Julio Reinecke

The production of anti-inflammatory cytokines in whole blood by physico-chemical induction

AbstractObjective and design: Cytokines such as interleukin-1 (IL-1) play an important role in degenerative musculo-skeletal diseases, including osteoarthritis, and a multitude of inflammatory disorders. Agents that inhibit the action of such cytokines have a high therapeutic potential in such diseases. Here we describe a new method for enhancing the production of the interleukin-l receptor antagonist (IL-1Ra) and other anti-inflammatory cytokines in whole blood. Material and methods: Human venous blood was incubated in the presence of CrSO4-treated glass beads. Serum was recovered and the concentrations of IL-1Ra and other relevant cytokines were measured by ELISA. Results: The interaction of the glass bead surface with cells in whole blood increased production of IL-1Ra and anti-inflammatory cytokines. Removal of the beads and centrifugation generated a serum preparation enriched in anti-inflammatory cytokines. This preparation is of therapeutic value in treating various inflammatory and degenerative disorders. Conclusions: The increased de novo production of anti-inflammatory cytokines by a direct physico-chemical induction of whole blood in the Orthokin system is feasible and offers an alternative, novel approach to treating mild to moderate OA and other orthopaedic conditions such as degenerative spine diseases.

Transfer of genes to chondrocytic cells of the lumbar spine: Proposal for a treatment strategy of spinal disorders by local gene therapy

Study Design. In the current study, chondrocytic cells from bovine intervertebral end plates were cultivated in vitro and modified genetically. Objective. The authors intended to perform isolation and cultivation of cells from bovine end plates of the spine. They also intended to show, in principle, the feasibility of introducing exogenous genes into chondrocytic cells from bovine intervertebral end plates by way of retroviral vectors. Summary of Background Data. The involvement of cytokines in the destruction of articular cartilage is established. It appears possible that similar mechanisms may play a role in intervertebral disc degeneration and other spinal disorders. Conventional medication and surgery of intervertebral disc degeneration addresses neither the pathophysiology nor the chronicity of the disease. Therapeutic proteins carry great potential as locally produced drugs after transfer of their cognate genes to the sites of interest. Methods. Vertebral end plate tissue was obtained from bovine os coccygis. Chondrocytic cells were isolated and cultured in vitro. The bacterial β‐galactosidase (LacZ) gene and, alternatively, the complementary DNA (DNA copy of the mRNA) of the human interleukin‐1 receptor antagonist were introduced into the isolated cells by retrovirus mediated gene transfer. β‐galactosidase activity was determined by staining, and interleukin‐1 receptor antagonist protein was quantitated by enzyme‐linked immunosorbent assay. Results. Isolation and cultivation of chondrocytic end plate cells is possible. Native cells continue to grow in culture for more than 2 months. Transfer of the β‐galactosidase gene to cultured cells resulted in ∼1% β‐galactosidase positive cells. Transfer of the interleukin‐1 receptor antagonist complementary DNA resulted in the production of 24 ng/ml/106 cells interleukin‐1 receptor antagonist protein in 48 hours. Conclusions. The introduction of exogenous therapeutic genes into cells from the intervertebral end plate opens the possibility for a local gene‐based treatment of intervertebral disc degeneration. This approach avoids some of the shortcomings of conventional drug‐ and surgery‐based treatments and has the potential to be specific, effective, and appropriate to the chronicity of the disease.

Autologous Conditioned Serum in the Treatment of Orthopedic Diseases The Orthokine ® Therapy

The common strategies for the treatment of patients with orthopedic diseases do not address the underlying pathogenesis. Several biologically based, local therapies aiming to influence the cytokine imbalance are either in development or in the initial stages of clinical use. A method based on exposure of blood leukocytes to pyrogenfree surfaces (e.g. glass spheres) elicits an accumulation of anti-inflammatory cytokines, including interleukin-1 receptor antagonist, and several growth factors, including insulin-like growth factor-1, platelet-derived growth factor, and transforming growth factor-β1, in the liquid blood phase. Based on these observations, a new therapy using cell-free, autologous conditioned serum (ACS) from the incubation of whole blood with glass spheres was developed. The injection of ACS into affected tissue (s) has shown clinical effectiveness and safety in animal models and studies, as well as in human clinical studies, for the treatment of osteoarthritis, lumbar stenosis, disc prolapse, and muscle injuries.

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.

Clinical Responses to Gene Therapy in Joints of Two Subjects with Rheumatoid Arthritis

This paper provides the first evidence of a clinical response to gene therapy in human arthritis. Two subjects with rheumatoid arthritis received ex vivo, intraarticular delivery of human interleukin-1 receptor antagonist (IL-1Ra) cDNA. To achieve this, autologous synovial fibroblasts were transduced with a retrovirus, MFG-IRAP, carrying IL-1Ra as the transgene, or remained as untransduced controls. Symptomatic metacarpophalangeal (MCP) joints were injected with control or transduced cells. Joints were clinically evaluated on the basis of pain; the circumference of MCP joint 1 was also measured. After 4 weeks, joints underwent surgical synovectomy. There were no adverse events in either subject. The first subject responded dramatically to gene transfer, with a marked and rapid reduction in pain and swelling that lasted for the entire 4 weeks of the study. Remarkably, joints receiving IL-1Ra cDNA were protected from flares that occurred during the study period. Analysis of RNA recovered after synovectomy revealed enhanced expression of IL-1Ra and reduced expression of matrix metalloproteinase-3 and IL-1beta. The second subject also responded with reduced pain and swelling. Thus, gene transfer to human, rheumatoid joints can be accomplished safely to produce clinical benefit, at least in the short term. Using this ex vivo procedure, the transgene persisted within the joint for at least 1 month. Further clinical studies are warranted.

Neurophysiologic Changes in Lumbar Nerve Root Inflammation in the Rat after Treatment with Cytokine Inhibitors: Evidence for a Role of Interleukin-1

Study Design The present study was designed to evaluate the effect of cytokine inhibitors in experimental allergic radicultis. Objective Evaluation of the effect of cytokine inhibitors in experimental allergic radicultis. Summary of Background Data A number of cytokines are known to be involved in hyperalgesia and may play a role in radicultis. Corticosteroids and other cytokine inhibitors antagonize their effects. Methods Experimental allergic radiculitis was induced in rats by injection of bovine myelin from the peripheral nervous system. The sham group subsequently received saline injections; the treatment groups received either prednisolone or interleukin‐1 receptor antagonist. Treatment effect was assessed on the basis of motor performance and neurophysiologic parameters. Results Treatment ameliorated the symptoms of experimental allergic radiculitis. Prednisolone appeared to be somewhat more effective than interleukin‐1 receptor antagonist. Conclusions Because interleukin‐1 receptor antagonist specifically blocks the effects of interleukin‐1 at its receptors, the present results imply that interleukin‐1 is a causal factor in the model of experimental radiculitis used. Its specificity and apparent lack of side effects make interleukin‐1 receptor antagonist an attractive candidate treatment for the human disease.

Die Rolle der Zytokine bei Knorpeldefekten und in der Knorpeltherapie

ZusammenfassungZytokine spielen eine Schlüsselrolle in der Pathogenese degenerativer aber auch inflammatorischer Gelenkerkrankungen wie der Osteoarthrose (OA) und rheumatoiden Arthritis (RA). Eine besondere Rolle wird dabei dem Interleukin-1 und dem TNF-α zugeschrieben. Bestimmte Zytokine, welche die Wirkung der katabolen Zytokine inhibieren, besitzen großes therapeutisches Potenzial und werden durch zahlreiche klinische Studien derzeit näher untersucht. Stand der Wissenschaft ist, dass proinflammatorische Zytokine den Knorpelabbau stimulieren und deren Blockade den Knorpel schützen kann.AbstractCytokines play a pivotal role in the pathogenesis of degenerative joint disease but also in inflammatory conditions as well as osteoarthritis (OA) and rheumatoid arthritis (RA). A key role is attributed to interleukin-1 and tumor necrosis factor-α. Certain cytokines that can inhibit the activity of catabolic cytokines have great therapeutic potential and are currently being investigated in numerous clinical studies. Available scientific findings indicate that proinflammatory cytokines stimulate cartilage breakdown and blockade of these cytokines can protect the cartilage.

Gene therapy for rheumatoid arthritis: clinical studies

Collectively, over 35 million Americans suffer from the various arthritidies with an annual cost estimated to be greater than 18 billion dollars annually [1]. Rheumatoid arthritis (RA) is a progressive, autoimmune disease that primarily affects diarthro-dial joints. It has a world-wide incidence of approximately four per 10000 in the population aged greater than 15 years, and causes significant increases in morbidity and even mortality in affected patients [2–4]. 150 000 patients are diagnosed with RA each year in the United States at a cost of approximately

Current Status of Gene Therapy for Rheumatoid Arthritis

30 000 per case [5]. Women are twice as likely to be affected as men, and the prevalence increases with age. Although the prevalence of RA varies across the globe, no etiologic agents have been unequivocally identified [6–9].

Method of producing interleukin-1 receptor antagonist in a syringe filled with blood

Despite the high prevalence of the disease, at present little effective pharmacological treatment of rheumatoid arthritis is available. Novel approaches utilising biological agents have resulted in the development of new antiarthritic and antiinflammatory agents, such as tumour necrosis factor-α (TNFα)-specific antibodies and interleukin-1 receptor antagonist (IL-1ra). Local gene therapy not only allows the pharmaceutical use of these biologicals, but also allows for continuous drug supply, which is necessary for chronic diseases like rheumatoid arthritis.We discuss the basics of rheumatoid arthritis therapy, candidate genes and possible gene transfer methods. A current clinical gene therapy trial is focusing on the IL-1 system using IL-1ra as a transgene. The transfer system, clinical protocol and preliminary results are described. After treatment of 11 patients we feel that gene therapy will offer potential as a new avenue to treat rheumatoid arthritis.