Biomedical subjects

Pamela G Robey

Publications and source records attributed to Pamela G Robey.

15 recordsLinked to original sources

PubMed2006

Sensitive and specific method for detecting G protein-coupled receptor mRNAs.

G protein-coupled receptors (GPCRs) mediate effects of extracellular signaling molecules in all the body's cells. These receptors are encoded by scarce mRNAs; therefore, detecting their transcripts with conventional microarrays is difficult. We present a method based on multiplex PCR and array detection of amplicons to assay GPCR gene expression with as little as 1 mug of total RNA, and using it, we profiled three human bone marrow stromal cell (BMSC) lines.

Cell Line↗

PubMed2006

Parathyroid-specific double knockout of Gq and G11 alpha-subunits leads to a phenotype resembling germline knockout of the extracellular Ca2+ -sensing receptor.

Germline knockout of the extracellular Ca2+ -sensing receptor (CaR) leads to a phenotype that includes severe hypercalcemia, hyperparathyroidism, relative hypocalciuria, skeletal abnormalities, retarded growth, and early postnatal death. To investigate the role of heterotrimeric G proteins in CaR signaling, we used cre/lox technology to delete the respective alpha-subunits of Gq and G11 selectively in parathyroid cells. Mice that were PTH-Cre(+/-); Gnaq(flox/flox); Gna11(-/-) (PTH-Galphaq/Galpha11 -double knockouts) were viable, but showed all the features of germline knockout of the CaR except hypocalcuria. Our results demonstrate the critical role of both Gq and G11 in mediating inhibition of PTH secretion by extracellular Ca2+.

Animals↗

PubMed2006

Pegvisomant for the treatment of gsp-mediated growth hormone excess in patients with McCune-Albright syndrome.

CONTEXT: GH excess affects approximately 20% of the patients with McCune-Albright syndrome (MAS). MAS is caused by sporadic, postzygotic, activating mutations in the GNAS gene, which codes for the cAMP-regulating protein, G(s)alpha (gsp oncogene). These same mutations are found in approximately one third of the sporadic cases of acromegaly. OBJECTIVE: We examined efficacy of the GH receptor antagonist, pegvisomant, in controlling gsp oncogene-mediated GH excess and skeletal disease (fibrous dysplasia of bone) associated with MAS. SETTING AND PATIENTS: Five MAS patients with GH excess were treated with 20 mg/d sc injection of pegvisomant for 12 wk in a randomized, double-blind, placebo-controlled crossover study at the National Institutes of Health. MAIN OUTCOME MEASURES: The primary measure of efficacy was normalization of IGF-I. Secondary outcome measures were reduction in serum IGF binding protein-3 (IGFBP-3), improvement of fatigue and sweating, and reduction in markers of bone metabolism and bone pain. RESULTS: Combined mean changes in serum IGF-I at 6 and 12 wk were -236.4 ng/ml (53%, P < 0.005) and -329.8 ng/ml (62%, P < 0.001), respectively. IGFBP-3 decreased by 0.8 mg/liter (24%, P < 0.01) and 2.9 mg/liter (37%, P < 0.005), respectively. There were no significant changes in signs and symptoms of acromegaly or markers of bone metabolism and bone pain, nor was there a significant change in pituitary size. Retrospective comparison of the degree of control achieved with pegvisomant vs. other medications (long-acting octreotide +/- dopamine agonist) in the same group showed that the two regimens were similarly effective. CONCLUSIONS: Pegvisomant effectively reduced IGF-I and IGFBP-3 levels in gsp-mediated GH excess but had no effect on fibrous dysplasia.

Bone and Bones↗

PubMed2006

The mechanical phenotype of biglycan-deficient mice is bone- and gender-specific.

Biglycan (bgn) is a small leucine-rich proteoglycan (SLRP) enriched in the extracellular matrix of skeletal tissues. While bgn is known to be involved in the growth and differentiation of osteoblast precursor cells and regulation of collagen fibril formation, it is unclear how these functions impact bone's geometric and mechanical properties, properties which are integral to the structural function of bone. Because the genetic control of bone structure and function is both local- and gender-specific and because there is evidence of gender-specific effects associated with genetic deficiencies, it was hypothesized that the engineered deletion of the gene encoding bgn would result in a cortical bone mechanical phenotype that was bone- and gender-specific. In 11-week-old C57BL6/129 mice, the cortical bone in the mid-diaphyses of the femora and tibiae of both genders was examined. Phenotypic changes in bgn-deficient mice relative to wild type controls were assayed by four-point bending tests to determine mechanical properties at the whole bone (structural) and tissue levels, as well as analyses of bone geometry and bone formation using histomorphometry. Of the bones examined, bgn deficiency most strongly affected the male tibiae, where enhanced cross-sectional geometric properties and bone mineral density were accompanied by decreased tissue-level yield strength and pre-yield structural deformation and energy dissipation. Because pre-yield properties alone were impacted, this implies that the gene deletion causes important alterations in mineral and/or the matrix/mineral ultrastructure and suggests a new understanding of the functional role of bgn in regulating bone mineralization in vivo.

Animals↗

PubMed2006

Skeletal site-specific characterization of orofacial and iliac crest human bone marrow stromal cells in same individuals.

Autologous grafts from axial and appendicular bones commonly used to repair orofacial bone defects often result in unfavorable outcome. This clinical observation, along with the fact that many bone abnormalities are limited to craniofacial bones, suggests that there are significant differences in bone metabolism in orofacial, axial and appendicular bones. It is plausible that these differences are dictated by site-specificity of embryological progenitor cells and osteogenic properties of resident multipotent human bone marrow stromal cells (hBMSCs). This study investigated skeletal site-specific phenotypic and functional differences between orofacial (maxilla and mandible) and axial (iliac crest) hBMSCs in vitro and in vivo. Primary cultures of maxilla, mandible and iliac crest hBMSCs were established with and without osteogenic inducers. Site-specific characterization included colony forming efficiency, cell proliferation, life span before senescence, relative presence of surface markers, adipogenesis, osteogenesis and transplantation in immunocompromised mice to compare bone regenerative capacity. Compared with iliac crest cells, orofacial hBMSCs (OF-MSCs) proliferated more rapidly with delayed senescence, expressed higher levels of alkaline phosphatase and demonstrated more calcium accumulation in vitro. Cells isolated from the three skeletal sites were variably positive for STRO 1, a marker of hBMSCs. OF-MSCs formed more bone in vivo, while iliac crest hBMSCs formed more compacted bone that included hematopoietic tissue and were more responsive in vitro and in vivo to osteogenic and adipogenic inductions. These data demonstrate that hBMSCs from the same individuals differ in vitro and in vivo in a skeletal site-specific fashion and identified orofacial marrow stromal cells as unique cell populations. Further understanding of site-specific properties of hBMSCs and their impact on site-specific bone diseases and regeneration are needed.

Adolescent↗

PubMed2006

Long-term outcome of optic nerve encasement and optic nerve decompression in patients with fibrous dysplasia: risk factors for blindness and safety of observation.

OBJECTIVE: Fibrous dysplasia (FD) of bone may occur solely as a skeletal condition or it may occur in association with extraskeletal manifestations, including growth hormone (GH) excess. Uncertainty exists as to the management of FD involving the optic nerves. In an effort to clarify management, the authors studied a large population of patients. METHODS: One hundred four patients underwent an evaluation that included review of records, endocrine testing, cranial computed tomography, and neuro-ophthalmological examination. RESULTS: Ninety-one of 104 patients had craniofacial FD; complete records were available for 87 patients (174 nerves). Seventeen percent of the optic nerves were less than 50% encased, 22% were 50 to 99% encased, and 61% were 100% encased. Twelve percent of the nerves that were 100% encased showed evidence of optic neuropathy, but 88% did not. The group with optic neuropathy was not older than the group without. Patients with GH excess were significantly more likely to have nerves that were 100% encased (relative risk, 4.1; 95% confidence interval, 1.5-11.1; P = 0.0017) and to have optic neuropathy (relative risk, 3.8; 95% confidence interval, 2.0-7.1; P = 0.0019). Six prophylactic optic nerve decompressions were performed; in five patients, vision was stable after surgery, and one patient was blind after surgery. Thirteen interventional optic nerve decompression procedures were performed; six of the 13 patients showed some improvement and seven of the 13 showed no improvement or worsened vision. CONCLUSION: The vast majority of optic nerves encased with FD do not exhibit symptoms of optic neuropathy and seem to be stable over time. GH excess is associated with increased risk of nerve encasement and optic neuropathy. Patients with craniofacial FD should be screened for GH excess, and optic nerve decompression should be performed only when there is objective evidence of progressive optic neuropathy.

Adolescent↗

PubMed2005

Extracellular matrix proteoglycans control the fate of bone marrow stromal cells.

Extracellular matrix glycoproteins and proteoglycans bind a variety of growth factors and cytokines thereby regulating matrix assembly as well as bone formation. However, little is known about the mechanisms by which extracellular matrix molecules modulate osteogenic stem cells and bone formation. Using mice deficient in two members of the small leucine-rich proteoglycans, biglycan and decorin, we uncovered a role for these two extracellular matrix proteoglycans in modulating bone formation from bone marrow stromal cells. Our studies showed that the absence of the critical transforming growth factor-beta (TGF-beta)-binding proteoglycans, biglycan and decorin, prevents TGF-beta from proper sequestration within the extracellular matrix. The excess TGF-beta directly binds to its receptors on bone marrow stromal cells and overactivates its signaling transduction pathway. Overall, the predominant effect of the increased TGF-beta signaling in bgn/dcn-deficient bone marrow stromal cells is a "switch in fate" from growth to apoptosis, leading to decreased numbers of osteoprogenitor cells and subsequently reduced bone formation. Thus, biglycan and decorin appear to be essential for maintaining an appropriate number of mature osteoblasts by modulating the proliferation and survival of bone marrow stromal cells. These findings underscore the importance of the micro-environment in controlling the fate of adult stem cells and reveal a novel cellular and molecular basis for the physiological and pathological control of bone mass.

Animals↗

PubMed2004

A novel technique based on a PNA hybridization probe and FRET principle for quantification of mutant genotype in fibrous dysplasia/McCune-Albright syndrome.

Somatic mutations are present in various proportions in numerous developmental pathologies. Somatic activating missense mutations of the GNAS gene encoding the Gs(alpha) protein have previously been shown to be the cause of fibrous dysplasia of bone (FD)/McCune-Albright syndrome (MAS). Because in MAS patients, tissues as diverse as melanocytes, gonads and bone are affected, it is generally accepted that the GNAS mutation in this disease must have occurred early in development. Interestingly, it has been shown that the development of an active FD lesion may require both normal and mutant cells. Studies of the somatic mosaic states of FD/MAS and many other somatic diseases need an accurate method to determine the ratio of mutant to normal cells in a given tissue. A new method for quantification of the mutant:normal ratio of cells using a PNA hybridization probe-based FRET technique was developed. This novel technique, with a linear sensitivity of 2.5% mutant alleles, was used to detect the percentage mutant cells in a number of tissue and cell culture samples derived from FD/MAS lesions and could easily be adapted for the quantification of mutations in a large spectrum of diseases including cancer.

Alleles↗

PubMed2004

Fibrous dysplasia in the spine: prevalence of lesions and association with scoliosis.

BACKGROUND: Lesions of fibrous dysplasia involving the spine and scoliosis are thought to be uncommon entities in patients with polyostotic fibrous dysplasia and McCune-Albright syndrome. By examining bone scans of a relatively large cohort of patients with these disorders, we sought to determine the prevalence of spinal involvement and any association with scoliosis. METHODS: Sixty-two patients with polyostotic fibrous dysplasia were studied. There were twenty-three male and thirty-nine female patients, and they had a mean age of twenty-five years (range, four to eighty years). Technetium-99m-methylene diphosphonate (MDP) bone scans of the patients were evaluated for evidence of increased uptake in the spine. The presence or absence of scoliosis or a level pelvis and the distribution of other lesions in the skeleton were noted. RESULTS: Thirty-nine (63%) of sixty-two patients were found to have seventy-six lesions of fibrous dysplasia in the spine. Fifty-four lesions (71%) demonstrated increased uptake in the posterior aspects of the spine. Most lesions were located in the lumbar spine (thirty-two lesions) and the thoracic spine (twenty-seven), with less frequent involvement in the sacrum (ten) and cervical spine (six). Twenty-five (40%) of the sixty-two patients had scoliosis; seventeen had a thoracolumbar curve; six, a lumbar curve; and two, a thoracic curve. Seven patients had curves that could not be accurately measured by bone scanning and, therefore, could not be classified. Thirty patients (48%) had no evidence of scoliosis. Thus, the prevalence of scoliosis in patients with polyostotic fibrous dysplasia was between 40% and 52%. There was a strong correlation between spinal lesions and scoliosis (p < 0.001) and pelvic asymmetry (p < 0.05). Back pain was an uncommon symptom. Two patients had a neurologic abnormality; neither abnormality was related to the location of the lesions or the curve. CONCLUSIONS: Spinal lesions and scoliosis may be more common in patients with polyostotic fibrous dysplasia than has been previously reported. Since there is a strong correlation between a spinal lesion and scoliosis, these patients should be screened clinically for scoliosis. LEVEL OF EVIDENCE: Prognostic study, Level II-1 (retrospective study). See Instructions to Authors for a complete description of levels of evidence.

Adolescent↗

PubMed2003

Fracture incidence in polyostotic fibrous dysplasia and the McCune-Albright syndrome.

UNLABELLED: In patients with polyostotic fibrous dysplasia of bone, the peak incidence of fractures is during the first decade of life, followed by a decrease thereafter. Phosphaturia is associated with an earlier incidence and increased frequency of fractures. INTRODUCTION: Fibrous dysplasia (FD) is a disorder involving either one (monostotic) or several bones (polyostotic FD [PFD] and sometimes is associated with cafe-au-lait hyperpigmentation of the skin and one or more hyperfunctioning endocrinopathies (McCune-Albright syndrome [MAS]). Both PFD and MAS are often associated with phosphaturia. Although fractures occur frequently in PFD/MAS, fracture incidence and the effect of age and co-existing metabolic abnormalities (endocrinopathy and/or phosphaturia) on fractures are ill defined. MATERIALS AND METHODS: We reviewed the medical records and examined the endocrine and phosphorus metabolism of 35 patients with PFD/MAS. We report on the age at which extremity fractures occurred and their location and treatment. The results of endocrine and phosphorus metabolism testing and associations between age of first fractures, number of fractures, fracture rate, and metabolic abnormalities were noted. RESULTS: The average follow-up was 14.2 years (range, 2-39 years), during which 172 fractures occurred. The number and sites of fractures were 103 femoral, 25 tibial, 33 humeral, and 11 forearm. Twenty-seven patients had PFD with one or more endocrinopathies and/or phosphaturia, and eight had PFD alone. The endocrinopathies included precocious puberty (n = 19), hyperthyroidism (n = 9), growth hormone excess (n = 6), and one patient each with Cushing syndrome and primary hyperparathyroidism. Twelve patients had phosphaturia. The peak rate of fractures occurred between 6 and 10 years of age and decreased thereafter. Patients with metabolic abnormalities sustained their first fracture at an earlier age (6.9 versus 16.6 years, p < 0.005) and had a higher lifetime rate of fractures (0.29 versus 0.08 fractures/year), relative to patients with PFD alone. Phosphaturia was the single metabolic dysfunction associated with both an earlier age of first fracture (5.1 versus 16.6 years, p < 0.05) and a greater lifetime fracture rate (0.35 versus 0.08 fractures/year, p < 0.05). CONCLUSIONS: The occurrence of extremity fractures in FD peaks between 6 and 10 years of age and declines thereafter. Fractures occur earlier and more frequently in the presence of phosphaturia. These data have implications for long-term prognosis, clinical management, and interpretation of therapeutic interventions.

Adolescent↗

PubMed2003

Dental characteristics of fibrous dysplasia and McCune-Albright syndrome.

OBJECTIVE: Fibrous dysplasia (FD) is a skeletal disorder often associated with McCune-Albright syndrome, a rare multisystem disorder caused by GNAS1 gene mutation. FD frequently affects the craniofacial bones, including the maxilla and the mandible; nevertheless, its effects on dental tissues and the implications for dental care remain unclear. The aim of this study was to characterize the dental features associated with FD and the reaction of affected bones to routine dental therapy. Study design Thirty-two patients with FD underwent dental evaluation and endocrine testing as part of the diagnosis of FD/McCune-Albright syndrome. Any dental anomalies were recorded, and the associations between endocrinopathies and dental anomalies were analyzed statistically by means of the paired t test. RESULTS: Eighty-four percent had FD in the maxilla and/or mandible; endocrine dysfunction; and/or renal phosphate wasting. The caries index scores were 2.9 (ages 4-17 years) and 9.6 (ages 18-50 years). Malocclusion (81%) and other prevalent dental anomalies (41%) included tooth rotation, oligodontia, and taurodontism. The expansion of the maxilla or mandible by FD did not distort the dental arch curvature, and routine dental therapies such as extractions, restorations, and orthodontic treatment did not exacerbate FD lesions. CONCLUSION: Maxillomandibular FD was associated with higher rates of caries and malocclusion than were present in healthy patients. Furthermore, patients with FD did not require special dental management and were able to undergo routine dental care without an exacerbation of FD lesions.

Adolescent↗

PubMed2003

Thyroid carcinoma in the McCune-Albright syndrome: contributory role of activating Gs alpha mutations.

McCune-Albright syndrome (MAS) is defined by the triad of café-au-lait skin pigmentation, polyostotic fibrous dysplasia, and hyperfunctioning endocrinopathies, such as precocious puberty, hyperthyroidism, GH excess, and Cushing's syndrome. This disorder is caused by sporadic, postzygotic activating mutations in the GNAS1 gene, which codes for the G(s)alpha protein in the cAMP signaling cascade. Nodular and diffuse goiters (with and without hyperthyroidism), as well as benign thyroid nodules, have been reported in association with MAS. Herein we report two cases of thyroid carcinoma in patients with MAS. The first is a case of papillary thyroid cancer detected incidentally during a hemithyroidectomy for hyperthyroidism in a 14-yr-old girl. The second is one of a 41-yr-old woman with long-standing MAS and an enlarging thyroid nodule, which was diagnosed as a clear cell thyroid carcinoma, a rare variant of thyroid cancer. Molecular analysis revealed that foci of malignancy and adjacent areas of hyperplasia and some areas of normal thyroid harbored activating mutations of Arg(201) in the GNAS1 gene. These findings suggest that the infrequent development of thyroid carcinoma in MAS patients involves additional mutational or epigenetic events.

Adenocarcinoma, Clear Cell↗

PubMed2003

Telomerase accelerates osteogenesis of bone marrow stromal stem cells by upregulation of CBFA1, osterix, and osteocalcin.

Telomerase activity can prevent telomere shortening and replicative senescence in human somatic cells. We and others have previously demonstrated that forced expression of telomerase in human bone marrow stromal stem cells (BMSSCs) was able to extend their life-span and enhance their bone-forming capability, without inducing malignant transformation. In this study, we determined that telomerase was able to accelerate calcium accumulation of human BMSSCs under osteogenic inductive conditions. Similarly, xenogeneic transplantation of telomerase-expressing BMSSCs (BMSSC-Ts) yielded ectopic bone formation at 2 weeks post-transplantation, 2-4 weeks earlier than typically seen with BMSSCs transfected with empty vector (BMSSC-Cs). Low-density DNA array analysis revealed that telomerase activity increases the expression of G1 regulating genes including cyclin D3, cyclin E1, E2F-4, and DP2, associated with hyperphosphorylation of retinoblastoma (pRb), leading to the extended proliferative capacity of BMSSC-Ts. Importantly, BMSSC-T transplants showed a higher number of human osteogenic cells at 8 weeks post transplantation compared with the BMSSC-C transplants, coupled with a significantly increased osteogenic capacity. One possible mechanism leading to accelerated osteogenesis by BMSSC-Ts may be attributed, at least in part, to the upregulation of the important osteogenic genes such as CBFA1, osterix, and osteocalcin in vitro. Taken together, these findings show that telomerase can accelerate cell cycle progression from G1-to-S phase and enhance osteogenic differentiation of BMSSCs, because of the upregulation of CBFA1, osterix, and osteocalcin.

Alu Elements↗

PubMed2002

Normal vision despite narrowing of the optic canal in fibrous dysplasia.

BACKGROUND: Fibrous dysplasia of bone frequently involves the anterior base of the cranium and results in encasement of the optic-nerve canals. It has been assumed that such encasement leads to constriction and eventual blindness. There is controversy about whether patients should be regularly monitored or whether they should undergo prophylactic decompression of the optic nerve. This question is of particular concern in patients with normal vision, since the risks associated with surgical decompression include blindness. METHODS: We studied 38 patients with fibrous dysplasia of the lesser wing of the sphenoid bone. The patients underwent a detailed neuro-ophthalmologic examination and computed tomography of the face and skull, reformatted to measure the extent of involvement of the optic canal and the area of the canals. The results were compared with those of 38 age- and sex-matched controls. RESULTS: Of the 38 patients, 15 were male and 23 female, and their mean age was 26 years. Twelve had polyostotic fibrous dysplasia, and 26 had the McCune-Albright syndrome. Sixty-seven optic canals were affected by fibrous dysplasia; in 49 of them (73 percent) there was complete encasement. The mean (+/-SD) areas of the right and left canals were 9.6+/-3.8 mm2 and 9.9+/-3.6 mm2, respectively, in the patients, as compared with 12.0+/-2.9 mm2 and 11.9+/-2.7 mm2 in the controls (P=0.009 for the comparison of the right areas and P=0.03 for the comparison of the left areas by the paired t-test). In all but two of the patients, the results of neuro-ophthalmologic examination were normal. In the two patients with monocular visual impairment, the areas of the optic canals were similar on the normal and abnormal sides. CONCLUSIONS: Encasement of the optic canal in fibrous dysplasia causes narrowing of the canal, but that in itself does not result in visual loss. Therefore, prophylactic decompression of the optic nerve does not appear to be indicated on the basis of the presence of fibrous dysplasia on diagnostic images alone, since it does not correlate with visual loss.

Adolescent↗

PubMed2002

Bone formation by human postnatal bone marrow stromal stem cells is enhanced by telomerase expression.

Human postnatal bone marrow stromal stem cells (BMSSCs) have a limited life-span and progressively lose their stem cell properties during ex vivo expansion. Here we report that ectopic expression of human telomerase reverse transcriptase (hTERT) in BMSSCs extended their life-span and maintained their osteogenic potential. In xenogenic transplants, hTERT-expressing BMSSCs (BMSSC-Ts) generated more bone tissue, with a mineralized lamellar bone structure and associated marrow, than did control BMSSCs. The enhanced bone-forming ability of BMSSC-Ts was correlated with a higher and sustained expression of the early pre-osteogenic stem cell marker STRO-1, indicating that telomerase expression helped to maintain the osteogenic stem cell pool during ex vivo expansion. These results show that telomerase expression can overcome critical technical barriers to the ex vivo expansion of BMSSCs, and suggest that telomerase therapy may be a useful strategy for bone regeneration and repair.

Adult↗