Search PubMed⌕ Search

Biomedical subjects

C H Evans

Publications and source records attributed to C H Evans.

At least 91 records · Page 5Linked to original sources
PubMed1999

[Gene transfer in the treatment of arthritis].

Current concepts in treating arthritis by gene transfer are described, including different vector systems and strategies of gene transfer into target cells. Promissing antiarthritic gene products are a variety of growth factors which facilitate increased matrix synthesis and mitogensis in articular chondrocytes. Furthermore, rheumatoid joint destruction can be treated genetically by the transfer of certain antiinflammatory cytokine genes, which provide locally high concentrations of the antiinflammatory gene product. First clinical trails using the IRAP gene (interleukin I receptor antagonist protein) to eliminate the inflammatory reaction caused by interleukin I in rheumatoid joints are on its way. In order to investigate potential improvement in cartilage regeneration retroviral TGF-beta gene transfer in rabbit articular chondrocytes has been carried out. The TGF-beta group showed an in vitro increase in collagen type II neosynthesis by 304%, compared to normal chondrocytes.

Animals↗
PubMed1999

A gene therapy approach to accelerating bone healing. Evaluation of gene expression in a New Zealand white rabbit model.

It has been demonstrated that BMPs, IGFs, and TGFbetas improve the process of bone healing in vivo. We have suggested the use of gene therapy as a possible way to deliver growth factors to fracture sites in order to improve repair. The aim of this study was to develop a minimally invasive gene therapy approach to treat bone injuries locally without damaging the local blood circulation. A segmental defect of 1.3 cm was created in the diaphysis of the femur in mature NZW rabbits. Internal fixation with 7-hole DCP plates and 2.7 mm screws was used to stabilize the bone. After building a chamber by tightly closing the muscles around the segmental defect, 0.5 ml of either saline solution or a collagen gel containing 1 x 10(10) particles of adenovirus carrying cDNA encoding either the bacterial beta-galactosidase gene (LacZ), or the firefly luciferase gene were injected into the gap. The control side received 0.5 ml of saline solution without virus particles. Bone marrow, cortical and trabecular bone and surrounding muscle were harvested from the injected femur and were analyzed for local gene expression through X-gal staining or measurement of local luciferase activity. To determine whether distant sites were transduced, tissue from the spleen, liver, and lung were harvested as well as bone, bone marrow and muscle from the contralateral diaphysis of the femur. The delivery of the adenoviral vector suspended in saline solution led to local transduction of the bone, bone marrow and the muscle surrounding the gap. No luciferase activity was found in the contralateral femur, lung, or spleen, and only transient luciferase activity was seen in the liver. While marker gene expression persisted within the surrounding soft tissues for at least 2 weeks, the expression in bone lasted up to 6 weeks. This study has shown that it is possible to use adenoviral vectors to transfer and express genes locally within a segmental defect. Gene expression persisted for several weeks, which may be already sufficient to accelerate repair.

Adenoviruses, Human↗
PubMed1999

[In vitro transduction of human osteoblast cell populations with retroviral vectors].

OBJECTIVES: The involvement of cytokines in degeneration and inflammation of human tissue is well established. Interleukin-1 (IL-1) is a major agent in the pathophysiology of periarticular bone resorption in rheumatoid arthritis and in osteoporosis. Because the use of recombinant cytokines and growth factors is limited due to their short half lives, techniques are needed to get a permanent release of these therapeutic proteins. The rational of this study was to show that retroviral transduction of human osteoblastic cells is possible in vitro using the marker gene LacZ and the potentially therapeutic gene encoding for human interleukin-1 receptor antagonist protein (IL-1Ra). Different transduction techniques were combined to improve the rate of transduction in vitro. METHODS: Osteoblastic cells were isolated from human spongious bone and cultured in vitro. The beta-galactosidase (LacZ) gene and the cDNA of IL-1Ra were introduced into the isolated cells by retrovirus mediated gene transfer. LacZ activity was determined by Xgal staining, IL-1Ra was measured quantitatively by ELISA. RESULTS: The transfer of retroviral IL-1Ra led to IL-1Ra expression of 8614 to 10,089 pg IRAP/50,000 cells/48 h. By combining different techniques to improve transduction, the X-gal staining established a rate of transduction of 60%. CONCLUSION: Our results demonstrate that retroviral transduction of human osteobalstic cells is possible in vitro, and leads to high levels of the synthesized transgene product. The rate of retroviral transduction can be accelerated in vitro.

Cell Line↗
PubMed1999

The role of gene therapy. Fact or fiction?

Current research in molecular biology and genetics has dramatically advanced the understanding of the cellular events involved in homeostasis, disease, injury, and healing processes of the tissues of the musculoskeletal system. Recently, genetic predispositions to diseases have been described which offer novel means to address musculoskeletal disorders. Growth factors and cytokines have been identified as key elements in both the injured and healing states. Gene therapy offers an elegant solution to the delivery of therapeutic proteins to the site of disease or injury.

Cytokines↗
PubMed1999

Potential treatment of osteoarthritis by gene therapy.

OA is common, debilitating, costly, incurable, and, in many cases, resistant to treatment. Novel approaches to therapy are clearly required. Progress in understanding the biology of cartilage and OA have led to our suggestion of a gene therapy approach to treatment. Genes whose products stimulate chondrogenesis or inhibit breakdown of the cartilaginous matrix are obviously candidates for therapeutic use. These genes may be transferred to the synovium or cartilage of affected joints by in vivo or ex vivo means using a variety of vectors. Transfer of such genes to chondroprogenitor cells is a particularly attractive approach.

Animals↗
PubMed1999

Adenovirus-mediated direct gene therapy with bone morphogenetic protein-2 produces bone.

The need to improve bone healing permeates the discipline of orthopedic surgery. Bone morphogenetic proteins (BMPs) are capable of inducing ectopic and orthotopic bone formation. However, the ideal approach with which to deliver BMPs remains unknown. Gene therapy to deliver BMPs offers several theoretical advantages over implantation of a recombinant BMP protein, including persistent BMP delivery and eliminating the need for a foreign body carrier. A replication defective adenoviral vector was constructed to carry the rhBMP-2 gene (AdBMP-2). The direct in vivo gene therapy approach was applied in both immunodeficient and immunocompetent animals to produce intramuscular bone as early as 2 weeks following injection. Radiographic and histologic analysis revealed radiodense bone containing mature bone marrow elements. Adenovirus-mediated delivery of a marker gene (beta-galactosidase) into control animals produced no bone but indicated the cells transduced with the AdBMP-2 vector. Furthermore, comparisons between immunodeficient and immunocompetent animals illustrated the magnitude and significance of the immune response. Gene therapy to deliver BMP-2 has innumerable potential clinical applications from bone defect healing to joint replacement prosthesis stabilization. This study is the first to establish the feasibility of in vivo gene therapy to deliver active BMP-2 and produce bone.

Adenoviridae↗
PubMed1999

Retrovirally transduced bone marrow stromal cells isolated from a mouse model of human osteogenesis imperfecta (oim) persist in bone and retain the ability to form cartilage and bone after extended passaging.

Bone marrow stromal cells isolated from a model of osteogenesis imperfecta (oim) mice, were transduced with a retrovirus (BAG) carrying the LacZ and neor genes after passage 21. The transduced cells retained the ability to express alkaline phosphatase activity in vitro when treated with recombinant human bone morphogenetic protein two (rhBMP-2), formed cartilage in vitro in aggregate cultures and formed bone in ceramic cubes after 6 weeks of implantation in nude mice. X-gal staining of ceramic cubes seeded with the transduced cells demonstrated the presence of LacZ-positive cells on the edges of bone and also in the lacunae of the newly formed bone 6 weeks after implantation. After infusion into femurs of oim mice, the transduced cells were detected in the marrow cavity and on the edges of the trabecular bone of the injected and contralateral femurs by X-gal staining and PCR analysis at 4, 10, 20, 30 and 40 days after injection. The LacZ gene was also detected in the lung and liver of the recipient mice at 4 and 10 days after injection but not at later time-periods. The present findings suggest that long-term cultured bone marrow stromal cells from osteogenesis imperfecta (OI) animals have the potential to traffic through the circulatory system, home to bone, form bone and continue to express exogenous genes. These findings open the possibility of using these cells as vehicles to deliver normal genes to bone as an alternative approach for the treatment of some forms of OI and certain other bone acquired and genetic diseases.

Alkaline Phosphatase↗
PubMed1999

Intermittent sub-ambient interstitial hydrostatic pressure as a potential mechanical stimulator for chondrocyte metabolism.

OBJECTIVE: Experimental findings have suggested that the metabolic activities of articular cartilage can be influenced by mechanical stimuli. Our mathematical analysis predicted that cyclic compressive loading may create periods of intermittent sub-ambient hydrostatic pressure within the cartilage extracellular matrix. Based on this mathematical analysis, the present study was aimed to investigate whether the intermittent sub-ambient hydrostatic pressure, created in the cartilage extracellular matrix during cyclic compression, has a stimulative effect on the biosynthesis of chondrocytes. METHOD: In order to test this hypothesis, the present study developed a custom-designed sub-ambient pressure generator to subject a monolayer culture of chondrocytes to an intermittent sub-ambient pressure. Using this pressure generator, the monolayer chondrocyte culture system was analyzed for 35S-sulfate and 3H-proline incorporation rates for biosynthesis of proteoglycan and collagenous/noncollagenous protein molecules, respectively. Northern analyses for aggrecan and type II collagen mRNAs were also performed. RESULTS: It was found that the intermittent sub-ambient pressure produced a 40% increase in proteoglycan and a 17% increase in non-collagenous protein synthesis during the pressurization period (P < 0.05). The collagenous protein synthesis was not affected by the intermittent sub-ambient pressure regimen used in this study. After the intermittent sub-ambient pressurization, the metabolic activities of the chondrocytes returned to normal (control level). The intermittent sub-ambient pressure also produced an increase in the mRNA signals for aggrecan. Therefore, we conclude that intermittent sub-ambient pressure may be one of the potential mechanical stimulators of chondrocytes in articular cartilage during dynamic compression.

Aggrecans↗
PubMed1999

Nitric oxide in osteoarthritis.

Activated articular chondrocytes produce large amounts of nitric oxide (NO), and there is increasing evidence that this is involved in the etiopathogenesis of osteoarthritis (OA). Because of its short half-life, the biological effects of endogenously produced NO are likely to occur locally within the cartilage. We have observed that inhibitors of NO synthases relieve the inhibition of matrix synthesis that otherwise occurs in response to IL-1. To avoid the use of inhibitors, we have recently transduced chondrocytes with the iNOS (NOS-2) gene and confirmed the ability of the endogenously produced NO to inhibit matrix synthesis. Despite the high levels of NO made by these cells, there was no evidence of apoptosis or other forms of cell death. NO was also shown to inhibit the production of TGF-beta(1)by cells treated with IL-1, as well as to decrease matrix production in response to IGF-1. The hypothesis that NO inhibits matrix production by interfering with important autocrine and paracrine factors should be entertained.

Chondrocytes↗
PubMed1999

Genetically augmented tissue engineering of the musculoskeletal system.

Recent advances in gene transfer technology permit the design of strategies to improve the outcome of orthopaedic tissue engineering by genetic means. Using ex vivo and in vivo strategies, genes have been transferred successfully to, and expressed within, numerous tissues of the musculoskeletal system, including articular cartilage, meniscus, intervertebral disc, bone, tendon, ligament, synovium, and muscle. With these technologies, various genes encoding modulatory species of ribonucleic acid or proteins such as growth factors, receptors, and transcription factors could be used in the context of genetically augmented tissue engineering. Proof of principle has been established in numerous animal models, and a human protocol for the transfer of genes to synovium already is underway. Progress so far permits cautious optimism of a successful outcome to these pursuits.

Animals↗
PubMed1999

Transfer of lacZ marker gene to the meniscus.

BACKGROUND: Lesions in the avascular two-thirds of the meniscus do not heal well and are of concern clinically. Various growth factors promote the synthesis of matrix by meniscal cells and thus have the potential to augment healing. However, their clinical application is severely hindered by problems with delivery. An attractive approach to overcoming such problems is to transfer genes that encode the growth factors in question to the site of the injury. As a prelude to this, we evaluated methods for delivering genes to the meniscus. METHODS: Gene transfer was evaluated in vitro and in vivo with a lacZ marker gene, which expresses the enzyme beta-galactosidase. Two types of vectors were tested: an adenovirus and a retrovirus. Monolayers of lapine, canine, and human meniscal cells, as well as intact lapine and human menisci, were used for the in vitro studies. Lesions were created in the menisci of rabbits and dogs for the in vivo studies. Gene transfer to the sites of the experimental meniscal lesions in vivo was accomplished in two ways. In the lapine model, a suspension of adenovirus carrying the lacZ marker gene was mixed with whole blood and the clot was inserted into the lesion. In the canine model, retrovirally transduced allogenic meniscal cells carrying the lacZ marker gene were embedded in collagen gels and transferred to the defects. The animals were killed at various time-points, and gene expression was evaluated by histological examination of sections stained with 5-bromo-4-chloro-indolyl-beta-D-galactose (X-gal), from which a blue chromagen is released in the presence of beta-galactosidase. RESULTS: Monolayer cultures of lapine, canine, and human meniscal cells were susceptible to genetic transduction by both adenoviral and retroviral vectors. In vitro gene transfer to intact human and lapine menisci proved possible both by direct, adenoviral, delivery and indirect, retroviral, delivery. Gene expression persisted for at least twenty weeks under in vitro conditions. With regard to the in vivo studies, gene expression persisted within the clot and in some of the adjacent meniscal cells for at least three weeks in the lapine defect model. In the canine defect model, gene expression persisted within the transplanted, transduced meniscal cells for at least six weeks. CONCLUSIONS: It is possible to transfer genes to sites of meniscal damage and to express them locally within the lesion for several weeks.

Animals↗
PubMed1999

Cytokines and the role they play in the healing of ligaments and tendons.

Cytokines are small proteins with the properties of locally acting hormones. They are essential for the healing of connective tissues following injury. Various cytokines are expressed endogenously during the healing responses of those ligaments and tendons which possess them, and there is evidence that the exogenous application of certain cytokines promotes healing. Because healing is a multistep process, several different cytokines may need to be applied at different times to achieve a satisfactory response. Sustained, localised delivery of the appropriate cytokines presents a technical challenge to their clinical use in the repair of ligaments and tendons. Slow release devices and gene transfer are being evaluated as strategies for obviating this limitation.

Animals↗
PubMed1999

Gene therapy of arthritis.

Genes encoding anti-arthritic products can be transferred to intra-or extraarticular sites where their expression suppresses various aspects of the pathophysiology of arthritis. A variety of viral and non-viral vectors can be used for the in vivo or ex vivo delivery of such genes. Promising pre-clinical data have resulted from the application of these strategies in several animal models of disease. Genes showing efficacy in this way include these encoding interleukin (IL) -1Ra, IL-1sR, TNFsR, transforming growth factor beta (TGF-beta), IL-13, Fas L, IL- 10 and vIL-10. Two human arthritis gene therapy protocols are underway in the USA and Germany. Both studies involve the ex vivo transfer of an IL-1Ra cDNA to the metacarpophalangeal joints of patients with rheumatoid arthritis. Progress in developing gene treatments for arthritis has been rapid, and permits optimism about their ability eventually to improve the treatment of this group of diseases.

Animals↗
PubMed1999

Adenoviral transduction of human osteoblastic cell cultures: a new perspective for gene therapy of bone diseases.

This article confirms the susceptibility of osteoblastic cells to adenoviral transduction. Osteoblasts were harvested from human cancellous bone. Cells were transduced, using various amounts of adenoviral vectors carrying the cDNA encoding interleukin-1 receptor antagonist (IL-1 Ra), or the marker genes beta-galactosidase and luciferase. Expression of the transgenes and the biological activity of IL-1 Ra produced by gene transfer were measured quantitatively in a time-course by ELISA. The rate of transduction was 100% after exposure to 1 x 10(7) infective particles of adeno-LacZ. No expression of IL-1Ra was seen after transduction with adeno-IL-1Ra at titers of 1 x 10(4) and less. However, after transduction at titers of 1 x 10(7), infective particles cells expressed IL-1 Ra consistently for 72 days, with levels up to 1 microg IL-1 Ra/1 x 10(6) cells/ 48 hours. None of the control samples expressed detectable levels of IL-1 Ra. The biological activity of the transgenic IL-1 Ra was demonstrated by its ability to suppress successfully IL-1-induced nitric oxide synthesis by rabbit articular chondrocytes. After transduction with 1 x 10(7) infective particles of the adenoluciferase vector, up to 81,000 Units transgenic luciferase/x 10(6) osteoblastic cells were measured 2 days after gene transfer. Our results show that adenovirus transduces osteoblastic cells at a high rate in vitro.

Adenoviruses, Human↗
PubMed1998

Adenovirus-mediated gene transfer to nucleus pulposus cells. Implications for the treatment of intervertebral disc degeneration.

STUDY DESIGN: In vitro and in vivo studies using a rabbit model were performed to determine the feasibility of adenovirus-mediated gene transfer to the intervertebral disc. OBJECTIVES: This study was conducted to determine whether it is possible to transfer genes to cells within the intervertebral disc by direct injection of an adenovirus and to determine the duration of gene expression obtained by this method. SUMMARY OF BACKGROUND DATA: Although growth factors have the potential to stimulate the regeneration of nucleus pulposus, sustained delivery of growth factors to a degenerated disc is clinically unfeasible with present technology. Novel approaches such as gene transfer should be investigated as possible solutions to this problem. METHODS: The lacZ marker gene was used to evaluate gene delivery to cells within intervertebral discs. For the in vitro study, cell cultures were established from the nucleus pulposus tissue of New Zealand white rabbits and infected with an adenovirus encoding the lacZ gene (Ad-lacZ). For the in vivo study, the anterior aspects of lumbar intervertebral discs were surgically exposed, and Ad-lacZ in saline solution was directly injected into the nucleus pulposus. An equal volume of saline only was injected into control discs. Expression of the transferred gene was detected by staining with 5-bromo-4-chloro-3-indolyl-beta-galactosidase (X-Gal). RESULTS: The in vitro experiments confirmed that nucleus pulposus cells were efficiently transduced by an adenoviral vector carrying the lacZ gene. In vivo injection of Ad-lacZ into the nucleus pulposus resulted in the transduction of a considerable number of cells. Marker gene expression in vivo persisted at an apparently undiminished level for at least 12 weeks. No staining was noted in control discs. CONCLUSIONS: The results show the feasibility of adenovirus-mediated gene transfer to the intervertebral disc. Expression of the marker gene persisted at least 12 weeks in vivo. This successful demonstration of exogenous gene transfer to the disc and sustained, long-term expression suggests that the adenoviral vector may be suitable for delivery of appropriate genes to the disc for the treatment of spinal disorders.

Adenoviridae↗
PubMed1998

Leukoregulin induction of protein expression in human orbital fibroblasts: evidence for anatomical site-restricted cytokine-target cell interactions.

The molecular basis for the profound inflammatory response and the accumulation of hyaluronan in orbital connective tissues seen in thyroid-associated ophthalmopathy is unknown. Moreover, the link between the orbital manifestations of Graves' disease and those in the pretibial skin, localized dermopathy, has yet to be established. We have reported recently that leukoregulin, an activated T lymphocyte-derived cytokine, dramatically induces hyaluronan synthesis and prostaglandin-endoperoxide H synthase 2 in human orbital fibroblasts in culture. In the current studies, utilizing giant two-dimensional gel electrophoresis, we find that orbital fibroblasts express constitutively a protein profile that distinguishes them from skin fibroblasts derived from the abdominal wall and from the pretibium. We further demonstrate that leukoregulin, when present in culture medium for 16 hr, up-regulates a set of orbital fibroblast proteins not present in untreated cultures or in fibroblasts from the abdominal wall. However, some of the same protein inductions are present in the pretibial fibroblasts. These leukoregulin-induced changes in protein expression are completely blocked by dexamethasone (10 nM). Our findings are the first to identify proteins that appear to be expressed and differentially regulated in an anatomical site-restricted manner in orbital and pretibial fibroblasts and seem to establish a molecular link between fibroblasts from the orbit and those in pretibial skin.

Binding Sites↗