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Biomedical subjects

C H Evans

Publications and source records attributed to C H Evans.

At least 19 recordsLinked to original sources
PubMed1999

Cyclic tensile stress exerts antiinflammatory actions on chondrocytes by inhibiting inducible nitric oxide synthase.

Continuous passive motion manifests therapeutic effects on inflamed articular joints by an as-yet-unknown mechanism. Here, we show that application of cyclic tensile stress (CTS) in vitro abrogates the catabolic effects of IL-1beta on chondrocytes. The effects of CTS are mediated by down-regulation of IL-1beta-dependent inducible NO production, and are directly attributed to the inhibition of inducible NO synthase (iNOS) mRNA expression and protein synthesis. The inhibition of iNOS induction by CTS is paralleled by abrogation of IL-1beta-induced down-regulation of proteoglycan synthesis. Furthermore, CTS inhibits iNOS expression and up-regulates proteoglycan synthesis at concentrations of IL-1beta frequently observed in inflamed arthritic joints, suggesting that the actions of CTS may be clinically relevant in suppressing the sustained effects of pathological levels of IL-1beta in vivo. These results are the first to demonstrate that mechanisms of the intracellular actions of CTS in IL-1beta-activated chondrocytes are mediated through inhibition of a key molecule in the signal transduction pathway that leads to iNOS expression.

Animals↗
PubMed1999

Direct adenoviral gene transfer of viral IL-10 to rabbit knees with experimental arthritis ameliorates disease in both injected and contralateral control knees.

IL-10, a cytokine produced primarily by macrophages, B lymphocytes, and Th2 cells, has both immunostimulatory and immunosuppressive properties. A homologue of IL-10 encoded by EBV, known as viral IL-10 (vIL-10), is also able to suppress the immune response, but may lack some of the immunostimulatory properties of IL-10. To evaluate the potential of vIL-10 to block the progression of rheumatoid arthritis, we have utilized a replication-defective adenovirus vector to deliver the gene encoding vIL-10 to the knee joints of rabbits with Ag-induced arthritis. Intraarticular expression of vIL-10 significantly reduced leukocytosis, cartilage matrix degradation, and levels of endogenous rabbit TNF-alpha, as well as the degree of synovitis, while maintaining high levels of cartilage matrix synthesis. Interestingly, an antiarthritic effect was also observed in opposing contralateral control knee joints that received only a marker gene. An adenoviral vector carrying the enhanced green fluorescent protein marker gene was used to demonstrate that a morphologically similar subset of cells infected in the injected knee joint are able to traffic to the uninjected contralateral knee joint. Our results suggest that direct, local intraarticular delivery of the vIL-10 gene may have polyarticular therapeutic effects.

Adenoviruses, Human↗
PubMed1999

Adenoviral transfer of the viral IL-10 gene periarticularly to mouse paws suppresses development of collagen-induced arthritis in both injected and uninjected paws.

Gene therapy is a promising new approach in the treatment of rheumatoid arthritis. Gene delivery to diseased joints offers the prospect of achieving high, local concentrations of a therapeutic gene product in a sustained manner, while minimizing exposure of nontarget organs. We report that a single administration of a modified adenovirus encoding the Epstein-Barr-derived homologue of IL-10 can suppress the development of disease for extended periods of time when injected locally within the periarticular tissue surrounding the ankle joints of mice with collagen type II-induced arthritis. Furthermore, we show that injection of an adenoviral vector carrying the IL-10 gene into a single paw can suppress development of arthritis in other, noninjected paws of the same individual. The systemic protection resulting from local gene therapy occurred in the absence of detectable levels of viral IL-10 in the serum. Circulating Ab levels to heterologous collagen were unaffected; however, treatment with viral IL-10 significantly suppressed the development of Abs to autologous mouse type II collagen. Thus, the treatment of a single joint by local delivery of the vIL-10 gene may protect multiple joints of the same individual while avoiding deleterious side effects often associated with systemic therapy.

Adenoviridae↗
PubMed1999

Inhibition of transforming growth factor beta production by nitric oxide-treated chondrocytes: implications for matrix synthesis.

OBJECTIVE: Nitric oxide (NO) is generated copiously by articular chondrocytes activated by interleukin-1beta (IL-1beta). If NO production is blocked, much of the IL-1beta inhibition of proteoglycan synthesis is prevented. We tested the hypothesis that this inhibitory effect of NO on proteoglycan synthesis is secondary to changes in chondrocyte transforming growth factor beta (TGFbeta). METHODS: Monolayer, primary cultures of lapine articular chondrocytes and cartilage slices were studied. NO production was determined as nitrite accumulation in the medium. TGFbeta bioactivity in chondrocyte- and cartilage-conditioned medium (CM) was measured with the mink lung epithelial cell bioassay. Proteoglycan synthesis was measured as the incorporation of 35S-sodium sulfate into macromolecules separated from unincorporated label by gel filtration on PD-10 columns. RESULTS: IL-1beta increased active TGFbeta in chondrocyte CM by 12 hours; by 24 hours, significant increases in both active and latent TGFbeta were detectable. NG-monomethyl-L-arginine (L-NMA) potentiated the increase in total TGFbeta without affecting the early TGFbeta activation. IL-1beta stimulated a NO-independent, transient increase in TGFbeta3 at 24 hours; however, TGFbeta1 was not changed. When NO synthesis was inhibited with L-NMA, IL-1beta increased CM concentrations of TGFbeta1 from 24-72 hours of culture. L-arginine (10 mM) reversed the inhibitory effect of L-NMA on NO production and blocked the increases in TGFbeta1. Anti-TGFbeta1 antibody prevented the restoration of proteoglycan synthesis by chondrocytes exposed to IL-1beta + L-NMA, confirming that NO inhibition of TGFbeta1 in IL-1beta-treated chondrocytes effected, in part, the decreased proteoglycan synthesis. Furthermore, the increase in TGFbeta and proteoglycan synthesis seen with L-NMA was reversed by the NO donor S-nitroso-N-acetylpenicillamide. Similar results were seen with cartilage slices in organ culture. The autocrine increase in CM TGFbeta1 levels following prior exposure to TGFbeta1 was also blocked by NO. CONCLUSION: NO can modulate proteoglycan synthesis indirectly by decreasing the production of TGFbeta1 by chondrocytes exposed to IL-1beta. It prevents autocrine-stimulated increases in TGFbeta1, thus potentially diminishing the anabolic effects of this cytokine in chondrocytes.

Animals↗
PubMed1999

Gene transfer of cytokine inhibitors into human synovial fibroblasts in the SCID mouse model.

OBJECTIVE: To investigate the effects of retrovirus-based gene delivery of inhibitory cytokines and cytokine inhibitors into human synovial fibroblasts in the SCID mouse model of rheumatoid arthritis (RA). METHODS: The MFG vector was used for gene delivery of tumor necrosis factor alpha receptor (TNFalphaR) p55, viral interleukin-10 (IL-10), and murine IL-10 into RA synovial fibroblasts. The effect on invasion of these cells into human articular cartilage and on perichondrocytic cartilage degradation was examined after 60 days of coimplantation into the SCID mouse. RESULTS: TNFalphaR p55 gene transfer showed only a limited effect on inhibition of RA synovial fibroblast invasiveness and cartilage degradation. In contrast, invasion of the RA synovial fibroblasts into the coimplanted cartilage was strongly inhibited by both viral and murine IL-10. Perichondrocytic cartilage degradation was not affected by either form of IL-10. CONCLUSION: The data show that cytokines can be successfully inserted into the genome of human RA synovial fibroblasts using a retroviral vector delivery system, and that the SCID mouse model of human RA is a valuable tool for examining the effects of gene transfer. In addition, inhibition of more than one cytokine pathway may be required to inhibit both synovial- and chondrocyte-mediated cartilage destruction in RA.

Animals↗
PubMed1999

Early expression of marker genes in the rabbit medial collateral and anterior cruciate ligaments: the use of different viral vectors and the effects of injury.

Gene therapy is a technique that may offer advantages over current methods of cytokine delivery to ligaments. To determine if implanted genes could be expressed in normal and injured knee ligaments, the medial collateral ligament and anterior cruciate ligament were studied in 18 rabbits. A retroviral ex vivo technique using allograft medial collateral ligament and anterior cruciate ligament fibroblasts and an adenoviral in vivo technique were compared as methods for delivering the LacZ marker gene to knee ligaments. Bilateral knee surgeries were performed, and the rabbits were equally divided into three groups. Group 1 received the retrovirus and the medial collateral ligament was ruptured, Group 2 received the adenovirus and the medial collateral ligament was ruptured, and Group 3 received the adenovirus and the medial collateral ligament was not injured. The anterior cruciate ligament was not injured in any group. The medial collateral and anterior cruciate ligaments of the right knees received 10(6) allografted, transduced ligament fibroblasts or 10(9) adenovirus particles, whereas the ligaments of the left knee received a similar volume of saline solution only. Equal numbers of rabbits were killed at 10 days, 3 weeks, and 6 weeks following the procedure. Ligament samples were stained with X-gal to detect the expression of the LacZ gene product, beta-galactosidase. LacZ gene expression was evident in ruptured and uninjured medial collateral ligaments as well as in the anterior cruciate ligament. The expression lasted between 10 days and 3 weeks in the medial collateral and anterior cruciate ligaments with use of the retrovirus and between 3 and 6 weeks in the medial collateral ligament and at least 6 weeks in the anterior cruciate ligament with the adenovirus. The length of gene expression in the ruptured and uninjured medial collateral ligaments did not differ. These preliminary studies indicate that gene transfer to normal and injured knee ligaments is possible.

Adenoviridae↗
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↗