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.

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.

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.

One-Year Follow-Up of COVID-19 Patients Indicates Substantial Assay-Dependent Differences in the Kinetics of SARS-CoV-2 Antibodies.

Determination of antibody levels against the nucleocapsid (N) and spike (S) proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are used to estimate the humoral immune response after SARS-CoV-2 infection or vaccination. Differences in the design and specification of antibody assays challenge the interpretation of test results, and comparative studies are often limited to single time points per patient. We determined the longitudinal kinetics of antibody levels of 145 unvaccinated coronavirus disease 2019 (COVID-19) patients at four visits over 1 year upon convalescence using 8 commercial SARS-CoV-2 antibody assays (from Abbott, DiaSorin, Roche, Siemens, and Technoclone), as well as a virus neutralization test (VNT).

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.

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.

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.

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.

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.

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.

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.

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.

Def6 Restrains Osteoclastogenesis and Inflammatory Bone Resorption.

Inflammatory bone resorption mediated by osteoclasts is a major cause of morbidity and disability in many inflammatory disorders, including rheumatoid arthritis (RA). The mechanisms that regulate osteoclastogenesis and bone resorption in inflammatory settings are complex and have not been well elucidated. In this study, we identify the immunoregulator differentially expressed in FDCP 6 homolog (Def6) as a novel inhibitor of osteoclastogenesis in physiological and inflammatory conditions.

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.

iRHOM2 is a critical pathogenic mediator of inflammatory arthritis.

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.

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.

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.

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-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.

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.

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.

Activation of the interferon-gamma signaling pathway in systemic lupus erythematosus peripheral blood mononuclear cells.

Objective: 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.

Methods: 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.

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.