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Poster Presentations
Abstracts P-1 to P-49
Posters will be on display throughout the symposium, but will be attended by their presenting authors as follows:
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BONE MARKERS IN DETECTION ON BONE METASTASES IN PROSTATE CANCER C. De la Piedra1*, N. A. Castro-Errecaborde2, C. Mendez-Davila1, C. Garcia- Moreno1, L. Rodriguez de Acuńa1, M. L. Traba1 1Biochemistry Laboratory, Fundacion Jimenez Diaz, Madrid, Spain 2Family Medicine, Hospital Clinico San Carlos, Madrid, Spain Early detection of bone metastases in prostatic carcinoma (PC) is very useful in treatment and prognosis of the disease. Although these metastases are mainly of esclerotic type, both osteoblastic and osteoclastic activities are affected. Sixty six non-treated patients with: benign prostate hyperplasia (n=21), PC in several stages without bone metastases (TXMO) (n=31), and PC in several stages with bone metastases (TXM1) (n=14) were studied. The aim of this work was to evaluate the sensitivity and specificity of a group of bone markers in order to discriminate between patients MO and M1. The following markers were studied: A) Bone formation: 1) Serum Bone Alkaline Phosphatase, IRMA (Tandem Ostase, Beckman); 2) Serum Procollagen I Aminoterminal Propeptide (PINP), RIA (Orion Diagnostica); B) Bone resorption: 1) Urinary Collagen I Aminoterminal Telopeptide (NTX), ELISA (Ostex); 2) Collagen I Carboxyterminal Telopeptide (CTX) : 2-A) urinary alfa-CTX, RIA (Osteometer), 2-B) serum beta-CTX, Elecsys (Roche); Collagen I Carboxyterminal Telopeptides (ICTP), RIA (Orion Diagnostica). In all cases, the levels of bone markers were significantly higher in group M1 than in group MO. A complete separation of groups MO and M1 was achieved with serum PINP and serum beta-CTX, with a sensitivity and specificity of 100%. (Grant FIS 98/0967). EVIDENCE OF INCREASED BONE TURNOVER IN BREAST CANCER PATIENTS WITH BONE METASTASES IN A RETROSPECTIVE STUDY I. Martinovic1*, D. Vrbanec2, M. Jurcic2, B. Belev2, V. Kusec3 1Department of Oncology and Radiotherapy, Clinical Hospital Centre, Zagreb, Croatia 2Department of Pathophysiology, Clinical Hospital Centre, Zagreb, Croatia 3Clinical Institute of Laboratory Diagnosis, Clinical Hospital Centre, Zagreb, Croatia This retrospective study involved an analysis of available data on tumor markers and bone markers of 28 patients with clinical evidence of breast cancer metastases. This included one or more sets of data on tumor markers (CEA, CA 15-3, MCA), bone formation markers (total and bone alkaline phosphatase, osteocalcin, procollagen), and bone resorption markers (telopeptide, pyridinolines). The data on bone markers were compared with those obtained from breast cancer patients without metastases and normal women of the same age range. In patients with bone metastases approximately 80 percent of the values on tumor markers (CEA, CA 15-3) and between 16-96 percent of the values on bone markers were elevated, predominantly bone resorption markers. Bone resorption was also significantly higher than in breast cancer patients without metastases than in normal women, as assessed by telopeptide and both telopeptide and pyridinolines, respectively. Statistically significant positive correlations between tumour markers (CEA, CA 15-3) and bone resorption markers indicated that bone destruction was the main bone action in the metastatic disease. Correlations between bone markers were positive, revealing involvement of both formation and resorption processes in the metastatic spread of breast carcinoma to the skeleton. Correspondingly, in a small sample a positive correlation of CEA and MCA with bone formation indicator procollagen was found. These results showed increased bone turnover with prevailing bone resorption in breast cancer patients with bone metastases. Bone resorption was higher than in breast cancer patients without metastases and normal women of the same age range. In addition, bone destruction might be related to the extent of the malignant disease. COMBINED USE OF HYDROXYLAPATITE CERAMICS AND DEMINERALIZED BONE MATRIX FOR SUBSTITUTION OF BONE DEFECTS (AN EXPERIMENTAL STUDY) V. V. Golovchenko1*, V. I. Luzin2, E. P. Berezhnoy2 1Department of Traumatology and Orthopedy, State Medical University, Lugansk, Ukraine 2Department of Normal Anatomy, State Medical University, Lugansk, Ukraine The experimental research was carried out on 72 white rats with initial mass 130- 150 grams. The purpose of the study was to determine the influence of a demineralized bone matrix in a combination with powdery hydroxylapatite ceramics (CERHAP-M) on reparative osteogenesis. A 2.2-millimeter circular tibial bony defect was made in proximal metaphysis and combination of demineralized bone matrix with powdery hydroxylapatite ceramics was placed on the defect. Were used control groups where defects were empty (the group 1) or where defects was filled by a only demineralized bone matrix (the group 2) or by a only CERHAP-M (the group 3). Six rats from each group were sacrificed on 15, 30, 60, and 90 days after grafting. On sections of a zone of the bone defects, painted by hematoxylin-eosine, were carried out morphometric analysis of biodegradation of particles of hydroxylapatite, and also counted an index of osteointegration. Besides, defined a volumetric contents of a trabecular bone in a zone near to zone of a defect. Histomorphometric research has shown, that at filling of a defect of CERHAP-M have detected, that this material has mainly osteoconductive properties. The demineralized bone matrix exhibited mainly osteoinductive activity. In both cases the osteogenesis did not pass cartilagenous stage of a bone formation. If at filling of defects of the demineralized bone matrix in combination from a hydroxyapatite ceramics the degree of a biodegradation of particles of ceramics increased (the square of particles of CERHAP-M was decreased). Together with it, in comparison with group 3 the index of osteointegration was enlarged, and the volume size of a trabecular bone to 15 and 30 days was more check on 21.76 % and 14.69 %. Thus, the combined application of demineralized bone matrix with hydroxylapatite ceramics for filling of defects is accompanied by improvement of tissue composition of a regenerate and optimizes processes of reparative regeneration of a bone. ENDOGLIN (CD105) EXPRESSION AND DISTRIBUTION ON HUMAN SARCOMA CELLS L. Postiglione1*, G. Di Domenico1, L. Del Vecchio2, S. Montagnani3, M. Macrģ2, G. Rossi1 1Dipartimento di Biologia e Patologia Cellulare e Molecolare 'L. Califano', Un. 'Federico II', Napoli, Italy 2Servizio di Immunoematologia, AORN 'A. Cardarelli', Napoli, Italy 3Dipartimento di Scienze Biomorfologiche e Funzionali, Un. 'Federico II', Napoli, Italy Endoglin (CD105) is a homodimeric transmembrane glycoprotein, primarly expressed on endothelial cells, and represents an accessory component of the TGF- beta receptor complex. Endoglin binds TGF-beta1 and beta3 with high affinity and recent studies have shown that it interacts with other components of the TGF-beta superfamily, including activin-A, BMP-2 and BMP-7. There are some evidences supporting the idea that Endoglin is involved in angiogenesis and represents a powerful marker of neovascularization. It has been demonstrated that CD105 is expressed in hemopoietic tumors, such as mielogenous leukemias (AML) and lymphoblastic leukemias (B-ALL), and the analysis of CD105 expression in the various subtypes indicates that it is always expressed by most immature subtypes and that it is absent on more differentiated ones. These data suggest that Endoglin expression might be modulated during the cancerogenesis and they lead to study CD105 expression in human solid tumors of different histotype. The aim of our study was to evaluate Endoglin expression and distribution on different human tumoral cells of connectival origin, by Western blotting, Flow cytometric analysis and Immunofluorescence microscopy, using a specific anti- CD105 mAb (mouse, SN-6, Valter Occhiena, Torino). The cells used in our study were: human undifferenziated osteosarcoma cells MG-63 and human differenziated osteosarcoma cells SaOS-2; human chondrosarcoma cells SW1353; human rhabdomiosarcoma cells A204, human fibrosarcoma cells Hs913T; human epitheloid sarcoma cells VA-ES-BJ. Human skin fibroblasts and human endothelial cells were used as a normal control. Results showed a different expression pattern of Endoglin on the different tumoral cell lines. CD105 protein was strongly expressed on several human sarcoma cells studied as MG-63 SW1353, Hs913T and VA-ES-BJ. Whereas, CD105 protein was almost absent on A204 and it showed a heterogeneous pattern expression on SaOS-2 cells, characterized by a double peak, by Flow cytometric analysis. These results indicate that Endoglin is differently expressed in sarcoma cells, showing a high level of expression on almost all human sarcoma cells, except the human rhabdomiosarcoma cells A204. These data strongly suggest that CD105 is a promising molecular target to set up innovative diagnostic and bioimmunotherapeutic approaches in human malignancies of connectival origin. VALIDITY OF BIOCHEMICAL BONE MARKER IN PATIENTS WITH MYELOMA MULTIPLEX M. S. Prebilic, Z. C. Orlic*, A. N. Duletic, I. Host, I. Prebilic, D. Petranovic Internal Clinic, University of Rijeka, Croatia Osteolysis, hypercalcaemia and pathologic fractures are common complications in patients with multiple myeloma. In 64 multiple myeloma patients we performed bone densitometry (DEXA) of lumbal spine and hip to analyse bone mass. Homeostasis of bone metabolism was evaluated by electrolytes (calcium, phosphorus, magnesium in serum and 24 h urine) creatinin, whole alkaline phosphatase, bone specific alkaline phosphatase and osteocalcine in serum and pyridinoline and deoxypyridinoline in urine. We compared those data with the same kind of analyses of 50 patients with osteoporosis type I of same age and sex. Patients with multiple myeloma had significantly higher level of collagen degradation products in urine compared with osteoporotic patients (p<0.05). The difference between of those two groups was statisticaly highest in patients with stage III multiple myeloma. We conclude that biochemical markers of bone destruction and hypercalcaemia are significant prognostic factors for bone loss which needs to be prevented before fractures appear. IMMUNOLOCALIZATION OF OSTEOCALCIN IN CALCIFIED MICROSPHERES IN LAMB VERTEBRAE AND MOUSE BONES USING PAP METHODS S. M. Shahtaheri1*, J. E. Aaron2, B. A. Oakley2 1Hamadan Medical School, Hamadan, Iran 2Department of Human Biology, University of Leeds, Leeds, UK Osteocalcin is a calcium binding protein with a high affinity for hydroxyapatite and uncertain role in calcification. In order to better understand the role of this bone specific protein in skeletal metabolism it is essential to determine its precise distribution. Using rabbit anti-bovine polyclonal antibody and anti-mouse affinity purified monoclonal antibody the distribution of the stain was examined in chick limbs, in foetal lamb vertebrae and in 5-days-old mouse calvarium and long bone respectively. Controls incubated without primary antibody were negative. In undecalcified preparations the antibody was located in osteoblasts and in discrete bands at the calcification front. In decalcified preparations the stain was also found in association with a proportion of osteocytes and in the region of the golgi body and outside the cells as stained granules. Further stain was distributed heterogenously throughout the mineral matrix (but not in the osteoid tissue) and the nature of the staining was granular. At all locations staining was associated with particles approximately 1 µm in diameter resembling the calcified microspheres which constitute the inorganic phase of immature and mature bone. It was concluded that osteocalcin was integral to these microspheres where it functions in the containment of calcium phosphate, influencing its form and biomechanical properties. INVOLVEMENT OF BETA-ACTIN IN THE EFFECTS OF FETAL CALF SERUM ON THE BIOSYNTHESIS AND SECRETION OF OSTEOPONTIN IN MG-63 CELLS IN CULTURE D. W. Liu1*, B. B. Vandahl2, S. Birkelund2, B. Melsen1 1Department of Orthodontics, Royal Dental College, Aarhus, Denmark 2Department of Medical Microbiology and Immunology, University of Aarhus, Aarhus, Denmark Living condition of cells in vitro determines their functional responses to mechanical stimuli. To look into the impacts of serum supplementation on the biosynthesis and secretion of osteopontin in relation to beta-actin filaments distribution in MG-63 cells, different concentrations (0%, 2%, 5% and 10%) of Fetal Calf Serum (FCS) supplemented MEM culture medium were approached. To pulse- label osteopontin, [35S]-methionine/cysterine was incorporated in culture medium (147 mCi/ml). By autoradiography and immunofluorenscence tests, it was revealed that both the 'intra-' and 'extra-' cellular components of the newly synthesized (labeled) osteopontin were drastically reduced when the serum supplementation was lowered from 10% as normally used down to 5% and below (p<0.05). Meanwhile, beta-actin staining exhibited an obvious change of the shape and size of the cells. As a conclusion, the shape and size of the cells (reflected by beta-actin expression and orientation) are actively involved in the biosynthesis and secretion of osteopontin in MG-63 cells. It therefore is inferred that a proper serum concentration in medium (not less than 5%) be chosen in in vitro mechanoconduction experiments. Since the lower serum supplementation simulates the reduction or occlusion of blood circulation in periodontal ligament when heavier (either orthodontic or traumatic) force is loaded to teeth, the findings in this study do suggest lighter force be used to move teeth in a way of protecting the PDL cells from being starved from blood supply in clinical orthodontics. EFFECTS PRODUCED BY DIHYDROTESTOSTERONE AND BICALUTAMIDE ON INTERLEUKIN-6 AND AMINOTERMINAL PROPEPTIDE OF PROCOLLAGEN I RELEASE BY HUMAN OSTEOBLASTS IN CULTURE. M. Lefort1*, M. Dķaz-Curiel2, C. De la Piedra1 1Biochemistry Laboratory, Fundacion Jimenez Diaz, Madrid, Spain 2Internal Medicine, Fundacion Jimenez Diaz, Madrid, Spain It is known the protective effect of androgens on bone mass, although the mecanisms involved are not fully understood. On the other hand, new non-steroidal antiandrogens like Bicalutamide (Casodex) have been developed, that exert an inhibitory action on prostatic cell growth, but they have the same positive effects than androgens on bone. The aim of this work was to investigate if the protective effect of dihydrotestosterone (DHT) and Bicalutamide (Bic) on bone mass is produced through the inhibition of interleukin-6 (IL-6) synthesis, known osteoclastic activator, by the osteoblast, both at constitutive and IL-1 stimulated level. The possible effects of DHT and Bic on aminoterminal propeptide of procollagen I (PINP) has also been studied. Osteoblasts were obtained from trabecular bone of male patients undergoing orthopedic surgery. The cells were cultured as described by Marie et al. (In Vitro Cell & Developm Biol 1989; 25:373-380) with some modifications. Osteoblasts at confluence were cultured in serum and phenol red-free DMEM containing 0.1 % BSA for 24 h and 10-7 M DHT (Fluka), 8 µg/ml Bic (Astra-Zeneca) or vehicle (ethanol) were added with or without IL-1beta (1ng/ml). After 24 h, levels of IL-6 (RIA, Medgenix) and PINP (RIA, Orion Diagnostica) were determined in culture medium. As expected, IL-1 induced an increase in IL-6 secretion. However, the constitutive or IL-1 stimulated IL-6 production were not altered by treatment with DHT or Bic. We did not find any significant effect of Bic or DHT on PINP levels. These findings suggest that the beneficial effects of DHT and Bic on bone mass may not be mediated by a decrease in osteoblastic IL-6 secretion or an increase in collagen production. Other mechanisms may be implicated in the effects of DHT and Bic on bone mass. Supported by ASTRA-ZENECA. DISORDERS BONE FORMATIONS IN IMPAIRMENT HEMOPOIESIS V. I. Rugal*, V. Gonchar, V. N. Kravets Russian Research Institute of Hematology and Transfusiology, St. Petersburg, Russia AIM. Earlier we demonstrated the importance of bone cells in regulation development hemopoietic precursors in normal hemopoiesis. The aim of this work was study of bone tissue in impairment hemopoietic function of bone marrow. METHODS. Functional and structural features cells of iliac bone have been characterized in 50 patients with hypoplasia hemopoiesis connected with defects of hemopoietic microenvironment. Cultural, morphometric and ultracytochemical methods were used. RESULTS. The organ culture of bone tissue showed high proliferative activity of cells precursors of bone marrow stroma. The morphometric investigation of the whole bone fragments showed the increase of value of bone trabeculs in hypoplastic conditions 1.4 volume increase, in aplasia 1.7 volume increase. The number of osteogenic cells per unit of square in histological preparation of iliac bone in hypoplasia was 1.5 volume increase, and in aplasia was 2.0 volume increase. The ultracytochemical analysis demonstrated the increase functional activity of the intramedullar and endosteal cells of bone fragments. In osteogenic and stromal cells were found pathological intranuclear bodies. CONCLUSIONS. The cells of bone tissue are the main part of hemopoietic microenvironment. Disorders of the structure and function osteogenic cells can be cause impairment both bone and development of hemopoietic precursors. Close relationship between bone cells and hemopoietic cells confirms the thesis about the existence of a common cell progenitor of the hemopoietic and bone tissue in adult. EFFECTS OF 17BETA-ESTRADIOL, TAMOXIFEN AND RALOXIFENE ON INSULIN GROWTH FACTOR I AND TRANSFORMING GROWTH FACTOR I EXPRESSION IN HUMAN OSTEOBLAST CULTURE C. Garcia-Moreno*, C. De la Piedra Biochemistry Laboratory, Fundacion Jimenez Diaz, Madrid, Spain It is known that estrogens exert a protective action on bone mass and that postmenopausal lack of estradiol leads to an accelerated bone loss, but the physiological mechanisms implicated in the estrogen actions on bone are not fully understood. Insulin-like growth factor (IGF-I), an anabolic protein, and transforming growth factor I (TGF-I), an inhibitor of osteoclast activity and recruitment, are two local factors synthesized by the osteoblasts, which play an important role in the remodeling coupling. The aim of this work was to investigate if the actions of 17beta- estradiol and raloxifene and tamoxifen (two selective estrogen receptor modulators) on bone were mediated through the induction of changes in the expression of IGF-I and/or TGF-I. For that purpose, we investigate the effects of 17beta-estradiol, raloxifene and tamoxifen on IGF-I and TGFbeta-I mRNA levels in a primary human osteoblast culture. Osteoblasts were obtained from seven different postmenopausal women undergoing orthopedic surgery. The cells were cultured as described by Marie et al. (In Vitro Cell & Developm Biol 1989; 25:373-380). Osteoblasts were preincubated for 24h in serum-free DMEM supplemented with 0.1% BSA. After preincubation, the culture medium was changed by DMEM supplemented with 0.1% BSA (control samples), and 17beta-estradiol, tamoxifen or raloxifene at 10-8M concentration were added to the culture. After 1, 3, 6, 24 or 30h, medium was removed and total RNA was isolated. After RNA extraction, IGF-I and TGFbeta-I mRNA levels were assessed by reverse transcription followed by polymerase chain- reaction (RT-PCR). Incubation with 10-8M 17beta-estradiol or 10-8M raloxifene, but not with 10-8M tamoxifen, increased significantly the constitutive levels of IGF-I mRNA after 24 hours and 30 hours of incubation. However, we did not find any significant effect of these three compounds on TGFbeta-I mRNA at any of the studied incubation times. These findings suggest that the effects of estradiol and raloxifene on bone mass may be mediated by an increase in IGF-I expression, but not by actions on TGFbeta-I. Tamoxifen effects on bone appear not be mediated by these cytokines. BMP-1 EXPRESSION IS DEVELOPMENTALLY REGULATED DURING IN VIVO OSTEOGENESIS IN THE RAT TIBIA: A STUDY BY IN SITU HYBRIDIZATION P. Manduca1*, S. Zanotti1, F. Galmozzi1, A. Favre2, O. Barbieri1, D. Di Martino1 1Dobig, University of Genova, Italy 2Ist.G.Gaslini, Genova, Italy We have investigated the expression of BMP-1 (procollagenase I), a proteinase involved in fibrillar collagen processing, which removes the carboxyl-terminal fragment from pro-collagen trimers of type I, II and III collagens. In osteogenic cells in culture BMP-1 produces the carboxyl-trimer (C3) from pro-collagen type I, a molecule recently proven to be chemoattractant for human endothelial and tumor cells. We here present evidence, obtained by in situ hybridization, that the expression of BMP-1 mRNA is developmentally regulated in the bone, cartilage and bone marrow compartments of the developing long bone (tibia) in fetal, new born and young animals. In bone cells, the expression of BMP-1 is maximal in lining osteoblasts in active phase of bone deposition and is associated also to osteoclasts. BMP-1 is expressed in the periosteum in modulated fashion during bone growth. The expression in the bone marrow is limited to areas (possibly clones of stromal cells) and some of these are specifically localized within the bone shaft microenvironment. Both proliferating and mature-hyperthrophic chondrocytes express BMP-1, although the location and kind of cells expressing it varies during development of the bone. Moreover, the expression of BMP-1 in the epiphysis shows marked asymmetries, being most expressed in the proximal epiphysis. NEW WAY OF BONE REGENERATION IN EXPERIMENT V. A. Gonchar*, V. I. Rugal, V. N. Kravets, F. Baranovsky, V. Michailov Institute of Haematology and Transfusiology, St. Petersburg, Russia AIM: To study peculiarities the regeneration of long tube-bones fractures by treatment Osteolin in experiment at rats. METHODS: Adult rats caused combine mechanic fractures of tibiae and fibiae. Experimental rats got 0.1 mg/kg immunoglobulins (Ig) sera of rabbits immunized by femur epiphyses from adult rats. Control rats got analogically same dose Ig sera nonimmune rabbits. Course of treatment included 3 injections with interval 48 hours. Hystological investigation made once a week from day of fracture. RESULTS: Control rats showed usual bone regeneration: to 3d week was formed callus with tipical osteo structure (OS) on all fracture zone (FZ) without bone marrow canal (BMC). Periosteum was presented several layers of fibroblastlike cells. Experimental rats already 1-st week showed total growth of cartilage tissue (CT) apparently bone marrow genesis and forming periostal muff. Simultaneously was formed central BMC which determined by osseal model without OS but with extracellular basophillia. In CT arised niduses of concellous bone (NCB). At 3rd week restored thickening FZ by CT layer and wide periostal zone. To 4th week compact osseal tissue (COT) with typical OS became predominated on sites where persisted NCB arised zones. Atypical cell were not found. CONCLUSION: Formation COT from CT is phylogeny ancient. Used Osteolin allowed to reactivate this genetic program in precurser cells common for haemopoetic, cartilage and osseous tissues. CHANGES IN GENE EXPRESSION IN HUMAN OSTEOBLASTS FROM YOUNG AND OLD DONORS IN DIFFERENT CULTURE CONDITIONS D. C. Ireland*, S. R. Beavan, S. Bord, J. E. Compston School of Clinical Medicine, University of Cambridge, Cambridge, UK Osteoporosis is a common disease of the elderly. Loss of biosynthetic ability by osteoblasts could contribute to the alterations in bone resorption and formation seen with ageing. We have examined the growth of osteoblasts from female donors aged one year (young donor, YD-hOBs) and 66 years (old donor, OD-hOBs) in different culture conditions. Markers of proliferation, differentiation, mineralization, senescence and apoptosis were measured in cells grown on plastic or collagen in medium with human serum, without (proliferation medium) and with (differentiation medium) hydrocortisone and beta-glycerophosphate. mRNAs for ER-alpha, ER-beta, COL1, ALP, OC, RANKL and OPG were measured by real-time RT-PCR. Mineralization was detected using von Kossa reagent, senescence using detection of beta-galactosidase activity at pH 6 and apoptosis using TUNEL and antibody against active caspase 3. YD-hOBs grew well in proliferation medium on both substrates. OD-hOBs grew more slowly than YD-hOBs in proliferation medium on plastic and remained in clumps. OD-hOBs cultured in proliferation or differentiation medium on collagen grew more quickly than on plastic and formed monolayers. OD-hOBS on collagen in differentiation medium formed mineralized nodules five days after reaching confluence whereas YD-hOBs did not. Cultures of OD-hOBs on plastic and collagen in both proliferation and growth medium showed very few positive cells when stained for apoptosis but a high proportion of positive cells when stained for senescence. Changes from days two to nine in mRNA levels in hOBs cultured on collagen in differentiation medium reflected the different rates of differentiation of the cell lines. ER-beta mRNA increased 18.5-fold (p<0.001) in OD-hOBs but decreased slightly in YD-hOBs. COL1 and ALP mRNA levels increased in YD-hOBs (5-fold and 9-fold (p<0.01)) but decreased 3.5-fold (p<0.001) and 1.3-fold (ns) in OD-hOBs. When 17- beta estradiol was added, COL1, ALP and OPG mRNAs were higher at both two and nine days in YD-hOBs than in controls (2-fold, 2-fold and 2.5-fold (p<0.05) at two days with saturating estradiol) but were not increased in OD-hOBs. The limited synthesis of COL1 mRNA by OD-hOBs and their altered sensitivity to estradiol may be attributable to their increased rate of differentiation and mineralization DO STEM CELLS AGE? H-V. Leskelä1,2*, S. Niskanen1, J. Risteli3, P. Lehenkari1,2 1Department of Anatomy and Cell Biology, University of Oulu, Finland 2Department of Surgery, University of Oulu, Finland 3Department of Clinical Chemistry, University of Oulu, Finland Human bone marrow contains both hematopoietic stem cells and non- hematopoietic stem cells. The latter are often referred to as mesenchymal stem cells or marrow stromal cells (MSCs) (1). MSCs are multipotent cells that can differentiate into multiple lineages including osteoblasts, adipocytes, chondrocytes, myocytes, astrocytes, oligodendrocytes and neurons (1). Alkaline phosphatase and type I collagen are associated with the bone cell phenotype and are actively expressed during the maturation of the osteoblast (2). During mineralization, calcium deposits are formed into the mature organic matrix and develop into bone-like nodules (3). In this study we investigated the ability of MSCs, from female patients at different ages, to differentiate into osteoblasts and the ability of these cells to mineralize extracellular matrix in vitro. Human MSCs were obtained from 18 women ranging in age from 43 to 90 years from the femur neck. For osteogenic differentation MSCs were cultured in the basal medium in the presence and absence of dexamethasone (100nM). Alkaline phosphatase activity and the amino-terminal propeptide of type I procollagen (PINP) concentration were determined at days 7, 14 and 21. For extracellular matrix mineralization MSCs were cultured in basal medium in the presence and absence of b- glycerophosphate (10mM). Matrix mineralization, associated with the synthesis of calcium, was demonstrated at days 21, 28, 35, 42 and 49 by determining calcium concentration. Dexamethasone treatment increased levels of alkaline phosphatase 2.3-fold and PINP 6.8-fold, when MSCs were cultured for 21 days. In addition, b- glycerophosphate stimulated calcium deposition 3.0-fold when cultured for 49 days. In our material the activity of alkaline phosphatase increased with age for women when MSCs cultured 21 days. Our data suggest that the MSCs osteogenic potential does not decrease in aging woman. This observation might partly explain why osteoporotic bone has clinically good healing capacity. (1) Colter et al., 2001; (2) Beresford et al., 1986; (3) Dippolito et al., 1999 INFLUENCE OF SOME NON-IONIZING ELECTROMAGNETIC WAVES ON MORPHOFUNCTIONAL STATE OF EPIPHYSEAL CARTILAGES OF PREADOLESCENT WHITE RATS V. I. Luzin Department of Normal Anatomy, State Medical University, Lugansk, Ukraine The research was carried out on 280 preadolescent white rats with initial mass 65- 70 grams. The animals were irradiated by electromagnetic (EM) waves with frequency 61.5 GigaHaertz in modes of impulse and continuous generation, Helium- Neon laser in modes 0.5 milliWatts/cm2 during 10 minutes and 15 milliWatts/cm2 during 2 minutes, and also by volume-combinative impulse EM fields with amplitude 0.04/0.01 Teslas ('direct' configuration) and 0.04/0.05 Teslas ('transversal' configuration). Morphometry of epiphyseal cartilages of a tibial bone carried out with usage of classification of V. Koveshnikov. It was established, that at long (30 and 90 days) exposure under experimental animals by EM waves of the highest frequency in all series marked the increase of common width of an epiphyseal cartilage with decrease of its osteogenic functions (first of all decrease the volumetric contents of a primary spongiosa in a zone of primary osteogenesis), the most expressive at impulsed regimes. Enlargement of an proximal tibial epiphyseal cartilage and all of its zones with disturbance of boneformed function accompanied influence of He-Ne laser begining from the 30th day of exposure (first of all decrease volumetric contents of an osteoblasts in a zone of primary osteogenesis). During the readaptation after 30-day's exposure the revealed faults were softed in a large degree; amplitude of deviations was directly dependent on power of radiation. At last, at irradiation by volum-combinative EM impulse fields was also detected the enlargement of an proximal tibial epiphyseal cartilage and all of its zones with decrease of volum contents of a primary spongiosa in a zone of osteogenesis, which is restored during the period of readaptation. In the group with 'direct' configuration of impulses the expressiveness of deviations during a readaptation was larger. The received data allow to suppose, that investigated low-intensive non-ionizing radiations have the similar mechanism of action on morphofunctional properties of epiphyseal cartilages of long tubular bones of preadolescent rats. MICROARRAY ANALYSIS OF LATE PHASE GENES DURING BMP2- INDUCED OSTEOBLAST DIFFERENTIATION B. L. T. Vaes1*, K. J. Dechering3, D. S. de Jong1, J. M. A. Hendriks1, E. J. J. van Zoelen2, W. Olijve1,3, W. T. Steegenga1 1Department of Applied Biology, University of Nijmegen, Nijmegen, The Netherlands 2Department of Cell Biology, University of Nijmegen, Nijmegen, The Netherlands 3Target Discovery Unit, N.V. Organon, Oss, The Netherlands Osteoblast differentiation can be described by three phases. Firstly a proliferation phase, followed by a phase in which extracellular matrix is deposited, and in the last phase this matrix becomes mineralized. The three phases are characterized by specific gene expression patterns, but only the transcription factor cbfa1 and the late phase gene osteocalcin are known to be osteoblast specific. For a more accurate description of osteoblast differentiation and the identification of novel osteoblast specific genes, microarrays were employed to analyze gene expression on a broad scale. To study osteoblast differentiation, the murine mesenchymal progenitor cell line C2C12 was used as a model system. When treated with bone morphogenetic protein-2 (BMP2), these cells differentiated into osteoblastic cells within six days, with up-regulation at the mRNA level of osteoblast markers such as alkaline phosphatase and osteocalcin. Microarray analysis was done with mouse cDNA arrays with which expression regulation of approximately 9500 genes can be measured. During a time course experiment, 343 genes were over twofold modulated on microarray. These genes, (180 up- and 163 down-regulated) were used for cluster analysis to order the genes according to similarity in expression profiles. Cluster analysis divided the genes into groups of distinct kinetic patterns, showing early, intermediate or late phase responses. Description of the dataset in terms of protein function elucidated subsequent transient regulation of sets of proteins involved in intracellular signalling (e.g. transcription factors), extracellular matrix formation (e.g. collagens), and other secreted proteins (e.g. extracellular messengers and proteases). A subset of 54 late phase genes was further studied to determine osteoblast specific induction by BMP2. Expression analysis in different cell types, revealed four novel genes and four ESTs that were preferentially induced in osteoblast-like cell types, and were identified as new late phase markers for in vitro osteoblast differentiation. The knowledge about temporal expression of large sets of genes and their function leads to a better understanding of osteoblast differentiation, function and bone development. In combination with specificity of expression regulation, this facilitates the search for key regulators of osteoblast development that in future might serve as drug targets to treat bone related disorders. CLONING AND CHARACTERIZATION OF THE 5' FLANKING REGION OF THE MURINE OSTEOPROTEGERIN GENE P. Nemeth*, F. Varga, K. Klaushofer Ludwig Boltzmann Institute of Osteology,4th Medical Dept., Hanusch-Hospital, Vienna, Austria Osteoprotegerin (OPG), as a decoy receptor, is well known to inhibit osteoclastogenesis and osteoclast function by preventing receptor activator of NF- kappaB ligand (RANKL) from binding to receptor activator of NF-kappaB (RANK). To gain more insight into the regulatory process of OPG we cloned and characterized the murine osteoprotegerin promoter. Cloning of the OPG 5' flanking region was done by genome walking. The 3' primers were located within the 5' untranslated transcript of the murine OPG mRNA. 5' primers were provided by Clontech, sequencing was done automatically. The correctness of the sequence was confirmed by reamplification of genomic mouse DNA. The primers were derived from the cloned promoter sequence. Electro mobility shift assay (EMSA) was performed with chemically synthesized oligo nucleotides labeled with polynucleotide kinase (Pharmacia) and in vitro translated Cbfa1 (ivtCbfa1) using an erythrocyte translation system (Promega). For transfection assays we cloned the putative promoter into SEAP2-basic reporter vector (Clontech). Transfections were done into both MC3T3-E1 and ST2 cell lines using liposomal transfer (Dosper, Roche). We isolated 1761 base pairs of the 5'flanking region next to the putative transcription start for the murine OPG mRNA (GenBank AB013899S1). Comparison with the human OPG promoter revealed only weak homology. Compared with the same part of the humane promoter we found three Cbfa1 binding sites. Referring to the transcription start of the OPG mRNA, the most distal Cbfa1 binding site was found to be homologue to the sequence of the collagen alpha (I) promoter. It bound ivtCbfa1 only very weak. The proximal Cbfa1 binding site was homologue to the osteopontin Cbfa1-binding site with a higher affinity to ivtCbfa1. The middle Cbfa1 binding site showed the highest affinity in the EMSA and presented a sequence that has not been described until now. Transfection of the murine OPG promoter construct into MC3T3-E1 and ST2 cells, showed a weak increase of the reporter gene expression, but was only significant in ST2 cells. In summary we isolated a 5'flanking region of the OPG gene, which contained 3 Cbfa1 binding sites capable of binding ivtCbfa1 and showed promoter activity in transfection assays into osteoblast like cells. ISOLATION OF A CDNA WITH HOMOLOGY TO CARBOXYPEPTIDASE D FROM OSTEOBLASTS M. Rumpler*, F. Varga, K. Klaushofer Ludwig Boltzmann Insitute of Osteology, Hanusch Hospital, 4th Medical Dept., Vienna, Austria Cell lines can change their phenotype and their morphology with ongoing culture time often accompanied by an increased formation of tightly sealed contacts. The differentiation of bone marrow stromal cells into osteoblasts can be inhibited by preventing cell adhesion. In ostoeblastic cells confluence is considered to be a transition point in the differentiation process. This implicates that the formation of cell-cell contacts, together with other regulatory influences, play an essential role in the differentiation process. We used Differential Display to find genes, which are regulated when osteoblast-like cells reach confluence. Total RNA was isolated from MC3T3-E1 cells seeded at 5,000 and 50,000 cells/cm2 in aMEM supplemented with 5% FCS and cultured for 3 days. Total RNA was reverse transcribed with an oligo-dT primer. Thereafter the cDNA was amplified using an arbitrary primer and an oligo-dT primer in a PCR. After gel electrophoresis differentially expressed bands were isolated, cloned and used for further investigations. RNA from mouse tissue was isolated using TRIZOL reagent, reverse transcribed and amplified in a PCR. One isolated cDNA fragment exhibited a length of 550 bp. Sequence analysis exhibited a homology to the carboxypeptidase D from mouse, human and rat in a distance of 149 bp. This homologue region belongs to the domain 3 of the carboxypeptidase D. The presence of this cDNA was verified in MC3T3-E1 cells, ST2 and NIH3T3 cells by PCR with gene specific primers. Treatment of the mouse stromal cell line ST2 with ascorbic acid led to an increased expression of this cDNA. In MC3T3-E1 cells its expression was increased by T3 treatment. The expression of this cDNA with homology to carboxypeptidase D was found in brain, lung, kidney, ribs, calvariae, heart and liver, but not in thymus, spleen and muscle. In summary, the finding of a new differentially expressed gene with a partial homology to a carboxypeptidase points to a function of this enzyme in osteoblasts. LOW CONCENTRATION OF CLODRONATE IN THE BONE RINSING SOLUTION CAUSES APOPTOSIS OF CULTURED OSTEOCLASTS M. I. Kellinsalmi1*, M. Hämäläinen1, P. Lehenkari1,2 1Department of Orthopaedics, Universital Hospital of Oulu, Finland 2Department of Anatomy and Cell Biology, University of Oulu, Finland Background of the study: Bisphosphonates are the most important group of antiresorptive drugs when situations related to excess bone resorption are treated. Our aim is to study the prevention of osteolysis in the border of the femoral stem and bone and also in the bone-cement border using non-amino bisphosphonate, clodronate, to find out the optimal concentration of clodronate in the rinsing solution and optimal rinsing time. To study these parameters we made osteoclast cultures with rat bone cells, which were grown in the bovine bone chips. Prior to culture the bone chips were rinsed using different concentrations of clodronate-saline solution. Results: We find out that concentrations 10 mM, 2 mM and 0.2 mM of clodronate lowered equally effectively the number of osteoclasts and the resorbtion area. The mean apoptosis index of the control group was 0.09. It was tripled in concentration 0.02 mM also the apoptosis index was increased to 0.49 when rinsing time was continued to three minutes. With 0.2 mM concentration the apoptosis index was the same whether the rinsing time was one or three minutes. Further more the apoptosis index increased only a little with concentration 2 mM being 0.73. Conclusion: The mechanism of action of clodronate found to be osteoclast specific; no effect on stromal cells could be observed even at high concentrations. Our data suggests that the decrease in osteoclast number was due to osteoclast apoptosis that was dose- and timedependently increased. Clodronate attached very tightly to the bone and already short rinsing using 0.02mM concentrations had a clear effect in the prevention of the bone resorbtion. Finally, we conclude that the optimal concentration of clodronate, when applied locally in the rinsing saline is 0.2 mM and the optimal rinsing time is one minute. THE IDENTIFICATION OF GENES REGULATED IN OSTEOCLAST DIFFERENTIATION BY ARRAY ANALYSIS T. Vaughan1*, C. J. Aitken2, M. A. LeRoy1, A. S. Mellick1, W. E. Simcock1, M. S. Kim1, G. C. Nicholson2, N. A. Morrison1 1Genomics Research Centre, School of Health Science, Griffith University, Gold Coast, Australia 2Department of Medicine, The University of Melbourne, Barwon Health, Geelong Hospital, Geelong, Australia Osteoclasteogenesis is regulated in vivo by the action of osteoblast/stromal cells which express the membrane bound receptor activator of NF-kappa B ligand (RANKL). In vitro the soluble form of RANKL can be used in combination with macrophage colony stimulating factor (M-CSF) to induce the production of osteoclast like cells from peripheral blood monocytes (PBMCs). PBMCs treated with M-CSF alone results in the production of macrophage like cells that can be employed as a control to examine the effects of RANKL on a range of gene targets. Total RNA from monocytes treated with M-CSF alone and treated with RANKL and M-CSF together was isolated at 8 hours, 12 hours, 24 hours, 8 days and 21 days after treatment. Differential display (DD) was performed on RNA (8 hours, 12 hours, 24 hours, 8 days treatment with RANKL) the DD products were pooled to generate a probe for screening a gene array system derived from a human osteoclast cDNA library. Hybridisation of cDNA against the array revealed additional regulated genes. Gene clones that showed significant regulation in monocytes treated with M-CSF and RANKL compared with monocytes treated with M-CSF represent genes that are targets for RANKL-specific regulation. Osteopontin, creatine kinase B and cystatin B were up regulated by the treatment of RANKL. Thymosin beta-4, proteasome ATPase and thioredoxin binding protein were down regulated by the treatment of RANKL. Relative fold differences observed in several genes identified from array analysis were confirmed using real time PCR. These include the osteoclast acidic protease cathepsin K, and the protease inhibitors cystatin B and cystatin C. Cathepsin K relative fold expression peaked at one week treatment at 224±1.15 and subsequently decreased with increasing time of exposure to RANKL. Cystatin C relative fold of expression peaked at two weeks RANKL treatment at 8.4±1.08 and cystatin B showed a steady increase in expression. Our hypothesis is that gene clones, which showed significant regulation of M-CSF and RANKL when compared with M-CSF alone, represent potential downstream targets of RANKL activation. Genes that are regulated by the action of RANKL during osteoclastogenesis may provide potential targets for novel anti-osteoporotic drugs. IMMUNOLOGICAL AND HISTOCHEMICAL DETERMINATION OF TRAP 5B TO MEASURE OSTEOCLAST ACTIVITY UNDER ANTI- OSTEOLYTIC TREATMENT A. T. Mehlhorn1*, H. Rechl1, A. W. Stemberger2, R. Gradinger1 1Department of Orthopaedic Surgery of the Technische Universität, Munich, Ismaningerstr. 22, 81675 Munich, Germany 2Department of Experimental Oncology of the Technische Universität, Munich, Ismaningerstr. 22, 81675 Munich, Germany Background: The tartrat resistant acid phosphatase 5b is regarded to be the most sensitive parameter of osteoclast function. Detection in serum is performed by a two- site enzyme linked immunoassay. We developed a method for localization and quantification of TRAP 5b activities in bone and compared the results with histological examinations of the corresponding bone areas. Methods: A total of 12 femoral heads divided in two different test groups were examined. The control group (Age: 70±7 y, m:f = 1:3, n=6) underwent a total hip arthroplasty without preoperatively receiving bone affecting drugs. Bisphosphonate group (Age: 63±11y, m:f = 1:3, n=6) received 10 mg/d Alendronate (n=5) or 5 mg/d Risedronate (n=1) because of manifest osteoporosis at least 50 days prior to operation. Samples of three defined areas were prepared from the specimen: the femoral head (A1), the femoral neck (A2) and the intertrochanteric area (A3). Half the bone chips were grinded, incubated in isotonic saline and the supernatant was analysed by Bone TRAP_ Assay (Medac Diagnostica, Germany). The remaining chips were fixed in buffered 2.4% formalin, cold-embedded in methylmethacrylat and stained with a mixture of naphtol-AS-BI phosphate substrate, L(+)-tartrat and Fast Red Violet LB. TRAP-positive areas and cells were counted in all sections. Results: The quantitative determination of TRAP 5b demonstrated in the bisphosphonate group a decrease in enzyme activity compared with the controls: 18% in area A3 (0.179±0.131 vs. 0.218±0.195 Unit/mg), 5% in A2 (0.210±0.140 vs. 0.218±0.083 Unit/mg) and 11% in A1 (0.318±0.158 vs. 0.359±0.297 Unit/mg). The histological examination confirmed these results, it showed a lower number of TRAP- positive lacunae in sections of the bisphosphonate group. Conclusion: Bisphosphonate treatment decreases TRAP 5b activity as a marker of osteoclast function in all areas of arthritic femoral heads. Thus, the described histochemical method and ELISA are suitable tools to evaluate the local impact of bisphosphonates on osteoclast activity. TARTRATE-RESISTANT ACID PHOSPHATASE 5B AND OSTEOCALCIN REFLECT THE BONE RESORPTION RATE IN IN VITRO HUMAN OSTEOCLAST CULTURES H. Ylipahkala*, K. K. Ivaska, T. A. Hentunen, H. K. Väänänen, J. M. Halleen Institute of Biomedicine, Department of Anatomy, University of Turku, Turku, Finland Osteoclasts secrete tartrate-resistant acid phosphatase (TRAP) 5b into the circulation as an active enzyme, suggesting that serum TRAP 5b activity may be a useful marker of bone resorption. In the serum, TRAP circulates in a high molecular weight complex that contains alpha-2-macroglobulin. Osteocalcin is the most abundant non-collagenous protein in bone matrix. Serum osteocalcin is generally considered as a marker of bone formation, but some osteocalcin may also be released during bone resorption. We have studied the secretion of active TRAP 5b and osteocalcin from resorbing osteoclasts using an in vitro human osteoclast culture where peripheral blood mononuclear cells were cultured on bovine bone slices and allowed to differentiate into bone-resorbing osteoclasts in the presence of RANKL, M-CSF, TNF-alpha and dexamethasone. TRAP 5b activity (BoneTRAP, SBA-Sciences, Oulu, Finland) and C- terminal cross-links of type I collagen (CrossLaps, Osteometer Biotech, Herlev, Denmark) were measured using commercial immunoassays, and total osteocalcin using an in-house immunoassay. TRAP complex was purified using gel filtration chromatography. Both medium TRAP 5b activity and total osteocalcin correlated significantly with medium CrossLaps and osteoclast number (r=0.9, p<0.0001 in all cases). These results suggest that TRAP 5b and bone matrix-derived osteocalcin are secreted from resorbing osteoclasts. Purified TRAP complex contained also calcium, suggesting that TRAP 5b may be secreted together with calcium released from bone matrix, and they may circulate in blood in the same complex together with alpha-2-macroglobulin. THE SIZE OF IN VITRO CULTURED OSTEOCLASTS WAS INCREASED AND THE OSTEOCLASTIC ACTIVITY DECREASED AFTER TREATMENT WITH MMP-INHIBITOR BB-94 V. V. Parikka*, H. K. Väänänen Institution of Biomedicine, University of Turku, Turku, Finland Matrix metalloproteinases (MMPs) are a group of enzymes capable of degrading extracellular matrix components. The exact roles of MMPs in bone biology are not understood, but they have been suggested to be required for the solubilization of bone matrix and possibly for osteoclast migration and anchorage. At least two members of the group, MMP-9 and MT1-MMP, have been demonstrated to be present in osteoclasts. In addition, MMPs of osteoblast lineage cells probably also affect osteoclasts. We have previously shown that bone resorption is disturbed in the presence of MMP-inhibitor batimastat (BB-94). In the present study we further analysed the effects of BB-94 on osteoclasts. A mixed rat bone cell population was cultured on bovine bone slices for three days. After the culture period cells were stained for tartrate-resistant acid phosphatase (TRAP) and with DNA-binding fluorochrome Hoechst 33258 to visualize nuclei. F-actin was stained with TRITC-conjugated phalloidin to detect actin rings. MMP-inhibitor BB-94 at 6microM concentration, did not significantly affect the number of osteoclasts. Instead, the average number of nuclei per osteoclast was significantly increased (7.41±0.36 versus 9.53±0.55). In addition, the number of mono- and binuclear TRAP-positive cells was lower on BB-94 treated slices. Phalloidin staining revealed that the formation of actin rings was dramatically decreased by BB-94. These result were reproduced with another MMP-inhibitor BB- 2516. Another major group of proteinases expressed in osteoclasts is cysteine proteinases. To confirm that the effect on osteoclast size was specific for MMP- inhibition, we studied the effect of cysteine proteinase inhibitor E-64 on osteoclasts. It had no effect on the size of osteoclasts even at 50microM concentration. To conclude, we demonstrated that the osteoclast size was increased and the osteoclastic activity decreased by MMP-inhibitor BB-94. We suggest that the observed phenomenon may be due to disturbed migration of osteoclasts. COMPLICATIONS IN LEG LENGTHENING PROCEDURE BY WAGNER APPARATUS IN CHILDREN: DIFFERING CONGENITAL AND ACQUIRED AETIOLOGY OF SHORTENING M. Baretic General Hospital 'Sveti Duh', University of Zagreb, Croatia Leg lengthening procedure is often accompanied by many complications. Connection between aetiology of abbreviation and complications was studied in 36 patients (average age 11 years and 2 months). All the patients were younger than 18 yrs without previous leg lengthening operative procedure made of any kind. They underwent 41 lower limb leg lengthening operations (33 femoral and 8 tibial) by Wagner apparatus. To the one of the children femur and tibia on the one leg were elongated and to the other one together femur and tibia on both legs were lengthened. Their medical data were collected retrospectively and analysed. Average preoperative leg length discrepancy was 5.4 cm: average lengthening achieved was 5 cm. Wagner apparatus was placed in situ in average during 5 months and 21 days. Children were divided by the aetiology of leg shortening in two groups; in 25 of them aetiology was congenital (congenital idiopathic hyoplasia, congenital hip dislocation consequences, coxa vara, mb. Ollier, mb. Klippel-Trenaunay, achondroplasia) and 11 of them had acquired aetiology of abbreviation (tumours resection effect, paresis et paraparesis consequences caused by polio, meningitis, myelomeningocoela, tumours resection effect, posttraumatic status). 22 elongations were followed by one or more complications (14 patients had joint contractures, 8 pin-track infections, 4 fractures, 2 too early consolidations, 1 pseudarthrosis). Complications were divided on heavier (an additional operative procedure was required) and easier (solved with physical therapy and antibiotics). It was presumed that the frequency of complications and their severity are influenced by aetiology of shortening. Statistical data evaluated by Hi square test show no significant difference. It seems that the aetiology does not play the role in prognosis of complication rate and severity during leg lengthening procedure by Wagner apparatus. Reoperations could be avoided by continuous physical therapy considering setting the operated leg under graduate pressure, rigorous pin-track toilette with microbiological sample taking and AO fixation with spongioplasty. VITAMIN D RECEPTOR GENE POLYMORPHISMS AND BONE MINERAL DENSITY IN POSTMENOPAUSAL WOMEN IN MALTA C. Vidal1*, M. Brincat2, A. Xuereb1,3 1Institute of Health Care, University of Malta, Malta 2Department of Obstetrics and Gynaecology, University of Malta Medical School, Malta 3Department of Pathology, University of Malta Medical School, Malta Osteoporosis is a disease with a strong genetic component. One of the genes that has been implicated in osteoporosis is the Vitamin D receptor (VDR) gene, although there have been conflicting results as to whether VDR gene polymorphisms influence bone mineral density (BMD). The aim of this study was to determine whether correlation exists between VDR gene polymorphisms and bone mineral density in postmenopausal women in Malta. 104 postmenopausal women (aged 48-62 years) were recruited for this study. Amplification of the desired target sequence of the VDR gene was carried out by PCR, and the subjects were genotyped on the presence (b) or absence (B) of the BsmI restriction enzyme cleavage site in the VDR gene. BMD at the lumbar spine and femur was measured using a Norland DEXA 486. Serum concentrations of procollagen Type 1 and urinary deoxypyridinoline crosslinks were used as biochemical markers of bone turnover. The genotype frequencies observed (BB=16.4%, Bb=51.9% and bb=31.7%) were similar to those observed in other Caucasian populations. BMD at both anatomical sites was observed to be highest in BB homozygotes, although the difference between the genotypes was not statistically significant (ANOVA p=0.768; 0.967 for lumbar spine and femur, respectively). The biochemical marker of bone formation was observed to be highest in the BB genotype, whereas the biochemical marker of bone resorption was found to be lowest in the BB group, although both these results were not statistically significant (ANOVA p=0.591; p=0.806 respectively). A non- statistically significant difference (Chi-square test p=0.624) was observed in the frequencies of VDR genotypes between normal individuals and a group of osteoporotic women at the lumbar spine (t-score<-2.5.) In conclusion, there does not seem to be an association between polymorphisms in the VDR gene and BMD in postmenopausal women in Malta. POLYMORPHISM IN THE VITAMIN D RECEPTOR GENE AND THEIR RELATIONSHIP WITH BONE LOSS IN POSTMENOPAUSAL WOMEN M. Dķaz-Curiel1*, J. Iborra2, M. Quesada3, J. Farrerons4, M. Sosa5, R. Pérez-Cano6, J. Gonzįlez-Macias7, A. Dķez-Pérez8, J. Cannata9 1Fundación Jimenez Dķaz, Madrid, Spain 2Novartis Farmacéutica, Spain 3Hospital Reina Sofia, Cordoba, Spain 4Hospital de la Santa Creu i Sant Pau, Barcelona, Spain 5Hospital Universitario Insular de las Palmas, Gran Canarias, Spain 6Hospital Universitario Virgen de la Macarena, Sevilla, Spain 7Hospital Central de Asturias, Asturias, Spain 8Hospital del Mar, Barcelona, Spain 9Hospital Marques de Valdecilla, Santander, Spain Bone mineral density (BMD), the major determinant of fracture risk, is under strong genetic control. Although polymorphisms of the vitamin D receptor (VDR) gene have been suggested to account for some genetic variation in bone mass, the influence of VDR genotypes on osteoporosis remains controversial. We examined the possible effect of these genotypes on postmenopausal bone loss. Patients and methods:We performed a multicenter trial in 211 postmenopausal women (mean age 54.3). VDR polymorphisms were determined by RFLP using Bsm I (intron 8), Apa I (intron 8) and Taq I (T1055-C) after PCR. For the hypothesis contrast and for every polymorphism, we have analysed the genotypes separately and after we have analysed grouped in unfavorable genotypes and their relationship with bone loss rate. We have also analysed the lumbar spine and femoral neck BMD. Bone mass was measured by DEXA. The percent change in BMD at 2 yr was used to classify subjects as slow bone loss (<OR=1.4%), intermediate (between 1.4 and 2.7%) and fast bone loss patients (estimated annual bone loss >2.7%). Results:The prevalence of genotypes were BB: 22.3%, Bb 40.2% and bb: 37.5%; AA 31.3%, Aa 41.1% and aa 27.7% and TT 48.2%, Tt 51.8% and tt 18.8 %. Distribution of different genotypes, grouped and ungrouped, was similar between the three groups of annual bone loss rate, neither did we find any significant association between the differents genotypes and bone mass loss. Conclusions:In this study, VDR genotype is not associated with the annual bone loss in postmenopausal women This study was supported by Novartis. INFLUENCE ALLELES OF VDR3, COL1A1 AND CALCR GENES ON SPEED OF LOSS OF BMD IN POSTMENOPAUSAL WOMEN M. V. Moskalenko1*, M. V. Aseev1, I. E. Zazerskaya2, V. S. Baranov1 1Lab. Pren. Diag., Ott's Inst. of Obst. & Gynecol. RAMS, St-Peterburg, Russia 2Pavlov's State Medical University, St-Peterburg, Russia Osteoporosis is a common disease affecting the majority of older postmenopausal women. This skeletal disease characterised by bone mineral density, which leads to impaired skeletal strength and increased susceptibility to fracture. A genetic contribution to osteoporosis is well documented, but genes and alleles variants conferring osteoporotic risk are largely undefined. The distribution of functionally uncompleted alleles of VDR, COL1A1 and CALCR genes in Russian population and in the group of the postmenopausal women was investigated. The first group was the control group (174 women from north-west region of Russia without osteoporosis). The second group was the group of 119 postmenopausal women (all of them had been in menopause for 4-5 years). After 12 months observation, we have allocated two groups of the postmenopausal women differing on speed of loss of BMD. T-criterion have decreased during 12 month in group of women with fast loss of BMD (10±6.38%). T-criterion have during 12 month in group of women with slow loss of BMD (2±8.23%). Genomic DNA from peripheral blood was extracted by standards methods with using proteinase K. The polymorphisms of VDR3, COL1A1 and CALCR genes were studied by PCR-RFLP method (polymorphism VDR3/Taq I, COL1A1/Apa I and CALCR/Alu I). The differences in allele combinations of VDR and Col1A1 genes between group of control group and group of postmenopausal women with slow and fast loss of BMD were proved. Frequencies of functionally impaired alleles of VDR gene (t allele) was 32.7% (first group), 13.9% (group with slow loss of BMD), 46.8% (group with fast loss of BMD) respectively. Frequencies of functionally impaired alleles of COL1A1 gene (s allele) was 17.9% (first group), 2.4% (group with slow loss of BMD), 30.8% (group with fast loss of BMD) respectively. Frequencies of functionally impaired alleles CALCR (T allele) of gene was 73.8% (first group), 66.7% (group with slow loss of BMD), 81.3% (group with fast loss of BMD) respectively. Analysis of VDR, Col1A1 and CALCR alleles frequencies in two groups of postmenopausal women proved significant association between BMD loss values (speed) and particular alleles of the corresponding genes in these patients (p<0.001). POLYMORPHISMS IN THE PROMOTER OF THE INTERLEUKIN-6 GENE ARE NOT ASSOCIATED WITH OSTEOPOROTIC FRACTURES OR BONE MASS B. L. Langdahl*, M. Carstens, L. Stenkjęr, E. F. Eriksen Dept. of Endocrinology, Aarhus University Hospital, Aarhus, Denmark Interleukin-6 (IL-6) is a potent stimulator of bone resorption and IL-6 levels are elevated in postmenopausal women compared to premenopausal or HRT treated women. It has previously been shown that an AT rich VNTR polymorphism in the 3' UTR of the IL-6 gene is associated with bone mineral density in both pre- and postmenopausal women. Three polymorphisms have been identified in the promoter region:G-597-A, G-572-C and G-174-C. G-174-C has been demonstrated to be associated with production and serum levels of IL-6. We therefore wanted to investigate if these polymorphisms affect the prevalence of osteoporotic fractures and bone mass in 342 osteoporotic patients and 369 normal controls. The polymorphisms were examined by RFLP using Mnl I, Aci I and Nla III after PCR. BMD was examined by DXA. The G-597-A polymorphism was not found in our population. The genotype distribution of G-572-C was 0% CC, 6.3% GC and 93.7% GG in osteoporotic patients and 0.8% CC, 7.7% GC and 91.5% GG in normal controls (chi-sq.=2.69, p=0.26). The genotype distribution of G-174-C was 26.5% GG, 54.4% GC and 19.2% CC in osteoporotic patients and 29.4% GG, 49.2% GC and 21.4% CC in normal controls (chi-sq.=1.43, p=0.49). Both polymorphisms were in Hardy-Weinberg equilibrium and in linkage equilibrium. Combining the genotypes resulted in 6 different haplotypes which were distributed similarly in osteoporotic patients and normal controls (chi-sq.=4.63, p=0.46). BMD of the lumbar spine (ls), femoral neck (fn) and total hip (th) in the different genotypes are presented below (mean±SD). BMD was not different between individuals with the different genotypes or combined genotypes at any site. In conclusion: Three polymorphisms in the promoter of the IL-6 gene are
not associated with osteoporotic fractures or BMD at the spine or hip.
INTERLEUKIN-6, TRANSFORMING GROWTH FACTOR BETA AND BONE TURNOVER DECREASE WITH POST-TRANSPLANT PERIOD V. Kusec1*, R. Smalcelj2, D. Rogic1, Z. Puretic2 1Clinical Institute of Laboratory Diagnosis, Clinical Hospital Centre, Zagreb, Croatia 2Dialysis unit, Clinical Hospital Centre, Zagreb, Croatia Disturbance of bone metabolism and renal osteodystrophy which develop in the course of chronic renal failure and continue after kidney transplantation are a major risk factor for the skeletal integrity. There is considerable evidence that bone turnover is increased and bone density decreased in kidney transplant recipients. Several cytokines involved in bone turnover, e.g. interleukin 6 (IL-6) and transforming growth factor beta (TGF beta) are also influenced by PTH, mostly secreted in excess in kidney transplant recipients. Involvement of IL-6 and TGF beta has not been demonstrated in bone disorders after kidney transplantation. The aim of this study was investigation of IL-6 and TGF beta concentrations in relationship to post- transplantation time and biochemical markers of bone turnover. This study comprised 19 patients within the first post-transplant year and 45 with post-transplant period of 15-175 months. None of the patients had rejection crisis at least three months before the commencement of the study. IL-6, TGB beta, intact PTH, bone alkaline phosphatase (BALP), osteocalcin (OC) and procollagen type I propeptide (P1CP) were measured in serum and deoxypyridinoline crosslinks (DPD) in urine by commercial ELISA kits. In 16 kidney transplant recipients within the first post- transplant year biochemical analyses were performed twice with a 3-4 month interval, and once in other subjects. IL-6, TGF-beta, BALP, OC and DPD were significantly higher (p<0.02 to p<0.0002) in the first post-transplant year than later period. Statistically significant negative correlation (p<0.03 to p<0.0009) with post- transplantation time was found for IL-6, BALP, OC, P1CP, DPD and for TGF-beta only in the first twelve months after transplantation. Also, a significant difference (p<0.01) was found between two measurements only for TGF beta in the first 12 months after transplantation. No difference was found between sexes or correlations of measured parameters with age. These results indicated that higher bone turnover in the first post-transplant year corresponded with higher levels of cytokines IL-6 and TGF beta, most probably involved in bone remodeling of kidney transplant recipients. Decrement of IL-6, TGF beta and bone turnover with post-transplantation time suggested cessation or even improvement of the existing bone disorder. GENE THERAPY OF HUMAN OSTEOSARCOMA CELLS K. Trieb Dept. of Orthopedics, University of Vienna, Austria The five-year survival rate in osteosarcoma patients could be increased frome 20% to about 70% due to the combination of surgery and neoadjuvant chemotherapy. In the remaining 25% the problem of none response to chemotherapy remains. Heat shock proteins (hsp) have been shown to play a role in tumor biology and drug resistance. Hsps are predictive and prognostic markers in many cancers. It is the aim of this study to investigate the effect of a stable transfection of osteosarcoma cells with hsp27 on the susceptibility to chemotherapeutic drugs and cell growth. The human osteosarcoma cell line HOS-85 was transfected with the expression vector pSG-2711, which contains the complete human hsp27 gene with its promotor, a SV 40 promotor and a neomycin resistance gene. The same vector without the hsp27 gene served as control. The transfection was documented by PCR analysis and Western Blot. The transfection of cells resulted in a lower proliferation rate, tested by 3H- thymidine incorporation. The effect of transfection on sensitivity to drugs depended on the substance used. Transfected cells were sensitiver to the toxic effects of adriamycin, bleomycin and cisplatin, whereas no difference could be observed for four other chemotherapeutica tested. This results show that the transfection of osteosarcoma cells can change the response to chemotherapeutic therapy. The increase in drug sensitiveness and the decreased proliferation may lead to a new therapeutic concept in the treatment of osteosarcoma. ASSOCIATION OF THE C-ALLELE OF THE C677T MUTATION IN THE METHYLENE TETRAHYDROFOLATE REDUCTASE GENE WITH AN INCREASED RISK OF OSTEOPOROTIC FRACTURES: A CASE CONTROL STUDY IN DANISH POSTMENOPAUSAL WOMEN H. L. Jorgensen1*, J. S. Madsen2, B. Madsen1, M. M. A. Saleh3, B. Abrahamsen4, M. Fenger1, J. B. Lauritzen3 1Department of Clinical Biochemistry, Hvidovre University Hospital, Denmark 2Department of Clinical Biochemistry, Odense University Hospital, Denmark 3Department of Orthopaedic Surgery, Hvidovre University Hospital, Denmark 4University Department of Endocrinology, Aarhus Amtssygehus, Denmark Background: Recently, a point mutation in the gene coding for methylene tetrahydrofolate reductase, MTHFR (C677T), giving rise to a thermolabile variant of MTHFR, has been shown to significantly influence lumbar spine BMD in Japanese women (Miyao M et al. Calcif Tissue Int 2000; 66: 190-194). The aim of this study is to investigate the influence of this mutation on peripheral measures of bone density and on the odds ratios for hip and wrist fracture in a case control study of Danish postmenopausal women. Methods: 74 women with wrist fracture, 41 women with hip fracture and 207 age- matched controls were included. All had broadband ultrasound attenuation (BUA) and speed of sound (SOS) measured at the heel as well as bone mineral density (BMD) measured by dual X-ray absorptiometry at the distal forearm. The MTHFR (C677T) genotypes were determined using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Results: Only two patients out of 21 with the TT genotype had a fracture (ratio of fracture patients to controls, Fx/Cont: 0.11) as opposed to 46 out of 142 with the CT genotype (Fx/Cont: 0.48) and 67 out of 159 (Fx/Cont: 0.73) with the CC genotype (chi-square (df=2): 9.8, p=0.007). Using logistic regression with age, height, weight, age at menopause, BUA, SOS, lower forearm BMD and the MTHFR genotypes as co-variates, the following odds ratios were found when comparing the individuals homozygotic for the C-allele with those homozygotic for the T-allele: Wrist fracture OR=3.93 [1.25;12.40, p=0.02], hip fracture OR=6.99 [1.35; 36.92, p=0.02] and the fracture types combined OR=4.33 [1.73;10.81, p=0.002]. Even when comparing the individuals homozygotic for the C-allele to the heterozygotic individuals, the effect remains significant: Wrist fracture OR=1.98 (1.12;3.52, p=0.02), hip fracture OR=2.64 (1.17;5.99, p=0.02) and the fracture types combined OR=2.08 [1.32;3.29, p=0.002). No significant association of the MTHFR (C677T) genotypes was found with BMD at the lower forearm or with ultrasound parameters measured at the calcaneus. Conclusion: The C-allele of the MTHFR (C677T) mutation is associated with a highly significant increase in the odds ratio of hip fracture and wrist fracture in Danish postmenopausal women. IDENTIFYING THE GENES UNDERLYING THE OSTEOPETROTIC TOOTHLESS AND INCISORS ABSENT MUTATIONS IN THE RAT L. Van Wesenbeeck1*, P. R. Odgren2, C. MacKay2, S. C. Jr. Marks2, W. Van Hul1 1Dept. of Medical Genetics, University of Antwerp, Belgium 2Dept. of Cell Biology, University of Massachusetts Medical School, Worcester, MA, USA The mammalian osteopetroses are a heterogeneous group of skeletal disorders characterized by an increased bone mass due to a reduced bone resorption. Two osteopetrotic mutations in the rat, toothless (tl) and incisors absent (ia) rat, affect different genes and impact skeletal biology in different ways. Whereas tl rats have severe, unremitting osteoclastopenia, ia rats have numerous small, non-functioning osteoclasts which recover over the first few postnatal months. The tl mutation impairs an osteoblast-derived, osteoclastogenic signal while the ia affects a gene product needed by the differentiated osteoclast for ruffled border formation and normal resorptive function. Despite the fact that the osteopetrotic mutants tl and ia have been extensively studied, the primary genetic defects underlying both conditions are still unknown. In order to identify these mutations, outcrosses were set up between the inbred strain, tl or ia, and the Brown Norway strain, generating F1 animals that are obligatory carriers of the osteopetrotic mutations. Next an intercross between the animals from the F1 generation was performed. At this moment we have 11 affected F2 animals of the tl rat and 26 affected F2 animals of the ia rat available for this study. An extended set of polymorphic DNA markers are currently being analysed on these rats to localize the underlying genes. This procedure already enabled us to exclude the TNFSF11 (also called TRANCE, RANKL, ODF and OPGL) gene for the tl rat, despite some phenotypic similarities between tl rats and TNFSF11 null mice. The chromosomal localization of the rat TNFSF11 gene in the proximal part of chromosome 15q was deduced from synteny between rat, human and mouse. Informative polymorphic markers spanning this region were analysed but the phenotype turned out not to cosegregate with the chromosome 15 markers, excluding the TNFSF11 gene as the disease causing gene. Currently we are checking both mutations for other candidate genes in parallel with an ongoing genome scan. ESTROGEN RECEPTOR ALPHA GENE POLYMORPHISM PREDICTS RADIOGRAPHIC OSTEOARTHRITIS OF THE KNEE IN ELDERLY MEN AND WOMEN A. P. Bergink1,2*, J. van Meurs1, P. Arp1, Y. Fang1, A. Hofman2, C. M. van Duijn2, J. P. T. M. van Leeuwen1, A. G. Uitterlinden1,2, H. A. P. Pols1,2 1Department of Internal Medicine, Erasmus Medical Centre, Rotterdam, The Netherlands 2Department of Epidemiology & Biostatistics, Erasmus Medical Centre, Rotterdam, The Netherlands Objective. Osteoarthritis (OA) is a common disabling disease in the elderly. Genetic influences have been shown to play an important role in the etiology, but the genes involved are ill defined. We studied the influence of two polymorphisms in the ERalpha gene on the prevalence of radiographic osteoarthritis (ROA) of the knee in the Rotterdam Study. Methods. The study group consisted of 1487 men and women drawn from the Rotterdam Study, a large population-based follow-up study of the elderly. The PvuII restriction fragment length polymorphisms (RFLP) and the XbaI RFLP in the first intron of the ERalpha gene were determined in relation to each other by direct molecular haplotyping. Radiographs of the knee were evaluated using the Kellgren score. ROA of the knee was defined as having a Kellgren score of two or higher at one or both knees. Results. Three different haplotype alleles were found: allele 1 (px, 54.2 %), allele 2 (PX, 33.6 %) and allele 3 (Px, 12.2%). Allele 2 was associated with an increase in the prevalence of knee ROA: in subjects without allele 2 the percentage of ROA was 21.8%, in those carrying one copy 24.2 % and in those with two copies 33.5%. The corresponding, for confounding factors adjusted, odds ratios were 1.3 (95% C.I.: 0.9 to 1.7) for heterozygous and 2.0 (1.3 to 3.1) for homozygous subjects. Separate analyses for men and women showed similar effects. When we considered the different characteristics of ROA, osteophytosis and joint space narrowing, the association between ROA and the ERalpha haplotype seemed to be driven by the osteophytosis. Conclusion. In both elderly men and women, variations in the ERalpha gene are associated with radiographic osteoarthritis of the knee. INFLUENCE OF ESTROGEN RECEPTOR ALPHA POLYMORPHISMS ON BONE MINERAL DENSITY AND EFFECTIVENESS OF ALENDRONATE TREATMENT IN PATIENTS WITH OSTEOPOROSIS B. Arko1*, J. Marc1, J. Prezelj2, A. Kocijancic2 1Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia 2Department of Endocrinology and Metabolic Diseases, Clinical Centre, Ljubljana, Slovenia BACKGROUND AND OBJECTIVES: Numerous studies have shown that bone mass and bone turnover are under genetic control. Because of the important role of estrogens in bone metabolism gene for estrogen receptor alpha (ER alpha) represents one of the candidate genes, which could be involved in the development of osteoporosis. The purpose of this study was to assess whether bone mineral density (BMD) and its change in response to alendronate treatment is related to PvuII and XbaI polymorphisms of the ER alpha gene in postmenopausal women. METHODS: 59 postmenopausal Slovenian women (aged 65.2±6.7 years) with osteoporosis were studied. BMDs at the lumbar spine and hip were measured by dual X-ray absorptiometry at the start and after one year of treatment with alendronate (Fosamax, MSD). Genomic DNA extracted from whole blood was amplified by polymerase chain reaction and genotypes determined after digestion with PvuII and XbaI restriction enzymes. RESULTS: The frequencies of PvuII and XbaI genotypes were: PP 13.6%, Pp 57.6%, pp 28.8% and XX 11.9%, Xx 54.2%, xx 33.9%, respectively. Of the possible nine haplotypes only six were found. Four of them had frequency higher than 5%: PpXx 50.8%, ppxx 27.1%, PPXX 11.9% and Ppxx 6.8%. Statistically significant association of XbaI polymorphism with baseline z-scores of lumbar spine (p=0.027) and hip (p=0.033) BMD was demonstrated. The highest z- score was in both cases associated with XX genotype and the lowest with xx genotype. In contrast, no association of PvuII polymorphism with baseline z-scores of BMDs at either site was found. The increase in lumbar spine and hip BMD after one year of therapy did not differ with respect to PvuII and XbaI genotypes. CONCLUSION: Our results suggest that polymorphisms of the ER-alpha gene may represent a genetic determinant of BMD but they probably do not contribute to the effectiveness of alendronate treatment. FOKI-POLYMORPHISMS OF THE VITAMIN D RECEPTOR GENE ARE ASSOCIATED WITH 25(OH)VITAMIN D3 LEVELS IN ELDERLY RESIDENTS OF AUSTRIAN NURSING HOMES B. M. Obermayer-Pietsch*, A. Fahrleitner, W. Li, P. Maritschnegg, J. C. Piswanger- Sölkner, C. M. Bonelli, G. Leb, H. Dobnig Dept. of Internal Medicine, Karl-Franzens-University Graz, Austria BACKGROUND: Vitamin D receptor gene alleles were reported to influence bone metabolism and possibly bone fractures. Low vitamin D levels and concomitant disturbances of bone metabolism and muscle function are known to be major problems in elderly people in nursing homes. In this study, we evaluated FokI and BsmI polymorphisms of the vitamin D receptor (VDR) gene and parameters of bone and mineral metabolism to evaluate possible associations with bone turnover markers, neuromuscular function and bone fractures. METHODS: We investigated 1254 elderly Caucasian residents (mean age 83±6 years) living in 95 Austrian nursing homes. Medical history, past bone fractures and muscle strength were recorded. Serum measurements included routine chemistry parameters, 25(OH)vitamin D3, PTH, sCTX, and osteocalcin. Patients with impaired liver or kidney function or primary hyperparathyroidism were excluded from the study. DNA was prepared from peripheral leukocytes and genotyped for the VDR BsmI and FokI polymorphisms. RESULTS: 25(OH) vitamin D3 levels were markedly low in this population (11.2±6 ng/ml, normal range [9-45]). Sixty-seven percent of the residents had vitamin D serum levels <9 ng/ml thus considered vitamin D-deficient and only 3% had levels above 25 ng/ml. Frequencies of VDR genotypes paralleled the general population. Patients with VDR FokI genotypes 'ff' had significantly lower vitamin D levels (p=0.02) as compared to other genotypes, whereas BsmI genotypes showed no differences in bone metabolism. CONCLUSIONS: Lack of vitamin D is a major problem in elderly residents of nursing homes. Disability, neurophysiological disturbances and low BMD contribute to the risk for bone fractures. FokI genotypes of the VDR may be involved in the regulation of bone and mineral metabolism. Therefore, VDR genotyping could contribute to define nursing homes residents at high risk for bone disease. A DIRECT, NON-EXTRACTION ENZYME IMMUNOASSAY FOR MEASUREMENT OF 25-HYDROXYVITAMIN D D. Laurie*, A. K. Barnes, M. J. Gardner, R. T. Duggan IDS Ltd, Boldon Business Park, Boldon, UK Measurement of serum or plasma 25-hydroxyvitamin D (25-OH D) is the preferred method for establishing a patient's vitamin D status, including identification of vitamin D insufficiency. Previously, all 25D measurements have required extraction of sample to remove protein prior to analysis. This has applied equally to all methods of analysis, including HPLC, protein binding assays and immunoassays. IDS has developed a displacer reagent that enables measurement of 25-hydroxyvitamin D by immunoassay using serum samples directly, without the need for extraction. The direct 25-OH D assay comprises dilution of 25µL of serum or plasma with 1mL of a reagent containing binding protein displacers and a vitamin D - biotin conjugate. A portion of the diluted sample (200µL) was added to an antibody-coated microtitre plate and incubated for 2 hours at room temperature. After a wash, avidin- peroxidase reagent (200µL) was added, incubated 30 minutes and washed. TMB substrate (200µL) was added and colour allowed to develop for 30 minutes. After addition of 0.5M HCl (100µL) the absorbance was recorded at 450nm. A calibration curve was prepared from the absorbance values for 25OH-D serum standards (4 - 250nM) using a 4-parameter curve fit. The sensitivity of the direct 25-OH D EIA (the concentration corresponding to the mean minus two standard deviations of the '0' calibrator) was less than 6nM. The assay gave good precision with samples; intra-assay precision was 4.6%, 3.1% and 5.0% at 22.1nM, 62.0nM and 118nM respectively; inter-assay precision was 7.5%, 11.5% and 8.6% at 20.7nM, 56.5nM and 104nM respectively. Linearity was good with a mean for observed / expected of 117% for samples diluted 1:2 and 1:4 with the '0' calibrator. Cross-reactivity (at 50% displacement) for 25-OH D2 was 74%. Correlation against an extraction radio immunoassay (IDS Gamma B RIA, AA- 35F1) was carried out with 530 patient samples. Samples were measured in both the direct 25-OH D EIA and the extraction RIA method in a back-to-back assay set up. The comparison of methods by Passing-Babcock gave an equation of: Direct 25-OH D EIA = 1.00 x 25-OH D RIA - 4.0 nM. A linear regression analysis gave a correlation coefficient of R2=0.80. This technology provides a rapid, precise and sensitive non-isotopic assay for the measurement of 25D without the need for samples to be extracted. IDENTIFICATION OF BONE MORPHOGENETIC PROTEINS IN URINE BY WESTERN BLOTTING AND ELISA V. Miljavac1*, L. Grgurevic1, D. Batinic2, S. Vukicevic1 1Laboratory for Mineralized Tissues, Department of Anatomy, School of Medicine, University of Zagreb, Croatia 2Clinical Institute for Laboratory Diagnosis, Clinical Hospital, Zagreb, Croatia Bone morphogenetic proteins are signaling molecules with numerous functions. They are members of the larger transforming growth factor beta superfamily, expressed not only in bone but also in most other adult tissues. BMP-7 appears to have more diffused distribution. There is no evidence on the presence of BMPs in urine. To detect BMP-7 in biological fluids we used a modified ELISA system based on the sandwiching of the antigen between a monoclonal mouse 1B12 BMP-7 antibody and biotin labeled BMP-7. The capture polyclonal rabbit anti-mouse antibody was coated directly onto 96-well microtiter plates and was incubated overnight at +4 degrees Celsius. Wells without BMP-7 were used as a control. Signal was generated with a streptavidin-HRP-conjugate and ortho-phenylene diamine as a substrate. Dilution curves showed good linearity and the detection limit of the assay was 3 nanog/ml. To provide efficient concentration of biological samples for SDS polyacrylamide electrophoresis and Western blot we used Centricon Centrifugal Filter Devices YM- 10 and vacuum centrifuge. Protein electrophoresis was carried out in polyacrylamide gels under conditions that ensure dissociation of the proteins into their individual polypeptide subunits and minimize aggregation. Electrophoretically separated components were transferred from a gel to membrane making Gel over Membrane Sandwich. Membrane was incubated in 5 percent (w/v) nonfat dried milk overnight at +4 deg C and then incubated with the BMP-7 antibody (Bunny 3330 plus 3331 Pool) in dilution 1 in 100. The level of BMP-7 in human urine was less than 10 nanog/ml. The level of BMP-7 in urine from rats with chronic renal failure without therapy was less then 3 nanog/ml. Urine from rats suffering from chronic renal failure and treated with BMP-7 (20 microg/ kg) had BMP-7 levels approximately 10 nanog/ml. Significant amount of BMP-7 was detected in urine of pregnant women. In addition to BMP-7, BMP-3 and BMP-6 were detected in urine of healthy subjects. In conclusion, detection of BMPs in urine could become an important assay for kidney and bone diseases. ANTI-INFLAMMATORY PROPERTIES OF BONE MORPHOGENETIC PROTEIN-7 MIGHT BE BENEFICIAL IN THE TREATMENT OF INTESTINAL BOWEL DISEASE I. Maric1*, L. J. Poljak2, S. Zoricic1, D. Bobinac1, D. Bosukonda3, K. Sampath3, S. Vukicevic2 1Department of Anatomy, Medical School, University of Rijeka, Rijeka, Croatia 2Department of Anatomy, Medical School, University of Zagreb, Zagreb, Croatia 3Creative BioMolecules, Hopkinton, Massachusetts 01748, USA Abundant expression of bone morphogenetic protein-7 (BMP-7) in the developing intestine, and the association of the chronic intestinal inflammation and the malignant intestinal cell phenotype with alterations in the SMAD signalling pathway argue for an important, unrecognized role of BMPs in the pathophysiology of the gut. In the present study we evaluated the effect of recombinant human BMP-7 for the treatment of trinitrobenzenesulfonic (TNB) acid induced inflammatory bowel disease (IBD) in rats. Bioavailability studies in normal rats indicate that circulating iodinated BMP-7 declines with a half-life of 30 min upon systemic administration. A significant portion of 3.6% and 4.6% of the administered BMP-7 dose/g tissue is targeted for BMP-7 receptors in the stomach and the ileum, respectively. We show that BMP-7 treated animals developed a much less severe form of colitis as determined by preserved macroscopic appearance, normal body weight, less diarrhea and rectal bleeding, lack of ulcers and adhesions, and reduced histological damage score when administered 1.5 h before or 24 h after colitis induction. Rats were killed 2, 5, 10, 14 and 30 days following TNB administration. Histochemical and molecular analyses demonstrate that BMP-7: a) preserves microarchitecture of the colon tissue, b) suppresses inflammation by downregulating the expression of intercellular adhesive molecule, and prevents the accumulation of neutrophils, c) stimulates wound repair and modulates mucosal immunity by downregulating TNF-alpha and TGF-beta, and by inhibiting IL-6 expression, d) modulates the expression of its specific ALK-2 receptor. Collectively, these results suggest that BMP-7 prevents and restores the loss of gut function associated with TNB induced colitis, by a mechanism which makes it unique among many other agents reported to be beneficial in the treatment of IBD. THYROID DYSFUNCTION AND BONE MINERAL DENSITY IN EARLY POSTMENOPAUSE J. Jelcic1*, V. Kusec2, T. Kifer1, M. Korsic1 1Department of Endocrinology, Internal Medicine Clinic, Clinical Hospital Centre, Zagreb, Croatia 2Clinical Institute of Laboratory Diagnosis, Clinical Hospital Centre, Zagreb, Croatia The most common form of primary osteoporosis occurs in the postmenopause with most of the bone mass lost in the early postmenopause. Hyperthyroidism is one of the major risk factors for secondary osteoporosis, due to increased bone turnover and accelerated bone loss. The aim of this study was to investigate bone density in postmenopausal women with thyroid dysfunction. This retrospective analysis comprised 42 hyperthyroid, 92 hypothyroid and 562 euthyroid patients aged 40-85 years. Bone mineral density (BMD) was measured at the lumbar spine and femoral neck by dual X-ray absorptiometry. Patients with hypothyroidism were significantly younger than those without thyroid disorder (59.7 yr. vs. 62.1 yr.) and had significantly higher BMD in the lumbar spine and hip. Analysis of data for age groups revealed that the statistically significant difference (p<0.005) for the lumbar spine and hip BMD was found only for the 51-55 year old patients. No difference existed between the other two pairs of patient groups, i.e. the euthyroid and hyperthyroid and the hypothyroid and the hyperthyroid. There was no difference in incidence of osteopenia (T-score<-1) and osteoporosis (T-score<-2.5) between the three patient groups. These results suggest a protective effect of hypothyroidism on the skeleton in the early postmenopause probably through decelerated bone turnover and bone loss. BONE MORPHOGENETIC PROTEIN-7 PROMOTES HEALING OF LONG BONE DEFECTS IN THE RAT FEMUR BY PREFABRICATED BONE GRAFTS CREATED IN THE EPIGASTRIC FLAP R. Zic1*, S. Martinovic2, V. Kusec3, Z. Vlajcic1, M. Jelic2, S. Vukicevic2 1Department of Surgery, Clinical Hospital Dubrava, Zagreb, Croatia 2Department of Anatomy, School of Medicine, University of Zagreb, Croatia 3Clinical Institute of Laboratory Diagnosis, Clinical Hospital Centre, Zagreb, Croatia The aim of this study was investigating conditions which promote healing of the long bone defects by means of prefabricated bone graft in the epigastric flap. Helistat (collagen sponge 0.5 cm) or demineralised bone matrix (0.5 cm from rat long bone diaphysis) were inserted in the epigastric flaps in rats under aseptic conditions. Two doses (low, 30 microg/ml and high 120 microg/ml) of BMP-7 were added to both Helistat and demineralised bone matrix. In the control group only implants were used. After 6 weeks the bone graft was transferred from the epigastric flap into 0.8 cm long defect in the femur and fixed with intramedullary K- wire. Two prefabricated bone grafts from each experimental and control groups were used for histological analysis. Eight weeks after surgery X-ray was performed and femurs harvested. Healing of the bone defect as assessed by x-rays was observed in animals treated with a high BMP-7 dose graft. Histologic analysis of the bone grafts showed that BMP-7 stimulated bone formation in both the Helistat and demineralised bone implants, but dose-effect was observed only for the Helistat implants. Histology of the femur specimens showed healing of bone defect with prefabricated implants only in the high dose BMP-7 groups (both Helistat and demineralised bone matrix) with optimal retention of the bony fragments. Demineralised bone matrix was found to be superior to Helistat in supporting bone formation both in the epigastric flap and in the femur bone defect. Non-union remained if prefabricated non-treated or low BMP-7 dose implants were transferred into the femur gap. Retention of bone defects was a primary technical problem in this study. In conclusion, results of this pilot study in rats suggest that BMP-7 prefabricated bone grafts accelerate healing of long bone defects. HYPOPHOSPHATEMIA REDUCES HYPERPARATHYROIDISM CAUSED BY VITAMIN D DEFICIENT DIET IN RATS FOLLOWING NON-CHANGES IN SERUM CALCIUM LEVELS R. Masuyama*, A. Daizo, M. Uehara, K. Suzuki. Tokyo University of Agriculture, Tokyo, Japan Objective: Parathyroid hormone (PTH) is mainly stimulated by the decline in extracelluler concentration of calcium with the responsible reflection of calcium sensor; located in parathyroid grand, which transfer the signal of hypocalcemia to intracelluler PTH production. On the other hands, active form of vitamin D strongly inhibits the production of PTH. Therefore, high levels of PTH are appeared in the status of vitamin D deficiency, accompany with low levels of blood calcium. The alteration of blood (serum) phosphate also concerns with the regulation of PTH secretion. The increase of serum phosphate by the diet formulated with high contents of phosphorus, induces the PTH secretion immediately without the decline in blood calcium concentration. Accordingly, it has been suggested that the serum phosphate is a factor of PTH stimulation. To determine the effect of serum phosphate on the PTH stimulation independent on other factors such as blood calcium and vitamin D status, whether the PTH stimulation was influenced by phosphorus depletion, we studied using the vitamin D deficient rats maintained normal blood calcium concentration with vitamin D free-high calcium diet. Materials and Methods: Five weeks old Wistar strain-vitamin D deficient rats (n=20) were kept with a vitamin D free-high calcium diet (phosphorus replete) for 50 days, the diet was changed to a phosphorus-restricted diet (vitamin D free) in half of these mice. At the day of 56th, vitamin D was started to be supplemented orally for the half-rats of each phosphorus replete or restricted group while the other half-rats were continued to be fed two kinds of vitamin D free diets. At the day of 63rd, aged 13 weeks old, blood samples were collected. Results: Calcium absorption was increased by each vitamin D supplementation or phosphorus restriction in the diets. Therefore, serum calcium levels were reflected from results of the absorption. Serum phosphorus was decreased by the phosphorus-restricted diet. Phosphorus restriction significantly reduced PTH levels stimulated by vitamin D deficient status, despite no remarkable changes were observed in serum calcium levels. Conclusion: PTH-secretion might be regulated with the levels of serum phosphorus independently of serum calcium and vitamin D alterations. EVIDENCE FOR A ROLE OF ESTRADIOL IN THE CONTROL OF BONE MINERALIZATION M. van Driel*, C. J. Buurman, M. Koedam, H. A. P. Pols, J. P. T. M. van Leeuwen Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands Both a reduced quantity and quality of bone increase the risk for osteoporotic fractures. The elasticity of bone and its resistance to fracture is related to the degree of mineralization. When mineralization reaches too high levels, bone has a decreasing ability to absorb impact energy, becomes more stiff and brittle and more susceptible to fractures. Postmenopausal estrogen deficiency reduces bone mass, but its effect on the degree of mineralization of the remaining bone is not clear. The aim of the current study was to assess the effects of estradiol on mineralization by human osteoblasts. For this the human osteoblast cell line SV-HFO, that proceeds through different stages of differentiation in culture, including extracellular matrix formation and mineralization, was cultured for 23 days in the continuous presence of 17beta-estradiol (10-10 - 10-7 M) or its antagonist ICI 182.780 (10-9 - 10-6 M). Mineralization and alkaline phosphatase activity were measured at different time points during differentiation. Calcium deposition in the extracellular matrix starts at day 12 of culture and increases during time. Both in 17beta-estradiol and ICI 182.780 treated cultures, a reduction of 10-20% was found in the total amount of calcium deposited during the whole culture period, with most pronounced effects at later time points (days 19-23). This was observed for both compounds at all concentrations tested except at 10-8 M. In addition, both 17beta-estradiol and ICI 182.780 reduced alkaline phosphatase activity by 10-20%, with most pronounced effects in the period preceding the peak activity of alkaline phosphatase (days 7-14). Interestingly, this reduction was the strongest at the same concentrations that most effectively reduce mineralization. No effects of 17beta-estradiol and ICI 182.780 were observed on the DNA amount of the cells during culture. Besides the known role in controlling bone turnover via regulation of osteoclast function, on basis of the current data a new role for estradiol can be postulated: control of the extent or set-point of mineralization, which potentially contributes to the quality of bone. The comparable effects of both 17beta-estradiol and ICI 182.780 point to reconsider the view of ICI 182.780 as an estradiol antagonist in bone. HEALING OF SKIN NECROSIS AND REGRESSION OF ANTICARDIOLIPIN ANTIBODIES ACHIEVED BY PARATHYROIDECTOMY IN A DIALYSED FEMALE PATIENT WITH CALCIFIC URAEMIC ARTERIOLOPATHY S. Sefer1*, R. Trotic2, V. Degoricija1, M. Vrsalovic1, I. Ratkovic-Gusic1, P. Kes1 1Department of Intermal Medicine, Sisters of Mercy University Hospital, Zagreb, Croatia 2Department of Otorhinolaryngology and Head and Neck Surgery,Sisters of Mercy University Hospital, Zagreb, Croatia Aim: To present the impact of parathyroidectomy on the spontaneous healing of necrotic lesions of the skin of the lower leg and on anticardiolipin antibodies regression in a 68 years old female dialysed patient with hyperparathyroidism and calcific-uraemic arteriolopathy (CUA). Methods: After the occurrence of initial lesions of the lower leg skin, the intact parathyroid (iPTH) level, calcium (Ca) and phosphorus (P) product were measured, and on two occasions at 6 weeks' intervals, the titer of anticardiolipin antibodies was determined, followed by a clinical monitoring of the progress of necrotic skin lesions. Two months after the occurrence of skin lesions, the patient's right leg was amputated below the knee due to gangrene, and a histopathological analysis of the bioptic skin tissue of the amputated lower leg was made. After parathyroidectomy, iPTH, Ca and P product were measured, and on two occasions at 6 weeks' intervals, anticardiolipin antibodies titer was determined. Results: Before parathyroidectomy, an increased iPTH level, Ca and P product, as well as on two occasions at 6 weeks' intervals an elevated IgG anticardiolipin antibodies titer were determined. The histopathological analysis of the bioptic skin tissue of the amputated right lower leg showed mural calcification of artery walls and a thrombotic occlusion of small arteries, arterioles and dermal capillaries involving epidermolysis. One week after parathyroidectomy, iPTH level and Ca and P product were within normal range. Two measurements at a 6 weeks' interval revealed no anticardiolipin antibodies. Eight weeks after parathyroidectomy, spontaneous healing of necrotic skin lesions of the left lower leg was observed. Conclusion: Regression of anticardiolipin antibodies, normalisation of Ca and P product, and healing of the skin lesions after parathyroidectomy pointed to the elevated PTH level as a crucial factor in the pathogenesis of CUA. EVALUATION OF BONE MINERAL DENSITY IN PATIENTS WITH PRIMARY HYPERPARATHYROIDISM BEFORE AND AFTER SURGICAL TREATMENT M. Misjak1*, V. Petric2, H. Tomic-Brzac3, V. Altabas1, D. Globlek2, M. Sharma1 1Department Of Endocrinology, Diabetes And Metabolic Diseases, University Hospital 'Sestre Milosrdnice', Zagreb, Croatia 2Department of Otorhinolaryngology, University Hospital 'Sestre Milosrdnice', Zagreb, Croatia 3Department of Nuclear Medicine, University Hospital Centre 'Rebro', Zagreb, Croatia Aim: A sustained elevation in the secretion of parathyroid hormone (PTH) in most patients with primary hyperparathyroidism (PHPT) results in increased bone remodeling, demineralization, loss of skeletal mass and osteoporosis itself. It is expected that surgical removal of the parathyroid gland(s) in question results in both the cure and an improvement in bone mineral density. Patients and methods: A clinical study was conducted involving 107 patients with PHPT hospitalized at our hospital in the period from 1994 to 2000. PHPT was documented with elevated serum calcium, ionized calcium and PTH values. After surgical treatment all parameters were normalized. Dual Energy X-ray Absorptiometry (DEXA) was performed before and one year after parathyroidectomy. The results were statistically assessed using the Student's T-test for paired samples. Results: The results showed a statistically significant increase in bone mineral density one year after parathyroidectomy (total hip: p=0.025; femoral neck: p=0.004; L2 - L4: p=0.04). Conclusion: In patients with primary hyperparathyroidism the bone mineral density significantly increase after surgical removal of hyperfunctional parathyroid gland. ESTROGEN RECEPTOR EXPRESSION AND MEGAKARYOCYTE DIFFERENTIATION ARE STIMULATED BY ESTROGEN S. Bord1*, E. Frith1,2, D. C. Ireland1, M. A. Scott2, J. I. O. Craig2, J. E. Compston1 1University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Cambridge, UK 2Department of Haematology, Addenbrook's Hospital, Cambridge, UK High-dose estrogen (E) has been shown to produce anabolic effects in the skeleton. The mechanisms mediating these effects are unclear but may involve interactions between cells on bone surfaces and in the bone marrow compartment. We have previously demonstrated that in vivo the megakaryocyte population in human bone marrow increases with E treatment suggesting a possible role in bone remodelling. To investigate if E stimulates megakaryocytopoiesis CD34+ cells were isolated from cord blood by magnetic bead technology (MACS) and cultured for 9 days in a serum free collagen based system supplemented with thrombopoietin, interleukin-3 and-6, plus or minus 10nM 17beta estradiol. Collagen films were dried, fixed and cells immunolocalised for gpllla (CD61, a marker of early megakaryocyte maturation), gpllb/llla (CD41, a MK specific antigen expressed by more mature MKs in CFU- MKs) and estrogen receptors (ER) alpha and beta using specific polyclonal antibodies in an indirect immunoperoxidase technique. Extent and intensity of staining were measured quantitatively by image analysis. Cells cultured in the presence of E formed more megakaryocyte colony forming units (CFU-MKs) than untreated cells. These were highly CD61 positive with a three- fold increase in expression in the E treated cells (p<0.05). CD41 was expressed in CFU-MKs only in cultures treated with E. Low-level nuclear ER beta expression was seen in many of the CD34+ cells. This was increased in the E treated cells with intense staining in the CFU-MKs. ER alpha expression was mainly confined to cells within the CFU-MKs. E stimulated the extent of ER alpha and ER beta expression six- (p<0.01) and four-fold (p<0.001) respectively compared to untreated cultures. These results demonstrate that in vitro E stimulates the colony forming potential of CD 34+ cells and differentiation and maturation to a more megakaryocytic phenotype. These increases together with the stimulated ER protein expression suggests that megakaryocytes may play a role in mediating the E-induced anabolic effects seen in bone. HISTOLOGICAL AND FUNCTIONAL STUDIES OF THE THYROID GLAND IN FEMALE RATS CHRONICALLY EXPOSED TO CADMIUM B. Pilat-Marcinkiewicz1*, M. Brzoska2, B. Sawicki1, J. Moniuszko-Jakoniuk2 1Department of Histology and Embryology, Medical Academy, Kilinskiego 1 Street., 15-230 Bialystok, Poland 2Department of Toxicology, Medical Academy, Mickiewicza 2C street., 15-222 Bialystok, Poland The effect of chronic (over a year) cadmium (Cd) administration (5 or 50mg Cd/l in drinking water) to young female rats on the structure and function of the thyroid was evaluated. Thyroid sections, obtained using paraffin technique, were stained with hematoxylin and eosin (H + E) and assessed in light microscopy. Serum levels of thyroid hormones: triiodothyronine (T3) and tetraiodothyronine (T4) and thyroid stimulating hormone (TSH) as well as Cd in the blood were determined. The major changes in the thyroid gland were observed after exposure to 50 mgCd/l. Degenerative changes in some follicles and a rise in the number of large follicles which were frequently lined with atypical epithelium were found in the thyroid. Serum T3 and TSH concentration was not affected by Cd, while T4 was reduced but only at the higher level of Cd exposure. The results show that chronic exposure to Cd changes the structure of thyroid gland and influences its function. BONE MASS OR BONE QUALITY IS THE MAJOR DETERMINANT OF FRACTURES IN PRIMARY HYPERPARATHYROIDISM? E. Csupor1*, J. Szucs1, E. Toth1, S. Meszaros1, V. Ferencz1, P. Lakatos1, E. V. McCloskey2, C. Horvath1 11st Department of Medicine, Faculty of Medicine, Semmelweis University, Budapest, Hungary 2WHO Collaborating Centre for Metabolic Bone Diseases, University of Sheffield, Sheffield, UK Primary hyperparathyroidism (pHPT) has different clinical forms. Most patients recurrently produces kidney stones while in others pHPT causes bone disease with pain and fractures. The aim of our study was to measure bone density in the renal and osseal forms of this disease and to estimate the role of bone mass in determining fracture occurence. 72 patients with pHPT were studied: 66 women plus 6 men, aged 61.8±14.5 ys, 37 with renal stones and 35 with osseal symptoms. 17 of 72 patients had previously fractures. BMD was measured at the spine and hip by DEXA and at the forearm by SPA. Immunoradiometry was used to determine serum intact PTH level. Statistical evaluation used Student's t-test, Chi2-test and correlation analysis. Fractures occured more frequently in osseal (12/35, 34.0%) than in renal group (5/37, 13.5%, p<0.05). BMD (as Z-score) was decreased at axial and peripheral sites of both groups. No differences were found in BMD between renal and osseal groups at the spine or hip while forearm density was lower in patients with bone symptoms (0.51±0.03 vs 0.63±0.03, p<0.05). However, patients with or without fractures had the same bone mineral density. Fracture incidence had no correlation with BMD, PTH or other biochemical variables. Our results clearly show that decreased mineral mass of bones is a general feature of primary hyperparathyroidism independently of its clinical symptoms. However, the lack of difference in BMD between fractured and non-fractured patients suggest bone mass to be not the only major determinant of bone fragility in this disease. Further research is needed to study other bone characteristics determining fracture risk as quality and structure of bones. REGULATION OF OSTEOPROTEGERIN (OPG) AND OSTEOCALCIN BY TRIIODOTHYRONINE (T3) AND 1,25-DIHYDROXVITAMIN D3 (1,25(OH)2D3) IN OSTEOBLAST LIKES CELLS WITH A DISTINCT PHENOTYPE F. Varga*, S. Spitzer, K. Klaushofer Ludwig Boltzmann Institute of Osteology, 4th Medical Dept., Hanusch Hospital, Vienna, Austria Recently, OPG, a secreted member of the TNF-alpha receptor super family, has been identified as an regulator of bone resorption and has been implicated in the pathogenesis of post menopausal osteoporosis and other metabolic bone diseases. As a soluble decoy receptor for RANKL, OPG prevents the interaction of RANKL with RANK and as a consequence the formation and activity of osteoclasts. Both 1,25(OH)2D3 and T3 are important for the development and maintenance of the skeleton. Low serum concentration of 1,25(OH)2D3 results in rickets but pharmacological levels lead to bone loss. On the molecular level 1,25(OH)2D3 increases the expression of RANKL. Hypothyroidism in children causes severe disturbances of bone growth and development while hyperthyroidism could lead to osteoporosis. The goal of this study was to find out whether T3 acts on the OPG/RANKL system. MC3T3-E1 cells and two subclones of these cells (kindly provided by RT Franceschi) were cultured in alphaMEM for 4 days and thereafter treated with T3, 1,25(OH)2D3 or both hormones. mRNA was isolated after 3 to 48 hours treatment time. Northern hybridisation was performed according to standard laboratory protocols. In MC3T3-E1 cells clone 4, an osteoblast like cell line with features of a mature osteoblastic phenotype, we found that T3 time and dose dependently increased the expression of OPG mRNA. OPG similar to osteocalcin was first detectable after 6 hours of treatment. In normal MC3T3-E1 cells we only found a weak regulation of OPG while in clone 30 there was no regulation. Osteocalcin, however, was regulated in all cell lines but with different intensity. 1,25(OH)2D3 dose dependently attenuated the T3 induced expression of both OPG and osteocalcin. Interestingly, we found a tight relationship between the T3 and 1,25(OH)2D3: at 100 nM T3, 10 nM 1,25(OH)2D3 attenuated, while 100 nM inhibited the expression of both genes. At 10 nM T3, 10 nM 1,25(OH)2D3 completely inhibited the expression of both proteins in osteoblasts. In summary, in differentiated osteoblast like cells, T3 increased the expression of OPG and osteocalcin, which effect was attenuated by co-treatment with 1,25(OH)2D3. We conclude, that thyroid hormone and calcitriol interact in the regulation of OPG and osteocalcin expression in osteoblasts. Abstract withdrawn.
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