Biomedical subjects

T J Chambers

Publications and source records attributed to T J Chambers.

At least 163 records · Page 9Linked to original sources

PubMed1989

Calcitonin receptors as markers for osteoclastic differentiation: correlation between generation of bone-resorptive cells and cells that express calcitonin receptors in mouse bone marrow cultures.

The osteoclast is the cell that resorbs bone. It is known to derive from hemopoietic precursors, but analysis of lineage and regulation of differentiation has been hampered by lack of a specific marker that enables identification of cells of osteoclastic phenotype. Previously used markers, such as multinuclearity, that are specific for osteoclasts in bone become less specific in culture. Uniquely among bone and bone marrow cells, osteoclasts possess abundant calcitonin (CT) receptors. We therefore tested the correlation between the generation of bone-resorptive function and the formation of CT receptor-positive cells from hemopoietic tissue in vitro. Without 1,25-dihydroxy-vitamin D3 [1,25-(OH)2D3], a hormone that induces osteoclastic differentiation in vitro, bone marrow cultures showed very little bone resorption, and only small numbers of CT receptor-positive cells developed. When 1,25-(OH)2D3 was added to the cultures, CT receptor-positive cells developed within 1 day and reached a peak after 7 days. Bone resorption commenced within 2 days of hormone addition. There was a strong parallelism between the cumulative number of CT receptor-positive cells and the extent of bone resorption. The capacity of cultures to generate bone-resorptive activity and CT receptor-positive cells declined progressively when 1,25-(OH)2D3 was added to hemopoietic tissue after a 7- to 21-day hormone-free incubation period. The number of CT receptor-positive cells in these cultures correlated strongly (r = 0.96) with bone resorption. The behavior of these cultures suggests that 1,25-(OH)2D3 acts to induce terminal differentiation of osteoclast precursors present in the cultures, and that precursor cell numbers decreased with increasing time in vitro. All of the CT receptor-positive cells in control cultures and all of those seen shortly after 1,25-(OH)2D3 addition were mononuclear, despite considerable bone resorption; the majority of CT receptor-positive cells remained mononuclear throughout the incubation period. This suggests that mononuclear cells with characteristics of osteoclasts exist that are able to excavate bone. CT receptor-positive cells slightly preceded the development of bone-resorptive function, implying that CT receptors develop before the acquisition of bone-resorptive capacity by osteoclasts. Peritoneal macrophages, blood mononuclear cells, and cells of the J774 macrophage cell line failed to either resorb bone or express CT receptors, even after incubation with 1,25-(OH)2D3 for 14 days. These results show a strong and specific correlation between the generation of bone-resorptive cells and CT receptor-positive cells, and suggest that CT receptor express

Animals↗

PubMed1989

Transcription of infectious yellow fever RNA from full-length cDNA templates produced by in vitro ligation.

Yellow fever (YF) virus is the prototype member of the flavivirus family, a diverse group of human and animal pathogens. A live-attenuated strain of YF virus, called 17D, has been used successfully for human vaccination for more than 50 years. In this report we describe the construction of full-length YF 17D cDNA templates that can be transcribed in vitro to yield infectious YF virus RNA. Because of the instability of full-length YF cDNA clones and their toxic effects on Escherichia coli, we developed a strategy in which full-length templates for transcription were constructed by in vitro ligation of appropriate restriction fragments. The YF virus recovered from cDNA was indistinguishable from the parental virus by several criteria. This system should facilitate the molecular genetic analysis of flavivirus replication and attenuation and may allow YF 17D to be used as a carrier for immunologically important epitopes from other disease agents.

Base Sequence↗

PubMed1988

The multinucleate cells in giant cell granulomas of the jaw are osteoclasts.

The giant cell granuloma of jaw is a well-vascularised lesion comprising a mononuclear cell infiltrate with a large number of giant cells. It has been suggested that the lesion is reparative in nature, rather than neoplastic, and that the giant cells are phagocytes accumulating in chronic reparative granulation tissue. However, the nature of the multinucleate giant cells never has been established. One possibility is that the constituent giant cells are osteoclasts. The authors assessed expression by the giant cells of several osteoclast-specific characteristics: excavation of bone; motility inhibition by calcitonin (CT); and binding of osteoclast specific monoclonal antibodies. Two tumors were disaggregated and incubated on slices of cortical bone in the presence and absence of CT. Both tumors were found to excavate bone, a function unique to osteoclasts. The giant cells also were responsive to CT, resulting in cytoplasmic quiescence and inhibition of bone resorption. Two osteoclast-specific monoclonal antibodies bound all the giant cells in one central and six peripheral tumors examined immunohistochemically. These results provide strong evidence for the osteoclastic nature of the giant cells. The presence of alkaline phosphatase-positive cells forming woven bone in giant cell granulomas suggests that osteoblasts are present in the lesion. As cells of osteoblastic lineage are known to regulate osteoclastic function, it may be that osteoblasts account for the characteristic infiltration of osteoclasts into giant cell granulomas of jaws, either as part of a reparative response by reactive osteoblasts or as an infiltrate induced by osteoblasts of aberrant function, as suggested for giant cell tumors of bone.

Antibodies, Monoclonal↗

PubMed1988

Mycobacterial meningomyelitis associated with human immunodeficiency virus infection.

A homosexual man, seropositive for human immunodeficiency virus, developed back and leg pain that evolved, over three weeks, into a T-10 anesthetic, areflexic paraplegia. Spinal fluid examination showed lymphocytosis, markedly elevated spinal fluid protein, and hypoglycorrhachia. A spinal cord biopsy specimen disclosed an intramedullary granuloma containing acid-fast bacilli. The patient was treated with antituberculous drugs and had no progression of neurologic deficit. He died, eight months after first becoming ill, of Klebsiella pyelonephritis and septicemia. Mycobacterial meningomyelitis is presently the only known acquired immunodeficiency syndrome-related myelopathy responsive to specific treatment.

Acquired Immunodeficiency Syndrome↗

PubMed1988

The regulation of osteoclastic development and function.

Cells of the osteoblastic lineage exert a dominant influence on osteoclastic bone resorption. They form a communicating network of osteocytes, surface osteocytes and osteoblasts that seems well placed to monitor the structure and performance of bone and to judge where bone formation or resorption is appropriate. Osteoblasts produce prostaglandins (PGs) which strongly inhibit osteoclastic resorption. None of the agents that stimulate resorption in intact bone, such as parathyroid hormone (PTH), interleukin 1 (IL-1), 1,25-(OH)2 vitamin D3 (1,25-(OH)2D3) or tumour necrosis factors, affects isolated osteoclasts, but all induce osteoblastic cells to produce osteoclastic resorption stimulatory activity (ORSA) that acts directly on osteoclasts. Osteoblasts seem to initiate resorption as well as stimulating or inhibiting it. Contact with bone mineral appears to be necessary: osteoclasts resorb mineralized but not unmineralized bone. All bone surfaces are lined by unmineralized organic material. Osteoblastic cells secrete neutral proteases, including collagenase, in response to hormonal stimulators of bone resorption. Incubation of osteoblasts, in the presence of PTH, on such surfaces or preincubation of the bone with collagenase predisposes bone to osteoclastic resorption. Agents that stimulate resorption in organ cultures seem to share these osteoblast-mediated mechanisms for induction and stimulation of resorption but 1,25-(OH)2D3 stimulates it through an additional mechanism. We have found that osteoclasts can be induced from haemopoietic tissue (including haemopoietic spleen cells) in the presence of 1,25-(OH)2D3--PTH and IL-1 have no effect in this system. Because osteoclasts lack receptors for 1,25-(OH)2D3 these results suggest either that osteoclast precursors lose 1,25-(OH)2D3 receptors during differentiation, or that a 1,25-(OH)2D3-responsive accessory cell in bone marrow induces osteoclastic differentiation in the presence of 1,25-(OH)2D3.

Animals↗

PubMed1988

Human macrophage colony-stimulating factor inhibits bone resorption by osteoclasts disaggregated from rat bone.

Colony stimulating factors (CSFs) regulate the survival, proliferation and differentiation of haemopoietic progenitor cells, as well as the functional activity of mature cells. Because the osteoclast is derived from haemopoietic tissue, and because osteoblastic cells produce CSFs, we tested the effects of several CSFs on bone resorption by osteoclasts disaggregated from neonatal rat long bone. We found that recombinant macrophage (M)-CSF was a potent inhibitor of bone resorption, causing significant inhibition at concentrations similar to those required to support the growth of macrophage colonies in agar. Unlike other inhibitors of osteoclastic resorption, M-CSF did not alter cytoplasmic motility in time-lapse recordings, suggesting that M-CSF may inhibit osteoclasts through a different transduction mechanism. None of the remaining cytokines tested (granulocyte-macrophage CSF, interleukin 3, interleukin 6, or interferon gamma) influenced bone resorption. M-CSF production may be a mechanism by which osteoblastic cells, which produce M-CSF, may regulate osteoclastic function. Alternatively, inhibition of osteoclastic resorption by a CSF that is responsible for amplification of the macrophage compartment may reflect a close lineage relationship between mononuclear phagocytes, in which M-CSF induces a diversion of lineage resources away from osteoclastic function.

Animals↗

PubMed1988

Effects of peptides from the calcitonin genes on bone and bone cells.

The calcitonin-calcitonin gene-related peptide (CGRP) gene complex encodes a family of novel peptides--calcitonin, CGRP and katacalcin. Whereas calcitonin is a circulating hormone involved in skeletal maintenance, the physiological function of CGRP still remains unclear. In the present study we have compared the biological activity of CGRP with that of calcitonin using three experimental systems. We have demonstrated that both peptides inhibit bone resorption by active rat osteoclasts and thus lower plasma calcium when injected into young rats. In both respects the CGRP homologues (rat, human alpha and human beta) were found to be 100- to 1000-fold less potent than human calcitonin. The effects of the CGRP peptides and calcitonin were only additive. Human CGRP (alpha) also caused a marked dose-dependent elevation of bone cyclic AMP levels in mice, somewhat like calcitonin. Though from our studies it would seem reasonably clear that CGRP is weakly agonistic for the calcitonin receptor on the osteoclast to produce effects on bone resorption and plasma calcium, it is still unclear whether the elevation of bone cyclic AMP simply represents an osteoclastic effect or an additional, more important, effect on osteoblasts. It is highly unlikely that CGRP may exert systemic effects on bone. Nevertheless, the peptide may be an important local regulator of bone cell function.

Animals↗

PubMed1987

Tumor necrosis factors alpha and beta induce osteoblastic cells to stimulate osteoclastic bone resorption.

Antigen- or mitogen-stimulated leukocytes release bone-resorbing activity into culture supernatants in vitro. Among the agents likely to be present in such supernatants are monocyte-derived tumor necrosis factor (TNF-alpha) and lymphocyte-derived tumor necrosis factor (TNF-beta) (lymphotoxin), both of which have recently been shown to stimulate bone resorption in organ culture. To identify the mechanism of action of these agents, we compared bone resorption by isolated osteoclasts with bone resorption by osteoclasts cocultured with osteoblastic cells, and with bone resorption by osteoclasts incubated with supernatants from osteoblastic cells, in the presence and absence of recombinant TNF-alpha and TNF-beta. We found that neither TNF-alpha nor TNF-beta had any significant effect on bone resorption by isolated osteoclasts, but in the presence of osteoblasts the agents caused a twofold to threefold stimulation of bone resorption. A similar degree of stimulation was achieved by supernatants from osteoblasts incubated with TNF before addition to osteoclasts, compared with supernatants to which TNF were added after osteoblast incubation. These experiments suggest that TNF-alpha and TNF-beta stimulate bone resorption through a primary effect on osteoblastic cells, which are induced by TNF to produce a factor that stimulates osteoclastic resorption. Half-maximal stimulation of resorption occurred at 1.5 X 10(-10) M and 2.5 X 10(-10) M for TNF-alpha and TNF-beta, respectively. This degree of potency is comparable to that of parathyroid hormone, the major physiologic systemic regulator of bone resorption, and suggests that the TNF may exert a significant influence on osteoclastic bone resorption in vivo.

Animals↗

PubMed1987

Hormonal regulation of acid phosphatase release by osteoclasts disaggregated from neonatal rat bone.

Osteoclasts disaggregated from neonatal rat long bones and incubated on plastic or glass substrates were found to release a considerable proportion of tartrate-resistant acid phosphatase into culture supernatants. Enzyme release was detectable in the supernatant medium of cultures containing as few as ten cells after 1 hr of incubation and proceeded in a linear manner for the ensuing 6 hr. Calcitonin (1 pg/ml) and cytochalasin B (5 micrograms/ml) inhibited release into the supernatant, suggesting that release represents enzyme secretion. Prostaglandin E1 induced transient inhibition followed by recovery; parathyroid hormone and 1,25(OH)2 vitamin D3 were without influence. Acid phosphatase release in these cultures shows a pattern of hormone responsiveness that coincides with the effects of these hormones on bone resorption by isolated osteoclasts. The extent of acid phosphatase release and its regulation by calciotropic hormones imply a central role for acid hydrolase secretion in osteoclastic bone resorption. The experimental system described in this study may facilitate analysis of the pharmacological hormonal and cellular regulation of osteoclastic function.

Acid Phosphatase↗

PubMed1987

Generation of osteoclasts in cultures of rabbit bone marrow and spleen cells.

The primary and specific function of the osteoclast is the resorption of bone. We have applied this criterion, and a monoclonal antibody that binds specifically to osteoclasts, to cultures of tissues that may contain osteoclastic precursors. Bone marrow and spleen cells were incubated for up to 4 weeks in the presence or absence of parathyroid hormone, interleukin 1, or 1,25(OH)2 vitamin D3, on plastic coverslips or slices of devitalised bone. Osteoclasts (as judged by the presence of resorption cavities and the appearance of monoclonal antibody-positive cells) did not develop in cultures incubated without added hormones, nor in cultures containing parathyroid hormone or interleukin 1, but were regularly observed when bone marrow cells were incubated with 1,25(OH)2 vitamin D3. Although multinucleate giant cells were common after incubation, especially in the presence 1,25(OH)2 vitamin D3, monoclonal antibody bound not to these cells but to a minor and distinctive population of mononuclear cells and cells of low multinuclearity. We found no excavations and no monoclonal antibody-positive cells after incubation of peritoneal macrophages with 1,25(OH)2D3. These results provide direct evidence of osteoclastic function arising in cultures of haemopoietic tissues.

Animals↗

PubMed1987

Calcitonin gene-related peptide inhibits osteoclastic bone resorption: a comparative study.

Besides the calcitonin (CT) precursor, the calcitonin gene also encodes another peptide--calcitonin gene-related peptide (CGRP). We have previously reported that CGRP lowers plasma calcium in the rat. In the present study we have evaluated the effect of CGRP on resorption of bone by isolated rat osteoclasts and have compared these effects to those produced by calcitonins from three species (salmon, pig, and human calcitonins). There was a significant inhibition of bone resorption with rat calcitonin gene-related peptide (rCGRP) at a 1000-fold higher dose than that used for human CT. This effect well explains the CT-like effect of CGRP seen in the in vivo rat CT bioassay. Our results suggest that though CGRP may not be involved in the hormonal control of plasma calcium, the peptide may be an important local regulator of bone cell function.

Animals↗

PubMed1987

1,25-Dihydroxyvitamin D3 stimulates rat osteoblastic cells to release a soluble factor that increases osteoclastic bone resorption.

Although 1,25-dihydroxyvitamin D3 stimulates osteoclastic bone resorption in vivo and in organ culture, the mechanism by which it effects this stimulation is unknown. We have recently found that the agent does not stimulate resorption by osteoclasts mechanically disaggregated from bone and incubated on slices of cortical bone. This suggests that the osteoclasts were removed by disaggregation from the influence of some cell type, present in intact bone, that mediates hormone responsiveness. We therefore tested the ability of osteoblastic cells derived from neonatal rat calvariae and of cloned, hormone-responsive osteosarcoma cells (UMR106) to restore hormone responsiveness to unresponsive populations of osteoclasts. We found that osteoblastic cells from both sources induced a two- to fourfold stimulation of osteoclastic bone resorption in the presence of 1,25-dihydroxyvitamin D3. Stimulation was observed at concentrations of 10(-10) M and above. Actinomycin D and cycloheximide did not affect bone resorption by osteoclasts incubated alone, but abolished the capacity of osteoblastic cells to stimulate osteoclastic resorption in the presence of 1,25-dihydroxyvitamin D3. When calvarial cells or osteoblastlike UMR cells were incubated with the hormone, they produced a factor in cell-free supernatants that stimulated bone resorption by disaggregated osteoclasts. These experiments suggest that 1,25-dihydroxyvitamin D3 stimulates bone resorption through a primary action on osteoblastic cells, that are induced by the hormone to produce a factor that stimulates osteoclastic bone resorption.

Animals↗

PubMed1987

A direct action of human calcitonin gene-related peptide on isolated osteoclasts.

The calcitonin gene encodes a small family of peptides: calcitonin, calcitonin gene-related peptide (CGRP) and katacalcin. Whereas calcitonin is concerned with skeletal maintenance, the function, if any, of katacalcin is still unknown. In the present study we have assessed resorption of human cortical bone substrate by isolated rat osteoclasts and have shown that CGRP acts directly on the osteoclast to inhibit bone resorption. The three CGRP peptides (rat, human(alpha) and human(beta) caused an almost equivalent decrease in osteoclastic bone resorption and were approximately 1000-fold less potent than human calcitonin in this respect. The responses of human calcitonin and human CGRP(alpha) were additive. Furthermore, prior treatment with trypsin to destroy receptors abolished the responsiveness of osteoclasts to CGRP and calcitonin. The carboxyl- and amino-terminal fragments of CGRP were found not to inhibit bone resorption, suggesting that the whole molecule of CGRP is necessary for biological activity. We have therefore suggested that the calcitonin-like effects of CGRP, seen both in vivo in the rat bioassay and in vitro in organ cultures, are due to the direct action of CGRP on the osteoclast, probably mediated through the calcitonin receptor. Though it is unlikely that CGRP is involved in the regulation of plasma calcium, the peptide may be an important local regulator of bone cell function.

Animals↗

PubMed1987

Molecular biology of the flaviviruses.

An overview of the molecular biology of the flaviviruses is presented. The members of this virus family are enveloped positive-strand RNA viruses capable of causing a number of important human diseases.

Animals↗

PubMed1986

Osteoblasts mediate interleukin 1 stimulation of bone resorption by rat osteoclasts.

A monocyte-derived factor with IL-1-like properties has recently been shown to cause resorption of bone in organ culture. We have investigated the action of IL-1 on disaggregated populations of osteoclasts, incubated alone or in the presence of osteoblastic cells, in an attempt to identify the target cell for IL-1 in bone, and to elucidate the mechanism by which IL-1 induces osteoclastic resorption. Osteoclasts were disaggregated from neonatal rat long bones and incubated on slices of human femoral cortical bone. Under these conditions, the majority of osteoclasts form distinctive excavations in the bone surface within 24 h, the volume of which can be quantified by computer-assisted morphometric and stereophotogrammetic techniques. IL-1 had no effect on bone resorption by osteoclasts alone, but when incubated in the presence of calvarial cells or cloned osteosarcoma cells, it induced a 3.8 (+/- 0.38)-fold increase in osteoclastic bone resorption, with significant enhancement at concentrations of greater than or equal to 30 pg/ml. The osteoblastic populations themselves did not resorb bone. The mechanism by which osteoblastic cells stimulate osteoclasts did not appear to depend upon PG synthesis; nor could we detect a diffusible substance in the medium of stimulated cocultures. These results indicate that IL-1 stimulates bone resorption through a primary action on osteoblasts, which are induced by IL-1 to transmit a short-range signal that stimulates osteoclastic bone resorption.

Animals↗

PubMed1986

Giant cell formation in rabbit long-term bone marrow cultures: immunological and functional studies.

A method for the long-term culture of rabbit newborn bone marrow has been developed. It is characterized by the rapid appearance of an adherent, adipocyte-containing stromal layer, proliferation of mature myeloid cells, and the formation of numerous, large multinucleate giant cells. By the combined use of morphological, immunological, and functional criteria these giant cells have been characterized as macrophage polykaryons and not osteoclastic giant cells. We conclude that long-term bone marrow culture in the rabbit favors the proliferation, maturation, and fusion of macrophage, but not osteoclast, precursors--new experimental models will have to be developed to enable the developmental biology of osteoclasts to be studied in the rabbit.

Animals↗

PubMed1986

Osteoblastic cells mediate osteoclastic responsiveness to parathyroid hormone.

Indirect evidence suggests that cells of the osteoblastic lineage may mediate augmented osteoclastic bone resorption induced by PTH. To test this suggestion, osteoclasts were disaggregated from neonatal rat long bones and incubated on slices of human femoral cortical bone. Resorption was measured by computer-assisted morphometric and stereophotogram-metric quantification of osteoclastic excavations, identified in the scanning electron microscope after culture. We compared the effect of PTH on bone resorption by osteoclasts incubated alone with the effect of the hormone on resorption by osteoclasts cocultured with osteoblastic cells. PTH had no effect on bone resorption by osteoclasts alone, but in the presence of any of three osteoblast-containing cell populations, or in the presence of cloned, hormone-responsive osteosarcoma cells, PTH caused a 2- to 4-fold increase in osteoclastic resorption. Significant stimulation was observed at 10(-4) IU/ml PTH. None of the osteoblastic cell populations caused morphologically detectable bone resorption in the absence of osteoclasts. These results indicate that PTH acts primarily on osteoblasts, which are induced by the presence of the hormone to stimulate osteoclastic bone resorption.

Animals↗

PubMed1986

Osteoblast-like cells in the presence of parathyroid hormone release soluble factor that stimulates osteoclastic bone resorption.

PTH stimulates osteoclastic bone resorption in vivo and in organ culture. We have previously found that if osteoclasts are disaggregated from bone and incubated on bone slices, PTH does not increase bone resorption, but does so if osteoblastic cells are added to the cultures. This suggests that PTH acts primarily on osteoblasts, which are induced by the presence of the hormone to stimulate osteoclastic bone resorption. In the present paper we describe investigations into the mechanism by which osteoblastic cells stimulate osteoclasts. We found that increased resorption could not be accounted for by changes in the bone substrate. Osteoblast-like cells (UMR106) incubated with PTH did, however, release a factor into the culture supernatant that stimulated osteoclastic bone resorption. This factor was stable for at least 7 days when stored at 4 C and survived freeze-thawing, but was inactivated by heating to 65 C for 30 min. Activity was lost entirely after dialysis using a Spectrapor membrane with a mol wt cut-off (MWCO) of 2000. The small size of the molecule was confirmed after ultrafiltration across Amicon filters YM2 and YC05. There was no loss of activity across YM2 (MWCO, 1000), but, in contrast, there was no stimulation in the conditioned medium after ultrafiltration across YC05 (MWCO, 500).

Animals↗