Publications by authors named "Laurens Liesenborghs"

26 Publications

  • Page 1 of 1

Early high antibody-titre convalescent plasma for hospitalised COVID-19 patients: DAWn-plasma.

Eur Respir J 2021 Aug 26. Epub 2021 Aug 26.

Department of Intensive Care Medicine, University Hospitals Leuven, and Department of Cellular and Molecular Medicine, Laboratory of Intensive Care Medicine, KU Leuven, Leuven, Belgium.

Background: Several randomised clinical trials have studied convalescent plasma (CP) for COVID-19 using different protocols, with different SARS-CoV-2 neutralising-antibody-titres, at different time-points and severities of illness.

Methods: In the prospective multicentre DAWN-plasma trial, adult patients hospitalised with COVID-19 were randomised to 4 units of open-label convalescent plasma combined with standard of care (intervention group) or standard of care alone (control group). Plasma from donors with neutralising-antibody-titres (NT50) ≥1/320 was the product of choice for the study.

Results: Between May 2nd, 2020 and January 26th, 2021, 320 patients were randomised to convalescent plasma and 163 patients to the control group according to a 2:1 allocation scheme. A median volume of 884 mL convalescent plasma (IQR 806-906 mL) was administered, and 80.68% of the units came from donors with neutralising-antibody-titres (NT50) ≥1/320. Median time from onset of symptoms to randomisation was 7 days. The proportion of patients alive and free of mechanical ventilation on Day 15 was not different between both groups (convalescent plasma: 83.74% (n=267) control: 84.05% (n=137) - Odds ratio 0.99 (0.59-1.66) - p-value=0.9772). The intervention did not change the natural course of antibody titres. The number of serious or severe adverse events was similar in both study arms, and transfusion-related side effects were reported in 19/320 patients in the intervention group (5.94%).

Conclusions: Transfusion of 4 units of convalescent plasma with high neutralising-antibody-titres early in hospitalised COVID-19 patients did not result in a significant improvement of the clinical status, or a reduced mortality.
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http://dx.doi.org/10.1183/13993003.01724-2021DOI Listing
August 2021

Venous Thromboembolism in Patients Discharged after COVID-19 Hospitalization.

Semin Thromb Hemost 2021 Jun 23;47(4):362-371. Epub 2021 Apr 23.

Department of Cardiovascular Diseases, University Hospitals Leuven, Leuven, Belgium.

Background:  Venous thromboembolism (VTE) is a frequent complication of COVID-19, so that the importance of adequate in-hospital thromboprophylaxis in patients hospitalized with COVID-19 is well established. However, the incidence of VTE after discharge and whether postdischarge thromboprophylaxis is beneficial and safe are unclear. In this prospective observational single-center study, we report the incidence of VTE 6 weeks after hospitalization and the use of postdischarge thromboprophylaxis.

Methods:  Patients hospitalized with confirmed COVID-19 were invited to a multidisciplinary follow-up clinic 6 weeks after discharge. D-dimer and C-reactive protein were measured, and all patients were screened for deep vein thrombosis with venous duplex-ultrasound. Additionally, selected high-risk patients received computed tomography pulmonary angiogram or ventilation-perfusion (V/Q) scan to screen for incidental pulmonary embolism.

Results:  Of 485 consecutive patients hospitalized from March through June 2020, 146 patients were analyzed, of which 39% had been admitted to the intensive care unit (ICU). Postdischarge thromboprophylaxis was prescribed in 28% of patients, but was used more frequently after ICU stay (61%) and in patients with higher maximal D-dimer and C-reactive protein levels during hospitalization. Six weeks after discharge, elevated D-dimer values were present in 32% of ward and 42% of ICU patients. Only one asymptomatic deep vein thrombosis (0.7%) and one symptomatic pulmonary embolism (0.7%) were diagnosed with systematic screening. No bleedings were reported.

Conclusion:  In patients who had been hospitalized with COVID-19, systematic screening for VTE 6 weeks after discharge revealed a low incidence of VTE. A strategy of selectively providing postdischarge thromboprophylaxis in high-risk patients seems safe and potentially effective.
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http://dx.doi.org/10.1055/s-0041-1727284DOI Listing
June 2021

Itraconazole for COVID-19: preclinical studies and a proof-of-concept randomized clinical trial.

EBioMedicine 2021 Apr 19;66:103288. Epub 2021 Mar 19.

Department of Cardiovascular Sciences and Clinical Department of Laboratory Medicine, KU Leuven, Belgium.

Background: The antifungal drug itraconazole exerts in vitro activity against SARS-CoV-2 in Vero and human Caco-2 cells. Preclinical and clinical studies are required to investigate if itraconazole is effective for the treatment and/or prevention of COVID-19.

Methods: Due to the initial absence of preclinical models, the effect of itraconazole was explored in a clinical, proof-of-concept, open-label, single-center study, in which hospitalized COVID-19 patients were randomly assigned to standard of care with or without itraconazole. Primary outcome was the cumulative score of the clinical status until day 15 based on the 7-point ordinal scale of the World Health Organization. In parallel, itraconazole was evaluated in a newly established hamster model of acute SARS-CoV-2 infection and transmission, as soon as the model was validated.

Findings: In the hamster acute infection model, itraconazole did not reduce viral load in lungs, stools or ileum, despite adequate plasma and lung drug concentrations. In the transmission model, itraconazole failed to prevent viral transmission. The clinical trial was prematurely discontinued after evaluation of the preclinical studies and because an interim analysis showed no signal for a more favorable outcome with itraconazole: mean cumulative score of the clinical status 49 vs 47, ratio of geometric means 1.01 (95% CI 0.85 to 1.19) for itraconazole vs standard of care.

Interpretation: Despite in vitro activity, itraconazole was not effective in a preclinical COVID-19 hamster model. This prompted the premature termination of the proof-of-concept clinical study.

Funding: KU Leuven, Research Foundation - Flanders (FWO), Horizon 2020, Bill and Melinda Gates Foundation.
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http://dx.doi.org/10.1016/j.ebiom.2021.103288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979145PMC
April 2021

Direct antivirals working against the novel coronavirus: azithromycin (DAWn-AZITHRO), a randomized, multicenter, open-label, adaptive, proof-of-concept clinical trial of new antivirals working against SARS-CoV-2-azithromycin trial.

Trials 2021 Feb 9;22(1):126. Epub 2021 Feb 9.

Katholieke Universiteit Leuven Universitaire Ziekenhuizen Leuven, Leuven, Belgium.

Background: The rapid emergence and the high disease burden of the novel coronavirus SARS-CoV-2 have created a medical need for readily available drugs that can decrease viral replication or blunt the hyperinflammatory state leading to severe COVID-19 disease. Azithromycin is a macrolide antibiotic, known for its immunomodulatory properties. It has shown antiviral effect specifically against SARS-CoV-2 in vitro and acts on cytokine signaling pathways that have been implicated in COVID-19.

Methods: DAWn-AZITHRO is a randomized, open-label, phase 2 proof-of-concept, multicenter clinical trial, evaluating the safety and efficacy of azithromycin for treating hospitalized patients with COVID-19. It is part of a series of trials testing promising interventions for COVID-19, running in parallel and grouped under the name DAWn-studies. Patients hospitalized on dedicated COVID wards are eligible for study inclusion when they are symptomatic (i.e., clinical or radiological signs) and have been diagnosed with COVID-19 within the last 72 h through PCR (nasopharyngeal swab or bronchoalveolar lavage) or chest CT scan showing typical features of COVID-19 and without alternate diagnosis. Patients are block-randomized (9 patients) with a 2:1 allocation to receive azithromycin plus standard of care versus standard of care alone. Standard of care is mostly supportive, but may comprise hydroxychloroquine, up to the treating physician's discretion and depending on local policy and national health regulations. The treatment group receives azithromycin qd 500 mg during the first 5 consecutive days after inclusion. The trial will include 284 patients and recruits from 15 centers across Belgium. The primary outcome is time from admission (day 0) to life discharge or to sustained clinical improvement, defined as an improvement of two points on the WHO 7-category ordinal scale sustained for at least 3 days.

Discussion: The trial investigates the urgent and still unmet global need for drugs that may impact the disease course of COVID-19. It will either provide support or else justify the discouragement of the current widespread, uncontrolled use of azithromycin in patients with COVID-19. The analogous design of other parallel trials of the DAWN consortium will amplify the chance of identifying successful treatment strategies and allow comparison of treatment effects within an identical clinical context.

Trial Registration: EU Clinical trials register EudraCT Nb 2020-001614-38 . Registered on 22 April 2020.
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http://dx.doi.org/10.1186/s13063-021-05033-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7871018PMC
February 2021

A single-dose live-attenuated YF17D-vectored SARS-CoV-2 vaccine candidate.

Nature 2021 02 1;590(7845):320-325. Epub 2020 Dec 1.

KU Leuven Department of Microbiology, Immunology and Transplantation, Rega Institute, Virology and Chemotherapy, Molecular Vaccinology and Vaccine Discovery, KU Leuven, Leuven, Belgium.

The expanding pandemic of coronavirus disease 2019 (COVID-19) requires the development of safe, efficacious and fast-acting vaccines. Several vaccine platforms are being leveraged for a rapid emergency response. Here we describe the development of a candidate vaccine (YF-S0) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that uses live-attenuated yellow fever 17D (YF17D) vaccine as a vector to express a noncleavable prefusion form of the SARS-CoV-2 spike antigen. We assess vaccine safety, immunogenicity and efficacy in several animal models. YF-S0 has an excellent safety profile and induces high levels of SARS-CoV-2 neutralizing antibodies in hamsters (Mesocricetus auratus), mice (Mus musculus) and cynomolgus macaques (Macaca fascicularis), and-concomitantly-protective immunity against yellow fever virus. Humoral immunity is complemented by a cellular immune response with favourable T helper 1 polarization, as profiled in mice. In a hamster model and in macaques, YF-S0 prevents infection with SARS-CoV-2. Moreover, a single dose conferred protection from lung disease in most of the vaccinated hamsters within as little as 10 days. Taken together, the quality of the immune responses triggered and the rapid kinetics by which protective immunity can be attained after a single dose warrant further development of this potent SARS-CoV-2 vaccine candidate.
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http://dx.doi.org/10.1038/s41586-020-3035-9DOI Listing
February 2021

A randomized, multicentre, open-label phase II proof-of-concept trial investigating the clinical efficacy and safety of the addition of convalescent plasma to the standard of care in patients hospitalized with COVID-19: the Donated Antibodies Working against nCoV (DAWn-Plasma) trial.

Trials 2020 Nov 27;21(1):981. Epub 2020 Nov 27.

University Hospitals Leuven (UZ Leuven), Leuven, Belgium.

Background: The COVID-19 pandemic has imposed an enormous burden on health care systems around the world. In the past, the administration of convalescent plasma of patients having recovered from SARS and severe influenza to patients actively having the disease showed promising effects on mortality and appeared safe. Whether or not this also holds true for the novel SARS-CoV-2 virus is currently unknown.

Methods: DAWn-Plasma is a multicentre nation-wide, randomized, open-label, phase II proof-of-concept clinical trial, evaluating the clinical efficacy and safety of the addition of convalescent plasma to the standard of care in patients hospitalized with COVID-19 in Belgium. Patients hospitalized with a confirmed diagnosis of COVID-19 are eligible when they are symptomatic (i.e. clinical or radiological signs) and have been diagnosed with COVID-19 in the 72 h before study inclusion through a PCR (nasal/nasopharyngeal swab or bronchoalveolar lavage) or a chest-CT scan showing features compatible with COVID-19 in the absence of an alternative diagnosis. Patients are randomized in a 2:1 ratio to either standard of care and convalescent plasma (active treatment group) or standard of care only. The active treatment group receives 2 units of 200 to 250 mL of convalescent plasma within 12 h after randomization, with a second administration of 2 units 24 to 36 h after ending the first administration. The trial aims to include 483 patients and will recruit from 25 centres across Belgium. The primary endpoint is the proportion of patients that require mechanical ventilation or have died at day 15. The main secondary endpoints are clinical status on day 15 and day 30 after randomization, as defined by the WHO Progression 10-point ordinal scale, and safety of the administration of convalescent plasma.

Discussion: This trial will either provide support or discourage the use of convalescent plasma as an early intervention for the treatment of hospitalized patients with COVID-19 infection.

Trial Registration: ClinicalTrials.gov NCT04429854 . Registered on 12 June 2020 - Retrospectively registered.
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http://dx.doi.org/10.1186/s13063-020-04876-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7691949PMC
November 2020

STAT2 signaling restricts viral dissemination but drives severe pneumonia in SARS-CoV-2 infected hamsters.

Nat Commun 2020 11 17;11(1):5838. Epub 2020 Nov 17.

Laboratory of Virology and Chemotherapy, Rega Institute, KU Leuven Department of Microbiology, Immunology and Transplantation, 3000, Leuven, Belgium.

Emergence of SARS-CoV-2 causing COVID-19 has resulted in hundreds of thousands of deaths. In search for key targets of effective therapeutics, robust animal models mimicking COVID-19 in humans are urgently needed. Here, we show that Syrian hamsters, in contrast to mice, are highly permissive to SARS-CoV-2 and develop bronchopneumonia and strong inflammatory responses in the lungs with neutrophil infiltration and edema, further confirmed as consolidations visualized by micro-CT alike in clinical practice. Moreover, we identify an exuberant innate immune response as key player in pathogenesis, in which STAT2 signaling plays a dual role, driving severe lung injury on the one hand, yet restricting systemic virus dissemination on the other. Our results reveal the importance of STAT2-dependent interferon responses in the pathogenesis and virus control during SARS-CoV-2 infection and may help rationalizing new strategies for the treatment of COVID-19 patients.
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http://dx.doi.org/10.1038/s41467-020-19684-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672082PMC
November 2020

Favipiravir at high doses has potent antiviral activity in SARS-CoV-2-infected hamsters, whereas hydroxychloroquine lacks activity.

Proc Natl Acad Sci U S A 2020 10 9;117(43):26955-26965. Epub 2020 Oct 9.

Drug Delivery & Disposition, Department of Pharmaceutical and Pharmacological Sciences, Katholieke Universiteit Leuven, 3000 Leuven, Belgium.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly spread around the globe after its emergence in Wuhan in December 2019. With no specific therapeutic and prophylactic options available, the virus has infected millions of people of which more than half a million succumbed to the viral disease, COVID-19. The urgent need for an effective treatment together with a lack of small animal infection models has led to clinical trials using repurposed drugs without preclinical evidence of their in vivo efficacy. We established an infection model in Syrian hamsters to evaluate the efficacy of small molecules on both infection and transmission. Treatment of SARS-CoV-2-infected hamsters with a low dose of favipiravir or hydroxychloroquine with(out) azithromycin resulted in, respectively, a mild or no reduction in virus levels. However, high doses of favipiravir significantly reduced infectious virus titers in the lungs and markedly improved lung histopathology. Moreover, a high dose of favipiravir decreased virus transmission by direct contact, whereas hydroxychloroquine failed as prophylaxis. Pharmacokinetic modeling of hydroxychloroquine suggested that the total lung exposure to the drug did not cause the failure. Our data on hydroxychloroquine (together with previous reports in macaques and ferrets) thus provide no scientific basis for the use of this drug in COVID-19 patients. In contrast, the results with favipiravir demonstrate that an antiviral drug at nontoxic doses exhibits a marked protective effect against SARS-CoV-2 in a small animal model. Clinical studies are required to assess whether a similar antiviral effect is achievable in humans without toxic effects.
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http://dx.doi.org/10.1073/pnas.2014441117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604414PMC
October 2020

Coagulation: At the heart of infective endocarditis.

J Thromb Haemost 2020 05 3;18(5):995-1008. Epub 2020 Feb 3.

Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.

Infective endocarditis is a life-threatening and enigmatic disease with a mortality of 30% and a pathophysiology that is poorly understood. However, at its core, an endocarditis lesion is mainly a fibrin and platelet blood clot infested with bacteria, clinging at the cardiac valves. Infective endocarditis therefore serves as a paradigm of immunothrombosis gone wrong. Immunothrombosis refers to the entanglement of the coagulation system with innate immunity and the role of coagulation in the isolation and clearance of invading pathogens. However, in the case of infective endocarditis, instead of containing the infection, immunothrombosis inadvertently creates the optimal shelter from the immune system and allows some bacteria to grow almost unimpeded. In every step of the disease, the coagulation system is heavily involved. It mediates the initial adhesion of bacteria to the leaflets, fuels the growth and maturation of a vegetation, and facilitates complications such as embolization and valve destruction. In addition, the number one cause of infective endocarditis, Staphylococcus aureus, has proven to be a true manipulator of immunothrombosis and thrives in the fibrin rich environment of an endocarditis vegetation. Considering its central role in infective endocarditis, the coagulation system is an attractive therapeutic target for this deadly disease. There is, however, a very delicate balance at play and the use of antithrombotic drugs in patients with endocarditis is often accompanied with a high bleeding risk.
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http://dx.doi.org/10.1111/jth.14736DOI Listing
May 2020

Von Willebrand factor and ADAMTS13 impact on the outcome of Staphylococcus aureus sepsis.

J Thromb Haemost 2020 03 22;18(3):722-731. Epub 2019 Dec 22.

Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.

Background: Previous clinical evidence correlates levels of von Willebrand factor (VWF) and its cleaving protease ADAMTS13 with outcome in septic patients. No previous studies addressed if VWF and ADAMTS13 affected the outcome of Staphylococcus aureus sepsis.

Objectives: We studied the role of VWF and ADAMTS13 in S. aureus sepsis both in patients and in mice.

Methods: VWF levels and ADAMTS13 activity levels were measured in plasma samples from 89 S. aureus bacteremia patients by chemiluminescent assays and were correlated with clinical sepsis outcome parameters. In wild-type mice and mice deficient in VWF and ADAMTS13, we investigated the outcome of S. aureus sepsis and quantified bacterial clearance and organ microthrombi.

Results: In patients with S. aureus bloodstream infections, high VWF levels and low ADAMTS13 activity levels correlated with disease severity and with parameters of inflammation and disseminated intravascular coagulation. In septic mice, VWF deficiency attenuated mortality, whereas ADAMTS13 deficiency increased mortality. Bacterial clearance was enhanced in VWF-deficient mice. The differences in mortality for the studied genotypes were associated with differential loads of organ microthrombi in both liver and kidneys.

Conclusions: In conclusion, this study reports the consistent relation of VWF, ADAMTS13 and their ratio to disease severity in patients and mice with S. aureus sepsis. Targeting VWF multimers and/or the relative ADAMTS13 deficiency that occurs in sepsis should be explored as a potential new therapeutic target in S. aureus endovascular infections.
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http://dx.doi.org/10.1111/jth.14686DOI Listing
March 2020

Staphylococcus aureus endocarditis: distinct mechanisms of bacterial adhesion to damaged and inflamed heart valves.

Eur Heart J 2019 10;40(39):3248-3259

Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, KU Leuven, Herestraat 49, Leuven, Belgium.

Aims: The pathogenesis of endocarditis is not well understood resulting in unsuccessful attempts at prevention. Clinical observations suggest that Staphylococcus aureus infects either damaged or inflamed heart valves. Using a newly developed endocarditis mouse model, we therefore studied the initial adhesion of S. aureus in both risk states.

Methods And Results: Using 3D confocal microscopy, we examined the adhesion of fluorescent S. aureus to murine aortic valves. To mimic different risk states we either damaged the valves with a surgically placed catheter or simulated valve inflammation by local endothelium activation. We used von Willebrand factor (VWF) gene-deficient mice, induced platelet and fibrinogen depletion and used several S. aureus mutant strains to investigate the contribution of both host and bacterial factors in early bacterial adhesion. Both cardiac valve damage and inflammation predisposed to endocarditis, but by distinct mechanisms. Following valve damage, S. aureus adhered directly to VWF and fibrin, deposited on the damaged valve. This was mediated by Sortase A-dependent adhesins such as VWF-binding protein and Clumping factor A. Platelets did not contribute. In contrast, upon cardiac valve inflammation, widespread endothelial activation led to endothelial cell-bound VWF release. This recruited large amounts of platelets, capturing S. aureus to the valve surface. Here, neither fibrinogen, nor Sortase A were essential.

Conclusion: Cardiac valve damage and inflammation predispose to S. aureus endocarditis via distinct mechanisms. These findings may have important implications for the development of new preventive strategies, as some interventions might be effective in one risk state, but not in the other.
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http://dx.doi.org/10.1093/eurheartj/ehz175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7963134PMC
October 2019

Assessment of the Dual Role of Clumping Factor A in S. Aureus Adhesion to Endothelium in Absence and Presence of Plasma.

Thromb Haemost 2018 Jul 17;118(7):1230-1241. Epub 2018 Jun 17.

Division of Cardiovascular Developmental Biology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium.

Adhesion of to endothelial cells (ECs) is paramount in infective endocarditis. Bacterial proteins such as clumping factor A (ClfA) and fibronectin binding protein A (FnbpA) mediate adhesion to EC surface molecules and (sub)endothelial matrix proteins including fibrinogen (Fg), fibrin, fibronectin (Fn) and von Willebrand factor (vWF). We studied the influence of shear flow and plasma on the binding of ClfA and FnbpA (including its sub-domains A, A, ABC, CD) to coverslip-coated vWF, Fg/fibrin, Fn or confluent ECs, making use of , expressing these adhesins heterologously. Global adherence profiles were similar in static and flow conditions. In the absence of plasma, binding to Fg increased with shear forces, whereas binding to fibrin did not. The degree of adhesion of to EC-bound Fn and of to EC-bound Fg, furthermore, was similar to that of to coated vWF domain A1, in the presence of vWF-binding protein (vWbp). Yet, in plasma, adherence to activated EC-vWF/vWbp dropped over 10 minutes by 80% due to vWF-hydrolysis by a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13 and that of likewise by > 70% compared to the adhesion in absence of plasma. In contrast, plasma Fg supported high binding to resting and activated ECs. Or, in plasma adhesion to active endothelium occurs mainly via two complementary pathways: a rapid but short-lived vWF/vWbp pathway and a stable integrin-coupled Fg-pathway. Hence, the pharmacological inhibition of ClfA-Fg interactions may constitute a valuable additive treatment in infective endocarditis.
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http://dx.doi.org/10.1055/s-0038-1660435DOI Listing
July 2018

Targeting Coagulase Activity in Staphylococcus aureus Bacteraemia: A Randomized Controlled Single-Centre Trial of Staphylothrombin Inhibition.

Thromb Haemost 2018 05 3;118(5):818-829. Epub 2018 Apr 3.

Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, University of Leuven, Leuven, Belgium.

Background: () bacteraemia is frequent and carries a high morbidity and mortality. Coagulases secreted by initiate blood coagulation by directly activating prothrombin. This pathogen-activated coagulation is insensitive to most antithrombotic drugs, with the exception of small molecule direct thrombin inhibitors (DTIs). DTIs inhibit the coagulase-prothrombin complex, or staphylothrombin, and improve outcome in preclinical models of infection.

Objective: A single-centre, randomized, controlled feasibility and safety trial of staphylothrombin inhibition with DTIs in patients with bacteraemia.

Patients And Methods: Consecutive eligible adult patients with positive blood cultures in the University Hospitals Leuven (Belgium) were randomized 1:1 to DTI (oral dabigatran 110 mg twice daily or intravenous argatroban according to activated partial thromboplastin time [aPTT]) for 7 to 10 days, or subcutaneous enoxaparin 40 mg once daily. Primary outcomes were feasibility and safety of DTI in patients with bacteraemia. Secondary outcomes include D-dimer evolution (day 0-4) as marker of coagulation activation; inflammatory and microbiological parameters; and clinical outcomes including metastatic infections.

Results: Thirty-one percent (94/303) of screened patients were enrolled. Dabigatran plasma levels inhibited staphylothrombin. Clinically relevant bleeding (5/47 vs. 5/47) and thrombotic (7/47 vs. 7/47) complications were similar in both groups. Coagulase inhibition with DTIs was associated with a trend towards faster D-dimer decrease at day 4 (-662 ± 249 ng/mL vs. -40 ± 213 ng/mL for DTI-treated patients vs. control;  = 0.06) and a numerically lower number of persistently positive blood cultures. No differences in inflammatory parameters or other clinical outcomes were observed.

Conclusion: Targeting staphylothrombin with DTIs is feasible in a subset of bacteraemic patients, with comparable safety to standard thromboprophylaxis. In future studies of staphylothrombin inhibition, feasibility can be further improved by rapid diagnostics and by strategies without concomitant anticoagulant effect.
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http://dx.doi.org/10.1055/s-0038-1639586DOI Listing
May 2018

Absence of Pear1 does not affect murine platelet function in vivo.

Thromb Res 2016 Oct 26;146:76-83. Epub 2016 Aug 26.

Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, University of Leuven, Leuven, Belgium. Electronic address:

Background: Platelet Endothelial Aggregation Receptor-1 (PEAR1) is a transmembrane platelet receptor that amplifies the activation of the platelet fibrinogen receptor (αIIbβ3) during platelet aggregation. In man, Pear1 polymorphisms are associated with changes in platelet aggregability. In this report, we characterized Pear1 expression and function in murine platelets.

Methods: Pear1 phosphorylation and signaling, platelet aggregation, α-degranulation and clot retraction were studied in WT and Pear1 platelets. The function of Pear1 in haemostasis and thrombosis was studied in a mouse tail vein bleeding and ferric chloride-induced mesenteric thrombosis model.

Results: Mature murine platelets express Pear1 on their membrane and clustering of Pear1 by anti-Pear1 antibodies triggered platelet aggregation. Pear1 was weakly phosphorylated during collagen-induced murine platelet aggregation and was translocated to the cytoskeleton. Absence of murine Pear1 impaired dextran sulfate-induced platelet aggregation, but did not impact collagen-, AYPGK and ADP-induced platelet aggregation, coupled to a lower Pear1 expression in murine than in human platelets and to weaker Pear1-mediated downstream signaling. Neither clot retraction nor α-degranulation was affected in Pear1 mice. Likewise, in vivo tests like the tail vein bleeding time and thrombus formation in mesenteric veins were similar in WT and Pear1 mice.

Conclusion: Murine platelet Pear1 shares a number of characteristics with human platelet PEAR1. Nevertheless, murine Pear1 contributes less to platelet function as does human PEAR1 and does not overtly impact haemostasis and thrombosis in mice.
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http://dx.doi.org/10.1016/j.thromres.2016.08.026DOI Listing
October 2016

Idarucizumab for dabigatran overdose.

Clin Toxicol (Phila) 2016 Sep 25;54(8):644-6. Epub 2016 May 25.

a Department of Cardiovascular Sciences , University of Leuven , Leuven , Belgium ;

Context: An overdose of oral anticoagulants represents a challenging scenario for emergency physicians. Dabigatran, an oral direct thrombin inhibitor, is increasingly used in place of warfarin. The lack of an antidote is a concern in patients who overdose on dabigatran, even though the drug can be eliminated with hemodialysis. Idarucizumab is an antibody fragment that binds dabigatran with high affinity. It reverses the anticoagulant effect of dabigatran within minutes and is approved for the reversal of dabigatran during emergency situations.

Case Details: We describe the use of idarucizumab in the management of a 68-year-old woman who was taking dabigatran 150 mg twice daily and ingested 125 capsules. Despite gastric lavage and administration of activated charcoal within two hours of drug intake, the activated partial thromboplastin time (aPTT) and prothrombin time (PT) remained prolonged. The administration of 5 g of intravenous idarucizumab promptly and completely reversed the anticoagulant activity of dabigatran as assessed by routine and specific coagulation assays (aPTT from to 75 to 26 s, PT from 26 to 11 s and diluted thrombin time from 92 to 27 s). The initially planned emergency hemodialysis was canceled.

Discussion: This case highlights the potential use of idarucizumab for the management of massive dabigatran overdoses.
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http://dx.doi.org/10.1080/15563650.2016.1187737DOI Listing
September 2016

Shear-Resistant Binding to von Willebrand Factor Allows Staphylococcus lugdunensis to Adhere to the Cardiac Valves and Initiate Endocarditis.

J Infect Dis 2016 Apr 6;213(7):1148-56. Epub 2016 Jan 6.

Center for Molecular and Vascular Biology, KU Leuven, Belgium.

Background: Staphylococcus lugdunensis is an emerging cause of endocarditis. To cause endovascular infections, S. lugdunensis requires mechanisms to overcome shear stress. We investigated whether platelets and von Willebrand factor (VWF) mediate bacterial adhesion to the vessel wall and the cardiac valves under flow.

Methods: S. lugdunensis binding to VWF, collagen, and endothelial cells was studied in a parallel flow chamber in the absence and presence of platelets. In vivo adhesion of S. lugdunensis was evaluated in a mouse microvasculature perfusion model and a new mouse model of endocarditis.

Results: Contrary to other coagulase-negative staphylococci, S. lugdunensis bound to VWF under flow, thus enabling its adhesion to endothelial cells and to the subendothelial matrix. In inflamed vessels of the mesenteric circulation, VWF recruited S. lugdunensis to the vessel wall. In a novel endocarditis mouse model, local inflammation and the resulting release of VWF enabled S. lugdunensis to bind and colonize the heart valves.

Conclusions: S. lugdunensis binds directly to VWF, which proved to be vital for withstanding shear forces and for its adhesion to the vessel wall and cardiac valves. This mechanism explains why S. lugdunensis causes more-aggressive infections, including endocarditis, compared with other coagulase-negative staphylococci.
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http://dx.doi.org/10.1093/infdis/jiv773DOI Listing
April 2016

Bacterial pathogens activate plasminogen to breach tissue barriers and escape from innate immunity.

Crit Rev Microbiol 2016 Nov 20;42(6):866-82. Epub 2015 Oct 20.

a Center for Molecular and Vascular Biology, KU Leuven , Leuven , Belgium.

Both coagulation and fibrinolysis are tightly connected with the innate immune system. Infection and inflammation cause profound alterations in the otherwise well-controlled balance between coagulation and fibrinolysis. Many pathogenic bacteria directly exploit the host's hemostatic system to increase their virulence. Here, we review the capacity of bacteria to activate plasminogen. The resulting proteolytic activity allows them to breach tissue barriers and evade innate immune defense, thus promoting bacterial spreading. Yersinia pestis, streptococci of group A, C and G and Staphylococcus aureus produce a specific bacterial plasminogen activator. Moreover, surface plasminogen receptors play an established role in pneumococcal, borrelial and group B streptococcal infections. This review summarizes the mechanisms of bacterial activation of host plasminogen and the role of the fibrinolytic system in infections caused by these pathogens.
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http://dx.doi.org/10.3109/1040841X.2015.1080214DOI Listing
November 2016

Staphylokinase Control of Staphylococcus aureus Biofilm Formation and Detachment Through Host Plasminogen Activation.

J Infect Dis 2016 Jan 1;213(1):139-48. Epub 2015 Jul 1.

Department of Rheumatology and Inflammation Research, Institute of Medicine.

Staphylococcus aureus biofilms, a leading cause of persistent infections, are highly resistant to immune defenses and antimicrobial therapies. In the present study, we investigated the contribution of fibrin and staphylokinase (Sak) to biofilm formation. In both clinical S. aureus isolates and laboratory strains, high Sak-producing strains formed less biofilm than strains that lacked Sak, suggesting that Sak prevents biofilm formation. In addition, Sak induced detachment of mature biofilms. This effect depended on plasminogen activation by Sak. Host-derived fibrin, the main substrate cleaved by Sak-activated plasminogen, was a major component of biofilm matrix, and dissolution of this fibrin scaffold greatly increased susceptibility of biofilms to antibiotics and neutrophil phagocytosis. Sak also attenuated biofilm-associated catheter infections in mouse models. In conclusion, our results reveal a novel role for Sak-induced plasminogen activation that prevents S. aureus biofilm formation and induces detachment of existing biofilms through proteolytic cleavage of biofilm matrix components.
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http://dx.doi.org/10.1093/infdis/jiv360DOI Listing
January 2016

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions.

J Vis Exp 2015 Jun 11(100):e52862. Epub 2015 Jun 11.

Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, KU Leuven.

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood. To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.
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http://dx.doi.org/10.3791/52862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4545207PMC
June 2015

Plasminogen activation by staphylokinase enhances local spreading of S. aureus in skin infections.

BMC Microbiol 2014 Dec 17;14:310. Epub 2014 Dec 17.

Center for Molecular and Vascular Biology, KU Leuven, Herestraat 49, Box 911, Leuven, Belgium.

Background: Staphylococcus aureus (S. aureus) is a frequent cause of skin and soft tissue infections. A unique feature of S. aureus is the combined presence of coagulases that trigger fibrin formation and of the plasminogen activator staphylokinase (SAK). Whereas the importance of fibrin generation for S. aureus virulence has been established, the role of SAK remains unclear. We studied the role of plasminogen activation by SAK in a skin infection model in mice and evaluated the impact of alpha-2-antiplasmin (α2AP) deficiency on the spreading and proteolytic activity of S. aureus skin infections. The species-selectivity of SAK was overcome by adenoviral expression of human plasminogen. Bacterial spread and density was assessed non-invasively by imaging the bioluminescence of S. aureus Xen36.

Results: SAK-mediated plasmin activity increased the local invasiveness of S. aureus, leading to larger lesions with skin disruption as well as decreased bacterial clearance by the host. Even though fibrin and bacterial surfaces protected SAK-mediated plasmin activity from inhibition by α2AP, the deficiency of α2AP resulted in increased bacterial spreading. SAK-mediated plasmin also induced secondary activation of gelatinases, shown both in vitro and in lesions from the in vivo model.

Conclusion: SAK contributes to the phenotype of S. aureus skin infections by enhancing bacterial spreading as a result of fibrinolytic and proteolytic activation.
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http://dx.doi.org/10.1186/s12866-014-0310-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4274676PMC
December 2014

Adhesion of Staphylococcus aureus to the vessel wall under flow is mediated by von Willebrand factor-binding protein.

Blood 2014 Sep 20;124(10):1669-76. Epub 2014 Jun 20.

Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, and.

Adhesion of Staphylococcus aureus to blood vessels under shear stress requires von Willebrand factor (VWF). Several bacterial factors have been proposed to interact with VWF, including VWF-binding protein (vWbp), a secreted coagulase that activates the host's prothrombin to generate fibrin. We measured the adhesion of S aureus Newman and a vWbp-deficient mutant (vwb) to VWF, collagen, and activated endothelial cells in a microparallel flow chamber. In vivo adhesion of S aureus was evaluated in the mesenteric circulation of wild-type (WT) and VWF-deficient mice. We found a shear-dependent increase in adhesion of S aureus to the (sub)endothelium that was dependent on interactions between vWbp and the A1-domain of VWF. Adhesion was further enhanced by coagulase-mediated fibrin formation that clustered bacteria and recruited platelets into bacterial microthrombi. In vivo, deficiency of vWbp or VWF as well as inhibition of coagulase activity reduced S aureus adhesion. We conclude that vWbp contributes to vascular adhesion of S aureus through 2 independent mechanisms: shear-mediated binding to VWF and activation of prothrombin to form S aureus-fibrin-platelet aggregates.
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http://dx.doi.org/10.1182/blood-2014-02-558890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7025350PMC
September 2014

Trends in serotype prevalence in invasive pneumococcal disease before and after infant pneumococcal vaccination in Belgium, 2002-2010.

Vaccine 2013 Mar 20;31(11):1529-34. Epub 2012 Dec 20.

Department of Internal Medicine, University Hospitals Leuven, Herestraat 49, 3000 Leuven, Belgium.

Objectives: We evaluated the effect of the infant 7-valent pneumococcal conjugate vaccine (PCV7) program on the serotype distribution in invasive pneumococcal disease in the Belgian population.

Methods: Serotyping was performed on 13,998 bacteraemic and pleural fluid isolates sent to the National Reference Laboratory between 2002 and 2010. We compared the distribution of serogroups (SGs) between the pre- (2002-2004) and post-PCV7 (2007-2010) era for children (<18 years), adults (18-59 years) and older individuals (≥60 years).

Results: The proportion of cases caused by PCV7-SGs in subjects <18 years decreased from 69% pre-PCV7 to 26% post-PCV7 (p<0.005) and the majority of cases caused by PCV7-SGs were caused by SG 19. Post-PCV7, the prevalence of PCV7-SGs decreased from 38% to 29% and from 57% to 35% in subjects in the age groups 18-59 and ≥60 years, respectively (p<0.005). Post-PCV7 the prevalence of SGs 1, 7 and 19 increased significantly in subjects aged <18 years. The increase of SG19 was caused by an increase of serotype 19A in this age group (p<0.005). After the introduction of infant PCV7 the largest rise in prevalence occurred for SGs 7, 12 and 22 (p<0.005) in the two older age categories. Post PCV7, the overall PCV13 and 23-valent pneumococcal polysaccharide vaccine coverage rates decreased from 85% to 69% and from 96% to 93%, respectively (p<0.005).

Conclusions: PCV7 has an impact on SG distribution of invasive pneumococcal disease isolates of vaccinated and unvaccinated subjects. SG replacement forms a major threat to the success of PCV7. PCV13, including several additional replacement serotypes (STs 1, 7F, 19A), represents an attractive alternative.
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http://dx.doi.org/10.1016/j.vaccine.2012.11.103DOI Listing
March 2013

Anti-PlGF inhibits growth of VEGF(R)-inhibitor-resistant tumors without affecting healthy vessels.

Cell 2007 Nov;131(3):463-75

Department for Transgene Technology and Gene Therapy, VIB, 3000, Leuven, Belgium.

Novel antiangiogenic strategies with complementary mechanisms are needed to maximize efficacy and minimize resistance to current angiogenesis inhibitors. We explored the therapeutic potential and mechanisms of alphaPlGF, an antibody against placental growth factor (PlGF), a VEGF homolog, which regulates the angiogenic switch in disease, but not in health. alphaPlGF inhibited growth and metastasis of various tumors, including those resistant to VEGF(R) inhibitors (VEGF(R)Is), and enhanced the efficacy of chemotherapy and VEGF(R)Is. alphaPlGF inhibited angiogenesis, lymphangiogenesis, and tumor cell motility. Distinct from VEGF(R)Is, alphaPlGF prevented infiltration of angiogenic macrophages and severe tumor hypoxia, and thus, did not switch on the angiogenic rescue program responsible for resistance to VEGF(R)Is. Moreover, it did not cause or enhance VEGF(R)I-related side effects. The efficacy and safety of alphaPlGF, its pleiotropic and complementary mechanism to VEGF(R)Is, and the negligible induction of an angiogenic rescue program suggest that alphaPlGF may constitute a novel approach for cancer treatment.
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http://dx.doi.org/10.1016/j.cell.2007.08.038DOI Listing
November 2007
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