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PubMed1996

Immunobiology of pseudorabies (Aujeszky's disease).

Aujeszky's Disease (AD), a serious illness of pigs causing significant economic losses in the pig industry, is caused by Pseudorabies Virus (PrV). PrV belongs to the alphaherpesvirus subfamily of the herpesviruses with a double-stranded DNA genome in an enveloped capsid capable of encoding approximately 70 proteins. For disease control, vaccination with live and killed vaccines is performed. Recently, 'marked' vaccines have become available for use in eradication programs based on the differentiation between infected and vaccinated animals. PrV is also used as a viral vector for the development of multivalent vaccines. Despite the effectiveness of PrV vaccines, relatively little is known about the immune response against PrV infection. Several viral envelope glycoproteins have been shown to represent targets for antibody responses, and a number of isolated glycoproteins as well as genetically engineered proteins were able to elicit protective immunity. The nature of the cellular immune response is even less defined. Using viral mutants genetically engineered to lack specific antigens, it has been shown that glycoprotein C (gC) acts as a target for cytotoxic T-lymphocytes, and gB, gC, gD, and gH appear to be involved in stimulation of in vitro proliferation of PBMC from immune animals. In addition, gB and gC have been implicated in recognition of infected cells by lymphokine-activated killer (LAK) cells. In summary, the data indicate a prominent role for viral envelope glycoproteins in eliciting humoral and cellular immune responses in the animal host. A complicating factor is the ability of PrV to productively infect cells of the hematopoietic system, which may impair immune responses and might also play a role in persistent or latent infection.

Animals↗

PubMed2006

Time-dependent modulation of alignment and differentiation of smooth muscle cells seeded on a porous substrate undergoing cyclic mechanical strain.

The orientation of cellular alignment in smooth muscle tissue engineering is directly related to optimal movement of engineered tissue when it is transplanted in vivo. Cyclic mechanical strain has been applied to modulate the alignment, proliferation, and differentiation of smooth muscle cells. This study was conducted to investigate the effects of cyclic mechanical strain on primary cultured myofibroblasts seeded onto three-dimensional polymeric scaffolds, and to determine the optimal mechanical treatment time required to produce artificial smooth muscle. The cells were primary cultured from rabbit esophageal smooth muscle layer, and a self-designed stretching chamber was used to modulate the cells on porous polyurethane (PU) scaffolds with 10% strain at a frequency of 1 Hz. The applied cyclic strain induced cellular alignment. In particular, cellular alignment perpendicular to the direction of strain was generated in the condition strained over 18 h. In terms of proliferation, the strained groups differed significantly from the statically cultured group, but no difference was observed between groups that were subjected to straining for different lengths of time. Quantitative analysis of alpha-smooth muscle actin (SMA) showed that differentiation was significantly promoted at 18 h of strain. Penetration of primary cultured cells into the pores of PU scaffolds was shown after cyclic strain application, especially in 18 and 24 h of strain. Consequently, it is expected that myofibroblast/scaffold hybrids, cyclically strained in the defined time course, could be practically applied to organize functional smooth muscle tissues having consistent cell alignment and up-regulated SMA.

Actins↗

PubMedDate not supplied

Comparison of three myofibroblast cell sources for the tissue engineering of cardiac valves.

The objective of this study was to evaluate the capacity of three clinically useful tissue sources: tricuspid valve leaflet (TVL), carotid artery (CA), and jugular vein (JV), to generate myofibroblasts for potential use in a tissue-engineered cardiac valve replacement. Tissue biopsies of clinically appropriate sizes obtained from juvenile sheep were used for this work. Cells obtained from all three tissue sources exhibited a myofibroblast phenotype in vitro, as demonstrated by their immunoreactivity with antibodies directed against vimentin, alpha-smooth muscle actin, fibronectin, and chondroitin sulfate. Protein synthesis characteristics were defined for the key extracellular matrix components: collagen, glycosaminoglycans, and elastin. Among the three sources, JV generated the highest numbers of cells, and JV cells produced the largest amount of collagen per cell. These data suggest that venous tissue, with its relative ease of accessibility, may generate myofibroblasts potentially useful for the interstitial cellular component of a tissue-engineered cardiac valve.

Actins↗

PubMed2007

Controlling tissue microenvironments: biomimetics, transport phenomena, and reacting systems.

The reconstruction of tissues ex vivo and production of cells capable of maintaining a stable performance for extended time periods in sufficient quantity for synthetic or therapeutic purposes are primary objectives of tissue engineering. The ability to characterize and manipulate the cellular microenvironment is critical for successful implementation of such cell-based bioengineered systems. As a result, knowledge of fundamental biomimetics, transport phenomena, and reaction engineering concepts is essential to system design and development. Once the requirements of a specific tissue microenvironment are understood, the biomimetic system specifications can be identified and a design implemented. Utilization of novel membrane systems that are engineered to possess unique transport and reactive features is one successful approach presented here. The limited availability of tissue or cells for these systems dictates the need for microscale reactors. A capstone illustration based on cellular therapy for type 1 diabetes mellitus via encapsulation techniques is presented as a representative example of this approach, to stress the importance of integrated systems.

Biological Transport, Active↗

PubMed2003

Pulsatile perfusion and cardiomyocyte viability in a solid three-dimensional matrix.

BACKGROUND: The manufacture of full thickness three-dimensional myocardial grafts by means of tissue engineering is limited by the impeded cellular viability in unperfused in vitro systems. We introduce a novel concept of pulsatile tissue culture perfusion to promote ubiquitous cellular viability and metabolism. METHODS: In a novel bioreactor we established pulsatile flow through the embedded three-dimensional tissue culture. Fibrin glue served as the ground matrix wherein neonatal rat cardiomyocytes were inoculated. Fluor-Deoxy-Glucose-Positron-Emission-Tomography (FDG-PET) and life/dead assays were employed for comparative studies of glucose uptake resp. cell viability. RESULTS: A solid 8 mm thick structure resulted. Cellular viability significantly increased in the perfused chambers. We observed centripetal migration of the embedded cardiomyocytes to the site of the core vessel. However, cellular viability was high in the periphery of the tissue block too. FDG-PET revealed enhanced metabolic activity in perfused chambers. CONCLUSIONS: The present concept is highly effective in enhancing cellular viability and metabolism in a three-dimensional tissue culture environment. It could be utilized for various co-culture systems and the generation of viable tissue grafts.

Animals↗

PubMed1999

Intrabody construction and expression III: engineering hyperstable V(H) domains.

The folding of immunoglobulin domains requires the formation of a conserved structural disulfide. Therefore, as a general rule, they cannot be functionally expressed in the reducing environment of the cellular cytoplasm. We have previously reported that stability engineering can lead to the cytoplasmic expression of functional immunoglobulin V(L) domains. Here we apply rational stability engineering by consensus sequence analysis to V(H) domains. Isolated V(H) domains tend to aggregate more easily than V(L) domains; they do not refold quantitatively and are generally more difficult to handle in vitro. To overcome these problems, we successfully predicted and experimentally verified several stabilizing point mutations in the V(H) domain of a designed, catalytic Fv fragment. The effect of single mutations was additive, and they could be combined in a prototype domain with significantly improved stability against chemical denaturation and a 20-fold increased half time of irreversible thermal denaturation, at physiological temperature. This stabilized, isolated V(H) domain could be expressed solubly in the reducing cellular cytoplasm of Escherichia coli, at a yield of approximately 1.2 mg/L of shake flask culture. It remains fully functional, as evidenced by the successful reconstitution of an esterolytic Fv fragment with the V(L) domain. This success provides further evidence that consensus sequence engineering is a rational, plannable route to the construction of intrabodies.

Amino Acid Sequence↗

PubMed2002

Challenges and opportunities for in vivo imaging in oncology.

Advances in genomics, proteomics and technology are changing medicine in fundamental ways. There are increasing clinical and laboratory requirements to obtain cellular and molecular information in vivo. This is particularly true in oncology, where the behavior of tumor cells is inextricably linked to their milieu. If cancer cells are removed from their microenvironment, their pattern of gene expression changes. Therefore, non-invasive, quantitative means of detecting gene and protein activity are essential. In vivo imaging is one methodology for achieving this. Marked advances in tracer methods for PET scanning or single-photon nuclear medicine techniques have occurred in the past few years. MRI contrast agents that reflect physiologic information are also being developed, although larger mass quantities of injectable material are required. The useful concept of "activatable agents" was pioneered in MRI. Similarly, ultrasound and computed tomography are being re-engineered to reflect information at the cellular level. In vivo optical imaging technologies have matured to the point where they are indispensable laboratory tools for small animal imaging. Human applications are in the feasibility testing stage, and the future for clinical optical imaging techniques looks bright. Merging these molecular imaging techniques with minimally or non-invasive image-guided therapeutic delivery techniques is a subsequent goal in the fight against cancer.

Humans↗

PubMed2006

Effect of spatial architecture on cellular colonization.

The spatial cell-material interaction remains vital issue in forming biodegradable scaffolds in Tissue Engineering. In this study, to understand the influence of spatial architecture on cellular behavior, 2D and 3D chitosan scaffolds of 50-190 kD and >310 kD MW were synthesized through air drying and controlled rate freezing/lypohilization technique, respectively. In addition, chitosan was emulsified with 19, 76, and 160 kD 50:50 poly lactide-co-glycolide (PLGA) using 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) as stabilizer. 2D and 3D scaffolds were formed by air drying and lyophilization as before. Tensile and compressive properties of films and scaffolds were analyzed in wet conditions at 37 degrees C. Alterations in the cell spreading, proliferation, and cytoskeletal organization of human umbilical vein endothelial cells (HUVECs) and mouse embryonic fibroblasts (MEFs) were studied. These results showed that the formed 3D chitosan scaffolds had interconnected open pore architecture (50-200 microm size). HUVECs and MEFs had reduced spreading areas and circular morphology on 2D chitosan membranes compared with 3D chitosan scaffolds. The fluorescence photomicrographs for actin (using Alexa Fluor 488 phalloidin) and cytoplasm staining (using carboxyfluorescein diacetate-succinimidyl ester) demonstrated that the cells spread within 3D chitosan matrix. 2D and 3D emulsified chitosan and chitosan/PLGA scaffolds reduced the spreading of HUVECs and MEFs even further. Proliferation results, analyzed via MTT-Formazan assay and BrdU uptake assay, correlated with the spreading characteristics. The reductions in cell spreading area on emulsified surfaces were not detrimental to the viability and endocytic activity but to proliferation. The observed alterations in cellular colonization are in part due to the substrate stiffness and surface topography. In summary, these results suggest a significant influence of spatial architecture on cellular colonization.

Cell Culture Techniques↗

PubMed2003

Creating alignment and anisotropy in engineered heart tissue: role of boundary conditions in a model three-dimensional culture system.

Electrical and mechanical anisotropy arise from matrix and cellular alignment in native myocardium. Generation of anisotropy in engineered heart tissue will be required to match native properties and will provide immediate opportunities to investigate the genesis and structural determinants of functional anisotropy. We investigated the influence of geometry and boundary conditions on fibroblast alignment in thin collagen gels. Consistent with previous reports, we found that human dermal fibroblasts align parallel to free edges in partially constrained gels; in contrast to at least one report, fibroblasts in fully constrained gels remained randomly aligned independent of geometry. These experiments allowed us to distinguish between two possible mechanisms for such alignment. Mean orientations that followed the shape of the free edges and stronger alignment nearest the free edges in gels with a variety of geometries suggested that cells align parallel to a local free boundary rather than to local lines of tension. These findings focus attention on the presence of voids and free surfaces such as the endocardium and epicardium, cleavage planes, and blood vessels in governing cell and fiber alignment in developing and remodeling myocardium, myocardial scar tissue, and engineered heart constructs.

Anisotropy↗

PubMed2006

Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells.

Zinc-finger nucleases are chimeric proteins consisting of engineered zinc-finger DNA-binding motifs attached to an endonuclease domain. These proteins can induce site-specific DNA double-strand breaks in genomic DNA, which are then substrates for cellular repair mechanisms. Here, we demonstrate that engineered zinc-finger nucleases function effectively in somatic cells of the nematode Caenorhabditis elegans. Although gene-conversion events were indistinguishable from uncut DNA in our assay, nonhomologous end joining resulted in mutations at the target site. A synthetic target on an extrachromosomal array was targeted with a previously characterized nuclease, and an endogenous genomic sequence was targeted with a pair of specifically designed nucleases. In both cases, approximately 20% of the target sites were mutated after induction of the corresponding nucleases. Alterations in the extrachromosomal targets were largely products of end-filling and blunt ligation. By contrast, alterations in the chromosomal target were mostly deletions. We interpret these differences to reflect the abundance of homologous templates present in the extrachromosomal arrays versus the paucity of such templates for repair of chromosomal breaks. In addition, we find evidence for the involvement of error-prone DNA synthesis in both homologous and nonhomologous pathways of repair. DNA ligase IV is required for efficient end joining, particularly of blunt ends. In its absence, a secondary end-joining pathway relies more heavily on microhomologies in producing deletions.

Animals↗

PubMed2001

Engineering orthogonal ligand-receptor pairs from "near drugs".

Cell-permeable small molecules are powerful tools for unraveling complex cellular pathways. We demonstrate that nuclear hormone receptors can be engineered through mutagenesis to create orthogonal ligand-receptor pairs to control transcription. Mutated residues in the retinoid X receptor (RXR) were chosen from structural analysis of RXR and the retinoic acid receptor (RAR) ligand binding domains. The potential ligands screened for activation of variant receptors are "near drugs"--compounds synthesized during structure-activity studies that are structurally similar to an approved drug yet inactive on the wild-type receptor. One variant, Q275C;I310M;F313I, is poorly activated by ligands for the wild-type receptor but is activated by a "near drug", fulfilling the criteria of an orthogonal ligand-receptor pair. These experiments demonstrate that nuclear hormone receptors are well suited to supply orthogonal ligand-receptor pairs for experimental biology, biotechnology, and gene therapy. Our findings also demonstrate the general principle that inactive compounds synthesized during drug discovery can be combined with mutant proteins to rapidly create new tools for controlling cellular processes.

Alitretinoin↗

PubMed2004

Stabilisation of cables of fibronectin with micromolar concentrations of copper: in vitro cell substrate properties.

We have previously described the production of large cables of fibronectin, a large extracellular matrix cell adhesion glycoprotein, which has a potential application in tissue engineering. Here we have stabilised these cables for longer survival and looked at their ultrastructural cell-substrate behaviour in vitro. Dissolution experiments showed that low concentrations of copper not only caused significant material stabilisation but left pores which could promote cell ingrowth, as we have previously reported with Fn-mats. Indeed, the greatest amount of cell ingrowth was observed for copper treated cables. Immunostaining showed S-100(+) multi-layers of cells around the edge of cables while ultrastructural analysis confirmed the presence of a mixture of fibroblasts and bipolar cells associated with fragments of basal lamina, which is a Schwann cell phenotype. Interestingly, the outermost layers of cells consisted of S-100(-) cells, presumed fibroblasts, apparently 'capping' the Schwann cells. Toxicity tests revealed that Schwann cells were only able to grow at the lowest concentration of copper used (1microM) while fibroblasts grew at all concentrations tested. These results could be used to design biomaterials with optimum properties for promoting cellular ingrowth and survival in tissue engineered grafts which may be used to improve peripheral nerve repair.

Animals↗

PubMed2006

Impact of fibrin glue and urinary bladder cell spraying on the in-vivo acellular matrix cellularization: a porcine pilot study.

PURPOSE: Urinary bladder tissue engineering utilizing autologous cell-seeded scaffolds requires enough bladder cells to populate a large surface area which may be difficult to obtain from abnormal bladders. We evaluated whether a fibrin glue spray technique enhances cell seeded acellular matrix (ACM) repopulation in a porcine bladder model. MATERIALS AND METHODS: Porcine urothelial and smooth muscle cells cultured from open bladder biopsy were sprayed with or without fibrin glue onto porcine bladder ACM. After 10 days in vitro, constructs were implanted onto porcine bladders (4/group) and harvested after 1 or 6 weeks for H&E and immunohistochemical staining. RESULTS: In vitro, fibrin glue was associated with more continuous cell growth and enhanced cellular organization, maintained particularly in the periphery in vivo, where both groups demonstrated central fibrosis. CONCLUSIONS: While fibrin glue enhanced cellular organization on ACM in vitro, central fibrosis in vivo suggests that factors supporting seeded cell survival are lacking.

Animals↗

PubMed1997

Intracerebral versus subcutaneous immunization with allogeneic fibroblasts genetically engineered to secrete interleukin-2 in the treatment of central nervous system glioma and melanoma.

OBJECTIVE: The purpose of this study was to determine the optimal route of delivery of gene therapy for an intracerebral (IC) tumor. In previous studies, treatment of an IC tumor with the IC administration of a cellular vaccine consisting of allogeneic fibroblasts genetically engineered to secrete cytokines prolonged survival. Systemic delivery of gene therapy is of significant clinical interest. METHODS: In this study, allogeneic fibroblasts engineered to secrete interleukin (IL)-2 (LM-IL-2 cells) were administered either subcutaneously or intracerebrally to C57BL/6 mice with IC glioma. In addition, fibroblasts genetically engineered to express (antibody-defined) melanoma-associated antigens and to secrete IL-2 (RLBA-IL-2) were injected either intracerebrally or subcutaneously into mice bearing IC melanoma. RESULTS: The results indicate a significant prolongation of survival in mice with IC glioma treated intracerebrally with LM-IL-2 cells, relative to the survival of mice with IC glioma treated subcutaneously with LM-IL-2 cells or untreated mice with glioma. The specific release of isotope from 51Cr-labeled glioma cells coincubated with spleen cells from animals treated either subcutaneously or intracerebrally with LM-IL-2 cells was significantly greater than the release of isotope from glioma cells coincubated with spleen cells from nonimmunized mice. In a similar fashion, the survival of mice with IC B16 melanoma immunized intracerebrally with RLBA-IL-2 cells was significantly longer than nonimmunized mice injected with B16 cells alone. In contrast, the survival of mice with IC melanoma treated by subcutaneous injection with RLBA-IL-2 cells was not significantly different than that of untreated mice. Using a 51Cr-release assay, the specific release of isotope from labeled B16 cells coincubated with spleen cells from mice immunized either intracerebrally or subcutaneously with RLBA-IL-2 cells was significantly higher than that of B16 cells coincubated with cells from nonimmunized mice. CONCLUSIONS: Direct IC administration of fibroblasts genetically engineered to secrete IL-2 was more effective in prolonging survival than peripheral subcutaneous administration in the treatment of mice with IC glioma or melanoma.

Animals↗

PubMed1998

Redesigning metabolic networks using mathematical programming

The design of new generation bioprocessing plants is increasingly dependent on the design of process-compatible microorganisms. The latter, whether through genetic or physiological manipulations, can be greatly assisted by metabolic engineering. An emerging powerful tool in metabolic engineering research is computer-assisted cell design using mathematical programming. In this work, the problem of optimizing cellular metabolic networks has been formulated as a Mixed Integer Nonlinear Programming (MINLP) model. The model can assist genetic engineers to identify which cellular enzymes should be modified, and the new levels of activity required to produce an optimal network. Results are presented from the tricarboxylic acid cycle in Dictyostelium discoideum. Copyright 1998 John Wiley & Sons, Inc.

Journal Article↗

PubMed2004

Potential tissue-engineering applications for neonatal surgery.

Tissue engineering attempts to build neotissue from its cellular building blocks. This neotissue can then be used for reconstructive surgical applications such as replacement of a congenitally abnormal heart valve or repair of a craniofacial abnormality. Since its inception in the late 1980s, tissue engineering has sparked the interests of physicians and scientists alike because of its great potential. Significant progress has been made in this burgeoning branch of science. This article reviews some of the ongoing preclinical and clinical tissue engineering research as it applies to neonatology.

Animals↗

PubMed2004

Micropatterning of proteins and mammalian cells on biomaterials.

Controlling the spatial organization of cells is vital in engineering tissues that require precisely defined cellular architectures. For example, functional nerves or blood vessels form only when groups of cells are organized and aligned in very specific geometries. Yet, scaffold designs incorporating spatially defined physical cues such as microscale surface topographies or spatial patterns of extracellular matrix to guide the spatial organization and behavior of cells cultured in vitro remain largely unexplored. Here we demonstrate a new approach for controlling the spatial organization, spreading, and orientation of cells on two micropatterned biomaterials: chitosan and gelatin. Biomaterials with grooves of defined width and depth were fabricated using a two-step soft lithography process. Selective attachment and spreading of cells within the grooves was ensured by covalently modifying the plateau regions with commercially available protein resistant triblock copolymers. Precise spatial control over cell spreading and orientation has been observed when human microvascular endothelial cells are cultured on these patterned biomaterials, suggesting the potential of this technique in creating tissue culture scaffolds with defined chemical and topographical features.

Biocompatible Materials↗

PubMed2005

Expressing engineered thymidylate kinase variants in human cells to improve AZT phosphorylation and human immunodeficiency virus inhibition.

The triphosphorylated form of the nucleoside analogue AZT (AZTTP) acts as a chain terminator during reverse transcription of the human immunodeficiency virus (HIV) genome. The bottleneck in the conversion of AZT to AZTTP is the phosphorylation of AZT monophosphate (AZTMP) by cellular thymidylate kinase. Human thymidylate kinase was engineered to exhibit highly improved activity for AZTMP to AZTDP conversion. It was demonstrated here that genetically modified human cells transiently expressing these enzyme variants show more than 10-fold higher intracellular concentrations of AZTDP and AZTTP. Stable clones expressing these enzymes appear to phosphorylate AZTMP less efficiently, but first experiments indicate they are still more potent in HIV inhibition than the parental cells. It was proposed that the concept of introducing into human cells a catalytically improved human enzyme, rather than an enzyme of viral, bacterial or yeast origin, may serve as a paradigm for ameliorating the metabolic activation of an established drug.

Genetic Engineering↗