Herniated lumbar discs were making spontaneously increased amounts of matrix metalloproteinases, nitric oxide, prostaglandin E2, and interleukin-6. These products may be involved intimately in the biochemistry of disc degeneration and the pathophysiology of radiculopathy. Their exact roles certainly need further investigation, but their mere presence implicates biochemical processes in intervertebral disc degeneration.

Objective. This study explored the therapeutic effect of interleukin‐1 receptor antagonist (IL‐1Ra), administered by gene transfer, on the progression of osteoarthritic (OA) lesions in an experimental dog model. Methods. Seventeen mature mongrel dogs were divided into 3 groups. Group 1 (n = 7) had an anterior cruciate ligament (ACL) section of the right knee through a stab wound incision. Groups 2 and 3 (n = 5 per group), had an ACL section of the right knee and partial synovectomy of the left knee. Each dog's synovium was subjected to enzymatic digestion, and the synovial fibroblasts were propagated in monolayer culture. Synovial cells from each dog were transduced in vitro using the retrovirus MFG with either the Escherichia coli β‐galactosidase (lac Z) gene (group 2) or the human IL‐1Ra gene (group 3). Two days after surgery, the dogs received intraarticular injections as follows: group 1 phosphate buffered saline (PBS) (2 ml); group 2 autologous cells (60 × 106 cells/2 ml of PBS) transduced with the lac Z gene; group 3 autologous cells transduced with the IL‐1Ra gene. Synovial fluid was aspirated at 2 weeks and 4 weeks. All dogs were euthanized at 4 weeks postsurgery. The right knees were dissected, and lesions were scored for macroscopic and microscopic changes. Synovial explants were dissected and representative specimens were used for histology or were cultured for 48 hours. The levels of IL‐1Ra in synovial fluid and synovial explant conditioned medium were measured by specific enzyme‐linked immunosorbent assay. Results. The level of IL‐1Ra in synovial fluid of group 3 was 202.8 ± 131.5 ng/ml (mean ± SEM) at 2 weeks and 2.8 ± 2.2 ng/ml at 4 weeks after surgery. Membrane explants isolated from dogs that received synovial cells transduced with the IL‐1Ra gene (group 3) actively produced IL‐1Ra (4.0 ± 2.0 ng/gm of tissue wet weight). The severity of OA cartilage lesions was similar in groups 1 and 2. In contrast, group 3 dogs had a marked reduction in macroscopic lesion severity on the tibial plateaus (P < 0.01 for grade; P < 0.04 for size) and femoral condyles. Moreover, the histologic lesion severity was decreased on both plateaus (P < 0.06) and condyles. Conclusion. This study showed that a local increase in IL‐1Ra production in OA knee joints by intraarticular injection of transduced synovial cells can reduce the progression of experimentally induced lesions.

No abstract

Gene therapy offers a radical different approach to the treatment of arthritis. Here we have demonstrated that two marker genes (lacZ and neo) and cDNA coding for a potentially therapeutic protein (human interleukin 1-receptor-antagonist protein; IRAP or IL-lra) can be delivered, by ex vivo techniques, to the synovial lining of joints; intraarticular expression of IRAP inhibited intraarticular responses to interleukin 1. To achieve this, lapine synoviocytes were first transduced in culture by retroviral infection. The genetically modified synovial cells were then transplanted by intraarticular I jection into the knee joints of rabbits, where they efficiently colonized the synovium. Assay of joint lavages confirmed the in vivo expression of biologically active human IRAP. With allografted cells, IRAP expression was lost by 12 days after transfer. In contrast, autografted synoviocytes continued to express IRAP for -5 weeks. Knee joints expressing human IRAP were protected from the leukocytosis that otherwise follows the intraarticular injection of recombinant human interleukin 1p3. Thus, we report the intraarticular expression and activity of a potentially therapeutic protein by genetransfer technology; these experiments demonstrate the feasibility of treating arthritis and other joint disorders with gene therapy.Arthritis is a chronic, debilitating condition affecting over 30 million Americans (1). Presently incurable, it remains the agent of considerable suffering and economic loss. Therapeutic intervention in arthritis is hindered by a number of factors, including difficulties in targeting drugs to joints. Proteins are particularly vulnerable to this restriction, which is of special concern, as many new agents with considerable antiarthritic potential are proteins. As an alternative to traditional methods of drug delivery, we have suggested the transfer oftherapeutic genes to the synovial lining ofjoints (2,3). Expression of these genes would overcome proteindelivery problems and lead to the intraarticular accumulation of the gene products at the site of disease, with reduced exposure of nontarget organs.Using the rabbit knee joint as a model system, we are therefore developing in vivo and ex vivo methods for delivering genes to joints. This model takes advantage of the similarity in size between the knee joint of the rabbit and the human proximal interphalangeal joint, a common site of rheumatoid arthritis. Moreover, there exists a large body of literature on the biology of the rabbit's knee, including well-established methods for synovial cell culture (e.g., refs. 4-6). Here we report the transfer to synovium of two marker genes and one potentially therapeutic gene by an ex vivo approach. Intraarticular expression of the interleukin 1-re-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. (rhIL-1,8). These results demonstrate the feasibility of ...

Although it has been reported that several growth factors modulate soft-tissue healing, the specific effects of growth factors on protein synthesis during ligament healing have not been widely investigated. In this study, we examined the effects of basic and acidic fibroblast growth factors, transforming growth factor beta 1, and epidermal growth factor on collagen and noncollagenous protein synthesis by cultured fibroblasts from medial collateral ligament and anterior cruciate ligament in vitro. Uptake of tritiated proline was used to measure synthesis of collagen and noncollagenous protein, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the type of collagens synthesized. Our data showed that transforming growth factor beta 1 increased both collagen and noncollagenous protein synthesis by medial collateral and anterior cruciate ligament fibroblasts on a dose-dependent basis. Collagen synthesis by cultured fibroblasts from the medial collateral and anterior cruciate ligaments was increased by treatment with transforming growth factor beta 1 by as much as approximately 1.5 times that of untreated controls. Although the response to transforming growth factor beta 1 by anterior cruciate ligament fibroblasts was equal to that by medial collateral ligament fibroblasts, the amounts of matrix proteins synthesized by anterior cruciate ligament fibroblasts were approximately half of that by medial collateral ligament fibroblasts. The increase was mostly in type-I collagen. Treatment of anterior cruciate ligament fibroblasts with epidermal growth factor increased collagen synthesis by approximately 25% but had little effect on medial collateral ligament fibroblasts. Neither basic nor acidic fibroblast growth factor increased either collagen or noncollagenous protein synthesis. These findings suggest that topical application of transforming growth factor beta 1, alone or in combination with epidermal growth factor, may have the potential to strengthen the ligament by increasing matrix synthesis during its remodeling and healing processes.

Objective. To assess the abilities of various vectors to transfer genes to the synovial lining of joints.Methods. Vectors derived from retrovirus, adenovirus, and herpes simplex virus as well as cationic liposomes and naked plasmid DNA were evaluated. Each construct contained the lac Z marker gene; and one retroviral construct, and one plasmid also contained a gene encoding human interleukin-1 receptor antagonist. Gene expression was under the control of the human cytomegalovirus promoter in all vectors except the retrovirus, where the endogenous 5' long terminal repeat was retained as the promoter. Cultures of rabbit synovial fibroblasts were exposed to these vectors and stained with X-gal to identify lac Z+ cells. Vectors were then injected directly into rabbits' knee joints, and gene transfer and expression were assessed by X-gal staining and polymerase chain reaction (PCR).Results. Adenovirus was a highly effective vector both in vitro and in vivo, with lac Z gene expression persisting for at least 28 days. However, an inflammatory response was noted in vivo. Cells infected in vitro and in vivo with herpes simplex virus also expressed the loc 2 gene at high levels, but expression was limited by cytotoxicity. Retroviruses, in contrast, were effective --~ _-

Growth factors have been shown to stimulate fibroblast division and thus may influence ligament healing. We analyzed the effects of individual growth factors on the proliferation of fibroblasts from the medial collateral and anterior cruciate ligaments of the rabbit in vitro in order to identify growth factors that might enhance proliferation of fibroblasts and to compare the responses of the fibroblasts from the two ligaments to these growth factors. Through measurement of the uptake of [3H]-thymidine into DNA, fibroblasts from these ligaments that had been treated with epidermal growth factor and basic fibroblast growth factor were found to proliferate nearly eight times more than control fibroblasts. Additionally, the fibroblasts of both ligaments proliferated at similar rates when exposed to platelet-derived growth factor-AA, platelet-derived growth factor-BB, basic fibroblast growth factor, insulin-like growth factor-1, and interleukin-1-alpha. However, epidermal growth factor and transforming growth factor-beta caused the fibroblasts from the medial collateral ligament to proliferate at a rate 1.3-1.4 times greater than that of fibroblasts from the anterior cruciate ligament. The reverse was true with acidic fibroblast growth factor, which stimulated the fibroblasts from the anterior cruciate ligament to proliferate at a rate 1.3-1.6 times greater than that of fibroblasts from the medial collateral ligament. This study demonstrated that growth factors can stimulate cell division in ligaments and may be effective in enhancing ligament healing but that these differences were not great enough to explain fully the clinical differences observed between healing of the medial collateral and anterior cruciate ligaments.

The addition of human recombinant interleukin-1beta (IL-1beta) to cultures of lapine articular chondrocytes provoked the synthesis of large amounts of NO and reduced the production of type-II collagen. NG-Monomethyl-l-arginine (L-NMA), an inhibitor of NO synthase, strongly suppressed the production of NO and partially relieved the inhibition of collagen synthesis in response to IL-1beta. The NO donor S-nitrosoacetylpenicillamine (SNAP), on the other hand, inhibited collagen production. IL-1 lowered the abundance of Col2A1 mRNA in an NO-independent manner. Collectively, these data indicate that IL-1 suppresses collagen synthesis at two levels: a pretranslational level which is NO-independent, and a translational or post-translational level which is NO-mediated. These effects are presumably specific as L-NMA and SNAP had no effect on total protein synthesis or on the distribution of newly synthesized proteins between the cellular and extracellular compartments. Prolyl hydroxylase is an important enzyme in the post-translational processing of collagen, and its regulation and cofactor requirements suggest possible sensitivity to NO. Extracts of cells treated with IL-1 or SNAP had lower prolyl hydroxylase activity, and L-NMA was partially able to reverse the effects of IL-1. These data suggest that prolyl hydroxylase might indeed be a target for NO. Because underhydroxylated collagen monomers fail to anneal into stable triple helices, they are degraded intracellularly. Inhibition of prolyl hydroxylase by NO might thus account for the suppressive effect of this radical on collagen synthesis.