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Biomedical subjects

P D Robbins

Publications and source records attributed to P D Robbins.

At least 109 records · Page 6Linked to original sources
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

Cyclin D1 associates with the TBP-associated factor TAF(II)250 to regulate Sp1-mediated transcription.

We have previously shown that Sp1-mediated transcription is stimulated by Rb and repressed by cyclin D1. The stimulation of Sp1 transcriptional activity by Rb is conferred, in part, through a direct interaction with the TBP-associated factor TAF(II)250. Here we investigated the mechanism(s) through which cyclin D1 represses Sp1. We examined the ability of cyclin D1 to regulate transcription mediated by Gal4-Sp1 fusion proteins, which contain the Gal4 DNA-binding domain and Sp1 trans-activation domain(s). The domain of Sp1 sufficient to confer repression by cyclin D1 was mapped to a region important for interaction with TAF(II)110. We further demonstrate that TAF(II)250-cyclin D1 complexes can be immunoprecipitated from mammalian and baculovirus-infected insect cells and that recombinant GST-TAF(II)250 (amino acids 1-434) associates with cyclin D1 in vitro. Moreover, the overexpression of Rb or CDK4 reduced the level of TAF(II)250-cyclin D1 complex. The amino terminus of cyclin D1 (amino acids 1-100) was sufficient for association with TAF(II)250 and for repressing Sp1-mediated transcription. Taken together, the results suggest that cyclin D1 may regulate transcription by interacting directly or indirectly with TAF(II)250.

3T3 Cells↗
PubMed1999

CD154 inhibits tumor-induced apoptosis in dendritic cells and tumor growth.

We have recently demonstrated that murine and human tumors induce apoptosis of dendritic cells (DC). Here, we evaluated the effect of CD40 ligation on the survival of tumor-associated DC and tumor growth. Retroviral transduction of MC38 colon carcinoma cells with the CD154 gene resulted in inhibition of tumor growth. This effect was abrogated in IL-12 knockout mice. Immunohistochemical analysis revealed an increase in CD11c+ (N418) and CD8+ but not NLDC-145+ cells in CD154-transfected tumors in wild-type mice. This increase was less pronounced in IL-12-deficient mice. In vitro, overexpression of CD154 on tumor cells significantly decreased the level of tumor-induced DC apoptosis. Surprisingly, the CD154-induced protection of DC from tumor-induced apoptosis was IL-12 independent in vitro, suggesting an IL-12-dependent and an IL-12-independent mechanism of CD154-induced anti-tumor immunity. Thus, our data suggest a new strategy to improve immunotherapy of cancer by protecting DC from tumor-induced apoptosis.

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

Genetic engineering of dendritic cells to express immunosuppressive molecules (viral IL-10, TGF-beta, and CTLA4Ig).

There is growing evidence that, in addition to their role as initiators of immune responses, dendritic cells (DC) can exhibit tolerogenic properties. Immature DC deficient in cell surface costimulatory/accessory molecules can prolong organ and pancreatic islet allograft survival, whereas in vitro manipulation of DC by exposure to a variety of factors (e.g., viral interleukin-10; CTLA4Ig) can confer tolerogenic properties on these cells. Genetic engineering of DC to express immunosuppressive molecules is, in theory, an attractive approach to the therapy of allograft rejection and possibly, autoimmune disorders.

Abatacept↗
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

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

Immunology of type 1 diabetes. Intervention and prevention strategies.

Type 1 diabetes is the outcome of a progressive and selective destruction of insulin-producing cells in the pancreatic islets of Langerhans. The precise cause and mechanism(s) that trigger the insulin-producing cell destruction are still unclear, although it is well accepted that an autoimmune process plays a central role in diabetes development among genetically susceptible children. Additionally, certain viral infections, especially those caused by Coxsackievirus B, have been associated with the onset of type 1 diabetes. Possible gene therapy-based prevention and intervention strategies are discussed, based on the most accepted models of type 1 diabetes pathogenesis.

Autoimmune Diseases↗
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

Co-delivery of T helper 1-biasing cytokine genes enhances the efficacy of gene gun immunization of mice: studies with the model tumor antigen beta-galactosidase and the BALB/c Meth A p53 tumor-specific antigen.

DNA-based immunization is currently being investigated as a new method for the induction of cellular and humoral immunity directed against viral disease and cancer. In the present study we characterized and compared the immune responses induced in mice following particle-bombardment of the skin ('gene gun' immunization) with those elicited by intracutaneous injection of a recombinant adenoviral vector. Using the well characterized beta-galactosidase (beta gal) model Ag system we find that both in vivo gene transfer systems elicit potent and long-lasting anti-beta gal-specific CD8+ and CD4+ T cell responses. However, gene gun immunization predominantly promotes the production of anti-beta gal antibodies of the gamma 1 isotype, indicative of a Th2-biased immune response, while intradermal injection of recombinant adenovirus primarily leads to the production of anti-beta gal gamma 2a antibodies, indicative of a Th1-biased immune response. Since viral infections are generally associated with the production of large amounts of IFN-alpha and IL-12, we investigated whether administration of expression plasmids encoding these Th1-associated cytokines along with antigen-encoding cDNA can influence the nature of the immune response resulting from gene gun immunization. We observed that co-delivery of IFN-alpha or IL-12 resulted in increased production of anti-beta gal gamma 2a antibodies. This suggests a shift towards a Th1 phenotype of the resulting immune response, thus mimicking a viral infection. Importantly, gene gun immunization of mice with a naturally occurring tumor antigen, the tumor-specific p53 mutant antigen expressed by the chemically induced BALB/c Meth A sarcoma, required co-delivery of IL-12 for the induction of effective antitumor immunity. These results have important implications for the design of clinically relevant gene gun immunization strategies for tumor immunotherapy.

Animals↗
PubMed1999

Adenoviral delivery of CTLA4Ig into myeloid dendritic cells promotes their in vitro tolerogenicity and survival in allogeneic recipients.

Dendritic cells (DC) are highly specialized antigen-presenting cells (APC) that initiate and modulate immune responses. They are essential for naive T cell activation, but may also play roles both in central and peripheral tolerance. Blockade of costimulatory pathways that provide the crucial second signal for lymphocyte activation is one strategy to augment the potential tolerogenicity of DC. Here, in vitro propagated DC were transduced using an adenoviral (Ad) vector to express the gene encoding cytotoxic T lymphocyte antigen 4-immunoglobulin (CTLA4lg), which blocks interaction of CD80 and CD86 on DC with CD28 on T cells. Supernatants of AdCTLA4lg-transduced DC strikingly inhibited mixed leukocyte reactions (MLR) induced by non-transduced DC. Whereas transduction of marker genes (LacZ or enhanced green fluorescence protein (EGFP)) did not alter their potent allostimulatory activity, DC transduced with CTLA4lg exhibited striking reductions in cell surface staining for CD86, but not MHC class II, and were poor stimulators of T cell proliferation and cytotoxic T lymphocyte (CTL) responses. In addition, they induced alloantigen-specific T cell hyporesponsiveness. They were detected, following local injection, in significantly increased numbers in the lymphoid tissue of unmodified allogeneic recipients. This is the first report of the functional properties of DC genetically engineered to express CTLA4lg.

Abatacept↗
PubMed1999

Potent antitumor effects mediated by local expression of the mature form of the interferon-gamma inducing factor, interleukin-18 (IL-18).

IL-18 is produced during the acute immune response by macrophages and immature dendritic cells. IL-18 receptors are induced on T cells and NK cells by IL-12 and together they enhance a cellular immune response. We constructed retroviral and adenoviral vectors encoding the mature bioactive murine IL-18 in order to examine their immune and antitumor effects in murine tumor models. Secretion of bioactive IL-18 from murine tumor cells was facilitated by transfecting them with recombinant viral vectors carrying the prepro leader sequence of human parathyroid hormone fused to the 5' end of the mature murine IL-18 cDNA. Direct injection of the IL-18 recombinant adenoviral vector (Ad.PTH.IL-18) into an established MCA205 murine fibrosarcoma completely eradicated tumor in all animals with concomitant induction of protective systemic immunity. Co-administration of systemic IL-12 provided synergistic antitumor effects when combined with peritumoral injections of Ad.PTH.IL-18 without apparent side-effects as we observed with systemic administration of IL-18. Depletion of asialo GM-1+ cells completely abrogated the antitumor effects of Ad.PTH.IL-18, suggesting a major role for NK cells in mediating the anti-tumor effects of IL-18. Peritumoral injection of Ad.PTH.IL-18 was also associated with reduced numbers of CD8+ cells found within the tumor (HBSS versus Ad.PTH.IL-18, P < 0.0001). This suggests that IL-18 could be utilized as an alternative cancer gene therapy especially when combined with systemic administration of IL-12.

Adenoviridae↗