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PubMed2003

In silico multicellular systems biology and minimal genomes.

The in vivo and in silico understanding of genomes and networks in cellular and multicellular systems is essential for drug discovery for multicellular diseases. In silico methodologies, when integrated with in vivo engineering methods, lay the groundwork for understanding multicellular organisms and their genomes. The quest to construct a minimal cell can be followed by designed, minimal multicellular organisms. In silico multicellular systems biology will be essential in the design and construction of minimal genomes for minimal multicellular organisms. Advanced methodologies come to light that can aid drug discovery. These novel approaches include multicellular pharmacodynamics and networked multicellular pharmacodynamics.

Animals↗
PubMed2002

New in vivo and ex vivo models for the experimental study of sheep scrapie: development and perspectives.

Sheep scrapie is a prototypical transmissible spongiform encephalopathy (TSE), and the most widespread of these diseases. Experimental study of TSE infectious agents from sheep and other species essentially depends on bioassays in rodents. Transmission of natural sheep scrapie to conventional mice commonly requires one or two years. In an effort to develop laboratory models in which investigations on the sheep TSE agent would be facilitated, we have established mice and cell lines that were genetically engineered to express ovine PrP protein and examined their susceptibility to the infection. A series of transgenic mice lines (tgOv) expressing the high susceptibility allele (VRQ) of the ovine PrP gene from different constructs was expanded. Following intracerebral inoculation with natural scrapie isolates, all animals developed typical TSE neurological signs and accumulated abnormal PrP in their brain. The survival time in the highest expressing tgOv lines ranged from 2 to 7 months, depending on the isolate. It was inversely related to the brain PrP content, and essentially unchanged on further passaging. Ovine PrP transgene expression thus enhanced scrapie disease transmission from sheep to mice. Such tgOv mice may bring new opportunities for analysing the natural variation of scrapie strains and measuring infectivity. As no relevant cell culture models for agents of naturally-occurring TSE exist, we have explored various strategies in order to obtain stable cell lines that would propagate the sheep agent ex vivo without prior adaptation to rodent. In one otherwise refractory rabbit epithelial cell line, a regulable expression of ovine PrP was achieved and found to enable an efficient replication of the scrapie agent in inoculated cultures. Cells derived from sheep embryos or from tgOv mice were also used in an attempt to establish permissive cell lines derived from the nervous system. Cells engineered to express PrP proteins of a specified sequence may thus represent a promising strategy to further explore, at the cellular level, various aspects of TSE diseases.

Alleles↗
PubMed2006

A CD33-specific single-chain immunotoxin mediates potent apoptosis of cultured human myeloid leukaemia cells.

A novel single-chain immunotoxin was constructed by combining a CD33-specific single chain Fv (scFv) antibody fragment with an engineered variant of Pseudomonas exotoxin A (ETA). The variant toxin carries the KDEL peptide at its C-terminus, a cellular peptide mediating improved retrograde transport to the endoplasmic reticulum. The purified recombinant fusion protein induced potent apoptosis of the human myeloid cell lines U937, HL-60 and THP-1. Up to 98% of U937 cells were eliminated after treatment for 72 h with a single dose of 500 ng/ml (c. 7 nmol/l). Killing was antigen-specific and occurred by apoptosis. A control protein, consisting of a CD19-specific scFv antibody fragment fused to the ETA-KDEL toxin, failed to induce death of the CD19-negative cell lines U937, HL-60 and THP-1. The CD33-ETA toxin also mediated apoptosis of fresh patient-derived acute myeloid leukaemia cells from bone marrow and peripheral blood. The pronounced antigen-restricted cytotoxicity of the novel fusion protein makes it a candidate for further evaluation of its therapeutic potential.

ADP Ribose Transferases↗
PubMed2004

Thin bio-artificial tissues in plane stress: the relationship between cell and tissue strain, and an improved constitutive model.

Constitutive models are needed to relate the active and passive mechanical properties of cells to the overall mechanical response of bio-artificial tissues. The Zahalak model attempts to explicitly describe this link for a class of bio-artificial tissues. A fundamental assumption made by Zahalak is that cells stretch in perfect registry with a tissue. We show this assumption to be valid only for special cases, and we correct the Zahalak model accordingly. We focus on short-term and very long-term behavior, and therefore consider tissue constituents that are linear in their loading response (although not necessarily linear in unloading). In such cases, the average strain in a cell is related to the macroscopic tissue strain by a scalar we call the "strain factor". We incorporate a model predicting the strain factor into the Zahalak model, and then reinterpret experiments reported by Zahalak and co-workers to determine the in situ stiffness of cells in a tissue construct. We find that, without the modification in this article, the Zahalak model can underpredict cell stiffness by an order of magnitude.

Animals↗
PubMed2004

The relationship between cell and tissue strain in three-dimensional bio-artificial tissues.

Continuum constitutive laws are needed to ensure that bio-artificial tissue constructs replicate the mechanical response of the tissues they replace, and to understand how the constituents of these constructs contribute to their overall mechanical response. One model designed to achieve both of these aims is the Zahalak model, which was modified by Marquez and co-workers to incorporate inhomogeneous strain fields within very thin tissues. When applied to reinterpret previous measurements, the modified Zahalak model predicted higher values of the continuum stiffness of fibroblasts than earlier estimates. In this work, we further modify the Zahalak model to account for inhomogeneous strain fields in constructs whose cell orientations have a significant out-of-plane component. When applied to reinterpret results from the literature, the new model shows that estimates of continuum cell stiffness might need to be revised upward. As in this article's companion, we updated the average cell strain by defining a correction factor ("strain factor"), based upon the elastic response. Three different cell orientation distributions were studied. We derived an approximate scaling model for the strain factor, and validated it against exact and self-consistent (mean-field) solutions from the literature for dilute cell concentrations, and Monte Carlo simulations involving three-dimensional finite element analyses for high cell concentrations.

Animals↗
PubMed2003

[Methodologies for isolation, expansion and differentiation of somatic stem cells(tissue stem cells)].

Recent studies have shown that pluriponten stem cells are also found in adult tissue. These adult stem cells has a great advantage over other stem cells sources, human embryos or human fetal tissue, that trouble many people on ethical grounds. While the adult stem cells have great potential for use in the cell therapy, we must pass numerous technical hurdles to reach a goal. Here we describe a subset of problems encountered in isolation and expansion of somatic stem cells for clinical application. As it is necessary to obtain the enough number of cells to supply the defect tissue and organ, we required purification and ex vivo expansion of stem cells, overcoming senescence. We also refer to the argument about plasticity of adult stem cells.

Adult↗
PubMed2002

[Use of phage libraries for the in vitro production of recombinant monoclonal antibodies of predetermined specificity].

The biotechnological generation of monoclonal antibodies of predetermined specificity has traditionally involved the production of hybridomas obtained by somatic cellular fusion of splenocytes from immunized animals with myeloma cell lines bearing selectable markers. Now, monoclonal antibodies could be genetically engineered thus bypassing all the natural systems for making antibodies. Filamentous bacteriophages provides a means to display and select large single chain fragments variable (scFv) repertoires created by cloning the natural rearranged V-immunoglobulin genes or introducing predetermined level of randomization into germline V-gene segments. In this article we demonstrated that by using a well characterized scFv phage synthetic library it is possible to generate in vitro recombinant human monoclonal antibodies directed to a large array of antigens showing different molecular weights, conformations and origins.

Antibodies, Monoclonal↗
PubMed1990

CD8 functions as an inhibitory ligand in mediating the immunoregulatory activity of CD8+ cells.

Antisense and sense transfection technologies were employed in cellular coculture systems to investigate the physiologic requirements for CD8-dependent immunoregulation. Our data indicate that cells bearing genetically engineered CD8 molecules incorporating a glycoinositolphospholipid membrane anchor, as well as fixed cells bearing natural CD8 molecules, retain specific, CD8-dependent immunoregulatory activity. These findings together support the novel concept that CD8, a molecule traditionally thought of as a receptor, can function as an inhibitory ligand. CD8-dependent inhibition was shown to induce nonresponsiveness, persisting for at least 24 h, in Ag-specific responders. Moreover, only cells undergoing primary, but not secondary, antigenic stimulation through their TCR were found to be susceptible to CD8-dependent inhibition. Both CD4+ and CD8+ responding T cells were inhibited by CD8+ modulatory cells. These functional analyses of inhibitory and responder cells in CD8-dependent inhibition lay the groundwork for developing artificial CD8-based immunomodulatory peptides and deciphering CD8's role in natural immunoregulation.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1996

Electromagnetic interference can cause hospital devices to malfunction, McGill group warns.

Electromagnetic interference (EMI) from sources such as television transmitters, police radios and cellular phones can cause medical monitors and other hospital devices to malfunction, says the principal investigator of a McGill biomedical engineering group set up in 1989 to study, predict and prevent such problems. The impact of equipment malfunction can range from mere inconvenience to serious problems. The research group advises physicians and other health care professionals to learn how to spot problems related to EMI and electromagnetic compatibility.

Electromagnetic Fields↗
PubMed1998

Biological therapy of ovarian cancer: current directions.

Despite recent advances in the chemotherapy of ovarian cancer, the development of alternative therapies that retain activity against drug-resistant tumors remains a high priority. Our knowledge regarding growth factors, cytokines, and the immune response continues to expand, and molecular biology has provided an increased diversity of reagents for clinical evaluation. This review focuses on regulatory targets in ovarian cancer, including Her2/neu (c-erbB2) and other growth factor receptors; interferons, interleukins, and other immunoregulatory cytokines; cellular adhesion molecules; antigen-specific T lymphocytes and adoptive immunotherapy; choice of monoclonal antibody reagents and advances in antibody engineering, including recombinant single-chain binding sites, chimeric proteins, radioconjugates, cytotoxic drug conjugates, immunotoxins, and bispecific antibodies. Although specific roles for biologic therapy in the management of ovarian cancer have yet to be defined, current priorities for clinical research are reviewed.

Antibodies, Monoclonal↗
PubMed2004

Engineered autolytic yeast strains secreting Kluyveromyces lactis beta-galactosidase for production of heterologous proteins in lactose media.

Secretion of the heterologous Kluyveromyces lactis beta-galactosidase into culture medium by several Saccharomyces cerevisiae osmotic-remedial thermosensitive-autolytic mutants was assayed and proved that new metabolic abilities were conferred since the constructed strains were able to grow in lactose-containing media. Cell growth became independent of a lactose-uptake mechanism. Higher levels of extra-cellular and intra-cellular beta-galactosidase production, lactose consumption and growth were obtained with the LHDP1 strain, showing a thermosensitive-autolytic phenotype as well as being peptidase-defective. The recombinant strain LHDP1 presented the highest beta-galactosidase yields from biomass and the lowest ethanol levels from lactose. This strain is effective for the heterologous production and release of K. lactis beta-galactosidase into the extra-cellular medium after osmotic shock.

Cheese↗
PubMed1999

Murine cytomegalovirus homologues of cellular immunomodulatory genes.

The study of 'molecular mimicry' or 'genetic piracy', with respect to the utilisation of cellular genes captured and modified during the course of virus evolution, has been an area of increasing research with the expansion in virus genome sequencing. Examples of cellular immunomodulatory genes which have been captured from hosts have been identified in a number of viruses. This review concentrates upon studies of murine cytomegalovirus (MCMV), investigating the functions of viral genes homologous to G protein-coupled receptors, MHC class I and chemokines. The study of recombinant MCMV engineered with specific disruptions of these genes has revealed their significance during virus replication and dissemination within the host. In the case of the latter two classes of genes, evidence suggests they interfere with cellular immune responses, although the detailed mechanisms underlying this interference have yet to be delineated.

Amino Acid Sequence↗
PubMed2004

Age and gender effect on the growth of bone marrow stromal cells in vitro.

Bone marrow harvested by aspiration contains connective tissue progenitor cells which can be selectively isolated and induced to express bone phenotype in vitro. The osteoblastic progenitor can be estimated by counting the number of cells attach using the haemacytometer. This study was undertaken to test the hypothesis that human aging is associated with a significant change on the number of osteoblastic progenitors in the bone marrow. Bone marrow aspirates were harvested from 38 patients, 14 men (age 11-70) and 24 women (age 10-70) and cultured in F12: DMEM (1:1). In total 15 bone marrow samples have been isolated from patients above 40 years old (men/women) of age. Fourteen (93.3%) of this samples failed to proliferate. Only one (6.7%) bone marrow sample from a male patient, aged 59 years old was successfully cultured. Seventy percent (16/23) of the samples from patient below than 40 years old were successfully cultured. However, our observation on the survival rate for cells of different gender from patient below 40 years old does not indicate any significant difference. From this study, we conclude that the growth of bone marrow stromal cells possibly for bone engineering is better from bone marrow aspirates of younger patient.

Adolescent↗
PubMed2006

Evaluation of chondrogenesis within PEGT: PBT scaffolds with high PEG content.

Porous poly(ethylene glycol) terephthalate:poly (butylene terephthalate) (PEGT:PBT) scaffolds with high PEG molecular weight (1000 g/mole) and PEGT content (60%) were fabricated using two different processes-paraffin templating and compression molding-for cartilage engineering applications. This polymer composition has previously been shown to enable chondrocyte adhesion and maintain differentiated phenotype in 2D monolayer culture. The influence of 3D polymer scaffold processing on the formation of cartilaginous tissue was studied by seeding primary immature bovine chondrocytes within cylindrical scaffolds in mixed flask reactors for 3 days, followed by cultivation in culture plates for a total of 10 or 24 days. Tissue-polymer constructs were evaluated morphologically by SEM and histology, and quantitatively for cellularity, total collagen, and glycosaminoglycan content, all of which remained statistically equivalent for each time point tested, irrespective of fabrication method. These data demonstrate that the polymers engineered for this study were able to support chondrogenesis independent of scaffold fabrication process, with the influence of pore architecture lessened by the highly hydrated scaffold microenvironments induced by high PEG content.

Animals↗
PubMed1999

Towards the production of salt-tolerant crops.

Crop production is affected by numerous environmental factors, with soil salinity and drought having the most detrimental effects. Attempts to improve yield under stress conditions by plant breeding have been unsuccessful, primarily due to the multigenic origin of the adaptive responses. The transfer of genes through genetic engineering of crop plants appears more feasible. Important adaptive mechanisms targeted for potential gene transfer would be the tonoplast Na+/H+ antiport, compatible solute synthesis and, regulation of water channel activity and expression, mechanisms involved in cellular osmoregulation. In this review we discuss recent advances in our understanding of these adaptive mechanisms.

Aquaporins↗
PubMed2006

Engineering RNA-based circuits.

Nucleic acids can modulate gene function by base-pairing, via the molecular recognition of proteins and metabolites, and by catalysis. This diversity of functions can be combined with the ability to engineer nucleic acids based on Watson-Crick base-pairing rules to create a modular set of molecular "tools" for biotechnological and medical interventions in cellular metabolism. However, these individual RNA-based tools are most powerful when combined into rational logical or regulatory circuits, and the circuits can in turn be evolved for optimal function. Examples of genetic circuits that control translation and transcription are herein detailed, and more complex circuits with medical applications are anticipated.

Animals↗
PubMed2004

From cell-ECM interactions to tissue engineering.

The extracellular matrix (ECM) consists of a complex mixture of structural and functional macromolecules and serves an important role in tissue and organ morphogenesis and in the maintenance of cell and tissue structure and function. The great diversity observed in the morphology and composition of the ECM contributes enormously to the properties and function of each organ and tissue. The ECM is also important during growth, development, and wound repair: its own dynamic composition acts as a reservoir for soluble signaling molecules and mediates signals from other sources to migrating, proliferating, and differentiating cells. Approaches to tissue engineering center on the need to provide signals to cell populations to promote cell proliferation and differentiation. These "external signals" are generated from growth factors, cell-ECM, and cell-cell interactions, as well as from physical-chemical and mechanical stimuli. This review considers recent advances in knowledge about cell-ECM interactions. A description of the main ECM molecules and cellular receptors with particular care to integrins and their role in stimulation of specific types of signal transduction pathways is also explained. The general principles of biomaterial design for tissue engineering are considered, with same examples.

Animals↗
PubMed1999

The CD14 ligands lipoarabinomannan and lipopolysaccharide differ in their requirement for Toll-like receptors.

Mammalian Toll-like receptor (TLR) proteins are new members of the IL-1 receptor family that participate in activation of cells by bacteria and bacterial products. Several recent reports indicate that TLR proteins mediate cellular activation by bacterial LPS via a signaling pathway that is largely shared by the type I IL-1 receptor. We previously showed that Chinese hamster ovary (CHO) fibroblasts engineered to express CD14 (CHO/CD14) were responsive to LPS, but not to a distinct CD14 ligand, mycobacterial lipoarabinomannan (LAM). These CHO/CD14 cells were subsequently found to possess a frame-shift mutation within the TLR2 gene which resulted in their inability to express functional TLR2 protein. Thus, we hypothesized that TLR2, but not TLR4, was necessary for LAM signaling. In this paper we show that CHO/CD14 cells engineered to express functional TLR2 protein acquired the ability to be activated by LAM. Similarly, overexpression of TLR2 in murine macrophages conferred enhanced LAM responsiveness. Together, our data demonstrate that the distinct CD14 ligands LAM and LPS utilize different TLR proteins to initiate intracellular signals. These findings suggest a novel receptor signaling paradigm in which the binding of distinct ligands is mediated by a common receptor chain, but cellular activation is initiated via distinct signal-transducing chains that confer ligand specificity. This paradigm contrasts with many cytokine receptor complexes in which receptor specificity is conferred by a unique ligand-binding chain but cellular activation is initiated via shared signal-transducing chains.

Animals↗