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5. Macromolecules produced by bone marrow stromal cells in diffusion chambers
252 reported by Aaron (Lancet i 229 1974) who additionally found reduced calcification fronts. Increased nonmineralizing osteoid surfaces are also found in crush fracture osteoporosis and correlate with negative calcium balance and kinetically determined resorption. The accumulation of trabecular osteoid surfaces of normal thickness appears to be an adverse prognostic indicator for cortical osteoporosis.
3. Vitamin D metabolism in oncogenous osteomalacia R Thakker,’ M Hewison,’ R Karmali,’ R Smith,t and JLH O’Riordan’ *Departnje,7t of Medicine, The Middlesex Hospital, London WIN SAA tThe Nufield Orthopaedic Centre, Oxford OX3 7LD Adult onset hypophosphataemic osteomalacia which occurs in associati?n with mesenchymal tumours is a rare disorder. Removal of the tumour corrects the metabolic disturbances, and it has therefore been postulated that the tumour either produces a phosphaturic factor or a factor that inhibits renal 1-hydroxylase activity. To further investigate this we studied the vitamin D metabolism in one such case. Case study: A 60 year old English lady, who was previously well, presented with a five year history of difficulty in walking. She was noted to have hypophosphataemia, a raised phosphate excretion index (PEI) (normal -0.09 to +0.09), a normal serum calcium, raised alkaline phosphatase and fractures of the ribs and pelvis. Bone biopsy revealed osteomalacia. Further investigation showed no evidence of malabsorption, aminoaciduria, myeloma or renal failure. Serum 25-hydroxy vitamin D3 was 8.9ngiml (normal 340ngiml) and the nPTH was 80pgiml (normal <120pg/ml). Treatment with oral la-hydroxy vitamin D3 and phosphate initially improved her condition but she relapsed after three years and developed a proximal myopathy. Treatment was then stopped and further investigation revealed that the circulating concentration of 1,25-dihydroxy vitamin D3 was undetectable (normal 20-65pgiml). She was also noted to be euphoric and to have lost her sense of smell. Computerised Tomography of the brain demonstrated the presence of an enhancing mass in the right frontal lobe. Excision of the tumour resulted in a rapid rise in 1,25-dihydroxy vitamin D3 to lOOpg/ml, and subsequently an increase in serum phosphate to 0.8mmol/L (normal 0.6-11.3mmoliL) and the restoration of a normal PEI (-0.09). Histology showed the tumour to be an haemangiopericytoma of meninges, a rare cause of tumour related osteomalacia shown to be associated with deficient circulating 1,25-dihydroxy vitamin D?.
4. 1,25(OHJ2D3 regulates c-myc expression in U937 cells R Karmali, A Bhalla, S Farrow, P Lydyard and JLH O’Riordan The Middlesex Hospital, London, UK Expression of c-myc gene is amplified in U937 cells and may account for their abnormal proliferation. Since
Abstracts from the Bone and Tooth Society Meeting 1,25(OH)?D3 is able to inhibit proliferation of U937cells, the effect of 1,25(OH)2D30n c-myc gene expression was therefore studied and was related to changes in cell proliferation and in cell phenotype induced by the hormone. U937 cells were cultured in the presence or the absence of 1,25(OH)*D, up to 72 hours. Cell proliferation was assessed by (?H]thymidine uptake; cell differentiation monitored by the binding of UCHMI, which defines a monocyte-related antigen. Changes in c-myc mRNA were assessed by dot-blot assays. In the presence of 1,25(OH)zDi (lo-“M), inhibition of cell proliferation could only be observed after 24 hours when it was decreased by 25%. By 72 hours, proliferation was reduced to 50% of control values. Cell differentiation, as assessed by UCHMl binding, occurred earlier. At 18 hours 45% of the cells were positive and this fraction increased further to 65% at 72 hours. However changes in c-myc mRNA levels could be detected even earlier. These were reduced to 53% of control values at 6 hours with a further decrease to 30% at 24 hours. It then remained at the same level up to 72 hours. Thus the effect of 1,25(OH)2Di on U937 cell involves first a genomic action as reflected by the decrease of c-myc transcription, which precedes phenotypicchanges and reduction in DNA synthesis.
5. Macromolecules produced by bone marrow stromal cells in diffusion chambers DE Ashhurst,* BA Ashton,t and M Owen$ *Drpar~rr7c17tof At7ntoq St Gcorgck Hospita! Medical Shoal, Lo77do77Swli DRE tDcpmfrt7enf of Rheu777nk7lo~y,Shropshire Orthopaedic Hospikl, Oswcstry, Salop SY70 7AG $MRC Bone Rcscnrcl7 Lohorntor~y,Nuffield Ortl~opartficCcntw, Oxford OX3 iLD When rabbit bone marrow cells are cultured in a diffusion chamber in the visceral cavity of an athymic mouse, most haemopoietic cells die in the first week, but the stromal cells proliferate and differentiate to produce a variety of connective tissue matrices. These have been characterized using histochemical and immunohistochemical techniques; specific polyclonal antibodies to rabbit collagens have been used. The stromal cells first produce a loose fibrous matrix with rather sparsely distributed fibrils mainly of type III collagen. By 2 weeks, some cells have differentiated and are alkaline phosphatase positive; the matrix around these cells contains types I and V collagens and it is mineralized later. More centrally some cells differentiate to produce a cartilaginous matrix which contains type II collagen. This matrix binds Alcian Blue at magnesium chloride concentrations of up to 0.6M which suggests that chondroitin and keratan sulphates are present. The distribution of the collagens is precisely localized; in this respect the matrices resemble the matrices produced de 170710 during embryonic development, rather than the heterogeneous matrices produced in the callus of healing fractures.
3. Vitamin D metabolism in oncogenous osteomalacia R Thakker,’ M Hewison,’ R Karmali,’ R Smith,t and JLH O’Riordan’ *Departnje,7t of Medicine, The Middlesex Hospital, London WIN SAA tThe Nufield Orthopaedic Centre, Oxford OX3 7LD Adult onset hypophosphataemic osteomalacia which occurs in associati?n with mesenchymal tumours is a rare disorder. Removal of the tumour corrects the metabolic disturbances, and it has therefore been postulated that the tumour either produces a phosphaturic factor or a factor that inhibits renal 1-hydroxylase activity. To further investigate this we studied the vitamin D metabolism in one such case. Case study: A 60 year old English lady, who was previously well, presented with a five year history of difficulty in walking. She was noted to have hypophosphataemia, a raised phosphate excretion index (PEI) (normal -0.09 to +0.09), a normal serum calcium, raised alkaline phosphatase and fractures of the ribs and pelvis. Bone biopsy revealed osteomalacia. Further investigation showed no evidence of malabsorption, aminoaciduria, myeloma or renal failure. Serum 25-hydroxy vitamin D3 was 8.9ngiml (normal 340ngiml) and the nPTH was 80pgiml (normal <120pg/ml). Treatment with oral la-hydroxy vitamin D3 and phosphate initially improved her condition but she relapsed after three years and developed a proximal myopathy. Treatment was then stopped and further investigation revealed that the circulating concentration of 1,25-dihydroxy vitamin D3 was undetectable (normal 20-65pgiml). She was also noted to be euphoric and to have lost her sense of smell. Computerised Tomography of the brain demonstrated the presence of an enhancing mass in the right frontal lobe. Excision of the tumour resulted in a rapid rise in 1,25-dihydroxy vitamin D3 to lOOpg/ml, and subsequently an increase in serum phosphate to 0.8mmol/L (normal 0.6-11.3mmoliL) and the restoration of a normal PEI (-0.09). Histology showed the tumour to be an haemangiopericytoma of meninges, a rare cause of tumour related osteomalacia shown to be associated with deficient circulating 1,25-dihydroxy vitamin D?.
4. 1,25(OHJ2D3 regulates c-myc expression in U937 cells R Karmali, A Bhalla, S Farrow, P Lydyard and JLH O’Riordan The Middlesex Hospital, London, UK Expression of c-myc gene is amplified in U937 cells and may account for their abnormal proliferation. Since
Abstracts from the Bone and Tooth Society Meeting 1,25(OH)?D3 is able to inhibit proliferation of U937cells, the effect of 1,25(OH)2D30n c-myc gene expression was therefore studied and was related to changes in cell proliferation and in cell phenotype induced by the hormone. U937 cells were cultured in the presence or the absence of 1,25(OH)*D, up to 72 hours. Cell proliferation was assessed by (?H]thymidine uptake; cell differentiation monitored by the binding of UCHMI, which defines a monocyte-related antigen. Changes in c-myc mRNA were assessed by dot-blot assays. In the presence of 1,25(OH)zDi (lo-“M), inhibition of cell proliferation could only be observed after 24 hours when it was decreased by 25%. By 72 hours, proliferation was reduced to 50% of control values. Cell differentiation, as assessed by UCHMl binding, occurred earlier. At 18 hours 45% of the cells were positive and this fraction increased further to 65% at 72 hours. However changes in c-myc mRNA levels could be detected even earlier. These were reduced to 53% of control values at 6 hours with a further decrease to 30% at 24 hours. It then remained at the same level up to 72 hours. Thus the effect of 1,25(OH)2Di on U937 cell involves first a genomic action as reflected by the decrease of c-myc transcription, which precedes phenotypicchanges and reduction in DNA synthesis.
5. Macromolecules produced by bone marrow stromal cells in diffusion chambers DE Ashhurst,* BA Ashton,t and M Owen$ *Drpar~rr7c17tof At7ntoq St Gcorgck Hospita! Medical Shoal, Lo77do77Swli DRE tDcpmfrt7enf of Rheu777nk7lo~y,Shropshire Orthopaedic Hospikl, Oswcstry, Salop SY70 7AG $MRC Bone Rcscnrcl7 Lohorntor~y,Nuffield Ortl~opartficCcntw, Oxford OX3 iLD When rabbit bone marrow cells are cultured in a diffusion chamber in the visceral cavity of an athymic mouse, most haemopoietic cells die in the first week, but the stromal cells proliferate and differentiate to produce a variety of connective tissue matrices. These have been characterized using histochemical and immunohistochemical techniques; specific polyclonal antibodies to rabbit collagens have been used. The stromal cells first produce a loose fibrous matrix with rather sparsely distributed fibrils mainly of type III collagen. By 2 weeks, some cells have differentiated and are alkaline phosphatase positive; the matrix around these cells contains types I and V collagens and it is mineralized later. More centrally some cells differentiate to produce a cartilaginous matrix which contains type II collagen. This matrix binds Alcian Blue at magnesium chloride concentrations of up to 0.6M which suggests that chondroitin and keratan sulphates are present. The distribution of the collagens is precisely localized; in this respect the matrices resemble the matrices produced de 170710 during embryonic development, rather than the heterogeneous matrices produced in the callus of healing fractures.