PubMed Health⌕ Search

Biomedical subjects

P D Robbins

Publications and source records attributed to P D Robbins.

At least 127 records · Page 7Linked to original sources
PubMed1999

Nitric oxide in osteoarthritis.

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

Chondrocytes↗
PubMed1999

Genetically augmented tissue engineering of the musculoskeletal system.

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

Animals↗
PubMed1999

Dendritic cells transduced with an adenovirus vector encoding Epstein-Barr virus latent membrane protein 2B: a new modality for vaccination.

Epstein-Barr virus (EBV) is a herpesvirus commonly associated with several malignancies, particularly in immunocompromised hosts. As a strategy for stimulating immunity against EBV for the treatment of EBV-associated tumors, we have genetically engineered dendritic cells (DC) to express EBV antigens, such as latent membrane protein 2B (LMP2B), using recombinant adenovirus vectors. CD8(+) T lymphocytes from HLA-A2.1(+), EBV-seropositive healthy donors were cultured with autologous DC infected with recombinant adenovirus vector AdEGFP, encoding an enhanced green fluorescent protein (EGFP), or AdLMP2B at a multiplicity of infection of 250. After 48 h, >95% of the DC were positive for EGFP expression as assessed by fluorescence-activated cell sorting analysis, indicating efficient gene transfer. AdLMP2-transduced DC were used to stimulate CD8(+) T cells. Responder CD8(+) T cells were tested for gamma interferon (IFN-gamma) release by enzyme-linked spot (ELISPOT) assay and cytotoxic activity. Prior to in vitro stimulation, the frequencies of T-cells directed against two HLA-A2-presented LMP2 peptides (LMP2 329-337 and LMP2 426-434) were very low as assessed by IFN-gamma spot formation (T-cell frequency, <0.003%). IFN-gamma ELISPOT assays performed at day 14 showed a significant (2-log) increase of the day 0 frequency of T cells reactive against the LMP2 329-337 peptide, from 0.003 to 0.3 (P < 0.001). Moreover, specific cytolytic activity was observed against the autologous EBV B-lymphoblastoid cell lines after 21 days of stimulation of T-cell responders with AdLMP2-transduced DC (P < 0.01). In summary, autologous mature DC genetically modified with an adenovirus encoding EBV antigens stimulate the generation of EBV-specific CD8(+) effector T cells in vitro, supporting the potential application of EBV-based adenovirus vector vaccination for the immunotherapy of the EBV-associated malignancies.

Adenoviruses, Human↗
PubMed1999

Rb inhibits the intrinsic kinase activity of TATA-binding protein-associated factor TAFII250.

The retinoblastoma tumor suppressor protein, Rb, interacts directly with the largest TATA-binding protein-associated factor, TAFII250, through multiple regions in each protein. To define the potential role(s) of this interaction, we examined whether Rb could regulate the intrinsic, bipartite kinase activity of TAFII250. Here, we report that Rb is able to inhibit the kinase activity of immunopurified and gel-purified recombinant TAFII250. Rb inhibits the autophosphorylation of TAFII250 as well as its phosphorylation of the RAP74 subunit of TFIIF in a dose-responsive manner. Inhibition of TAFII250 kinase activity involves the Rb pocket (amino acids 379 to 928) but not its amino terminus. In addition, Rb appears to specifically inhibit the amino-terminal kinase domain of TAFII250 through a direct protein-protein interaction. We further demonstrate that two different tumor-derived Rb pocket mutants, C706F and Deltaex22, are functionally defective for kinase inhibition, even though they are able to bind the amino terminus of TAFII250. Our results suggest a novel mechanism of transcriptional regulation by Rb, involving direct interaction with TAFII250 and inhibition of its ability to phosphorylate itself, RAP74, and possibly other targets.

Animals↗
PubMed1999

Transfer of lacZ marker gene to the meniscus.

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

Animals↗
PubMed1999

Gene therapy of arthritis.

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

Animals↗
PubMed1999

Targeting autoimmune diabetes with gene therapy.

The autoimmune nature of insulin-dependent, or type 1, diabetes targets the beta-cells of the pancreas for destruction and results in a lifelong commitment to insulin replacement therapy. Although the number of formulations and dosing of insulin have become more sophisticated and more efficient in recent years, insulin therapy alone is unable to prevent nephropathy, retinopathy, or vascular and heart disease, which still occur in a large number of patients. Different approaches have been attempted to eliminate the requirement of exogenous insulin administration. Historically, these have included pancreatic and islet transplants, which were later combined with treatments intended to halt the destructive process directed against the islets. Despite significant advances made in all of these areas, each approach faces a hostile immunological response that frequently ends with the loss of the islets. Gene therapy-based approaches add a new dimension to the efforts aimed at specifically blocking the immunological attack against the islets in genetically at-risk individuals (autoimmunity) or the immunological response against transplanted allogeneic islets (rejection). This new technology may have an important role in the therapy and cure of type 1 diabetes.

Animals↗
PubMed1999

Adenoviral gene transfer of the interleukin-1 receptor antagonist protein to human islets prevents IL-1beta-induced beta-cell impairment and activation of islet cell apoptosis in vitro.

The beta-cells in the pancreatic islets of Langerhans are the targets of autoreactive T-cells and are destroyed in type 1 diabetes. Macrophage-derived interleukin-1beta (IL-1beta) is important in eliciting beta-cell dysfunction and initiating beta-cell damage in response to microenvironmental changes within islets. In particular, IL-1beta can impair glucose-stimulated insulin production in beta-cells in vitro and can sensitize them to Fas (CD95)/FasL-triggered apoptosis. In this report, we have examined the ability to block the detrimental effects of IL-1beta by genetically modifying islets by adenoviral gene transfer to express the IL-1 receptor antagonist protein. We demonstrate that adenoviral gene delivery of the cDNA encoding the interleukin-1 receptor antagonist protein (IL-1Ra) to cultured islets results in protection of human islets in vitro against IL-1beta-induced nitric oxide formation, impairment in glucose-stimulated insulin production, and Fas-triggered apoptosis activation. Our results further support the hypothesis that IL-1beta antagonism in in situ may prevent intra-islet proinsulitic inflammatory events and may allow for an in vivo gene therapy strategy to prevent insulitis and the consequent pathogenesis of diabetes.

Adenoviridae↗
PubMed1999

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

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

Adenoviruses, Human↗
PubMed1998

Phenotype, function, and in vivo migration and survival of allogeneic dendritic cell progenitors genetically engineered to express TGF-beta.

BACKGROUND: Administration of donor bone marrow (BM)-derived dendritic cell (DC) progenitors (DCp) that are major histocompatibility complex (MHC) class II+ but costimulatory molecule (CD40, CD80, CD86)-deficient can prolong mouse heart allograft survival This is associated with microchimerism and inhibition of antidonor cytotoxic T lymphocyte (CTL) activity. Genetic modification of these donor antigen-presenting cells to express an immunosuppressive molecule(s) may enhance their in vivo survival and potential tolerogenicity. METHODS: The surface phenotype of B10(H-2b) DCp before and after gene transfer using replication-deficient adenoviral (Ad) vectors was determined by monoclonal antibody (mAb) staining and flow cytometry. Transforming growth factor-beta (TGF-beta) production was quantitated by enzyme-linked immunosorbent assay. Allostimulatory activity of the gene-transduced DCp was ascertained by mixed leukocyte reaction (MLR) and CTL induction. To assess their in vivo migratory activity and survival, the transduced cells were injected subcutaneously into one hind footpad of C3H (H-2k) mice. Tissues (draining popliteal lymph nodes [LN], spleens, and thymi) were removed 1, 2, 7, and 14 days later and stained for donor MHC class II using anti-LA(b) mAb in an immunohistochemical procedure. The mean number of IAb+ cells per unit area was determined. RESULTS: Transduction with a control Ad vector (Ad-LacZ) at 50 multiplicity of infection slightly increased CD40 and CD86 expression and up-regulated the poor allostimulatory activity of the DCp assessed by MLR and CTL responses. These effects on function were negated in Ad-TGF-beta1-transduced cells. After their injection into mouse footpads, the gene-transduced IAb+ cells were observed in maximal numbers in the popliteal LN at day 1 and in marginal zones and T-dependent areas of spleens (peak at day 7) but were rare in thymi. Transduction with Ad-LacZ reduced the numbers of IAb+ cells identified in both LN and spleens at all time points postinjection, suggesting that the vector alone affected DC life span in allogeneic recipients. TGF-beta1 transgene expression not only fully prevented the reduction in DC induced by Ad transduction alone, but also increased numbers and prolonged the survival of donor cells in the spleen, as shown by a two-to fivefold increase in IAb+ cells at days 2-14 compared with control (Ad-LacZ-transduced) DC. CONCLUSION: BM-derived DCp can be transduced efficiently to express TGF-beta1 using an Ad vector. They exhibit very poor allostimulatory activity and similar migration characteristics in vivo to unmodified DCp. Survival of TGF-beta gene-transduced DC, however, is enhanced significantly compared with unmodified and (especially) control Ad-LacZ gene-transduced DC. Genetic engineering of donor DC to express the immunosuppressive molecule TGF-beta promotes their survival in allogeneic hosts and may potentiate their previously reported tolerogenicity.

Adenoviridae↗
PubMed1998

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

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

Adenoviridae↗
PubMed1998

Development of approaches to improve cell survival in myoblast transfer therapy.

Myoblast transplantation has been extensively studied as a gene complementation approach for genetic diseases such as Duchenne Muscular Dystrophy. This approach has been found capable of delivering dystrophin, the product missing in Duchenne Muscular Dystrophy muscle, and leading to an increase of strength in the dystrophic muscle. This approach, however, has been hindered by numerous limitations, including immunological problems, and low spread and poor survival of the injected myoblasts. We have investigated whether antiinflammatory treatment and use of different populations of skeletal muscle-derived cells may circumvent the poor survival of the injected myoblasts after implantation. We have observed that different populations of muscle-derived cells can be isolated from skeletal muscle based on their desmin immunoreactivity and differentiation capacity. Moreover, these cells acted differently when injected into muscle: 95% of the injected cells in some populations died within 48 h, while others richer in desmin-positive cells survived entirely. Since pure myoblasts obtained from isolated myofibers and myoblast cell lines also displayed a poor survival rate of the injected cells, we have concluded that the differential survival of the populations of muscle-derived cells is not only attributable to their content in desmin-positive cells. We have observed that the origin of the myogenic cells may influence their survival in the injected muscle. Finally, we have observed that myoblasts genetically engineered to express an inhibitor of the inflammatory cytokine, IL-1, can improve the survival rate of the injected myoblasts. Our results suggest that selection of specific muscle-derived cell populations or the control of inflammation can be used as an approach to improve cell survival after both myoblast transplantation and the myoblast-mediated ex vivo gene transfer approach.

Animals↗
PubMed1998

Direct adenovirus-mediated gene transfer of interleukin 1 and tumor necrosis factor alpha soluble receptors to rabbit knees with experimental arthritis has local and distal anti-arthritic effects.

Adenoviral vectors were used to deliver genes encoding a soluble interleukin 1 (IL-1)-type I receptor-IgG fusion protein and/or a soluble type I tumor necrosis factor alpha (TNFalpha) receptor-IgG fusion protein directly to the knees of rabbits with antigen-induced arthritis. When tested individually, knees receiving the soluble IL-1 receptor had significantly reduced cartilage matrix degradation and white blood cell infiltration into the joint space. Delivery of the soluble TNFalpha receptor was less effective, having only a moderate effect on white blood cell infiltration and no effect on cartilage breakdown. When both soluble receptors were used together, there was a greater inhibition of white blood cell infiltration and cartilage breakdown with a considerable reduction of synovitis. Interestingly, anti-arthritic effects were also seen in contralateral control knees receiving only a marker gene, suggesting that sustained local inhibition of disease activity in one joint may confer an anti-arthritic effect on other joints. These results suggest that local intra-articular gene transfer could be used to treat systemic polyarticular arthritides.

Adenoviridae↗
PubMed1998

Anti-inflammatory effect of transforming growth factor-beta1 in myoblast transplantation.

BACKGROUND: The inflammatory reaction that occurs during the 5 days after transplantation led at 3 days to the death of 70% of injected myoblasts. Use of anti-inflammatory agents appeared to be a possible way to increase myoblast survival. The application of gene transfer techniques to cell transplantation offers the potential for the prevention of inflammatory reaction. METHODS: In this study, transforming growth factor-beta1 (TGF-beta1) gene was introduced in myoblasts with a retroviral vector to permit the secretion of this anti-inflammatory cytokine. Survival of (1) infected myoblasts expressing TGF-beta1 or (2) normal myoblasts transplanted with genetically modified cloned myoblasts was compared with survival of normal myoblasts. RESULTS: Expression of TGF-beta1 by myoblasts or by cotransplanted cells decreased myoblast mortality after 3 days by roughly 20% (66.0+/-3.0% in control vs. 46.3+/-4.2% and 46.2+/-5.9%). The increase of myoblast survival by TGF-beta1 expression was correlated with a lower polymorphonuclear cell and macrophage infiltration in muscles compared with control. In addition, cytotoxicity of neutrophils against myoblasts was assayed in vitro. The oxidation of myoblasts by activated neutrophils was decreased after infection of the myoblasts with the TGF-beta1 retroviral vector. CONCLUSIONS: These data demonstrate that the insertion of TGF-beta1 decreases inflammatory reaction observed after myoblast transplantation and thus prolongs their survival.

Animals↗
PubMed1998

IFN-gamma-inducing factor/IL-18 administration mediates IFN-gamma- and IL-12-independent antitumor effects.

We evaluated the mechanism of the antitumor effects of mouse rIFN-gamma-inducing factor/IL-18 protein on the growth of mouse tumor cell lines in vivo. Mice received IL-18 before or after challenge with CL8-1, a mouse melanoma cell line. Both regimens significantly suppressed tumor growth and reduced the number of mice with growth of tumor from 60% (3/5) to 20% (1/5). Furthermore, IL-18 administered before and after tumor inoculation completely abrogated the establishment of CL8-1 in all animals. IL-18 administration also significantly suppressed the growth of MCA205, a sarcoma cell line, even when treatment was delayed to 7 days following tumor inoculation. Although IL-18/IL-12 combination therapy had the most significant and immediate antitumor effects, many mice so treated succumbed with markedly elevated serum IFN-gamma levels. The antitumor effects of IL-18 were abrogated almost completely when NK cells were eliminated using anti-asialo GM1 Ab administration, but only marginally impaired in IFN-gamma or IL-12 gene-disrupted mice. Immunohistochemical staining revealed that the number of the CD8+ T cells, but not CD4+ T cells, found at the tumor site was reduced in animals treated with IL-18. These results indicate that IL-18 has potent antitumor effects mediated by CD4+ T cells and NK cells, but in IFN-gamma- and IL-12-independent pathways.

Adjuvants, Immunologic↗
PubMed1998

Blocking cytokines with genes.

The pathophysiology of rheumatoid arthritis (RA) is primarily driven by proinflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor alpha. Several biological agents have been identified that effectively block the activity of these cellular messengers, and administration of these agents to animal models of RA and human patients has been found to have therapeutic benefit. The application of gene therapy for the treatment of RA and other articular diseases is being explored to overcome current limitations with delivery of therapeutics to joint tissues. To date, laboratory research has focused on two main areas: (1) evaluation of gene delivery approaches and (2) identification of therapeutic gene products. Considerable progress has been reported with the use of local gene delivery to synovial cells by both in vivo and ex vivo methods and by systemic administration of gene delivery vectors via the circulation. Gene products that have therapeutic efficacy in animal models of RA include: IL-1 receptor antagonist, soluble IL-1 receptor I, soluble tumor necrosis factor receptor II, viral IL-10, and transforming growth factor beta, among others. The success of these laboratory studies has led to the implementation of a Phase I clinical trial to asses the safety and feasibility of using gene therapy in the treatment of RA.

Animals↗