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Steven C Ghivizzani

Publications and source records attributed to Steven C Ghivizzani.

At least 19 recordsLinked to original sources
PubMed2006

Exogenous glucosamine globally protects chondrocytes from the arthritogenic effects of IL-1beta.

The effects of exogenous glucosamine on the biology of articular chondrocytes were determined by examining global transcription patterns under normal culture conditions and following challenge with IL-1beta. Chondrocytes isolated from the cartilage of rats were cultured in several flasks either alone or in the presence of 20 mM glucosamine. Six hours later, one-half of the cultures of each group were challenged with 10 ng/ml IL-1beta. Fourteen hours after this challenge, RNA was extracted from each culture individually and used to probe microarray chips corresponding to the entire rat genome. Glucosamine alone had no observable stimulatory effect on the transcription of primary cartilage matrix genes, such as aggrecan, collagen type II, or genes involved in glycosaminoglycan synthesis; however, glucosamine proved to be a potent, broad-spectrum inhibitor of IL-1beta. Of the 2,813 genes whose transcription was altered by IL-1beta stimulation (P < 0.0001), glucosamine significantly blocked the response in 2,055 (approximately 73%). Glucosamine fully protected the chondrocytes from IL-1-induced expression of inflammatory cytokines, chemokines, and growth factors as well as proteins involved in prostaglandin E2 and nitric oxide synthesis. It also blocked the IL-1-induced expression of matrix-specific proteases such as MMP-3, MMP-9, MMP-10, MMP-12, and ADAMTS-1. The concentrations of IL-1 and glucosamine used in these assays were supraphysiological and were not representative of the arthritic joint following oral consumption of glucosamine. They suggest, however, that the potential benefit of glucosamine in osteoarthritis is not related to cartilage matrix biosynthesis, but is more probably related to its ability to globally inhibit the deleterious effects of IL-1beta signaling. These results suggest that glucosamine, if administered effectively, may indeed have anti-arthritic properties, but primarily as an anti-inflammatory agent.

Animals↗
PubMed2005

The 3rd International Meeting on Gene Therapy in Rheumatology and Orthopaedics.

The 3rd International Meeting on Gene Therapy in Rheumatology and Orthopaedics was held in Boston, Massachusetts, USA in May 2004. Keystone lectures delivered by Drs Joseph Glorioso and Inder Verma provided comprehensive, up-to-date information on all major virus vectors. Other invited speakers covered the application of gene therapy to treatment of arthritis, including the latest clinical trial in rheumatoid arthritis, as well as lupus and Sjögren's syndrome. Applications in mesenchymal stem cell biology, tissue repair, and regenerative medicine were also addressed. The field has advanced considerably since the previous meeting in this series, and further clinical trials seem likely.

Genetic Therapy↗
PubMed2005

Gene transfer to human joints: progress toward a gene therapy of arthritis.

This article describes the clinical application of gene therapy to a nonlethal disease, rheumatoid arthritis (RA). Intraarticular transfer of IL-1 receptor antagonist (IL-1Ra) cDNA reduces disease in animal models of RA. Whether this procedure is safe and feasible in humans was addressed in a phase I clinical study involving nine postmenopausal women with advanced RA who required unilateral sialastic implant arthroplasty of the 2nd-5th metacarpophalangeal (MCP) joints. Cultures of autologous synovial fibroblasts were established and divided into two. One was transduced with a retrovirus carrying IL-1Ra cDNA; the other provided untransduced, control cells. In a dose escalation, double-blinded fashion, two MCP joints were injected with transduced cells, and two MCP joints received control cells. One week later, injected joints were resected and examined for evidence of successful gene transfer and expression by using RT-PCR, ex vivo production of IL-1Ra, in situ hybridization, and immunohistochemistry. All subjects tolerated the protocol well, without adverse events. Unlike control joints, those receiving transduced cells gave positive RT-PCR signals. Synovia that were recovered from the MCP joints of intermediate and high dose subjects produced elevated amounts of IL-1Ra (P = 0.01). Clusters of cells expressing high levels of IL-1Ra were present on synovia of transduced joints. No adverse events occurred. Thus, it is possible to transfer a potentially therapeutic gene safely to human rheumatoid joints and to obtain intraarticular, transgene expression. This conclusion justifies additional efficacy studies and encourages further development of genetic approaches to the treatment of arthritis and related disorders.

Aged↗
PubMed2005

Gene-induced chondrogenesis of primary mesenchymal stem cells in vitro.

Adult mesenchymal stem cells (MSCs) have the capacity to differentiate into various connective tissues such as cartilage and bone following stimulation with certain growth factors. However, less is known about the capacity of these cells to undergo chondrogenesis when these proteins are delivered via gene transfer. In this study, we investigated chondrogenesis of primary, bone marrow-derived MSCs in aggregate cultures following genetic modification with adenoviral vectors encoding chondrogenic growth factors. We found that adenoviral-mediated expression of TGF-beta1 and BMP-2, but not IGF-1, induced chondrogenesis of MSCs as evidenced by toluidine blue metachromasia and immunohistochemical detection of type II collagen. Chondrogenesis correlated with the level and duration of expressed protein and was strongest in aggregates expressing 10-100 ng/ml transgene product. Transgene expression in all aggregates was highly transient, showing a marked decrease after 7 days. Chondrogenesis was inhibited in aggregates modified to express >100 ng/ml TGF-beta1 or BMP-2; however, this was found to be partly due to the inhibitory effect of exposure to high adenoviral loads. Our findings indicate that parameters such as these are important functional considerations for adapting gene transfer technologies to induce chondrogenesis of MSCs.

Adenoviridae↗
PubMed2005

Stem-like cells in bone sarcomas: implications for tumorigenesis.

Bone sarcomas are a clinically and molecularly heterogeneous group of malignancies characterized by varying degrees of mesenchymal differentiation. Despite advances in medical and surgical management, survival rates for high-grade tumors have remained static at 50% to 70%. Tumor stem cells have been recently implicated in the pathogenesis of other heterogeneous, highly malignant tumors. We demonstrate here the existence of a small subpopulation of self-renewing bone sarcoma cells that are capable of forming suspended spherical, clonal colonies, also called "sarcospheres," in anchorage-independent, serum-starved conditions. These bone sarcoma cells as well as tissue specimens express activated STAT3 and the marker genes of pluripotent embryonic stem (ES) cells, Oct 3/4 and Nanog. Expression levels of Oct 3/4 and Nanog are greater in sarcospheres than in adherent cultures. A subset of bone sarcoma cells displays several surface markers of mesenchymal stem cells (Stro-1, CD105, and CD44) as well as attributes of mesodermal, ectodermal, and endodermal differentiation. Although previously documented in brain and breast tumors, our results support the extension of the cancer stem cell hypothesis to include tumors of mesenchymal lineage. Furthermore, they suggest the participation of ES cell homeobox proteins in non-germ cell tumorigenesis.

Bone Neoplasms↗
PubMed2004

Evaluation of Ad-BMP-2 for enhancing fracture healing in an infected defect fracture rabbit model.

The objective of this study was to evaluate the use of adenoviral transfer of the BMP-2 gene (Ad-BMP-2) for enhancing healing in an infected defect fracture model. A femoral defect stabilized with plates and screws was surgically created in sixty-four skeletally mature New Zealand white rabbits. Experimental groups were: (1) non-infected Ad-luciferase (Ad-LUC, NONLUC), (2) non-infected Ad-BMP-2 (NONBMP), (3) infected Ad-LUC (INFLUC), and (4) infected Ad-BMP-2 (INFBMP). A sclerosing agent was applied to the ends of the bone at surgery to facilitate the development of osteomyelitis. Fracture healing was evaluated radiographically and histologically. Data were analyzed using an ANOVA, with statistical significance set as p<0.05. Rabbits in the non-infected and infected groups that were treated with Ad-BMP-2 had earlier initial- and bridging-callus formation, and a higher overall external callus grade compared to rabbits in the Ad-LUC groups. Rabbits in the Ad-LUC groups had more defect ossification compared to rabbits in the Ad-BMP-2 groups. There was a trend for rabbits in the Ad-BMP-2 group that were euthanized at 2 and 4 weeks after surgery to have more bone and cartilage compared to rabbits in the Ad-LUC group. The results of this study suggest that Ad-BMP-2 enhances the early stages of healing in an infected defect fracture. The results of our study were not as favorable as those reported in previous studies because animals healed by a large bridging callus and not by defect ossification. This could have been a result of the sclerosing agent, which may have damaged the cells in the defect.

Adenoviridae↗
PubMed2004

The 2003 Nicolas Andry Award. Orthopaedic gene therapy.

We review progress in the field of orthopaedic gene therapy since the concept of using gene transfer to address orthopaedic problems was initiated approximately 15 years ago. The original target, arthritis, has been the subject of two successful Phase I clinical trials, and additional human studies are pending in rheumatoid arthritis and osteoarthritis. The repair of damaged musculoskeletal tissues also has proved to be a fruitful area of research, and impressive enhancement of bone healing has been achieved in preclinical models. Rapid progress also is being made in the use of gene transfer to improve cartilage repair, ligament healing, and restoration of various additional tissues, including tendon and meniscus. Other applications include intervertebral disc degeneration, aseptic loosening, osteoporosis, genetic diseases, and orthopaedic tumors. Of these various orthopaedic targets of gene therapy, tissue repair is likely to make the earliest clinical impact because it can be achieved with existing technology. Tissue repair may become one of the earliest clinical successes for gene therapy as a whole. Orthopaedics promises to be a leading discipline for the use of human gene therapy.

Animals↗
PubMed2004

In vitro gene transfer to chondrocytes and synovial fibroblasts by adenoviral vectors.

The major requirement of a successful gene transfer is the efficient delivery of an exogenous therapeutic gene to the appropriate cell type with subsequent high or regulated levels of expression. In this context, viral systems are more efficient than nonviral systems, giving higher levels of gene expression for longer periods. For the application of osteoarthritis (OA), gene products triggering anti-inflammatory or chondroprotective effects are of obvious therapeutic utility. Thus, their cognate genes are candidates for use in the gene therapy of OA. In this chapter, we describe the preparation, the use, and the effect of the transduction of chondrocytes or synovial fibroblasts with an adenoviral vector encoding the cDNA for glutamine: fructose-6-phosphate amidotransferase (GFAT). This is intended to serve as an example of a technology that can be used to evaluate the biological effects of overexpression of other cDNAs.

Adenoviridae↗
PubMed2003

A comparative study of the inhibitory effects of interleukin-1 receptor antagonist following administration as a recombinant protein or by gene transfer.

Anakinra, the recombinant form of IL-1 receptor antagonist (IL-1Ra), has been approved for clinical use in the treatment of rheumatoid arthritis as the drug Kineret trade mark, but it must be administered daily by subcutaneous injection. Gene transfer may offer a more effective means of delivery. In this study, using prostaglandin E2 production as a measure of stimulation, we quantitatively compared the ability of anakinra, as well as that of IL-1Ra delivered by gene transfer, to inhibit the biologic actions of IL-1beta. Human synovial fibroblast cultures were incubated with a range of doses of anakinra or HIG-82 cells genetically modified to constitutively express IL-1Ra. The cultures were then challenged with recombinant human IL-1beta either simultaneously with addition of the source of IL-1Ra or 24 hours later. In a similar manner, the potencies of the two sources of IL-1Ra were compared when human synovial fibroblasts were challenged with IL-1beta produced constitutively by genetically modified cells. No significant difference in inhibitory activity was observed between recombinant protein and IL-1Ra provided by the genetically modified cells, under static culture conditions, even following incubation for 4 days. However, under culture conditions that provided progressive dilution of the culture media, striking differences between these methods of protein delivery became readily apparent. Constitutive synthesis of IL-1Ra by the genetically modified cells provided sustained or increased protection from IL-1 stimulation over time, whereas the recombinant protein became progressively less effective. This was particularly evident under conditions of continuous IL-1beta synthesis.

Animals↗
PubMed2003

Adverse effects of adenovirus-mediated gene transfer of human transforming growth factor beta 1 into rabbit knees.

To examine the effect of transforming growth factor (TGF)-beta1 on the regulation of cartilage synthesis and other articular pathologies, we used adenovirus-mediated intra-articular gene transfer of TGF-beta1 to both naïve and arthritic rabbit knee joints. Increasing doses of adenoviral vector expressing TGF-beta1 were injected into normal and antigen-induced arthritis rabbit knee joints through the patellar tendon, with the same doses of an adenoviral vector expressing luciferase injected into the contralateral knees as the control. Intra-articular injection of adenoviral vector expressing TGF-beta1 into the rabbit knee resulted in dose-dependent TGF-beta1 expression in the synovial fluid. Intra-articular TGF-beta1 expression in both naïve and arthritic rabbit knee joints resulted in significant pathological changes in the rabbit knee as well as in adjacent muscle tissue. The observed changes induced by elevated TGF-beta1 included inhibition of white blood cell infiltration, stimulation of glycosaminoglycan release and nitric oxide production, and induction of fibrogenesis and muscle edema. In addition, induction of chondrogenesis within the synovial lining was observed. These results suggest that even though TGF-beta1 may have anti-inflammatory properties, it is unable to stimulate repair of damaged cartilage, even stimulating cartilage degradation. Gene transfer of TGF-beta1 to the synovium is thus not suitable for treating intra-articular pathologies.

Adenoviridae↗
PubMed2003

Lentiviral-mediated gene delivery to synovium: potent intra-articular expression with amplification by inflammation.

Clinical translation of gene-based therapies for arthritis could be accelerated by vectors capable of efficient intra-articular gene delivery and long-term transgene expression. Previously, we have shown that lentiviral vectors transduce rat synovium efficiently in vivo. Here, we evaluated the functional capacity of transgene expression provided by lentiviral-mediated gene delivery to the joint. To do this, we measured the ability of a lentiviral vector containing the cDNA for human interleukin-1 receptor antagonist (LV-hIL-1Ra) to suppress intra-articular responses to IL-1beta. Groups of rats were injected in one knee with 5 x 10(7) infectious units of LV-IL-1Ra. After 24 h, a range of doses of fibroblasts (3 x 10(3), 10(4), 3 x 10(4), or 10(5) cells) genetically modified to overexpress IL-1beta was injected into both knees. Intra-articular delivery of LV-hIL-1Ra strongly prevented swelling in all treated knees, even in those receiving the greatest dose of IL-1beta(+) cells. Cellular infiltration, cartilage erosion, and invasiveness of inflamed synovium were effectively prevented in LV-hIL-1Ra-treated knees and were significantly inhibited in contralateral joints. Beneficial effects were also observed systemically in the lentivirus-treated animals. Interestingly, intra-articular expression of the IL-1Ra transgene was found to increase in relation to the number of IL-1beta(+) cells injected. Further experiments using GFP suggest this is due to the proliferation of cells, stably modified by the integrative lentivirus, in response to inflammatory stimulation.

Animals↗
PubMed2002

In vivo gene delivery to synovium by lentiviral vectors.

The delivery of anti-arthritic genes to the synovial lining of joints is being explored as a strategy for the treatment of rheumatoid arthritis. In this study, we have investigated the use of VSV-G pseudotyped, HIV-1-based lentiviral vectors for gene delivery to articular tissues. Recombinant lentivirus containing a beta-galactosidase/neomycin resistance fusion gene under control of the elongation factor (EF) 1alpha promoter efficiently transduced human and rat synoviocytes and chondrocytes in cell culture. When directly injected into the knees of rats, this vector transduced synovial lining cells, but not other articular tissues such as cartilage. We also constructed a lentiviral vector containing the human interleukin-1 receptor antagonist (IL1RA) cDNA and examined transgene expression in vitro and in vivo following injection into the knee joints of rats. In immunocompetent animals, intra-articular IL1RA expression was high and persisted, at a sharply declining rate, for approximately 20 days. In immunocompromised rats, however, lentivirus-mediated intra-articular expression of human IL1RA was found to persist for at least 6 weeks. Extra-articular expression of the transgene was minimal. These results indicate that lentiviral vectors are capable of efficient in vivo gene transfer to synovium and merit further investigation as a means of providing long-term expression for gene-based treatments of arthritis.

Animals↗