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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.

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.

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.

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.

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 &gt, 0.99, and coefficients of variation for repeatability and reproducibility were &lt, 5% and &lt, 10%, respectively. A normally distributed reference range of 47.0&ndash, 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&reg, FXIII Activity, a functional ammonia release assay, and the HemosIL&trade, 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.

Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection. While first-generation antibody tests have primarily provided qualitative results with low specificity, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. Here, we describe two quantitative ELISA antibody tests based on the SARS-CoV-2 spike receptor-binding domain and the nucleocapsid protein. Comparative expression in bacterial, insect, mammalian and plant-based platforms enabled the identification of new antigen designs with superior quality and high suitability as diagnostic reagents. Both tests scored excellently in clinical validations with multi-centric specificity and sensitivity cohorts and showed unprecedented correlation with SARS-CoV-2 neutralization titers. Orthogonal testing increased assay specificity to 99.8%, thereby enabling robust serodiagnosis in low-prevalence settings. 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.

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.Evaluate a simple, semiquantitative ADAMTS-13 activity screening test for early identification/exclusion of TTP.Plasma from 220 patients with suspected thrombotic microangiopathy at three reference centers were tested with TECHNOSCREENScreen 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.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.

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

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.

ObjectivesBone 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.MethodsWe 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.ResultsWe 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.ConclusionTherefore, non-classical monocytes are pivotal cells in arthritis tissue damage and a possible target for therapeutically intervention for the prevention of inflammatory joint damage.

Background The thrombocytopenic purpura (TTP) is a life-threatening condition characterized by microvascular thrombosis where it is of great importance to shorten the time to the treatment. TTP can be caused by congenital or acquired deficiency of ADAMTS13 (an enzyme that cleaves the von Willebrand factor). Deficiency leads to the accumulation of ultra-large von Willebrand multimers, which causes platelet aggregation and the formation of microthrombi. TTP laboratory findings include thrombocytopenia, hemolytic anemia, the presence of schistocytes and a markedly reduced activity of ADAMTS13. ADAMTS13 activity can be analyzed using an ELISA method. We have evaluated whether a new semi-quantitative method, TECHNOSCREEN from Technoclone, can be used as a screening method to shorten the time for patient diagnosis. Method We analyzed 43 unidentified samples with known ADAMTS 13 activity, which consisted of 20 samples with an ADAMTS13 activity Results and conclusion The results showed good agreement between the existing ELISA and the newTECHNOSCREEN method. Sensitivity was 100% and specificity 71% if the ELISA is used as a gold standard. In May 2019, the TECHNOSCREEN method was introduced as a first-line analysis. This has resulted in faster patient diagnosis as well as saving laboratory staff time. The TECHNOSCREEN assay takes about 1 hour to perform compared to about 5 hours for the ELISA. To exactly quantify ADAMTS13 activity or detect the presence of antibodies, the ELISAs are performed approximately once a week. Disclosures Binder: Technoclone: Employment. Antovic:Shire: Other: Grants and Personal Fees; Roche: Other: Personal Fees; Stago: Other: Personal Fees; Werfen: Other: Personal Fees; Siemens: Other: Personal Fees; Sobi: Other: Personal Fees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees; CSL Behring: Other: Personal Fees.

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.

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-1β) 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-1β 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-1β from human monocytes and mouse macrophages. Importantly, using macrophages from mice deficient in components of the NALP3 inflammasome, we show PMMA-induced IL-1β 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.

To investigate interferon-gamma (IFNgamma) signaling in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) by analyzing IFNgamma receptor (IFNgammaR) expression, STAT-1 expression and phosphorylation, and the regulation of IFNgamma-inducible genes.Fluorocytometry was used to investigate expression of STAT-1, pSTAT-1, CD95, HLA-DR, class I major histocompatibility complex (MHC), IFNgamma-inducible 10-kd protein (IP-10), monokine induced by IFNgamma (Mig), and IFNgammaR in PBMCs from SLE patients and healthy individuals. STAT-1 phosphorylation was determined by fluorocytometry and Western blotting after stimulation with IFNalpha or IFNgamma. Quantitative polymerase chain reaction was used to assess messenger RNA (mRNA) expression of the IFNgamma-inducible genes IP-10 and Mig shortly after preparation or after stimulation with IFNgamma in monocytes.STAT-1 expression was increased in PBMCs from SLE patients and correlated significantly with disease activity and with the IFN-inducible expression of CD95 and HLA-DR. STAT-1 expression also showed a trend toward association with class I MHC expression. In addition, the expression of other IFNgamma-inducible genes, such as IP-10 or Mig, was increased in SLE monocytes. While STAT-1 phosphorylation in SLE PBMCs and PBMCs from healthy individuals was similar after IFNalpha stimulation, incubation with IFNgamma induced STAT-1 phosphorylation only in SLE lymphocytes. Moreover, SLE monocytes showed a considerably higher increase in pSTAT-1 expression upon IFNgamma stimulation than monocytes from healthy individuals. Increased responsiveness of SLE monocytes to IFNgamma was also confirmed on the mRNA level, where expression of the IFN-inducible, STAT-1-dependent genes IP-10 and Mig was more efficiently increased in SLE cells. However, IFNgammaR was similarly expressed on SLE lymphocytes and monocytes and those from healthy individuals.In addition to supporting the role of IFNs in SLE immunopathogenesis in general, the findings of the present study support a role of IFNgamma in this disease.

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.

iRHOM2, encoded by the gene Rhbdf2, regulates the maturation of the TNF-α convertase (TACE), which controls shedding of TNF-α and its biological activity in vivo. TACE is a potential target to treat TNF-α–dependent diseases, such as rheumatoid arthritis, but there are concerns about potential side effects, because TACE also protects the skin and intestinal barrier by activating EGFR signaling. Here we report that inactivation of Rhbdf2 allows tissue-specific regulation of TACE by selectively preventing its maturation in immune cells, without affecting its homeostatic functions in other tissues. The related iRHOM1, which is widely expressed, except in hematopoietic cells, supported TACE maturation and shedding of the EGFR ligand TGF-α in Rhbdf2-deficient cells. Remarkably, mice lacking Rhbdf2 were protected from K/BxN inflammatory arthritis to the same extent as mice lacking TACE in myeloid cells or Tnfa-deficient mice. In probing the underlying mechanism, we found that two main drivers of K/BxN arthritis, complement C5a and immune complexes, stimulated iRHOM2/ TACE-dependent shedding of TNF-α in mouse and human cells. These data demonstrate that iRHOM2 and myeloid-expressed TACE play a critical role in inflammatory arthritis and indicate that iRHOM2 is a potential therapeutic target for selective inactivation of TACE in myeloid cells.

In recent years the determination of ADAMTS-13 activity in plasma has become an important diagnostics test in the differentiation of Thromobocytopenic Purpura (TTP) patients from those with other forms of Thrombotic micoangiopathies (TMA). Moreover, it is also useful for monitoring the effectiveness of treatment in such patients. The standard laboratory test for ADAMTS-13 Activity is an ELISA assay, which has an assay which requires 4 hours to process. This assay tends to be performed by specialized laboratories, so samples are usually batch tested. Therefore for a physician to receive a patient ADAMTS-13 activity result can range from 24 hours to as much as one week. This delay in reporting could have a negative impact on patient care. We have developed a rapid and simple assay which would allow the physician to have an initial reportable result for ADAMTS-13 Activity within 1 hour from the blood draw. The assay is based on flow through technology, which allows for simple, rapid processing. Plasma samples are pre-incubated with a substrate which is cleaved by ADAMTS-13 present in the plasma. This pre-treated sample is applied to the single test unit which contains a membrane with immobilized antibodies specific for the cleaved substrate. As the sample flows through the membrane into an absorbent pad the cleaved substrate binds to capture antibody. A secondary biotinylated antibody is used to detect the membrane bound complex. Using a Streptavidin-gold conjugate a red colour is produced. The colour intensity is proportional to the level of ADAMTS-13 activity in the plasma sample and compared with a colour standard card, allowing semi-quantitative analysis of the patient sample. The assay can be performed at room temperature without specialized laboratory equipment and minimal training is needed to perform the test. The assay processing time is under 30 minutes. The test is designed for evaluation of a single patient sample, with a positive control run in parallel. The designed assay cut-off value of 10% ADAMTS-13 Activity is clinically relevant for differentiation of TTP patients. A 100% correlation was observed when TTP patient samples (n=20) were run in the TECHNOZYM ADATMS-13 ELISA and this assay, as summarized in the figure below. This rapid and simple assay is versatile and suitable as a cost effective screening assay enabling quick turn-around for ADAMTS-13 activity reporting. Figure Shows a graphically representation of the method comparison between single test and ELISA results. Figure. Shows a graphically representation of the method comparison between single test and ELISA results. Disclosures Binder: Technoclone Herstellung von Diagnostika und Arzneimitteln GmbH: Employment. Griffiths:Technoclone Herstellung von Diagnostika und Arzneimitteln GmbH: Employment. Vetr:Technoclone Herstellung von Diagnostika und Arzneimitteln GmbH: Employment.

Background Bone destruction in rheumatoid arthritis is mediated by osteoclasts, which are derived from precursor cells of the myeloid lineage. Although there is much known about mature osteoclasts, the identity of osteoclast precursor populations during arthritis is poorly understood. Blood monocytes can be subdivided in classical inflammatory monocytes (CD115 + Ly6C high CCR2 + ) and non-classical resident monocytes (CD115 + Ly6C -/low CCR2 – ) and especially classical monocytes have been implicated in mediating tissue damage in autoimmunity. Objectives To investigate the role of different monocyte populations in joint destruction. Methods HTNFtg mice were clinically scored once per week for grip strength and swelling. In addition, blood was collected every other week starting on week 4. Mice were sacrificed at week 10 - blood, spleen and bone marrow were collected for flow cytometry analysis. K/BxN Arthritis was induced in wild type mice, blood and spleen were collected 14 days after disease induction. HTNFtg/CCR2 –/– and hTNFtg mice were analyzed histologically. Different monocyte subsets were Facs-sorted and cultured in the presence of RANKL and MCSF to induce osteoclasts. RNA sequencing of RANKL stimulated osteoclast precursor cells was performed. Results Here we show that hTNFtg mice lacking CCR2, which lack circulating classical inflammatory monocytes, show enhanced local bone erosion and osteoclast generation in chronic TNF driven arthritis. When we correlated the number of the two monocyte subsets in blood with histological signs of joint destruction the number of inflammatory monocytes did not correlate at all with those parameters. In contrast, the number of non-classical monocytes in blood significantly correlated with the extent of tissue damage in both hTNFtg arthritis and also K/BxN serum transfer arthritis. Histological examination revealed that while all infiltrating monocytes express CD115, only a small fraction of these cells express Ly6C, suggesting that the synovial infiltrate predominantly consists of Ly6C -/low monocytes. Upon sorting resident and from blood, we demonstrate that resident Ly6C -/low monocytes are more potent to form osteoclasts ex vivo than classical Ly6C high monocytes. Genome-wide transcriptome profiling revealed increased expression of genes which are required for pre-osteoclast fusion in RANKL-stimulated resident Ly6C -/low monocytes. Conclusions Non classical resident monocytes possess particular osteoclastogenic potential and their numbers in blood correlate with histological parameters of joint destruction in two different models of inflammatory arthritis. Therefore these cells may provide a biomarker for erosive inflammatory arthritis and even a possible target for therapeutically intervention. Disclosure of Interest None declared

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.

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-1β) 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-1β 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-1β from human monocytes and mouse macrophages. Importantly, using macrophages from mice deficient in components of the NALP3 inflammasome, we show PMMA-induced IL-1β 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.

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.

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.

The role of tumour necrosis factor (TNF) in the induction and maintenance of human rheumatoid arthritis is well established. However, the role of its two receptors, especially of TNFR2, is not sufficiently understood. The authors have previously demonstrated that lack of TNFR2 on haematopoietic cells leads to increased osteoclastogenesis in vitro and in …