S. Meghji

Stimulation of human buccal mucosa fibroblasts in vitro by betel-nut alkaloids.

Oral submucous fibrosis (OSF) is characterized by excessive collagen production by mucosal fibroblasts and is associated with the habitual chewing of betel-nuts (Areca catechu); nut extracts stimulate fibroblast activity in vitro. The metabolism of arecoline, the major alkaloid in the nut, by human buccal mucosa fibroblasts in vitro was investigated; alkaloid metabolites extracted from culture media were analysed by gas chromatography and thin-layer chromatography. [3H]-arecoline was metabolized predominantly to [3H]-arecaidine and this was accompanied by a concentration-dependent stimulation of collagen synthesis and cell proliferation. Arecaidine was a more potent stimulator than arecoline. The rate of hydrolysis of a series of synthetic arecaidine esters (methyl, ethyl, butyl, propyl and pentyl) by fibroblasts was closely correlated with the extent of stimulation of collagen synthesis. Thus fibroblasts are responsive to the major metabolite of arecoline and hydrolysis of the ester group may be necessary for this action. Exposure of buccal mucosa fibroblasts to these alkaloids in vivo may contribute to the accumulation of collagen in OSF.

The potent bone-resorbing mediator of Actinobacillus actinomycetemcomitans is homologous to the molecular chaperone GroEL.

Actinobacillus actinomycetemcomitans is a Gram-negative bacterium implicated in the pathology of localized juvenile periodontitis, a condition involving rapid destruction of alveolar bone. We have established that gentle extraction of this bacterium in saline releases a proteinaceous fraction (which we have termed surface-associated material [SAM] which has potent osteolytic activity in the murine calvarial bone resorption assay. Fractionation of the SAM has now revealed that activity is associated with a 62-kD protein. This bone-resorbing activity can be blocked by a monoclonal antibody (raised to the whole bacterium) that is claimed to recognize a protein homologous to the Escherichia coli molecular chaperone GroEL. Purification of this bone-resorbing protein to homogeneity has been achieved by a combination of anion exchange, gel filtration, and ATP-affinity chromatography and the NH2-terminal sequence shows > 95% homology to E. coli GroEL. This GroEL homologue is found in the SAM of A. actinomycetemcomitans but is not found in the osteolytically active SAM from other Gram-negative or Gram-positive bacteria. The GroEL protein from E. coli, but not from Mycobacterium tuberculosis and Mycobacterium leprae, also showed activity in the bone resorption assay. We believe this to be the first observation that a molecular chaperone has the capacity to stimulate the breakdown of connective tissue.

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.

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.

An in-vitro comparison of human fibroblasts from normal and oral submucous fibrosis tissue

Fibroblasts cultured in vitro from normal buccal tissue and from tissue from oral submucous fibrosis (OSF) associated with betel-nut chewing showed no significant difference in their rates of proliferation in culture, nor in the rate at which they hydrolysed the betel nut alkaloid arecoline to arecaidine. Basal rates of collagen synthesis were slightly higher in the OSF cells but, on addition of arecoline, the rate of collagen synthesis in normal and OSF cells was stimulated to the same level.

MURINE OSTEOBLASTS RELEASE BONE-RESORBING FACTORS OF HIGH AND LOW-MOLECULAR WEIGHTS - STIMULATION BY MECHANICAL DEFORMATION

Murine calvarial osteoblasts in monolayer culture were found to constitutively produce bone-resorbing factors; mechanical deformation significantly increased the synthesis and/or release of these factors. In short-term cultures (2 h) the resorptive activity was largely dialysable, indicating a relative molecular mass (Mr) less than 2000. Intermittent mechanical deformation stimulated the synthesis of these low Mr factors irrespective of serum conditions. Continuous deformation, however, was without effect. When the culture period was extended to 24 h, bone resorptive activity was stimulated by both intermittent and continuous deformation in the presence of 10% serum. This activity was dialysable. Over this same period in cultures with 2% serum, intermittent deformation also produced a non-dialysable bone-resorbing factor. We also cultured osteoblasts for 72 h in serum-free conditions and deformed the cells intermittently. Fractionation of the medium by high pressure liquid chromatography (HPLC) resolved three peaks of bone resorptive activity: peak I (Mr 50-60,000); peak II (Mr 5-20,000); and peak III (Mr less than 1000). Only peaks II and III were stimulated by mechanical deformation. These bone-resorbing factors remain as yet poorly characterized, but none of the activity in the HPLC fractions was attributable to interleukin-1 or prostaglandin E2.

Interleukin-1: The principal osteolytic cytokine produced by keratocysts

Fragments of keratocysts removed at operation were maintained in explant culture and the media were assayed for the biological activity of the potent osteolytic cytokines--interleukin (IL)-1, interleukin (IL)-6 and tumour necrosis factor (TNF). Media were also assayed for their ability to stimulate bone resorption. All six cysts examined released IL-1 and IL-6 bioactivity but TNF bioactivity was unmeasurable. Dialysed cyst media stimulated bone resorption and this could be completely inhibited by a monospecific antibody which neutralized IL-1 alpha and IL-1 beta. Immunohistochemical staining of cryostat sections of keratocysts revealed the presence of IL-1 alpha and IL-6 in cyst epithelial cells but not in other cell types. Sections did not react with antibodies to IL-1 beta or TNF. It is therefore proposed that IL-1 alpha is the major osteolytic cytokine produced by keratocysts and that IL-6 and IL-1 may contribute to keratocyst growth by promoting epithelial cell proliferation and bone resorption, respectively.

Heterogeneity of bone resorbing factors produced by unstimulated murine osteoblasts in vitro and in response to stimulation by parathyroid hormone and mononuclear cell factors.

The bone resorbing activity of factors released from monolayer cultures of osteoblasts (OB) was examined by measurement of calcium released by neonatal mouse calvaria in vitro. Unstimulated conditioned media (CM) were found to contain significant bone resorbing activity, which was partially inhibited by indomethacin, dexamethasone and nordihydroguaiaretic acid. Ultrafiltration of CM (molecular weight cut-off of 5000) revealed bone resorbing activity in the filtrate and retentate. Fractionation of the CM by high-performance liquid chromatography revealed four major peaks of bone resorbing activity. Stimulation of the OB by mononuclear cell factor and parathyroid hormone significantly increased the synthesis and/or release of these factors with a relatively greater increase of lipid-soluble, low molecular-weight activity. These results suggested an important role for relatively small non-popular mediators in hormonally stimulated bone resorption.

Stimulation of bone collagen and non-collagenous protein synthesis by products of 5 : and 12-lipoxygenase : determination by use of a simple quantitative assay

The influence of 5- and 12-lipoxygenase products on the rate of collagen and non-collagenous protein (NCP) synthesis by murine calvarial explants has been investigated using a new assay based on the resistance of native collagen to degradation by pepsin. The reproducibility and simplicity of this assay allows the quantitative estimation of the rate of bone formation in large numbers of cultures. Hydroxyeicosatetraenoic acids (HETEs) stimulated both the rate of collagen and NCP synthesis with maximal stimulation occurring at 10-100 pM. All leukotrienes stimulated collagen synthesis. LTB4, C4 and D4 showed similar dose-responses with maximal activity occurring at 100 pM. LTE4 was less potent only showing activity at 1-10 nM. Only LTD4 demonstrated the capacity to stimulate NCP synthesis with significant stimulation being seen at 10 nM. The extreme sensitivity of bone collagen and NCP synthesis to lipoxygenase products suggests that these mediators may play a physiological role in bone remodelling.