ABSTRACTS P-1 to P-46
Posters will be on display throughout the symposium, but
will be attended by their presenting authors as follows:
Cancer and Metabolic Bone Diseases other than Osteoporosis
THE DIFFERENT KINETICS OF BONE MARKERS IN NORMAL AND DELAYED FRACTURE-HEALING OF LONG BONES
M. Herrmann1*, D. Klitscher1, T. Georg2, J. Frank3, I. Marzi3, W. Herrmann1
1Department of Clinical Chemistry and Laboratory Medicine, University Hospital of the Saarland, Homburg/Saar, Germany
2Institute of Medical Biometrics, Epidemiology and Medical Informatics, University of the Saarland, Homburg/Saar, Germany
3Department of Trauma-, Hand- and Reconstructive Surgery, University Hospital, Frankfurt/Main, Germany
Background: The prediction of delayed fracture-healing is still an unresolved problem in traumatology. Biochemical bone markers such as Osteocalcin (OC), bone alkaline phosphatase (bone-ALP) and serum ß-crosslaps (beta-CTx) might help to predict delayed fracture-healing.
Methods: We investigated 14 patients with operatively treated crural or femoral shaft fractures for 1 year. Patients were visited on day 0, 7, 14, 28, 42, 60, 90, 180 and 365. All visitations included the evaluation of a standardized clinical score of fracture- healing and venous blood sampling for the estimation of OC, bone-ALP and beta- CTx. Fracture-healing was classified as normal if the fracture was healed completely after 6 months and score level 2 (of 4) was attained after 2 months. If one of these criteria was not fulfilled delayed fracture-healing was assumed.
Results: According to our criteria 10 patients healed normally and 4 patients showed delayed fracture-healing. OC base line levels were similar in the two groups (16,96 ng/ml vs. 15,73 ng/ml). Normal fracture-healing was characterized by a first increase of OC on day 28 and maximum values on day 60, while in delayed fracture- healing, OC began to rise and peaked 1 month later. OC maxima were equal in the two groups (30,52 ng/ml vs. 30,37 ng/ml). Bone-ALP base line levels were three times higher in delayed than in normal fracture-healing (60,91 U/l vs. 20,02 U/l). After operative treatment bone-ALP increased. This increase tended to be later in patients with delayed fracture-healing. No effects were seen for beta-CTx.
Conclusion: Sequential measurement of OC on day 0, (28), 42 and 60 might help to predict delayed fracture-healing. Larger studies are needed to confirm our results.
INHIBITION OF BMP-MEDIATED OSTEOINDUCTION BY PLEIOTROPHIN: POSSIBLE RELEVANCE TO FIBRODYSPLASIA OSSIFICANS PROGRESSIVA?
H. I. Roach1*, R. S. Tare1, R. O. C. Oreffo1, E. M. Shore2, F. S. Kaplan2
1University Orthopaedics, University of Southampton, General Hospital, Southampton, UK
2Dept. of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, USA
Fibrodysplasia ossificans progressiva (FOP) is a rare and devastating disease, in which major striated muscles progressively are replaced by bone, frequently following trauma. The causes relate to inappropriate expression of BMPs, specifically BMP-4, but no defects have been identified in the genes for BMPs, BMP-receptors or the BMP-inhibitor noggin. Hence the disease remains a clinical and biological mystery.
We are investigating the roles of pleiotrophin (PTN), a putative osteotrophic factor, during bone development. We have demonstrated that PTN is produced by osteoblasts and stored in the bone matrix. Furthermore, PTN is chemotactic for osteogenic cells and enhances osteogenic differentiation,(1,2,3) consistent with a role in bone remodelling. The aims of this study were to examine whether PTN is osteoinductive and/or whether it influences BMP-mediated osteoinduction. To test for osteoinductive capacity, we used pre-myoblastic C2C12 cells, which are exquisitely sensitive to osteoinduction by BMPs. In the presence of 100 ng/ml BMP-2, ~50% of C2C12 cells became positive for alkaline phosphatase (ALP) after two days, indicative of osteoinduction. By contrast, PTN failed to induce ALP activity at concentrations up to 100 ng/ml, demonstrating that PTN was not osteoinductive. Furthermore, when C2C12 cells were co-treated with PTN and BMP-2, PTN inhibited the BMP-mediated osteoinduction by ~80%, an effect that was apparent at concentrations of PTN as low as 0.05 pg/ml. However, when PTN was added to C2C12 cells after osteoinduction by BMP-2, PTN stimulated further osteogenic differentiation, suggesting that the stage of PTN inhibition of osteoinduction is very specific.
Examination of PTN expression in skeletal muscle demonstrated high levels of PTN protein in muscle, suggesting that PTN could regulate osteogenesis in this tissue. PTN is thought to acts as a necessary accessory protein or co-factor for a variety of primary signalling factors.(1,2) These findings could be of considerable importance in developing treatments for patients with FOP. The remarkable inhibition of BMP- mediated osteoinduction together with the presence of PTN in muscle suggest that PTN is a potential candidate for further investigations of the role of PTN in relation to FOP.
1.J Bone Miner Res(2002) 17:2009-2020. 2.Biochem Biophys Res Comm(2002) 298:324-332. 3.J Bone Miner Res(2000) 15:1179
THE BISPHOSPHONATE RISEDRONATE INHIBITS BOTH ANGIOGENESIS AND BONE METASTASIS FORMATION IN VIVO
P. Fournier1, S. Boissier1, C. M. Serre1, M. Colombel1, R. Phipps2, F. H. Ebetino2, P. Clezardin1*
1INSERM, Lyon, France
2Procter & Gamble Pharmaceuticals, Cincinnati, Ohio, USA
In the present study, we have investigated the effects of the nitrogen-containing bisphosphonate risedronate (RIS) on angiogenesis and tumor growth in vivo. Risedronate, dose-dependently reduced endothelial cell survival and inhibited capillary-like tube formation in vitro. We have previously shown that bisphosphonates specifically accumulate in rat kidney and prostate tissues but not in other nonmineralized tissues (Fournier et al., Cancer Res., 2002). Castration induces the regression of the vasculature in the prostate and testosterone treatment of castrated rats causes a rapid vascular regrowth in the prostate. The effect of RIS on the re- vascularization of the prostate gland induced by testosterone in castrated rats was therefore tested. A daily s.c. treatment of castrated rats with testosterone + RIS (20 and 100 mg/kg/day) for 6 days reduced the prostate weight (20% inhibition, P = 0.01) compared to that observed with testosterone alone. In addition, morphometric analysis of immunostained blood vessels in prostate tissue sections demonstrated that RIS treatment induced a 45% reduction of the mean vessel area (testosterone + RIS: 865 ± 122 mm2 vs testosterone: 1561 ± 174 mm2, P = 0.009). In sharp contrast, clodronate did not inhibit testosterone-induced prostate regrowth and revascularization when used at a dose up to 20 mg/kg/day.
Angiogenesis is associated with tumor growth and metastasis progression. The effects of RIS were therefore studied on the growth of s.c. tumor xenografts and the formation of bone metastases induced by human MDA-MB-231/B02 breast cancer cells stably transfected to express the green fluorescent protein. Continuous treatment of nude mice with RIS (150 mg/kg/day) did not inhibit the growth of s.c. tumors. In contrast, treatment of animals bearing bone metastases with RIS at a similar dose almost completely inhibited the formation of osteolytic lesions (as judged by radiography) and substantially reduced the extent of skeletal tumor burden (as judged by fluorescence imaging).
Overall, our results indicate that RIS inhibits angiogenesis and metastasis formation in tissues where it accumulates.
IDIOPATHIC HYPERCALCIURIA(IH) IS THE LEADING CAUSE OF THE VERTEBRAL FRACTURES IN HUMAN BEINGS: A TEN-YEAR PROSPECTIVE STUDY ON 2.753 FEMALE AND 153 MALE PATIENTS WITH IH
M. Bevilacqua*, V. Righini, L. Foddis, L. Baruffaldi, R. Toscano, G. Baldi, T. Vago
Sacco University Hospital, Milan, Italy
Background and Aims. Idiopathic hypercalciuria (>300 mg/die in male, 250 mg/die in female) is the leading cause of nephrolithiasis (~ 50%). Hypercalciuric patients are also characterized by decreased lumbar and femoral bone density. Increased (~ 4 fold) incidence of vertebral fractures has been observed in nephrolithiasic subjects in Rochester (USA): however it is unknown if vertebral fractures are linked to nephrolithiasis (i.e. by spontaneous reduction of calcium untake) and/or hypercalciuria.
This study was designed to quantitatively investigate the relationship between hypercalciuria and fractures.
Materials and Methods. We prospectively studied the incidence of hypercalciuria in 13.259 pts consecutively admitted to our facilities in the decade 1991-2001.
Patients were otherwise healty men (689) and post-menopausal women (12.570) who attended the out patients clinic for minor age-related problems. Routine analysis included lumbar and femoral BMD (Hologic 2000 until 1998, then Delphi), lateral D2-L5 X-rays, 1-84 PTH, ionized calcium, urinary NTX, 25 (OH) Vit D, alkaline phosphatase, 24 hr urinary calcium and creatinine to exclude secondary hypercalciuria.
Results. Incidence of hypercalciuria was 21,9% in female (2.753) and 22,2% in male (153). After exclusion of absorptive hypercalciuria (by the application of a dietist-assisted low-calcium diet for 1 month and exclusion of patients with 2 hr calcium/creatinine mol/mol < 0,36), we obtained 1.232 female (9,8% of female) and 47 male (6,8% of male) with fasting (idiopathic) hypercalciuria.
The total number of vertebral fractures was 1.946, 1.257 in female and 689 in male.
In female with fractures, 233 were normocalciuric (2,4% of female) [D5-12= 162, L1-5 = 101] and 262 were hypercalciuric (21,3% of female; p<0,001 vs normocalciuric) [D5-12= 197, L1-5= 128].
In male 16 normocalciuric pts had al least 1 fracture (3% of male) [D5-12= 26, L1- 5= 9] and 40 hypercalciuric (85,1%; p<0,001) [D5-12= 35, L1-5= 13].
Conclusion. Idiopathic hypercalciuria is a leading cause of vertebral fractures in female and is the main cause of fractures in men.
BISPHOSPHONATES INDUCE APOPTOSIS IN STROMAL TUMOR CELLS OF GIANT CELL TUMOR OF BONE
Y. Y. Cheng1, L. L. Huang1*, K. M. Lee2, K. H. K. Li3, S. M. Kumta1
1Dept. of Orthopaedics & Traumatology, the Chinese University of Hong Kong
2Leehysan Clinical Research Laboratory, the Chinese University of Hong Kong
3Department of Pediatrics, the Chinese University of Hong Kong
Giant cell tumor of bone (GCT) is a benign but locally aggressive neoplasm of bone characterized by massive bone destruction at the epiphysis of long bones. The stromal cells are the main neoplastic components of this tumor and regulate the formation of osteoclastic-like giant cells ultimately responsible for bone destruction. Bisphosphonates prevent bone resorption by inhibiting osteoclast activity, promoting osteoclast apoptosis, and they can also induce apoptosis of primary neoplastic cells such as breast and prostate cancer. We hypothesized that bisphosphonates may induce apoptosis not only in giant cells but also neoplastic stromal cells of GCT, and such systemic treatment may mitigate osteolysis in GCT patients. Eight GCT primary cell cultures were treated with zolendronate, pamidronate or alendronate for 48 hours with different dosage (3, 30, 150 mM) and subjected to apoptosis assay by flow cytometry following fluorescent Annexin-V labeling. Surgical specimens were collected from 15 patients with GCT before and after pamidronate administration at dosage of 90 mg given weekly for 4-8 weeks. TUNEL assay was used to study the morphological changes associated with apoptosis and an image analysis program was used to calculate the percentage of apoptotic cells in representative sections. All three bisphosphonates significantly induced stromal tumor cell apoptosis in the cultures. Zolendronate being the most potent inducing 20% apoptosis at 30mM, followed by alendronate 13% and pamidronate 11%. Significant apoptosis was observed in both stromal tumor cells and multinucleated giant cells in GCT patients followed pamidronate treatment. Our observations suggest that bisphosphonates induce apoptosis in both stromal tumor cells and multinucleated giant cells and these drugs may be useful adjuvant in the treatment of GCT of bone.
A CASE OF TYPE II AUTOSOMAL DOMINANT OSTEOPETROSIS PRESENTING WITH UNUSUAL CLINICAL MANIFESTATIONS: GENETIC, CLINICAL AND CELLULAR STUDY
A. Taranta1*, C. Letizia2, S. Migliaccio1,3, E. Delfini2, E. D'Erasmo2, M. Iacobini4, M. Roggini4, O. M. E. Albagha5, S. H. Ralston5, A. Teti1
1Dept of Experimental Medicine, Univ of L'Aquila, L'Aquila, Italy
2Dept of Clinical Science, Univ of Rome La Sapienza, Rome, Italy
3Dept Medical Pathophysiology, Univ of Rome La Sapienza, Rome, Italy
4Dept of Pediatrics, Univ of Rome La Sapienza, Rome, Italy
5Dept of Medicine and Therapeutics, Univ of Aberdeen Medical School, Aberdeen, Scotland, UK
Autosomal Dominant Osteopetrosis (ADO) is classified in two subclasses, type I and II, primarily on the basis of radiological features. Type II ADO displays sclerosis of the skull base and pelvis, vertebral endplate thickening, cranial nerve palsies, mandibular osteomyelitis, multiple fractures and delayed healing. Genetically, heterozygous mutations of the osteoclast-specific chloride channel gene, ClCN7, underlies type II ADO. We have studied a 16-year old male patient, with radiologically unclassifiable ADO, which was genetically identified as type II. The patient was found to harbour a novel mutation in the ClCN7 gene, consisting in a heterozygous C/A transition in exon 25, codon 788, predicted to lead to replacement of alanine (GCC) with asparagine (GAC) in the C-terminal domain of the protein. This genotype was not found in 50 control chromosomes. Radiographic analysis and bone mineral densitometry demonstrated generalised osteosclerosis and increase of vertebral and pelvis bone mass, albeit with normal mineral content values and absence of pathologic fracture. The patient suffered from recurrent bronchitis and otitis, and right ear deafness. Deafness appeared independent of acoustic nerve compression syndrome as no narrowing of the auditory nerve canal was found. Rather, hearing impairment appeared to be caused by compression of the left frontal lobe, due to an arachnoid cyst. He had increased levels of the bone formation markers bone alkaline phoshatase isoenzyme, osteocalcin and N-terminal type I collagen telopeptide/creatine ratio. Other biochemical findings and acid-base balance were normal, except serum levels of creatine kinase and lactic dehydrogenase. At the cellular level, cultured osteoclasts, generated from peripheral blood monocytic fraction of the patient, showed increased mobility, with lamellipodia, membrane ruffling and motile pattern of podosome distribution, all of which could have contributed to functional impairment of bone resorption. In conclusion, we have studied a case of type II ADO, with unusual radiological and clinical manifestations, finding a novel mutation of the CLCN7 gene which originated a mild phenotype similar to type I ADO. We also illustrated that factors other than auditory nerve compression can contribute to deafness in this patient, and underscored that bone formation markers are consistently increased in osteopetrosis.
OSTEOPROTEGERIN (OPG)/RECEPTOR ACTIVATOR OF NF-KB LIGAND (RANKL) BALANCE IN PATHOLOGIC OSTEOLYSIS : RT- PCR STUDY AND SERUM ANALYSIS
E. Grimaud1, L. Soubigou1, S. Couillaud1, N. Passuti2, F. Rédini1*, F. Gouin2, D. Heymann1
1Lab. Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, Faculté de Médecine, Nantes, France
2Orthopaedic Department, Nantes Hospital, Nantes, France
Introduction: Pathologic osteolysis can be considered as a consequence of a disturbance in the mechanisms that govern the bone remodeling such as the communication between osteoclasts and osteoblasts. OPG and RANKL are newly discovered molecules that are greatly involved in such communications. RANKL, expressed as a membrane-associated protein by osteoblast/stromal cells is essential for osteoclast via its receptor RANK. OPG is an osteoblast-secreted decoy receptor that inhibits osteoclast differentiation through its binding to RANKL. The goal of the present study is the analysis of the OPG/RANKL balance during human osteolysis associated to various etiologies.
Methods: Sixty biopsies collected from patients (31 women, 29 men ; mean age 51.2 years, range 15-80) with pathologic osteolysis (primitive bone tumors, bone metastasis, aseptic prosthetic loosening) referred to the Department of Orthopedic Surgery (Nantes Hospital) were included in this study. Sixteen biopsies of healthy tissues harvested on epiphysis resected because of total hip prosthesis or bone tumors (bone, cartilage, capsule skeletal muscle) were used as control tissues (13 women, 3 men ; mean age 65.8 , range 23-80). The osteolysis degree was evaluated according to the Merle d'Aubigné classification. The OPG and RANKL expressions were analyzed by semi-quantitative RT-PCR. At the same time, OPG and RANKL levels were determined using ELISA tests in serum of each patient and compared to 9 control serum.
Results: The RANKL levels were significantly increased in serum of patients suffering from osteolysis compared to the control group (297.3+237.6 pg/ml and 106+77.3 pg/ml respectively; p < 0.05). No significant difference in serum OPG was observed between control group (216.6+75.6 pg/ml) and osteolytic diseases (323.5+234.9 pg/ml). OPG and RANKL transcripts were detected in all samples studied. Whereas no significant difference of OPG and RANKL expression levels can be determined between control group and osteolytic disorders, the ratio RANKL/OPG significantly increased in high grade osteolysis (1.43+1.88 compared to 0.27+0.24; p < 0.01 in the control).
Conclusion: For the first time, an increase of serum RANKL levels is evidenced in osteolytic situations. These results show that imbalance of RANKL/OPG is involved in osteolytic pathologies in each group studied.
BONE REMODELLING MARKERS IN HYPOPHOSPHATASIA
S. J. Iqbal*, J. Mahabir, D. Manning, S. Holland, T. Davies
Department of Chemical Pathology, Leicester Royal Infirmary, Leicester, England
Hypophosphatasia is a rare inherited disorder characterised by low serum levels of tissue non specific alkaline phosphatase (TNSALP) associated with a range of skeletal abnormalities. We investigated to see if the low alkaline phosphatase production from osteoblasts would have secondary effect on other osteoblastic products and whether this may affect bone remodelling.
In four families, 9 subjects, (4F, 5M; age range 24-52 yrs) with hypophosphatasia osteocalcin/bone gla protein (BGP) carboxy terminal of procollagen type 1 (P1CP) and urinary deoxypyridinoline, creatinine (DPYD/cr), carboxy terminal telopeptide of type 1 collagen (1CTP) were measured using E1A, hydroxyproline/Cr (OHP/cr) using a colorimetric method. Results (mean;SD): total alkaline phosphatase (rr 40-130 iu/L)
19; 7. BGP (ng/ml) 21.4; 10.5, P1CP (mg/L) 151; 66, 1CTP (mg/L) 11.3; 14.2, DPD/cr (nmol/mmol) 4.0; 1.5, OHP/cr (mmol/mmol) 9.7; 4.9. All results were normal except 1CTP levels which were elevated in two of the index cases with clinical bone disease (42.4, 12.9, normal range 1.8-5.0 mg/L). BGP was correlated with; P1CP (r = 0.61 NS), OHP/cr (r= 0.71,
P < 0.05), DPD v 1CTP (r = 0.80, P < 0.05). There did not appear to be
any global osteoblastic failure in these subjects.
DIRECT ANTI-TUMOR EFFECTS OF ZOLEDRONATE IN RAT OSTEOSARCOMA. THERAPEUTIC RELEVANCE OF ITS ASSOCIATION WITH IFOSFAMIDE
D. Heymann1, F. Blanchard1, P. Coipeau1, C. Charrier1, S. Couillaud1, J. P. Thiery2, F. Gouin3, F. Rédini1*
1EE 99-01, Faculté de Médecine, Nantes, France
2Hôpital Paul-Brousse, Villejuif, France
3Orthopaedic department, Nantes Hospital, Nantes, France
Background: Local growth of osteosarcoma involves destruction of host bone by proteolytic mechanisms and/or host osteoclast activation. Nitrogen-containing bisphosphonates (N-BP) as zoledronate inhibit the mevalonate pathway, which results in the inhibition of osteoclast function and the induction of apoptosis in osteoclasts and tumor cells alike. The use of animal models has demonstrated that N-BP reduce skeletal tumor burden in myeloma, breast and prostate cancers. In vitro findings revealed that the combination of zoledronic acid with standard anticancer drugs results in synergistic apoptotic effects on myeloma and breast cancer cell lines.
Objectives: The purpose of this study was to evaluate zoledronate efficacy on osteosarcoma growth, in association with the standard anticancer drug ifosfamide.
Methods: Experiments were performed using a rat transplantable model of osteosarcoma. 4 week-old male Sprague-Dawley rats were transplanted with tumor fragments, placed in contiguous to the exposed tibia metaphysis surface. Tumors develop in 4-6 weeks and are associated with lung metastases in 80-85% of cases. Four series of 7 rats were used, treated or not by zoledronate (100 microg/kg, at day 7, 14, 21 and 28 after tumor implantation) and associated or not with ifosfamide (30 millig/kg; at day 13, 14 and 15). 35 days after tumor implantation, animals were killed and lung metastases, tumor development, radiological and histological studies were assessed. Complementary studies of zoledronate effects were performed in vitro, using the OSRGA cell line isolated from the same rat osteosarcoma tumor.
Results and Conclusion: Zoledronate was very effective not only in limiting the formation of osteolytic lesions induced by osteosarcoma, but also in reducing the local tumor growth (75%), as compared to the untreated rats. In vitro studies demonstrated that zoledronate (10-5 M) inhibited OSRGA proliferation by 60%. The association of ifosfamide with zoledronate reduced the tumor development more efficiently than ifosfamide alone. This study revealed for the first time the direct anti-tumor effect of zoledronate on primary bone tumor and its potentiality to reinforce the local tumor growth inhibition induced by ifosfamide.
This work was supported by Novartis Laboratories (Rueil-Malmaison, France).
SERUM TARTRATE-RESISTANT ACID PHOSPHATASE 5B AS A MARKER OF BONE METASTASES IN PROSTATE CANCER
R. Maddison1*, P. A. Kyd1, M. Swinn2, A. Fairney3
1Dept of Chemical Pathology, St Mary's Hospital, London
2Dept of Urology, St Mary's Hospital, London
3Imperial College Faculty of Medicine, London
Prostate cancer is the most common carcinoma in men, often complicated by metastases which are usually assessed by bone scintigraphy, an expensive technique with limited availability. Metastases are predominantly osteosclerotic, though with evidence of increased resorption, and various bone markers have been studied as indicators for bone metastasis. A recently-introduced method for serum tartrate - resistant acid phosphatase TRAP 5b ( BoneTRAP, SBA, Oulu Finland), is osteoclast- specific, independent of liver and renal function and relatively convenient. This study evaluates its clinical utility in the detection of bone metastases in prostate cancer, and compares TRAP5b with conventional serum bone markers of formation (bone alkaline phosphatase, BALP) and resorption (serum C-telopeptide, CTx).
Serum TRAP 5b, BALP ( Metra) and CTx (Nordic Bioscience) were measured in men assigned to the following groups based on bone scintigraphy, biopsy, Gleason score and serum PSA : benign prostatic hypertrophy (n=17), prostate cancer without metastases (n=28) and prostate cancer with metastases (n=27). A local reference range derived from 52 men aged 40-90 years with no known prostate disease gave TRAP5b values of 0.8-5.7U/L. TRAP 5b was significantly elevated in patients with bone metastases compared with those without ( 7.1 (1.27) mean (SEM) v 3.4(0.3) U/L, p < 0.001) and with benign prostatic hypertrophy (2.6(0.2)) U/L, p < 0.0001). There was a significant correlation of TRAP 5b with BALP (p < 0.0001), with serum CTx (p < 0.0001) and with PSA (p < 0.01), but not with Gleason Score. ROC analysis showed TRAP5b (AUC 0.75(0.07)) to have similar diagnostic utility in detecting bone metastases to BALP (AUC 0.79(0.06)) and serum CTx (AUC 0.74(0.07)).
We conclude that TRAP 5b appears to have a potential use for the detection of early metastases in prostate cancer.
MEASUREMENT OF OPG, RANKL, AND TRAP5B IN THE SERUM OF ELDERLY PATIENTS WITH ACTIVE PAGET'S DISEASE
B. H. Durham*, A. Daroszewkska, W. D. Fraser
The Royal Liverpool University Hospital, Liverpool, UK
We have measured osteoprotegrin [OPG], the receptor activator of nuclear factor kappaB ligand [RANKL] and the osteoclast derived tartrate - resistant acid phosphatase 5b[TRAP5b]. in a cohort of 64 elderly patients with active Paget's disease, F31, M33, mean age 74.5yr range 55-88 yr. After venepuncture serum was separated as soon as possible and stored at -70°C until analysed , all assays were run at the same time so that the sera did not need re-thawing. OPG and RANKL ELISA kits were from Biomedica , Vienna, Austria and TRAP5b ELISA kit from SBA Sciences, Turku, Finland. In the RANKL assay only the soluble unbound fraction is measured.There was no significant differences between men and women in any of the assays so the cohort of patients was considered as one group. Mean, SD, range for OPG was 3.6,1.7, 0.7-8.9 pmol/L, for RANKL 7.4,4.7, 1.3-25.9 pmol/L and for TRAP5b 5.7,1.8,2.2-15.2 U/L. The ratio OPG/RANKL had a mean of 0.65,SD 0.42, range 0.13-1.50, there was no significant relationship between either OPG or RANKL and TRAP5b, the concentration of OPG was equal to, or greater than that of RANKL in 15/64 [23%] of patients. In our cohort of active Paget's patients OPG values were significantly lower compared with those of age-related normals 3.6±1.7 v 5.5±1.8 [p<0.05]; 8/64 [12.5%] were lower than the age-related reference range, whereas RANKL was significantly raised 7.4±4.7 v 3.8±2.0 [p<0.01] with 28/64 [44%] greater than an age-related reference range. TRAP5b was also raised 5.7±1.8 v 4.6±0.9 [p<0.05]with 17/64[26%] of values greater than an age-related reference range. The measurement of OPG, RANKL, and TRAP5b should advance our knowledge of osteoclastogenesis and particularly the communication between osteoblasts and osteoclasts in Paget's disease.
CIRCULATING FGF-23 LEVELS CORRELATES WITH RENAL FUNCTION IN PATIENTS WITH CHRONIC KIDNEY DISEASE, BUT DOES NOT CHANGE IN RESPONSE TO VARIATION IN PHOSPHATE INTAKE IN HEALTHY VOLUNTEERS
T. E. M. Larsson1*, R. Marsell1, U. Nisbeth1, H. Jueppner2, O. Ljunggren1, K. B. Jonsson3
1Dept of Medical Sciences, University Hospital, Uppsala, Sweden
2Endocrine Unit, Massachussetts General Hospital, Harvard Medical School, Boston, USA
3Dept of Surgical Sciences, University Hospital, Uppsala, Sweden
Hyperphosphatemia is a risk factor for the development of secondary hyperparathyroidism and cardiovascular complications in chronic kidney disease (CKD). Fibroblast Growth Factor 23 (FGF-23) is a recently discovered protein that is mutated in the inherited phosphate wasting disorder autosomal dominant hypophosphatemic rickets and may represent a novel hormonal regulator of phosphate metabolism. We therefore hypothesized that FGF-23 levels are affected by hyperphosphatemic disease and change in response to different levels of phosphate intake.
Using an immunometric assay against the C-terminal portion of FGF-23, serum levels of FGF-23 were determined in 20 patients in early renal failure (creatinine range 155-724 mmol/l), in 33 patients on dialysis treatment and in 30 patients with a functioning renal graft. Furthermore, six healthy males were treated with statin based phosphate binders in combination with low phosphate diet intake during two days followed by three days of inorganic phosphate treatment. FGF-23 levels were determined at multiple time points.
FGF-23 serum levels were significantly elevated in patients with CKD. FGF-23 levels in the dialysis group were 59600 ±20500 RU/ml, in the pre-dialysis group 2010 ±890 RU/ml but near normal in the transplanted group (230 ±42 RU/ml). There was a strong correlation between creatinine and FGF-23 levels. Significant independent correlations was also seen between FGF-23 and phosphate, calcium and PTH. No changes in serum FGF-23 levels were observed in the volunteers following treatment with phosphate binders or inorganic phosphate, although phosphate clearance changed significantly.
Circulating FGF-23 is significantly elevated in patients with CKD and correlates with renal clearance function. FGF-23 levels could not be altered by phosphate deprivation or phosphate loading in healthy volunteers.
PREVALENCE OF VITAMIN D DEFICIENCY IN PATIENTS WITH OSTEOGENESIS IMPERFECTA
G. Chung1*, R. W. Keen1,2
1Metabolic Unit, Royal National Orthopaedic Hospital, Stanmore, Middlesex, United Kingdom
2Bone and Mineral Centre, Department of Medicine, University College London, London, United Kingdom
There is growing interest in bisphosphonate treatment for patients with osteogenesis imperfecta (OI) although an adequate vitamin D status is recommended prior to initiating treatment. Lifestyle restrictions and impaired mobility of OI patients may limit exposure to sunlight, a major source of vitamin D, thus the purpose of this study was to determine the vitamin D status of patients with OI and to evaluate correlation between vitamin D levels and both PTH levels and BMD.
Patients attending a metabolic bone service underwent clinical evaluation and biochemical assessment. Data was collected on age, sex, racial origin, OI type and date of attendance. Blood samples were taken and analysed for serum 25(OH) vitamin D level, serum PTH level and routine bone profile. Lumbar spine and hip BMD were measured using a Hologic QDR-Delphi. Vitamin D deficiency was defined as a serum 25(OH) vitamin D level less than 25 nmol/l and insufficiency as a level between 25-50 nanomol/l.
Data is included on 31 patients (19 female, 12 male) with mean age of 34 years (range 7-79 years). The majority had type I OI (21 type I, 2 type III and 8 type IV). The mean serum 25(OH) vitamin D level was 45.1 nanomol/l (SD=21.6 nmol/l). 25(OH) vitamin D levels were <25 nanomol /l in 23% of patients (n=7), between 25- 50 nanomol/l in 42 % (n=11) and >50 nanomol/l in 35% (n=13). There was a weak but positive correlation between serum 25(OH) vitamin D and lumbar spine BMD (R2=0.17, p=0.03), although no significant correlation was seen at the hip. In 2 of the 7 patients with vitamin D deficiency and in 1 of the 11 patients with vitamin D insufficiency there was evidence of secondary hyperparathyroidism.
This study has demonstrated that low serum levels of 25(OH) vitamin D are common in OI patients, with nearly two thirds being either vitamin D deficient or insufficient. Clinicians should be aware of this finding, and OI patients should have their vitamin D status assessed to consider either formal supplementation or dietary modification. This is particularly important for those OI patients commencing bisphosphonate treatment.
PRO-COLLAGEN I COOH-TERMINAL TRIMER INDUCES DIRECTIONAL MIGRATION AND METALLOPROTEINASES EXPRESSION IN BREAST CANCER CELLS
D. Palmieri1, S. Zanotti1, G. L. Casartelli2, K. Buschiazzo1, P. Manduca1*
1Genetica, Dipartimento di Oncologia, Biologia e Genetica Università di Genova, Genova, Italy
2Istituto per la Ricerca sul Cancro, Genova, Italy
The conditioned medium (CM) by mature osteoblasts, induces directional migration of breast and prostatic carcinomas, melanoma and endothelial cells. The carboxyl terminal trimer of procollagen type I (C3) purified from CM of mature osteoblasts is the chemotactic agent responsible of migration of endothelial cells. We here report that C3 is also a chemoattractant for the breast carcinoma cell line MDA MB231.The directional migration induced by C3 is also accompanied by enhanced expression of Metalloproteinases (MMP)-2 and -9 and by their proteolytic activation. No significative change in the level of tissue inhibitor (TIMP)-1 and increased level of TIMP-2 were detected. The function of beta1 and beta3 integrins are required for induction of MMPs by C3. Membrane MMP-14 is costitutively expressed, unchanged upon exposure to C3. Specific antisera against MMP -2, -9 or -14 and addition of TIMP-1 or of GM6001 inhibit C3 induced directional migration suggesting the involvement of MMPs in the process. Urokinase (uPA) and its receptor are also costitutively expressed and unchanged upon exposure to C3. The specific antibody against uPA and addition of plasminogen activator inhibitor inhibit directional migration. Treatment with Pertussis toxin inhibits C3 induced cell migration and MMP-2 and -9 expression, implying the requirement of heterotrimeric G-protein mediated pathway(s) signaling. Chemotaxis induced by C3 is inhibited also by treatment with Genistein , an inhibitor of Phosphotyrosine Kinases, by antibody against integrin chain alpha6, beta1 and beta3, and by Wortmannin, an inhibitor of Phosphatidylinositol-3 kinase. Thus, more than one receptor family and molecular mediators might be involved in the acquisition of migratory phenotype in MDA MB231 upon induction by C3. Moreover C3 is mitogenic but not apoptotic for MDA MB231.
Presently C3 is the only agent known to induces directional migration specifically of endothelial and breast carcinoma cells. In vivo, high pro-collagen type I production by the fibroblasts in the tumor stroma is often associated with tumor progression. It is possible that the C3 formed in pro-collagen processing might promote a favorable microenvironment for tumor progression by stimulating tumor cells viability, migration and pericellular proteolysis, and by attracting endothelial cells, thus favoring also tumor angiogenesis.
EVALUATION OF CORTICAL BONE BY PERIPHERAL QCT IN PERITONEAL DIALYSIS (PD)PATIENTS
A. L. Negri*, R. Barone, C. Lombas, C. Bogado, J. R. Zanchetta
Instituto de Investigaciones Metabolicas, Buenos Aires, Argentina
Peripheral QCT allows the non-invasive evaluation of cortical and trabecular bone separately as well as the geometrical properties of the radius. We investigated cortical bone by pQCT in 22 patients (6 males and 16 females) on maintenance PD; comparisons were made with 28 normal controls.pQCT(XCT 9690, Stratec, Pforheim, Germany) was performed at the proximal radius of the non-dominant forearm (15% the length of the ulna from endplate). We evaluated Total and cortical bone mineral density (TBMD, cBMD), Total and cortical area (TA,cA), cortical thikness (cThK) endosteal and periosteal perimeters and buckling ratio. Intact PTH levels were measured by IRMA. correlations were made with age, total time in dialysis and serum iPTH. DP patients had a marked decrease in cThK(1.90 vs 2.95 mm;p<.0001)and marked increase in endosteal perimeter (31.2 vs 23.9 mm;p<.0001) buckling ratio [r/cThK] (3.81 vs 2.21;p<.0001)and TA. TBMD and cBMD correlated negatively with total time in dialysis (p<.01); no correlations were found between cA, cBMD and cThK with iPTH. Age correlated positively with TA, endosteal and periosteal perimeter and negatively with cBMD. Our results show a cortical thinning of the radius with cortical parameters correlating with total time in dialysis and that bone adaptations to aging (pripherization) occur despite disturbances in endocrine- metabolic environment of dialysis.
ANTI-RESORPTIVE POTENCY ALONE DOES NOT PREDICT THE EFFICACY OF BISPHOSPHONATES IN THE RAT ADJUVANT MODEL OF RHEUMATOID ARTHRITIS
J. M. Meyer, M. D. Francis, F. H. Ebetino*
Procter & Gamble Pharmaceuticals, Cincinnati, Ohio, USA
The rat adjuvant arthritis model is similar to human rheumatoid arthritis and is characterized by severe intra- & peri-articular inflammation & bone lysis. Bisphosphonates (BPs) are known to bind to hydroxyapatite and inhibit bone resorption. We have shown that some BPs, such as risedronate (ActonelTM) are effective at inhibiting cartilage lesion size and severity and osteophytosis in the guinea pig model of primary osteoarthritis. This efficacy did not correlate with anti-resorptive potency. Using the rat adjuvant arthritis model, we evaluated bisphosphonates with varying degrees of anti-resorptive potency for their anti-inflammatory efficacy as measured by paw volume & inhibition of bone resorption.
Modified Freund's adjuvant (MFA, 4.4mg/kg) was injected into the tail of Lewis rats (prophylactic) or Sprague-Dawley (therapeutic) rats. For prophylactic studies, daily subcutaneous (sc) injections of treatment started on the day of MFA injection. For prophylactic studies, daily treatment began on Day 10, after disease was established. Treatment continued until Day 24.
In general, there is not a strong relationship between antiresorptive potency and anti-arthritic activity. This suggests there may be an additional activity of BPs that contribute to the anti-inflammatory effect seen in this model, and some BPs may be a useful therapy for RA, even beyond their ability to inhibit bone resorption.
OSTEOPROTEGERIN DETECTION BY IMMUNOHISTOCHEMISTRY IN BONE BIOPSIES OF PATIENTS WITH RENAL OSTEODYSTROPHY
P. Ballanti1*, G. Silvestrini1, G. Coen2, M. S. Fischer2, S. Calabria2, S. Berni1, M. Leopizzi1, E. Bonucci1
1Department of Experimental Medicine and Pathology, 'La Sapienza' University of Rome, Rome, Italy
2Department of Clinical Sciences, 'La Sapienza' University of Rome, Rome, Italy
Osteoprotegerin (OPG) and Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL) are important mediators that regulate the effects on osteoclasts of calcitropic hormones and cytokines. In vitro, calcitriol increases and parathyroid
hormone (PTH) reduces OPG production in osteoblastic-like cells. Renal osteodystrophy (ROD) is mainly due to alterations of vitamin D and PTH serum levels, affecting several cytokines. This study was carried out to assess OPG immunohistochemical presence and distribution in bone samples of patients with different ROD patterns.
Fifteen haemodialysis patients (8 males, 7 females; 56 ±10 years of age) underwent a transiliac biopsy. After fixation, bone specimens were longitudinally divided. One half was embedded in glycolmethacrylate, without decalcification, for histological diagnosis and histomorphometry. The other half was decalcified and embedded in paraffin; for each sample, three sections about 3 micrometers thick were cut at intervals of 20 micrometers for OPG immunohistochemistry. After pretreatment with 0.1% Tween 20, a polyclonal OPG antibody (Santa Cruz Biotechnology; 1:100) was used.
ROD was histologically classified as: hyperparathyroidism (HP; n=8), osteomalacia (OM; n=2), mixed ROD (n=2), adynamic bone disease (ABD; n=2); one patient had normal bone. Focusing attention to the endosteal envelope, in all patients OPG was present in the cytoplasm of all cells of the osteoblastic phenotype: plump and/or flat osteoblasts in contact with osteoid seams, lining cells bordering quiescent surfaces and, in HP and mixed ROD, elongated cells of peritrabecular fibrosis. Positivity was chiefly evident in plump osteoblasts due to their abundant cytoplasm. As shown by osteoblastic surface (Ob.S/BS, %), these cells were increased in high turnover ROD (HP: 18.59 ±14.33; mixed ROD: 9.40 ±6.18) and reduced in low turnover ROD (OM: 0.20 ±0.20; ABD: 0.34 ±0.48), in comparison with the case with normal bone (4.63).
These results suggest higher production of OPG in high than in low turnover ROD. They are in agreement with previous findings (Coen et al., NDT 2002; 17:233- 8)showing higher serum OPG in HP and mixed ROD than in ABD. Further studies are in progress to assess whether types of ROD are characterized by alterations of OPG/RANKL ratio.
EXPRESSION OF ALPHAVBETA3 IN CHINESE OVARY HAMSTER (CHO) CELLS INHIBITS IN VIVO OSTEOLYTIC BONE METASTASES
A. Funari*, I. Recchia, C. DiGiacinto, F. Marampon, M. Longo, A. Teti, N. Rucci
Department of Experimental Medicine, University of L'Aquila, L'Aquila, Italy
AlphaVbeta3 integrin is involved in adhesion, migration, proliferation and survival of selected cell types. Consistent evidence supports its involvement in tumorigenesis, but its role in metastatic spreading remains controversial. AlphaVbeta3 is a relevant integrin in bone, therefore we asked whether it played a role in the development of in vivo bone metastases. We demonstrated the ability of the CHO tumour cell line to induce osteolytic lesions in female BALB-c nu/nu mice subjected to heart injection. We then employed the CHO parental cell line (CHOp) and a CHO subclone stably transfected with the human beta3 integrin subunit (CHOalphaVbeta3). Integrin profile by FACS confirmed expression of functional alphaVbeta3 receptor only in the transfected cells. Unexpectedly, a significantly reduced incidence of bone metastases in mice injected with CHOalphaVbeta3 cells was observed relative to CHOp cells, albeit with no changes in lesion size and histological appearance. Similar life expectation was observed. However, mice injected with CHOp cells displayed an increment of renal metastases relative to those injected with CHOalphaVbeta3 cells, with no changes in other organs. We also noted no significant increase in the incidence of solid tumours in mice subcutaneously injected with CHOalphaVbeta3 cells relative to CHOp, with no obvious differences in tumour volume and angiogenesis. In vitro studies failed to evidence changes in proliferation and in migration through gelatine substrates between the two cell lines. In contrast, CHOp cells displayed a higher ability to invade matrigel matrices, and their supernatants contained greater uPA activity and pro-MMP9 levels relative to CHOalphaVbeta3 cells, with equal levels of Tissue Inhibitor of Metalloproteinase (TIMP)-1/2. Adhesion assays to various substrates showed selective ability of the CHOalphaVbeta3 cells to attach to the anti- alphaVbeta3 antibody LM609, enhanced adhesion to collagen I and fibrinogen, and reduced adhesion to fibronectin compared to CHOp cells. In conclusion our findings suggest that the expression of the alphaVbeta3 integrin in CHO cells does not significantly affect their ability to grow in vivo and in vitro, but leads to a reduced onset of bone and renal metastases, with a mechanism likely associated with a drop in adhesion to fibronectin and in invasion ability.
THE N-TERMINAL PROPEPTIDE OF TYPE I PROCOLLAGEN-(PINP ) AND TARTRATE RESISTANT ACID PHOSPHATASE-(TRAP-5B) AS BONE TURNOVER MARKERS IN DIALYSED PATIENTS
Z. Nowak*, M. Konieczna, Z. Wankowicz
Central Hospital of Military Institute of Medicine, Warsaw, Poland
Non invasive measurement of bone turnover markers especially PINP and TRAP 5b has been considered as a potentially useful tool of diagnosis of renal osteodystrophy (ROD) in dialysed patients.
The aim of the study was to compare clinical usefulness of PINP and TRAP 5b with the standard marker of ROD which is iPTH.
We studied 80 patients: 58 on hemodialysis (HD) for 43 ± 25 months (36M, 22F aged 59 ± 25 yr.) and 22 on continuous ambulatory peritoneal dialysis (CAPD) for 47 ± 28 months (11M. 11F, aged 53± 23 yr.). According to the level of iPTH the whole group was divided in 3 subgroups: low (group1), medium (group2) and high turnover (group3) ROD.
The following parameters were determined in serum: PINP, TRAP 5b, iPTH, Ca, P. PINP was measured using radioimmuno assay (Orion Diagnostica Finland); TRAP 5b activity was measured using assay Bone TRAP(SBA Finland). Intact PTH was measured using immunoradiometric assay (Incstar USA).
We found significant correlation between iPTH and PINP ( r= 0,7124; p<0,001) and between iPTH and TRAP ( r= 0,6872, p < 0,001). In patients with high turnover ROD-(group 3.) significantly higher values of PINP and TRAP were found in comparison with group 1 of patients with low turnover ROD.(42±20 vs.283±138 p<0,001 and 7,3± vs. 2,8± p < 0,001 respectively). There ware not significant differences between values of, P, Ca in group1. vs. group 3. We also didn't find significant correlation between values of PINP, TRAP and gender, age and method of dialysotherapy.
In conclusion this results indicate the usefulness of PINP and TRAP 5b as markers of bone turnover in dialysed patients.
THE SHORT LIVED EXOSTOSIS INDUCED SURGICALLY VERSUS THE LASTING GENETIC HEREDITARY MULTIPLE EXOSTOSES
M. Trebicz-Geffen1, Z. Nevo1*, Z. Evron1, N. Posternak1, T. Glaser1, M. Fridkin3, K. Kollander4, D. Robinson1,2
1Department of Clinical Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv, 69978, Israel
2Orthopedic Department, Assaf Harofe Medical Center, Zeriffin, 70300, Israel
3Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot, 76100, Israel
4Orthopedic Oncology Department, Tel Aviv Sourasky Medical Center, Tel Aviv, 64239, Israel
Hereditary osteochondromas (HME), are often caused by mutation in the EXT1 gene. The lesions are typified by formation of a 'pseudo' growth plate like lesion growing at 60 degrees to the normal growth direction of the bone. Such lesions can be mimicked surgically by reverting the polarity of the zone of LaCroix. The current study attempts to compare the pathology between EXT1 gene expression in humans and surgically created osteochondroma in a rat model. Rats were operated on conducting an inversion of a 60-degree span of the ring of LaCroix, as described by Delgado. The created osteochondroma was assessed by histological techniques. The surgically created lesions contain only a few left FGF receptor 3 (FGFR3) located in the perichondrium.
The shortage, as well as disturbed migration routes of the residual mesenchymal stem cells in surgically created osteochondromas leads eventually to resorption of the pathological elements.
EXT1 is a type II transmembrane glycoprotein localized in the endoplasmic reticulum and Golgi apparatus, which is characteristic of glycosyltransferases, (heparan sulfate polymerase). The EXT1 as components of the heparan sulfate biosynthesis pathway suggested that HME probably results from insufficient presentation of cell-surface heparan sulfate during bone development, causing both abnormal migration and deviated activation of FGF family of receptors.
Our finding shows that in HME patients the levels of pericellular heparan sulfate are lower compared to solitary exostosis and control.
The EXT1 protein of HME is migrating and bands at a lower molecular weight then the EXT1 protein of Solitary and control in western blot analysis. This means that the EXT1 protein in HME patients is abnormal .
In conclusion, in the genetic cases of osteochondromas there is a compensation attempt in the elevation of mRNA levels of EXT1 in HME patients compared to Solitary patients and controls, while in the surgically created osteochondromas there is a lack the massive and continueous population of mesenchymal stem cells with the Bcl2 expression. However, the small residual mesenchymal cell population is giving rise to a short-lived exostosis that disappears eventually due to spontaneous resorption.
These findings could suggest a feasibility to replace the surgical treatment of these patients by trying to cure the HME malady by a gene therapy approach.
BONE LOSS AFTER RENAL TRANSPLANTATION IN RELATION TO IMMUNOSUPPRESSIVE THERAPY
C. Ejersted1*, J. D. Jensen2, B. Jespersen3, C. Bistrup3, L. Mosekilde4, T. Torfing1, L. Lund-Olsen5, J. Carstens2, K. Brixen1
1Dep. of Endocrinology, Odense University Hospital, Odense, Denmark
2Dep. of Nephrology, Aarhus University Hospitals, Aarhus , Denmark
3Dep. of Nephrology, Odense University Hospital, Odense, Denmark
4Dep. of Endocrinology, Aarhus University Hospitals, Aarhus , Denmark
5Dep. of Radiology, Aarhus University Hospitals, Aarhus , Denmark
Renal transplantation is a well-established treatment of end-stage renal disease. After renal transplantation, a decline in bone mass has been described and is thought to be due to the use of immunosuppressive agents.
In this study, bone mineral density (BMD) of lumbar spine and femoral neck and whole body bone mineral content (BMC) where measured by dual energy x-ray absorptiometry at start, 3, 6, 12, and 24 months after renal transplantation. Patients have been included from two different cities in Denmark having the same criteria for renal transplantation but using different immunosuppressive therapy: in Odense a combination of calcineurin inhibitor and mycophenolate (group I) and in Aarhus a combination of prednisolone, calcineurin inhibitor, and azathioprine (group II). Sixty patients have been included; and so far 13 and 10 patients of the 2 groups, respectively, have been followed for 1 year. Because of anti-rejection prednisolone therapy 3 patients from group I have been excluded from this analysis. Group I consisted of male/female n=5/5, mean age 42.4 years, group II consisted of male/female n= 7/3, mean age 45.5 years. No differences concerning BMD of lumbar spine, femoral neck, or whole body BMC where measured between the 2 groups neither in start value or at any of the other measuring points. Within the groups a slight decrease in BMD in percent of start value of lumbar spine was found in group II (mean (SEM) at 0, 3, 6, and 12 months for group II: 100 (0), 98.5 (1.6), 95.0 (2.0), 95.2 (1.9) %), but not in group I (mean (SEM) at 0, 3, 6, and 12 months: 100 (0), 96.1 (1.2), 96.8 (1.9), 99.4 (1.6) %, p-value 0.01 and 0.10, respectively.
In conclusion, our preliminary data shows no differences between the groups concerning bone loss in relation to immunosuppressive therapy. Further data is being collected, however, and will be presented at the 30th European Symposium on Calcified Tissues.
CHROMOSOME ABERRATIONS IN HUMAN LYMPHOCYTES AFTER TOTAL HIP REPLACEMENT: A PROSPECTIVE STUDY
S. Bernardini*, I. D. Learmonth, C. P. Case
Bristol Implant Research Centre, Southmead Hospital, Bristol, UK
Based on previous studies of metal on polyethylene joint replacements showing an increasing number of chromosomal aberrations (aneuploidy and translocations) in patients at revision arthroplasty, a prospective study of the long-term risk of joint replacement is being performed. Peripheral blood lymphocytes from patients with different types of joint replacements were analyzed to look at the development of aneuploidy and chromosomal translocations with changes in metal exposure over time. The metals involved include chrome, titanium, cobalt, nickel, vanadium, aluminium and molybdemum. Genetic polymorphism can alter therapeutic responses and toxicity to certain xenobiotics and may also be a factor that influences a patient's susceptibility to certain diseases. NAT2 polymorphism was compared in 60 healthy subjects and 60 patients with Total Hip Replacement (THR). NAT2 alleles (*4, *5, *6, and *7) were determined by polymerase chain reaction method.
Blood samples from at least 60 patients with total hip replacement will be cultured and analyzed by comparing cytogenetic and metal levels preoperatively and at 6 months, 1 year, and 2 years post operatively. Fluorescent in situ hybridization (FISH) techniques to paint individual chromosomes allow the detection of individual translocations involving different chromosomes. Chromosomes 1,2 and 3 (23.9% of the whole genome) is painted to detect potentially 40.2 % of all stable chromosome translocations between painted and unpainted chromosomes from 600 cells of each patient. The levels of chromium, cobalt, molybdenum and nickel were measured by High Resolution ICP-MS (Inductively Coupled Plasma Mass Spectrometry).
The level of chromosome translocations and of aneuploidy was increased after metal on metal hip arthroplasty but only in a minority of patients by 2 years postoperatively. The level of chromium and cobalt in the peripheral blood was also increased.
There is some limited evidence that the frequency of chromosomal damage in peripheral lymphocytes may also help in predicting subsequent cancer risk in healthy individuals. Data have suggested that chromosomal aberrations are associated with cancer risk. It appears that chromosomal aberrations might be a valid predictor of cancer occurrence at the group level also in non-smokers and in subjects with no apparent occupational exposure to carcinogens.
USE OF FISSION YEAST FOR INVESTIGATION OF CHROMOSOMAL DAMAGE INDUCED BY METALS USED IN ORTHOPAEDIC SURGERY
S. Bernardini1*, C. J. Norbury2, C. P. Case1
1University of Bristol, Southmead Hospital, Bristol, UK
2University of Oxford, Sir William Dunn School of Pathology, Oxford, UK
Chromium alloys are widely used in joint replacement surgery. Failed prostheses can liberate particulate and soluble metal wear debris that causes chromosomal damage to human cells in vivo and in vitro. Interestingly the orthopaedic alloy titanium vanadium aluminium causes a five-fold increase in aneuploidy. In contrast, the orthopaedic alloy cobalt chrome causes a three-fold increase in both chromosome translocations and aneuploidy. We have explored the effects of metal ions as well as metal liberated from orthopaedic alloys on the fission yeast Schizosaccharomyces pombe. This genetically tractable model system allows the rapid identification of conserved molecular mechanisms relevant to chromosome damage in human cells.
Mutants defective in homologous recombinational repair (rhp51D) were hypersensitive to Cr (VI) during short-term exposure in liquid culture. These results are in line with a recently reported requirement for homologous recombination in the repair of Cr (VI)-induced lesions in Saccharomyces cerevisiae, which is only distantly related to S. pombe. Further investigation showed that S. pombe rad3D mutants, which are defective in DNA structure checkpoint signalling, were also hypersensitive to Cr (VI). Together, these data suggest that Cr (VI)-induced DNA damage is sensed by a rad3 -dependent pathway and repaired by a recombination-dependent mechanism. By analogy, responses to Cr (VI)-induced damage in human cells may depend on homologous recombinational repair, as well as on ATM/ATR, protein kinases that are orthologs of S. pombe Rad3.
Fission yeast represents a convenient system for further dissection of the mechanism of toxicity of Cr (VI), for example through the use of simple assays to measure mitotic recombination and chromosome loss rates. Additional data indicate that S. pombe can be used as a convenient tool with which to screen clinically relevant metal alloys for their capacity to induce chromosomal aberrations.
EARLY TREATMENT DOES NOT PREVENT SKELETAL CHANGES IN X-LINKED HYPOPHOSPHATEMIC RICKETS
O. Makitie*, A. Doria, A. Daneman, E. Sochett
The Hospital for Sick Children, University of Toronto, Toronto, Canada
X-linked hypophosphatemic rickets (XLH) is characterized by hypophosphatemia, rickets and impaired growth. Despite oral phosphate and 1,25(OH)2D3 treatment many patients have suboptimal bone healing. The aim of this study was to assess the impact of timing of treatment on skeletal changes.
Radiographic severity of the rickets was retrospectively estimated in skeletal radiographs of 19 well-controlled patients with XLH. The radiographs were reviewed at diagnosis, at the end of first treatment year and at latest prepubertal time point available for analysis. The findings were graded as normal, normal/mild, mild, mild/moderate, moderate, moderate/severe or severe rickets. For statistical analysis these seven grades of severity were given a numeric value ranging from 0 (=normal) to 6 (=severe). Patients were divided into two groups based on the age at treatment onset (Group 1, <1.0 years, Group 2, >1.0 years).
At treatment onset the patients in Group 1 had radiologically milder rickets as compared to patients in Group 2; the median score for the severity of rickets was 2.0 (±0.3) (±SE) for Group 1 (mild) and 4.5 (±0.3) for Group 2 (between moderate and moderate/severe) (P=0.0002). At the end of the first treatment year the median score for Group 1 was unchanged, and improved to 4.0 (±0.4) in Group 2 (P=0.052). At prepuberty (median age 10.4 years) the median scores were 4.0 (±0.5) and 5.0 (±0.7) for Groups 1 and 2, respectively (P=0.27). Degree of severity of the rickets at treatment onset, but not after that, correlated positively with the age (r=0.65, P=0.0055) at treatment onset: the later the diagnosis the more marked the ricketic changes in the radiographs.
These data suggest that treatment commenced in early infancy, as compared to treatment commenced after age 1.0 year, results in slightly improved skeletal outcome but does not completely prevent radiographic signs of rickets even in well-controlled patients with XLH.
DIAGNOSTIC VALUE OF DUAL ENERGY X RAY ABSORPTIOMETRY IN RENAL OSTEODYSTROPHY
S. Mazzaferro1*, P. Ballanti2, G. Matera1, G. Otranto1, G. Barresi1, C. Albanese3
1Department of Clinical Science, University 'La Sapienza' of Rome, Italy
2Department of Experimental Medicine and Pathology
3Department of Radiology
Absorptiometry techniques, including DEXA (Dual Energy X-Ray Absorptiometry), are being used in ROD (Renal Osteodystrophy), but paradigms for BMD (Bone Mineral Density) values have been established only for osteoporosis (OP). In our study we verified the correspondence between DEXA and bone biopsy in
25 pts (51±11 y.o.; 11M/14F; dialysis since 102±66 months) through biochemistries (Ca, P, Alkaline Phosphatase (AP), PTH, Osteocalcin (BGP)), Lumbar (L) and Femoral (N) DEXA, and transiliac bone biopsy (bone histology and, in 17/25, static histomorphometry). Mean values (±SD) were: PTH: 604±491 pg/ml; AP: 204±151 mU/ml; BGP: 426±723 ng/ml; Ca: 9,8±1.3 mg/dl; P: 5.7±1.7 mg/dl; BMD-L 0.93±0.24 g/cm2; BMD-N 0.70±0.16 g/cm2. ROD diagnosis were: 13 prevailing hyperparathyroidism; 7 Mild; 3 Mixed; 1 Osteomalacia and 1 Adynamic Bone. T- Scores (L and N) were used to obtain 3 groups of pts: OP (>-2.5DS), Osteopenia (<- 2.5>-1DS), or Normal BMD. For both sites (L and N) we did not observe the prevailing of any histologic diagnosis in any group. Patients with DEXA-OP had higher PTH, bone resorption and endosteal fibrosis as compared to DEXA-Normals (ANOVA p<0.03) but not different Bone Volume, Osteoid Volume and Calcified Volume. Negative correlations were found between PTH and L (r=-0.49, p<.05) and N (r=-0.7,p<.01) T-Scores and between these BMD parameters and semiquantitative evaluation of resorption (p<.05) and fibrosis (p<.05). PTH and AP correlated positively with resorption and fibrosis (p<.01). DEXA-L and N were also negatively correlated with Osteoid Surface (p<.05), Osteoclastic Surface (p<.01) and Osteoclasts Number (p<.01), but not with Bone Volume, Osteoid Volume and Calcified Volume. Our results suggest that DEXA-L or F: 1. Do not allow to distinguish between types of ROD; 2. Are not strictly correlated with bone volumes (calcified or not); 3. Are related to the severity of bone lesion, independently of its type. We conclude that in ROD the lower is the DEXA-BMD, the greater is the severity of the underlying bone lesion, independently of its type.
PAMIDRONATE INDUCES CELL DEATH OF STROMAL CELLS OF GIANT CELL TUMORS
J. D. Doppelt1, S. S. Chang1, F. Y. Lee1*, H. Z. Zhang2, R. J. Winchester2
1Department of Orthopaedic Surgery, Columbia University, USA
2Center for Autoimmune and Molecular Diseases, Department of Pediatrics, Columbia Univeristy, USA
INTRODUCTION: Giant cell tumor of bone (GCT) is a primary neoplasm consisting of stromal cells and large, multinucleated giant cells that are phenotypically similar to osteoclasts. In this study we show that Pamidronate, a nitrogen-containing BP, induces apoptosis in cultured GCT stromal cells as indicated by morphological characteristics and flow cytometry measurements of annexin V binding.
METHODS: Specimens were freshly minced with scissors in Dulbecco's minimum essential medium (DMEM) producing a cell suspension with small fragments of tissue. After 3-4 passages, primary GCT cell cultures mainly consisting of proliferating stromal cells were briefly trypsinized and transferred to petri dishes. Zero, 50, 100, or 200 mM Pamidronate disodium was added to the culture medium when the cells reached 70-80% confluency. Five ml of annexin V-FITC (A.G. Scientific) was added to 195 ml of cell suspension and incubated in the dark for 10 minutes. The cells were pelleted and washed with PBS and resuspended again in 190 ml of binding buffer. Ten ml of 20 mg/ml propidium iodide (A.G. Scientific) was added to the cell suspension and flow cytometry was performed within 30 minutes using a FACS Calibur (Becton Dickson). Monoparametric cytograms of annexin V- FITC fluorescence (FL1) versus number of events were created using the CellQuest program gating for living cells and excluding dead cells on the basis of their propidium iodide uptake.
RESULTS: Pamidronate decreases cell proliferation and induces cell death in cultured GCT stromal cells. Exposure to all 3 concentrations of bisphosphonate produces morphological changes and a decrease in the number of adherent cells after only 24 hours of treatment compared to controls. Similarly cultured and treated normal fibroblasts are not affected by Pamidronate, providing evidence of specificity in the drug's action. Analysis of annexin V binding by flow cytometry shows that Pamidronate treatment increases the occurrence of apoptosis.
DISCUSSION: The tumor-induced osteolysis seen with GCT might be treated with Pamidronate based on its ability to both inhibit giant cell mediated bone resorption as well as induce apoptosis in the neoplastic stromal cells.
SERUM TARTRATE-RESISTANT ACID PHOSPHATASE 5B AND OSTEOCALCIN IN HUMAN TYPE II AUTOSOMAL DOMINANT OSTEOPETROSIS
S. L. Alatalo1*, K. K. Ivaska1, S. G. Waguespack2, M. J. Econs3, H. K. Väänänen1, J. M. Halleen1
1Institute of Biomedicine, Department of Anatomy, University of Turku, Turku, Finland
2Department of Endocrine Neoplasia and Hormonal Disorders, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA
3Departments of Medicine and Medical and Molecular Genetics, Indiana University, Indianapolis, IN, USA
Autosomal dominant osteopetrosis type II (ADO2) is an uncommon metabolic bone disorder characterized by decreased bone resorption and significantly increased bone mass. The mutated gene in ADO2 is chloride channel 7 (ClCN7), which is needed for acidification of resorption lacunae. Patients with ADO2 have increased numbers of large ineffective osteoclasts and elevated serum levels of tartrate-resistant acid phosphatase (TRACP) and the BB isoenzyme of creatine kinase (CK-BB). We investigated the serum levels of the osteoclast-derived 5b isoform of TRACP (TRACP 5b) and the bone formation marker osteocalcin in ADO2 patients with known ClCN7 mutations. Ten ADO2 families participated in the current study. Based on radiographs, family history, and genotyping for ClCN7 gene mutations, 232 subjects were divided into three groups: clinically affected, carriers (with the ClCN7 mutation but no clinical evidence of disease), and healthy controls. The participants were further divided into children (aged from 2 to 18 years) and adult (aged >18 years) groups. The results are shown in table 1. Serum TRACP 5b levels were significantly elevated in affected patients compared with age-matched controls in both age groups, the values being several orders of magnitude higher than in any bone diseases with increased bone resorption. These results suggest that in ADO2, serum TRACP 5b reflects the number of osteoclasts rather than their activity, and that the extremely high serum TRACP 5b level is a specific indicator of the disease. Serum total osteocalcin levels were unchanged in children, but significantly decreased in adult carriers and significantly elevated in adult affected patients, suggesting that the balance in bone turnover may be disturbed in ADO2.
Table 1. Serum TRACP 5b and osteocalcin levels in ADO2 patients, carriers and healthy controls
IN VIVO MODEL OF BONE METASTASIS IN HUMAN PROSTATE CANCER
A. Angelucci1, N. Rucci1, C. Festuccia1, G. L. Gravina1, P. Muzi1, L. Ventura3, A. Teti1, M. Bologna1,2*
1Department of Experimental Medicine, University of L'Aquila, L'Aquila, Italy
2Department of Basic and Applied Biology, University of L'Aquila, Italy
3Department of Pathology, San Salvatore Hospital, L'Aquila, Italy
Prostate cancer is frequently associated with bone metastases, which are considered so far the main cause of morbidity and mortality for this tumour. To better investigate this process, animal models of bone and bone marrow metastases need to be developed. However, experimental prostate cancer bone metastases are difficult to form in vivo, and some typical clinical patterns remain irreproducible. In our study, we injected prostate cancer cells in the left cardiac ventricle of nude mice, thus reproducing the basic biological phenomenon of tumour cell spreading in the arterious blood stream. Mice were monitored by x-ray analysis and about 1/2 of them revealed osteolytic lesions 40 days after tumor cell injection. Histological characterization of bone bioptic specimens revealed metastatic tumour cells especially in the diaphysis of posterior limbs, with large areas of bone and cartilage degradation. Immunohistochemical analysis of a wide panel of antigens revealed a high positivity for matrix proteases, including uPA, MMP1, MMP3, and MMP9, suggesting a role of these proteases in the osteolytic process.
Further characterization of these tumours was performed in cultured cells from bone metastasis explants. One of the resulting cell population, termed PCb1, demonstrated a more invasive phenotype compared to parental PC3. PCb1 cells crossed more easily the matrigel barrier compared to PC3 cells in the presence of the bone chemotactic factor osteopontin or of the osteoblast conditioned media, but not in the presence of NIH 3T3 fibroblast conditioned media. This phenomenon was partially due to an increased capacity of PCb1 to release proteolitic enzymes relative to the original PC3 cells.
This study demonstrates that heart injection of prostate cancer cells in nude mice may represent a good experimental model to investigate the pathophysiology of bone and bone marrow metastases in vivo.
OSTEOPONTIN SUPPORTS PROLIFERATION INDUCED BY EPIDERMAL GROWTH FACTOR IN HUMAN PROSTATE CANCER CELLS
A. Angelucci1*, C. Festuccia1, G. L. Gravina1, P. Muzi1, L. Bonghi1, M. Bologna1,2
1Department of Experimental Medicine, University of L'Aquila, L'Aquila, Italy
2Department of Basic and Applied Biology, University of L'Aquila, L'Aquila, Italy
Prostate cancer progression is characterized by its predisposition to acquire an androgen-independent phenotype and to metastasize to bone. Although extracellular matrix and growth factors have been demonstrated to play a critical role in the development of prostate cancer metastases, the mechanisms underlying their action remain largely unknown. LNCaP cell line is a suitable model in the study of prostate cancer progression because it shows a strong responsiveness in its growth and survival to androgens and to growth factors. In our study we tested the LNCaP growth ability in steroid-free culture conditions in response to osteopontin, a non-collageneous matrix protein, localized in large amount in the bone. Several studies have demonstrated that osteopontin in many physiological and pathological situations is able to show not only a structural role but mainly a chemokine-like behaviour. In the LNCaP cell model osteopontin stimulates clonal and cell growth in serum-free medium but this effect is visible only in presence of EGF. The addition to culture medium of Ly294002, a PI3 kinase inhibitor, completely abolished the proliferation stimulus. Proliferation induced by osteopontin is accompanied by a sustained activation of EGF-receptor whose phosphorilation is detectable up to 12 h after treatment in association with EGF. The colocalization of integrin beta1, a ligand of osteopontin, and EGFR on the cellular membrane, suggests that the association of these receptors may be the principal mechanism involved in the long-term activation of EGFR. In conclusion our data describe a new possible mechanism responsible for the establishment of bone metastases and for the generation of androgen-independent cellular clones.
CLODRONATE IN HORMONE REFRACTORY PROSTATE CANCER
R. K. Tahtela1*, T. Kylmala2, M. Kurkilahti1, S. Atula1, I. Elomaa3, T. Tammela2
1Leiras Oy, Clinical Research, Helsinki, Finland
2Tampere University Hospital, Tampere, Finland
3Helsinki University Hospital, Helsinki, Finland
Bone metastases from prostate cancer are mainly of sclerotic type, but also osteoclastic lesions are seen. Excessive bone formation leads to formation of woven bone, whereas increased bone resorption is believed to be the reason for bone pain. Clodronate has been shown to reduce pain, and to decrease bone turnover in patients with advanced prostate cancer, but administered with estramustine it causes impaired bone mineralization.
This study investigated the efficacy of intravenous (iv) clodronate (BONEFOS), followed by oral clodronate, in reducing bone resorption, and the effect of clodronate on osteoid in patients with hormone refractory prostate cancer and bone metastases, bone histomorphometry from tumour-free bone, and serum markers of bone turnover and calcium metabolism as efficacy variables.
Twenty patients were randomised to receive clodronate, 600 mg iv for 3 days and 2400 mg oral daily for 3 months, or similar placebo. Histomorphometric parameters assessed were eroded surface (ES/BS), osteoclast number (N.Oc), bone formation rate (BFR), and mineral apposition rate (MAR), measured at study entry and end. Serum markers, measured at several time-points, were type I collagen carboxy-terminal telopeptides S-ICTP and S-CTx-beta for bone resorption, amino-terminal propeptide of type I procollagen (S-PINP) and osteocalcin (S-Osteoca) for bone formation, and calcium (S-Cacor), phosphate (S-Pi), and calcitriol (S-1,25(OH)2-D) for calcium metabolism.
Histomorphometric indices and type I collagen derived markers indicated increased bone turnover at study entry. Clodronate decreased N.Oc and increased ES/BS, in the placebo group N.Oc slightly increased. BFR increased in both groups, but less in the clodronate group. Clodronate increased MAR in line with the increase in BFR, in the placebo group MAR increase was small compared to BFR increase. Iv clodronate decreased S-CTx-beta and increased S-Osteoca, at study end they returned to baseline levels. S-PINP increased up to one month, and decreased below the baseline level at study end in the clodronate group. In the placebo group these markers did not change. Clodronate induced an increase of vitamin D, whereas in the placebo group vitamin D decreased.
To conclude, clodronate caused a decrease in bone resorption, it prevented inappropriate bone formation, and it did not cause osteomalacia.
CLODRONATE PREVENTS BREAST CANCER-INDUCED OSTEOLYSIS IN A MOUSE MODEL
E. Aho*, M. Suominen, C. Malmström, T. Österman, P. Isaksson, S-M. Käkönen, P. Lakkakorpi, S. Hokkanen, R. Hannuniemi
Biomedical Research Center, Leiras Oy, Turku, Finland
Oral clodronate (Bonefos®) at a dose of 1600 mg daily was recently shown to prevent the occurrence of skeletal metastases in primary breast cancer patients (Powles et al., J Clin Oncol 2002; 20:3219). The aim of the present study was to investigate the preventive effects of clodronate (50 mg/kg/d) in a pre-clinical mouse model that resembles the characteristics of osteolytic bone metastasis in human breast cancer. Furthermore, the dose-response of preventive clodronate treatment (10, 20, and 40 mg/kg/d) was studied in the same setting.
Bone metastases were caused by MDA-MB-231 human breast cancer cells that were inoculated into the left cardiac ventricle of 5-week-old nude mice and bisphosphonates (BP; risedronate 0.2 mg/kg/d as a reference) were administered s.c. for 21 days after the inoculation. On day 22, the mice were sacrificed and radiographed, followed by densitometric measurements by pQCT and histomorphometric analyses. BP-treated groups were compared to untreated positive (cancer cell inoculation) and negative (saline inoculation) controls.
Statistically significant inhibition of tumor-induced bone loss by both clodronate and risedronate was observed in X-ray studies and histomorphometry. The number of osteoclasts was in both treatment groups lower than in the positive control group, and in the clodronate group it was lower than in the risedronate group. In the dose- response study, clodronate reduced the area of osteolytic lesions as well as the tumor- induced cortical and trabecular bone loss at all doses when determined by radiography, densitometry and histomorphometry. At the site of the osteolytic metastases, the values of densitometric parameters were significantly lower in the untreated tumor-inoculated mice compared to the negative controls. However, no differences were observed at the mid-diaphyseal bone where no tumor tissue was present. This suggests that the osteolytic effects are local in this model.
In conclusion, the present data indicate that clodronate prevents tumor-induced osteolysis when determined by radiography, densitometry and histomorphometry. The data support clinical findings and suggest that clodronate offers a potent treatment for the prevention of bone destruction in breast cancer.
EXTRACELLULAR MATRIX DEGRADING PROTEINASES IN PANNUS INVASION IN RHEUMATOID ARTHRITIS
M. Ainola1*, J. Mandelin1, M. Liljestrom1, G. Ma1, Z-K. Chen1, Y. T. Konttinen2
1Department of Anatomy, Biomedicum, University of Helsinki, Finland
2Department of Medicine, Helsinki University Hospital, Helsinki, Finland
Distinctive features of rheumatoid arthritis (RA) comprise continuous inflammation of synovium, in which synovial membrane expands on cartilage leading to pannus formation. Pannus formation and appearance of proteolytic enzymes cause articular cartilage and bone destruction which lead to erosions and permanent joint damage.
Matrix metalloproteinases (MMP) are involved in degradation of extracellular matrix (ECM) and especially MMP-3 production in pannus tissue is greatly increased compare to synovial tissue of RA and osteoarthritis. Monocyte derived tumour- associated trypsinogens (TATs) and tumour-associated trypsin inhibitor (TATI) are present in both pannus and synovial tissue. The potential roles of TATs are in proteolytical activation of proMMPs and degradation of cartilage and ECM. Copynumbers of TATI mRNA versus b-actin are half of TAT copies. TATI inhibits cell migration and invasion probably by inhibition of TAT, which together form a complex. ADAMs (a disintegrin and a metalloproteinase) are membrane-anchored proteinases involved in cell-matrix interactions. The incidence of ADAM-12 in synovial tissue indicates its possible role in pannus formation by remodelling of synovial tissue and activation of other proteinases.
CONCURRENT PAGET'S DISEASE OF BONE AND PRIMARY HYPERPARATHYROIDISM
C. Leens, J-P. Devogelaer*
Department of Rheumatology, Saint-Luc University Hospital, Université catholique de Louvain, B-1200 Brussels, Belgium
Primary hyperparathyroidism (PHPT) is a classical, though not frequently acknowledged condition associated with Paget's disease of bone (PD). The aim of this study was to determine whether in 2002, the association still holds and which factors could favour it. We have therefore reviewed the medical records of 117 patients (66 M, 51 F, aged 58-97 y), regularly followed at the outpatient clinic. IPTH (IRMA, NV < 60 pg/ml) had been measured at least once, more than 3 months apart from antiresorbing therapy in 75 patients (64.1 %). In 25 patients (33.3 %), iPTH was found to be superior to the upper limit of normal. Among these patients, 11 had normal levels of 25OHvit D (14.7 %) and 14 (18.7 %) had low levels of 25OHvit D. Resistance to intravenous pamidronate, defined as a drop of alkaline phosphatase
activity smaller than 25 % after 6 months, was found in 23 patients (16 M, 7 F), out of which 13 (8 M, 5 F) had elevated levels of iPTH (2 M with hypovitaminosis D, 1 F with pagetic osteosarcoma). 33 patients suffered from a polyostotic form of PD (23 M, 10 F), out of which 12 (8 M, 4 F) had elevated iPTH, with 2 F only with normal 25OHvit D levels. 17 patients were both polyostotic and resistant to pamidronate (12 M, 5 F), out of which 9 (6 M, 3 F) had elevated iPTH, with 1 F only with normal 25OHvit D levels. In 3 patients, with initially low levels of 25OHvit D, later normalized, iPTH remained elevated.
In conclusion, high iPTH plasma levels are a frequent finding in our patients suffering from PD. Hypovitaminosis D could constitute a favouring factor in 45 % of cases. However, if we consider exclusively PD patients with normal 25OHvit D levels, up to 12 % have high levels of iPTH, a potential harbinger of PHPT. This is particularly the case in resistant and polyostotic PD patients.
BIOLOGICAL EFFECTS OF GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (GM-CSF) ON HUMAN SARCOMA CELL LINES TROUGH REGULATION OF ITS RECEPTOR (CDW116) AND ENDOGLIN (CD105) EXPRESSION
G. Di Domenico1, L. Del Vecchio2, G. Di Spigna1, P. Ladogana1, E. M. Bruno1, M. Turano1, A. Scognamiglio3, G. Rossi1, L. Postiglione1*
1Dipartimento di Biologia e patologia Cellulare e Molecolare 'L.Califano', Università 'Federico II', Napoli, Italy
2Servizio di Immunoematologia, AORN 'A.Cardarelli', Napoli, Italy
3Cattedra di Reumatologia, Università 'Federico II', Napoli, Italy
Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) is a cytokine active on hematopoietic cells. The GM-CSF receptor (GM-CSFR) has been molecularly identified; it is composed of two chains , termed alpha and beta , of 80 and 120 KDa molecular weight, respectively. Recently we have shown that the human osteosarcoma cell line SaOS-2 express the GM-CSFRalpha (CDW116) and that GM- CSF reduces the proliferation and induces an osteoblastic differentiation of these cells. Furthermore, we have reported that Endoglin (CD105), a TGF-beta receptor complex binding protein, is differently expressed in human carcinoma and sarcoma cells, showing high levels of expression on human sarcoma cells and low expression levels on human carcinoma cells.
In the present study, we have investigated the GM-CSF production and secretion in the culture medium; CDW116 and CD105 expression on human sarcoma cell lines of different histotypes and its regulation by GM-CSF (200 ng/ml). Furthermore, we have analysed cell growth of the human sarcoma cells stimulated and not by GM-CSF. The cells employed in our studies were: MG-63 and SaOS-2 (osteosarcoma), SW1353 and H-EMC-SS (chondrosarcoma), A204 and TE671 (rhabdomyosarcoma), HT1080 and Hs913T (fibrosarcoma). Human endothelial cells (HECV) were used as a positive control of CDW116 and CD105 protein expression. The analysis of GM-CSF production was performed by mRNA analysis whereas, in order to measure GM-CSF secreted in the culture medium, commercially available ELISA kits were used. CDW116 and CD105 protein were analysed by Western blotting, using specific mAbs. Flow cytometric analysis was performed to quantify CDW116 and CD105 production on the cell membrane and cytoplasm. Furthermore, the mRNA analysis of GM-CSFRalpha and Endoglin was performed using specific primers for PCR amplification of reverse-transcribed cellular mRNA. Finally, cell proliferation was estimated by 3H-Thymidine incorporation.
The results show that all the lines express CDW116 and CD105; GM-CSF seem to regulate this expression in almost all the lines. In fact, GM-CSF up-regulate CDW116 in HECV, H-EMC-SS, SW1353, and down-regulate CDW116 in SaOS-2, TE671 and Hs913T. Furthermore, GM-CSF up-regulate CD105 in HECV, and down-regulate CD105 in others cell lines. Cell proliferation too seem to be differently regulated by GM-CSF. These data show that the human sarcoma lines express CDW116 and CD105 and that GM-CSF can to differently regulate this expression and cellular proliferation.
BONE TURNOVER EVALUATION BY USING THE BIOLOGICALLY ACTIVE FRAGMENT OF PARATHYROID HORMONE IN RENAL OSTEODYSTROPHY
A. Montagnani1*, S. Gonnelli1, M. Mangeri1, C. Cepollaro1, M. B. Franci1, E. Gaggiotti2, G. Bellucci3, R. Nuti1
1Department of Internal Medicine, Endocrine-Metabolic Science and Biochemistry , University of Siena, Italy
2Unit of Nephrology, Siena, Italy
3Unit of Nefrology, Grosseto, Italy
The assay commonly used for intact parathyroid hormone (iPTH) recognizes the fragment PTH(1-84) as well as the large C-terminal PTH fragment. In vitro studies showed that C-PTH fragments result either inactive or antagonist to PTH(1-84) action.
Recent studies reported conflicting results on the discrimination of bone turnover in dialysis patients by using PTH(1-84), and namely PTH(1-84)/C-PTH ratio.
The aim of the present study was to investigate the usefulness of the new method to measure PTH(1-84) in evaluating bone turnover in patients on dialysis.
A fasting blood sample was obtained from 140 patients on chronic dialysis and from 120 healthy subjects as control group. Serum levels of calcium, phosphorus, bone alkaline phoshatase (BALP) and N-terminal peptide of type I collagen (NTx) were measured in all subjects. In the same population PTH peptides were assessed using both the traditional assay for iPTH (N-tact PTH SP IRMA Kit- DiaSorin) and a new method (IRMA, Scantibodies Duo PTH. Inc.) specific for PTH(1-84). The levels of C-PTH was calculated by subtracting PTH(1-84) from iPTH. Patients were classified into high (HT) or normal-low (NLT) bone turnover groups on the basis of BALP and NTx. The ability in predicting bone turnover was evaluated by performing a multiple logistic regression analysis. PTH(1-84), C-PTH and iPTH were significantly higher in dialysis patients than in controls. All the three fragments of PTH were highly correlated each other. Among dialysed patients 75 patients were included in NLT group and 20 in HT group. These latter showed iPTH, PTH(1-84) and C-PTH significantly (P<0.001) higher than NLT patients. On the contrary no difference was found for PTH(1-84)/C-PTH ratio. PTH(1-84) and iPTH showed similar ability in the prediction of bone turnover, in particular, PTH(1-84) and iPTH classified correctly the 78% and 84% of cases, respectively. Instead, PTH(1-84)/C- PTH ratio did not show an adequate discriminatory ability. In conclusion, the present study points out that in dialysis patients the use of the new method for the assessment of active fragment of PTH does not result more useful than iPTH in discriminating bone turnover in renal osteodystrophy.
MARKERS OF BONE TURNOVER AND DISEASE PROGRESSION IN PROSTATE CANCER
R. A. Hannon1*, J. M. Wells2, N. Hoyle3, F. C. Hamdy2, R. Eastell1
1Bone Metabolism Group, Division of Clinical Sciences (North), University of Sheffield, Sheffield, UK
2Academic Urology Unit, University of Sheffield, Sheffield, UK
3Roche Diagnostics GmbH, Penzberg, Germany
Markers of bone turnover are elevated in metastatic bone disease and may be useful in tracking disease progression in prostate cancer. The aims of this study were to determine which markers of bone turnover best indicated disease status in newly diagnosed patients and indicated relapse after treatment, with androgen ablation, in patients with advanced disease.
Newly diagnosed patients were classified as having localised disease (T1/2)(n = 30, mean age 64.6 years) or nonlocalised disease (T3/4) (n = 27, mean age 74.2 years). Patients treated with androgen ablation were classified as responders (n = 12, mean age 75.8 years), borderline responders (n = 11, mean age 76.2 years) or refractory ( n = 25, mean age 74.8 years).
Serum formation markers measured were osteocalcin (OC), procollagen type I N terminal propeptide (PINP) and bone alkaline phosphatase(Bone ALP) . Serum resorption markers measured were C terminal cross-linking telopeptide of type I collagen (betaCTX), C terminal cross-linking telopeptide of type I collagen generated by MMPs (CTX-MMP), and tartrate resistant acid phosphatase 5b (TRACP 5b) Table shows median concentrations.
PINP, bone ALP, beta CTX and CTX-MMP are elevated in patients where disease is already advanced at diagnosis. In refractory patients, bone ALP and CTX-MMP are significantly increased, compared to responders. We conclude that different markers maybe useful in assessing skeletal involvement at different stages of disease. However, longitudinal studies are required to investigate whether they predict disease progression.
EXPRESSION OF BONE MORPHOGENETIC PROTEINS IN HUMAN PROSTATE CANCER WITH SKELETAL METASTASES
D. Bobinac1*, J. Spanjol2, S. Zoricic1, I. Maric1, G. Dordevic3, Z. Fuckar1
1Deaprtment of Anatomy, School of Medicine, University of Rijeka, Croatia
2Clinical Hospital Rijeka, Croatia
3Department of Pathology, School of Medine, University of Rijeka, Croatia
Prostatic adenocarcinoma is one of the most common cancers in men and preferentially metastasized to bone. Majority of skeletal metastases are osteoblastic in nature. These osteoblastic metastases show extensive new bone formation with possible involvement of the solubile growth factors such as bone morphogenetic proteins (BMPs) secreted by tumor cells. Bone morphogenetic proteins (BMPs) were originally extracted from bone as factors that induce cartilage and bone formation in vivo. They are multifunctional regulators of cell proliferation, differentiation and apoptosis. Human prostate cancer cells are known to produce several growth regulatory factors, including BMPs. The presence of BMPs in primary prostate carcinomas could be linked to the increased osteoblastic activity seen in skeletal metastasis. We investigated the expression of different BMPs in 25 histopathologically diagnosed prostate cancer samples. All examined patients had multiple skeletal metastases as shown by a positive bone scan. Tumor specimens were moderately or poorly differentiated cancer, according to the Gleason score. Paraffin- embedded samples were analyzed immunohistochemically using the polyclonal anti- BMP-2/4, -3, -5, -6, and -7 antibodies. The results demonstrated that adenocarcinoma specimens showed positive cytoplasmatic staining for BMP-2/4, -3, -5, and -6 in epithelial cancer cells. Also, we found BMP-7 positive nuclear staining. According to expression level of BMPs, BMP-2/4 is positive in 70-80% tumor cells, BMP-3 in 30- 50% tumor cells, BMP-5 in 50-70% tumor cells and BMP-6 in 40-60% tumor cells. Our findings suggest that BMPs may hold potential role in prostate carcinoma pathogenesis and developing of skeletal metastasis.
BONE MORPHOGENETIC PROTEIN-7 EXPRESSION IN TUMORAL CALCINOSIS AND BROWN TUMOR OF SECUNDARY HYPERPARATHYROIDISM
S. Zoricic1*, G. Dordevic2, D. Bobinac1
1Department of Anatomy, School of Medicine, University of Rijeka, Croatia
2Department of Pathology, School of Medicine, University of Rijeka, Croatia
Bone morphogenetic proteins (BMPs) are family of multifunctional growth and differentiation factors that were identified based on their ability to induce endochondral bone development at extraskeletal sites. A member of this family, BMP- 7 acts locally and has predominant effect on bone cell growth and tissue differentiation. In the postnatal life, BMP-7 is expressed predominantly in kidney but it was also found in osteoblasts, bone matrix, periost, hypertrophic chondrocites, centers of ossification and bone regeneration processes. Therefore, there is a potential possibility that BMP-7 could be involved in the pathogenesis of the renal osteodystrophy. BMPs were found in various pathological lesions in skeletal tissues such as myositis ossificans, fibrodysplasia ossificans progressiva, osteogenic sarcoma. We investigated tumoral calcifications and brown tumor of secondary hyperparathyreoidismus in patients with end-stage renal disease. The intention was to investiagate BMP-7 protein expression in both types of osteodystrophic lesions. In both lesions, multinucleated giant osteoclast-like cells showed moderate cytoplasmatic expression of BMP-7. Mononuclear stromal cells of brown tumor also express BMP-7. Within areas of bone resorption, there is a positive immunohistochemical reaction in cytoplasms of osteoblasts and bone matrix around osteocytes. Accordingly, we report another bone and soft tissue lesion with BMP-7 expression.
THE BIOLOGICAL RELEVANCE OF PTH FRAGMENTS: CORRELATION WITH BONE TURNOVER MARKERS IN PATIENTS WITH IMPAIRED RENAL FUNCTION
A. Fahrleitner-Pammer1*, G. Wirnsberger2, P. Krisper2, H. Holzer2, G. Leb1, B. Obermayer-Pietsch1, H. Dobnig1
1Division of Endocrinology, Department of Internal Medicine, Karl-Franzens University, Graz, Austria
2Division of Nephrology, Department of Internal Medicine, Karl-Franzens University, Graz, Austria
In patients with chronic renal failure PTH levels are commonly used to identify different subtypes of renal osteodystrophy, although several 2nd generation PTH assays do not measure exclusively the full length biological active PTH (1-84) but also inactive (7-84) fragments. There is evidence that in patients with renal impairment 7- 84 PTH fragments accumulate, leading to an overestimation of bone turnover with consecutive overtreatment possibly resulting in adynamic bone disease. Aim of the current study was to compare the results of a 2nd (iPTH [7-84]; Nichols Diagnostics) and 3rd generation assay ( duoPTH [CAP & CIP]/CAP[1-84]/CIP[7-84]; Scantibodies Laboratory) in relationship to bone turnover markers.
We investigated 109 patients with impaired renal function who received no bone active medication. Blood samples were analysed for serum chemistry and bone markers (sCTX, TRAP 5b, bALP, osteocalcin iPTH, duoPTH, CAP, CIP). Cyclase Activating PTH measuring only the 1-84 PTH is the biologically active PTH whereas Cyclase Inactive PTH (7-84) binds competetively to the PTH-receptor and may inhibit bone resorption. Calculation of the CAP/CIP ratio was performed and related to the different bone remodelling stages.
IntactPTH, duoPTH, CAP and CIP showed a strong correlation with each other (r- values 0.78-0.88) and serum creatinine (r-values 0.55-0.63). sCTX and osteocalcin increased with diminishing kidney function whereas TRAP 5b and bALP were not related to serum creatinine levels and showed a strong correlation with the CAP/CIP ratio (TRAP 5b: r=0.48; bALP: r=0.59) as well as CAP (TRAP 5b:r=0.38; bALP: r=0.31). These results were consistent after adjustment for renal function. Osteocalcin and sCTX were not related to CAP/CIP ratio but to CAP and in contrast to the kidney- independent markers also to iPTH, CIP and duoPTH.
At least in patients with renal impairment 1-84 PTH (CAP) differs significantly from iPTH and duoPTH levels. The CAP/CIP ratio correlates well to TRAP 5b and bALP, whereas sCTX and osteocalcin show a strong dependence on renal function. 3rd generation PTH assays distinguishing between 1-84 PTH (CAP) and 7-84 PTH (CIP) seem to be helpful in identifying different subtypes of renal osteodystrophy. TRAP 5b and bALP in combination with the CAP/CIP ratio could serve as potential surrogate-markers for bone turnover if our results can be confirmed by histomorphometric analyses.
FAMILIAL HYPERCALCEMIA HYPOCALCIURIA SYNDROME (FHH): NEW INACTIVATING MUTATION (DELGATT) AT EXON 3 OF CASR GENE IN AN ITALIAN FAMILY
A. Falchetti*, F. Del Monte, F. Marini, F. Franceschelli, V. Ghinoi, L. Masi, M. L. Brandi
Department of Internal Medicine, University of Florence, Florence, Italy
FHH syndrome (OMIM 145980) is characterized by hypercalcemia, inappropriately normal PTH circulating levels and inadequate low urinary excretion of calcium respect to the serum calcium values. It is inherited with autosomal dominant manner. Inactivating point mutations of extracellular domain of CASR gene, on chromosome 3q13.3-q21, are mainly responsible for the above-described metabolic abnormalities. CASR protein is particularly expressed at the parathyroid and the renal level, belonging to the superfamily of G-protein coupled receptors and regulating both PTH secretion, according to the extracellular variations of calcium, and urinary excretion of calcium itself. Differential diagnosis with moderate forms of primary hyperparathyroidism (pHPT) is not always easy. Thus, patients affected by FHH are sometimes erroneously reported as pHPT subjects and consequently addressed to surgeon to undergo useless parathyroidectomy unresolving the hypercalcemic state. Recently, a 31 years old male patient has been referred to Our Center for a counseling with a diagnosis of pHPT. Several evaluations of calcium-phosphorous metabolism exhibited hypercalcemia (10.6-11.2 mg/dl), phosphoremia within the normal range, magnesium at the upper limits of range (2.38-2.45 mg/dl), inadequately normal PTH levels (55, 62 pg/ml) and urinary excretion calcium levels within 134 and 252 mg/24 hours. Suspecting an FHH case we performed mutational analysis of CASR gene on DNA obtained from peripheral blood sample, by DNA sequencing (ABI Prism 310). Such analysis revealed the deletion of 4 bp (GATT) in exon 3, with consequent 'frame- shift' of the DNA reading code determining the presence of an immediate downstream stop codon. The mutation has been also confirmed by polyacrilamide gel electrophoresis of the specific PCR product by evidencing a heterozygous state (2 bands) in the affected individual when compared to the homozygosity of exon 3 wild- type control DNA (1 band). The same deletion has been subsequently identified also in constitutive DNA of 'asymptomatic' mother and brother, actually under clinical investigation. This represents the first deletion of exon 3 of CASR gene reported up to date in FHH patients and it confirms the importance of genetic analysis that can strongly help clinician in doubtful cases of hypercalcemia, especially in young people, avoiding a useless surgery in affected patients.
HYPOXIA IS A MAJOR STIMULATOR OF OSTEOCLAST FORMATION FROM HUMAN PERIPHERAL BLOOD
T. R. Arnett1*, H. Massey1, J. C. Utting1, I. R. Orriss1, A. M. Flanagan2
1Department of Anatomy & Developmental Biology, University College London, London, UK
2Department of Histopathology, University College London, London, UK
Because hypoxia increases osteoclast (OC) formation in mouse marrow cultures, we wished to determine whether this phenomenon might be relevant to human pathophysiological bone loss, eg in fractures, tumours, inflammation and ischaemia. Human PBMCs were cultured on 5mm ivory discs (200,000 cells/disc) for 7 or 14d in MEM with 15% FCS, 20 ng/ml M-CSF and 0 - 30 ng/ml RANKL. After overnight seeding in 96-multiwells, discs were transferred to 25 cm2 flasks gassed with 2% or 20% O2 (plus 5% CO2; balance N2); 1 ml medium / disc was used to ensure excess buffering capacity. Flasks were re-gassed daily and medium was acidified to pH 7.0
with 10 mmol/l HCl for the final 2d to activate resorption pit formation by OC generated. Medium pH, pO2 and pCO2 were monitored by blood gas analyser. Discs were immunostained to visualise OC using anti-vitronectin receptor MAb 23C6. The area covered by 23C6-positive OC on discs was measured by automated analysis of scanned images of ivory discs and the area covered by resorption pits was determined by dot counting morphometry using reflected light microscopy. OC areas in 14d / 20% O2 cultures with 0, 1, 10 & 30 ng/ml RANKL were 0.00±0.00, 0.07±0.00, 0.10±0.03 & 0.09±0.02 mm2, respectively; corresponding pit areas were 0.0±00.00, 0.02±0.01, 0.03±0.01 & 0.04±0.02 mm2. However, for 14d cultures in 2% O2, OC areas were 0.18±0.05, 1.70±0.33, 2.88±0.53 & 3.17±0.10 mm2, respectively; corresponding pit areas were 0.02±0.04, 0.49±0.12, 0.83±0.15 & 1.05±0.28 mm2; values are means ± SEM (n=6). Medium pH was unaltered by O2 level. Stimulation of OC number and pit formation were also elicited by 2% O2 after 7d culture, albeit at levels that were ~10% of those observed in 14d cultures. Overall, hypoxia caused ~25-30-fold upregulation of OC and pit formation. The effect of hypoxia was to increase resorption by increasing formation, rather than activity of OC, in agreement with results from mouse marrow experiments. However, in vivo (or when in vitro buffering is limited), hypoxia causes tissue acidosis, which will increase OC resorptive activity. These findings indicate a mechanism for the control of bone resorption of general importance.
TREATMENT OF A CONGENITAL PSEUDARTHROSIS OF THE TIBIA BY RECOMBINANT BONE MORPHOGENETIC PROTEIN-7 (OP-1 DEVICE)
D. Anticevic1*, M. Jelic1,2, S. Vukicevic2
1Department of Orthopaedic Surgery, School of Medicine, University of Zagreb, Croatia
2Department of Anatomy, School of Medicine, University of Zagreb, Croatia
Three and a half year old boy with Type II (Crawford) CPT and antero-lateral angulation for 20 deg of the left leg was initially surgically treated in another institution with resection the Ilizarov external frame. He never walked until the age of four. Subsequently, he had additional twelve surgeries including corticotomy, intramedullary nailing with an ankle transfixation and several autogenous iliac crest spongioplasties. He first came at our Clinic at the age of 13. He could never walk without crutches and had an orthoprothesis due to six centimeters shortening of the lower leg. Radiographs revealed a hyperthrophic non-union with an antero-lateral angulation. Fibula was hypoplastic and deficient in the middle segment. The ankle joint was positioned at 25 deg of valgus.
At surgery, the OP-1 device was carefully placed on the contact area between both tibial ends adding also autogenous cancellous bone. The tibial fragments and the foot were fixed with the Ilizarov device and at the proximal tibial third corticotomy was done. Two weeks following surgery weight bearing was initiated. Gradual elongation of the proximal part of the tibia at rate of 1 mm/day was continued. Eight months later, the Ilizarov device was removed.
Three weeks following surgery a significant amount of new bone was radiographically observed. Five months following surgery the non-union was completely healed. The Ilizarov device was kept in place for additional few months. One year following surgery both lower limbs were of equal length with corrected valgus ankle deformity. The patient was protected with bivalved splint and fully weight bearing, using one crutch while out-door walking.
In conclusion, the time to complete the tibial union was five months. In a single surgical procedure congenital tibial pseuadrthrosis healing, equalization of lower leg length and the correction of the ankle valgus was obtained. Therefore, in patients with CPT adjuvant local application of OP-1 (BMP-7) could significantly stimulate the tibial bone union and fully restore a lower limb weight-bearing.
This is the first child with a long standing CPT and multiple unsuccessful surgeries treated with a recombinant bone morphogenetic protein to initiate bone regeneration and repair.
SEVERE PARAPLEGIA CAUSED BY PAGET'S DISEASE SUCCESSFULLY TREATED WITH ALENDRONATE - CASE REPORT, FOLLOW UP OF FIVE YEARS
D. Krpan1*, J. Buljan Culej2, D. Dogan1, K. Orsolic1, Z. Lajtman3
1General Hospital Sv. Duh, University Medical School, Zagreb, Croatia
2Department of Anatomy, School of Medicine, University of Zagreb, Zagreb, Croatia
3General Hospital Merkur, University Medical School, Zagreb, Croatia
We report a clinical case of unusual clinical and radiological features of Paget's disease in a 42-year old, previously healthy caucasian women who suffered of severe paraplegia. Clinical symptoms appeared suddenly as a parastesia and weakening of the legs and developed to complete paraplegia during next two months. Extended diagnostic evaluation subsequently done, demonstrated serum alkaline phosphatase levels 50% higher then normal (145 mmol/l) gamma globulin values slightly higher and other biochemical parameters in the normal range. NMR of the vertebrae showed compression myelopathy of vertebral bodies in region th XII caused by the proliferation of vertebral body. X-ray of frontal bones, vertebrae and the proximal femur showed hyper mineralization and demineralization zones. Whole-body skeletal scintigraphy was atypical, and was not of diagnostic value. Ascendant lumbar myelography showed stop of the contrast flow in the region of th XII and L I. Diagnostic evaluation including repeatable bone biopsies and pathology assessment did not prove neoplastic disease. Since diagnostic evaluation did not explain the cause of the disease, no therapy was applied and the patient was completely immobilized during a year and half, before she came to our clinic, where, transiliacal bone biopsy and histomorphometry has been performed and confirmed the diagnosis of Paget's disease.
ZOLEDRONATE TREATMENT IN A PATIENT WITH SEVERE OSTEOGENESIS IMPERFECTA
J. Popovic*, J. D. Jacobson
Children's Mercy Hospital/University of Missouri/Kansas City School of Medicine, USA
Osteogenesis Imperfecta (OI), or brittle bone disease, is an inherited connective tissue disorder. It is characterized by osteopenia, fractures, progressive deformity, loss of mobility and chronic bone pain. Osteogenesis Imperfecta is caused by quantitative or qualitative defect in collagen synthesis. Children with severe forms suffer recurrent fractures resulting in limb and spine deformities and restricted ambulation. Recent experiences with the bisphosphonate group of drugs indicate that anti-resorptive therapy may reduce fracture frequency, increase bone density, promote remodeling of the bones, reduce chronic pain and improve mobility in affected individuals. Intravenous cyclical Pamidronate has been proven to be very effective and beneficial therapy for the individuals with OI.
Zoledronate, third generation bisphosphonate is new more potent drug in this class. It is more convenient and easier to administer and more effective in inhibiting skeletal morbidity.
We present 19 3/12 year old female with severe OI, bone pain, fragility and a history of frequent multiple fractures. She was diagnosed with OI at birth. She was lost for follow up for few years. The family was not aware of the benefits of bisphosphonate therapy in individuals with OI. Her height (74 cm) was average for the 1 year and weight (15.7 kg) for 4.5 years old respectively. She achieved menarche at age 17 years and had irregular menstrual cycles. The N-telopetide (NTX), osteocalcin and alkaline phosphatase levels were elevated indicating high bone turnover and resorption. Total body DEXA scan revealed Z score of -5.0 SD.
She received two Zoledronate infusions and immediately reported less bone pain and decreased rate of fractures. She had no significant changes in serum calcium levels or side effects related to Zoledronate therapy. The NTX level decreased 5-fold after the first Zoledronate infusion. Oral Alendronate therapy was introduced after the first Zoledronate infusion. She reports no additional fractures and only occasional bone pain. In addition, growth hormone therapy was prescribed to improve the height in a view of delayed bone age.
Therapeutic effects and tolerance to Zoledronate therapy were excellent. Controlled trials are needed to assure long-term safety and efficacy of Zoledronate therapy in patients with OI.
HEMANGIOPERICYTOMA OF THE FOOT- CASE REPORT
M. E. Domzalski*, J. Fabis
Medical University in Lodz, Poland
Hemangiopericytoma is extremly rare in the bone. This tumour seems particularly occur in the long bones. Since only few cases have been described up to now in the literature, we decided to present an unique localisations of such a tumour - the foot.
Case report :
26 years old female admited to the hospital due to deformation and pain of the left foot which started 3 years ago. After X- ray and MRI examination she was scheduled for surgical biopsy and the sample for histological examination was collected. histological results showed malignant form of the hemangiopericytoma. Subsequent diagnosis was composed of ultrasound of the involved area , scyntigraphic examination of the skeleton and CT of the lungs. Even though the first results suggested malignant but limited tumour, in the scyntigraphic examination a new metastatic focus was found in the distal epiphysis of tibia. Patient was scheduled for the amputation above that region with subsequent radiotherapy. Within 1 year follow- up we did not find the evidence of new metastasis. We realise, however, the course and prognosis of this disease are unpredictible and some cases metastasized, even many years after treatment. Therefore very careful follow- up examination is neccessary and final assessment is possible only after many years.
DETERMINANTS OF BONE MINERAL DENSITY IN PATIENTS ON DIALYSIS. A CROSS-SECTIONAL AND LONGITUDINAL STUDY
M. Aarup1*, C. Ejersted1, A. P. Hermann2, B. Jespersen1, K. Brixen2
1Department of Nephrology
2Department of Endocrinology, Odense University Hospital, Denmark
Chronic hemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD) may be complicated with renal osteodystrophy and fractures. The aim of the present study was to compare BMD and bone loss in patients on HD or CAPD.
In clinical routine, DXA scans are offered to all patients in the dialysis unit. Thus, data on 146 patients on HD and 28 on CAPD were available. Information on renal disease, diabetes, dialysis, previous renal allo-transplantation, cumulative predinisolon dosage, HRT or P-pills, tobacco consumption, and biochemistry were extracted from the patient files. Data from 303 healthy controls were used for comparison. DXA of the lumbar spine and femoral neck was performed using a Hologic-QDR 2000 scanner. Serum alkaline phosphatase (AP), parathyroid hormone (PTH), ionized calcium, and phosphate were measured by standard methods.
BMD of the spine (Z-score) was not significantly reduced in the patients while BMD of the femoral neck was significantly reduced (-0.51 ± 1.37, p<0.001). No difference in BMD of the spine or femoral neck or any of the biochemical parameters was found between patients on HD and CAPD. In multiple regression analysis, age at initiation of dialysis (R=0.38, p<0.001), body weight (R=0.39, p<0.001), and sex (R=0.15, p=0.07) were associated with BMD-spine. Similarly, age at initiation of dialysis (R=0.36, p<0.001), body weight (R=0.57, p<0.001), and sex (R=0.15, p=0.08) were associated with BMD-neck. In contrast, duration of dialysis, diabetes, cumulative prednisolon dosage, or smoking did not predict BMD.
In 66 patients on HD and 7 on CAPD, follow-up DXA was performed after 0.7-1.9 years. A significant decrease in BMD-spine (-1.2 ± 4.7 %/year, p<0.05) and BMD- neck (-2.3 ± 6.3 /year, p<0.01) was seen in patients on dialysis even after correcting for the normal age-related bone loss. Only serum PTH (R=-0.38) and AP (R=-0.35) predicted the rate of bone loss significantly (p<0.05).
We conclude, that patients on both HD and CAPD have reduced femoral neck BMD. Moreover, the age at initiation of dialysis seems to be an important predictor for BMD. In patients on dialysis, secondary hyperparathyroidism predicts bone loss.