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Biomedical subjects

P D Robbins

Publications and source records attributed to P D Robbins.

At least 91 records · Page 5Linked to original sources
PubMed2000

Insulinlike growth factor-I gene therapy applications for cartilage repair.

Cartilage function after resurfacing with cell-based transplantation procedures or during the early stages of arthritic disease may be bolstered by the addition of growth factor genes to the transplanted tissue. Insulinlike growth factor-I maintains chondrocyte metabolism in normal cartilage homeostasis and has been shown to improve cartilage healing in vivo. Given the relatively short half-life of insulinlike growth factor-I in biologic systems, however, maintenance of effective concentrations of this peptide has necessitated either very high initial doses or repeated treatment. Delivery of the insulinlike growth factor-I gene, using a deleted adenovirus vector, specifically targeting graftable articular chondrocytes, bone marrow-derived chondroprogenitor cells, or synovial lining cells, may provide more durable insulinlike growth factor-I fluxes to articular tissues. Cultured equine articular chondrocytes, mesenchymal stem cells, synovial explants, and synovial intimal cells were readily transfected with an E1-deleted adenoviral vector containing equine insulinlike growth factor-I coding sequence. Optimal viral concentrations for effective transduction were 100 multiplicities of infection in synoviocytes, 500 multiplicities of infection in chondrocytes, and 1000 multiplicities of infection in mesenchymal stem cells. Production of insulinlike growth factor-I ligand varied from 65 ng/mL to 246 ng/mL in medium from chondrocytes and synovial explants, respectively. For chondrocytes, these concentrations were sufficient to produce significant stimulation of cartilage matrix gene expression and subsequent proteoglycan production. Moreover, cells in infected cultures maintained a chondrocytic phenotype and continued to express elevated insulinlike growth factor-I levels during 28 days of monolayer culture. Minimal synthetic activity, other than insulinlike growth factor-I ligand synthesis, was evident in synovial cultures. These experiments suggest several avenues for insulinlike growth factor-I supplementation of articular cartilage, including preimplantation adenoviral-insulinlike growth factor gene transfer to chondrocytes or chondroprogenitor cells, and direct injection of adenoviral-insulinlike growth factor to transfect the synovial structures in situ.

Adenoviridae↗
PubMed2000

Using gene therapy to protect and restore cartilage.

Numerous gene products have the potential to help protect cartilage from degradation and to repair cartilage that has become damaged as a result of disease or injury. The genes that encode these products thus may serve as chondroprotective and chondroregenerative medicines. To bring these agents into clinical use, it is necessary to screen candidate genes for efficacy under in vitro and in vivo conditions, to determine the best cells to target, and to develop appropriate gene transfer technologies. As discussed in the current review, progress has been made in each of these areas. Various viral and nonviral vectors are able to deliver genes to synoviocytes, articular chondrocytes, and mesenchymal stem cells. There also is evidence to suggest that ex vivo and in vivo approaches can be used for gene transfer to articular cartilage, synovium, and meniscus. Moreover various cytokine antagonists and growth factors have been shown to protect cartilage and stimulate chondrogenesis. In vivo methods and strategies that target synovium may be useful in a chondroprotective mode but because they do not increase the number of chondrogenic cells within lesions, they may be ill-equipped to repair large defects. Ex vivo methods however, provide cells and genes. It also is important to distinguish the treatment of isolated lesions occurring as a result of injury from the treatment of lesions resulting from underlying disease processes. Additional development of these approaches should result in clinically useful genetic methods for the protection and regeneration of cartilagenous tissues.

Animals↗
PubMed2000

Potential applications of gene therapy to the treatment of intervertebral disc disorders.

Gene therapy involves the transfer of genes to cells such that the recipient cells express these genes and thereby synthesize the ribonucleic acid and protein that they encode. Recent investigations suggest that gene therapy may have potential applications in the treatment of intervertebral disc disorders, particularly those associated with disc degeneration. The successful in vivo transfer of therapeutic genes to target cells within the intervertebral disc in clinically relevant animal models is one example of the rapid progress that is being made. The purpose of the current review is to address several important technical issues, including choice of vectors and gene delivery strategy and the characteristics of the target tissues, which are relevant to future clinical applications of gene therapy for the treatment of intervertebral disc disorders. It already is apparent from the growing literature that gene therapy has the potential of becoming a valuable clinical treatment mode for intervertebral disc disorders in the twenty-first century.

Adenoviridae↗
PubMed2000

Gene therapy approaches for treating rheumatoid arthritis.

Current gene therapy approaches for treating rheumatoid arthritis have made use of gene transfer technology as an improved delivery system for emerging proteins and other biologicals whose activities may have therapeutic value. Preclinical research has focused on two primary directions, evaluation of methods of gene delivery and identification of gene products with antiarthritic potential. Although there are reports involving systemic gene delivery, the bulk of effort has focused on local, intraarticular administration using ex vivo and in vivo methods. Viral-based vectors, including adenovirus, adeno-associated virus and herpes simplex virus have the greatest efficiency of gene delivery after intraarticular injection and are capable of generating relevant levels of gene products in several animal models of disease. However, there are limitations to existing generations of these systems that currently preclude their clinical application. Those gene products found to be efficacious in animal models of rheumatoid arthritis include proteins that specifically block the activity of the primary inflammatory cytokines, and include interleukin-1 receptor antagonist and soluble receptors for tumor necrosis factor and interleukin-1. Delivery and expression of genes encoding certain cytokines such as interleukins -4, -10, and -13 and viral interleukin-10, that block synthesis of inflammatory mediators and downregulate aspects of cellular and humoral immune pathways have been found beneficial. Although significant progress has been made, leading to Phase I clinical trials, there remain several hurdles to the routine practice of gene therapy for treatment of rheumatoid arthritis.

Animals↗
PubMed2000

Clinical trials in the gene therapy of arthritis.

Gene therapy clinical trials raise important safety issues that complicate their design and require extensive preclinical testing. Human protocols for the treatment of arthritis and most other orthopaedic and rheumatologic indications are complicated additionally by the perception that they are largely acquired, nonlethal conditions. Taking these considerations into account, the first such human study used the local, ex vivo delivery of a gene whose product, the interleukin-1 receptor antagonist, has an outstanding safety profile. This gene was delivered to the metacarpophalangeal joints of postmenopausal women 1 week before these joints were removed during total joint replacement surgery. In addition to providing an additional safety cushion, the surgical removal of the genetically modified joints made available large amounts of tissue to examine for evidence of successful gene transfer and gene expression. This Phase I safety study was approved at the local and federal levels, and its funding was contingent on the establishment of an external monitoring board. This trial now has been completed and a Phase II, efficacy study is being planned. A similar study has begun in Dusseldorf, Germany and results from the first two patients are similar to the results of the American patients. Permission has been given for two additional human trials, one in the United States and one in the Netherlands, in which a gene encoding herpes thymidine kinase will be transferred to the joints of patients with rheumatoid arthritis who then will be administered gancyclovir. This procedure aims to treat the disease by producing a genetic synovectomy. Additional development of human gene therapies for arthritis and other orthopaedic and rheumatic conditions will be aided by the successful completion of these studies.

Animals↗
PubMed2000

Recombinant adenovirus induces maturation of dendritic cells via an NF-kappaB-dependent pathway.

Recombinant adenovirus (rAd) infection is one of the most effective and frequently employed methods to transduce dendritic cells (DC). Contradictory results have been reported recently concerning the influence of rAd on the differentiation and activation of DC. In this report, we show that, as a result of rAd infection, mouse bone marrow-derived immature DC upregulate expression of major histocompatibility complex class I and II antigens, costimulatory molecules (CD40, CD80, and CD86), and the adhesion molecule CD54 (ICAM-1). rAd-transduced DC exhibited increased allostimulatory capacity and levels of interleukin-6 (IL-6), IL-12p40, IL-15, gamma interferon, and tumor necrosis factor alpha mRNAs, without effects on other immunoregulatory cytokine transcripts such as IL-10 or IL-12p35. These effects were not related to specific transgenic sequences or to rAd genome transcription. The rAd effect correlated with a rapid increase (1 h) in the NF-kappaB-DNA binding activity detected by electrophoretic mobility shift assays. rAd-induced DC maturation was blocked by the proteasome inhibitor Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) or by infection with rAd-IkappaB, an rAd-encoding the dominant-negative form of IkappaB. In vivo studies showed that after intravenous administration, rAds were rapidly entrapped in the spleen by marginal zone DC that mobilized to T-cell areas, a phenomenon suggesting that rAd also induced DC differentiation in vivo. These findings may explain the immunogenicity of rAd and the difficulties in inducing long-term antigen-specific T-cell hyporesponsiveness with rAd-transduced DC.

Adenoviridae↗
PubMed2000

Forskolin-induced apical membrane insertion of virally expressed, epitope-tagged CFTR in polarized MDCK cells.

Channel gating of the cystic fibrosis transmembrane conductance regulator (CFTR) is activated in response to cAMP stimulation. In addition, CFTR activation may also involve rapid insertion of a subapical pool of CFTR into the plasma membrane (PM). However, this issue has been controversial, in part because of the difficulty in distinguishing cell surface vs. intracellular CFTR. Recently, a fully functional, epitope-tagged form of CFTR (M2-901/CFTR) that can be detected immunologically in nonpermeabilized cells was characterized (Howard M, Duvall MD, Devor DC, Dong J-Y, Henze K, and Frizzell RA. Am J Physiol Cell Physiol 269: C1565-C1576, 1995; and Schultz BD, Takahashi A, Liu C, Frizzell RA, and Howard M. Am J Physiol Cell Physiol 273: C2080-C2089, 1997). We have developed replication-defective recombinant adenoviruses that express M2-901/CFTR and used them to probe cell surface CFTR in forskolin (FSK)-stimulated polarized Madin-Darby canine kidney (MDCK) cells. Virally expressed M2-901/CFTR was functional and was readily detected on the apical surface of FSK-stimulated polarized MDCK cells. Interestingly, at low multiplicity of infection, we observed FSK-stimulated insertion of M2901/CFTR into the apical PM, whereas at higher M2-901/CFTR expression levels, no increase in surface expression was detected using indirect immunofluorescence. Immunoelectron microscopy of unstimulated and FSK-stimulated cells confirmed the M2-901/CFTR redistribution to the PM upon FSK stimulation and demonstrates that the apically inserted M2-901/CFTR originates from a population of subapical vesicles. Our observations may reconcile previous conflicting reports regarding the effect of cAMP stimulation on CFTR trafficking.

Adenoviridae↗
PubMed2000

Genetic approaches for biologic therapy of cancer.

Recently, there has been an increase in the types of biological therapeutic approaches developed for the treatment of cancer. This rapid advance in the biological therapy of cancer is due in part to advances in the field of molecular and cell biology as well as in the development of gene transfer systems. In particular, a better understanding of the mechanism of antigen presentation and T lymphocyte activation has resulted in the development of new immunotherapeutic strategies targeting tumor-associated antigens (TAA). The discovery of dendritic cells (DC) as potent antigen presenting cells and the development of methods for their use in immunotherapeutic regimens has led to novel approaches for treating cancer. Furthermore, the identification of genes encoding TAA and their peptide products, which are recognized by T lymphocytes in the context of major histocompatibility complexes class I and class II molecules, has led to the development of DNA-based vaccines against defined tumor antigens. Cytokines have been shown to be important adjuvant tools for immunization protocols by directing a T helper response favorable for an adequate cytotoxic T lymphocyte-mediated immune response. Novel gene transfer technologies have made it possible to employ a wide range of gene delivery systems, either viral or nonviral based, in anticancer therapies. Current immunotherapeutic strategies, including the use of DC transduced with genes coding for tumor antigens and cytokines delivered by recombinant viral vectors, have shown promise in animal tumor models.

Journal Article↗
PubMed2000

Gene therapy for juvenile rheumatoid arthritis?

Gene therapy shows promise as a means of improving the treatment of a number of rheumatic diseases, including adult rheumatoid arthritis (RA). We summarize these developments and discuss the merits of extending such approaches to the treatment of juvenile RA. The special issues that arise when treating children by experimental new procedures predicate a cautious approach using strategies first shown to be safe in adults. Patients with severe, erosive, nonremitting pauciarticular juvenile RA might be suitable first candidates for gene therapy.

Arthritis, Juvenile↗
PubMed2000

Cartilage injury and repair.

Articular cartilage is a complex structure that, once damaged, has little capacity for permanent repair. The problem lies in the inability of the body to regenerate tissue with the appropriate macromolecular constituents and architecture of normal hyaline cartilage. Although full-thickness defects are capable of stimulating a repair response, the resulting fibrocartilage is inferior and cannot withstand long-term, repetitive use. Numerous surgical approaches that involve penetration of subchondral bone offer short-term to moderate-term relief of symptoms, whereas other approaches have seen significant improvement through transplantation of osteochondral and periosteal tissue and implantation of autologous chondrocytes. Despite these procedures, articular cartilage damage continues to be an unmet clinical problem. Improvements in biochemical and molecular biologic techniques may allow advances in the understanding of chondrocyte and cartilage biology and may provide innovative and novel approaches to stimulating the repair of articular cartilage through biologic means.

Animals↗
PubMed2000

Potential applications of gene therapy in sports medicine.

Gene therapy has the potential to improve greatly the management of sports medicine injuries. By the expeditious introduction of the appropriate genes to sites of injury, it should prove possible to initiate healing responses in those tissues that normally fail to heal, to accelerate healing in tissues which do naturally heal, and to improve the quality of the repair tissue. In addition, genes with anti-inflammatory properties offer novel possibilities for the control of chronic, sports related inflammation. None of these applications of gene therapy in sports medicine is yet in the clinic. It would seem, however, only a matter of time before the first clinical trials emerge. Their acceptance will be facilitated by the two human protocols that are presently underway for the treatment of rheumatoid arthritis. The present status of sports medicine gene therapy is summarized in Table 3.

Athletic Injuries↗
PubMed2000

The Marshall R. Urist Young Investigator Award. Orthopaedic applications of gene therapy. From concept to clinic.

Gene therapy offers new possibilities for the clinical management of orthopaedic conditions that are difficult to treat by traditional surgical or medical means. To bring the potential of this novel technology into the clinic, a research program was initiated that aimed to identify orthopaedically useful genes and develop methods for delivering them to suitable sites under conditions in which gene expression remains at therapeutic levels for the appropriate periods of time; this program is now 10 years old. Rheumatoid arthritis was selected as the lead disease. Preclinical studies evaluating the local and systemic delivery of numerous different genes by in vivo and ex vivo methods in murine and lapin models led to the development of a human gene therapy protocol for arthritis. In this protocol, a gene encoding the human interleukin-1 receptor antagonist protein is transferred to the metacarpophalangeal joints of female patients with rheumatoid arthritis. The first patient was treated this way in July 1996. This is not only the first orthopaedic application of human gene therapy, but also the first use of gene therapy approved for a nonlethal disease. In addition to providing additional therapeutic options for the treatment of rheumatoid arthritis, the experimental data from this study suggest that gene transfer approaches may improve the treatment of osteoarthritis, the repair of cartilage, ligaments, tendons, menisci, intervertebral discs and bone, and the management of disorders such as osteoporosis and osteogenesis imperfecta. They also show promise as a means for developing novel and improved animal models of orthopaedic diseases. If the current rate of progress continues, wide clinical application of gene therapy in various orthopaedic indications should occur within the next 5 to 10 years.

Animals↗
PubMed1999

Modulation of the biologic activity of the rabbit intervertebral disc by gene therapy: an in vivo study of adenovirus-mediated transfer of the human transforming growth factor beta 1 encoding gene.

STUDY DESIGN: In vivo studies using a rabbit model to determine the biologic effects of direct, adenovirus-mediated transfer of a therapeutic gene to the intervertebral disc. OBJECTIVES: 1) To deliver an exogenous therapeutic gene to rabbit lumbar intervertebral discs in vivo, 2) to quantify the resulting amount of gene expression, and 3) to determine the effect on the biologic activity of the discs. SUMMARY OF BACKGROUND DATA: Although growth factors such as transforming growth factor beta 1 appear to have promising therapeutic properties, there currently is no practical method for sustained delivery of exogenous growth factors to the disc for the management of certain chronic types of disease (e.g., disc degeneration). A possible solution is to modify the disc cells genetically through gene transfer such that the cells manufacture the desired growth factors endogenously on a continuous basis. METHODS: Saline, with or without virus, was injected directly into lumbar discs of 22 skeletally mature female New Zealand white rabbits. Group 1 (n = 11) received the adenovirus construct Ad/CMV-hTGF beta 1 containing the therapeutic human transforming growth factor beta 1-encoding gene. Group 2 (n = 6) received adenovirus containing the luciferase marker gene. Group 3 (n = 5) received saline only. The rabbits were killed 1 week after injection. Immunohistochemical staining for human transforming growth factor beta 1 was performed on the disc tissues of one rabbit from Group 1. Nucleus pulposus tissues from the remaining rabbits were cultured in serumless medium. Bioassays were performed to determine human transforming growth factor beta 1 production and proteoglycan synthesis. RESULTS: Discs injected with Ad/CMV-hTGF beta 1 exhibited extensive and intense positive immunostaining for transforming growth factor beta 1. The nucleus pulposus tissues from the discs injected with Ad/CMV-hTGF beta 1 exhibited a 30-fold increase in active transforming growth factor beta 1 production, and a 5-fold increase in total (active + latent) transforming growth factor beta 1 production over that from intact control discs (P < 0.05). Furthermore, these tissues exhibited a 100% increase in proteoglycan synthesis compared with intact control tissue, which was statistically significant (P < 0.05). CONCLUSIONS: The results of this study suggest that the intervertebral disc is an appropriate site for adenovirus-mediated transfer of exogenous genes and subsequent production of therapeutic growth factors. Gene therapy therefore may have useful applications for study of the basic science of the intervertebral disc and for clinical management of degenerative disc disease.

Adenoviridae↗
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

Induction of systemic and therapeutic antitumor immunity using intratumoral injection of dendritic cells genetically modified to express interleukin 12.

Bone marrow-derived dendritic cells (BM-DCs) retrovirally transduced with genes encoding murine interleukin (IL)-12 stably expressed bioactive IL-12 protein at high levels. Intratumoral injection with IL-12 gene-modified BM-DCs resulted in regression of day 7 established weakly immunogenic tumors (MCA205, B16, and D122). This antitumor effect was substantially better than that of IL-12-transduced syngeneic fibroblasts or nontransduced BM-DCs. Furthermore, intratumoral injection with IL-12-transduced dendritic cells (DCs) induced specific TH1-type responses to the tumor in regional lymph nodes and spleen at levels greater than those of IL-12-transduced fibroblasts or nontransduced BM-DCs. Trafficking studies confirmed that intratumorally injected IL-12-transduced DCs, but not fibroblasts, could migrate to the draining lymph node to the same extent as nontransduced BM-DCs. This strategy designed to deliver genetically modified DCs to tumor sites is associated with systemic and therapeutic antitumor immunity and is an alternative approach to those that use delivery of DCs loaded with tumor antigen. These results support the clinical application of IL-12 gene-modified DCs in patients with cancer.

Animals↗
PubMed1999

Differential antitumor effects of administration of recombinant IL-18 or recombinant IL-12 are mediated primarily by Fas-Fas ligand- and perforin-induced tumor apoptosis, respectively.

Systemic administration of rIL-18 protein to mice significantly suppresses the growth of murine tumor cell lines. The antitumor effect of IL-18 appears to be primarily mediated by asialo GM1+ cells. Since IL-18 enhances Fas ligand (FasL) expression on NK cell lines, the IL-18 antitumor effects could be mediated by FasL-induced cross-linking of Fas and subsequent tumor apoptosis. To address this question, rIL-18 or rIL-12 was administered to animals bearing the CL8-1 melanoma inoculated intradermally into wild type (wt), lymphoproliferation gene (lpr) (Fas deficient), or generalized lymphoproliferative disease gene (gld) (FasL deficient) mice. Although rIL-12 treatment retained significant antitumor effects in gld and lpr mice, those of rIL-18 administration were completely abrogated in gld but not lpr or wt mice. In vitro cytotoxicity was significantly enhanced against NK-sensitive YAC-1 cells and CL8-1 cells by rIL-18 administration to wt mice, but not to gld mice. Furthermore, rIL-18 administration augmented the cytotoxicity of liver lymphocytes harvested from perforin-deficient mice, whereas rIL-12 administration did not. Consistent with the role of this pathway, rIL-18 administration also up-regulates the expression of FasL mRNA in splenocytes. Lysis of CL8-1 cells induced by anti-Fas agonistic Ab was enhanced about 1.4-fold by IFN-gamma, a cytokine that is induced by IL-18 in vitro and in vivo. We conclude that the antitumor effect of IL-18 is exerted predominantly through a Fas-dependent pathway. The perforin pathway, however, appears to be the predominant cytolytic pathway mediating IL-12 antitumor effects.

Adjuvants, Immunologic↗
PubMed1999

Lessons learned from gene transfer approaches.

Recent technological advances allow the transfer of genes to the synovial lining of joints. As well as opening novel opportunities for therapy, these techniques provide valuable new tools for the study of synovitis and other aspects of the biology of joints in health and disease. This article reviews briefly the results of experiments in which selected genes have been transferred to the knee joints of healthy rabbits and rabbits with antigen-induced arthritis.

Animals↗