Biomedical subjects
Publications and source records attributed to D W Howat.
Using disulphide cysteine-based inhibitors as lead structures, this communication describes our strategy for identifying more stable, potent antagonists of the alpha4beta1 integrin. These studies ultimately discovered potent, low molecular weight inhibitors based on D-thioproline-L-tyrosine.
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In a short-term culture system, polymorphonuclear neutrophils (PMNs) were shown to cause significant loss of glycosaminoglycan (GAG) from cartilage. In addition, a lysate of these cells, and in particular cells stimulated with a phorbol ester, greatly enhanced this loss of GAG. This loss could be partially or completely inhibited by the addition of cell-free fluid exudate to the system. In other long-term culture experiments, IL-1a was shown to enhance fibroblast-induced GAG loss from cartilage. In this system too the breakdown was inhibited by cell-free fluid exudate. Initial characterization of the protective agent or agents shows them to be heat-stable at 56 degrees C and to be non-dialysable. It is proposed that in the chronic inflammatory joint disease, rheumatoid arthritis, the fluid phase of an inflammatory response may have a protective effect against the underlying pathological processes.
Rats developing experimental allergic encephalomyelitis have been treated with intra-venous injections of Platelet-Activating Factor on days 5, 6 and 7 post adjuvant injection. Other rats have been treated with one of two different PAF antagonists or vehicle. The animals injected with PAF on day 5 post adjuvant developed a more severe form of the disease at an earlier time point than did control animals. Animals treated with the PAF antagonists did not develop the disease to any great extent. Treatment with the vehicle had no effect compared to control. These results implicate PAF in the aetiology of this model of multiple sclerosis and may suggest a role for PAF antagonists in the treatment of this disease.
The effects of recombinant interleukin-1 alpha (rIL-1 alpha) and indomethacin on the glycosaminoglycan (GAG) content of rat femoral head cartilage (FHC) were studied in vitro. Net GAG loss was observed from cartilage cultured in the presence of indomethacin plus rIL-1 alpha. Indomethacin and rIL-1 alpha alone caused no net loss of GAG from cartilage unless the cartilage was cultured in the presence of fibroblasts.
Endothelin-1 (ET-1), when injected intradermally in rats concurrently with FMLP, did not suppress cell accumulation 6 h later. In view of the fact that ET-1 can antagonize mediators of increased vascular permeability, it is proposed that this is further evidence that fluid and cell accumulation at an inflammatory site are separate processes.
The effects of heparin and cortisone were investigated in a model of experimental pannus-mediated cartilage degradation. Rat femoral head cartilages were implanted bilaterally (sc.) into female mice either non-wrapped or wrapped in cotton. Animals were treated with tap water (p.o.), heparin 1000 Units (p.o.), cortisone 2 mg/kg (s.c) and heparin-cortisone combined. After 14 days, the implants were removed and analysed for exudate volume, granulation-tissue dry weight and cartilage glycosamino-glycan (GAG) content. In the heparin-treated animals there was a significant (p less than 0.001) increase in the granulation-tissue dry weight, whilst combined treatment significantly (p less than 0.001) reduced both exudate volume and cartilage degradation. It is possible that the effects on angiogenesis may indicate novel treatment for the growth of pannus and cartilage breakdown.
Cartilage breakdown products have been tested for their effects on the locomotion of rat polymorphonuclear neutrophils in vitro. Chondroitin sulphate, keratan sulphate and hyaluronic acid were used in checkerboard migration assays to differentiate chemotaxis from chemokinesis. All test substances showed both chemokinetic and chemotactic activities. The same substances were then injected intradermally to determine their effects on vascular permeability and leukocyte accumulation in vivo. There was no significant effect on vascular permeability 30 minutes after injection as measured by a dye-leakage method. Histological examination of skin sections taken 6 hours after injection showed modest accumulations of polymorphonuclear neutrophils. It is suggested that cartilage breakdown products may account for the persistence of polymorphonuclear neutrophils in some chronic inflammatory joint diseases.
Ultrastructural studies of one week old cotton-induced granulation tissue revealed the presence of polymorphonuclear neutrophils (PMN), macrophages and mast cells, whilst the granulation tissue from two week old cotton implants contained fibroblasts, myofibroblasts and abundant collagen fibres. Similarly, the bovine adrenal capsule contained both fibroblasts and myofibroblasts. It is proposed that the bovine adrenal capsule and two week cotton-induced granulation tissue could be utilized as in-vitro models to study the mechanism(s) involved in myofibroblast contraction.
Rat femoral head cartilages (FHCs) in culture for six days retained their glycosaminoglycan (GAG) content and were not affected by recombinant human interleukin-la (IL-1), conditioned media from concanavalin-A stimulation of sensitized lymphocytes (lymphocyte media), or conditioned media from lymphokine-stimulated macrophages (macrophage media). Similarly FHCs cultured with macrophages in the presence of IL-1, lymphocyte medium or macrophage medium did not suffer a loss of GAG. However, FHCs cultured with fibroblasts showed a substantial loss of GAG. This fibroblast-mediated cartilage degradation was significantly increased by IL-1 and macrophage media respectively. Stimulation of fibroblasts by lymphocyte media failed to increase the loss of GAG. It is proposed that cartilage degradation in inflammatory joint disease is brought about by a sequential cell-to-cell interaction via cytokines leading ultimately to stimulation of fibroblast-mediated cartilage degradation.
The oxidative biochemistry of the adjuvant-inflamed rat air pouch has been studied using the techniques of quantitative cytochemistry. Highly significant increases in the maximal activities of glucose-6-phosphate dehydrogenase, lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase were observed. These changes are similar to those that have been reported by others in both human rheumatoid synovial tissue and the synovial tissue of rabbits with allergic arthritis. It is suggested that modulation of these changes by putative therapeutic agents may form the basis for a readily quantifiable drug screening programme.
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A perplexing feature of rheumatoid arthritis is the increase in the number of synovial lining cells with no mitotic activity. This feature has been investigated in the rabbit model. Rabbits with the established condition were injected into the affected joint with tritiated thymidine and killed either up to 24 hours later, or at 3 or 7 days. The location of labelled cells, detected by autoradiography, showed the label predominantly in the stroma in the former, and mainly in the lining cells in the latter, indicating that the lining cells were derived by recruitment from active cells deep in the stroma.
The experimental model of inflammatory arthritis in the rabbit has been used to study the possible origin of the apparent synovial lining cell hyperplasia. A small amount of 3H-thymidine was injected into the joints and the fate of the labelled cells investigated by autoradiography. At times up to 24 h after the injection, most of the labelled cells occurred in the sub-lining region; this proportionality was reversed when the joints were sampled at later times. These results indicate that at least much of the increase of synovial lining cells may be derived from cells from deeper in the synovial tissue which move to the synovial surface.
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