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OBJECTIVE: To investigate the kinetics of bony spur formation and the relationship of bony spur formation to synovial inflammation and bone erosion in 2 rat arthritis models, and to address whether bony spur formation depends on the expression of tumor necrosis factor alpha (TNFalpha) or RANKL. METHODS: Analysis of the kinetics of synovial inflammation, bone erosion, osteoclast formation, and growth of bony spurs was performed in rat collagen-induced arthritis (CIA) and adjuvant-induced arthritis (AIA). In addition, inhibition experiments were performed to assess whether inhibition of TNFalpha and RANKL by pegylated soluble TNF receptor type I (pegTNFRI) and osteoprotegerin (OPG), respectively, affected bony spur formation. RESULTS: Bony spurs emerged from the periosteal surface close to joints, and initial proliferation of mesenchymal cells was noted as early as 3 days and 5 days after onset of CIA and AIA, respectively. Initiation of bony spur formation occurred shortly after the onset of inflammation and bone erosion. Neither pegTNFRI nor OPG could significantly halt the osteophytic responses in CIA and AIA. CONCLUSION: These results suggest that bony spur formation is triggered by inflammation and initial structural damage in these rat models of inflammatory arthritis. Moreover, emergence of bony spurs depends on periosteal proliferation and is not affected by inhibition of either TNFalpha or RANKL. Bony spur formation can thus be considered a process that occurs independent of TNFalpha and RANKL and is triggered by destructive arthritis. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To investigate whether the bone-preserving effects of a RANKL antagonist or a tumor necrosis factor (TNF) antagonist could be further improved by the addition of a bone anabolic agent in inflammatory arthritis. METHODS: Lewis rats with either adjuvant-induced arthritis (AIA) or collagen-induced arthritis (CIA) were treated for 10 days with PEGylated soluble tumor necrosis factor receptor type I (PEG sTNFRI), interleukin-1 receptor antagonist (IL-1Ra), osteoprotegerin (OPG), parathyroid hormone (PTH), or combinations of these agents starting on day 4 after disease onset. Treatment effects were assessed clinically, radiologically, and histologically, and by morphometry for the extent of paw swelling, bone erosive changes, and synovial inflammation. RESULTS: Paw swelling and synovial inflammation were significantly inhibited by PEG sTNFRI in AIA and CIA, and by IL-1Ra in CIA. OPG and PTH had no significant effect on these parameters. Analysis of bone erosion revealed a significant bone-sparing effect of monotherapy with PEG sTNFRI or OPG in both models, whereas IL-1Ra was only effective in CIA. PTH treatment alone did not show a bone-protective effect in either model. With the combination of PEG sTNFRI and PTH, erosion scores (-74% in AIA and -61% in CIA versus controls) were significantly lower than those elicited by PEG sTNFRI alone (-41% and -29%, respectively, versus controls). Similar results were also obtained with the combination of OPG and PTH (-88% in AIA and -73% in CIA, compared with -70% and -55%, respectively, with OPG monotherapy). Coadministration of IL-1Ra and PTH had no synergistic bone-sparing effect. Morphometric analysis revealed that the addition of PTH to PEG sTNFRI or OPG resulted in higher bone volume and higher osteoblast numbers in both AIA and CIA. CONCLUSION: The bone-protective effects resulting from RANKL or TNF antagonism can be further improved by the addition of a bone anabolic agent. [ABSTRACT FROM AUTHOR]

At present, in Russia every fifth desired pregnancy terminates prematurely; the main cause of pregnancy loss are sexually transmitted infections (STIs). The chielf culprit, human papillomavirus (HPV) infection, contributes to significant changes in immune response and development of autoimmune disorders, therefore the combined treatment of genital infections should include drugs of proven immunomodulating activity. The purpose of this study was to examine the efficacy of Roncoleukin (interleukin-2 recombinant human) in the combined treatment of urogenital infections in women with history of recurrent pregnancy loss (RPL) who were planning pregnancy. Materials and Methods: We observed 226 women (aged 19-39 years) with history of RPL and urogenital infection caused by bacterial-viral associations diagnosed on the basis of medical history, clinical, instrumental and laboratory investigations. The comprehensive therapy was directed at identified infectious agents, comprehensively. As immunomodulatory therapy, all patients received Roncoleukin (LLC Biotech, Russia). The study showed that the presence of HPV was detected in 83.5% of cases. According to clinical symptoms and colposcopy. There were detected single and multiple condylomata of the vulva, vagina and cervix (91.8%). The cytological examination of smears obtained from the cervical canal and the surfaces of cervix, vagina and vulva showed koilocytes (76.5%), dyskeratocytes (43.1%), cells with parakeratosis (53.1%), pronounced dystrophy (28.3%), manifestations of CIN I-III (33.4%); the PCR confirmed HPV presence in 98.8% of the cases. After 30 days of stage I of the full treatment course the control colposcopic monitoring showed reduction of condylomatosis foci in 23.7% of women, in 47.6% -- decrease of the white patches' area with complete absence of cells with signs of CIN at a control cytological examination. The control PCR detected HPV in less than half of the cases (34.3%). At stage II, in parallel with the irrigation of the cervical canal and the urethra with the Roncoleukin solution we conducted destruction of infection foci using radiowave surgery. In 88.9% of patients, another session of radiowave surgery was not required. The dynamic assessment of the colposcopic data showed that reparation occurred in the shortest possible time. Dynamic control cytological examination of the smear shows the normal levels of koilocytes and dyskeratocytes. There were no signs of cell degeneration. The advance dynamic PCR analysis for HPV showed 100.0% negative results. During the following year, 165 (73.0%) women achieved the desired pregnancy, which resulted in the births of healthy infants carried to term (average body weight 3300±350 g, average Apgar score of 8.9). The remaining patients are at the planning stage of pregnancy. We conclude that optimization of human papillomavirus genital infection treatment in women with recurrent miscarriages may require combined two-step therapy using Roncoleukin in the period of time prior to pregnancy planning. [ABSTRACT FROM AUTHOR]

Obesity and type 2 diabetes (T2D) remain major global healthcare challenges, and developing therapeutics necessitates using nonhuman primate models. Here, we present a transcriptomic and proteomic atlas of all the major organs of cynomolgus monkeys with spontaneous obesity or T2D in comparison to healthy controls. Molecular changes occur predominantly in the adipose tissues of individuals with obesity, while extensive expression perturbations among T2D individuals are observed in many tissues such as the liver and kidney. Immune-response-related pathways are upregulated in obesity and T2D, whereas metabolism and mitochondrial pathways are downregulated. Moreover, we highlight some potential therapeutic targets, including SLC2A1 and PCSK1 in obesity as well as SLC30A8 and SLC2A2 in T2D. Our study provides a resource for exploring the complex molecular mechanism of obesity and T2D and developing therapies for these diseases, with limitations including lack of hypothalamus, isolated islets of Langerhans, longitudinal data, and body fat percentage., Competing Interests: Declaration of interests All authors are current or former employees of Amgen., (Copyright © 2023. Published by Elsevier Inc.)

Background: Fracture repair involves the reactivation of developmental signaling cascades, including Wnt signaling that stimulates bone formation and bone regeneration. Rodent data indicate that dual inhibition of the Wnt signaling antagonists sclerostin and Dickkopf-1 (DKK1) increases callus bone volume and strength while increasing bone mass systemically., Methods: We evaluated the effects of 16 weeks of subcutaneously administered carrier solution (vehicle, VEH), anti-sclerostin antibody (Scl-Ab), anti-DKK1 antibody (DKK1-Ab), or Scl-Ab plus DKK1-Ab combination therapy (COMBO) on ulnar osteotomy healing in nonhuman primates (cynomolgus monkeys; 20 to 22 per group)., Results: Scl-Ab and COMBO therapy increased systemic markers of bone formation versus VEH, with COMBO leading to synergistic increases versus Scl-Ab or DKK1-Ab monotherapies. The COMBO and Scl-Ab groups showed reduced serum markers of bone resorption versus VEH. The COMBO and DKK1-Ab groups exhibited greater callus bone mineral density (BMD), torsional stiffness, and torsional rigidity versus VEH. Lumbar vertebrae from the Scl-Ab and COMBO groups showed greater BMD and bone formation rate versus VEH, and the femoral mid-diaphysis of the Scl-Ab and COMBO groups showed greater periosteal and endocortical bone formation rates versus VEH., Conclusions: DKK1-Ab increased BMD and strength at the ulnar osteotomy site, Scl-Ab increased bone formation and BMD at uninjured skeletal sites, and Scl-Ab plus DKK1-Ab combination therapy induced all of these effects, in some cases to a greater degree versus 1 or both monotherapies. These results in nonhuman primates suggest that DKK1 preferentially regulates bone healing while sclerostin preferentially regulates systemic bone mass., Clinical Relevance: Combination therapy with antibodies against sclerostin and DKK1 may offer a promising therapeutic strategy for both fracture treatment and fracture prevention., Competing Interests: Disclosure: The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article ( http://links.lww.com/JBJS/H512 )., (Copyright © 2023 by The Journal of Bone and Joint Surgery, Incorporated.)

Influenza remains a major cause of death and disability with limited treatment options. Studies of acute lung injury have identified angiopoietin-2 (Ang-2) as a key prognostic marker and a potential mediator of Acute respiratory distress syndrome. However, the role of Ang-2 in viral pneumonia remains poorly defined. This study characterized the time course of lung Ang-2 expression in severe influenza pneumonia and tested the therapeutic potential of Ang-2 inhibition. We inoculated adult mice with influenza A (PR8 strain) and measured angiopoietin-1 (Ang-1), Ang-2, and Tie2 expressions during the evolution of inflammatory lung injury over the first 7 days post-infection (dpi). We tested a peptide-antibody inhibitor of Ang-2, L1-7, administered at 2, 4, and 6 dpi and measured arterial oxygen saturation, survival, pulmonary edema, inflammatory cytokines, and viral load. Finally, we infected primary human alveolar type II epithelial (AT2) cells grown in air-liquid interface culture with influenza and measured Ang-2 RNA expression. Influenza caused severe lung injury between 5 and 7 dpi in association with increased Ang-2 lung RNA and a dramatic increase in Ang-2 protein in bronchoalveolar lavage. Inhibition of Ang-2 improved oxygenation and survival and reduced pulmonary edema and alveolar-capillary barrier permeability to protein without major effects on inflammation or viral load. Finally, influenza increased the expression of Ang-2 RNA in human AT2 cells. The increased Ang-2 levels in the airspaces during severe influenza pneumonia and the improvement in clinically relevant outcomes after Ang-2 antagonism suggest that the Ang-1/Ang-2 Tie-2 signaling axis is a promising therapeutic target in influenza and potentially other causes of viral pneumonia., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Heart failure with reduced ejection fraction (HFrEF) constitutes 50% of HF hospitalizations and is characterized by high rates of mortality. To explore the underlying mechanisms of HFrEF etiology and progression, we studied the molecular and cellular differences in four chambers of non-failing (NF, n = 10) and HFrEF (n = 12) human hearts. We identified 333 genes enriched within NF heart subregions and often associated with cardiovascular disease GWAS variants. Expression analysis of HFrEF tissues revealed extensive disease-associated transcriptional and signaling alterations in left atrium (LA) and left ventricle (LV). Common left heart HFrEF pathologies included mitochondrial dysfunction, cardiac hypertrophy and fibrosis. Oxidative stress and cardiac necrosis pathways were prominent within LV, whereas TGF-beta signaling was evident within LA. Cell type composition was estimated by deconvolution and revealed that HFrEF samples had smaller percentage of cardiomyocytes within the left heart, higher representation of fibroblasts within LA and perivascular cells within the left heart relative to NF samples. We identified essential modules associated with HFrEF pathology and linked transcriptome discoveries with human genetics findings. This study contributes to a growing body of knowledge describing chamber-specific transcriptomics and revealed genes and pathways that are associated with heart failure pathophysiology, which may aid in therapeutic target discovery.

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) regulate glucose and energy homeostasis. Targeting both pathways with GIP receptor (GIPR) antagonist antibody (GIPR-Ab) and GLP-1 receptor (GLP-1R) agonist, by generating GIPR-Ab/GLP-1 bispecific molecules, is an approach for treating obesity and its comorbidities. In mice and monkeys, these molecules reduce body weight (BW) and improve many metabolic parameters. BW loss is greater with GIPR-Ab/GLP-1 than with GIPR-Ab or a control antibody conjugate, suggesting synergistic effects. GIPR-Ab/GLP-1 also reduces the respiratory exchange ratio in DIO mice. Simultaneous receptor binding and rapid receptor internalization by GIPR-Ab/GLP-1 amplify endosomal cAMP production in recombinant cells expressing both receptors. This may explain the efficacy of the bispecific molecules. Overall, our GIPR-Ab/GLP-1 molecules promote BW loss, and they may be used for treating obesity., Competing Interests: The authors declare no competing interests. All authors are employees and shareholders of Amgen. A patent related to this work has been filed: International Application No. PCT/US18/13918., (© 2021 Amgen Inc.)

In search of more efficacious and safe pharmacological treatments for atrial fibrillation (AF), atria-selective antiarrhythmic agents have been promoted that target ion channels principally expressed in the atria. This concept allows one to engage antiarrhythmic effects in atria, but spares the ventricles from potentially proarrhythmic side effects. It has been suggested that cardiac small conductance Ca 2+ -activated K + (SK) channels may represent an atria-selective target in mammals including humans. However, there are conflicting data concerning the expression of SK channels in different stages of AF, and recent findings suggest that SK channels are upregulated in ventricular myocardium when patients develop heart failure. To address this issue, RNA-sequencing was performed to compare expression levels of three SK channels ( KCNN1 , KCNN2 , and KCNN3 ) in human atrial and ventricular tissue samples from transplant donor hearts (no cardiac disease), and patients with cardiac disease in sinus rhythm or with AF. In addition, for control purposes expression levels of several genes known to be either chamber-selective or differentially expressed in AF and heart failure were determined. In atria, as compared to ventricle from transplant donor hearts, we confirmed higher expression of KCNN1 and KCNA5 , and lower expression of KCNJ2 , whereas KCNN2 and KCNN3 were statistically not differentially expressed. Overall expression of KCNN1 was low compared to KCNN2 and KCNN3 . Comparing atrial tissue from patients with AF to sinus rhythm samples we saw downregulation of KCNN2 in AF, as previously reported. When comparing ventricular tissue from heart failure patients to non-diseased samples, we found significantly increased ventricular expression of KCNN3 in heart failure, as previously published. The other channels showed no significant difference in expression in either disease. Our results add weight to the view that SK channels are not likely to be an atria-selective target, especially in failing human hearts, and modulators of these channels may prove to have less utility in treating AF than hoped. Whether targeting SK1 holds potential remains to be elucidated., Competing Interests: TN, MS, and SR were employed by Amgen Inc., at the time of submission of the manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Darkow, Nguyen, Stolina, Kari, Schmidt, Wiedmann, Baczkó, Kohl, Rajamani, Ravens and Peyronnet.)

In 2003, we briefly reported the remarkable osteopathy of a 12-year-old boy who at age two months began fracturing his limbs with subsequent hyperplastic callus formation and expansion and fusion of appendicular bones. By age ten years he had coalesced his lumbosacral spine, pelvis, femurs, and leg and foot bones as a single structure. Computed tomography of expanded bone revealed a thin cortical shell, diminished irregular trabeculae, and cystic areas. Histopathology featured foci of woven bone, densely packed osteocytes, cartilage, fibrovascular tissue, and massive fat deposition in the marrow space lacking hematogenous precursor cells. Bone turnover markers indicated accelerated remodeling and the few radiographically assessable appendicular bones improved during brief adherence to alendronate therapy. Following puberty, serum multiplex biomarker profiling confirmed accelerated bone turnover. At age 23 years, macrospecimens from leg amputation revealed ossification along capsular tissue together with hyaline cartilage degeneration. Concurrently, the life-long course of this same disorder was delineated in an unrelated woman until her death at age 51 years. Both patients demonstrated the radiographic hallmarks and harbored the heterozygous point mutation (c.-14C>T) in the 5'-UTR of IFITM5 associated with osteogenesis imperfecta type V (OI-V). Herein, we detail the clinical, radiological, histopathological, biochemical, and molecular findings and discuss the etiology and pathogenesis of this extraordinary osteopathy that we call coalescing expansile skeletal disease., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Heart failure (HF) with preserved ejection fraction (HFpEF) constitutes half of all HF but lacks effective therapy. Understanding of its myocardial biology remains limited because of a paucity of heart tissue molecular analysis., Methods: We performed RNA sequencing on right ventricular septal endomyocardial biopsies prospectively obtained from patients meeting consensus criteria for HFpEF (n=41) contrasted with right ventricular septal tissue from patients with HF with reduced ejection fraction (HFrEF, n=30) and donor controls (n=24). Principal component analysis and hierarchical clustering tested for transcriptomic distinctiveness between groups, effect of comorbidities, and differential gene expression with pathway enrichment contrasted HF groups and donor controls. Within HFpEF, non-negative matrix factorization and weighted gene coexpression analysis identified molecular subgroups, and the resulting clusters were correlated with hemodynamic and clinical data., Results: Patients with HFpEF were more often women (59%), African American (68%), obese (median body mass index 41), and hypertensive (98%), with clinical HF characterized by 65% New York Heart Association Class III or IV, nearly all on a loop diuretic, and 70% with a HF hospitalization in the previous year. Principal component analysis separated HFpEF from HFrEF and donor controls with minimal overlap, and this persisted after adjusting for primary comorbidities: body mass index, sex, age, diabetes, and renal function. Hierarchical clustering confirmed group separation. Nearly half the significantly altered genes in HFpEF versus donor controls (1882 up, 2593 down) changed in the same direction in HFrEF; however, 5745 genes were uniquely altered between HF groups. Compared with controls, uniquely upregulated genes in HFpEF were enriched in mitochondrial adenosine triphosphate synthesis/electron transport, pathways downregulated in HFrEF. HFpEF-specific downregulated genes engaged endoplasmic reticulum stress, autophagy, and angiogenesis. Body mass index differences largely accounted for HFpEF upregulated genes, whereas neither this nor broader comorbidity adjustment altered pathways enriched in downregulated genes. Non-negative matrix factorization identified 3 HFpEF transcriptomic subgroups with distinctive pathways and clinical correlates, including a group closest to HFrEF with higher mortality, and a mostly female group with smaller hearts and proinflammatory signaling. These groupings remained after sex adjustment. Weighted gene coexpression analysis yielded analogous gene clusters and clinical groupings., Conclusions: HFpEF exhibits distinctive broad transcriptomic signatures and molecular subgroupings with particular clinical features and outcomes. The data reveal new signaling targets to consider for precision therapeutics.

Cardiometabolic syndrome has become a global health issue. Heart failure is a common comorbidity of cardiometabolic syndrome. Successful drug development to prevent cardiometabolic syndrome and associated comorbidities requires preclinical models predictive of human conditions. To characterize the heart failure component of cardiometabolic syndrome, cardiometabolic, metabolic, and renal biomarkers were evaluated in lean and obese ZSF1 19- to 32-week-old male rats. Histopathological assessment of kidneys and hearts was performed. Cardiac function, exercise capacity, and left ventricular gene expression were also analyzed. Obese ZSF1 rats exhibited multiple features of human cardiometabolic syndrome by pathological changes in systemic renal, metabolic, and cardiovascular disease circulating biomarkers. Hemodynamic assessment, echocardiography, and decreased exercise capacity confirmed heart failure with preserved ejection fraction. RNA-seq results demonstrated changes in left ventricular gene expression associated with fatty acid and branched chain amino acid metabolism, cardiomyopathy, cardiac hypertrophy, and heart failure. Twelve weeks of growth differentiation factor 15 (GDF15) treatment significantly decreased body weight, food intake, blood glucose, and triglycerides and improved exercise capacity in obese ZSF1 males. Systemic cardiovascular injury markers were significantly lower in GDF15-treated obese ZSF1 rats. Obese ZSF1 male rats represent a preclinical model for human cardiometabolic syndrome with established heart failure with preserved ejection fraction. GDF15 treatment mediated dietary response and demonstrated a cardioprotective effect in obese ZSF1 rats., Competing Interests: All authors are employees and stockholders of Amgen Inc., with no other competing interests to disclose. This relationship does not alter our adherence to PLOS ONE policies on sharing data and materials.

Romosozumab (EVENITY™ [romosozumab-aqqg in the US]) is a humanized monoclonal antibody that inhibits sclerostin and has been approved in several countries for the treatment of osteoporosis in postmenopausal women at high risk of fracture. Sclerostin is expressed in bone and aortic vascular smooth muscle (AVSM). Its function in AVSM is unclear but it has been proposed to inhibit vascular calcification, atheroprogression, and inflammation. An increased incidence of positively adjudicated serious cardiovascular adverse events driven by an increase in myocardial infarction and stroke was observed in romosozumab-treated subjects in a clinical trial comparing alendronate with romosozumab (ARCH; NCT01631214) but not in a placebo-controlled trial (FRAME; NCT01575834). To investigate the effects of sclerostin inhibition with sclerostin antibody on the cardiovascular system, a comprehensive nonclinical toxicology package with additional cardiovascular studies was conducted. Although pharmacodynamic effects were observed in the bone, there were no functional, morphological, or transcriptional effects on the cardiovascular system in animal models in the presence or absence of atherosclerosis. These nonclinical studies did not identify evidence that proves the association between sclerostin inhibition and adverse cardiovascular function, increased cardiovascular calcification, and atheroprogression., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: James R Turk, Marina Stolina, Denise Dwyer, Sheetal V Kumar, Emily M de Koning, Yusheng Qu, and Michael Engwall are employees and stockholders of Amgen Inc. AMD, JY, KL, LW, CG, YDH, and RWB are former Amgen employees.. Melanie Felx, Gabrielle Boyd, Jean-Guy Bienvenu, Aurore Varela, and Martin Guillot are employees of Charles River Laboratories Montreal ULC, which received funding from Amgen Inc., Astellas, and UCB Pharma to conduct this research. Gill Holdsworth and Alison Wolfreys are employees and stockholders of UCB Pharma., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Bruck syndrome (BRKS) is the rare disorder that features congenital joint contractures often with pterygia and subsequent fractures, also known as osteogenesis imperfecta (OI) type XI (OMIM # 610968). Its two forms, BRKS1 (OMIM # 259450) and BRKS2 (OMIM # 609220), reflect autosomal recessive (AR) inheritance of FKBP10 and PLOD2 loss-of-function mutations, respectively. A 10-year-old girl was referred with blue sclera, osteopenia, poorly-healing fragility fractures, Wormian skull bones, cleft soft palate, congenital fusion of cervical vertebrae, progressive scoliosis, bell-shaped thorax, restrictive and reactive pulmonary disease, protrusio acetabuli, short stature, and additional dysmorphic features without joint contractures. Iliac crest biopsy after alendronate treatment that improved her bone density revealed low trabecular connectivity, abundant patchy osteoid, and active bone formation with widely-spaced tetracycline labels. Chromosome 22q11 deletion analysis for velocardiofacial syndrome, COL1A1 and COL1A2 sequencing for prevalent types of OI, and Sanger sequencing of LRP5, PPIB, FKBP10, and IFITM5 for rare pediatric osteoporoses were negative. Copy number microarray excluded a contiguous gene syndrome. Instead, exome sequencing revealed two missense variants in PLOD2 which encodes procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (lysyl hydroxylase 2, LH2); exon 8, c.797G>T, p.Gly266Val (paternal), and exon 12, c.1280A>G, p.Asn427Ser (maternal). In the Exome Aggregation Consortium (ExAC) database, low frequency (Gly266Val, 0.0000419) and absence (Asn427Ser) implicated both variants as mutations of PLOD2. The father, mother, and sister (who carried the exon 12 defect) were reportedly well with normal parental DXA findings. BRKS2, characterized by under-hydroxylation of type I collagen telopeptides compromising their crosslinking, has been reported in at least 16 probands/families. Most PLOD2 mutations involve exons 17-19 (of 20 total) encoding the C-terminal domain with LH activity. However, truncating defects (nonsense, frameshift, splice site mutations) are also found throughout PLOD2. In three reports, AR PLOD2 mutations are not associated with congenital contractures. Our patient's missense defects lie within the central domain of unknown function of PLOD2. In our patient, compound heterozygosity with PLOD2 mutations is associated with a clinical phenotype distinctive from classic BRKS2 indicating that when COL1A1 and COL1A2 mutation testing is negative for OI without congenital contractures or pterygia, atypical BRKS should be considered., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Antiresorptive agents, such as bisphosphonates and denosumab, are frequently used for the management of osteoporosis. Indeed, both medications decrease the risk of osteoporotic fractures; however, these medications are associated with rare but potentially severe side effects, such as osteonecrosis of the jaw (ONJ). ONJ, defined as an area of exposed bone in the maxillofacial region that lasts for 8 weeks, often presents with significant pain and infection and can lead to serious complications. Interestingly, other treatments for osteoporosis have been developed, such as antibodies against the osteocyte-secreted protein, sclerostin. Sclerostin functions to inhibit the Wnt signaling cascade, leading to inhibition of bone formation. In clinical trials, a sclerostin antibody (romosozumab, Amgen Inc., UCB Brussels) increases bone formation and lowers the risk of osteoporotic fractures. However, in conjunction with increased osteoblastic activity, a reduction in bone resorption markers is observed. This antiresorptive effect raises the concern of possible ONJ development in patients treated with sclerostin antibodies. Here, utilizing ligature-induced experimental periodontitis (EP), we evaluated the effects of sclerostin inhibition on the development of ONJ-like lesions in ovariectomized rats. Beginning 8 weeks post-ovariectomy, rats were treated for 22 weeks with weekly injections of vehicle (Veh), 200 μg/kg zoledronic acid (ZA), a potent bisphosphonate at 100-fold the osteoporosis dose, or 5 mg/kg sclerostin antibody (Scl-Ab) at the osteoporotic dose. EP was initiated at week 12 and maintained for the remainder of the study. Scl-Ab treatment transiently increased serum P1NP, a bone formation marker, increased BV/TV, and decreased eroded surfaces in lumbar vertebrae. ZA-treated rats developed histologic features of ONJ, whereas Veh-treated controls did not. Scl-Ab animals lost less periodontal bone in sites with EP. However, these animals presented with no histologic signs of ONJ. In conclusion, sclerostin inhibition enhanced structural bone parameters, without inducing ONJ-like lesions, in ovariectomized rats with EP. © 2018 American Society for Bone and Mineral Research., (© 2018 American Society for Bone and Mineral Research.)

Gnathodiaphyseal dysplasia (GDD; OMIM #166260) is an ultra-rare autosomal dominant disorder caused by heterozygous mutation in the anoctamin 5 (ANO5) gene and features fibro-osseous lesions of the jawbones, bone fragility with recurrent fractures, and bowing/sclerosis of tubular bones. The physiologic role of ANO5 is unknown. We report a 5-year-old boy with a seemingly atypical and especially severe presentation of GDD and unique ANO5 mutation. Severe osteopenia was associated with prenatal femoral fractures, recurrent postnatal fractures, and progressive bilateral enlargement of his maxilla and mandible beginning at ~2months-of-age that interfered with feeding and speech and required four debulking operations. Histopathological analysis revealed benign fibro-osseous lesions resembling cemento-ossifying fibromas of the jaw without psammomatoid bodies. A novel, de novo, heterozygous, missense mutation was identified in exon 15 of ANO5 (c.1553G>A; p.Gly518Glu). Our findings broaden the phenotypic and molecular spectra of GDD. Fractures early in life with progressive facial swelling are key features. We assessed his response to a total of 7 pamidronate infusions commencing at age 15months. Additional reports must further elucidate the phenotype, explore any genotype-phenotype correlation, and evaluate treatments., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Etelcalcetide, a novel peptide agonist of the calcium-sensing receptor, prevents vascular calcification in a rat model of renal insufficiency with secondary hyperparathyroidism. Vascular calcification occurs frequently in patients with chronic kidney disease (CKD) and is a consequence of impaired mineral homeostasis and secondary hyperparathyroidism (SHPT). Etelcalcetide substantially lowers parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) levels in SHPT patients on hemodialysis. This study compared the effects of etelcalcetide and paricalcitol on vascular calcification in rats with adenine-induced CKD and SHPT. Uremia and SHPT were induced in male Wistar rats fed a diet supplemented with 0.75% adenine for 4 weeks. Rats were injected with vehicle, etelcalcetide, or paricalcitol for 4 weeks from the beginning of adenine diet. Rats fed an adenine-free diet were included as nonuremic controls. Similar reductions in plasma PTH and parathyroid chief cell proliferation were observed in both etelcalcetide- and paricalcitol-treated rats. Serum calcium and phosphorus were significantly lower in etelcalcetide-treated uremic rats and was unchanged in paricalcitol-treated rats. Both serum FGF23 and aortic calcium content were significantly lower in etelcalcetide-treated uremic rats compared with either vehicle- or paricalcitol-treated uremic rats. The degree of aortic calcium content for etelcalcetide-treated rats was similar to that in nonuremic controls and corroborated findings of lack of histologic aortic mineralization in those groups. In conclusion, etelcalcetide and paricalcitol similarly attenuated progression of SHPT in an adenine rat model of CKD. However, etelcalcetide differentially prevented vascular calcification, at least in part, due to reductions in serum FGF23, calcium, and phosphorus levels.

Sustained elevation of parathyroid hormone (PTH) is catabolic to cortical bone, as evidenced by deterioration in bone structure (cortical porosity), and is a major factor for increased fracture risk in chronic kidney disease (CKD). Etelcalcetide (AMG 416), a novel peptide agonist of the calcium-sensing receptor, reduces PTH levels in subtotal nephrectomized (Nx) rats and in hemodialysis patients with secondary hyperparathyroidism (SHPT) in clinical studies; however, effects of etelcalcetide on bone have not been determined. In a rat model of established SHPT with renal osteodystrophy, etelcalcetide or vehicle was administered by subcutaneous (s.c.) injection to subtotal Nx rats with elevated PTH (>750pg/mL) once per day for 6weeks. Sham-operated rats receiving vehicle (s.c.) served as non-SHPT controls. Prior to treatment, significant increases in serum creatinine (2-fold), blood urea nitrogen (BUN, 3-fold), PTH (5-fold), fibroblast growth factor-23 (FGF23; 13-fold) and osteocalcin (12-fold) were observed in SHPT rats compared to non-SHPT controls. Elevations in serum creatinine and BUN were unaffected by treatment with vehicle or etelcalcetide. In contrast, etelcalcetide significantly decreased PTH, FGF23 and osteocalcin, whereas vehicle treatment did not. Cortical bone porosity increased and bone strength decreased in vehicle-treated SHPT rats compared to non-SHPT controls. Cortical bone structure improved and energy to failure was significantly greater in SHPT rats treated with etelcalcetide compared to vehicle. Mineralization lag time and marrow fibrosis were significantly reduced by etelcalcetide. In conclusion, etelcalcetide reduced bone turnover, attenuated mineralization defect and marrow fibrosis, and preserved cortical bone structure and bone strength by lowering PTH in subtotal Nx rats with established SHPT., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Results of prior studies suggest that fibroblast growth factor 21 (FGF21) may be involved in bone turnover and in the actions of peroxisome proliferator-activated receptor (PPAR) α and γ in mice. We have conducted independent studies to examine the effects of FGF21 on bone homeostasis and the role of FGF21 in PPARα and γ actions. High-fat-diet-induced obesity (DIO) mice were administered vehicle or recombinant human FGF21 (rhFGF21) intraperitoneally at 0 (vehicle), 0.1, 1, and 3 mg/kg daily for 2 weeks. Additional groups of DIO mice received water or 10 mg/kg rosiglitazone daily. Mice treated with rhFGF21 or rosiglitazone showed expected metabolic improvements in glucose, insulin, and lipid levels. However, bone loss was not detected in rhFGF21-treated mice by dual-energy X-ray absorptiometry (DXA), micro-CT, and histomorphometric analyses. Mineral apposition rate, a key bone formation parameter, was unchanged by rhFGF21, while significantly decreased by rosiglitazone in DIO mice. Bone resorption markers, OPG/RANKL mRNA expression, and histological bone resorption indices were unchanged by rhFGF21 or rosiglitazone. Bone marrow fat was unchanged by rhFGF21, while increased by rosiglitazone. Furthermore, FGF21 knockout mice did not show high bone mass phenotype. Treatment with PPARα or PPARγ agonists caused similar metabolic effects in FGF21 knockout and wild-type mice. These results contrast with previous findings and suggest that FGF21 is not critical for bone homeostasis or actions of PPARα and PPARγ. © 2016 American Society for Bone and Mineral Research., (© 2016 American Society for Bone and Mineral Research.)

Widely distributed osteosclerosis is an unusual radiographic finding with multiple causes. A 42-year-old premenopausal Spanish woman gradually acquired dense bone diffusely affecting her axial skeleton and focally affecting her proximal long bones. Systemic lupus erythematosus (SLE) diagnosed in adolescence had been well controlled. She had not fractured or received antiresorptive therapy, and she was hepatitis C virus antibody negative. Family members had low bone mass. Lumbar spine bone mineral density (BMD) measured by dual-photon absorptiometry (DPA) at age 17 years, while receiving glucocorticoids, was 79% the average value of age-matched controls. From ages 30 to 37 years, dual-energy X-ray absorptiometry (DXA) BMD Z-scores steadily increased in her lumbar spine from +3.8 to +7.9, and in her femoral neck from -1.4 to -0.7. Serum calcium and phosphorus levels were consistently normal, 25-hydroxyvitamin D (25OHD) <20 ng/mL, and parathyroid hormone (PTH) sometimes slightly increased. Her reduced estimated glomerular filtration rate (eGFR) was 38 to 55 mL/min. Hypocalciuria likely reflected positive mineral balance. During increasing BMD, turnover markers (serum bone-specific alkaline phosphatase [ALP], procollagen type 1 N propeptide [P1NP], osteocalcin [OCN], and carboxy-terminal cross-linking telopeptide of type 1 collagen [CTx], and urinary amino-terminal cross-linking telopeptide of type 1 collagen [NTx and CTx]) were 1.6- to 2.8-fold above the reference limits. Those of bone formation seemed increased more than those of resorption. FGF-23 was slightly elevated, perhaps from kidney disease. Serum osteoprotegerin (OPG) and TGFβ1 levels were normal, but sclerostin (SOST) and receptor activator of nuclear factor kappa-B ligand (RANKL) were elevated. Serum multiplex biomarker profiling confirmed a high level of SOST and RANKL, whereas Dickkopf-1 (DKK-1) seemed low. Matrix metalloproteinases-3 (MMP-3) and -7 (MMP-7) were elevated. Iliac crest biopsy revealed tetracycline labels, no distinction between thick trabeculae and cortical bone, absence of peritrabecular fibrosis, few osteoclasts, and no mastocytosis. Then, for the past 3 years, BMD Z-scores steadily decreased. Skeletal fluorosis, mastocytosis, myelofibrosis, hepatitis C-associated osteosclerosis, multiple myeloma, and aberrant phosphate homeostasis did not explain her osteosclerosis. Mutation analysis of the LRP5, LRP4, SOST, and osteopetrosis genes was negative. Microarray showed no notable copy number variation. Perhaps her osteosclerosis reflected an interval of autoimmune-mediated resistance to SOST and/or RANKL. © 2016 American Society for Bone and Mineral Research., (© 2016 American Society for Bone and Mineral Research.)

Inhibition of the Wnt antagonist sclerostin increases bone mass in patients with osteoporosis and in preclinical animal models. Here we show increased levels of the Wnt antagonist Dickkopf-1 (DKK-1) in animals treated with sclerostin antibody, suggesting a negative feedback mechanism that limits Wnt-driven bone formation. To test our hypothesis that co-inhibition of both factors further increases bone mass, we engineer a first-in-class bispecific antibody with single residue pair mutations in the Fab region to promote efficient and stable cognate light-heavy chain pairing. We demonstrate that dual inhibition of sclerostin and DKK-1 leads to synergistic bone formation in rodents and non-human primates. Furthermore, by targeting distinct facets of fracture healing, the bispecific antibody shows superior bone repair activity compared with monotherapies. This work supports the potential of this agent both for treatment and prevention of fractures and offers a promising therapeutic approach to reduce the burden of low bone mass disorders.

Sclerostin antibody (Scl-Ab) and parathyroid hormone (PTH) are bone-forming agents that have different modes of action on bone, although a study directly comparing their effects has not been conducted. The present study investigated the comparative quantitative effects of these two bone-forming agents over time on bone at the organ, tissue, and cellular level; specifically, at the level of the osteoblast (Ob) lineage in adolescent male and female rats. Briefly, eight-week old male and female Sprague-Dawley rats were administered either vehicle, Scl-Ab (3 or 50mg/kg/week subcutaneously), or human PTH (1-34) (75 μg/kg/day subcutaneously) for 4 or 26 weeks. The 50mg/kg Scl-Ab and the PTH dose were those used in the respective rat lifetime pharmacology studies. Using robust stereological methods, we compared the effects of these agents specifically at the level of the Ob lineage in vertebrae from female rats. Using RUNX2 or nestin immunostaining, location, and morphology, the total number of osteoprogenitor subpopulations, Ob, and lining cells were estimated using the fractionator or proportionator estimators. Density estimates were also calculated referent to total bone surface, total Ob surface, or total marrow volume. Scl-Ab generally effected greater increases in cancellous and cortical bone mass than PTH, correlating with higher bone formation rates (BFR) at 4 weeks in the spine and mid-femur without corresponding increases in bone resorption indices. The increases in vertebral BFR/BS at 4 weeks attenuated with continued treatment to a greater extent with Scl-Ab than with PTH. At 4 weeks, both Scl-Ab and PTH effected equivalent increases in total Ob number (Ob.N). Ob density on the formative surfaces (Ob.N/Ob.S) remained similar across groups while mineral apposition rate (MAR) was significantly higher with Scl-Ab at week 4, reflecting an increase in individual Ob vigor relative to vehicle and PTH. After 26 weeks, Scl-Ab maintained BFR/BS with fewer Ob and lower Ob.N/Ob.S by increasing the Ob footprint (bone surface area occupied by an Ob) and increasing MAR, compared with PTH. The lower Ob.N and Ob.N/Ob.S with Scl-Ab at 26 weeks were associated with decreased osteoprogenitor numbers compared with both vehicle and PTH, an effect not evident at week 4. Osteoprogenitor numbers were generally positively correlated with Ob.N across groups and timepoints, suggesting dynamic coordination between the progenitor and Ob populations. The time-dependent reductions in subpopulations of the Ob lineage with Scl-Ab may be integral to the greater attenuation or self-regulation of bone formation observed at the vertebra, as PTH required more Ob at the formative site with correlative increased numbers of progenitors compared with Scl-Ab indicating potentially greater stimulus for progenitor pool proliferation or differentiation., (Copyright © 2015. Published by Elsevier Inc.)

Background: Patients with kidney disease have disordered bone and mineral metabolism, including elevated serum concentrations of fibroblast growth factor-23 (FGF23). These elevated concentrations are associated with cardiovascular and all-cause mortality. The objective was to determine the effects of the calcimimetic cinacalcet (versus placebo) on reducing serum FGF23 and whether changes in FGF23 are associated with death and cardiovascular events., Methods and Results: This was a secondary analysis of a randomized clinical trial comparing cinacalcet to placebo in addition to conventional therapy (phosphate binders/vitamin D) in patients receiving hemodialysis with secondary hyperparathyroidism (intact parathyroid hormone ≥300 pg/mL). The primary study end point was time to death or a first nonfatal cardiovascular event (myocardial infarction, hospitalization for angina, heart failure, or a peripheral vascular event). This analysis included 2985 patients (77% of randomized) with serum samples at baseline and 2602 patients (67%) with samples at both baseline and week 20. The results demonstrated that a significantly larger proportion of patients randomized to cinacalcet had ≥30% (68% versus 28%) reductions in FGF23. Among patients randomized to cinacalcet, a ≥30% reduction in FGF23 between baseline and week 20 was associated with a nominally significant reduction in the primary composite end point (relative hazard, 0.82; 95% confidence interval, 0.69-0.98), cardiovascular mortality (relative hazard, 0.66; 95% confidence interval, 0.50-0.87), sudden cardiac death (relative hazard, 0.57; 95% confidence interval, 0.37-0.86), and heart failure (relative hazard, 0.69; 95% confidence interval, 0.48-0.99)., Conclusions: Treatment with cinacalcet significantly lowers serum FGF23. Treatment-induced reductions in serum FGF23 are associated with lower rates of cardiovascular death and major cardiovascular events., Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00345839., (© 2015 American Heart Association, Inc.)

The effects of up to 26 weeks of sclerostin antibody (Scl-Ab) treatment were investigated in ovariectomized (OVX) rats. Two months after surgery, 6-month-old osteopenic OVX rats were treated with vehicle or Scl-Ab (25 mg/kg, sc, one time per week) for 6, 12, or 26 weeks. In vivo dual-energy x-ray absorptiometry analysis demonstrated that the bone mineral density of lumbar vertebrae and femur-tibia increased progressively through 26 weeks of Scl-Ab treatment along with progressive increases in trabecular and cortical bone mass and bone strength at multiple sites. There was a strong correlation between bone mass and maximum load at lumbar vertebra, femoral neck, and diaphysis at weeks 6 and 26. Dynamic histomorphometric analysis showed that lumbar trabecular and tibial shaft endocortical and periosteal bone formation rates (BFR/BS) increased and peaked at week 6 with Scl-Ab-treatment; thereafter trabecular and endocortical BFR/BS gradually declined but remained significantly greater than OVX controls at week 26, whereas periosteal BFR/BS returned to OVX control levels at week 26. In the tibia metaphysis, trabecular BFR/BS in the Scl-Ab treated group remained elevated from week 6 to week 26. The osteoclast surface and eroded surface were significantly lower in Scl-Ab-treated rats than in OVX controls at all times. In summary, bone mass and strength increased progressively over 26 weeks of Scl-Ab treatment in adult OVX rats. The early gains were accompanied by increased cortical and trabecular bone formation and reduced osteoclast activity, whereas later gains were attributed to residual endocortical and trabecular osteoblast stimulation and persistently low osteoclast activity.

Among the high bone mass disorders, the osteopetroses reflect osteoclast failure that prevents skeletal resorption and turnover, leading to reduced bone growth and modeling and characteristic histopathological and radiographic findings. We report an 11-year-old boy with a new syndrome that radiographically mimics osteopetrosis (OPT), but features rapid skeletal turnover. He presented at age 21 months with a parasellar, osteoclast-rich giant cell granuloma. Radiographs showed a dense skull, generalized osteosclerosis and cortical thickening, medullary cavity narrowing, and diminished modeling of tubular bones. His serum alkaline phosphatase was >5000 IU/L (normal <850 IU/L). After partial resection, the granuloma re-grew but then regressed and stabilized during 3 years of uncomplicated pamidronate treatment. His hyperphosphatasemia transiently diminished, but all bone turnover markers, especially those of apposition, remained elevated. Two years after pamidronate therapy stopped, bone mineral density (BMD) Z-scores reached +9.1 and +5.8 in the lumbar spine and hip, respectively, and iliac crest histopathology confirmed rapid bone remodeling. Serum multiplex biomarker profiling was striking for low sclerostin. Mutation analysis was negative for activation of lipoprotein receptor-related protein 4 (LRP4), LRP5, or TGFβ1, and for defective sclerostin (SOST), osteoprotegerin (OPG), RANKL, RANK, SQSTM1, or sFRP1. Microarray showed no notable copy number variation. Studies of his nonconsanguineous parents were unremarkable. The etiology and pathogenesis of this unique syndrome are unknown., (© 2014 American Society for Bone and Mineral Research.)

Sclerostin (Scl) is an osteocyte protein that decreases bone formation, and its inhibition by neutralizing antibodies (Scl-Ab) increases bone formation, mass and strength. We investigated the effects of Scl-Ab in mature ovariectomized (OVX) rats with a mechanistic focus on longer-term responses of osteoclasts, osteoblasts and osteocytes. Four-month-old Sprague-Dawley rats had OVX or sham surgery. Two months later, sham controls received sc vehicle while OVX rats received vehicle (OVX-Veh) or Scl-Ab (25mg/kg) once weekly for 6 or 26weeks followed by necropsy (n=12/group). Terminal blood was collected for biochemistry, non-adherent marrow cells were harvested from femurs for ex vivo osteoclast formation assays, and vertebrae and tibiae were collected for dynamic histomorphometry and mRNA analyses. Scl-Ab treatment led to progressively thicker but fewer trabeculae in the vertebra, leading to increased trabecular bone volume and reduced trabecular surfaces. Scl-Ab also increased cortical bone volume in the tibia, via early periosteal expansion and progressive endocortical contraction. Scl-Ab significantly reduced parameters of bone resorption at week 6 relative to OVX-Veh controls, including reduced serum TRACP-5b, reduced capacity of marrow cells to form osteoclasts ex vivo, and >80% reductions in vertebral trabecular and tibial endocortical eroded surfaces. At week 26, serum TRACP-5b and ex vivo osteoclast formation were no longer reduced in the Scl-Ab group, but eroded surfaces remained >80% lower than in OVX-Veh controls without evidence for altered skeletal mRNA expression of opg or rankl. Scl-Ab significantly increased parameters of bone formation at week 6 relative to OVX-Veh controls, including increases in serum P1NP and osteocalcin, and increased trabecular, endocortical and periosteal bone formation rates (BFRs). At week 26, surface-referent trabecular BFR remained significantly increased in the Scl-Ab group versus OVX-Veh controls, but after adjusting for a reduced extent of trabecular surfaces, overall (referent-independent) trabecular BFR was no longer significantly elevated. Similarly, serum P1NP and osteocalcin were no longer significantly increased in the Scl-Ab group at week 26. Tibial endocortical and periosteal BFR were increased at week 6 in the Scl-Ab group versus OVX-Veh controls, while at week 26 only endocortical BFR remained increased. The Scl-Ab group exhibited significant increments in skeletal mRNA expression of several osteocyte genes, with sost showing the greatest induction in both the tibia and vertebra. We propose that Scl-Ab administration, and/or the gains in bone volume that result, may have increased osteocytic expression of Scl as a possible means of regulating gains in bone mass., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Systemic administration of a sclerostin neutralizing antibody (Scl-Ab) has been shown to enhance fracture callus density and strength in several animal models. In order to further evaluate the potential of Scl-Ab to improve healing in a bone defect model,we evaluated Scl-Ab in a 3mm femoral defect in young male outbred rats. Scl-Ab was given either continuously for 6 or 12 weeks after surgery or with 2 weeks of delay for 10 weeks. Bone formation was assessed by radiographs, µ-CT, and histology. Complete bony union was achieved in only a few defects after 12 weeks of healing (Scl-Ab treated 5/30, vehicle treated 1/15). µ-CT evaluation demonstrated a significant increase in the BV/TV in the defect in the delayed treatment group (65%, p<0.05), but a non-significant increase in the continuous group (35%, p = 0.11) compared to control. However, both regimens induced an anabolic response in the bone proximal and distal to the defect and in the un-operated femurs. We demonstrate that treatment with Scl-Ab can enhance bone repair in a bone defect and in the surrounding host bone, but lacks the osteoinductive activity to heal it. This agent seems to be most effective in bone repair scenarios where there is cortical integrity., (© 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Receptor activator of NF-κB ligand (RANKL) plays a key role in osteoclast-induced bone resorption across a range of degenerative bone diseases, and its specific inhibition has been recently approved as a treatment for women with postmenopausal osteoporosis at high or increased risk of fracture in the United States and globally. In the present study, we generated transgenic mice (TghuRANKL) carrying the human RANKL (huRANKL) genomic region and achieved a physiologically relevant pattern of RANKL overexpression in order to establish novel genetic models for assessing skeletal and extraskeletal pathologies associated with excessive RANKL and for testing clinical therapeutic candidates that inhibit human RANKL. TghuRANKL mice of both sexes developed early-onset bone loss, and the levels of huRANKL expression were correlated with bone resorption and disease severity. Low copy Tg5516 mice expressing huRANKL at low levels displayed a mild osteoporotic phenotype as shown by trabecular bone loss and reduced biomechanical properties. Notably, overexpression of huRANKL, in the medium copy Tg5519 line, resulted in severe early-onset osteoporosis characterized by lack of trabecular bone, destruction of the growth plate, increased osteoclastogenesis, bone marrow adiposity, increased bone remodeling, and severe cortical bone porosity accompanied by decreased bone strength. An even more severe skeletal phenotype developed in the high copy Tg5520 founder with extensive soft tissue calcification. Model validation was further established by evidence that denosumab, an antibody that inhibits human but not murine RANKL, fully corrected the hyper-resorptive and osteoporotic phenotypes of Tg5519 mice. Furthermore, overexpression of huRANKL rescued osteopetrotic phenotypes of RANKL-defective mice. These novel huRANKL transgenic models of osteoporosis represent an important advance for understanding the pathogenesis and treatment of high-turnover bone diseases and other disease states caused by excessive RANKL., (© 2014 American Society for Bone and Mineral Research.)