Vaccine development against dengue and Japanese encephalitis: report of a World Health Organization meeting.
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Dengue↗
Biomedical subjects
T J Chambers
Publications and source records attributed to T J Chambers.
Cyclical etidronate prevents spinal bone loss in early post-menopausal women.
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Bone Density↗
Induction of NO and prostaglandin E2 in osteoblasts by wall-shear stress but not mechanical strain.
The nature of the stimulus sensed by bone cells during mechanical usage has not yet been determined. Because nitric oxide (NO) and prostaglandin (PG) production appear to be essential early responses to mechanical stimulation in vivo, we used their production to compare the responsiveness of bone cells to strain and fluid flow in vitro. Cells were incubated on polystyrene film and subjected to unidirectional linear strains in the range 500-5,000 microstrain (microepsilon). We found no increase in NO or PGE2 production after loading of rat calvarial or long bone cells, MC3T3-E1, UMR-106-01, or ROS 17/2.8 cells. In contrast, exposure of osteoblastic cells to increased fluid flow induced both PGE2 and NO production. Production was rapidly induced by wall-shear stresses of 148 dyn/cm2 and was observed in all the osteoblastic populations used but not in rat skin fibroblasts. Fluid flow appeared to act through an increase in wall-shear stress. These data suggest that mechanical loading of bone is sensed by osteoblastic cells through fluid flow-mediated wall-shear stress rather than by mechanical strain.
3T3 Cells↗
Angiotensin II is generated from angiotensin I by bone cells and stimulates osteoclastic bone resorption in vitro.
During bone resorption, osteoclasts are closely associated with endothelial cells. The latter are able to produce several agents that regulate bone resorption. In view of the increasing evidence that angiotensin II, which can be generated by endothelial cells, has actions outside the traditional renin-angiotensin system, we tested the effect of angiotensin II on bone resorption Angiotensin II showed no effect either on osteoclast formation or on bone resorption by isolated osteoclasts. However, in co-cultures of osteoclasts with calvarial or MC3T3-E1 osteoblastic cells, and in osteoclastic cultures co-cultured with other bone cells obtained by prolonged sedimentation, angiotensin II stimulated bone resorption to a similar degree to that observed with 1,25(OH)2 vitamin D3. Stimulation of resorption was noted at concentrations of 10(-7) M and above. We found that angiotensin I also stimulated bone resorption in co-cultures of osteoclasts with osteoblastic cells, and that this action was inhibited by inhibitors of angiotensin-converting enzyme. These results identify angiotensin I and II as potent stimulators of osteoclastic bone resorption, and raise the possibility that bone might contain a tissue-renin-angiotensin system that might play a role in the regulation of bone resorption.
Angiotensin I↗
Estrogen suppresses activation but enhances formation phase of osteogenic response to mechanical stimulation in rat bone.
We used a model whereby mechanical stimulation induces bone formation in rat caudal vertebrae, to test the effect of estrogen on this osteogenic response. Unexpectedly, estrogen administered daily throughout the experiments (8-11 d) suppressed, and ovariectomy enhanced, mechanically induced osteogenesis. Osteogenesis was unaffected by the resorption-inhibitor pamidronate, suggesting that the suppression of bone formation caused by estrogen was not due to suppression of resorption. We found that estrogen did not significantly reduce the proportion of osteocytes that were induced by mechanical stimulation to express c-fos and IGF-I mRNA; and estrogen suppressed mechanically induced osteogenesis whether administration was started 24 h before or 24 h after loading. This suggests that estrogen acts primarily not on the strain-sensing mechanism itself, but on the osteogenic response to signals generated by strain-sensitive cells. We also found that when estrogen administration was started 3 d after mechanical stimulation, by which time osteogenesis is established, estrogen augmented the osteogenic response. This data is consistent with in vitro evidence for estrogen responsiveness in two phenotypically distinct bone cell types: stromal cells, whose functional activities are suppressed, and osteoblasts, which are stimulated, by estrogen.
Animals↗
Mutagenesis of the N-linked glycosylation sites of the yellow fever virus NS1 protein: effects on virus replication and mouse neurovirulence.
The flavivirus nonstructural glycoprotein NS1 is highly conserved and contains two N-linked glycosylation sites which are both utilized for addition of oligosaccharides during replication in cell culture. NS1 has been shown to contain epitopes for protective antibodies; however, its roles in virus replication and pathogenesis remain unknown. To study the function of NS1 during yellow fever virus replication, six mutant viruses which lack either one or both glycosylation sites and another one containing silent mutations at both sites were generated by site-directed mutagenesis. Mutants lacking the second glycosylation site and those bearing silent mutations were similar to the parental virus in their cell culture properties. Ablation of the first or both glycosylation sites generated mutants exhibiting small plaque phenotypes, decreased virus yields, reduced cytopathic effects, impaired NS1 secretion, and depressed RNA accumulation. In addition, mutants lacking the first or both glycosylation sites exhibited significant reduction in mouse neurovirulence after intracerebral inoculation. These defects appear to result from the lack of N-linked glycans rather than the introduction of deleterious amino acid substitutions or disruption of cis-acting RNA elements important for RNA replication. These results suggest an important role for NS1 in flavivirus RNA replication and pathogenesis.
Animals↗
IL-10 modulates formation of osteoclasts in murine hemopoietic cultures.
IL-10, originally described as a cytokine synthesis inhibitory factor produced by T cells, has recently been found to suppress osteoblastic differentiation in mouse bone marrow cultures. Since osteoblastic cells exert a major influence on the production and regulation of osteoclasts, the cells that resorb bone, this suggests that the cytokine might play a role in the regulation of bone resorption. We, therefore, tested the actions of the cytokine on osteoclast formation and function. We found no effect of IL-10 on the resorptive function of mature osteoclasts, either when isolated or when incubated in the presence of osteoblastic cells. However, IL-10 suppressed bone resorption in bone marrow cultures and in cocultures of bone marrow stromal cell lines with hemopoietic spleen cells. In both systems, suppression of bone resorption was associated with a substantial reduction in the ratio of calcitonin receptor-positive cells to macrophages, suggesting that IL-10 exerts a reciprocal action on the differentiation of osteoclasts and macrophages from their shared precursor. This reciprocal action is very similar to that associated with the addition of macrophage CSF to hemopoietic cultures, and we found that IL-10 increased the expression of mRNA for macrophage CSF in bone marrow cultures. This potent inhibition of osteoclast formation by IL-10 suggests that IL-10 might play a role in the modulation of bone loss in inflammatory disorders.
Animals↗
Bone cells required for osteoclastic resorption but not for osteoclastic differentiation.
It is generally considered that osteoblastic cells are essential for osteoclast formation. We tested the ability of hemopoietic tissue to differentiate osteoclastic characteristics in the absence of osteoblastic cells. We found that large numbers of calcitonin-receptor positive (CTRP) cells can be induced by prostaglandin E2 and 1,25(OH)2 vitamin D3, in cultures of hemopoietic mouse spleen. Moreover, spleen stromal cells were equivalent to bone marrow stromal cells in CTRP-cell induction. The spleen CTRP cells did not resorb bone, but were rapidly induced to full resorptive activity upon osteoblast addition. Thus, bone cells may not be essential for osteoclast formation, but rather are required to activate and regulate the resorptive function of mature osteoclasts.
Animals↗
Molecular aspects of osteoclast function.
In this article we have overviewed recent important advances in understanding the molecular mechanisms involved in osteoclastic bone resorption. Specifically, new findings relating to osteoclast activation and the process of bone resorption are reviewed and a current overall model of how osteoclasts resorb bone is presented. Controversial research topics concerning the regulation of osteoclast activity are also critically discussed.
Adenosine Triphosphatases↗
Reduction in dynamic indices of cancellous bone formation in rat tail vertebrae after caudal neurectomy.
We have previously noted that a relatively large load (150 N) is required to induce a strain on the cortex of rat vertebrae similar to that induced on weight-bearing bones by normal mechanical usage. It seems unlikely that the musculature of the tail normally imposes loads of this magnitude, and this suggests that the quantity of bone in caudal vertebrae is maintained at a higher level than would be expected for the mechanical environment to which it is exposed. This high bone mass could represent a genetically determined minimum, or could be maintained through increased sensitivity to mechanical stimuli. To distinguish between these two possibilities, we denervated the tails of 13-week-old rats by neurectomy at L6, and assessed the response of the caudal vertebrae to mechanical disuse. We found that caudal neurectomy caused a reduction in the cancellous bone formation rate in the eighth caudal vertebrae to 12% of that seen in sham-operated animals. The cancellous bone formation rate in the thoracic vertebrae of neurectomized rats, which are not mechanically disused by caudal neurectomy, was not significantly reduced. This suggests that the cancellous bone formation rate in vertebrae is maintained by substantially less intense mechanical environments than those prevailing in weight-bearing bones, raising the possibility that bones may differ in their sensitivity to mechanical strain.
Animals↗
Colitis causes bone loss in rats through suppression of bone formation.
BACKGROUND & AIMS: Idiopathic inflammatory bowel disease (IBD) is associated with bone loss in more than 30% of cases. Nevertheless, the pathogenesis of the bone loss is uncertain. The aim of this study was to investigate the bone loss and underlying mechanisms using an animal model of IBD. METHODS: Severe colitis was induced by intrarectal administration of a hapten, 2,4,6-trinitrobenzenesulfonic acid (TNBS). Severity of IBD was graded macroscopically and histologically. Bone histomorphometry was performed on cancellous bone of the tibiae. RESULTS: A florid transmural colitis was observed 3 weeks after administration of TNBS. In these animals, there was considerable cancellous bone loss of 33% compared with age-matched, pair-fed control animals. This was associated with a marked suppression of the cancellous bone formation rate to < 30% of that in control animals. Thereafter, bone formation rate increased in parallel with healing of colitis. Twelve weeks after TNBS administration, the persistent increase in bone formation rate was associated with return of bone volume to control levels. CONCLUSIONS: The data suggest that bone loss can occur rapidly in colitis and is associated with suppression of bone formation. This study also shows that the bone loss that occurs during TNBS-induced colitis is reversible.
Animals↗
Aminohexane bisphosphonate suppresses bone turnover in postmenopausal women more rapidly than oestrogen-gestagen therapy.
Although animal studies suggest that there may be major differences between the effects of bisphosphonates and ovarian hormones on skeletal metabolism, whether this also holds for their actions in patients is unknown. To address this question, we compared the effects of 12 weeks treatment with HRT on bone turnover markers in osteopenic postmenopausal women with those of an amino-bisphosphonate. Women within 15 yr of the menopause, with a lumbar and/or femoral neck bone mineral density 1 S.D. below the predicted value, received either oestradiol valerate 2 mg and dydrogesterone 5 mg (E/D; n = 16) or aminohexane bisphosphonate 400 mg (AHBP; n = 9). Urine and serum samples were collected on two separate occasions before starting treatment, and 1, 2, 4, 8 and 12 weeks afterwards. To assess bone resorption, we measured the urinary deoxypyridinoline/creatinine ratio (DPD/crea), while serum alkaline phosphatase (ALP), osteocalcin and C-terminal propeptide of type I collagen (CICP) were analysed to assess bone formation. Repeated measures analysis of variance revealed a highly significant decrease in DPD/crea over the treatment period. Furthermore, this pattern of response differed significantly between the two treatment groups, since DPD/crea was maximally suppressed within 2 weeks of starting AHBP, while E/D showed little decrease until 8 weeks. AHBP was also found to suppress ALP, osteocalcin and CICP more rapidly than E/D, the former reducing these markers by 8 weeks, while E/D caused little inhibition even by 12 weeks. We conclude that, in the doses used in this study, AHBP appears to suppress bone turnover significantly more rapidly than E/D, suggesting that clinically important differences may exist in the effects of bisphosphonates and ovarian hormones on bone metabolism.
Adult↗
Replication of yellow fever virus in the mouse central nervous system: comparison of neuroadapted and non-neuroadapted virus and partial sequence analysis of the neuroadapted strain.
Serial passage of yellow fever virus (YF17D) in mouse brain enhances neurovirulence, causing a reduction in survival time after intracerebral inoculation of adult mice. To study the biological and genetic basis for this phenomenon, we compared neurovirulence properties of the neuroadapted Porterfield strain (PYF) to a YF17D strain generated from a full-length YF cDNA template (YF5.2iv). Adult mice were infected by olfactory bulb inoculation, which results in widespread distribution of virus throughout the central nervous system. Although PYF and YF5.2iv spread rapidly throughout the neuraxis, maximal titres of PYF in the brain and spinal cord were 1000- to 10,000-fold higher than those of YF5.2iv. Paralysis and death occurred earlier with the PYF strain. Several cDNA clones of the E/NS1 region of the PYF strain were sequenced. Three predicted amino acid changes were consistently observed in the envelope protein of the PYF strain compared to YF5.2iv. Common substitutions were also identified in NS1 and NS2A. The potential contribution of these genetic differences to neurovirulence was evaluated by generating recombinant, intertypic PYF/YF5.2iv viruses. Physical signs of disease and mean spinal cord titres after inoculation of one recombinant were not different from the YF5.2iv parent. Our data indicate that PYF and YF5.2iv differ significantly in their virulence properties, however, common amino acid substitutions in the E/NS1 region of the PYF strain do not determine its enhanced neurovirulence. Other regions of the viral genome may contribute dominant effects on the virulence properties of the PYF strain.
Animals↗
Androgens contribute to the stimulation of cancellous bone formation by ovarian hormones in female rats.
We investigated whether androgens, for which the ovaries are the major source in female rats, contribute to the stimulation of cancellous bone formation by ovarian hormones in female rats. Ovariectomized animals were administered 5alpha-dihydrotestosterone (DHT; 10 and 100 microgram/kg) by daily subcutaneous injection for 13 days, after which histomorphometric analysis was performed at the proximal tibial metaphysis. To prevent ovariectomy from stimulating bone turnover, and hence complicating the interpretation of changes in bone formation, animals were also given the resorption inhibitor 3-amino-1-hydroxypropylidene-1-bisphosphonate. We found that ovariectomy markedly suppressed cancellous bone formation, which was partially prevented by DHT (100 microgram/kg). To further address whether androgens contribute to the stimulation of bone formation by ovarian hormones, we treated intact and ovariectomized female rats with the androgen antagonist flutamide (15 mg/kg/day) for 28 days. Whereas flutamide had no effect in ovariectomized rats, it significantly reduced cancellous bone formation in intact animals. We conclude that, in the female rat, androgens contribute under physiological conditions to the stimulation of cancellous bone formation by ovarian hormones.
Animals↗
Osteocytic expression of mRNA for c-fos and IGF-I: an immediate early gene response to an osteogenic stimulus.
We analyzed the expression, during the osteogenic response of bone to mechanical stimulation, of insulin-like growth factor I (IGF-I), a growth factor implicated in bone formation, and c-fos, a protooncogene in which disordered regulation specifically affects bone. Both genes were strongly expressed in osteocytes of mechanically stimulated but not control bones within 30 min of the osteogenic stimulus. IGF-I mRNA expression increased up to 6 h, was restricted to osteocytes, and was strongly suppressed by indomethacin. Although early IGF-I mRNA expression was resistant to cycloheximide, there was a degree of suppression after 6 h, raising the possibility that IGF-I expression might be prolonged by autocrine mechanisms. c-fos mRNA was increased both in osteocytes and on bone surfaces. At both sites, c-fos expression was transient, prolonged by cycloheximide, and was strongly stimulated even in the presence of indomethacin. Thus osteocytes respond to mechanical stimulation with immediate prolonged expression of IGF-I and immediate transient expression of c-fos, implicating osteocytes in the osteogenic response to mechanical stimulation. Moreover, the different spatial distribution and indomethacin sensitivity of c-fos and IGF-I gene expression suggest that at least two signaling pathways are activated in osteocytes during this process.
Animals↗
Nitric oxide is an early mediator of the increase in bone formation by mechanical stimulation.
We tested the ability of NG-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthase (NOS), to suppress the osteogenic response in a recently developed model of mechanically induced osteogenesis. L-NMMA was given either as a single intraperitoneal dose, 15 min before the episode of mechanical stimulation, or as four doses every 6 h, commencing 2 h after loading. Administration of L-NMMA before loading completely prevented the increase in cancellous bone formation by mechanical stimulation. This suppression was largely lost when L-NMMA was administered after loading. Thus the response is likely to be due to activation of a preexistent constitutive NOS in bone cells during or very soon after mechanical stimulation. Suppression of the osteogenic response by L-NMMA was prevented by coadministration of L-arginine but not by the inactive isomer, D-arginine. These changes in cancellous bone were mirrored by similar changes on the corticoendosteal and periosteal bone surfaces. These data suggest that early release of NO is a key signal in the transduction of mechanical stimuli into subsequent bone formation.
Animals↗
Sequential analysis of gene expression after an osteogenic stimulus: c-fos expression is induced in osteocytes.
We have recently developed an experimental model whereby mechanical stimulation induces osteogenesis in the caudal vertebrae of rats. We used this model to assess expression of genes induced by mechanical loading. Bulk preparations of mRNA extracted after loading did not show > 2-fold increases in expression of mRNA for matrix proteins or growth factors in Northern blotting analysis. c-jun was undectable. However, c-fos showed a 4-fold increase in expression within 60 mins of loading, before returning to control levels by 4 hrs. This increase was associated with intense signals in in situ hybridization, not seen in any nonloaded vertebrae, for c-fos over cortical osteocytes: thus osteocytes respond to mechanical loading with c-fos expression so strongly as to be visible even in the bulk RNA preparations. The results represent persuasive evidence for a role for osteocytes, and for c-fos, in the osteogenic response of bone to mechanical stimulation.
Animals↗
The effect of hepatocyte growth factor on the behaviour of osteoclasts.
Hepatocyte growth factor (HGF) stimulates the growth, motility and morphogenesis of a variety of cell types, including hemopoietic progenitors. We found that HGF is a potent inhibitor of bone resorption by isolated rat osteoclasts. However, in the presence of the osteoblastic cell line UMR 106, it stimulated osteoclastic resorption. HGF also increased osteoclastic motility and spread area, over a similar concentration range. We detected no effect on osteoclast formation or survival. Our data suggest that HGF may be involved in the recruitment of osteoclasts to sites of bone resorption, but that during migration resorptive functions are suppressed. Once on resorptive sites, the osteoclast response to HGF is modulated by osteoblastic cells and the bone resorptive activity of osteoclasts may be stimulated accordingly.
Animals↗