Generalized Degenerative Joint Disease in Osteoprotegerin (Opg) Null Mutant Mice.

Bone structure is modulated by the interaction between receptor activator of nuclear factor-κB (RANK) and RANK ligand (RANKL). Osteoprotegerin (OPG), a decoy receptor for RANKL, modifies osteoclast-mediated bone resorption directly and spares articular cartilage indirectly in rodents with immune-mediated arthritis by preventing subchondral bone destruction. The OPG/RANKL balance also seems to be critical in maintaining joint integrity in osteoarthritis, a condition featuring articular bone and cartilage damage in the absence of profound inflammation. The current study explored the role of OPG in sparing articular cartilage by evaluating joint lesions in adult C57BL/6J mice lacking osteoprotegerin (Opg (-) (/-)). At 3, 5, 7, 9, and 12 months of age, both sexes of Opg (-) (/-) mice developed severe degenerative joint disease (DJD) characterized by progressive loss of cartilage matrix and eventually articular cartilage. Lesions developed earlier and more severely in Opg (-) (/-) mice relative to age-matched, wild-type (Opg (+) (/+)), or heterozygous (Opg (+) (/-)) littermates (P ≤ .05). The femorotibial joint was affected bilaterally at 3 months, while other key weight-bearing diarthrodial joints (eg, coxofemoral, scapulohumeral, humeroradioulnar) were affected later and unilaterally. Cortical bone in subchondral plates and long bone diaphyses of Opg (-) (/-) mice but not Opg (+/+) or Opg (+) (/-) animals was osteoporotic by 3 months of age (P ≤ .05); the extent of porosity was less than the degree of DJD. Closure of the physes in long bones (P ≤ .05) and cartilage retention in the femoral primary spongiosa (P ≤ .05) affected chiefly Opg (-) (/-) mice. These data suggest that OPG plays an essential direct role in maintaining cartilage integrity in the articular surfaces and physes.

Enhanced mineralization potential of vascular cells from SM22α-Rankl (tg) mice.

Vascular calcification, prevalent in diabetes and chronic kidney disease, contributes to morbidity and mortality. To investigate the effect of receptor activator of NF-kB ligand (RANKL) on vascular calcification in vivo, transgenic mice, where RANKL expression was targeted to vascular smooth muscle cells using the SM22α promoter (SM22α-Rankl ( tg )), were created. Sixteen-month-old male SM22α-Rankl ( tg ) mice had higher body weight and higher serum calcium levels but lower lumbar bone mineral density (BMD) compared with age- and gender-matched wild-type (WT) littermates. BMD of long bones, body fat (percent of weight) of the leg, and serum levels of phosphate and RANKL were not significantly different. No significant differences in these parameters were observed in female mice. Histological analysis did not reveal calcium deposits in the aortic roots of SM22α-Rankl ( tg ) mice. To analyze the osteoblastic differentiation and mineralization potentials of vascular cells, aortic smooth muscle cells (SMCs) were isolated and cultured. Results showed that SM22α-Rankl ( tg ) SMCs had higher baseline alkaline phosphatase (ALP) activity but not baseline matrix calcification. When induced by the PKA agonist forskolin, ALP activity was greater in SM22α-Rankl ( tg ) than in WT SMCs. Real-time RT-qPCR revealed higher baseline expression of ALP and ankylosis genes but lower osteoprotegerin gene in SM22α-Rankl ( tg ) SMCs. Matrix mineralization induced by inorganic phosphate or forskolin was greater in SM22α-Rankl ( tg ) than in WT SMCs. Treatment of these cells with the ALP inhibitor levamisole abolished forskolin-induced matrix mineralization but not inorganic phosphate-induced matrix mineralization. These findings suggest that RANKL overexpression in the vasculature may promote mineralization potential.

Soluble RANKL induces high bone turnover and decreases bone volume, density, and strength in mice.

Receptor activator for nuclear factor-kappa B ligand (RANKL) is an essential mediator of osteoclastogenesis. We hypothesized that administration of soluble RANKL to mice would result in high turnover and deleterious effects on both cortical and trabecular bone. For 10 days, 10-week-old C57BL/6J female mice (n = 12/group) were given twice-daily subcutaneous injections of human recombinant RANKL (0.4 or 2 mg/kg/day) or inert vehicle (VEH). Bone turnover was greatly accelerated by RANKL, as evidenced by the 49-84% greater levels of serum TRAP-5b (bone resorption marker) and 300-400% greater levels of serum alkaline phosphatase (bone formation marker). RANKL resulted in significantly greater endocortical bone erosion surface (79-83%) and periosteal bone formation rate (64-87%) vs. VEH. Microcomputed tomographic (microCT) analysis of the proximal tibia indicated a reduction in trabecular volume fraction (-84%) for both doses of RANKL. Cortical bone geometry and strength were also negatively influenced by RANKL. MicroCT analysis of the femoral diaphysis indicated significantly lower cortical bone volume (-10% to -13%) and greater cortical porosity (8-9%) relative to VEH. Biomechanical testing of the femur diaphysis revealed significantly lower maximum bending load (-19% to -25%) vs. VEH. Bone strength remained correlated with bone mass, independent of RANKL stimulation of bone turnover. These findings are consistent with the hypothesis that soluble RANKL could be an important etiologic factor in pathologic bone loss. RANKL also has potential utility as a model for studying the consequences of high bone turnover on bone quality and strength in animals.

Skeletal deterioration induced by RANKL infusion: a model for high-turnover bone disease.

UNLABELLED: RANKL was administered continuously to rats for 28 days to investigate its potential as a disease model for the skeletal system. Bone turnover rates, bone material, structural and mechanical properties were evaluated. RANKL infusion caused overall skeletal complications comparable to those in high bone-turnover conditions, such as postmenopausal osteoporosis. INTRODUCTION: RANKL is an essential mediator for osteoclast development. No study has examined in detail the direct skeletal consequences of excess RANKL on bone turnover, mineralization, architecture, and vascular calcification. We, therefore, administrated soluble RANKL continuously into mature rats and created a bone-loss model. METHODS: Six-month-old Sprague-Dawley (SD) rats were assigned to three groups (n = 12) receiving continuous administration of saline (VEH) or human RANKL (35 microg/kg/day, LOW or 175 microg/kg/day, HI) for 28 days. Blood was collected routinely during the study. At sacrifice, hind limbs and aorta were removed and samples were analyzed. RESULTS: High dose RANKL markedly stimulated serum osteocalcin and TRAP-5b levels and reduced femur cortical bone volume (-7.6%) and trabecular volume fraction (BV/TV) at the proximal tibia (-64% vs. VEH). Bone quality was significantly degraded in HI, as evidenced by decreased femoral percent mineralization, trabecular connectivity, and increased endocortical bone resorption perimeters. Both cortical and trabecular bone mechanical properties were reduced by high dose RANKL. No differences were observed in the mineral content of the abdominal aorta. CONCLUSIONS: Continuous RANKL infusion caused general detrimental effects on rat skeleton. These changes are comparable to those commonly observed in high-turnover bone diseases such as postmenopausal osteoporosis.

The receptor activator of nuclear factor-kappaB ligand inhibitor osteoprotegerin is a bone-protective agent in a rat model of chronic renal insufficiency and hyperparathyroidism.

Osteoprotegerin (OPG) acts by neutralizing the receptor activator of nuclear factor-kappaB ligand (RANKL), the primary mediator of osteoclast differentiation, function, and survival. We examined whether OPG could affect the bone loss associated with chronic kidney disease (CKD) in a rodent model of CKD and secondary hyperparathyroidism (SHPT). SHPT was induced in rats by 5/6 nephrectomy (5/6 Nx) and a 1.2% P/0.6% Ca(2+) diet. Starting 1 week after 5/6 Nx, rats were treated with vehicle (veh) or OPG-Fc (3 mg/kg, intravenously) every 2 weeks for 9 weeks. At baseline, 3, 6, and 9 weeks, blood was taken and bone mineral density (BMD) and bone mineral content (BMC) were assessed by dual-energy X-ray absorptiometry. Serum parathyroid hormone (sPTH) levels reached 912 pg/ml in 5/6 Nx rats vs. 97 pg/ml in shams at 9 weeks. OPG-Fc had no effect on sPTH or Ca(2+) levels throughout the 9-week study, indicating that SHPT was a renal effect independent of bone changes. At 3 weeks, 5/6 Nx-veh rats had osteopenia compared with sham-veh rats and 5/6 Nx-OPG-Fc rats had significantly higher percent changes in whole-body BMC, leg BMD, and lumbar BMD versus 5/6 Nx-veh rats. By 6-9 weeks, elevated sPTH was associated with reversal of bone loss and osteitis fibrosa in the proximal tibial metaphysis. OPG-Fc decreased this sPTH-driven high bone turnover, resulting in augmented thickness of proximal tibial trabeculae in 5/6 Nx rats. Thus, RANKL inhibition with OPG-Fc can block the deleterious effects of continuously elevated sPTH on bone, suggesting that RANKL may be an important therapeutic target for protecting bone in patients with CKD and SHPT.

CD4+CD45RBHi T cell transfer induced colitis in mice is accompanied by osteopenia which is treatable with recombinant human osteoprotegerin.

BACKGROUND AND AIMS: Transfer of CD4+CD45RBHi T cells into semi syngeneic immunodeficient mice represents a model of inflammatory bowel disease (IBD). As patients with IBD often suffer from osteopenia, we studied if this T cell transfer in mice results in osteopenia in addition to colitis, and if treatment with osteoprotegerin (OPG) has effects on the bone mineral density of T cell transferred mice. We also investigated whether osteopenia was due to malabsorption as a result of a dysregulated digestive tract or as a consequence of the inflammatory process. METHODS: CD4+CD45RBHi or CD4+CD45RBLo T cells (4 x 10(5)) were sorted from CB6F1 and transferred into C.B.17 scid/scid mice. Recipient mice were treated with human IgG1 Fc (control) or Fc-OPG three times per week in a prophylactic regimen as well as a therapeutic regimen (after 10% body weight loss) and were evaluated for osteopenia and colitis. RESULTS: Mice that received CD4+CD45RBHi T cells developed osteopenia (as indicated by decreased bone density accompanied by decreased osteoblasts and increased osteoclasts) and colitis (as indicated by histological changes in the large intestine). Mice that received CD4+CD45RBLo T cells developed neither osteopenia nor colitis. All animals consumed, on average, the same amount of food and water over the course of the study. Prophylactic treatment with Fc-OPG increased bone density in mice that received either CD4+CD45RBHi or CD4+CD45RBLo T cells but had no effects on the gastrointestinal tract. Fc-OPG treatment of osteopenic mice with established IBD caused the normalisation of bone density. Osteopenia in CD4+CD45RBHi T cell recipients was accompanied by hypoparathyroidism that was partially normalised by treatment with Fc-OPG. CD4+CD45RBHi T cell recipients also had a bone marrow inflammatory cell infiltrate expressing tumour necrosis factor alpha which was unaffected by treatment with Fc-OPG. CONCLUSIONS: CD4+CD45RBHi T cell transfer results in osteopenia in addition to colitis. Evidence suggests that this osteopenia was induced by inflammatory cell infiltration and not by malabsorption of calcium. Recombinant human osteoprotegerin effectively treated the osteopenia. OPG may be a useful therapeutic option for treating osteopenia in patients with IBD.

Osteoclast numbers in Lewis rats with adjuvant-induced arthritis: identification of preferred sites and parameters for rapid quantitative analysis.

This study defined the best site for quantifying osteoclasts in male Lewis rats with mycobacteria-induced adjuvant arthritis. Hind paw sections of normal and arthritic rats (n = 6 per group) taken 7 days after disease onset were stained for osteoclasts using an anti-human cathepsin K primary antibody. Erosions and osteoclasts were assessed using semiquantitative scores (entire section) and quantitative measures (in calcaneus, navicular tarsal, and tibia). Bone area in arthritic rats was significantly reduced (P

Skeletal muscle adaptations to microgravity exposure in the mouse.

To investigate the effects of microgravity on murine skeletal muscle fiber size, muscle contractile protein, and enzymatic activity, female C57BL/6J mice, aged 64 days, were divided into animal enclosure module (AEM) ground control and spaceflight (SF) treatment groups. SF animals were flown on the space shuttle Endeavour (STS-108/UF-1) and subjected to approximately 11 days and 19 h of microgravity. Immunohistochemical analysis of muscle fiber cross-sectional area revealed that, in each of the muscles analyzed, mean muscle fiber cross-sectional area was significantly reduced (P < 0.0001) for all fiber types for SF vs. AEM control. In the soleus, immunohistochemical analysis of myosin heavy chain (MHC) isoform expression revealed a significant increase in the percentage of muscle fibers expressing MHC IIx and MHC IIb (P < 0.05). For the gastrocnemius and plantaris, no significant changes in MHC isoform expression were observed. For the muscles analyzed, no alterations in MHC I or MHC IIa protein expression were observed. Enzymatic analysis of the gastrocnemius revealed a significant decrease in citrate synthase activity in SF vs. AEM control.

OPG and PTH-(1-34) have additive effects on bone density and mechanical strength in osteopenic ovariectomized rats.

PTH is a potent bone anabolic factor, and its combination with antiresorptive agents has been proposed as a therapy for osteoporosis. We tested the effects of PTH, alone and in combination with the novel antiresorptive agent OPG, in a rat model of severe osteopenia. Sprague Dawley rats were sham-operated or ovariectomized at 3 months of age. Rats were untreated for 15 months, at which time ovariectomy had caused significant decreases in bone mineral density in the lumbar vertebrae and femur. Rats were then treated for 5.5 months with vehicle (PBS), human PTH-(1-34) (80 microg/kg), rat OPG (10 mg/kg), or OPG plus PTH (all three times per wk, sc). Treatment of ovariectomized rats with OPG or PTH alone increased bone mineral density in the lumbar vertebrae and femur, whereas PTH plus OPG caused significantly greater and more rapid increases than either therapy alone (P < 0.05). OPG significantly reduced osteoclast surface in the lumbar vertebrae and femur (P < 0.05 vs. sham or ovariectomized), but had no effect on osteoblast surface at either site. Ovariectomy significantly decreased the mechanical strength of the lumbar vertebrae and femur. In the lumbar vertebrae, OPG plus PTH was significantly more effective than PTH alone at reversing ovariectomy-induced deficits in stiffness and elastic modulus. These data suggest that OPG plus PTH represent a potentially useful therapeutic option for patients with severe osteoporosis.

Osteoprotegerin inhibits osteolysis and decreases skeletal tumor burden in syngeneic and nude mouse models of experimental bone metastasis.

Certain malignancies, including breast cancer, frequently metastasize to bone, where the tumor cells induce osteoclasts to locally destroy bone. Osteoprotegerin (OPG), a member of the tumor necrosis factor receptor family, is a negative regulator of osteoclast differentiation, activation, and survival. We tested the ability of recombinant OPG to inhibit tumor-induced osteoclastogenesis, osteolysis, and skeletal tumor burden in two animal models. In a syngeneic model, mouse colon adenocarcinoma (Colon-26) cells were injected into the left ventricle of mice. Treatment with OPG dose-dependently decreased the number and area of radiographically evident lytic bone lesions, which, at the highest dose, were undetectable. Histologically, OPG also decreased skeletal tumor burden and tumor-associated osteoclasts. In a nude mouse model, OPG treatment completely prevented radiographic osteolytic lesions caused by human MDA-MB-231 breast cancer cells. Histologically, OPG decreased skeletal tumor burden by 75% and completely eradicated MDA tumor-associated osteoclasts. In both models, OPG had no effect on metastatic tumor burden in a panel of soft tissue organs. These data indicate that OPG may be an effective therapy for preventing osteolysis and decreasing skeletal tumor burden in patients with bone metastasis.

Adenoviral delivery of osteoprotegerin ameliorates bone resorption in a mouse ovariectomy model of osteoporosis.

Osteoprotegerin (OPG) regulates bone resorption by inhibiting osteoclast formation, function, and survival. The current studies employed a mouse ovariectomy (OVX) model of estrogen deficiency to investigate gene therapy with OPG as a means of preventing osteoporosis. Young adult females injected with a recombinant adenoviral (Ad) vector carrying cDNA of either full-length OPG or a fusion protein combining the hOPG ligand-binding domain with the human immunoglobulin constant domain (Ad-hOPG-Fc) developed serum OPG concentrations exceeding the threshold needed for efficacy. However, elevated circulating OPG levels were sustained for up to 18 months only in mice given Ad-hOPG-Fc. Administration of Ad-hOPG-Fc titers between 10(7) and 10(9) pfu yielded dose-dependent increases in serum OPG. Mice subjected to OVX or sham surgery followed by immediate treatment with Ad-hOPG-Fc had significantly more bone volume with reduced osteoclast numbers in axial and appendicular bones after 4 weeks. In contrast, animals given OVX and either a control vector or vehicle had significantly less bone than did comparably treated sham-operated mice. This study demonstrates that a single adenoviral gene transfer can produce persistent high-level OPG expression and shows that gene therapy to provide sustained delivery of OPG may prove useful in treating osteoporosis.

Osteoprotegerin reverses osteoporosis by inhibiting endosteal osteoclasts and prevents vascular calcification by blocking a process resembling osteoclastogenesis.

High systemic levels of osteoprotegerin (OPG) in OPG transgenic mice cause osteopetrosis with normal tooth eruption and bone elongation and inhibit the development and activity of endosteal, but not periosteal, osteoclasts. We demonstrate that both intravenous injection of recombinant OPG protein and transgenic overexpression of OPG in OPG(-/-) mice effectively rescue the osteoporotic bone phenotype observed in OPG-deficient mice. However, intravenous injection of recombinant OPG over a 4-wk period could not reverse the arterial calcification observed in OPG(-/-) mice. In contrast, transgenic OPG delivered from mid-gestation through adulthood does prevent the formation of arterial calcification in OPG(-/-) mice. Although OPG is normally expressed in arteries, OPG ligand (OPGL) and receptor activator of NF-kappaB (RANK) are not detected in the arterial walls of wild-type adult mice. Interestingly, OPGL and RANK transcripts are detected in the calcified arteries of OPG(-/-) mice. Furthermore, RANK transcript expression coincides with the presence of multinuclear osteoclast-like cells. These findings indicate that the OPG/OPGL/RANK signaling pathway may play an important role in both pathological and physiological calcification processes. Such findings may also explain the observed high clinical incidence of vascular calcification in the osteoporotic patient population.

RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism.

We have generated RANK (receptor activator of NF-kappaB) nullizygous mice to determine the molecular genetic interactions between osteoprotegerin, osteoprotegerin ligand, and RANK during bone resorption and remodeling processes. RANK(-/-) mice lack osteoclasts and have a profound defect in bone resorption and remodeling and in the development of the cartilaginous growth plates of endochondral bone. The osteopetrosis observed in these mice can be reversed by transplantation of bone marrow from rag1(-/-) (recombinase activating gene 1) mice, indicating that RANK(-/-) mice have an intrinsic defect in osteoclast function. Calciotropic hormones and proresorptive cytokines that are known to induce bone resorption in mice and human were administered to RANK(-/-) mice without inducing hypercalcemia, although tumor necrosis factor alpha treatment leads to the rare appearance of osteoclast-like cells near the site of injection. Osteoclastogenesis can be initiated in RANK(-/-) mice by transfer of the RANK cDNA back into hematopoietic precursors, suggesting a means to critically evaluate RANK structural features required for bone resorption. Together these data indicate that RANK is the intrinsic cell surface determinant that mediates osteoprotegerin ligand effects on bone resorption and remodeling as well as the physiological and pathological effects of calciotropic hormones and proresorptive cytokines.

Osteoprotegerin prevents and reverses hypercalcemia in a murine model of humoral hypercalcemia of malignancy.

Osteoprotegerin (OPG), a novel, secreted tumor necrosis factor receptor family member that inhibits osteoclast formation and activity was examined for its activity in a syngeneic tumor model of humoral hypercalcemia of malignancy. Normal mice bearing Colon-26 tumors develop increases in both parathyroid hormone-related protein (PTHrP) expression and plasma PTHrP, marked hypercalcemia, and increased bone resorption. OPG, given either at the onset of hypercalcemia or after it had occurred, blocked tumor-induced increases in bone resorption and hypercalcemia and rapidly normalized blood ionized calcium. In tumor-bearing mice, OPG treatments reduced osteoclast activity from approximately 2-fold above normal into the subphysiological range but had no effects on tumor size, tumor-induced cachexia, or PTHrP levels. The potent effects of OPG in this humoral hypercalcemia of malignancy model suggest a potential therapeutic role for OPG in the prevention and treatment of this disorder.

Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand.

Bone remodelling and bone loss are controlled by a balance between the tumour necrosis factor family molecule osteoprotegerin ligand (OPGL) and its decoy receptor osteoprotegerin (OPG). In addition, OPGL regulates lymph node organogenesis, lymphocyte development and interactions between T cells and dendritic cells in the immune system. The OPGL receptor, RANK, is expressed on chondrocytes, osteoclast precursors and mature osteoclasts. OPGL expression in T cells is induced by antigen receptor engagement, which suggests that activated T cells may influence bone metabolism through OPGL and RANK. Here we report that activated T cells can directly trigger osteoclastogenesis through OPGL. Systemic activation of T cells in vivo leads to an OPGL-mediated increase in osteoclastogenesis and bone loss. In a T-cell-dependent model of rat adjuvant arthritis characterized by severe joint inflammation, bone and cartilage destruction and crippling, blocking of OPGL through osteoprotegerin treatment at the onset of disease prevents bone and cartilage destruction but not inflammation. These results show that both systemic and local T-cell activation can lead to OPGL production and subsequent bone loss, and they provide a novel paradigm for T cells as regulators of bone physiology.

A chimeric form of osteoprotegerin inhibits hypercalcemia and bone resorption induced by IL-1beta, TNF-alpha, PTH, PTHrP, and 1, 25(OH)2D3.

Osteoprotegerin (OPG) is a secreted protein that inhibits osteoclast formation and activity and appears to be a critical regulator of bone mass and metabolism. In the current study, mice were challenged with various cytokines and hormones (interleukin-1beta, tumor necrosis factor-alpha, parathyroid hormone, parathyroid hormone-related protein, and 1alpha,25-dihydroxyvitamin D3) that are known to increase bone resorption and cause hypercalcemia and treated concurrently with either a recombinant chimeric Fc fusion form of human OPG, with enhanced biological activity (cOPG) (2.5 mg/kg/day) or vehicle. Mice receiving these bone-resorbing factors became hypercalcemic by day 3 after commencing treatment and had increased bone resorption as evidenced by elevated osteoclast numbers on day 5. Concurrent cOPG treatment prevented hypercalcemia (p < 0.05) and maintained osteoclast numbers in the normal range (p < 0.001). The demonstration that cOPG can inhibit bone resorption suggests that this molecule may be useful in the treatment of diseases including hyperparathyroidism, humoral hypercalcemia of malignancy, osteoporosis, and inflammatory bone disease, which are characterized, in part, by increases in osteoclastic bone resorption.

TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling.

Bone resorption and remodeling is an intricately controlled, physiological process that requires the function of osteoclasts. The processes governing both the differentiation and activation of osteoclasts involve signals induced by osteoprotegerin ligand (OPGL), a member of tumor necrosis factor (TNF) superfamily, and its cognate receptor RANK. The molecular mechanisms of the intracellular signal transduction remain to be elucidated. Here we report that mice deficient in TNF receptor-associated factor 6 (TRAF6) are osteopetrotic with defects in bone remodeling and tooth eruption due to impaired osteoclast function. Using in vitro assays, we demonstrate that TRAF6 is crucial not only in IL-1 and CD40 signaling but also, surprisingly, in LPS signaling. Furthermore, like TRAF2 and TRAF3, TRAF6 is essential for perinatal and postnatal survival. These findings establish unexpectedly diverse and critical roles for TRAF6 in perinatal and postnatal survival, bone metabolism, LPS, and cytokine signaling.

Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand.

A receptor that mediates osteoprotegerin ligand (OPGL)-induced osteoclast differentiation and activation has been identified via genomic analysis of a primary osteoclast precursor cell cDNA library and is identical to the tumor necrosis factor receptor (TNFR) family member RANK. The RANK mRNA was highly expressed by isolated bone marrow-derived osteoclast progenitors and by mature osteoclasts in vivo. Recombinant OPGL binds specifically to RANK expressed by transfected cell lines and purified osteoclast progenitors. Transgenic mice expressing a soluble RANK-Fc fusion protein have severe osteopetrosis because of a reduction in osteoclasts, similar to OPG transgenic mice. Recombinant RANK-Fc binds with high affinity to OPGL in vitro and blocks osteoclast differentiation and activation in vitro and in vivo. Furthermore, polyclonal Ab against the RANK extracellular domain promotes osteoclastogenesis in bone marrow cultures suggesting that RANK activation mediates the effects of OPGL on the osteoclast pathway. These data indicate that OPGL-induced osteoclastogenesis is directly mediated through RANK on osteoclast precursor cells.

OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis.

The tumour-necrosis-factor-family molecule osteoprotegerin ligand (OPGL; also known as TRANCE, RANKL and ODF) has been identified as a potential osteoclast differentiation factor and regulator of interactions between T cells and dendritic cells in vitro. Mice with a disrupted opgl gene show severe osteopetrosis and a defect in tooth eruption, and completely lack osteoclasts as a result of an inability of osteoblasts to support osteoclastogenesis. Although dendritic cells appear normal, opgl-deficient mice exhibit defects in early differentiation of T and B lymphocytes. Surprisingly, opgl-deficient mice lack all lymph nodes but have normal splenic structure and Peyer's patches. Thus OPGL is a new regulator of lymph-node organogenesis and lymphocyte development and is an essential osteoclast differentiation factor in vivo.

SPECT abnormalities in Landau-Kleffner syndrome.

Five right-handed children with acquired aphasia elipepsy syndrome (Landau-Kleffner, LKS), were investigated with 99(m)TcHMPAO single photon emission computed tomography (SPECT) and the results were correlated with their EEGs and clinical history. The childrens' ages ranged from 2 to 5 years and the aphasia had been present for 6 to over 12 months. No clinical seizure had ever been onserved in the younger two children and their waking EEGs showed infrequent central spikes. Both children had areas of low intensity on SPECT, involving the left temporal lobe in one and the right temporal lobe in the other, which has also been reported in children with congenital dysphasia who have normal EEGs. The three older children presented with frequent generalized seizures, with the aphasia occurring 3-6 months later. The SPECT scans in these children were performed either in the ictal state, or when electrographic seizure activity was very frequent on EEG. All three children had hyperintense foci on SPECT involving the left posterior temporal region corresponding to Wernickes area. We conclude that LKS may be initially a unilateral seizure disorder of Wernickes area, with EEG discharges in the contralateral hemisphere representing propagation from the unilateral focus. Copyright 1999 Harcourt Publishers Ltd.