Brian Henderson

A Study of the Uptake of Toluidine Blue O by Porphyromonas gingivalis and the Mechanism of Lethal Photosensitization

The purpose of the study was to determine the distribution of the photosensitizer toluidine blue O (TBO) within Porphyromonas gingivalis and the possible mechanism(s) involved in the lethal photosensitization of this organism. The distribution of TBO was determined by incubating P. gingivalis with tritiated TBO (3H‐TBO) and fractionating the cells into outer membrane (OM), plasma membrane (PM), cytoplasmic proteins, other cytoplasmic constituents and DNA. The percentage of TBO in each of the fractions was found to be, 86.7, 5.4, 1.9, 5.7 and 0.3%, respectively. The involvement of cytotoxic species in the lethal photosensitization induced by light from a helium‐neon (HeNe) laser and TBO was investigated by using deuterium oxide (D2O), which prolongs the lifetime of singlet oxygen, and the free radical and singlet oxygen scavenger L‐tryptophan. There were 9.0 log10 and 2 log10 reductions in the presence of D2O and H2O (saline solutions), respectively, at a light dose of 0.44 J (energy density = 0.22 J/cm2), suggesting the involvement of singlet oxygen. Decreased kills were attained in the presence of increasing concentrations of L‐tryptophan. The effect of lethal photosensitization on whole cell proteins was determined by measuring tryptophan fluorescence, which decreased by 30% using 4.3 J (energy density = 4.3 J/ cm2) of light. Effects on the OM and PM proteins were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. There was evidence of change in the molecular masses of several PM proteins and OM proteins compared to controls. There was evidence of damage to the DNA obtained from irradiated cells. Scanning electron microscopic studies showed that there was coaggre‐gation of P. gingivalis cells when sensitized and then exposed to laser light. These results suggest that lethal photosensitization of P. gingivalis may involve changes in OM and/or PM proteins and DNA damage mediated by singlet oxygen.

Effect of dosimetric and physiological factors on the lethal photosensitization of Porphyromonas gingivalis in vitro.

The aims of this study were to (1) determine the effect of dosimetric and physiological factors on the lethal photosensitization of Porphyromonas gingivalis using tolui‐dine blue O (TBO) and light from a helium/neon (HeNe) laser; (2) determine the influence of sensitizer concentration, preirradiation time, serum and growth phase on sensitizer uptake by P. gingivalis. The dosimetric factors studied were concentration of TBO, light dose and preirradiation time. The physiological factors were presence of serum, pH and bacterial growth phase. Sensitizer uptake by P. gingivalis under various conditions was determined using tritiated TBO (3H‐TBO). In the presence of TBO, a light dose‐dependent increase in kill was attained (100% kill at 4.4 J). There was no significant effect on the numbers killed when TBO was increased from 12.5 to 50 µg/mL. An increase in preirradiation time gave slightly increased kills. High kills were achieved at all three pH (6.8–8.0). Although kills were substantial in the presence of serum, they were significantly less than those obtained in the presence of saline. Cells in all three growth phases were susceptible to lethal photosensitization, although stationary phase cells were slightly less susceptible. Maximum uptake of TBO occurred within 60 s and uptake in serum was less than in saline. The uptake by the log phase cells was greater at lower concentrations of sensitizer (50 µg/mL), compared to the other two phases.

Inhibition of interleukin-1-induced synovitis and articular cartilage proteoglycan loss in the rabbit knee by recombinant human interleukin-1 receptor antagonist

Intra-articular injection of interleukin-1 (IL-1) into the knee joints of rabbits produces a synovitis associated with the loss of proteoglycan from the matrix of articular cartilage. This experimental finding supports the hypothesis that IL-1 is a possible mediator of the pathology of inflammatory joint diseases and suggests that antagonism of IL-1 could offer a therapeutic approach to these diseases. It has recently been reported that culture of human monocytes on adherent IgG stimulates these cells to synthesize a specific inhibitor of IL-1 bioactivity (IL-1ra) that acts as a receptor antagonist with lymphocytes and mesenchymal cells. We have now shown that intravenous injection of IL-1ra into rabbits given an intra-articular injection of recombinant IL-1 beta not only inhibits the entry of leukocytes into the synovial lining and joint cavity but blocks the ability of IL-1 to cause loss of proteoglycan from articular cartilage. This ability of IL-1ra to inhibit IL-1-induced arthritis in the rabbit reveals that this protein has appropriate pharmacokinetic and pharmacodynamic properties and further strengthens the belief that it may be a useful therapeutic agent.

Anti-proliferative and cytotoxic activity of surface-associated material from periodontopathogenic bacteria

The easily solubilized surface-associated material from three bacterial species associated with periodontal diseases, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Eikenella corrodens, produced dose-dependent inhibition of thymidine incorporation by human fibroblasts, the human monocytic cell line U937 and guinea pig epidermal cells. In contrast, lipopolysaccharides from A. actinomycetemcomitans and P. gingivalis were either inactive or substantially less active over the dose range tested. One of the constituents of surface-associated material from a non-leucotoxic strain of A. actinomycetemcomitans was highly cytotoxic to human peripheral blood polymorphonuclear cells, with 50% killing from less than 1 ng/ml. A constituent of the surface-associated material from P. gingivalis was approximately one log order less active. The lipopolysaccharides from these bacteria were at least three log orders less active in neutrophil killing. These findings add weight to the hypothesis that easily solubilized exopolymers from periodontopathogens play a major part in the pathology of periodontal diseases.

Modulation of cytokine function: therapeutic applications.

Publisher Summary This chapter illustrates that cytokines are important therapeutic targets in a range of diseases and has given an overview of current approaches being used to modify the activity of “pathological” cytokines. Anticytokine therapy is now in the clinic with blocking antibodies, antagonists, and inhibitors of release being clinically evaluated in phase II/III trials and other modalities (e.g., anti-LPS antibodies), waiting, uncomfortably, on the U.S. Food and Drug Administration (FDA) decision to license. Largely, with protein products, this can depend on the skills of the molecular biologist and the production engineers and the vagaries of the human immune system. Proteins (antibodies, soluble receptors, and natural antagonists) represent the first phase of anticytokine therapy. These may have use in the treatment of acute conditions (e.g., septic shock) and life-threatening diseases such as cancer. The choice among the various proteinaceous drugs depends on their efficacy and cost. However, there are still many problems to be overcome in the development of cytokine-modulatory drugs, and it is only with the second phase of development of nonpeptide agents that one can confidently predict that anticytokine reagents will be clinically used for the more common but nonfatal diseases, such as rheumatoid arthritis and osteoarthritis.

Control of the human cell cycle by a bacterial protein, gapstatin

The oral gram-negative bacterium Actinobacillus actinomycetemcomitans is a major pathogen in human periodontal disease. Saline extraction releases a range of surface-associated components from this bacterium, including one which exhibits potent anti-proliferative activity as assessed by its capacity to inhibit DNA synthesis by human and other mammalian cells. Cultures incubated with this bacterial fraction for a prolonged period comprise a high proportion of cells containing a 4n level of DNA. Studies using hydroxyurea-synchronized cultures showed that cells treated with the surface-associated fraction were arrested in the G2 phase of the cell cycle and did not enter mitosis. This G2/M blockade was observed only when the bacterial fraction was added to the cells during early S phase. Our data also suggest that the active bacterial component binds to surface receptors expressed by the human cells and may act by a novel mechanism which involves down-regulation of cyclin B1 expression. The anti-proliferative activity of the bacterial fraction, purified by a combination of ammonium sulphate precipitation, HPLC anion exchange and gel filtration, has been shown to be an 8 kDa protein, which we have called gapstatin. Purified gapstatin was shown to be responsible for the the inhibitory effects of the surface-associated fraction on mammalian cells.

The application of quantitative cytochemistry to the study of diseases of the connective tissues.

The connective tissues are a complex organisation of tissues, cells and intercellular materials spread throughout the body and are subject to a large number of diseases. Such complexity makes the study of the metabolism of the connective tissues in health and more particularly in disease states difficult if one uses conventional biochemical methodology. Fortunately the techniques of quantitative cytochemistry, as developed in recent years, have made it possible to study the metabolism of even such complex and refractory connective tissues as bone. Using properly validated assays of enzyme activity in unfixed sections from various tissues a number of the diseases of the connective tissues have been studied. For example the synovia from patients with rheumatoid arthritis and related conditions have been studied using these techniques and marked alterations in the metabolism of the synovial lining cell population of this tissue have been demonstrated. These alterations in metabolism are believed to be related to the destruction of cartilage and bone found in such diseases. Investigations of the metabolism of the chondrocytes of articular cartilage in a strain of mice which spontaneously develops osteoarthritis has revealed a lack of certain key enzymes of carbohydrate metabolism in precisely those areas where degradation of the matrix of articular cartilage begins suggesting a causal relationship between these events. These same techniques have been used to study the cellular kinetics and metabolism of the dermis and epidermis in the disfiguring disease, psoriasis. The metabolism of healing bone fractures, the diagnosis and treatment of the mucopolysaccharidoses and the metabolic effects of currently used anti-inflammatory and anti-rheumatic drugs have also been examined. Perhaps the most exciting aspect of these studies has been the development and use of the technique of the cytochemical bioassay (CBA) to study hormonally mediated diseases of the connective tissues. Such studies have recently shed new light on the molecular lesion in pseudohypoparathyroidism. Though still in their relative infancy the studies described in this review show the potential inherent in the use of quantitative cytochemistry for the study of diseases of the connective tissues.

The contribution made by cytochemistry to the study of the metabolism of the normal and rheumatoid synovial lining cell (synoviocyte)

The synovial lining cell, or synoviocyte, is found in the synovium (also called the synovial lining or synovial membrane) lining the capsule of the synovial joints, bursae and tendon sheaths. Most of our knowledge of these cells comes from the studies of the synovium of the joints and this review will concern itself solely with this tissue. The relationship between the synoviocyte, synovium and synovial joint is shown in Fig. 1.

Microbiochemical analysis of changes in proteoglycan and collagen in joint tissues during the development of antigen-induced arthritis in the rabbit.

Microbiochemical assays of the proteoglycan and collagen content of articular cartilage and synovial lining have been performed on tissue sections taken from rabbits with antigen-induced arthritis. This experimental arthritis is a close analogue of the natural disease-rheumatoid arthritis. Animals were killed at intervals during the first 21 days following induction of arthritis to assess changes in the composition of the extracellular matrices of the synovial lining and articular cartilage during the early development of this experimental lesion. In confirmation of earlier studies these microbiochemical assays revealed a rapid and significant loss of proteoglycan from the articular cartilage. This loss was, however, not uniform but was restricted to the intermediate zone of the cartilage. Over the period studied, there was only a slight loss of proteoglycan from the superficial zone of the cartilage facing the joint cavity. These findings demonstrate that, at least in this model, cartilage proteoglycan loss is not due to the action of proteases present in the synovial fluid. Moreover it suggests that the chondrocytes in the mid-zone of the cartilage are responsive to those signals stimulating proteoglycan breakdown. There was no significant loss of collagen from the cartilage over the time period of this study. The synovial lining from arthritic joints, in contrast, showed a progressive increase in both proteoglycan and collagen.

Increase in the activity of lysosomal acid hydrolases in the chondrocytes of arthritic joints of rabbits with experimental allergic arthritis

SummaryUndecalcified cryostat sections of the cartilage-covered ends of the femurs have been prepared from the control and inflamed knees of rabbits with experimental allergic arthritis. The activities of two lysosomal hydrolases, naphthylamidase and β-glucuronidase, were assayed in the chondrocytes of the articular cartilage in such sections by scanning and integrating microdensitometry. The activities of both enzymes were found to increase significantly in the chondrocytes of the inflamed joints.