Peter Brouckaert

Ghent University, Gand, Flanders, Belgium

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Publications (176)776.47 Total impact

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    ABSTRACT: Hepatocyte growth factor (HGF) and its receptor MET represent validated targets for cancer therapy. However, HGF/MET inhibitors being explored as cancer therapeutics exhibit cytostatic activity rather than cytotoxic activity, which would be more desired. In this study, we engineered an antagonistic anti-MET antibody that, in addition to blocking HGF/MET signaling, also kills MET-overexpressing cancer cells by antibody-dependent cellular cytotoxicity (ADCC). As a control reagent, we engineered the same antibody in an ADCC-inactive form that is similarly capable of blocking HGF/MET activity, but in the absence of any effector function. In comparing these two antibodies in multiple mouse models of cancer, including HGF-dependent and -independent tumor xenografts, we determined that the ADCC-enhanced antibody was more efficacious than the ADCC-inactive antibody. In orthotopic mammary carcinoma models, ADCC enhancement was crucial to deplete circulating tumor cells and to suppress metastases. Prompted by these results, we optimized the ADCC-enhanced molecule for clinical development generating an antibody (ARGX-111) with improved pharmacological properties. ARGX-111 competed with HGF for MET binding, inhibiting ligand-dependent MET activity; down-regulated cell surface expression of MET, curbing HGF-independent MET activity; and engaged NK cells to kill MET-expressing cancer cells, displaying MET-specific cytotoxic activity. ADCC assays confirmed the cytotoxic effects of ARGX-111 in multiple human cancer cell lines and patient-derived primary tumor specimens, including MET-expressing cancer stem-like cells. Together, our results show how ADCC provides a therapeutic advantage over conventional HGF/MET signaling blockade, and generate proof-of-concept for ARGX-111 clinical testing in MET-positive oncological malignancies. Copyright © 2015, American Association for Cancer Research.
    Cancer Research 07/2015; DOI:10.1158/0008-5472.CAN-15-0356 · 9.28 Impact Factor
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    ABSTRACT: H2S is known to confer cardioprotection, however, the pathways mediating its effects in vivo remain incompletely understood. The purpose of the present study is to evaluate the contribution of cGMP-regulated pathways in the infarct-limiting effect of H2S in vivo. Anesthetized rabbits were subjected to myocardial ischemia (I)/reperfusion (R) and infarct size was determined in control or H2S exposed groups. NaHS (an agent that generates H2S) increased cardiac cGMP and reduced the infarct size. The cGMP-dependent protein kinase (PKG)-I inhibitor DT2 abrogated the protective effect of NaHS, while the control peptide TAT or L-nitroarginine methyl ester (L-NAME) did not alter the effect of NaHS. Moreover, the KATP channel inhibitor glibenclamide partially reversed the effects of NaHS, while inhibition of mitochondrial KATP did not modify the NaHS response. NaHS enhanced phosphorylation of phospholamban (PLN), in a PKG-dependent manner. To further investigate the role of PLN in H2S-mediated cardioprotection, wild type and PLN KO mice underwent I/R. NaHS did not exert cardioprotection in PLN KO mice. Unlike what was observed in rabbits, genetic or pharmacological inhibition of eNOS abolished the infarct-limiting effect of NaHS in mice. Our findings demonstrate i) that administration of NaHS induces cardioprotection via a cGMP/PKG/PLN pathway and ii) contribution of NO to the H2S response is species-specific. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.
    Cardiovascular Research 04/2015; 106(3). DOI:10.1093/cvr/cvv129 · 5.81 Impact Factor
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    ABSTRACT: The risk for cardiovascular morbidity and mortality is increased in chronic kidney disease; in this process micro-inflammation plays an essential role. Responsible mechanisms remain to a large extent unidentified. In this pilot study transcriptome analysis of peripheral blood monocytes was used to identify in an unprejudiced manner which factors could be discriminative for cardiovascular disease in patients with chronic kidney disease on hemodialysis. Forty gender- and age-matched, non-diabetic, non-smoking subjects with CRP < 20 mg/L were recruited: 9 healthy controls, 11 patients with eGFR > 60 mL/min/1.73m2 and a history of cardiovascular event (CVE), 10 patients with chronic kidney disease stage 5 on hemodialysis without previous cardiovascular event (CKD5HD) and 10 with a previous cardiovascular event (CKD5HD/CVE). Monocytes were isolated and their mRNA was submitted to focused transcriptome analysis using a macroarray platform containing ca. 700 genes associated with macrophage functional capacity. The macroarray data indicated 9 genes (8 upregulated and 1 downregulated) with a significant differential expression in CKD5HD/CVE vs. CVE alone, after excluding genes differentially expressed in CKD5HD vs. For FCGR3A (CD16) and CX3CR1 (chemokine receptor) the upregulation vs. control and vs. CVE could be confirmed by quantitative RT-PCR for all CKD5HD patients. Furthermore, CX3CR1 relative expression on monocytes correlated with CRP. Flow cytometric analysis of purified monocytes confirmed a significant increase in the percentage of CD16 positive monocytes in all CKD5HD patients vs. control and CVE. The present study indicates the importance of a specific pro-inflammatory monocyte subpopulation, positive for CD16 and the co-expressed chemokine receptor, CX3CR1, discriminative for CKD5HD patients.
    PLoS ONE 04/2015; 10(4):e0121750. DOI:10.1371/journal.pone.0121750 · 3.23 Impact Factor
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    ABSTRACT: IntroductionBecause of their nitric oxide (NO)-donating capacities, oxime derivatives have shown to offer some therapeutic perspective for the treatment of erectile dysfunction (ED) as well as cardiovascular diseases. However, to date the in vivo effect of these oximes on erectile function remains unknown. In many disease states oxidative stress occurs, impairing NO-mediated relaxations. Hence the influence of oxidative stress on oxime-induced effects is also of interest.AimsThis study aimed to evaluate the in vivo effect of formaldoxime (FAL) and formamidoxime (FAM) on blood pressure and intracavernosal pressure (ICP); and to examine the role of soluble guanylyl cyclase (sGC) and the influence of oxidative stress on the FAL and FAM responses.Methods Blood pressure and ICP were monitored in vivo after resp. intravenous or intracavernosal injection of FAL and FAM. Moreover isometric tension was measured in vitro on isolated mice corpora cavernosa (CC), thoracic aorta, and femoral artery in organ baths. The role of sGC was investigated using transgenic mice lacking the alpha 1 subunit of sGC.Main Outcome MeasuresMean arterial pressure (MAP) and ICP were measured after FAL/FAM injection. In vitro relaxation of CC strips was evaluated in response to addition of FAL/FAM.ResultsIn vivo both FAL and FAM elicit a dose-dependent lowering of blood pressure (maximal ΔMAP: 33.66 ± 4.07 mm Hg [FAL] and 20.43 ± 2.06 mm Hg [FAM] ) as well as an increase of ICP (maximal increase of ICP/MAP: 70.29 ± 2.88% [FAL] and 52.91 ± 8.61% [FAM] ). The FAL/FAM effect is significantly lower in knockout vs. wild-type mice. Oxidative stress has an inhibitory effect on corporal NO-mediated relaxations induced by electrical field stimulation, acetylcholine, and sodium nitroprusside whereas the responses to 8-(4-chlorophenylthio)-guanosine 3′,5′-cyclic monophosphate sodium salt, FAL and FAM were not influenced.Conclusions Oximes induce erection which is mediated by sGC. The oxime-induced relaxations are resistant to oxidative stress, which increases their therapeutic potential for the treatment of ED. Pauwels B, Boydens C, Brouckaert P, and Van de Voorde, J. Oximes Induce Erection and Are Resistant to Oxidative Stress. J Sex Med **;**:**–**.
    Journal of Sexual Medicine 03/2015; 12(4). DOI:10.1111/jsm.12846 · 3.15 Impact Factor
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    ABSTRACT: Enhanced arginase-induced arginine consumption is believed to play a key role in the pathogenesis of sickle cell disease-induced end organ failure. Enhancement of arginine availability with L-arginine supplementation exhibited less consistent results; however, L-citrulline, the precursor of L-arginine, may be a promising alternative. In this study, we OPEN ACCESS Nutrients 2015, 7 5218 determined the effects of L-citrulline compared to L-arginine supplementation on arginine-nitric oxide (NO) metabolism, arginine availability and microcirculation in a murine model with acutely-enhanced arginase activity. The effects were measured in six groups of mice (n = 8 each) injected intraperitoneally with sterile saline or arginase (1000 IE/mouse) with or without being separately injected with L-citrulline or L-arginine 1 h prior to assessment of the microcirculation with side stream dark-field (SDF)-imaging or in vivo NO-production with electron spin resonance (ESR) spectroscopy. Arginase injection caused a decrease in plasma and tissue arginine concentrations. L-arginine and L-citrulline supplementation both enhanced plasma and tissue arginine concentrations in arginase-injected mice. However, only the citrulline supplementation increased NO production and improved microcirculatory flow in arginase-injected mice. In conclusion, the present study provides for the first time in vivo experimental evidence that L-citrulline, and not L-arginine supplementation, improves the end organ microcirculation during conditions with acute arginase-induced arginine deficiency by increasing the NO concentration in tissues.
    Nutrients 01/2015; 7(7):5217-5238. DOI:10.3390/nu7075217 · 3.15 Impact Factor
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    ABSTRACT: The red wine polyphenols resveratrol and quercetin are known for their vasorelaxant and antioxidant capacity, which is assumed to rely on the activation of the nitric oxide (NO)/soluble guanylyl cyclase (sGC) pathway. Vasodilators as well as antioxidants can regulate penile erection and be beneficial for the treatment of erectile dysfunction (ED). The goal of this study was to evaluate the NO/sGC dependency of the relaxant effect of resveratrol and quercetin on mice aorta and corpora cavernosa (CC), as well as to explore their influence on oxidative stress-induced ED. Isolated mice aorta and CC were mounted for isometric tension recordings into organ baths. Cumulative concentration-response curves were constructed for resveratrol and quercetin in the absence/presence of inhibitors of the NO/sGC pathway. In addition, in CC the effect of resveratrol and quercetin was studied on NO-mediated relaxations using acetylcholine (Ach), sodium nitroprusside (SNP), and electrical field stimulation (EFS). In certain experiments, corporal tissues were exposed to oxidative stress using palmitic acid (PA, 0.5 mM). Corporal responses to resveratrol and quercetin were measured in the presence/absence of inhibitors of different molecular pathways. The effect of resveratrol and quercetin incubation on Ach-, SNP-, or EFS-mediated responses was explored in the presence/absence of PA. While both polyphenols are potent vasodilators of mice aorta, only resveratrol relaxes mice CC. The relaxation response to resveratrol on aorta was diminished in sGCα1 (-/-) mice, but not on CC. The polyphenols did not influence Ach-, SNP-, or EFS-mediated relaxations as such. Resveratrol, but not quercetin, was able to significantly reverse PA-induced decrease of EFS relaxations. The red wine compound resveratrol, but not quercetin, relaxes isolated mice CC concentration-dependently through mechanisms independent of the NO/sGC pathway. Resveratrol is a more potent antioxidant than quercetin, being able to restore decreased neuronal NO responses in mice CC. Boydens C, Pauwels B, Decaluwé K, Brouckaert P, and Van de Voorde J. Relaxant and antioxidant capacity of the red wine polyphenols, resveratrol and quercetin, on isolated mice corpora cavernosa. J Sex Med **;**:**-**. © 2014 International Society for Sexual Medicine.
    Journal of Sexual Medicine 12/2014; 12(2). DOI:10.1111/jsm.12786 · 3.15 Impact Factor
  • Nitric oxide : biology and chemistry / official journal of the Nitric Oxide Society; 11/2014
  • Nitric Oxide 11/2014; 42C:120-121. DOI:10.1016/j.niox.2014.09.066 · 3.18 Impact Factor
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    ABSTRACT: Rationale: Inspiratory resistive breathing (RB), encountered in obstructive lung diseases, induces lung injury. Soluble Guanylyl Cyclase (sGC)/cGMP pathway is downregulated in chronic and acute animal models of RB such as asthma, COPD and in endotoxin-induced acute lung injury. Objectives: i) To characterize the effects of increased concurrent inspiratory and expiratory resistance in mice, via tracheal banding, ii) To investigate the contribution of the sGC/cGMP pathway in in RB-induced lung injury. Methods and Main Results: Anesthetized C57BL/6 mice underwent RB achieved by restricting tracheal surface area to 50% (tracheal banding). RB for 24 hours resulted in increased bronchoalveolar lavage fluid cellularity and protein content, in marked leukocyte infiltration in the lungs and perturbed respiratory mechanics (increased tissue resistance and elasticity, shifted static pressure volume curve right and downwards, decreased static compliance), consistent with the presence of acute lung injury. RB downregulated the sGC expression in the lung. All manifestations of lung injury caused by RB were exacerbated by the administration of the sGC inhibitor, ODQ, or when RB was performed using sGC α1 knockout mice. Conversely, restoration of sGC signalling by prior administration of the sGC activator BAY 58-2667 prevented the RB-induced lung injury. Strikingly, direct pharmacological activation of sGC with BAY 58-2667 24h post-RB reversed, within 6 hours, the established lung injury. Conclusions: These findings raise the possibility that pharmacological targeting of the sGC/cGMP axis could be used to ameliorate lung dysfunction in obstructive lung diseases.
    American Journal of Respiratory Cell and Molecular Biology 10/2014; 52(6). DOI:10.1165/rcmb.2014-0092OC · 4.11 Impact Factor
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    ABSTRACT: Nitric oxide (NO) is a gaseotransmitter, which is involved in many signalling processes in health and disease. Three enzymes generate NO from L-arginine, with citrulline formed as a by-product: neuronal NO synthase (nNOS or NOS1), endothelial NOS (eNOS or NOS3) and inducible NOS (iNOS or NOS2). NO is a ligand of soluble guanylyl cyclase (sGC), an intracellular heterodimer enzyme that catalyzes the conversion of guanosine triphosphate (GTP) to cyclic GMP (cGMP). cGMP further activates protein kinase G that eventually reduces the smooth muscle tone in bronchi or vessels. Phosphodiesterase 5 (PDE5) degrades cGMP to GMP. However, NO reacts with superoxide anion (O2-), leading to formation of the proinflammatory molecule peroxynitrite. Under physiological conditions, NO plays a homeostatic bronchoprotective role in healthy subjects. In obstructive airway diseases, NO can be beneficial by its bronchodilating effect, but could also be detrimental by the formation of peroxynitrite. Since asthma and COPD are associated with increased levels of exhaled NO, chronic inflammation and increased airway smooth muscle tone, the NO/sGC/cGMP pathway could be involved in these highly prevalent obstructive airway diseases. Here we review the involvement of NO, NO synthases, guanylyl cyclases, cGMP and phophodiesterase-5 in asthma and COPD and potential therapeutic approaches to modulate this pathway.
    Pulmonary Pharmacology &amp Therapeutics 10/2014; 29(1). DOI:10.1016/j.pupt.2014.07.004 · 2.57 Impact Factor
  • AIDS Research and Human Retroviruses 10/2014; 30 Suppl 1(S1):A63. DOI:10.1089/aid.2014.5114.abstract · 2.46 Impact Factor
  • International Journal of Stroke 10/2014; 9:26-26. · 2.87 Impact Factor
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    ABSTRACT: BackgroundMAPK-activated protein kinase 2 (MK2) plays a pivotal role in the cell response to (inflammatory) stress. Among others, MK2 is known to be involved in the regulation of cytokine mRNA metabolism and regulation of actin cytoskeleton dynamics. Previously, MK2-deficient mice were shown to be highly resistant to LPS/d-Galactosamine-induced hepatitis. Additionally, research in various disease models has indicated the kinase as an interesting inhibitory drug target for various acute or chronic inflammatory diseases.ResultsWe show that in striking contrast to the known resistance of MK2-deficient mice to a challenge with LPS/D-Gal, a low dose of tumor necrosis factor (TNF) causes hyperacute mortality via an oxidative stress driven mechanism. We identified in vivo defects in the stress fiber response in endothelial cells, which could have resulted in reduced resistance of the endothelial barrier to deal with exposure to oxidative stress. In addition, MK2-deficient mice were found to be more sensitive to cecal ligation and puncture-induced sepsis.Conclusions The capacity of the endothelial barrier to deal with inflammatory and oxidative stress is imperative to allow a regulated immune response and maintain endothelial barrier integrity. Our results indicate that, considering the central role of TNF in pro-inflammatory signaling, therapeutic strategies examining pharmacological inhibition of MK2 should take potentially dangerous side effects at the level of endothelial barrier integrity into account.
    BMC Physiology 09/2014; 14(1):5. DOI:10.1186/s12899-014-0005-1
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    ABSTRACT: Background Cinaciguat (BAY 58-2667), an NO- and heme-independent sGC activator, was shown to be more effective when the heme-group of sGC is oxidized in vascular tissue. In apo-sGC mice (sGCβ1His105Pheknockin) both sGC isoforms (sGCα1β1 and sGCα2β1) are heme-deficient and can no longer be activated by NO; these mice, showing decreased gastrointestinal nitrergic relaxation and decreased gastric emptying, can be considered as a model to study the consequence of heme-oxidation in sGC. Our aim was to compare the influence of cinaciguat, on in vitro muscle tone of gastrointestinal tissues, and on gastric emptying in WT and apo-sGC mice.Methods Gastrointestinal smooth muscle strips were mounted in organ baths for isometric force recording and cGMP levels were determined by enzyme immunoassay. Protein levels of sGC subunits were assessed by immunoblotting. Gastric emptying was determined by phenol red recovery.Key ResultsAlthough protein levels of the sGC subunits were lower in gastrointestinal tissues of apo-sGC mice, cinaciguat induced concentration-dependent relaxations and increased cGMP levels in apo-sGC fundus and colon to a similar or greater extent than in WT mice. The sGC inhibitor ODQ increased cinaciguat-induced relaxations and cGMP levels in WT fundus and colon. In apo-sGC antrum, pylorus and jejunum, cinaciguat was not able to induce relaxations. Cinaciguat did not improve delayed gastric emptying in apo-sGC mice.Conclusions & InferencesCinaciguat relaxes the fundus and colon efficiently when sGC is in the heme-free condition; the non-effect of cinaciguat in pylorus explains its inability to improve the delayed gastric emptying in apo-sGC mice.
    Neurogastroenterology and Motility 09/2014; 26(11). DOI:10.1111/nmo.12424 · 3.42 Impact Factor
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    ABSTRACT: Early detection and start of appropriate treatment are highly correlated with survival of sepsis and septic shock, but the currently available predictive tools are not sensitive enough to identify patients at risk. Linear (time and frequency domain) and nonlinear (unifractal and multiscale complexity dynamics) measures of beat-to-beat interval variability were analyzed in two mouse models of inflammatory shock to determine if they are sensitive enough to predict outcome. University research laboratory. Blood pressure transmitter-implanted female C57BL/6J mice. IV administration of tumor necrosis factor (n = 11) or lipopolysaccharide (n = 14). Contrary to linear indices of variability, unifractal dynamics, and absolute heart rate or blood pressure, quantification of complex beat-to-beat dynamics using multiscale entropy was able to predict survival outcome starting as early as 40 minutes after induction of inflammatory shock. Based on these results, a new and clinically relevant index of multiscale entropy was developed that scores the key features of a multiscale entropy profile. Contrary to multiscale entropy, multiscale entropy scoring can be followed as a function of time to monitor disease progression with limited loss of information. Analysis of multiscale complexity of beat-to-beat dynamics at high temporal resolution has potential as a sensitive prognostic tool with translational power that can predict survival outcome in systemic inflammatory conditions such as sepsis and septic shock.
    Critical care medicine 04/2014; DOI:10.1097/CCM.0000000000000299 · 6.15 Impact Factor
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    A Cauwels · E Rogge · B Vandendriessche · S Shiva · P Brouckaert
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    ABSTRACT: Systemic inflammatory response syndromes (SIRS) may be caused by both infectious and sterile insults, such as trauma, ischemia-reperfusion or burns. They are characterized by early excessive inflammatory cytokine production and the endogenous release of several toxic and damaging molecules. These are necessary to fight and resolve the cause of SIRS, but often end up progressively damaging cells and tissues, leading to life-threatening multiple organ dysfunction syndrome (MODS). As inflammasome-dependent cytokines such as interleukin-1β are critically involved in the development of MODS and death in SIRS, and ATP is an essential activator of inflammasomes in vitro, we decided to analyze the ability of ATP removal to prevent excessive tissue damage and mortality in a murine LPS-induced inflammation model. Our results indeed indicate an important pro-inflammatory role for extracellular ATP. However, the effect of ATP is not restricted to inflammasome activation at all. Removing extracellular ATP with systemic apyrase treatment not only prevented IL-1β accumulation but also the production of inflammasome-independent cytokines such as TNF and IL-10. In addition, ATP removal also prevented systemic evidence of cellular disintegration, mitochondrial damage, apoptosis, intestinal barrier disruption and even mortality. Although blocking ATP receptors with the broad-spectrum P2 purinergic receptor antagonist suramin imitated certain beneficial effects of apyrase treatment, it could not prevent morbidity or mortality at all. We conclude that removal of systemic extracellular ATP could be a valuable strategy to dampen systemic inflammatory damage and toxicity in SIRS.
    Cell Death & Disease 03/2014; 5(3):e1102. DOI:10.1038/cddis.2014.70 · 5.18 Impact Factor
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    ABSTRACT: Moyamoya is a cerebrovascular condition characterized by a progressive stenosis of the terminal part of the internal carotid arteries (ICAs) and the compensatory development of abnormal "moyamoya" vessels. The pathophysiological mechanisms of this condition, which leads to ischemic and hemorrhagic stroke, remain unknown. It can occur as an isolated cerebral angiopathy (so-called moyamoya disease) or in association with various conditions (moyamoya syndromes). Here, we describe an autosomal-recessive disease leading to severe moyamoya and early-onset achalasia in three unrelated families. This syndrome is associated in all three families with homozygous mutations in GUCY1A3, which encodes the α1 subunit of soluble guanylate cyclase (sGC), the major receptor for nitric oxide (NO). Platelet analysis showed a complete loss of the soluble α1β1 guanylate cyclase and showed an unexpected stimulatory role of sGC within platelets. The NO-sGC-cGMP pathway is a major pathway controlling vascular smooth-muscle relaxation, vascular tone, and vascular remodeling. Our data suggest that alterations of this pathway might lead to an abnormal vascular-remodeling process in sensitive vascular areas such as ICA bifurcations. These data provide treatment options for affected individuals and strongly suggest that investigation of GUCY1A3 and other members of the NO-sGC-cGMP pathway is warranted in both isolated early-onset achalasia and nonsyndromic moyamoya.
    The American Journal of Human Genetics 02/2014; 94(3). DOI:10.1016/j.ajhg.2014.01.018 · 10.99 Impact Factor
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    ABSTRACT: Overexpression of CD70 has been documented in a variety of solid and hematological tumors, where it is thought to play a role in tumor proliferation and evasion of immune surveillance. Here, we describe ARGX-110, a defucosylated IgG1 monoclonal antibody (mAb) that selectively targets and neutralizes CD70, the ligand of CD27. ARGX-110 was generated by immunization of outbred llamas. The antibody was germlined to 95% human identity, and its anti-tumor efficacy was tested in several in vitro assays. ARGX-110 binds CD70 with picomolar affinity. In depletion studies, ARGX-110 lyses tumor cells with greater efficacy than its fucosylated version. In addition, ARGX-110 demonstrates strong complement-dependent cytotoxicity and antibody-dependent cellular phagocytosis activity. ARGX-110 inhibits signaling of CD27, which results in blocking of the activation and proliferation of Tregs. In a Raji xenograft model, administration of the fucosylated version of ARGX-110 resulted in a prolonged survival at doses of 0.1 mg/kg and above. The pharmacokinetics of ARGX-110 was tested in cynomolgus monkeys; the calculated half-life is 12 days. In conclusion, ARGX-110 is a potent blocking mAb with a dual mode of action against both CD70-bearing tumor cells and CD70-dependent Tregs. This antibody is now in a Phase 1 study in patients with advanced malignancies expressing CD70 (NCT01813539).
    mAbs 12/2013; 6(2). DOI:10.4161/mabs.27398 · 4.73 Impact Factor
  • Journal of Critical Care 12/2013; 28(6):e27–e28. DOI:10.1016/j.jcrc.2013.07.004 · 2.19 Impact Factor
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    ABSTRACT: Sepsis and septic shock result from an exacerbated systemic inflammatory reaction to infection. Their incidence is rising, and they have recently become the main cause of death in intensive care units. Septic shock is defined as sepsis accompanied by life-threatening refractory hypotension, for which excessive nitric oxide (NO), produced by inducible NO synthase iNOS, is thought responsible. LPS, a vital outer membrane component of Gram-negative bacteria, mimics most of the septic effects and is widely used as a model for septic shock. TLR4 is the signal-transducing receptor for LPS, evidenced by the resistance of TLR4-deficient C3H/HeJ and C57BL/10ScNJ mice. As expected, we found that TLR4 deficiency precludes LPS-induced cytokine production, independent of the purity of the LPS preparation. However, various conventional LPS preparations induced NO in TLR4-deficient mice to the same level as in control animals, while ultrapure LPS did not, indicating the presence of NO-producing contaminant(s). Nevertheless, despite identical iNOS induction pattern and systemic NO levels, the contaminant does not cause hypotension, hypothermia, or any other sign of morbidity. Using mice deficient for TLR2, TRL3, TLR4, TRL2x4, TLR9, MyD88 or TRIF, we found that the contaminant signals via TLR2 and MyD88. In conclusion, conventional LPS preparations generally used in endotoxic shock research contain TLR2 agonists that induce iNOS and high levels of systemic NO as such, and synergize with LPS towards the production of pro-inflammatory cytokines, morbidity and mortality. Surprisingly, the excessive iNOS-derived systemic NO production induced by impure LPS in TLR4(-/-) is not accompanied by hypotension or morbidity.
    Nitric Oxide 11/2013; 36. DOI:10.1016/j.niox.2013.11.001 · 3.18 Impact Factor

Publication Stats

3k Citations
776.47 Total Impact Points

Institutions

  • 1986–2015
    • Ghent University
      • • VIB Department of Molecular Biomedical Research (DMBR)
      • • Department of Biology
      • • Department of Molecular Biotechnology
      Gand, Flanders, Belgium
  • 2013
    • Harvard Medical School
      • Department of Anesthesia
      Boston, Massachusetts, United States
  • 1995–2013
    • Vlaams Instituut voor Biotechnologie
      • • Inflammation Research Center (IRC)
      • • Laboratory for Molecular Cancer Biology
      Gand, Flemish, Belgium
  • 2006
    • Universitair Ziekenhuis Ghent
      • Department of Radiotherapy
      Gand, Flemish, Belgium
  • 2001
    • University of California, San Diego
      San Diego, California, United States
  • 2000
    • Roche Institute of Molecular Biology
      Nutley, New Jersey, United States