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Stuart H Ralston

Publications and source records attributed to Stuart H Ralston.

52 records · Page 3Linked to original sources
PubMed2004

Identification of biphenylcarboxylic acid derivatives as a novel class of bone resorption inhibitors.

UNLABELLED: A novel class of biphenylcarboxylic acid derivatives are described that inhibit osteoclastic bone resorption in vitro by promoting osteoclast apoptosis and that prevent ovariectomy-induced bone loss in vivo. The compounds act by a novel mechanism that seems to be distinct from existing antiresorptive drugs. INTRODUCTION: Many common bone diseases such as osteoporosis, Paget's disease, and cancer-associated bone disease are characterized by excessive bone loss caused by increased osteoclastic activity. Successful treatment of these diseases is based on osteoclast inhibition. The osteoclast inhibitory drugs that are currently available fall into relatively few mechanistic classes, indicating the need to identify novel antiresorptives. Here we describe a series of biphenylcarboxylic acid derivatives that have potent inhibitory effects on osteoclastic bone resorption in vitro and on ovariectomy-induced bone loss in vivo. MATERIALS AND METHODS: Compounds were tested for inhibitory effects on bone resorption in vitro using mouse osteoblast-bone marrow co-cultures, isolated rabbit osteoclasts, and mouse osteoclasts generated from bone marrow. Some experiments were also performed on human osteoclasts generated from peripheral blood mononuclear cells. We also investigated the effects of specific compounds on ovariectomy-induced bone loss in vivo in mice. RESULTS: One of the most potent compounds identified was the butanediol ester of biphenyl carboxylic acid (ABD056), which inhibited osteoclast formation in mouse osteoblast-bone marrow co-cultures by 50% (IC50) at a concentration of 26 microM and in macrophage-colony stimulating factor (M-CSF)- and RANKL-stimulated mouse bone marrow cultures with an IC50 of 8 microM. Mechanistic studies showed that ABD056 caused osteoclast apoptosis and inhibited TNFalpha-induced NF-kappaB activation. No inhibitory effects on osteoblast growth or differentiation were observed at concentrations of up to 100 microM. When administered to mice at doses of 5 and 10 mg/kg/day, ABD056 prevented ovariectomy-induced bone loss. CONCLUSIONS: Butanediol biphenylcarboxylic acid derivatives represent a new class of antiresorptive drug that might be of therapeutic value in the prevention and treatment of diseases characterized by osteoclast activation such as osteoporosis, cancer-associated bone disease, and Paget's disease of bone.

Alkaline Phosphatase↗
PubMed2003

Genotype-phenotype relationship in human ATP6i-dependent autosomal recessive osteopetrosis.

Autosomal-recessive osteopetrosis is a severe genetic disease caused by osteoclast failure. Approximately 50% of the patients harbor mutations of the ATP6i gene, encoding for the osteoclast-specific a3 subunit of V-ATPase. We found inactivating ATP6i mutations in four patients, and three of these were novel. Patients shared macrocephaly, growth retardation and optic nerve alteration, osteosclerotic and endobone patterns, and high alkaline phosphatase and parathyroid hormone levels. Bone biopsies revealed primary spongiosa lined with active osteoblasts and high numbers of tartrate-resistant acid phosphatase (TRAP)-positive, a3 subunit-negative, morphologically unremarkable osteoclasts, some of which located in shallow Howship lacunae. Scarce hematopoietic cells and abundant fibrous tissue containing TRAP-positive putative osteoclast precursors were noted. In vitro osteoclasts were a3-negative, morphologically normal, with prominent clear zones and actin rings, and TRAP activity more elevated than in control patients. Podosomes, alphaVbeta3 receptor, c-Src, and PYK2 were unremarkable. Consistent with the finding in the bone biopsies, these cells excavated pits faintly stained with toluidine blue, indicating inefficient bone resorption. Bone marrow transplantation was successful in all patients, and posttransplant osteoclasts showed rescue of a3 subunit immunoreactivity.

Acid Phosphatase↗
PubMed2003

Genetic determinants of susceptibility to osteoporosis.

Osteoporosis has a strong genetic component, and clinical studies have shown that heritable factors play a key role in regulating bone mineral density, ultrasound properties of bone, skeletal geometry, and bone turnover and contribute to the pathogenesis of osteoporotic fracture. In most cases, osteoporosis is caused by the combined effects of several different genes and their interaction with environmental influences, but it can occasionally occur as the result of mutations in a single gene. Genes that have been implicated in the regulation of bone mass in humans include the genes encoding lipoprotein receptor-related protein 5, sclerostin, transforming growth factor beta-1, collagen Ialpha1, vitamin D receptor, tumor necrosis factor receptor 2, and the estrogen receptor alpha. From a clinical standpoint, advances in knowledge about the genetic basis of osteoporosis are important because they offer the prospect of developing genetic markers for the assessment of fracture risk and the opportunity to identify molecules that will be used as targets for the design of new drugs for the prevention and treatment of bone disease.

Animals↗
PubMed2003

Genetic determinants of susceptibility to osteoporosis.

Major advances in our knowledge about the genetic determinants of osteoporosis have been made in recent years. They include the discovery of genes responsible for monogenic bone diseases associated with abnormal bone mass; the identification of quantitative trait loci for bone mass in the general population and in mice; and the characterisation of several candidate genes for osteoporosis. Information from these studies is being used to design new drugs for osteoporosis and to develop genetic markers for fracture risk assessment.

Animals↗
PubMed2003

A mutation affecting the latency-associated peptide of TGFbeta1 in Camurati-Engelmann disease enhances osteoclast formation in vitro.

Camurati-Engelmann disease (CED) is a rare autosomal dominant disorder characterized by bone pain and osteosclerosis affecting the diaphysis of long bones. CED is caused by various missense mutations in the TGFB1 gene that encodes TGFbeta1, the most common of which is an arginine-cysteine amino acid change at codon 218 (R218C) in the latency-associated peptide domain of TGFbeta1. We studied osteoclast formation in vitro from peripheral blood mononuclear cells obtained from three related CED patients harboring the R218C mutation, in comparison with one family-based and several unrelated controls. Osteoclast formation was enhanced approximately 5-fold (P < 0.001) and bone resorption approximately 10-fold (P < 0.001) in CED patients, and the increase in osteoclast formation was inhibited by soluble TGFbeta type II receptor. Total serum TGFbeta1 levels were similar in affected and unaffected subjects, but concentrations of active TGFbeta1 in conditioned medium of osteoclast cultures was higher in the three CED patients than in the unaffected family member. We concluded that the R218C mutation increases TGFbeta1 bioactivity and enhances osteoclast formation in vitro. The activation of osteoclast activity noted here is consistent with clinical reports that have shown biochemical evidence of increased bone resorption as well as bone formation in CED.

Adult↗
PubMed2003

Phenotypic characterization of early onset Paget's disease of bone caused by a 27-bp duplication in the TNFRSF11A gene.

Three different insertion mutations in the TNFRSF11A gene affecting the signal peptide of RANK have been described. An 18-bp duplication at position 84 (84dup18) is associated with the clinical syndrome of familial expansile osteolysis (FEO), whereas a 15-bp duplication at the same site (84dup15) causes the syndrome of expansile skeletal hyperphosphatasia (ESH). Here we report the phenotype of patients harboring a 27-bp duplication at position 75 (75dup27) in RANK. Affected individuals had hearing impairment and tooth loss beginning in the second or third decade. Radiographs of affected bones showed lytic and sclerotic lesions with bony enlargement and deformity. Serum alkaline phosphatase levels were elevated between 2 and 17 times above the normal range. Most patients had pelvic and skull involvement, and all had involvement of the mandible and maxilla. Most patients also had bony enlargement of the small joints of the hands, and one developed hypercalcemia during a period of immobilization. We conclude that the 75dup27 mutation of RANK causes a Paget's disease of bone-like phenotype that is distinct from, but which overlaps with, FEO and ESH. A particularly striking feature was involvement of the mandible and maxilla, but it remains to be seen if this is a specific feature of the 75dup27 mutation until further kindreds with this mutation are reported.

Age of Onset↗
PubMed2002

Transforming growth factor-beta 1 mutations in Camurati-Engelmann disease lead to increased signaling by altering either activation or secretion of the mutant protein.

Transforming growth factor-beta1 (TGF-beta1) is secreted as a latent precursor, consisting of a homodimer of the latency-associated peptide and the mature peptide. TGFbeta-1 can only exert its many functions after going from this latent to an active state, in which the binding site of the mature peptide for its receptor is no longer shielded by the latency-associated peptide. We and others reported that mutations in TGFB1 cause Camurati-Engelmann disease, a rare bone disorder. Until now, seven mutations have been published. In this study, we investigate the effect of the LLL12-13ins, Y81H, R218C, H222D, and C225R mutations on the functioning of TGF-beta1 in vitro. A luciferase reporter assay specific for TGF-beta-induced transcriptional response with wild type and mutant TGF-beta1 constructs showed a positive effect of all mutations on TGF-beta1 activity. By way of enzyme-linked immunosorbent assay, we found that in the R218C, H222D, and C225R mutant constructs, this effect is caused by an increase in active TGF-beta1 in the medium of transfected cells. The LLL12-13ins and Y81H mutations on the contrary have a profound effect on secretion; a decreased amount of TGF-beta1 is secreted, but the increased luciferase activity shows that the intracellular accumulation of (aberrant) TGF-beta1 can initiate an enhanced transcriptional response, suggesting the existence of an alternative signaling pathway. Our data indicate that the mutations in the signal peptide and latency-associated peptide facilitate TGF-beta1 signaling, thus causing Camurati-Engelmann disease.

Adult↗
PubMed2002

Domain-specific mutations in sequestosome 1 (SQSTM1) cause familial and sporadic Paget's disease.

Paget's disease of bone (PDB) is a common disorder characterized by focal abnormalities of increased and disorganized bone turnover. Genetic factors are important in the pathogenesis of PDB, and in previous studies, we and others identified a locus for familial PDB by genome-wide search on 5q35-qter (PDB3). The gene encoding sequestosome 1 (SQSTM1/p62) maps to within the PDB3 critical region, and recent studies have identified a proline-leucine amino acid change at codon 392 of SQSTM1 (P392L) in French-Canadian patients with PDB. We conducted mutation screening of positional candidate genes in the PDB3 locus in patients with PDB, and also identified mutations in the gene encoding SQSTM1 as a common cause of familial and sporadic PDB. Three different mutations were found, all affecting the highly conserved ubiquitin-binding domain. The most common mutation was the P392L change in exon 8, which was found in 13 of 68 families (19.1%). Another mutation-a T insertion that introduces a stop codon at position 396 in exon 8-was found in four (5.8%) families. A third mutation affecting the splice donor site in intron 7 was found in one (1.5%) family. The P392L mutation was also found in 15 of 168 (8.9%) of patients with sporadic PDB and 0 of 160 of age- and sex-matched controls (P<0.0001). These studies confirm that mutations affecting the ubiquitin-binding domain of SQSTM1 are a common cause of familial and sporadic Paget's disease of bone.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Linkage disequilibrium between polymorphisms in the human TNFRSF1B gene and their association with bone mass in perimenopausal women.

Osteoporosis is a multifactorial disease with a strong genetic component characterized by reduced bone density and increased fracture risk. A candidate locus for regulation of hip bone mineral density (BMD) has been identified on chromosome 1p36 by linkage analysis. One of the positional and functional candidate genes located within this region is the tumour necrosis factor receptor superfamily member 1B (TNFRSF1B). In order to investigate whether allelic variation in TNFRSF1B contributes to regulation of bone mass, we studied several polymorphisms of this gene in a population based cohort study of 1240 perimenopausal women from the UK. We studied a T676G change in exon 6 (196: Met-Arg) and three SNPs (G593A, T598G, and T620C) in the 3'UTR of the gene. The 3'UTR SNPs were in strong linkage disequilibrium (LD) with each other (P<0.00001), and the exon 6 SNP was in LD with G593A and T598G (P<0.00001). We found no association between T676G alleles and BMD at the spine or hip. However, haplotype analysis showed that subjects homozygous for the A593-T598-C620 haplotype (n=85) had femoral neck BMD values 5.7% lower than those who did not carry the haplotype (n=1155; P<0.00008) and this remained significant after correcting for confounding factors and multiple testing (P<0.0009). Regression analysis showed that the ATC haplotype accounted for 1.2% of the population variance in hip BMD and was the second strongest predictor after body weight. In summary, our work supports the view that allelic variation in the 3'UTR of TNFRSF1B gene contributes to the genetic regulation of bone mass, with effects that are specific for femoral neck BMD.

3' Untranslated Regions↗
PubMed2002

Pamidronate causes apoptosis of plasma cells in vivo in patients with multiple myeloma.

Anti-resorptive bisphosphonates, such as pamidronate, are an effective treatment for osteolytic disease and hypercalcaemia in patients with multiple myeloma, but have also been shown to cause apoptosis of myeloma cell lines in vitro. In this study, we found that a single infusion of pamidronate, in 16 newly diagnosed patients with multiple myeloma, caused a marked increase in apoptosis of plasma cells in vivo in 10 patients and a minimal increase in four patients (P < 0.05). The nitrogen-containing bisphosphonates pamidronate and zoledronic acid also induced apoptosis of authentic, human bone marrow-derived plasma cells in vitro. Apoptosis of plasma cells in vitro was probably caused by inhibition of the mevalonate pathway and loss of prenylated small GTPases, as even low concentrations (>or= 1 micro mol/l) of zoledronic acid caused accumulation of unprenylated Rap1A in cultures of bone marrow mononuclear cells in vitro. GGTI-298, a specific inhibitor of geranylgeranyl transferase I, also induced apoptosis in human plasma cells in vitro, suggesting that geranylgeranylated proteins play a role in signalling pathways that prevent plasma cell death. Our results suggest that pamidronate may have direct and/or indirect anti-tumour effects in patients with multiple myeloma, which has important implications for the further development of the more potent nitrogen-containing bisphosphonates, such as zoledronic acid, in the treatment of myeloma.

Aged↗
PubMed2002

Increased bone resorption in the critically ill: association with sepsis and increased nitric oxide production.

OBJECTIVE: Cytokines stimulate nitric oxide production in bone, and high concentrations of cytokine-induced nitric oxide inhibit bone resorption in vitro. This has led to the suggestion that nitric oxide may protect against bone loss in inflammatory and infectious diseases. In this study, we sought to determine whether nitric oxide generated as the result of sepsis was associated with suppression of bone resorption in vivo. DESIGN: Observational study. SETTING: Adult intensive care unit of a university hospital. PATIENTS: We studied 20 consecutive patients who had been admitted to the intensive care unit because of sepsis and three who had been admitted because of trauma. Controls were 29 patients with noninflammatory musculoskeletal conditions. INTERVENTIONS: Standard clinical care. MEASUREMENTS AND MAIN RESULTS: Bone resorption was assessed by measurement of urinary pyridinoline and deoxypyridinoline as a ratio to urinary creatinine. Nitric oxide production was assessed by measuring the ratio of the nitric oxide breakdown products nitrate and nitrite to urinary creatinine. Urinary nitrate and nitrite/creatinine values were significantly higher in intensive care patients with sepsis (mean +/- sem, 0.164 +/- 0.053 micromol/mmol) than in intensive care patients with trauma (0.066 +/- 0.008) and controls (0.079 +/- 0.007; p =.007 between groups). Urinary pyridinoline/creatinine values were increased in intensive care patients with sepsis (553.8 +/- 193 nmol/mmol) and trauma (238 +/- 32) compared with controls (44.7 +/- 2.6; p =.001 between groups), and similar differences between the groups were observed for deoxypyridinoline/creatinine values: intensive care patients with sepsis, 86.4 +/- 24.0; intensive care patients with trauma, 46 +/- 4.2; and controls, 10.3 +/- 0.7 (p =.001). CONCLUSIONS: Critically ill patients with sepsis have increased nitric oxide production and increased bone resorption, whereas trauma patients have increased bone resorption in the presence of normal nitric oxide production. High concentrations of nitric oxide generated during the course of infection do not afford significant protection against accelerated bone resorption.

Adult↗
PubMed2002

Single nucleotide polymorphism detection: allelic discrimination using TaqMan.

Candidate gene studies are one of the most widely used approaches in the dissection of the genetic basis of disease. High-throughput methods for genotyping single nucleotide polymorphisms (SNPs) are necessary to perform large-scale association studies. We describe the use of the TaqMan or 5' nuclease allelic discrimination assay for genotyping polymorphisms of the collagen I alpha 1 (COLIA1) and vitamin D receptor (VDR) genes. The basis for the assay is an allele specific oligonucleotide probe, labelled with a fluorescent reporter dye and a quencher dye, which is cleaved during the amplification process generating an increase in the intensity of fluorescence related to the accumulation of PCR product which is measured directly in the reaction well. Suitable for the discrimination of alleles differing by a single base change, this technique is robust, accurate, cost effective, and sufficiently high-throughput for a medium sized laboratory performing association analyses.

Alleles↗
PubMed2002

Genetic control of susceptibility to osteoporosis.

Osteoporosis is a common disease with a strong genetic component. Twin studies have shown that genetic factors play an important role in regulating bone mineral density (BMD), ultrasound properties of bone, skeletal geometry, and bone turnover as well as contributing to the pathogenesis of osteoporotic fracture itself. These phenotypes are determined by the combined effects of several genes and environmental influences, but occasionally, osteoporosis or unusually high bone mass can occur as the result of mutations in a single gene. Examples are the osteoporosis-pseudoglioma syndrome, caused by inactivating mutations in the lipoprotein receptor-related protein 5 gene and the high bone mass syndrome, caused by activating mutations of the same gene. Genome-wide linkage studies in man have identified loci on chromosomes 1p36, 1q21, 2p21, 5q33-35, 6p11-12, and 11q12-13 that show definite or probable linkage to BMD, but so far, the causative genes remain to be identified. Linkage studies in mice have similarly identified several loci that regulate BMD, and a future challenge will be to investigate the syntenic loci in humans. A great deal of research has been done on candidate genes; among the best studied are the vitamin D receptor and the collagen type I alpha 1 gene. Polymorphisms of vitamin D receptor have been associated with bone mass in several studies, and there is evidence to suggest that this association may be modified by dietary calcium and vitamin D intake. A functional polymorphism affecting an Sp1 binding site has been identified in the collagen type I alpha 1 gene that predicts osteoporotic fractures independently of bone mass by influencing collagen gene regulation and bone quality. An important problem with most candidate gene studies is small sample size, and this has led to conflicting results in different populations. Some researchers are exploring the use of meta-analysis to try and address this issue and gain an accurate estimate of effect size for different polymorphisms in relation to relevant clinical endpoints, such as BMD and fracture. From a clinical standpoint, advances in knowledge about the genetic basis of osteoporosis are important, because they offer the prospect of developing genetic markers for the assessment of fracture risk and the opportunity to identify molecules that will be used as targets for the design of new drugs for the prevention and treatment of bone disease.

Animals↗
PubMed2002

Association of polymorphisms of the estrogen receptor alpha gene with bone mineral density and fracture risk in women: a meta-analysis.

The contribution of genetic polymorphisms to bone mineral density (BMD) and fracture risk in women is a controversial topic. We evaluated the effect of the XbaI and PvuII polymorphisms of the estrogen receptor a to BMD and fracture risk in a meta-analysis, including published data and additional information from investigators. Five thousand eight hundred thirty-four women from 30 study groups were analyzed with fixed and random effects models. The PvuII polymorphism was not associated with BMD at any skeletal site examined and 95% CIs exclude effects over 0.015 g/cm2 for both the femoral neck and the lumbar spine. Conversely, XX homozygotes (women carrying two copies of the gene variant without an XbaI restriction site) consistently had higher BMD than other subjects. The magnitude of the effect was similar in the femoral neck and lumbar spine (0.014 g/cm2 [95% CI, 0.003-0.025] and 0.015 g/cm2 [95% CI, 0.000-0.030], respectively; no between-study heterogeneity for either). Total body BMD was also significantly higher in XX homozygotes (by 0.039 g/cm2 and 0.029 g/cm2 compared with Xx and xx, respectively). Available data on fractures suggested a protective effect for XX (odds ratio [OR], 0.66 [95% CI, 0.47-0.93] among 1591 women), but not PP (OR, 0.93 [95% CI, 0.72-1.18] among 2,229 women). In summary, we have found that XX homozygotes may have higher BMD and also a decreased risk of fractures when compared with carriers of the x allele, whereas the PvuII polymorphism is not associated with either BMD or fracture risk.

Adult↗
PubMed2002

Genetic and environmental determinants of peak bone mass in young men and women.

Peak bone mass is an important risk factor for the development of osteoporosis in later life. Previous work has suggested that genetic, intrauterine, and environmental factors all contribute to the regulation of bone mass, but the ways in which they interact with each other to do so remain poorly understood. In this study, we investigated the relationship between peak bone mass and polymorphisms of the vitamin D receptor (VDR), estrogen receptor (ER) a, and collagen type Ialpha1 (COLIA1) genes in relation to other factors such as birth weight, lifestyle diet, and exercise in a population-based cohort of 216 women and 244 men in their early 20s. Stepwise multiple regression analysis showed that body weight was the strongest predictor of bone mineral density (BMD) in women, accounting for 16.4% of the variance in spine BMD and 8.4% of the variance in femoral neck BMD. Other significant predictors were VDR genotype (3.8%) and carbohydrate intake (1.6%) at the spine and vitamin D intake (3.4%) and ER genotype (3.4%) at the femoral neck. Physical activity was the strongest predictor of BMD in men, accounting for 6.7% of the variance at the spine and 5.1% at the hip. Other significant predictors were body weight (5%) and ER PvuII genotype (2.8%) at the spine and weight (3.4%) and alcohol intake (2%) at the femoral neck. Birth weight was not a significant predictor of BMD at either site but COLIA1 genotype significantly predicted birth weight in women, accounting for 4.3% of the variance. We conclude that peak bone mass is regulated by an overlapping but distinct set of environmental and genetic influences that differ in men and women. However, much of the variance in BMD was unexplained by the variables studied here, which suggests that either most of the genes that regulate BMD remain to be discovered or major environmental influences on BMD exist that have not yet been identified.

Adult↗