Sajeda Meghji

Hypoxia is a Major Stimulator of Osteoclast Formation and Bone Resorption

Hypoxia is known to act as a general stimulator of cells derived from marrow precursors. We investigated the effect of oxygen tension on the formation and function of osteoclasts, the cells responsible for bore resorption, which are of promonocytic origin. Using 7‐ and 13‐day cultures of mouse marrow cells on ivory discs, we found that reducing oxygen tension from the ambient atmospheric level of 20% by increasing the proportion of nitrogen caused progressive increases in the formation of multinucleated osteoclasts and resorption pits. Peak effects occurred in 2% oxygen, where stimulations of resorption up to 21‐fold were measured. Significant stimulations of osteoclast formation and resorption were observed even in severely hypoxic cultures gassed with 0.2% oxygen. Short‐term cultures of cells disaggregated from rat bones indicated that hypoxia did not alter the resorptive activity of mature osteoclasts, but reduced their survival or adherence. In 3‐day organ cultures of mouse calvarial bones, exposure to 2% oxygen resulted in maximal, fivefold stimulation of osteoclast‐mediated calcium release, an effect equivalent to that of prostaglandin E2 (PGE2), a reference osteolytic agent. Hypoxia also caused a moderate acidosis in calvarial cultures, presumably as a result of increased anaerobic metabolism; this observation is significant because osteoclast activation is dependent on extracellular acidification. Our experiments reveal a previously‐overlooked mechanism of considerable potential importance for the regulation of bone destruction. These findings may help explain the bone loss associated with a wide range of pathological states involving local or systemic hypoxia, and emphasize the importance of the vasculature in bone.

The stimulation of bone formation in vitro by therapeutic ultrasound

A controlled study was performed to evaluate the effects of different ultrasound (US) intensities on 5-day-old mouse calvaria bone in tissue culture. A special technique to apply the US was developed, and the following parameters were measured: collagen and noncollagenous protein (NCP) synthesis (bone formation), and temperature change. It was found that ultrasound at 0.1 W/cm2 (SATA), pulsed 1:4, 3 MHz, 5 min, significantly stimulates bone formation (i.e., the synthesis of collagen and NCP) (p < 0.001 and p < 0.01). However, pulsed ultrasound at higher doses (1.0-2.0 W/cm2 (SATA), pulsed 1:4, 3 MHz, 5 min) significantly inhibited the synthesis of both collagen and NCP (p < 0.05). The temperature measurements showed a maximum rise of 1.8 degrees C [at 2.0 W/cm2 (SATA)] and no detected rise at 0.1 W/cm2 (SATA), suggesting that the effects in this study were primarily nonthermal. These results may reflect the healing effect of US on fractures and osteoradionecrosis and reinforces the use of low intensity US regimens [0.1 W/cm2 (SATA)] in clinical practice.

The role of endotoxin and cytokines in the pathogenesis of odontogenic cysts

Odontogenic cysts arise from tooth-forming epithelial residues. The stimulus for the formation of radicular cysts is thought to be endotoxin released from the infected necrotic tooth pulp. However, in keratocysts and follicular cysts, such a stimulus is not present. In order to investigate what drives the cyst epithelium to proliferate, explant media and fluids from 16 radicular cysts, eight keratocysts and seven follicular cysts and explant media from four specimens of non-inflamed gingival tissue were examined for the presence of endotoxin and cytokines. Cyst fluids were also cultured for 72 h in anaerobic and aerobic conditions to detect micro-organisms. Endotoxin from three different bacteria, cytokines [interleukin-(IL) 1 alpha, IL-1 beta and IL-6] as well as prostaglandin E2 (PGE2) were tested in an epithelial cell-proliferation assay. As the cyst epithelium is supported by a connective tissue capsule, the effect of fibroblast culture media on epithelial cell proliferation was also investigated. The results showed significantly higher concentrations of endotoxin in radicular cyst fluid than in the keratocyst or the follicular cyst. None of the cyst fluids contained micro-organisms. Immunoassays demonstrated the presence of IL-1 alpha and -6 in all fluids and explants tested; IL-1 beta was only found in the inflammatory radicular cysts. However, reverse transcriptase-polymerase chain reaction showed that mRNAs for IL-1 alpha, -1 beta and -6 were present in all cyst types. Proliferation studies indicated that endotoxin and the cytokines had a mitogenic effect on epithelia at low concentrations; PGE2 had very little effect at low concentrations, and had an inhibitory effect at high concentrations. Cyst fibroblast culture media had a mitogenic effect on the epithelia that was enhanced by the presence of endotoxin.

Staphylococcus aureus fibronectin binding proteins are essential for internalization by osteoblasts but do not account for differences in intracellular levels of bacteria

ABSTRACT Staphylococcus aureus is a major pathogen of bone that has been shown to be internalized by osteoblasts via a receptor-mediated pathway. Here we report that there are strain-dependent differences in the uptake of S. aureus by osteoblasts. An S. aureus septic arthritis isolate, LS-1, was internalized some 10-fold more than the laboratory strain 8325-4. Disruption of the genes for the fibronectin binding proteins in these two strains of S. aureus blocked their ability to be internalized by osteoblasts, thereby demonstrating the essentiality of these genes in this process. However, there were no differences in the capacity of these two strains to bind to fibronectin or osteoblasts. Analysis of the kinetics of internalization of the two strains by osteoblasts revealed that strain 8325-4 was internalized only over a short period of time (2 h) and to low numbers, while LS-1 was taken up by osteoblasts in large numbers for over 3 h. These differences in the kinetics of uptake explain the fact that the two strains ofS. aureus are internalized by osteoblasts to different extents and suggest that in addition to the fibronectin binding proteins there are other, as yet undetermined virulence factors that play a role in the internalization process.

Stimulation of bone resorption by lipoxygenase metabolites of arachidonic acid

We have studied the effect of leukotrienes, (LT): B4, C4, D4 and E4 and the hydroxyeicosatetraenoic acids (HETEs) 5-HETE and 12-HETE on bone resorption in vitro. Resorption was measured by colorimetric assay of calcium released from neonatal mouse calvaria maintained in organ culture for 72h. All the LTs and HETEs stimulated bone resorption, with optimum responses at picomolar or nanomolar concentrations. The responses were biphasic, with a decreasing effect at higher concentrations. In contrast, prostaglandin E2 (PGE2) stimulated resorption only at 10nM and above. Indomethacin partially inhibited resorption by LTB4, LTC4 and LTD4, but did not affect resorption stimulated by LTE4, 5-HETE and 12-HETE. These results indicate that lipoxygenase products of arachidonic acid are highly potent bone resorbing factors and may play an important role in the localised bone loss associated with inflammatory lesions.

Extracellular ADP is a powerful osteolytic agent: evidence for signaling through the P2Y(1) receptor on bone cells.

There is increasing evidence that extracellular nucleotides act on bone cells via P2 receptors. This study investigated the action of ADP and 2‐methylthioADP, a potent ADP analog with selectivity for the P2Y1 receptor, on osteoclasts, the bone‐resorbing multinuclear cells. Using three different assays, we show that ADP and 2–methylthioADP at nanomolar to submicromolar levels caused up to fourfold to sixfold increases in osteoclastic bone resorption. On mature rat osteoclasts, cultured for 1 day on polished dentine disks, peak effects on resorption pit formation were observed between 20 nM and 2 µΜ of ADP. The same concentrations of ADP also stimulated osteoclast and resorption pit formation in 10–day mouse marrow cultures on dentine disks. In 3–day explant cultures of mouse calvarial bones, the stimulatory effect of ADP on osteoclast‐mediated Ca2+ release was greatest at 5–50 µΜ and equivalent to the maximal effects of prostaglandin E2. The ADP effects were blocked in a nontoxic manner by MRS 2179, a P2Y1 receptor antagonist. Using in situ hybridization and immunocytochemistry, we found evidence for P2Y1 receptor expression on both osteoclasts and osteoblasts;thus, ADP could exert its actions both directly on osteoclasts and indirectly via P2Y1 receptors on osteoblasts. As a major ATP degradation product, ADP is a novel stimulator of bone resorption that could help mediate inflammatory bone loss in vivo.—Hoebertz, A., Meghji, S., Burnstock, G., Arnett, T. Extracellular ADP is a powerful osteolytic agent: evidence for signaling through the P2Y1 receptor on bone cells.—Hoebertz, A., Meghji, S., Burnstock, G., Arnett, T. Extracellular ADP is a powerful osteolytic agent: evidence for signaling through the P2Y1 receptor on bone cells. FASEB J. 15, 1139–1148 (2001)

Inhibition of HDAC activity by ITF2357 ameliorates joint inflammation and prevents cartilage and bone destruction in experimental arthritis.

Inhibition of histone deacetylases (HDAC) has been shown to modulate gene expression and cytokine production after stimulation with several stimuli. In the present study, the antiinflammatory effect of a potent HDACi, ITF2357, was explored in different experimental models of arthritis. In addition, the bone protective effect of ITF2357 was investigated invitro. Treatment of acute arthritis (Streptococcus pyogenes cell wall (SCW) arthritis) with ITF2357 showed that joint swelling and cell influx into the joint cavity were reduced. Furthermore, the chondrocyte metabolic function was improved by treatment of ITF2357. The production of proinflammatory cytokines by synovial tissue was reduced after ITF2357 treatment. To examine the effect of HDAC inhibition on joint destruction, ITF2357 was applied to both rat adjuvant arthritis and mouse collagen type II arthritis. ITF2357 treatment both ameliorates the severity scores in arthritis models and prevents bone destruction. In an in vitro bone destruction assay, ITF2357 was highly effective at a dose of 100 nmol/L. In conclusion, inhibition of HDAC prevents joint inflammation and cartilage and bone destruction in experimental arthritis.

DUAL ELEVATION OF CYCLIC-AMP AND INOSITOL PHOSPHATES IN RESPONSE TO MECHANICAL DEFORMATION OF MURINE OSTEOBLASTS

Mechanical deformation of bone cells was thought to be mediated via prostaglandin production and the cyclic AMP pathway. We present evidence that the phosphoinositide pathway is also activated by mechanical stress. We find that inositol phosphate production, but not glycerophosphoinositol production, is elevated, and the activation of adenylate cyclase is relatively small. These results are not compatible with the proposal that mechanical deformation of bone cells acts solely via prostaglandin synthesis.

The Escherichia coli Chaperonin 60 (groEL) Is a Potent Stimulator of Osteoclast Formation

Chaperonins (cpns) are intracellular oligomeric protein complexes that fold and refold proteins in a catalytic manner and aid in the transmembrane transport of cellular proteins. We reported previously that the lipopolysaccharide‐free recombinant cpn60 of Escherichia coli (groEL) is able to stimulate the breakdown of murine calvarial bone in culture and showed that such resorption is potently inhibited by an inhibitor of the enzyme cyclo‐oxygenase and to a lesser extent by inhibitors of 5‐lipoxygenase. In this study, we have investigated the effects of groEL on the resorptive activity and formation of osteoclasts in culture. In low density, osteoclast‐containing cultures from neonatal rats incubated for 24 or 96 h on dentine discs, groEL (1–1000 ng/ml) stimulated resorption pit formation up to 4‐fold, but this effect was essentially dependent on cell number. Using 12‐day cultures of mouse bone marrow to assess osteoclast recruitment, groEL (1–1000 ng/ml) caused a dramatic dose‐dependent stimulation of the formation of tartrate‐resistant acid phosphatase‐positive multinucleated cells and the resorption of the dentine on which bone marrow cells were cultured. Osteoclast formation elicited by groEL was almost completely abolished by indomethacin, an inhibitor of cyclo‐oxygenase, but was unaffected by inhibitors of 5‐lipoxygenase, suggesting that prostaglandins but not leukotrienes may mediate the action of groEL on osteoclastogenesis. It is possible that bacterial cpn60s such as groEL may play a role in the osteolysis associated with bone infections. Whether endogenous (“self”) chaperonins have a role in other bone loss disorders, such as osteoporosis, is an intriguing possibility.

Killing of the Yeast and Hyphal Forms of Candida albicans Using a Light-Activated Antimicrobial Agent

Abstract. Oral infections due to Candida albicans are a common occurrence in patients with acquired immunodeficiency syndrome (AIDS). The purpose of this investigation was to determine whether the yeast and hyphal forms of the organism could be killed using the light-activated antimicrobial agent toluidine blue O (TBO). Three variables were investigated: TBO concentration, laser light dose and pre-irradiation time (PIT). Irradiation with light from a helium neon (HeNe) gas laser used in conjunction with the photosensitiser TBO resulted in substantial kills of both the yeast and hyphal forms. Killing was light dose-dependent with 42 J being the most effective dose. The optimum PIT for the yeast form was 5 min, whereas killing of the hyphal form was not affected by PIT. The results of this study have shown that both forms of C. albicans are susceptible to lethal photosensitisation using TBO in conjunction with HeNe laser light, suggesting the possibility that this approach could be useful for eliminating the organism from diseased lesions in vivo.