Biomedical subjects

T J Chambers

Publications and source records attributed to T J Chambers.

At least 73 records · Page 4Linked to original sources

PubMed1995

Macrophage inflammatory protein-1 alpha and IL-8 stimulate the motility but suppress the resorption of isolated rat osteoclasts.

Cells of the osteoblastic lineage play a major role in the regulation of osteoclastic bone resorption. Recent studies have demonstrated production of chemokines by osteoblastic cells. Although these phagocyte-stimulating and proinflammatory cytokines act as chemoattractants and activators for other members of the hemopoietic lineage, their actions on osteoclasts have not been characterized. We found that macrophage inflammatory protein-1 alpha (MIP-1 alpha) and IL-8 inhibited bone resorption by rat osteoclasts, primarily through reduction in the proportion of osteoclasts resorbing bone, a pattern of inhibition previously observed in response to macrophage CSF (M-CSF). MIP-2, RANTES, MIP-1 beta, and monocyte chemotactic protein-1 were without effect on resorption. MIP-1 alpha and IL-8, but not the other chemokines, also stimulated osteoclastic motility and increased the osteoclast spread area in a dose-dependent manner, over the same concentration range as that which inhibited bone resorption. In addition, MIP-1 alpha induced osteoclast orientation in a gradient of the chemokine, and stimulated osteoclast migration. We detected no effect of chemokines on osteoclast formation or survival. Our data suggest that chemokines can promote osteoclast orientation and migration, processes that might be involved in chemotaxis; it seems appropriate that resorptive functions should be suppressed during migration. Because chemokines are proinflammatory, their actions on osteoclasts might represent mechanisms by which bone resorption is modulated by the inflammatory process when this occurs in bone. However, given that chemokines are increasingly recognized to be multifunctional and that they are produced by cells of the osteoblastic lineage, they may also be components of the physiologic regulation of bone resorption.

Animals↗

PubMed1995

The role of reactive oxygen intermediates in osteoclastic bone resorption.

Osteoclasts have been shown to produce reactive oxygen intermediates (ROI) and it has been suggested that ROI are involved in the process of bone resorption. ROI have also been shown to play a central role in the activation of the multisubunit transcription factor NF-kappa B that enhances the transcription of genes encoding defence and signaling proteins. Therefore, we have assessed the effect of pyrrolidine dithiocarbamate (PDTC), an oxygen-radical scavenger and metal chelator that is a selective and potent inhibitor of NF-kappa B activation, on osteoclastic bone resorption in the bone slice assay. PDTC (0.001-0.1 mM) dose-dependently and non-cytotoxically inhibited osteoclast activity with an IC50 of 0.01 mM. PDTC (0.01 mM) caused no change in the ratio of resorption pit area to resorption pit depth as measured by Lasertec confocal microscopy, indicating that ROI are not involved in the resorptive process per se. This view is supported by time-course studies showing that addition of PDTC or N-acetyl cysteine (NAC; an ROI scavenger, but not metal chelator), 6 hr after the start of the assay had no significant effect on subsequent bone resorption. Desferal (100 microM), a chelator of iron and other metal ions, had no significant effect on bone resorption, indicating (along with the results with NAC) that ROI-scavenging rather than metal chelation is responsible for inhibition of osteoclastic bone resorption by PDTC. Taken together these results indicate that ROI produced by osteoclasts in the bone slice assay are not involved in the process of bone resorption, but are important during osteoclast activation for bone resorption, possibly being involved in activation of the transcription factor NF-kappa B.

Acetylcysteine↗

PubMed1995

Stimulation of bone formation by dynamic mechanical loading of rat caudal vertebrae is not suppressed by 3-amino-1-hydroxypropylidene-1-bisphosphonate (AHPrBP).

We have recently developed an experimental system whereby pins inserted into the seventh and ninth caudal vertebrae of rat tails are used to load the eighth caudal vertebrae (C-8) in compression. In this model, a single 5-min period of dynamic loading, sufficient to induce strains within the range to which bones are exposed under physiological circumstances, stimulates lamellar bone formation in the cancellous bone of the vertebrae. The rapidity with which the increase in bone formation was induced raised the possibility that this bone formation might have occurred without prior resorption. To test the role of bone resorption in the response of the bone to mechanical stimulation, we compared the anabolic response to a single period of loading, of rats treated with 3-amino-1-hydroxypropylidene-1-bisphosphonate (AHPrBP) or vehicle. We found that mechanical loading caused a significant increase in dynamic and static indices of bone formation. The same indices were unaffected by AHPrBP, while the bone formation rate in the tibiae was reduced by AHPrBP. These results suggest that the increased bone formation induced by mechanical stimulation in the cancellous bone of rat vertebrae is not dependent on bone resorption.

Animals↗

PubMed1995

Mutagenesis of the yellow fever virus NS2B/3 cleavage site: determinants of cleavage site specificity and effects on polyprotein processing and viral replication.

The determinants of cleavage site specificity of the yellow fever virus (YF) NS3 proteinase for its 2B/3 cleavage site have been studied by using site-directed mutagenesis. Mutations at residues within the GARR decreases S sequence were tested for effects on cis cleavage of an NS2B-3(181) polyprotein during cell-free translation. At the P1 position, only the conservative substitution R-->K exhibited significant levels of cleavage. Conservative and nonconservative substitutions were tolerated at the P1' and P2 positions, resulting in intermediate levels of cleavage. Substitutions at the P3 and P4 positions had no effects on cleavage efficiency in the cell-free assay. Processing at other dibasic sites was studied by using transient expression of a sig2A-5(356) polyprotein. Cleavage at the 2B/3 site was not required for processing at downstream sites. However, increased accumulation of high-molecular-weight viral polyproteins was generally observed for mutations which reduced cleavage efficiency at the 2B/3 site. Several mutations were also tested for their effects on viral replication. Virus was not recovered from substitutions which blocked or substantially reduced cleavage in the cell-free assay, suggesting that efficient cleavage at the 2B/3 site is required for flavivirus replication.

Amino Acid Sequence↗

PubMed1995

Increased insulin-like growth factor I mRNA expression in rat osteocytes in response to mechanical stimulation.

We recently developed an experimental model whereby a single 10-min episode of mechanical stimulation induces bone formation in the eighth caudal vertebra of 13-wk-old rats. We used this model to relate the kinetics of the bone-forming response, as measured by administration of fluorescent markers, to an in situ hybridization analysis of changes in mRNA for two matrix proteins (type I collagen and osteocalcin) and a growth factor implicated in the regulation of bone formation [insulin-like growth factor I (IGF-I). We found that increased fluorochrome labeling was accompanied by an increase in the proportion of trabecular bone surfaces on which transcripts for collagen type I and osteocalcin were detectable, from < 3 to 25% 72 h after loading. IGF-I expression on trabecular surfaces showed a slightly earlier increase. We also noted intense hybridization for IGF-I in osteocytes in the diaphyseal cortex and in metaphyseal trabeculae. This was observed only in loaded bones, within 6 h of loading, and became undetectable in trabecular osteocytes 48 h and cortical osteocytes 120 h after loading. This is the first identification of a specific mRNA species in osteocytes after mechanical stimulation. Its production before the increase in transcription of matrix protein mRNA, and before the transcription of IGF-I mRNA in bone surface cells, represents persuasive evidence for a role for osteocytes, and for IGF-I, in the osteogenic response of bone to mechanical stimulation.

Animals↗

PubMed1995

Localisation of mRNA for collagenase in osteocytic, bone surface and chondrocytic cells but not osteoclasts.

Osteoclasts resorb the extracellular matrix of bone by secreting protons and enzymes into a circumpherentially sealed compartment between the osteoclast and the bone surface. Although the lysosomal cysteine proteinases play a major role in matrix degradation by osteoclasts, collagenase (matrix metalloproteinase-1, EC 3.4.24.7) is also required for osteoclastic bone resorption, and may be directly involved in collagen degradation in the hemivacuole. We assessed the effects of inhibitors of cysteine proteinases and collagenase on bone resorption by osteoclasts isolated from rodent bone. We found that while inhibition of cysteine proteinases strongly suppressed osteoclastic resorption, inhibitors of collagenase were without effect on the number, size, or demineralised fringe of excavations. We could find no evidence of expression of mRNA for collagenase in rat osteoclasts by in situ hybridisation, but found that it was expressed by chondrocytes, bone surface cells and osteocytes adjacent to osteoclasts. The distribution of these cells, and the correlation between increased collagenase production and increased stimulation of osteoclastic resorption in vitro by bone cells, suggests that these cells might be involved in the regulation of bone resorption in situ, and that collagenase production might play a role in this process.

Animals↗

PubMed1995

Distinct effects of ovarian transplantation and exogenous 17 beta-oestradiol on cancellous bone of osteopenic ovariectomized rats.

Although 17 beta-oestradiol (E2) is known to prevent bone loss, prolonged administration of E2 is unable to reverse this in female rats rendered osteopenic by ovariectomy. To determine whether this reflects a failure to replace other components of ovarian function involved in bone metabolism, we compared the effects of administering E2 to osteopenic ovariectomized (ovx) rats with those of ovarian transplantation. Ovariectomy was performed in female rats. After 13 weeks, by which time marked bone loss had occurred, one group of ovx animals received ovaries from donor rats, and, after a delay of 2 weeks to allow oestrus cycles to return, a further group received E2 5 micrograms.kg-1.day-1 for 9 weeks. The dose of E2 was chosen as that which in preliminary studies restored mean serum E2 levels to that of intact female rats. The study was terminated 24 weeks after ovariectomy. Both E2 and ovarian transplantation largely restored indices of oestrogenic exposure in ovx rats to those of sham-ovx animals. Animals receiving ovarian transplants also showed a small increase in serum progesterone and full restoration of serum testosterone. However, while ovarian transplantation also returned indices of cancellous bone metabolism to those of sham-ovx animals, there was little increase in bone volume. Interestingly, exogenous E2 caused a greater increase in cancellous bone volume than ovarian transplantation but also caused more marked suppression of bone formation, as assessed at the end of the study. In conclusion, exogenous E2 and ovarian transplantation exerted distinct effects on skeletal metabolism in osteopenic ovx rats, although the basis for this difference is currently unclear.

Animals↗

PubMed1994

Mutagenesis of the yellow fever virus NS2A/2B cleavage site: effects on proteolytic processing, viral replication, and evidence for alternative processing of the NS2A protein.

The yellow fever virus NS2B-3 proteinase mediates cleavages within the nonstructural region at a consensus sequence defined by G/ARR decreases S/G and also at an alternative site within the NS4A region. To determine the importance of specific residues within the consensus sequence for cleavage at the 2A/2B site, amino acid substitutions were introduced at each of the P4, P3, P2, P1, and P1' positions and the effects on proteolytic processing of a sig2A-5(356) polyprotein were examined using a vaccinia virus-T7 transient expression system. At the P1 and P1' positions, only the conservative substitutions P1:R-->K and P1':S-->G allowed efficient cleavage, suggesting that basic and small aliphatic amino acids are preferred at the P1 and P1' positions, respectively. At the P2 position, a preference for a basic amino acid was observed. In contrast, the P3 and P4 positions tolerated nonconservative substitutions and at P4 both enhancement and reduction in cleavage efficiency was observed. Evidence for cleavage at an alternative site within NS2A, defined by the sequence QK decreases T (NS2A residues 189-191) was obtained. Cleavage at this site, designated at NS2A alpha, is dependent upon an active NS2B-3 proteinase. To examine the effects of reduced cleavage efficiency at the 2A/2B and NS2A alpha cleavage sites on viral replication, mutations at each or both of these sites were incorporated into a full-length YF-17D cDNA template. RNA transcripts containing mutations which abolish cleavage were noninfectious whereas virus was recovered from several clones with mutations allowing partial cleavage at 2A/2B. However, some of these mutants exhibited a small plaque phenotype as well as reductions in RNA-specific infectivity and virus yield.

Amino Acid Sequence↗

PubMed1994

Automated three-dimensional characterization of osteoclastic resorption lacunae by stereoscopic scanning electron microscopy.

The use of stereoscopic scanning electron microscopy to analyze quantitatively the topography of excavations made by osteoclasts in slices of devitalized cortical bone was evaluated. Using this innovative technique, the need mechanically to tilt the specimen stage to gather three-dimensional information is obviated by instead tilting the electron beam both to produce real-time stereo pairs and to gather measurement data. Based on the comparison of two images of microscopic areas, cross-correlation is used to determine the image shift between the features in the stereo pair. This information is then used dynamically to correct the height of the tilt axis and lens focus in a feedback loop, generating a quantitative measurement of height difference. With this approach, relative heights of individual points, line profiles, area topography maps, and three-dimensional reconstructions of excavations were obtained rapidly and at high resolution. This approach combines the advantage in topographic data acquisition of confocal microscopy with the advantages of the increased resolution and focal depth of scanning electron microscopy (SEM). The technique should facilitate not only the topographic analysis of osteoclastic excavations in bone slices at high resolution but also the three-dimensional analysis of the structure of bone tissue.

Bone Resorption↗

PubMed1994

Effect of administration and subsequent cessation of buserelin on cancellous bone of female rats.

Although hormone replacement therapy is effective in preventing postmenopausal bone loss, it fails to cause a return of bone mass to normal in patients with established osteoporosis. Similarly, in the ovariectomized rat, estrogen administration protects the skeleton from bone loss but fails to reverse this once it has occurred. However, physiologically produced sex steroids may, in contrast to conventional methods of sex steroid administration, be capable of restoring bone mass in osteopenic states. To investigate this question, we analyzed the effect of treatment with the LHRH agonist buserelin for varying durations, and subsequent cessation thereof, on histomorphometric indices of rat cancellous bone. Female rats 13 weeks old were given daily SC injections of vehicle or buserelin as follows: vehicle days 1-90; vehicle days 1-150; vehicle days 1-60, buserelin days 61-90; vehicle days 1-60, buserelin days 61-90, vehicle days 91-150; vehicle days 1-30, buserelin days 31-90; vehicle days 1-30, buserelin days 31-90, vehicle days 91-150; buserelin days 1-90; buserelin days 1-90, vehicle days 91-150. At the end of the treatment period, animals were killed, tibiae removed, and histomorphometric indices assessed at the secondary spongiosa of the proximal metaphysis. Analysis of vaginal smears confirmed that buserelin rapidly suppressed ovulation, which quickly returned once treatment was stopped. We found that administration of buserelin for 30, 60, or 90 days reduced cancellous bone volume because of a reduction in both the number and thickness of trabeculae.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

A comparison of bone mineral density between Caucasian, Asian and Afro-Caribbean women.

1. We analysed the lumbar spine (L2-L4) and femoral neck bone mineral density results of Caucasian (n = 2232), Asian (Indian sub-continent) (n = 153) and Afro-Caribbean (n = 102) women referred for bone densitometry over a 30 month period. To assess the risk of osteoporosis, the results of Caucasian and Asian women were compared with those of a reference Caucasian population supplied by Lunar. 2. Subject characteristics were similar in all three groups, other than expected ethnic differences in stature and weight. We found that lumbar spine and femoral neck bone mineral density in Caucasians was lower than in Afro-Caribbeans, but higher than in Asians. Consistent with this, bone mineral density was also lower in Asians as compared with the reference Caucasian population, both at the lumbar spine and femoral neck. As a consequence, a higher proportion of Asian women were classified as being at increased risk of osteoporosis than Caucasian women. 3. Since ethnic differences in skeletal size might influence bone mineral density, we also obtained values for bone mineral content in Caucasian and Asian women that were corrected for projected skeletal area, and weight and years since menopause, using regression equations derived from the Caucasian study population. After this analysis, the difference in bone mineral content between Caucasians and Asians at the lumbar spine disappeared, while that at the femoral neck persisted. 4. We conclude that the assessment of risk of osteoporosis in Asian women by comparing bone mineral density with a reference Caucasian population may have limited validity because of the influence of skeletal size on such measurements.

Absorptiometry, Photon↗

PubMed1994

Indomethacin has distinct early and late actions on bone formation induced by mechanical stimulation.

The capacity of bone to adapt its architecture in response to changing mechanical demands is well recognized. However, the mechanisms by which mechanical stimuli are translated into new bone formation are poorly understood. Prostaglandins (PGs) may play a role. We therefore tested the effect of indomethacin on the cancellous bone formation induced by mechanical stimulation in the 8th caudal vertebrae of adult rats. Rats were given indomethacin 3 h before loading, 3 h before loading and daily thereafter, 6 h after loading, or 6 h after loading and daily thereafter. The increase in bone formation caused by loading was suppressed by a single dose of indomethacin if given before but not after loading. Daily administration of indomethacin suppressed the mechanical response, even when started after loading. These results suggest that PGs are essential for the transduction of mechanical stimuli into bone formation, and also that there may be two distinct phases of PG dependency in the response of bone to mechanical loading: an early phase associated with the immediate loading period and a later phase associated with osteogenic interactions entrained by the early phase.

Animals↗

PubMed1994

5 alpha-Dihydrotestosterone partially restores cancellous bone volume in osteopenic ovariectomized rats.

Although androgens are thought to be important for skeletal maintenance in females and males, little is known about the mechanisms involved. To investigate this question further, we examined the effects of administering 0.01, 0.1, or 1.0 mg/kg 5 alpha-dihydrotestosterone (DHT) for 60 days on the skeleton of ovariectomized rats. Treatment was delayed until 90 days after ovariectomy to enable bone loss to stabilize. We found that ovariectomy markedly reduced cancellous bone volume of the proximal tibial metaphysis due to a combination of loss and thinning of trabeculae. Cancellous bone volume was partially restored by all doses of DHT, with trabecular thickness, but not number, returning to that of sham-operated animals. DHT also stimulated longitudinal bone growth and endosteal and periosteal bone formation and suppressed histomorphometric indexes of cancellous bone resorption. This suggests that DHT influences skeletal metabolism in osteopenic ovariectomized rats both by stimulating bone formation and suppressing resorption, although it is unclear which, if any, of these actions predominate at cancellous sites.

Animals↗

PubMed1994

Intermittent retinoic acid in combination with continuous oestradiol-17 beta increases cancellous bone volume in osteopaenic ovariectomized rats.

Although short-term administration of oestradiol-17 beta (OE2) stimulates cancellous bone formation in the rat, this is replaced by a tendency to suppression after prolonged treatment. Hence, in rats rendered osteopaenic by ovariectomy, OE2 administration fails either to induce a sustained increase in bone formation or to restore bone volume. A possible explanation for this failure is that OE2 also inhibits bone resorption, secondarily suppressing bone formation through coupling mechanisms. We therefore investigated whether the effects of OE2 treatment might be modified by intermittently stimulating bone resorption with retinoic acid (120mg/kg daily) for 4 out of every 20 days. We found, in a preliminary experiment using intact animals, that intermittent retinoic acid reduced cancellous bone volume, consistent with previously documented stimulation of bone resorption by retinoic acid. Rats were then rendered osteopaenic by ovariectomy, and given vehicle, retinoic acid and/or OE2. We found that animals treated with intermittent retinoic acid and OE2 showed a substantial increase in cancellous bone volume compared with ovariectomized animals treated with vehicle, retinoic acid alone or OE2 alone. Therefore, intermittent retinoic acid appears to cause a net increase in bone formation over resorption when given to ovariectomized animals in conjunction with OE2. We conclude that the effects of OE2 on cancellous bone are modified by intermittent treatment with retinoic acid, resulting in a substantial increase in bone volume.

Animals↗

PubMed1994

The rate of cancellous bone formation falls immediately after ovariectomy in the rat.

We have recently found that administration of oestradiol-17 beta (OE2) to rats stimulates trabecular bone formation. It is not known, however, whether oestrogen has a similar action on bone formation rate under physiological circumstances. Oestrogen is known to suppress bone resorption, and oestrogen-deficient states in the rat, as in humans, are associated with an increase in bone resorption that entrains an increase in bone formation. To see if the latter masks a relative reduction in bone formation, due to oestrogen deficiency, we measured bone formation very early after ovariectomy, before the resorption-induced increase in bone formation becomes established. To do this, rats were administered fluorochrome labels before and after ovariectomy, spaced at weekly intervals in the first, and 3-day intervals in the second experiment. In both experiments there was a decrease in indices of bone formation in the labelling interval immediately following ovariectomy such that, using the shorter fluorochrome intervals, the mineral apposition rate fell to 69%, the double-labelled surface to 45%, and the bone formation rate to 36% of sham-ovariectomized levels. The reduction was not sustained in the subsequent label intervals, presumably masked by the increase in bone formation attributable to increased resorption. These results suggest that if bone formation is assessed before this resorption-entrained increase in bone formation occurs, oestrogen deficiency is associated with a reduction in dynamic indices of bone formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

The effects of ovarian transplantation on bone loss in ovariectomized rats.

Although hormone replacement therapy can prevent postmenopausal bone loss, it does not restore bone mass to normal in patients with established osteoporosis. This might reflect a failure to reproduce certain aspects of gonadal function. One method of investigating this possibility would be to examine the effect of ovarian transplantation on the skeleton of osteopaenic ovariectomized rats. However, ovarian transplantation may not fully restore ovarian function to normal, and it is not known whether transplanted ovaries reproduce the action of native ovaries on the skeleton. Therefore, we investigated whether renal capsular or subcutaneous ovarian transplants prevent the effects of ovariectomy on histomorphometric indices of rat tibiae over 44 days. Daily vaginal smears showed that oestrous cycles returned in all but two of 25 animals receiving ovarian transplants. We found that ovarian transplantation prevented the reduction in cancellous bone volume following ovariectomy. While trabecular number was reduced in ovariectomized animals receiving renal capsular ovarian transplants compared to intact animals, trabecular thickness was increased in both transplant groups. Ovarian transplantation also prevented the increase in cancellous and cortical bone formation, cancellous bone resorption and longitudinal growth rate caused by ovariectomy. We conclude that restoration of ovarian function by ovarian transplantation largely prevents the effects of ovariectomy on histomorphometric indices of rat tibiae, suggesting that transplanted ovaries can substitute for the action of native ovaries on the skeleton.

Animals↗

PubMed1994

Histomorphometric changes in the skeleton of postmenopausal women with low bone mineral density treated with percutaneous estradiol implants.

OBJECTIVE: To identify the effects of percutaneous estradiol (E2) implants on bone histology and bone mass of postmenopausal women with low bone mineral density. METHODS: Sixteen postmenopausal women with low bone mineral density were treated with 75-mg E2 implants. Each had iliac crest bone biopsies performed, following double-tetracycline labeling, before treatment and 1 year later. Dual energy x-ray absorptiometry of the lumbar spine and proximal hip was also performed before and after 1 year of therapy. Serum E2 and FSH were measured after 1 year. RESULTS: There were significant reductions in the osteoid volume, osteoid surface, eroded surface, and activation frequency following treatment. There was a statistically insignificant increase in the median bone volume from 11.3 to 15.8%. The median percentage increase in bone density at the lumbar spine was 14.4% and at the total hip 5.3%. The median post-treatment serum E2 level was 570 pmol/L. CONCLUSIONS: Estradiol implants reduce bone turnover in the iliac crest without significantly increasing trabecular bone volume after 1 year. The increases in bone density at the spine and hip may be explained by increased mineralization within the existing trabecular bone. However, the iliac crest may not represent the effect of estrogen at sites susceptible to osteoporotic fracture. If resorption is suppressed more than formation, then new bone would be deposited to account for the increase in bone density.

Aged↗

PubMed1993

Purification and characterization of a tripeptidyl peptidase I from human osteoclastomas: evidence for its role in bone resorption.

Tripeptidyl peptidase I (EC 3.4.14.9), which cleaves tripeptides from the N-terminus of synthetic substrates, has been purified from human osteoclastomas (a bone tumor containing large numbers of normal osteoclasts). The enzyme has an M(r) of 48 kDa but forms aggregates with an M(r) of about 700 kDa. The tripeptidyl peptidase has an acidic pH optimum (approximately pH 5.0), suggesting that it has a lysosomal localization and prefers substrates with a hydrophobic amino acid in the P1 position. There is an absolute requirement for a nonsubstituted N-terminus. The enzyme is inhibited by reagents which modify serine and histidine residues. Lysosomal tripeptidyl peptidase is known to be capable of cleaving Gly-Pro-X triplets from synthetic collagen-like polypeptides. Ala-Ala-Phe-CH2Cl, a potent inhibitor of osteoclastoma tripeptidyl peptidase, inhibits osteoclastic bone resorption in an in vitro test system. This suggests that tripeptidyl peptidase I, secreted by osteoclasts, is involved at some stage in the degradation of bone collagen.

Amino Acid Sequence↗