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Background: Immune thrombotic thrombocytopenic purpura (i-TTP) is a life-threatening thrombotic microangiopathy linked to ADAMTS-13 deficiency. It has long been assumed that the activation of endothelial cells is the triggering factor for the thrombotic thrombocytopenic purpura crisis. Circulating endothelial cells (CECs) have been shown to be a biomarker of vascular damage and are associated with the clinical severity of i-TTP. However, the mechanisms leading to endothelial cell detachment remain unclear., Objectives: We investigated junctional destabilization the mechanisms underlying cell detachment in thrombotic thrombocytopenic purpura., Methods: We quantified CECs in i-TTP patients and investigated the effect of plasmas in vitro by measuring phosphorylation and internalization of vascular endothelial (VE)-Cadherin and in vivo in a vascular permeability model., Results: In plasma from i-TTP patients, we show that CEC count is associated with severity and correlated to intracellular calcium influx (P < .01). In vitro, serum from i-TTP patients induced stronger detachment of human umbilical vein endothelial cells than serum from control patients (P < .001). Plasma from i-TTP patients induced a higher calcium-dependent phosphorylation (P < .05) and internalization (P < .05) of VE-cadherin compared with plasma from control patients. This effect could be reproduced by immunoglobulin (Ig)G fraction isolated from patient plasma and, in particular, by the F(ab)'2 fragments of the corresponding IgG. In addition, subcutaneous injection of i-TTP plasma into mice resulted in higher vascular permeability than plasma from control patients. An inhibitor of endothelial calcium influx, ITF1697, normalized this increase in permeability., Conclusion: Our results suggest that plasma-induced endothelial activation also leads to an increase in vascular permeability. They contribute to the understanding of the mechanisms behind the presence of elevated CECs in patients' blood by linking endothelial activation to endothelial injury., Competing Interests: Declaration of competing interests P.P. is a member of the scientific advisory boards of Ablynx-Sanofi. The other authors declare no competing financial interests., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Background: Laboratory diagnosis of immune-mediated thrombotic thrombocytopenic purpura (iTTP) remains challenging when ADAMTS-13 activity ranges between 10% and 20%. To prevent misdiagnosis, open ADAMTS-13 conformation gained clinical attention as a novel biomarker, especially to diagnose acute iTTP in patients with diagnostic undecisive ADAMTS-13 activity. Plasma ADAMTS-13 conformation analysis corrects for ADAMTS-13 antigen, with both parameters being characterized in enzyme-linked immunosorbent assay (ELISA)-based reference assays requiring expert technicians., Objectives: To design ADAMTS-13 antigen and conformation assays on automated, easy-to-use fiber optic surface plasmon resonance (FO-SPR) technology to promote assay accessibility and diagnose challenging iTTP patients., Methods: ADAMTS-13 antigen and conformation assays were designed on FO-SPR technology. Plasma of 20 healthy donors and 20 acute iTTP patients were quantified, and data from FO-SPR and ELISA reference assays were compared., Results: Following assay design, both antigen and conformation FO-SPR assays were optimized and characterized, presenting strong analytical sensitivity (detection limit of 0.001 μg/mL) and repeatability (interassay variation of 14.4%). Comparative analysis suggested positive correlation (Spearman r of 0.92) and good agreement between FO-SPR and ELISA assays. As expected, FO-SPR assays showed a closed or open ADAMTS-13 conformation in healthy donors and acute iTTP patients, respectively., Conclusion: Both ADAMTS-13 antigen and conformation assays were transferred onto automated, easy-to-use FO-SPR technology, displaying potent analytical sensitivity and reproducibility. ADAMTS-13 antigen and conformation were determined for healthy donors and acute iTTP patients showing strong correlation with ELISA reference. Introducing FO-SPR technology in clinical context could support routine diagnosis of acute iTTP patients, notably when ADAMTS-13 activity fluctuates between 10% and 20%., Competing Interests: Declaration of competing interests J.L. is a member of the Board of Directors of FOx Biosystems. Q.B., C.D., T.B., E.T., G.K., B.S.J., A.V., P.C., C.T., S.F.D.M., and K.V. have no conflicts of interest to disclose., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Abstract: Thrombotic thrombocytopenic purpura (TTP), a rare but fatal disease if untreated, is due to alteration in von Willebrand factor cleavage resulting in capillary microthrombus formation and ischemic organ damage. Interleukin-1 (IL-1) has been shown to drive sterile inflammation after ischemia and could play an essential contribution to postischemic organ damage in TTP. Our objectives were to evaluate IL-1 involvement during TTP and to test the efficacy of the recombinant IL-1 receptor antagonist, anakinra, in a murine TTP model. We retrospectively measured plasma IL-1 concentrations in patients with TTP and controls. Patients with TTP exhibited elevated plasma IL-1α and -1β concentrations, which correlated with disease course and survival. In a mouse model of TTP, we administered anakinra (IL-1 inhibitor) or placebo for 5 days and evaluated the efficacy of this treatment. Anakinra significantly reduced mortality of mice (P < .001). Anakinra significantly decreased TTP-induced cardiac damage as assessed by blood troponin concentrations, evaluation of left ventricular function by echocardiography, [18F]fluorodeoxyglucose positron emission tomography of myocardial glucose metabolism, and cardiac histology. Anakinra also significantly reduced brain TTP-induced damage evaluated through blood PS100b concentrations, nuclear imaging, and histology. We finally showed that IL-1α and -1β trigger endothelial degranulation in vitro, leading to the release of von Willebrand factor. In conclusion, anakinra significantly reduced TTP mortality in a preclinical model of the disease by inhibiting both endothelial degranulation and postischemic inflammation, supporting further evaluations in humans., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Objective: IL-1β is a leaderless cytokine with poorly known secretory mechanisms that is barely detectable in serum of patients, including those with an IL-1β-mediated disease such as systemic juvenile idiopathic arthritis (sJIA). Leukocyte microvesicles (MVs) may be a mechanism of IL-1β secretion. The first objective of our study was to characterize IL-1β-positive MVs obtained from macrophage cell culture supernatants and to investigate their biological functions in vitro and in vivo . The second objective was to detect circulating IL-1β-positive MVs in JIA patients., Methods: MVs were purified by serial centrifugations from PBMCs, or THP-1 differentiated into macrophages, then stimulated with LPS ± ATP. MV content was analyzed for the presence of IL-1β, NLRP3 inflammasome, caspase-1, P2X7 receptor, and tissue factor (TF) using ELISA, Western blot, or flow cytometry. MV biological properties were studied in vitro by measuring VCAM-1, ICAM-1, and E-selectin expression after HUVEC co-culture and factor-Xa generation test was realized. In vivo , MVs' ability to recruit leukocytes in a murine model of peritonitis was evaluated. Plasmatic IL-1β-positive MVs were studied ex vivo in 10 active JIA patients using flow cytometry., Results: THP-1-derived macrophages stimulated with LPS and ATP released MVs, which contained NLRP3, caspase-1, and the 33-kDa precursor and 17-kDa mature forms of IL-1β and bioactive TF. IL-1β-positive MVs expressed P2X7 receptor and released soluble IL-1β in response to ATP stimulation in vitro . In mice, MVs induced a leukocyte peritoneal infiltrate, which was reduced by treatment with the IL-1 receptor antagonist. Finally, IL-1β-positive MVs were detectable in plasma from 10 active JIA patients., Conclusion: MVs shed from activated macrophages contain IL-1β, NLRP3 inflammasome components, and TF, and constitute thrombo-inflammatory vectors that can be detected in the plasma from active JIA patients., Competing Interests: The 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 © 2023 Cambon, Rebelle, Bachelier, Arnaud, Robert, Lagarde, Muller, Tellier, Kara, Leroyer, Farnarier, Vallier, Chareyre, Retornaz, Jurquet, Tran, Lacroix, Dignat-George and Kaplanski.)

Background: Thrombotic thrombocytopenic purpura (TTP) is characterized by severe ADAMTS-13 activity deficiency (<10%). Diagnostic testing is challenging because of unavailability, high cost, and expert technician requirement of ADAMTS-13 enzyme assays. Cost-effective, automated fiber-optic surface plasmon resonance (FO-SPR) platforms show potential for developing diagnostic tests. Yet, FO-SPR has never been explored to measure enzymatic activities., Objectives: To develop an easy-to-use ADAMTS-13 activity assay utilizing optical fibers to rapidly diagnose TTP., Methods: The ADAMTS-13 activity assay was designed and optimized using FO-SPR technology based on a previously described enzyme-linked immunosorbent assay setup. A calibration curve was generated to quantify ADAMTS-13 activity in plasma of healthy donors and patients with acute immune-mediated TTP (iTTP), hemolytic uremic syndrome, or sepsis. ADAMTS-13 activity data from FO-SPR and fluorescence resonance energy transfer-based strategies (FRETS)-VWF73 reference assays were compared., Results: After initial assay development, optimization improved read-out magnitude and signal-to-noise ratio and reduced variation. Further characterization demonstrated a detection limit (6.8%) and inter-assay variation (Coefficient of variation, 7.2%) that showed good analytical sensitivity and repeatability. From diverse plasma samples, only plasma from patients with acute iTTP showed ADAMTS-13 activities below 10%. Strong Pearson correlation ( r  = 0.854) between FO-SPR and reference FRETS-VWF73 assays were observed for all measured samples., Conclusions: A fast ADAMTS-13 activity assay was designed onto automated FO-SPR technology. Optimization resulted in sensitive ADAMTS-13 activity measurements with a detection limit enabling clinical diagnosis of TTP within 3 hours. The FO-SPR assay proved strong correlation with the reference FRETS-VWF73 assay. For the first time, this assay demonstrated the capacity of FO-SPR technology to measure enzymatic activity in pre-clinical context., (© 2023 The Author(s).)

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is characterized by a severe ADAMTS13 deficiency due to the presence of anti-ADAMTS13 auto-antibodies, with subsequent accumulation of circulating ultra-large von Willebrand factor (VWF) multimers. The role of endothelial cell activation as a trigger of the disease has been suggested in animal models but remains to be demonstrated in humans. We prospectively obtained plasma from the first plasma exchange of 25 patients during iTTP acute phase. iTTP but not control plasma, induced a rapid VWF release and P-selectin exposure on the surface of dermal human micro-vascular endothelial cell (HMVEC-d), associated with angiopoietin-2 and endothelin-1 secretion, consistent with Weibel-Palade bodies exocytosis. Calcium (Ca2+) blockade significantly decreased VWF release, whereas iTTP plasma induced a rapid and sustained Ca2+ flux in HMVEC-d which correlated in retrospect, with disease severity and survival in 62 iTTP patients. F(ab)'2 fragments purified from the immunoglobulin G fraction of iTTP plasma mainly induced endothelial cell activation with additional minor roles for circulating free heme and nucleosomes, but not for complement. Furthermore, two anti-ADAMTS13 monoclonal antibodies purified from iTTP patients' B cells, but not serum from hereditary TTP, induced endothelial Ca2+ flux associated with Weibel-Palade bodies exocytosis in vitro, whereas inhibition of endothelial ADAMTS13 expression using small intering RNA, significantly decreased the stimulating effects of iTTP immunoglobulin G. In conclusion, Ca2+-mediated endothelial cell activation constitutes a "second hit" of iTTP, is correlated with the severity of the disease and may constitute a possible therapeutic target.

Thrombotic thrombocytopenic purpura (TTP) is a severe thrombotic microangiopathy. The current pathophysiologic paradigm suggests that the ADAMTS13 deficiency leads to Ultra Large-Von Willebrand Factor multimers accumulation with generation of disseminated microthrombi. Nevertheless, the role of endothelial cells in this pathology remains an issue. In this review, we discuss the various clinical, in vitro and in vivo experimental data that support the important role of the endothelium in this pathology, suggesting that ADAMTS13 deficiency may be a necessary but not sufficient condition to induce TTP. The "second hit" model suggests that in TTP, in addition to ADAMTS13 deficiency, endogenous or exogenous factors induce endothelial activation affecting mainly microvascular cells. This leads to Weibel-Palade bodies degranulation, resulting in UL-VWF accumulation in microcirculation. This endothelial activation seems to be worsened by various amplification loops, such as the complement system, nucleosomes and free heme.

Background: Public health surveillance relies on the collection of data, often in near-real time. Recent advances in natural language processing make it possible to envisage an automated system for extracting information from electronic health records., Objective: To study the feasibility of setting up a national trauma observatory in France, we compared the performance of several automatic language processing methods in a multiclass classification task of unstructured clinical notes., Methods: A total of 69,110 free-text clinical notes related to visits to the emergency departments of the University Hospital of Bordeaux, France, between 2012 and 2019 were manually annotated. Among these clinical notes, 32.5% (22,481/69,110) were traumas. We trained 4 transformer models (deep learning models that encompass attention mechanism) and compared them with the term frequency-inverse document frequency associated with the support vector machine method., Results: The transformer models consistently performed better than the term frequency-inverse document frequency and a support vector machine. Among the transformers, the GPTanam model pretrained with a French corpus with an additional autosupervised learning step on 306,368 unlabeled clinical notes showed the best performance with a micro F 1 -score of 0.969., Conclusions: The transformers proved efficient at the multiclass classification of narrative and medical data. Further steps for improvement should focus on the expansion of abbreviations and multioutput multiclass classification., (©Gabrielle Chenais, Cédric Gil-Jardiné, Hélène Touchais, Marta Avalos Fernandez, Benjamin Contrand, Eric Tellier, Xavier Combes, Loick Bourdois, Philippe Revel, Emmanuel Lagarde. Originally published in JMIR AI (https://ai.jmir.org), 12.01.2023.)

Sustained eutrophication of the aquatic environment by the remobilization of legacy phosphorus (P) stored in soils and sediments is a prevailing issue worldwide. Fluxes of P from the sediments to the water column, referred to as internal P loading, often delays the recovery of water quality following a reduction in external P loads. Here, we report on the vertical distribution and geochemistry of P, lanthanum (La), iron (Fe) and carbon (C) in the culturally eutrophied Lake Bromont. This lake underwent remediation treatment using La modified bentonite (LMB) commercially available as Phoslock™. We investigated the effectiveness of LMB in decreasing soluble reactive phosphorus (SRP) availability in sediments and in reducing dissolved fluxes of P across the sediment-water interface. Sediment cores were retrieved before and after LMB treatment at three sites representing bottom sediment, sediment influenced by lakeside housing and finally littoral sediment influenced by the lake inflow. Sequential extractions were used to assess changes in P speciation. Depth profiles of dissolved porewater concentrations were obtained after LMB treatment at each site. Results indicate that SRP extracted from the sediments decreased at all sites, while total extracted P (P TOT ) bound to redox-sensitive metal oxides increased. 31 P NMR data on P extract reveals that 20-43% of total solid-phase P is in the form of organic P (P org ) susceptible to be released via microbial degradation. Geochemical modelling of porewater data provides evidence that LaPO 4(s) mineral phases, such as rhabdophane and/or monazite, are likely forming. However, results also suggest that La 3+ binding by dissolved organic carbon (DOC) hinders La-phosphate precipitation. We rely on thermodynamic modelling to suggest that high Fe 2+ would bind to DOC instead of La 3+ , therefore promoting P sequestrations by LMB under anoxic conditions.

Competing Interests: Declaration of Competing Interest The authors report no proprietary or commercial interest in any product mentioned or concept discussed in this article.

During periods such as the COVID-19 crisis, there is a need for responsive public health surveillance indicators related to the epidemic. To determine the performance of keyword-search algorithm in call reports to emergency medical communication centers (EMCC) to describe trends in symptoms during the COVID-19 crisis. We retrospectively retrieved all free text call reports from the EMCC of the Gironde department (SAMU 33), France, between 2005 and 2020 and classified them with a simple keyword-based algorithm to identify symptoms relevant to COVID-19. A validation was performed using a sample of manually coded call reports. The six selected symptoms were fever, cough, muscle soreness, dyspnea, ageusia and anosmia. We retrieved 38,08,243 call reports from January 2005 to October 2020. A total of 8539 reports were manually coded for validation and Cohen's kappa statistics ranged from 75 (keyword anosmia) to 59% (keyword dyspnea). There was an unprecedented peak in the number of daily calls mentioning fever, cough, muscle soreness, anosmia, ageusia, and dyspnea during the COVID-19 epidemic, compared to the past 15 years. Calls mentioning cough, fever and muscle soreness began to increase from February 21, 2020. The number of daily calls reporting cough reached 208 on March 3, 2020, a level higher than any in the previous 15 years, and peaked on March 15, 2020, 2 days before lockdown. Calls referring to dyspnea, anosmia and ageusia peaked 12 days later and were concomitant with the daily number of emergency room admissions. Trends in symptoms cited in calls to EMCC during the COVID-19 crisis provide insights into the natural history of COVID-19. The content of calls to EMCC is an efficient epidemiological surveillance data source and should be integrated into the national surveillance system., (© 2021. Società Italiana di Medicina Interna (SIMI).)

Background: Takayasu arteritis (TAK) is a large vessel vasculitis resulting in artery wall remodeling with segmental stenosis and/or aneurysm formation. Mast cells (MCs) are instrumental in bridging cell injury and inflammatory response., Objectives: This study sought to investigate the contribution of MCs on vessel permeability, angiogenesis, and fibrosis in patients with TAK., Methods: MC activation and their tissue expression were assessed in sera and in aorta from patients with TAK and from healthy donors (HDs). In vivo permeability was assessed using a modified Miles assay. Subconfluent cultured human umbilic vein endothelial cells and fibroblasts were used in vitro to investigate the effects of MC mediators on angiogenesis and fibrogenesis., Results: This study found increased levels of MC activation markers (histamine and indoleamine 2,3-dioxygenase) in sera of patients with TAK compared with in sera of HDs. Marked expression of MCs was shown in aortic lesions of patients with TAK compared with in those of noninflammatory aorta controls. Using Miles assay, this study showed that sera of patients with TAK significantly increased vascular permeability in vivo as compared with that of HDs. Vessel permeability was abrogated in MC-deficient mice. MCs stimulated by sera of patients with TAK supported neoangiogenesis (increased human umbilic vein endothelial cell proliferation and branches) and fibrosis by inducing increased production of fibronectin, type 1 collagen, and α-smooth muscle actin by fibroblasts as compared to MCs stimulated by sera of HD., Conclusions: MCs are a key regulator of vascular lesions in patients with TAK and may represent a new therapeutic target in large vessel vasculitis., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by an autoantibody-mediated deficiency in ADAMTS13. In healthy individuals, ADAMTS13 has a folded conformation in which the central spacer (S) domain interacts with the C-terminal CUB domains. We recently showed that ADAMTS13 adopts an open conformation in iTTP and that patient immunoglobulin G antibodies (IgGs) can open ADAMTS13. Anti-ADAMTS13 autoantibodies in patients with iTTP are directed against the different ADAMTS13 domains, but almost all patients have autoantibodies binding to the cysteine/spacer (CS) domains. In this study, we investigated whether the autoantibodies against the CS and CUB domains can disrupt the S-CUB interaction of folded ADAMTS13, thereby opening ADAMTS13. To this end, we purified anti-CS and anti-CUB autoantibodies from 13 patients with acute iTTP by affinity chromatography. The successfully purified anti-CS (10/13 patients) and anti-CUB (4/13 patients) autoantibody fractions were tested further in our ADAMTS13 conformation enzyme-linked immunosorbent assay to study whether they could open ADAMTS13. Interestingly, all purified anti-CS fractions (10/10 patients) were able to open ADAMTS13. On the other hand, only half of the purified anti-CUB fractions (2/4 patients) opened ADAMTS13. Our finding highlights that anti-CS autoantibodies that open ADAMTS13 are a common feature of the autoimmune response in iTTP., (© 2021 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by inhibitory and/or clearing anti-ADAMTS-13 (A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats, member 13) autoantibodies. To determine the presence and total level of anti-ADAMTS-13 autoantibodies, commercial and in-house developed ELISAs are performed. However, different ELISA methods vary in relation to the presentation of recombinant (r)ADAMTS-13 and the detection method of the anti-ADAMTS-13 autoantibodies. Currently, the influence of those different approaches on anti-ADAMTS-13 autoantibody titers is not known., Objectives: To assess the influence of different ADAMTS-13 presentation- and autoantibody detection methods on anti-ADAMTS-13 autoantibody titers in ELISA., Materials/methods: Anti-ADAMTS-13 autoantibody titers from 18 iTTP patients were determined using four different set-ups of anti-ADAMTS-13 autoantibody ELISAs. The ELISAs varied in the used presentation of rADAMTS-13 (directly coated full-length rADAMTS-13, directly coated rMDTCS and rT2C2, or antibody-captured full-length rADAMTS-13) and the detection antibodies (polyclonal anti-human IgG or monoclonal anti-human IgG 1-4 antibodies)., Results: Strong correlations between the different anti-ADAMTS-13 autoantibody ELISA approaches were observed, when using polyclonal anti-human IgG detection antibodies recognizing all IgG subclasses similarly, independent of the method of rADAMTS-13 presentation. Anti-ADAMTS-13 autoantibody titers correlated less when using a mixture of monoclonal anti-human IgG 1-4 , because not all IgG subclasses were recognized with similar affinities., Conclusion: Anti-ADAMTS-13 autoantibody levels using different methods of rADAMTS-13 presentation strongly correlate. However, the levels of anti-ADAMTS-13 autoantibodies are highly dependent on the detection antibody used, which should detect all IgG subclasses (IgG 1-4 ) equally well., (© 2021 International Society on Thrombosis and Haemostasis.)

Objective: We assessed the performance of the ratio of peripheral arterial oxygen saturation to the inspired fraction of oxygen (SpO 2 /FiO 2 ) to predict the ratio of partial pressure arterial oxygen to the fraction of inspired oxygen (PaO 2 /FiO 2 ) among patients admitted to our emergency department (ED) during the SARS-CoV-2 outbreak., Methods: We retrospectively studied patients admitted to an academic-level ED in France who were undergoing a joint measurement of SpO 2 and arterial blood gas. We compared SpO 2 with SaO 2 and evaluated performance of the SpO 2 /FiO 2 ratio for the prediction of 300 and 400 mmHg PaO 2 /FiO 2 cut-off values in COVID-19 positive and negative subgroups using receiver-operating characteristic (ROC) curves., Results: During the study period from February to April 2020, a total of 430 arterial samples were analyzed and collected from 395 patients. The area under the ROC curves of the SpO 2 /FiO 2 ratio was 0.918 (CI 95% 0.885-0.950) and 0.901 (CI 95% 0.872-0.930) for PaO 2 /FiO 2 thresholds of 300 and 400 mmHg, respectively. The positive predictive value (PPV) of an SpO 2 /FiO 2 threshold of 350 for PaO 2 /FiO 2 inferior to 300 mmHg was 0.88 (CI95% 0.84-0.91), whereas the negative predictive value (NPV) of the SpO 2 /FiO 2 threshold of 470 for PaO 2 /FiO 2 inferior to 400 mmHg was 0.89 (CI95% 0.75-0.96). No significant differences were found between the subgroups., Conclusions: The SpO 2 /FiO 2 ratio may be a reliable tool for hypoxemia screening among patients admitted to the ED, particularly during the SARS-CoV-2 outbreak., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare., (Copyright © 2021. Published by Elsevier Inc.)

Objectives: During periods such as the COVID-19 crisis, there is a need for responsive public health surveillance indicators in order to monitor both the epidemic growth and potential public health consequences of preventative measures such as lockdown. We assessed whether the automatic classification of the content of calls to emergency medical communication centers could provide relevant and responsive indicators., Methods: We retrieved all 796,209 free-text call reports from the emergency medical communication center of the Gironde department, France, between 2018 and 2020. We trained a natural language processing neural network model with a mixed unsupervised/supervised method to classify all reasons for calls in 2020. Validation and parameter adjustment were performed using a sample of 39,907 manually-coded free-text reports., Results: The number of daily calls for flu-like symptoms began to increase from February 21, 2020 and reached an unprecedented level by February 28, 2020 and peaked on March 14, 2020, 3 days before lockdown. It was strongly correlated with daily emergency room admissions, with a delay of 14 days. Calls for chest pain and stress and anxiety, peaked 12 days later. Calls for malaises with loss of consciousness, non-voluntary injuries and alcohol intoxications sharply decreased, starting one month before lockdown. No noticeable trends in relation to lockdown was found for other groups of reasons including gastroenteritis and abdominal pain, stroke, suicide and self-harm, pregnancy and delivery problems., Discussion: The first wave of the COVID-19 crisis came along with increased levels of stress and anxiety but no increase in alcohol intoxication and violence. As expected, call related to road traffic crashes sharply decreased. The sharp decrease in the number of calls for malaise was more surprising., Conclusion: The content of calls to emergency medical communication centers is an efficient epidemiological surveillance data source that provides insights into the societal upheavals induced by a health crisis. The use of an automatic classification system using artificial intelligence makes it possible to free itself from the context that could influence a human coder, especially in a crisis situation. The COVID-19 crisis and/or lockdown induced deep modifications in the population health profile.

Immune Thrombotic Thrombocytopenic Purpura (iTTP) is a rare but severe disease with a mortality rate of almost 100 % in the absence of adequate treatment. iTTP is caused by a severe deficiency in ADAMTS13 activity due to the production of inhibitory antibodies. Age has been shown to be a major prognostic factor. iTTP patients in the elderly (60yo and over) have more frequent organ involvement, especially heart and kidney failures compared with younger patients. They also have non-specific neurologic symptoms leading to a delayed diagnosis. Factors influencing this impaired survival among older patients remain unknown so far. Alteration of the functional capacity of involved organs could be part of the explanation as could be the consequences of vascular aging. In fact, severe ADAMTS13 deficiency is necessary but likely not sufficient for iTTP physiopathology. A second hit leading to endothelial activation is thought to play a central role in iTTP. Interestingly, the mechanisms involved in endothelial activation may share common features with those involved in vascular aging, potentially leading to endothelial dysfunction. It could thus be interesting to better investigate the causes of mid- and long-term mortality among older iTTP patients to confirm whether inflammation and endothelial activation really impact vascular aging and long-term mortality in those patients, in addition to their presumed role at iTTP acute phase. If so, further insights into the mechanisms involved could lead to new therapeutic targets., (Copyright © 2021 Société Nationale Française de Médecine Interne (SNFMI). Published by Elsevier Masson SAS. All rights reserved.)

Background: Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by anti-ADAMTS13 autoantibodies inducing a severe deficiency of ADAMTS13. Epitope mapping studies on samples obtained during acute iTTP episodes have shown that the iTTP immune response is polyclonal, with almost all patients having autoantibodies targeting the spacer domain of ADAMTS13., Objectives: To identify the immunogenic hotspots in the spacer domain of ADAMTS13., Patients/methods: A library of 11 full-length ADAMTS13 spacer hybrids was created in which amino acid regions of the spacer domain of ADAMTS13 were exchanged by the corresponding region of the spacer domain of ADAMTS1. Next, the full-length ADAMTS13 spacer hybrids were used in enzyme-linked immunosorbent assay to epitope map anti-spacer autoantibodies in 138 samples from acute and remission iTTP patients., Results: Sixteen different anti-spacer autoantibody profiles were identified with a similar distribution in acute and remission patients. There was no association between the anti-spacer autoantibody profiles and disease severity. Almost all iTTP samples contained anti-spacer autoantibodies against the following three regions: amino acid residues 588-592, 602-610, and 657-666 (hybrids E, G, and M). Between 31% and 57% of the samples had anti-spacer autoantibodies against amino acid regions 572-579, 629-638, 667-676 (hybrids C, J, and N). In contrast, none of the samples had anti-spacer autoantibodies against amino acid regions 556-563, 564-571, 649-656, and 677-685 (hybrids A, B, L, and O)., Conclusion: We identified three hotspot regions (amino acid regions 588-592, 602-610, and 657-666) in the spacer domain of ADAMTS13 that are targeted by anti-spacer autoantibodies found in a large cohort of iTTP patients., (© 2020 International Society on Thrombosis and Haemostasis.)

Autoantibodies are key biomarkers in clinical diagnosis of autoimmune diseases routinely detected by enzyme-linked immunosorbent assays (ELISAs). However, the complexity of these assays is limiting their use in routine diagnostics. Fiber optic-surface plasmon resonance (FO-SPR) can overcome these limitations, but improved surface chemistries are still needed to guarantee detection of autoantibodies in complex matrices. In this paper, we describe the development of an FO-SPR immunoassay for the detection of autoantibodies in plasma samples from immune-mediated thrombotic thrombocytopenic purpura (iTTP) patients. Hereto, hexahistidine-tagged recombinant ADAMTS13 (rADAMTS13-His 6 ) was immobilized on nitrilotriacetic acid (NTA)-coated FO probes chelated by cobalt (Co(III)) and exposed to anti-ADAMTS13 autoantibodies. Initial studies were performed to optimize rADAMTS13-His 6 immobilization and to confirm the specificity of the immunoassay for detection of anti-ADAMTS13 autoantibodies with FO-SPR. The performance of the immunoassay was then evaluated by comparing Co(III)- and nickel (Ni(II))-NTA stabilized surfaces, confirming the stable immobilization of the antigen in Co(III)-NTA-functionalized FO probes. A calibration curve was prepared with a dilution series of a cloned human anti-ADAMTS13 autoantibody in ADAMTS13-depleted plasma resulting in an average interassay coefficient of variation of 7.1% and a limit of detection of 0.24 ng/mL. Finally, the FO-SPR immunoassay was validated using seven iTTP patient plasma samples, resulting in an excellent correlation with an in-house-developed ELISA ( r = 0.973). In summary, the specificity and high sensitivity in combination with a short time-to-result (2.5 h compared to 4-5 h for a regular ELISA) make the FO-SPR immunoassay a powerful assay for routine diagnosis of iTTP and with extension for any other autoimmune disease.

Recently, we showed that ADAMTS13 circulates in an open conformation during the acute phase of immune-mediated thrombotic thrombocytopenic purpura (iTTP). Although the cause of this conformational change remains elusive, ADAMTS13 is primarily closed in iTTP patients in remission with ADAMTS13 activity >50% and undetectable anti-ADAMTS13 autoantibodies, as well as after rituximab treatment, suggesting a role for anti-ADAMTS13 autoantibodies. Therefore, immunoglobulin G from 18 acute iTTP patients was purified and added to closed ADAMTS13 in healthy donor plasma. This resulted in open ADAMTS13 in 14 of 18 (78%) samples, proving that anti-ADAMTS13 autoantibodies can induce an open ADAMTS13 conformation. To further elucidate the conformation of ADAMTS13 in iTTP patients, we studied a novel iTTP patient cohort (n = 197) that also included plasma samples from iTTP patients in remission in whom ADAMTS13 activity was <50%. The open ADAMTS13 conformation was found during acute iTTP, as well as in patients in remission with ADAMTS13 activity <50% and in half of the patients with ADAMTS13 activity >50%, although free anti-ADAMTS13 autoantibodies were not always detected. Thus, open ADAMTS13 is a hallmark of acute iTTP, as well as a novel biomarker that can be used to detect subclinical iTTP in patients in remission. Finally, a long-term follow-up study in 1 iTTP patient showed that the open conformation precedes a substantial drop in ADAMTS13 activity. In conclusion, we have shown that anti-ADAMTS13 autoantibodies from iTTP patients induce an open ADAMTS13 conformation. Most importantly, an open ADAMTS13 conformation is a biomarker for subclinical iTTP and could become an important tool in TTP management., (© 2020 by The American Society of Hematology.)

Background: In immune-mediated thrombotic thrombocytopenic purpura (iTTP), patients develop an immune response against the multidomain enzyme ADAMTS13. ADAMTS13 consists of a metalloprotease (M) and disintegrin-like (D) domain, 8 thrombospondin type 1 repeats (T1-T8), a cysteine-rich (C), a spacer (S), and 2 CUB domains (CUB1-2). Previous epitope mapping studies have used relatively large overlapping ADAMTS13 fragments., Objectives: We aimed at developing small nonoverlapping ADAMTS13 fragments to fine map anti-ADAMTS13 autoantibodies in iTTP patients., Methods: A library of 16 ADAMTS13 fragments, comprising several small (M, DT, C, S, T2-T5, T6-T8, CUB1, CUB2), and some larger fragments with overlapping domains (MDT, MDTC, DTC, CS, T2-T8, CUB1-2, MDTCS, T2-C2), were generated. All fragments, and ADAMTS13, were expressed as a fusion protein with albumin domain 1, and purified. The folding of the fragments was tested using 17 anti-ADAMTS13 monoclonal antibodies with known epitopes. An epitope mapping assay using small ADAMTS13 fragments was set up, and validated by analyzing 18 iTTP patient samples., Results: Validation with the monoclonal antibodies demonstrated that single S and CUB1 were not correctly folded, and therefore CS and CUB1-2 fragments were selected instead of single C, S, CUB1, and CUB2 fragments. Epitope mapping of antibodies of patients with iTTP confirmed that 6 nonoverlapping ADAMTS13 fragments M, DT, CS, T2-T5, T6-T8, and CUB1-2 were sufficient to accurately determine the antibody-binding sites., Conclusion: We have developed a tool to profile patients with iTTP according to their anti-ADAMTS13 antibodies for a better insight in their immune response., (© 2020 Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

The mechanisms regulating inflammation in large vessels vasculitis (LVV) are poorly understood. Interleukin 33 (IL-33) has been shown to license innate and adaptive immunity by enhancing Th2 cytokines production. We aimed to examine the role of IL-33 in the immunomodulation of T cell activation in LVV. T cell homeostasis and cytokines production were determined in peripheral blood from 52 patients with giant cell arteritis (GCA) and 50 healthy donors (HD), using Luminex assay, flow cytometry, quantitative RT-PCR and by immunofluorescence analysis in inflammatory aorta lesions. We found increased level of IL-33 and its receptor ST2/IL-1R4 in the serum of patient with LVV. Endothelial cells were the main source of IL-33, whereas Th2 cells, Tregs and mast cells (MC) express ST2 in LVV vessels. IL-33 had a direct immunomodulatory impact by increasing Th2 and Tregs. IL-33 and MC further enhanced Th2 and regulatory responses by inducing a 6.1 fold increased proportion of Tregs (p = 0.008). Stimulation of MC by IL-33 increased indoleamine 2 3-dioxygenase (IDO) activity and IL-2 secretion. IL-33 mRNA expression was significantly correlated with the expression of IL-10 and TGF-β within aorta inflammatory lesions. To conclude, our findings suggest that IL-33 may exert a critical immunoregulatory role in promoting Tregs and Th2 cells in LVV.

Background: The biological diagnosis of immune-mediated thrombotic thrombocytopenic purpura (iTTP) is based on determination of ADAMTS13 activity (<10%) and anti-ADAMTS13 autoantibodies. ADAMTS13 antigen levels are not routinely measured in iTTP patients, but studies have shown that antigen levels are a valuable prognostic factor., Objectives: To (a) report the validation of our in-house developed ADAMTS13 antigen enzyme-linked immunosorbent assay (ELISA) and determine ADAMTS13 antigen in a large cohort of healthy donor and iTTP patient plasma samples; and (b) to investigate whether ADAMTS13 antigen determination is not disturbed by the presence of anti-ADAMTS13 autoantibodies., Methods: Our in-house ADAMTS13 antigen ELISA was validated in terms of sensitivity, repeatability, and reproducibility. ADAMTS13 antigen levels were determined in plasma samples from 423 healthy donors and 112 acute iTTP patients. Purified IgGs from iTTP patients were added to normal human plasma to determine whether anti-ADAMTS13 autoantibodies hampered ADAMTS13 antigen determination., Results: Our in-house ADAMTS13 antigen ELISA has a detection limit of 3% and low intra-assay (coefficient of variation, %CV < 10%) and inter-assay (%CV < 18%) variability. ADAMTS13 antigen levels were significantly reduced (P < .0001) in acute iTTP patients (15 ± 18%) compared to healthy donors (101 ± 18%). The anti-ADAMTS13 autoantibodies in plasma of iTTP patients did not impede ADAMTS13 antigen determinations using our in-house ELISA., Conclusions: Our in-house ADAMT13 antigen ELISA is a powerful tool to correctly determine ADAMTS13 antigen levels in iTTP patients, which supports routine ADAMTS13 antigen measurements in these patients to have better insight into disease prognosis., (© 2020 International Society on Thrombosis and Haemostasis.)

Introduction: Recent research suggests that up to 20% of minor trauma patients admitted to the emergency department (ED) will suffer from non-specific chronic conditions over the subsequent several months. Thus, the present study assessed the correlates of symptoms that persisted at 4 months after an ED visit and, in particular, evaluated the associations between these symptoms and self-reported stress levels at ED admission and discharge., Method: This study was a prospective observational investigation conducted in the ED of Bordeaux University Hospital that included patients admitted for minor trauma. All participants were contacted by phone 4 months after presentation at the ED to assess the occurrence of post-concussion-like symptoms (PCLS)., Results: A total of 193 patients completed the follow-up assessment at 4 months; 5.2% of the participants suffered from post-traumatic stress disorder (PTSD) and 24.5% suffered from PCLS. A multivariate analysis revealed an association between PCLS and stress level at discharge from the ED (odds ratios [OR]: 2.85, 95% confidence interval [CI]: 1.10-7.40)., Conclusions: The risk of PCLS at 4 months after an ED visit for a minor injury increased in association with the level of stress at discharge from the ED. These results may improve the quality of life for the millions of patients who experience a stressful injury event every year., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Diffuse alveolar hemorrhage (DAH) is a life-threatening complication of systemic lupus erythematosus (SLE) and systemic vasculitis. Although initially described to have antibacterial properties, increasing evidence suggests that neutrophil extracellular traps (NETs) have a detrimental role in both autoimmune diseases and acute lung injury. We investigated whether NETs could be detected in a murine model of pristane-induced lupus DAH and contribute to lung injury. Such NETs might constitute a therapeutic target. NETs were characterized by immunofluorescence staining of DNA, neutrophil elastase and citrullinated histones. Evaluation of lung injury was performed by haematoxylin-eosin staining and a quantification program. Clinical status of the mice was assessed by measurement of arterial oxygen saturation and survival curves after recombinant human deoxyribonuclease-1 (Rh-DNase-1) inhalations or polymorphonuclear neutrophil (PMN) depletion. Pristane was found to promote NETs formation in vitro and in vivo. Treatment of mice with Rh-DNase-1 inhalations cleared NETs and reduced lung injury. Clinical status improved significantly, with increased arterial oxygenation and survival. Following PMN depletion, NETs were absent with a subsequent reduction of lung injury and improved arterial oxygenation. These results support a pathogenic role of PMNs and NETs in lung injury during pristane-induced DAH. Targeting NETs with Rh-DNase-1 inhalations could constitute an interesting adjuvant therapy in human DAH., (Copyright © 2019. Published by Elsevier Ltd.)

Background: The outcome and functional trajectory of older persons admitted to intensive care (ICU) unit remain a true question for critical care physicians and geriatricians, due to the heterogeneity of geriatric population, heterogeneity of practices and absence of guidelines., Aim: To describe the 1-year outcome, prognosis factors and functional trajectory for older people admitted to ICU., Methods: In a prospective 1-year cohort study, all patients aged 75 years and over admitted to our ICU were included according to a global comprehensive geriatric assessment. Follow-up was conducted for 1 year survivors, in particular, ability scores and living conditions., Results: Of 188 patients included [aged 82.3 ± 4.7 years, 46% of admissions, median SAPS II 53.5 (43-74), ADL of Katz's score 4.2 ± 1.6, median Barthel's index 71 (55-90), AGGIR scale 4.5 ± 1.5], the ICU, hospital and 1-year mortality were, respectively, 34, 42.5 and 65.5%. Prognosis factors were: SAPS 2, mechanical ventilation, comorbidity (Lee's and Mc Cabe's scores), disability scores (ADL of Katz's score, Barthel's index and AGGIR scale), admission creatinin, hypoalbuminemia, malignant haemopathy, cognitive impairment. One-year survivors lived in their own home for 83%, with a preserved physical ability, without significant variation of the three ability assessed scores compared to prior ICU admission., Conclusion: The mortality of older people admitted to ICU is high, with a significant impact of disabilty scores, and preserved 1-year survivor independency. Other studies, including a better comprehensive geriatric assessment, seem necessary to determine a predictive "phenotype" of survival with a "satisfactory" level of autonomy.

Epidemiological and experimental studies indicate that early vascular dysfunction occurs in low-birth-weight subjects, especially preterm (PT) infants. We recently reported impaired angiogenic activity of endothelial colony-forming cells (ECFCs) in this condition. We hypothesized that ECFC dysfunction in PT might result from premature senescence and investigated the underlying mechanisms. Compared with ECFCs from term neonates (n = 18), ECFCs isolated from PT (n = 29) display an accelerated senescence sustained by growth arrest and increased senescence-associated β-galactosidase activity. Increased p16(INK4a) expression, in the absence of telomere shortening, indicates that premature PT-ECFC aging results from stress-induced senescence. SIRT1 level, a nicotinamide adenine dinucleotide-dependent deacetylase with anti-aging activities, is dramatically decreased in PT-ECFCs and correlated with gestational age. SIRT1 deficiency is subsequent to epigenetic silencing of its promoter. Transient SIRT1 overexpression or chemical induction by resveratrol treatment reverses senescence phenotype, and rescues in vitro PT-ECFC angiogenic defect in a SIRT1-dependent manner. SIRT1 overexpression also restores PT-ECFC capacity for neovessel formation in vivo. We thus demonstrate that decreased expression of SIRT1 drives accelerated senescence of PT-ECFCs, and acts as a critical determinant of the PT-ECFC angiogenic defect. These findings lay new grounds for understanding the increased cardiovascular risk in individuals born prematurely and open perspectives for therapeutic strategy.

Objective: Low birth weight (LBW) is a risk factor for hypertension at adulthood. Endothelial progenitor cells (EPCs) dysfunction has been characterized in LBW neonates. We hypothesized that changes in soluble, plasma pro- or anti-angiogenic factors are associated with EPCs dysfunction and impaired angiogenesis in LBW neonates., Method: Venous umbilical cord blood was collected from 42 normal, term neonates and 75 LBW neonates. Cord blood endothelial colony forming cells (ECFC) from control patients were cultured in the presence of 10% of serum obtained from both groups., Results: The proliferation and the migration of ECFC were significantly reduced when cultured with 10% of serum of LBW neonates compared to serum of control neonates. Matrigel invasion assay was not significantly altered. Umbilical vein plasma VEGF concentration was significantly reduced in LBW neonates while that of sVEGFR and PF4 were significantly higher. Addition of VEGF corrected the inhibitory effect of LBW serum on normal ECFC proliferation., Conclusions: Serum obtained from LBW babies contains factors that exhibit an antiangiogenic effect on ECFC proliferation and migration. VEGF/sVEGF/PF4 pathway seems to be involved in the EPCs dysfunction in LBW neonates.

Smooth muscle cells (SMCs) participate to the regulation of peripheral arterial resistance and blood pressure. To assume their function, SMCs differentiate throughout the normal vascular development from a synthetic phenotype towards a fully differentiated contractile phenotype by acquiring a repertoire of proteins involved in contraction. In human fetal muscular arteries and umbilical arteries (UAs), no data are available regarding the differentiation of SMCs during the last trimester of gestation. The objective of this study was to characterize the phenotype of SMCs during this gestation period in human UAs. We investigated the phenotype of SMCs in human UAs from very preterm (28-31 weeks of gestation), late preterm (32-35 weeks) and term (37-41 weeks) newborns using biochemical and immunohistochemical detection of α-actin, smooth muscle myosin heavy chain, smoothelin, and non-muscle myosin heavy chain. We found that the number of SMCs positive for smoothelin in UAs increased with gestational age. Western blot analysis revealed a higher content of smoothelin in term compared to very preterm UAs. These results show that SMCs in human UAs gradually acquire a fully differentiated contractile phenotype during the last trimester of gestation and thus that premature birth is associated with not fully differentiated contractile SMCs in human UAs., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Low birth weight (LBW) is associated with increased risk of cardiovascular diseases at adulthood. Nevertheless, the impact of LBW on the endothelium is not clearly established. We investigate whether LBW alters the angiogenic properties of cord blood endothelial colony forming cells (LBW-ECFCs) in 25 preterm neonates compared with 25 term neonates (CT-ECFCs). We observed that LBW decreased the number of colonies formed by ECFCs and delayed the time of appearance of their clonal progeny. LBW dramatically reduced LBW-ECFC capacity to form sprouts and tubes, to migrate and to proliferate in vitro. The angiogenic defect of LBW-ECFCs was confirmed in vivo by their inability to form robust capillary networks in Matrigel plugs injected in nu/nu mice. Gene profile analysis of LBW-ECFCs demonstrated an increased expression of antiangiogenic genes. Among them, thrombospondin 1 (THBS1) was highly expressed at RNA and protein levels in LBW-ECFCs. Silencing THBS1 restored the angiogenic properties of LBW-ECFCs by increasing AKT phosphorylation. The imbalance toward an angiostatic state provide a mechanistic link between LBW and the impaired angiogenic properties of ECFCs and allows the identification of THBS1 as a novel player in LBW-ECFC defect, opening new perspectives for novel deprogramming agents.

The tumor necrosis factor-alpha (TNF) converting enzyme (ADAM17) is a metalloprotease that cleaves several transmembrane proteins, including TNF and its receptors (TNFR1 and TNFR2). We recently showed that the shedding activity of ADAM17 is sequestered in lipid rafts and that cholesterol depletion increased the shedding of ADAM17 substrates. These data suggested that ADAM17 activity could be regulated by cholesterol movements in the cell membrane. We investigated if the membrane cholesterol efflux induced by high-density lipoproteins (HDLs) was able to modify the shedding of ADAM17 substrates. HDLs added to different cell types, increased the ectodomain shedding of TNFR2, TNFR1, and TNF, an effect reduced by inhibitors active on ADAM17. The HDLs-stimulated TNF release occurred also on cell-free isolated plasma membranes. Purified apoA1 increased the shedding of TNF in an ABCA1-dependent manner, suggesting a role for the cholesterol efflux in this phenomenon. HDLs reduced the cholesterol and proteins (including ADAM17) content of lipid rafts and triggered the ADAM17-dependent cleavage of TNF in the non-raft region of the membrane. In conclusion, these data demonstrate that HDLs alter the lipid raft structure, which in turn activates the ADAM17-dependent processing of transmembrane substrates., ((c) 2007 Wiley-Liss, Inc.)

Neutral sphingomyelinase (nSMase), the initial enzyme of the sphingolipid signaling pathway, is thought to play a key role in cellular responses to tumor necrosis factor alpha (TNF-alpha), such as inflammation, proliferation, and apoptosis. The mechanism of TNF-alpha-induced nSMase activation is only partly understood. Using biochemical, molecular, and pharmacological approaches, we found that nSMase activation triggered by TNF-alpha is required for TNF-alpha-induced proliferation and in turn requires a proteolytic cascade involving furin, membrane type 1 matrix metalloproteinase (MT1-MMP), and MMP2, and leading finally to extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and DNA synthesis, in smooth muscle cells (SMC) and fibroblasts. Pharmacological and molecular inhibitors of MMPs (batimastat), furin (alpha1-PDX inhibitor-transfected SMC), MT1-MMP (SMC overexpressing a catalytically inactive MT1-MMP), MMP2 (fibroblasts from MMP2(-/-) mice), and small interfering RNA (siRNA) strategies (siRNAs targeting furin, MT1-MMP, MMP2, and nSMase) resulted in near-complete inhibition of the activation of nSMase, sphingosine kinase-1, and ERK1/2 and of subsequent DNA synthesis. Exogenous MT1-MMP activated nSMase and SMC proliferation in normal but not in MMP2(-/-) fibroblasts, whereas exogenous MMP2 was active on both normal and MMP2(-/-) fibroblasts. Altogether these findings highlight a pivotal role for furin, MT1-MMP, and MMP2 in TNF-alpha-induced sphingolipid signaling, and they identify this system as a possible target to inhibit SMC proliferation in vascular diseases.

The tumor necrosis factor-alpha (TNF) converting enzyme (ADAM17) is a metalloprotease-disintegrin responsible for the cleavage of several biologically active transmembrane proteins. However, the substrate specificity of ADAM17 and the regulation of its shedding activity are still poorly understood. Here, we report that during its transport through the Golgi apparatus, ADAM17 is included in cholesterol-rich membrane microdomains (lipid rafts) where its prodomain is cleaved by furin. Consequently, ADAM17 shedding activity is sequestered in lipid rafts, which is confirmed by the fact that metalloproteinase inhibition increases the proportion of ADAM17 substrates (TNF and its receptors TNFR1 and TNFR2) in lipid rafts. Membrane cholesterol depletion increases the ADAM17-dependent shedding of these substrates demonstrating the importance of lipid rafts in the control of this process. Furthermore, ADAM17 substrates are present in different proportions in lipid rafts, suggesting that the entry of each of these substrates in these particular membrane microdomains is specifically regulated. Our data support the idea that one of the mechanisms regulating ADAM17 substrate cleavage involves protein partitioning in lipid rafts.

ADAM-17 is a metalloprotease-disintegrin responsible for the ectodomain shedding of several transmembrane proteins. Using the yeast two-hybrid system, we showed that ADAM-17 interacts with the Four and Half LIM domain 2 protein (FHL2), a LIM domain protein that is involved in multiple protein-protein interaction. We demonstrated that this interaction involved the amino-acid sequence of ADAM-17 from position 721 to739. In the cardiomyoblast cells H9C2, ADAM-17 and FHL2 colocalize with the actin-based cytoskeleton and we showed that FHL2 binds both ADAM-17 and the actin-based cytoskeleton. We found that mainly the mature form of ADAM-17 associates with the cytoskeleton, although the maturation of ADAM-17 by furin is not necessary for its binding to the cytoskeleton. Interestingly, less ADAM-17 was detected at the surface of wild-type mouse macrophages compared to FHL2 deficient macrophages. However, wild-type cells have a higher ability to release ADAM-17 substrates under PMA stimulation. Altogether, these results demonstrate a physical and functional interaction between ADAM-17 and FHL2 that implies that FHL2 has a role in the regulation of ADAM-17.

Our group has recently demonstrated (Gesta, S., Simon, M., Rey, A., Sibrac, D., Girard, A., Lafontan, M., Valet, P., and Saulnier-Blache, J. S. (2002) J. Lipid Res. 43, 904-910) the presence, in adipocyte conditioned-medium, of a soluble lysophospholipase d-activity (LPLDact) involved in synthesis of the bioactive phospholipid lysophosphatidic acid (LPA). In the present report, LPLDact was purified from 3T3F442A adipocyte-conditioned medium and identified as the type II ecto-nucleotide pyrophosphatase phosphodiesterase, autotaxin (ATX). A unique ATX cDNA was cloned from 3T3F442A adipocytes, and its recombinant expression in COS-7 cells led to extracellular release of LPLDact. ATX mRNA expression was highly up-regulated during adipocyte differentiation of 3T3F442A-preadipocytes. This up-regulation was paralleled by the ability of newly differentiated adipocytes to release LPLDact and LPA. Differentiation-dependent up-regulation of ATX expression was also observed in a primary culture of mouse preadipocytes. Treatment of 3T3F442A-preadipocytes with concentrated conditioned medium from ATX-expressing COS-7 cells led to an increase in cell number as compared with concentrated conditioned medium from ATX non-expressing COS-7 cells. The specific effect of ATX on preadipocyte proliferation was completely suppressed by co-treatment with a LPA-hydrolyzing phospholipase, phospholipase B. Finally, ATX expression was found in mature adipocytes isolated from mouse adipose tissue and was substantially increased in genetically obese-diabetic db/db mice when compared with their lean siblings. In conclusion, the present work shows that ATX is responsible for the LPLDact released by adipocytes and exerts a paracrine control on preadipocyte growth via an LPA-dependent mechanism. Up-regulations of ATX expression with adipocyte differentiation and genetic obesity suggest a possible involvement of this released protein in the development of adipose tissue and obesity-associated pathologies.

The prevalence of human immunodeficiency virus among haemodialysis patients is of interest and this article discusses the management of 25 patients between 1985 and 1998 in Paris.

We report the case of a 25 year-old man presenting Reiter's syndrome (urethritis, conjunctivitis and oligoarthritis). This clinical triad is a particular expression of reactive arthritis. A genital or enteric infection can be responsible for the onset. Presence of HLA B27 histocompatibility antigen is a genetic factor favoring the development of Reiter's syndrome. Many organs or systems can be affected. In addition to eradication of the initiating infection, treatment is mainly symptomatic and management is multidisciplinary.

The authors report 4 cases of ossification of the posterior vertebral ligament. In 3 cases, the cervical spine was involved (mainly C4-C6), two included thoracic lesions. Three cases showed signs of spinal cord compression. Only one case was operated on. The authors review the literature. The authors discuss the classification, and the etiology, together with the possible relationship between the disease and ankylosing hyperostosis. Finally the pathogenesis is studied: the authors believe that spinal ischemia plays a very important role.