effective vaccines are still some way off. Having directed research towards a specific organism or disease there are still many options available as to the scientific strategy to adopt. As discussed in this review it may be possible to consider subunits, synthetic antigens and live (attenuated or heterologous) organisms as possible vaccines.(ABSTRACT TRUNCATED AT 400 WORDS)

Adjuvants, Immunologic↗
PubMed2006

Fluorescence molecular tomography in the presence of background fluorescence.

Fluorescence molecular tomography is an emerging imaging technique that resolves the bio-distribution of engineered fluorescent probes developed for in vivo reporting of specific cellular and sub-cellular targets. The method can detect fluorochromes in picomole amounts or less, imaged through entire animals, but the detection sensitivity and imaging performance drop in the presence of background, non-specific fluorescence. In this study, we carried out a theoretical and an experimental investigation on the effect of background fluorescence on the measured signal and on the tomographic reconstruction. We further examined the performance of three subtraction methods based on physical models of photon propagation, using experimental data on phantoms and small animals. We show that the data pre-processing with subtraction schemes can improve image quality and quantification when non-specific background florescence is present.

Algorithms↗
PubMed2004

A mouse model for evaluation of capillary perfusion, microvascular permeability, cortical blood flow, and cortical edema in the traumatized brain.

Genetically engineered mice have successfully been used to investigate molecular and cellular mechanisms associated with cell dysfunction following brain trauma. Such animals may also offer a possibility to investigate mechanisms involved in posttraumatic hemodynamic alterations. The objective of the study was to establish a mouse model in which important hemodynamic alterations following trauma could be analyzed. C57/BL6 male mice were subjected to controlled cortical impact injury (CCI) or sham-injury. Distribution of blood flow was estimated by determining number of perfused capillaries using FITC-dextran as an intravascular marker. Cortical blood flow was measured using [(14)C]-iodoantipyrine, brain water content (BWC) was measured using a wet vs. dry weight method, and permeability surface area product (PS) was estimated by the transfer constant for [(51)Cr]-EDTA. Number of perfused capillaries in the contusion area was progressively reduced during the first 24 h following trauma by at most 60% relative to a value of 329 +/- 61/mm(2) in sham-injured animals. Blood flow in the contusion area decreased simultaneously by at most 50% relative to a control value of 1.8 +/- 0.4 mL.min(-1).g(-1), and was reduced further in subregions within the contusion area. BWC in the injured hemisphere increased from 79.3 +/- 0.5% at control to at most 79.9 +/- 0.6% at 24 h post trauma. PS in the injured hemisphere increased by 71% at 3 h post trauma relative to a control value of 0.45 +/- 0.1 microL.min(-1).g(-1), and was close to control at 24 h. The present study demonstrates that brain trauma in addition to a reduction in cortical blood flow, reduces number of perfused capillaries, which most likely affects exchange of nutrients and fluid. The CCI in mouse is likely to be a useful tool to elucidate mechanisms involved in hemodynamic alterations following brain trauma.

Animals↗
PubMed2003

Size-exclusion chromatography purification of high-titer vesicular stomatitis virus G glycoprotein-pseudotyped retrovectors for cell and gene therapy applications.

Vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped replication-defective retroviral particles are pantropic and amenable to concentration to high titer by ultracentrifugation. These features have allowed development of effective retroviral transduction protocols for stem cells in vitro as well as for tissue engineering in vivo. However, retroparticle ultracentrifugation protocols will also copellet cellular and subcellular debris released from retroviral producer cell lines during vector manufacture. We have analyzed concentrated vector preparations by chromatography and have found that a significant amount of genomic DNA released from producer cells coconcentrates with retroviral particles. In an effort to generate high-purity retroparticle preparations, devoid of subcellular contaminants and contaminating genomic DNA, we have developed a process using size-exclusion chromatography combined with host cell nucleic acid digestion and concentration by ultrafiltration. The procedure allowed for a final recovery of 19 +/- 0.4% infectious viral particles from unfractionated starting material, with an average retroparticle concentration of 7.7 x 10(7) +/- 1.5 x 10(6)/ml. The intact virus is of high purity, >90% as determined by anion-exchange high-performance liquid chromatography. Retroparticle structure appeared intact as determined by negative stain electron microscopy and purified virus was functional and allowed for efficient transduction of primary human bone marrow stromal cells in vitro. In conclusion, we have developed a VSV-G retrovector purification process that can be applied to large-scale retroviral production ideal for cell and gene therapy applications.

Cells, Cultured↗
PubMed2002

Naturally occurring scaffolds for soft tissue repair and regeneration.

Cell growth supports (i.e., scaffolds) that provide a conducive environment for normal cellular growth, differentiation, and angiogenesis are important components of tissue engineered grafts because rapid integration with the host is essential for long-term graft viability. While many of these scaffold materials are synthetic biodegradable polymers, others are naturally derived from mammalian tissue sources. Naturally occurring scaffold materials include small intestinal submucosa, acellular dermis, amniotic membrane tissue, cadaveric fascia, and the bladder acellular matrix graft. Upon implantation, these materials elicit a host-tissue response that initiates angiogenesis, encourages tissue deposition and culminates in restoration of structure and function specific to the grafted site. The sources, the methods of procurement and processing, and the effects of these naturally occurring materials on angiogenesis and tissue deposition are reviewed.

Animals↗
PubMed1995

Optimizing the composition of bone marrow for allogenic transplantation.

Graft versus host disease, graft failure, and disease relapse remain significant impediments to successful allogeneic transplantation. Ex vivo engineering of the graft offers the opportunity to modify the cellular composition to overcome these problems. This review describes recent developments that employ this approach.

Blood Cells↗
PubMed2006

Deinococcus radiodurans engineered for complete toluene degradation facilitates Cr(VI) reduction.

Toluene and other fuel hydrocarbons are commonly found in association with radionuclides at numerous US Department of Energy sites, frequently occurring together with Cr(VI) and other heavy metals. In this study, the extremely radiation-resistant bacterium Deinococcus radiodurans, which naturally reduces Cr(VI) to the less mobile and less toxic Cr(III), was engineered for complete toluene degradation by cloned expression of tod and xyl genes of Pseudomonas putida. The recombinant Tod/Xyl strain showed incorporation of carbon from 14C-labelled toluene into cellular macromolecules and carbon dioxide, in the absence or presence of chronic ionizing radiation. The engineered bacteria were able to oxidize toluene under both minimal and complex nutrient conditions, and recombinant cells reduced Cr(VI) in sediment microcosms. As such, the Tod/Xyl strain could provide a model for examining the reduction of metals coupled to organic contaminant oxidation in aerobic radionuclide-contaminated sediments.

Chromium↗
PubMed2004

Cofilin promotes actin polymerization and defines the direction of cell motility.

A general caging method for proteins that are regulated by phosphorylation was used to study the in vivo biochemical action of cofilin and the subsequent cellular response. By acute and local activation of a chemically engineered, light-sensitive phosphocofilin mimic, we demonstrate that cofilin polymerizes actin, generates protrusions, and determines the direction of cell migration. We propose a role for cofilin that is distinct from its role as an actin-depolymerizing factor.

Actin Depolymerizing Factors↗
PubMed2004

Scaffold topography alters intracellular calcium dynamics in cultured cardiomyocyte networks.

Structural and functional changes ensue in cardiac cell networks when cells are guided by three-dimensional scaffold topography. We report enhanced synchronous pacemaking activity in association with slow diastolic rise in intracellular Ca2+ concentration ([Ca2+]i) in cell networks grown on microgrooved scaffolds. Topography-driven changes in cardiac electromechanics were characterized by the frequency dependence of [Ca2+]i in syncytial structures formed of ventricular myocytes cultured on microgrooved elastic scaffolds (G). Cells were electrically paced at 0.5-5 Hz, and [Ca2+]i was determined using microscale ratiometric (fura 2) fluorescence. Compared with flat (F) controls, the G networks exhibited elevated diastolic [Ca2+]i at higher frequencies, increased systolic [Ca2+]i across the entire frequency range, and steeper restitution of Ca2+ transient half-width (n = 15 and 7 for G and F, respectively, P < 0.02). Significant differences in the frequency response of force-related parameters were also found, e.g., overall larger total area under the Ca2+ transients and faster adaptation of relaxation time to pacing rate (P < 0.02). Altered [Ca2+]i dynamics were paralleled by higher occurrence of spontaneous Ca2+ release and increased sarcoplasmic reticulum load (P < 0.02), indirectly assessed by caffeine-triggered release. Electromechanical instabilities, i.e., Ca2+ and voltage alternans, were more often observed in G samples. Taken together, these findings 1) represent some of the first functional electromechanical data for this in vitro system and 2) demonstrate direct influence of the microstructure on cardiac function and susceptibility to arrhythmias via Ca(2+)-dependent mechanisms. Overall, our results substantiate the idea of guiding cellular phenotype by cellular microenvironment, e.g., scaffold design in the context of tissue engineering.

Animals↗
PubMed2002

Mast cell chymase degrades apoE and apoA-II in apoA-I-knockout mouse plasma and reduces its ability to promote cellular cholesterol efflux.

OBJECTIVE: Mast cell chymase is a chymotryptic heparin proteoglycan-bound neutral protease that exerts its activity in extracellular fluids. We studied the effect of chymase on the apolipoprotein compositions and the abilities of plasmas from apolipoprotein (apo)A-I-knockout (A-I-KO) and wild-type (C57BL/6J) mice to stimulate efflux of cellular cholesterol from mouse macrophage foam cells. METHODS AND RESULTS: The A-I-KO apolipoproteins compared with the wild-type (apoA-I, apoA-II, apoA-IV, and apoE) showed total lack of apoA-I, unaltered apoA-II, an absence of apoA-IV, and an increase of apoE. Despite these major differences, the 2 plasmas induced similar high-affinity efflux of cholesterol from the foam cells. Quantitative analysis of chymase-treated plasmas revealed (1) in A-I-KO plasma, complete loss of apoE and apoA-II, and (2) in wild-type plasma, slight reduction of apoA-I associated with complete depletion of the minor pre-beta-high density lipoprotein fraction, strong reduction of apoA-II, and complete depletion of apoA-IV and apoE. Both proteolyzed plasmas had lost the ability to induce cellular cholesterol efflux with high affinity. Addition of discoidal pre-beta-migrating reconstituted high density lipoprotein particles containing human apoA-I or apoA-II to the chymase-treated A-I-KO plasma fully restored its cholesterol efflux-inducing ability, indicating functional replacement of the proteolyzed apoE and apoA-II. Thus, chymase degraded all the nondeleted apolipoproteins of the A-I-KO plasma involved in the high-affinity efflux of cellular cholesterol. CONCLUSIONS: This is the first indication that genetically engineered mice could be used as models for examining the hypothesis that extracellular proteases are involved in the development of atherosclerosis by inhibiting the apolipoprotein-mediated removal of macrophage cholesterol.

Animals↗
PubMed1996

Mathematical simulation of unidirectional tissue formation: in vitro transanastomotic endothelialization model.

In vitro transanastomotic endothelialization was studied using a mathematical model with the Fisher equation. The Fisher equation is a nonlinear parabolic equation which has a cell migration term and a population growth term. The mathematical model was used to simulate recent experiments performed to investigate quantitatively the unidirectional formation of a bovine endothelial cell monolayer in vitro. The two parameters included in the equation were estimated using a trial and error method in which the calculated solutions of the various values of the two parameters are compared with the experimental data and the best fit pair is adopted: one parameter, D, which represents the unidirectional cell migration rate and the other, k, which represents the population growth rate. The calculated solutions fit the experimental data well. We also simulated the healing of a mechanically disrupted endothelial monolayer sheet. The significance of cellular biomechanics in tissue formation and design of tissue-engineered devices is discussed.

Animals↗