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Immunodeficient rats have more bone and are better protected from castration or ovariectomy induced bone loss than normal rats
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Abstracts / Bone 44 (2009) S253–S338
P187 Osteoclast activity is regulated by the extracellular matrix protein fibronectin eliciting different signaling pathways A. Gramouna,*, D.P. Trebecb, N. Azizia, J. Sodeka, M.F. Manolsona a Faculty of Dentistry b Department of Biochemistry, University of Toronto, Toronto, Canada Osteoclasts (OCs), the bone resorbing multinucleated cells, are formed by fusion of mononuclear precursors. The ECM proteins, vitronectin (VN), fibronectin (FN) and osteopontin (OPN), all implicated in arthritis, interact with OCs through the surface receptors known as integrins. Aim: To determine the effects of VN, FN and OPN on OC formation and resorptive activity. Methods: RAW 264.7 cells were plated and differentiated (using 100 ng/ml RANKL) on dishes or Osteologic™ slides precoated with 0.01–20 μg/ml FN, VN, and OPN; concentrations shown not to affect cell proliferation. Results: After 96 h of differentiation, OC number on VN and FN remained significantly lower than those on OPN and the uncoated controls. The number of mononuclear TRAP+ cells (preOCs) at 48 h and large OCs (>10 nuclei) at 96 h showed that while FN did not affect preOC number, it significantly decreased OC multinucleation compared to the uncoated control. OPN had the opposite effect. When 20 μg/ml of FN was added to cultures plated on OPN on day 0 or on day 2 of differentiation, OC number was reduced by 40% indicating that FN was able to inhibit OPN's stimulatory effect on OC formation by preventing preOC fusion. Resorption studies revealed that both FN and OPN increased the total area of resorption and resorptive activity/OC by 40% compared to VN and the uncoated control groups. Further, FN increased the released TRAP5b/OC in cultures. Sealing zone formation (assessed by counting the number of actin rings) was 70% higher on FN than on any of the other experimental groups. Nitric oxide (NO) levels (as measured by Griess reagent) were significantly elevated by FN but not in any of the other groups. This increase in NO levels was associated with an increase in interleukin 1β (IL1β). IL1β levels were reduced in the presence of the NO-synthase inhibitor lNG-monomethyl Arginine in a dose dependant manner. Conclusions: Despite its inhibitory effect on preOC fusion and OC formation, FN significantly enhanced resorption/OC and due to an increase of the activity of the formed OCs. FN's induction of NO and IL1β, could both explain the increased OC activity. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.613
P188 Immunodeficient rats have more bone and are better protected from castration or ovariectomy induced bone loss than normal rats C.M. Bagi⁎, E.R. Berryman, M.R. Moalli, C.J. Andresen Global Science and Technology, Pfizer Inc, Groton, USA Introduction. Athymic “nude” mice and rats are commonly used for human tissue engrafted preclinical research models. Similar to athymic mice, athymic or “nude” rats lack a normal thymus and are largely deficient in functionally mature T cells. The interaction of osteoblasts and osteoclasts with cells of the immune system has been suggested as a contributing factor in the development of osteoporoses. Recent evidence from studies in animal models suggests that T cells play a significant role in bone loss following estrogen deficiency. The goal of this study was to investigate whether the lack of functional T cells in nude rodents prevents bone loss induced by castration (Ctx) or ovariectomy (Ovx). Methods. A pilot study was done to establish the immune phenotype of immunodeficient or “nude” (NIH:rnu) and non-immu-
nodeficient or “normal” Sprague-Dawley (CD) rats using flow cytometry analysis of lymphocyte subsets. The main study included the following age-matched study groups: Taconic NIH:rnu female sham, female Ovx, male sham and male Ctx; CD female sham, female OVX, male sham, male Ctx. The CD rats were selected as a positive control for hormone ablation induced bone loss since these changes are well documented in this strain. Bone mass and structure was assessed with micro-CT and serum biomarkers of bone metabolism at various time points throughout the course of the 12 week study. Results. Flow cytometry data demonstrated that normal CD rats have significantly higher values for NKT, T, CD8+, CD4+ and CD25+ cells than nude rats while the nude rats have higher B and NK cell values than the normal CD rats. Both male and female sham nude rats have higher trabecular number compared to sham CD. Immunodeficient sham female rats have higher trabecular number and BMD relative to female sham CD rats. Ctx or Ovx caused only moderate bone loss in immunodeficient rats. Conclusion. Our data provide additional evidence that sex steroids regulate bone metabolism through T cell function, and that lack of functional T cells protects skeleton from bone loss induced by Ctx or Ovx. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.614
P189 Prognostic potential of precise molecular diagnosis of autosomal recessive osteopetrosis C. Sobacchia,*, M.M. Guerrinia, B. Cassanib, A. Pangrazioa, A. Frattinic, P. Vezzonia, A. Villaa a Human Genome, CNR-ITB b Human Genome, IRCCS Istituto Clinico Humanitas, Rozzano c Human Genome, CNR-ITB, Segrate, Italy Autosomal Recessive Osteopetrosis (ARO) is a rare genetic bone disease in which a deficit in bone resorption by osteoclasts leads to increased bone density, fragile bones susceptible to fractures, reduced marrow space, anemia, hepatosplenomegaly, cranial nerves compression, blindness and/or deafness. The disease is often lethal within the first decade of life unless treated with Hematopoietic Stem Cell Transplantation (HSCT) from an HLA-matched donor, which should be performed as soon as possible in order to prevent the establishment of irreversible lesions. In the last years the identification of several genes responsible for ARO in man (TCIRG1, ClCN7, OSTM1, PLEKHM1, RANKL and RANK) has clearly shown that this disease is genetically heterogeneous. In our large cohort of ARO patients, the evaluation of molecular and clinical data has led to the establishment of genotype-phenotype correlations and has highlighted the presence of two subsets of patients which do not benefit from HSCT: those bearing mutations in either ClCN7 or OSTM1, whose severe neurological defects often lead to death despite HSCT; and those bearing mutations in RANKL, which is expressed by osteoblasts, i.e. cells of mesenchymal, and not hematopoietic origin. On the other hand, patients with mutations in either TCIRG1 or RANK can rescue their bone defect after transplant engraftment, while it is likely that HSCT would not be appropriate to treat PLEKHM1-dependent patients due to their mild phenotype. These data demonstrate that a precise and early molecular diagnosis of ARO patients can help clinicians to identify and establish the proper therapeutic approach for a better prognosis. Moreover, in order to improve the clinical management of patients, new cellular approaches have to be considered. We have already shown that in utero transplantation (IUT) of HSC in oc/oc mice can greatly improve the quality of life and the survival of transplanted animals, suggesting that a similar
Abstracts / Bone 44 (2009) S253–S338
P187 Osteoclast activity is regulated by the extracellular matrix protein fibronectin eliciting different signaling pathways A. Gramouna,*, D.P. Trebecb, N. Azizia, J. Sodeka, M.F. Manolsona a Faculty of Dentistry b Department of Biochemistry, University of Toronto, Toronto, Canada Osteoclasts (OCs), the bone resorbing multinucleated cells, are formed by fusion of mononuclear precursors. The ECM proteins, vitronectin (VN), fibronectin (FN) and osteopontin (OPN), all implicated in arthritis, interact with OCs through the surface receptors known as integrins. Aim: To determine the effects of VN, FN and OPN on OC formation and resorptive activity. Methods: RAW 264.7 cells were plated and differentiated (using 100 ng/ml RANKL) on dishes or Osteologic™ slides precoated with 0.01–20 μg/ml FN, VN, and OPN; concentrations shown not to affect cell proliferation. Results: After 96 h of differentiation, OC number on VN and FN remained significantly lower than those on OPN and the uncoated controls. The number of mononuclear TRAP+ cells (preOCs) at 48 h and large OCs (>10 nuclei) at 96 h showed that while FN did not affect preOC number, it significantly decreased OC multinucleation compared to the uncoated control. OPN had the opposite effect. When 20 μg/ml of FN was added to cultures plated on OPN on day 0 or on day 2 of differentiation, OC number was reduced by 40% indicating that FN was able to inhibit OPN's stimulatory effect on OC formation by preventing preOC fusion. Resorption studies revealed that both FN and OPN increased the total area of resorption and resorptive activity/OC by 40% compared to VN and the uncoated control groups. Further, FN increased the released TRAP5b/OC in cultures. Sealing zone formation (assessed by counting the number of actin rings) was 70% higher on FN than on any of the other experimental groups. Nitric oxide (NO) levels (as measured by Griess reagent) were significantly elevated by FN but not in any of the other groups. This increase in NO levels was associated with an increase in interleukin 1β (IL1β). IL1β levels were reduced in the presence of the NO-synthase inhibitor lNG-monomethyl Arginine in a dose dependant manner. Conclusions: Despite its inhibitory effect on preOC fusion and OC formation, FN significantly enhanced resorption/OC and due to an increase of the activity of the formed OCs. FN's induction of NO and IL1β, could both explain the increased OC activity. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.613
P188 Immunodeficient rats have more bone and are better protected from castration or ovariectomy induced bone loss than normal rats C.M. Bagi⁎, E.R. Berryman, M.R. Moalli, C.J. Andresen Global Science and Technology, Pfizer Inc, Groton, USA Introduction. Athymic “nude” mice and rats are commonly used for human tissue engrafted preclinical research models. Similar to athymic mice, athymic or “nude” rats lack a normal thymus and are largely deficient in functionally mature T cells. The interaction of osteoblasts and osteoclasts with cells of the immune system has been suggested as a contributing factor in the development of osteoporoses. Recent evidence from studies in animal models suggests that T cells play a significant role in bone loss following estrogen deficiency. The goal of this study was to investigate whether the lack of functional T cells in nude rodents prevents bone loss induced by castration (Ctx) or ovariectomy (Ovx). Methods. A pilot study was done to establish the immune phenotype of immunodeficient or “nude” (NIH:rnu) and non-immu-
nodeficient or “normal” Sprague-Dawley (CD) rats using flow cytometry analysis of lymphocyte subsets. The main study included the following age-matched study groups: Taconic NIH:rnu female sham, female Ovx, male sham and male Ctx; CD female sham, female OVX, male sham, male Ctx. The CD rats were selected as a positive control for hormone ablation induced bone loss since these changes are well documented in this strain. Bone mass and structure was assessed with micro-CT and serum biomarkers of bone metabolism at various time points throughout the course of the 12 week study. Results. Flow cytometry data demonstrated that normal CD rats have significantly higher values for NKT, T, CD8+, CD4+ and CD25+ cells than nude rats while the nude rats have higher B and NK cell values than the normal CD rats. Both male and female sham nude rats have higher trabecular number compared to sham CD. Immunodeficient sham female rats have higher trabecular number and BMD relative to female sham CD rats. Ctx or Ovx caused only moderate bone loss in immunodeficient rats. Conclusion. Our data provide additional evidence that sex steroids regulate bone metabolism through T cell function, and that lack of functional T cells protects skeleton from bone loss induced by Ctx or Ovx. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.614
P189 Prognostic potential of precise molecular diagnosis of autosomal recessive osteopetrosis C. Sobacchia,*, M.M. Guerrinia, B. Cassanib, A. Pangrazioa, A. Frattinic, P. Vezzonia, A. Villaa a Human Genome, CNR-ITB b Human Genome, IRCCS Istituto Clinico Humanitas, Rozzano c Human Genome, CNR-ITB, Segrate, Italy Autosomal Recessive Osteopetrosis (ARO) is a rare genetic bone disease in which a deficit in bone resorption by osteoclasts leads to increased bone density, fragile bones susceptible to fractures, reduced marrow space, anemia, hepatosplenomegaly, cranial nerves compression, blindness and/or deafness. The disease is often lethal within the first decade of life unless treated with Hematopoietic Stem Cell Transplantation (HSCT) from an HLA-matched donor, which should be performed as soon as possible in order to prevent the establishment of irreversible lesions. In the last years the identification of several genes responsible for ARO in man (TCIRG1, ClCN7, OSTM1, PLEKHM1, RANKL and RANK) has clearly shown that this disease is genetically heterogeneous. In our large cohort of ARO patients, the evaluation of molecular and clinical data has led to the establishment of genotype-phenotype correlations and has highlighted the presence of two subsets of patients which do not benefit from HSCT: those bearing mutations in either ClCN7 or OSTM1, whose severe neurological defects often lead to death despite HSCT; and those bearing mutations in RANKL, which is expressed by osteoblasts, i.e. cells of mesenchymal, and not hematopoietic origin. On the other hand, patients with mutations in either TCIRG1 or RANK can rescue their bone defect after transplant engraftment, while it is likely that HSCT would not be appropriate to treat PLEKHM1-dependent patients due to their mild phenotype. These data demonstrate that a precise and early molecular diagnosis of ARO patients can help clinicians to identify and establish the proper therapeutic approach for a better prognosis. Moreover, in order to improve the clinical management of patients, new cellular approaches have to be considered. We have already shown that in utero transplantation (IUT) of HSC in oc/oc mice can greatly improve the quality of life and the survival of transplanted animals, suggesting that a similar