Determination of Anti-Xa Inhibitor Plasma Concentrations Using a Universal Edoxaban Calibrator.

A universal calibrator for the determination of all anti-Xa inhibitors would support laboratory processes. We aimed to test the clinical performance of an anti-Xa assay utilizing a universal edoxaban calibrator to determine clinically relevant concentrations of all anti-Xa inhibitors. Following a pilot study, we enrolled 553 consecutive patients taking rivaroxaban, edoxaban, or apixaban from nine study centers in a prospective cross-sectional study. The Technochrom® anti-Xa assay was conducted using the Technoview® edoxaban calibrator. Using ultra-high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS), anti-Xa inhibitor drug concentrations were determined. Sensitivities and specificities to detect three clinically relevant drug concentrations (30 µgL-1, 50 µgL-1, 100 µgL-1) were determined. Overall, 300 patients treated with rivaroxaban, 221 with apixaban, and 32 with edoxaban were included. The overall correlation coefficient (rs) was 0.95 (95% CI 0.94, 0.96). An area under the receiver operating characteristic curve of 0.96 for 30 µgL-1, 0.98 for 50 µgL-1, and 0.99 for 100 µgL-1 was found. The sensitivities were 92.3% (95% CI 89.2, 94.6), 92.7% (89.4, 95.1), and 94.8% (91.1, 97.0), respectively (specificities 82.2%, 93.7%, and 94.4%). In conclusion, the clinical performance of a universal, edoxaban-calibrated anti-Xa assay was solid and most drug concentrations were predicted correctly.

ADAMTS13 Activity: Screening Test Protocol.

Accurate estimation of ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif, member 13) activity level is necessary for diagnosis and management of thrombotic microangiopathies (TMA). In particular, it permits distinction between thrombotic thrombocytopenic purpura (TTP) and other TMAs, prompting disorder appropriate treatment. Manual and automated quantitative assays of ADAMTS13 activity are commercially available, some providing results within less than an hour, but they require specialist equipment and personnel and tend to only be available in specialized diagnostic facilities. Technoscreen ADAMTS13 Activity is a rapid, commercially available, semiquantitative screening test employing flow-through technology and an ELISA activity assay principle. It is a simple to perform screening tool, not requiring specialist equipment or personnel. The colored end point is compared to a reference color chart containing four color intensity indicators corresponding to ADAMTS13 activity levels of 0, 0.1, 0.4, or 0.8 IU/mL. Reduced levels detected in the screening test should be confirmed by quantitative assay. The assay lends itself to use in nonspecialized laboratories, remote, and point-of-care settings.

ADAMTS13 Activity Measurement by ELISA and Fluorescence Resonance Energy Transfer Assay.

Accurate estimation of ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif, member 13) activity level is crucial in the diagnostic setting of differentiation between thrombotic thrombocytopenic purpura (TTP) and other thrombotic microangiopathies. The original assays were too cumbersome and time-consuming for use in the acute situation, and treatment was often based on clinical findings alone, with confirmatory laboratory assays following days or weeks later. Rapid assays are now available that can generate results fast enough to impact on immediate diagnosis and management. Assays based on fluorescence resonance energy transfer (FRET) or chemiluminescence principles can generate results in less than an hour, although they require specific analytical platforms. Enzyme-linked immunosorbent assays (ELISA) can generate results in about 4 h, but do not require specialized equipment beyond ELISA plate readers that are in regular use in many laboratories. The present chapter describes principles, performance, and practical aspects of an ELISA and a FRET assay, for quantitative measurement of ADAMTS13 activity in plasma.

ADAMTS13 Antibody and Inhibitor Assays.

A finding of an ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif, member 13) activity level of <10% of normal is usually sufficient to distinguish thrombotic thrombocytopenic purpura (TTP) from other thrombotic microangiopathies. TTP can be congenital or acquired, the most common form being acquired immune-mediated TTP caused by autoantibodies than inhibit ADAMTS13 function and/or increase its clearance. Basic 1 + 1 mixing tests can detect the presence of inhibitory antibodies, and quantification can be achieved with Bethesda-type assays that measure loss of function in a series of mixtures of test plasma and normal plasma. Not all patients present with inhibitory antibodies, and here the ADAMTS13 deficiency may be caused by clearing antibodies alone, which are not detectable in functional assays. ELISA assays are commonly used to detect clearing antibodies via capture with recombinant ADAMTS13. Since they also detect inhibitory antibodies, they are the preferred assay, although they cannot distinguish between inhibitory and clearing antibodies. The present chapter describes principles, performance, and practical aspects of a commercial ADAMTS13 antibody ELISA and a generic approach to Bethesda-type assays for detecting inhibitory ADAMTS13 antibodies.

Thrombin generation assays are versatile tools in blood coagulation analysis: A review of technical features, and applications from research to laboratory routine.

Thrombin is the pivotal enzyme in the biochemistry of secondary hemostasis crucial to maintaining homeostasis of hemostasis. In contrast to routine coagulation tests (PT or aPTT) or procoagulant or anticoagulant factor assays (e.g. fibrinogen, factor VIII, antithrombin or protein C), the thrombin generation assay (TGA), also named thrombin generation test (TGT) is a so-called "global assay" that provides a picture of the hemostasis balance though a continuous and simultaneous measurement of thrombin formation and inhibition. First described in the early 1950s, as a manual assay, efforts have been made in order to standardize and automate the assay to offer researchers, clinical laboratories and the pharmaceutical industry a versatile tool covering a wide range of clinical and non-clinical applications. This review describes technical options offered to properly run TGA, including a review of preanalytical and analytical items, performance, interpretation, and applications in physiology research and pharmacy.

Clinical use of thrombin generation assays.

Determining patient's coagulation profile, i.e. detecting a bleeding tendency or the opposite, a thrombotic risk, is crucial for clinicians in many situations. Routine coagulation assays and even more specialized tests may not allow a relevant characterization of the hemostatic balance. In contrast, thrombin generation assay (TGA) is a global assay allowing the dynamic continuous and simultaneous recording of the combined effects of both thrombin generation and thrombin inactivation. TGA thus reflects the result of procoagulant and anticoagulant activities in blood and plasma. Because of this unique feature, TGA has been widely used in a wide array of settings from both research, clinical and pharmaceutical perspectives. This includes diagnosis, prognosis, prophylaxis, and treatment of inherited and acquired bleeding and thrombotic disorders. In addition, TGA has been shown to provide relevant information for the diagnosis of coagulopathies induced by infectious diseases, comprising also disturbance of the coagulation system in COVID-19, or for the assessment of early recurrence in breast cancer. This review article aims to document most clinical applications of TGA.

A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests.

BACKGROUND: Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. METHODS: We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests' broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification. FINDINGS: Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus. INTERPRETATION: We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms. FUNDING: WWTF, Project No. COV20-016; BOKU, LBI/LBG.

Recombinant ADAMTS13 reduces abnormally up-regulated von Willebrand factor in plasma from patients with severe COVID-19.

Thrombosis affecting the pulmonary and systemic vasculature is common during severe COVID-19 and causes adverse outcomes. Although thrombosis likely results from inflammatory activation of vascular cells, the mediators of thrombosis remain unconfirmed. In a cross-sectional cohort of 36 severe COVID-19 patients, we show that markedly increased plasma von Willebrand factor (VWF) levels were accompanied by a partial reduction in the VWF regulatory protease ADAMTS13. In all patients we find this VWF/ADAMTS13 imbalance to be associated with persistence of ultra-high-molecular-weight (UHMW) VWF multimers that are highly thrombogenic in some disease settings. Incubation of plasma samples from patients with severe COVID-19 with recombinant ADAMTS13 (rADAMTS13) substantially reduced the abnormally high VWF activity, reduced overall multimer size and depleted UHMW VWF multimers in a time and concentration dependent manner. Our data implicate disruption of normal VWF/ADAMTS13 homeostasis in the pathogenesis of severe COVID-19 and indicate that this can be reversed ex vivo by correction of low plasma ADAMTS13 levels. These findings suggest a potential therapeutic role for rADAMTS13 in helping restore haemostatic balance in COVID-19 patients.

Clinical Validation of an Automated Fluorogenic Factor XIII Activity Assay Based on Isopeptidase Activity.

Hereditary factor XIII (FXIII) deficiency is a rare autosomal bleeding disorder which can cause life-threatening bleeding. Acquired deficiency can be immune-mediated or due to increased consumption or reduced synthesis. The most commonly used screening test is insensitive, and widely used quantitative assays have analytical limitations. The present study sought to validate Technofluor FXIII Activity, the first isopeptidase-based assay available on a routine coagulation analyser, the Ceveron s100. Linearity was evidenced throughout the measuring range, with correlation coefficients of >0.99, and coefficients of variation for repeatability and reproducibility were <5% and <10%, respectively. A normally distributed reference range of 47.0-135.5 IU/dL was derived from 154 normal donors. Clinical samples with Technofluor FXIII Activity results between 0 and 167.0 IU/dL were assayed with Berichrom® FXIII Activity, a functional ammonia release assay, and the HemosIL™ FXIII antigen assay, generating correlations of 0.950 and 0.980, respectively. Experiments with a transglutaminase inhibitor showed that Technofluor FXIII Activity can detect inhibition of enzymatic activity. No interference was exhibited by high levels of haemolysis and lipaemia, and interference by bilirubin was evident at 18 mg/dL, a level commensurate with severe liver disease. Technofluor FXIII Activity is a rapid, accurate and precise assay suitable for routine diagnostic use with fewer interferents than ammonia release FXIII activity assays.

Feasibility of an automated coagulation factor XIIIa test using its isopeptidase activity.

Plasma transglutaminase FXIII provides mechanical and biochemical stability to blood clots. Congenital or acquired deficiency may be associated with bleeding diathesis and requires therefore careful monitoring. The precise automated measurement of a large number of plasma samples can provide new insights regarding the clinical relevance of certain FXIII levels. There is still the unmet diagnostic need for a reliable high-throughput method. Here we report the development and feasibility study of a promising prototype, adapting the precise FXIIIa isopeptidase assay principle on the optimized automated Ceveron s100 platform.

A multi-center evaluation of TECHNOSCREEN® ADAMTS-13 activity assay as a screening tool for detecting deficiency of ADAMTS-13.

BACKGROUND: Quantifying A disintegrin-like and metalloprotease with thrombospondin type 1 motif, member 13 (ADAMTS-13) activity enhances thrombotic thrombocytopenic purpura (TTP) diagnosis but most assays are time consuming, technically demanding, and mainly available in reference centers. OBJECTIVE: Evaluate a simple, semiquantitative ADAMTS-13 activity screening test for early identification/exclusion of TTP. PATIENTS/METHODS: Plasma from 220 patients with suspected thrombotic microangiopathy at three reference centers were tested with TECHNOSCREEN® ADAMTS13 activity screening test in comparison with TECHNOZYM® ADAMTS-13 activity ELISA at two centers, and in-house fluorescence resonance energy transfer assay at the third center. The screening test indicates if ADAMTS-13 activity is at one of four level-indicator points: 0, 0.1, 0.4, or 0.8 IU/mL. RESULTS: Screen results were interpreted as binary data in that ADAMTS-13 activity was above or below the 0.1 IU/mL TTP clinical threshold. Combining all sites' data, the screen exhibited 88.7% sensitivity, 90.4% specificity, 74.6% positive predictive value, and 96.2% negative predictive value, comparable to published data for quantitative assays. Five samples with quantitative results below the threshold gave screen readings of 0.1 IU/mL and seven marginally above the threshold gave screen readings of zero. All would warrant plasma exchange while the level is quantified. Nine samples with normal/near normal results gave screens of zero and confirmatory quantifications would prompt early treatment withdrawal, as is current practice. One sample generated screen/quantitative results of 0.4/0.00 IU/mL respectively and was the only clear false-negative. CONCLUSIONS: The screening test provides more rapid ADAMTS-13 level evaluation than most currently available assays. Its simple operation renders it suitable for adoption in routine or specialist laboratory environments.

Non-classical monocytes as mediators of tissue destruction in arthritis.

OBJECTIVES: Bone destruction in rheumatoid arthritis is mediated by osteoclasts (OC), which are derived from precursor cells of the myeloid lineage. The role of the two monocyte subsets, classical monocytes (expressing CD115, Ly6C and CCR2) and non-classical monocytes (which are CD115 positive, but low in Ly6C and CCR2), in serving as precursors for OC in arthritis is still elusive. METHODS: We investigated CCR2-/- mice, which lack circulating classical monocytes, crossed into hTNFtg mice for the extent of joint damage. We analysed monocyte subsets in hTNFtg and K/BxN serum transfer arthritis by flow cytometry. We sorted monocyte subsets and analysed their potential to differentiate into OC and their transcriptional response in response to RANKL by RNA sequencing. With these data, we performed a gene ontology enrichment analysis and gene set enrichment analysis. RESULTS: We show that in hTNFtg arthritis local bone erosion and OC generation are even enhanced in the absence of CCR2. We further show the numbers of non-classical monocytes in blood are elevated and are significantly correlated with histological signs of joint destruction. Sorted non-classical monocytes display an increased capacity to differentiate into OCs. This is associated with an increased expression of signal transduction components of RANK, most importantly TRAF6, leading to an increased responsiveness to RANKL. CONCLUSION: Therefore, non-classical monocytes are pivotal cells in arthritis tissue damage and a possible target for therapeutically intervention for the prevention of inflammatory joint damage.

Fluoroquinolones impair tendon healing in a rat rotator cuff repair model: a preliminary study.

BACKGROUND: Recent studies suggest that fluoroquinolone antibiotics predispose tendons to tendinopathy and/or rupture. However, no investigations on the reparative capacity of tendons exposed to fluoroquinolones have been conducted. HYPOTHESIS: Fluoroquinolone-treated animals will have inferior biochemical, histological, and biomechanical properties at the healing tendon-bone enthesis compared with controls. STUDY DESIGN: Controlled laboratory study. METHODS: Ninety-two rats underwent rotator cuff repair and were randomly assigned to 1 of 4 groups: (1) preoperative (Preop), whereby animals received fleroxacin for 1 week preoperatively; (2) pre- and postoperative (Pre/Postop), whereby animals received fleroxacin for 1 week preoperatively and for 2 weeks postoperatively; (3) postoperative (Postop), whereby animals received fleroxacin for 2 weeks postoperatively; and (4) control, whereby animals received vehicle for 1 week preoperatively and for 2 weeks postoperatively. Rats were euthanized at 2 weeks postoperatively for biochemical, histological, and biomechanical analysis. All data were expressed as mean ± standard error of the mean (SEM). Statistical comparisons were performed using either 1-way or 2-way ANOVA, with P < .05 considered significant. RESULTS: Reverse transcriptase quantitative polymerase chain reaction (RTqPCR) analysis revealed a 30-fold increase in expression of matrix metalloproteinase (MMP)-3, a 7-fold increase in MMP-13, and a 4-fold increase in tissue inhibitor of metalloproteinases (TIMP)-1 in the Pre/Postop group compared with the other groups. The appearance of the healing enthesis in all treated animals was qualitatively different than that in controls. The tendons were friable and atrophic. All 3 treated groups showed significantly less fibrocartilage and poorly organized collagen at the healing enthesis compared with control animals. There was a significant difference in the mode of failure, with treated animals demonstrating an intrasubstance failure of the supraspinatus tendon during testing. In contrast, only 1 of 10 control samples failed within the tendon substance. The healing enthesis of the Pre/Postop group displayed significantly reduced ultimate load to failure compared with the Preop, Postop, and control groups. There was no significant difference in load to failure in the Preop group compared with the Postop group. Pre/Postop animals demonstrated significantly reduced cross-sectional area compared with the Postop and control groups. There was also a significant reduction in area between the Preop and control groups. CONCLUSION: In this preliminary study, fluoroquinolone treatment negatively influenced tendon healing. CLINICAL RELEVANCE: These findings indicate that there was an active but inadequate repair response that has potential clinical implications for patients who are exposed to fluoroquinolones before tendon repair surgery.

Tumor necrosis factor-inhibiting therapy preferentially targets bone destruction but not synovial inflammation in a tumor necrosis factor-driven model of rheumatoid arthritis.

OBJECTIVE: To investigate how tumor necrosis factor (TNF)-inhibiting therapy affects bone destruction and inflammation in a TNF-driven mouse model of rheumatoid arthritis. METHODS: In order to evaluate the influence of TNF on osteoclastogenesis in vitro, different concentrations of TNF were added to spleen cell-derived monocytes in the absence or presence of different concentrations of RANKL. In addition, the effects of TNF inhibition on osteoclast precursors as well as local bone destruction in vivo were assessed by treating TNF-transgenic mice with different doses of adalimumab. RESULTS: TNF stimulated osteoclastogenesis mainly by increasing the number of osteoclast precursor cells in vitro. This TNF effect was independent of the presence of RANKL. In the hTNF-transgenic mouse model of destructive arthritis, low-dose TNF-inhibiting therapy with adalimumab had no effect on synovial inflammation but significantly inhibited local bone destruction and the generation of osteoclasts. This inhibition was accompanied by a reduction in the number of c-Fms-positive osteoclast precursor cells in the bone marrow and a reduction of the osteoclast precursor pools in the blood and inflamed synovial membrane of hTNF-transgenic mice. CONCLUSION: Low-dose TNF-inhibiting therapy significantly reduces bone erosions by reducing the number of circulating and joint-invading osteoclast precursors. This effect is uncoupled from its antiinflammatory action.

Orthopedic wear debris mediated inflammatory osteolysis is mediated in part by NALP3 inflammasome activation.

Activation of myeloid cells by orthopedic particulate debris is a key event in the pathogenesis of periprosthetic osteolysis and implant loosening after total joint replacement (TJR). Several lines of evidence implicate NACHT, LRR, and PYD domains-containing protein 3 (NALP3) inflammasome-mediated production of interleukin 1 beta (IL-1ß) in the pathogenesis of clinical disorders ascribable to foreign particulate materials, including asbestos, silica, and urate crystals. Recent reports indicate that orthopedic polymer products and metallic particulates and ions may activate the same pathway. Here, we investigated the contribution of the NALP3 inflammasome to the pathogenesis of peri-implant osteolysis. Pharmaceutical and genetic perturbations of caspase-1 and inflammasome components were used to assess the role of the NALP3 inflammasome in IL-1ß production and osteoclast formation by human monocytes and mouse macrophages in response to polymethylmethacrylate (PMMA) particle phagocytosis. The role of caspase-1 in a mouse calvarial model of particle-mediated osteolysis was assessed using µCT. Phagocytosis of PMMA particles induces caspase-1 dependent release of IL-1ß from human monocytes and mouse macrophages. Importantly, using macrophages from mice deficient in components of the NALP3 inflammasome, we show PMMA-induced IL-1ß production is strictly dependent on these components. Mice lacking caspase-1, the sole effector of the NALP3 inflammasome, show reduced orthopedic wear particle-induced calvarial osteolysis compared to wild-type controls. Absence of NALP3 inflammasome components fails to alter osteoclast formation in vitro. Our findings identify the NALP3 inflammasome as a critical mediator of orthopedic wear-induced osteolysis and as a viable therapeutic target for the treatment of periprosthetic osteolysis.

Bone remodelling in inflammatory arthritis.

The inflammatory arthropathies that include rheumatoid arthritis, the seronegative spondyloarthropathies and systemic lupus erythematosus are characterised by marked alterations in the architecture and structural integrity of peri-articular bone; however, the pattern and natural history of the skeletal changes differs in these conditions. In part, this can be attributed to differences in the primary anatomical site of the inflammation, but also there is evidence that there are differences in the biological properties and products produced by inflammatory tissues. This review will focus on recent advances in the understanding of the cellular and molecular mechanisms that contribute to the differential pattern of articular bone remodelling in these prototypical inflammatory forms of arthritis.

Antiinflammatory effects of tumor necrosis factor on hematopoietic cells in a murine model of erosive arthritis.

OBJECTIVE: To investigate the mechanisms leading to the influx of inflammatory hematopoietic cells into the synovial membrane and the role of tumor necrosis factor receptor I (TNFRI) and TNFRII in this process in an animal model of rheumatoid arthritis (RA). METHODS: We performed bone marrow transplantations in human TNF-transgenic mice using hematopoietic cells from wild-type, TNFRI(-/-), TNFRII(-/-), and TNFRI/II(-/-) mice as donors and assessed the severity of arthritis histologically. Generation of osteoclasts from the different genotypes was analyzed in vitro and in vivo. Apoptosis was analyzed using annexin V staining as well as TUNEL assays. RESULTS: Despite lacking responsiveness to TNF in their hematopoietic compartment, mice not only developed full-blown erosive arthritis but even showed increased joint destruction when compared with mice with a TNF-responsive hematopoietic compartment. We demonstrated different roles of the 2 different TNFRs in the regulation of these processes. The absence of TNFRI on hematopoietic cells did not affect joint inflammation but markedly attenuated erosive bone destruction via reduced synovial accumulation of osteoclast precursors. In contrast, the absence of TNFRII on hematopoietic cells increased joint inflammation as well as erosive bone destruction via the regulation of osteoclast precursor apoptosis. CONCLUSION: Our findings indicate that selective blockade of TNFRI, leaving the antiinflammatory effects of TNFRII unaltered instead of unselectively blocking TNF, might be advantageous in patients with RA.

Interleukin-1 mediates long-term hippocampal dentate granule cell loss following postnatal viral infection.

Viral infections of the developing CNS can cause long-term neuropathological sequela through undefined mechanisms. Proinflammatory cytokines such as IL-1beta have gained attention in mediating neurodegeneration in corticohippocampal structures due to a variety of insults in adults, though there is less information on the developing brain. Little is known concerning the spatial-temporal pattern of IL-1beta induction in the developing hippocampus following live virus infection, and there are few studies addressing the long-term consequences of this cytokine induction. We report that infection of rats with lymphocytic choriomeningitis virus on postnatal day 4 induces IL-1beta protein in select regions of the hippocampus on 6, 15, 21, and 45 days after infection. This infection resulted in a 71% reduction of dentate granule cell neurons by the time the rats reached mid-adulthood. We further investigated the causative role of IL-1 in this dentate granule cell loss by blocking IL-1 activity using an IL-1ra-expressing adenoviral vector administered at the time of infection. Blockade of IL-1 abrogated the infection-associated neuron loss in this vivo model. Considering that IL-1 can be triggered by multiple perinatal insults, our findings suggest that early therapy with anti-inflammatory agents that block IL-1 may be effective for reducing adulthood neuropathology.

Estrogen-dependent and C-C chemokine receptor-2-dependent pathways determine osteoclast behavior in osteoporosis.

Understanding the mechanisms of osteoclastogenesis is crucial for developing new drugs to treat diseases associated with bone loss, such as osteoporosis. Here we report that the C-C chemokine receptor-2 (CCR2) is crucially involved in balancing bone mass. CCR2-knockout mice have high bone mass owing to a decrease in number, size and function of osteoclasts. In normal mice, activation of CCR2 in osteoclast progenitor cells results in both nuclear factor-kappaB (NF-kappaB) and extracellular signal-related kinase 1 and 2 (ERK1/2) signaling but not that of p38 mitogen-activated protein kinase or c-Jun N-terminal kinase. The induction of NF-kappaB and ERK1/2 signaling in turn leads to increased surface expression of receptor activator of NF-kappaB (RANK, encoded by Tnfrsf11a), making the progenitor cells more susceptible to RANK ligand-induced osteoclastogenesis. In ovariectomized mice, a model of postmenopausal osteoporosis, CCR2 is upregulated on wild-type preosteoclasts, thus increasing the surface expression of RANK on these cells and their osteoclastogenic potential, whereas CCR2-knockout mice are resistant to ovariectomy-induced bone loss. These data reveal a previously undescribed pathway by which RANK, osteoclasts and bone homeostasis are regulated in health and disease.