Publications by authors named "Gerry A F Nicolaes"

75 Publications

Structural anomalies in a published NMR-derived structure of IRAK-M.

J Mol Graph Model 2021 Nov 5;111:108061. Epub 2021 Nov 5.

Amsterdam UMC, University of Amsterdam, Medical Biochemistry, Meibergdreef 9, 1105 AZ, Amsterdam, the Netherlands; Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Universiteitssingel 50, 6229 ER, Maastricht, the Netherlands. Electronic address:

Signaling by Toll-Like Receptors and the Interleukin-1 Receptor (IL1-R) involves intracellular binding of MyD88, followed by assembly of IL1-R Associated Kinases (IRAKs) into the so-called Myddosome. Using NMR, Nechama et al. determined the structure of the IRAK-M death domain monomer (PDBid: 5UKE). With this structure, they performed a docking study to model the location of IRAK-M in the Myddosome. Based on this, they present a molecular basis for selectivity of IRAK-M towards IRAK1 over IRAK2 binding. When we attempted to use 5UKE as a homology modeling template, we noticed that our 5UKE-based models had structural issues, such as disallowed torsion angles and solvent exposed tryptophans. We therefore analyzed the NMR ensemble of 5UKE using structure validation tools and we compared 5UKE with homologous high-resolution X-ray structures. We identified several structural anomalies in 5UKE, including packing issues, frayed helices and improbable side chain conformations. We used Yasara to build a homology model, based on two high resolution death domain crystal structures, as an alternative model for the IRAK-M death domain (atomic coordinates, modeling details and validation are available at https://swift.cmbi.umcn.nl/gv/service/5uke/). Our model agrees better with known death domain structure information than 5UKE and also with the chemical shift data that was deposited for 5UKE.
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http://dx.doi.org/10.1016/j.jmgm.2021.108061DOI Listing
November 2021

Histone H3 Cleavage in Severe COVID-19 ICU Patients.

Front Cell Infect Microbiol 2021 10;11:694186. Epub 2021 Sep 10.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, Netherlands.

The severity of coronavirus disease 19 (COVID-19) is associated with neutrophil extracellular trap (NET) formation. During NET formation, cytotoxic extracellular histones are released, the presence of which is linked to the initiation and progression of several acute inflammatory diseases. Here we study the presence and evolution of extracellular histone H3 and several other neutrophil-related molecules and damage-associated molecular patterns (DAMPs) in the plasma of 117 COVID-19-positive ICU patients. We demonstrate that at ICU admission the levels of histone H3, MPO, and DNA-MPO complex were all significantly increased in COVID-19-positive patients compared to control samples. Furthermore, in a subset of 54 patients, the levels of each marker remained increased after 4+ days compared to admission. Histone H3 was found in 28% of the patients on admission to the ICU and in 50% of the patients during their stay at the ICU. Notably, in 47% of histone-positive patients, we observed proteolysis of histone in their plasma. The overall presence of histone H3 during ICU stay was associated with thromboembolic events and secondary infection, and non-cleaved histone H3 was associated with the need for vasoactive treatment, invasive ventilation, and the development of acute kidney injury. Our data support the validity of treatments that aim to reduce NET formation and additionally underscore that more targeted therapies focused on the neutralization of histones should be considered as treatment options for severe COVID-19 patients.
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http://dx.doi.org/10.3389/fcimb.2021.694186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8461091PMC
September 2021

Design, synthesis, and biological evaluation of novel pyrrolidinone small-molecule Formyl peptide receptor 2 agonists.

Eur J Med Chem 2021 Dec 2;226:113805. Epub 2021 Sep 2.

LifeArc, Accelerator Building, Open Innovation Campus, Stevenage, UK.

A series of Formyl peptide receptor 2 small molecule agonists with a pyrrolidinone scaffold, derived from a combination of pharmacophore modelling and docking studies, were designed and synthesized. The GLASS (GPCR-Ligand Association) database was screened using a pharmacophore model. The most promising novel ligand structures were chosen and then tested in cellular assays (calcium mobilization and β-arrestin assays). Amongst the selected ligands, two pyrrolidinone compounds (7 and 8) turned out to be the most active. Moreover compound 7 was able to reduce the number of adherent neutrophils in a human neutrophil static adhesion assay which indicates its anti-inflammatory and proresolving properties. Further exploration and optimization of new ligands showed that heterocyclic rings, e.g. pyrazole directly connected to the pyrrolidinone scaffold, provide good stability and a boost in the agonistic activity. The compounds of most interest (7 and 30) were tested in an ERK phosphorylation assay, demonstrating selectivity towards FPR2 over FPR1. Compound 7 was examined in an in vivo mouse pharmacokinetic study. Compound 7 may be a valuable in vivo tool and help improve understanding of the role of the FPR2 receptor in the resolution of inflammation process.
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http://dx.doi.org/10.1016/j.ejmech.2021.113805DOI Listing
December 2021

Conformational plasticity of ADAMTS13 in hemostasis and autoimmunity.

J Biol Chem 2021 10 27;297(4):101132. Epub 2021 Aug 27.

Department of Molecular Hematology, Sanquin-Academic Medical Center Landsteiner Laboratory, Amsterdam, the Netherlands; Department of Experimental Vascular Medicine, Amsterdam UMC, Amsterdam, the Netherlands. Electronic address:

A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) is a multidomain metalloprotease for which until now only a single substrate has been identified. ADAMTS13 cleaves the polymeric force-sensor von Willebrand factor (VWF) that unfolds under shear stress and recruits platelets to sites of vascular injury. Shear force-dependent cleavage at a single Tyr-Met peptide bond in the unfolded VWF A2 domain serves to reduce the size of VWF polymers in circulation. In patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP), a rare life-threatening disease, ADAMTS13 is targeted by autoantibodies that inhibit its activity or promote its clearance. In the absence of ADAMTS13, VWF polymers are not adequately processed, resulting in spontaneous adhesion of blood platelets, which presents as severe, life-threatening microvascular thrombosis. In healthy individuals, ADAMTS13-VWF interactions are guided by controlled conversion of ADAMTS13 from a closed, inactive to an open, active conformation through a series of interdomain contacts that are now beginning to be defined. Recently, it has been shown that ADAMTS13 adopts an open conformation in the acute phase and during subclinical disease in iTTP patients, making open ADAMTS13 a novel biomarker for iTTP. In this review, we summarize our current knowledge on ADAMTS13 conformation and speculate on potential triggers inducing conformational changes of ADAMTS13 and how these relate to the pathogenesis of iTTP.
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http://dx.doi.org/10.1016/j.jbc.2021.101132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8449270PMC
October 2021

Evolution of NETosis markers and DAMPs have prognostic value in critically ill COVID-19 patients.

Sci Rep 2021 08 3;11(1):15701. Epub 2021 Aug 3.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, P.O. Box 616, 6200 MD, Maastricht, the Netherlands.

Coronavirus disease 19 (COVID-19) presents with disease severities of varying degree. In its most severe form, infection may lead to respiratory failure and multi-organ dysfunction. Here we study the levels and evolution of the damage associated molecular patterns (DAMPS) cell free DNA (cfDNA), extracellular histone H3 (H3) and neutrophil elastase (NE), and the immune modulators GAS6 and AXL in relation to clinical parameters, ICU scoring systems and mortality in patients (n = 100) with severe COVID-19. cfDNA, H3, NE, GAS6 and AXL were increased in COVID-19 patients compared to controls. These measures associated with occurrence of clinical events and intensive care unit acquired weakness (ICUAW). cfDNA and GAS6 decreased in time in patients surviving to 30 days post ICU admission. A decrease of 27.2 ng/mL cfDNA during ICU stay associated with patient survival, whereas levels of GAS6 decreasing more than 4.0 ng/mL associated with survival. The presence of H3 in plasma was a common feature of COVID-19 patients, detected in 38% of the patients at ICU admission. NETosis markers cfDNA, H3 and NE correlated well with parameters of tissue damage and neutrophil counts. Furthermore, cfDNA correlated with lowest p/f ratio and a lowering in cfDNA was observed in patients with ventilator-free days.
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http://dx.doi.org/10.1038/s41598-021-95209-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8333321PMC
August 2021

ptFVa ( Venom-Derived Factor Va) Retains Structural Integrity Following Proteolysis by Activated Protein C.

Arterioscler Thromb Vasc Biol 2021 08 24;41(8):2263-2276. Epub 2021 Jun 24.

Division of Thrombosis and Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, the Netherlands (M.S., K.L.C., P.H.R., M.H.A.B.).

[Figure: see text].
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http://dx.doi.org/10.1161/ATVBAHA.121.316038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8288481PMC
August 2021

Structure-function of anticoagulant TIX-5, the inhibitor of factor Xa-mediated FV activation.

J Thromb Haemost 2021 07 1;19(7):1697-1708. Epub 2021 May 1.

Amsterdam UMC, University of Amsterdam, Center for Experimental and Molecular Medicine, Amsterdam Infection and Immunity Institute, Amsterdam, The Netherlands.

Background: The prothrombinase complex consists of factors Xa (FXa) and Va (FVa) on an anionic phospholipid surface and converts prothrombin into thrombin. Both coagulation factors require activation before complex assembly. We recently identified TIX-5, a unique anticoagulant tick protein that specifically inhibits FXa-mediated activation of FV. Because TIX-5 inhibited thrombin generation in blood plasma, it was concluded that FV activation by FXa contributes importantly to coagulation.

Objective: We aimed to unravel the structure-function relationships of TIX-5.

Method: We used a structure model generated based on homology with the allergen Der F7.

Results: Tick inhibitor of factor Xa toward FV was predicted to consist of a single rod formed by several beta sheets wrapped around a central C-terminal alpha helix. By mutagenesis we could show that two hydrophobic loops at one end of the rod mediate the phospholipid binding of TIX-5. On the other end of the rod an FV interaction region was identified on one side, whereas on the other side an EGK sequence was identified that could potentially form a pseudosubstrate of FXa. All three interaction sites were important for the anticoagulant properties of TIX-5 in a tissue factor-initiated thrombin generation assay as well as in the inhibition of FV activation by FXa in a purified system.

Conclusion: The structure-function properties of TIX-5 are in perfect agreement with a protein that inhibits the FXa-mediated activation on a phospholipid surface. The present elucidation of the mechanism of action of TIX-5 will aid in deciphering the processes involved in the initiation phase of blood coagulation.
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http://dx.doi.org/10.1111/jth.15329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8360041PMC
July 2021

Growth Arrest-Specific Factor 6 (GAS6) Is Increased in COVID-19 Patients and Predicts Clinical Outcome.

Biomedicines 2021 Mar 26;9(4). Epub 2021 Mar 26.

Department of Cell Death and Proliferation, IIBB-CSIC, IDIBAPS, 08036 Barcelona, Spain.

Background: Growth arrest-specific factor 6 (GAS6) and the Tyro3, AXL, and MERTK (TAM) receptors counterbalance pro-inflammatory responses. AXL is a candidate receptor for SARS-CoV-2, particularly in the respiratory system, and the GAS6/AXL axis is targeted in current clinical trials against COVID-19. However, GAS6 and TAMs have not been evaluated in COVID-19 patients at emergency admission.

Methods: Plasma GAS6, AXL, and MERTK were analyzed in 132 patients consecutively admitted to the emergency ward during the first peak of COVID-19.

Results: GAS6 levels were higher in the SARS-CoV-2-positive patients, increasing progressively with the severity of the disease. Patients with initial GAS6 at the highest quartile had the worst outcome, with a 3-month survival of 65%, compared to a 90% survival for the rest. Soluble AXL exhibited higher plasma concentration in deceased patients, without significant differences in MERTK among SARS-CoV-2-positive groups. GAS6 mRNA was mainly expressed in alveolar cells and AXL in airway macrophages. Remarkably, THP-1 human macrophage differentiation neatly induces AXL, and its inhibition (bemcentinib) reduced cytokine production in human macrophages after LPS challenge.

Conclusions: Plasma GAS6 and AXL levels reflect COVID-19 severity and could be early markers of disease prognosis, supporting a relevant role of the GAS6/AXL system in the immune response in COVID-19.
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http://dx.doi.org/10.3390/biomedicines9040335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8065652PMC
March 2021

PAD4 takes charge during neutrophil activation: Impact of PAD4 mediated NET formation on immune-mediated disease.

J Thromb Haemost 2021 07 12;19(7):1607-1617. Epub 2021 May 12.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands.

Background: Peptidyl arginine deiminase 4 (PAD4) is an enzyme that converts arginine into citrulline. PAD4 is expressed in neutrophils that, when activated, can drive the formation of neutrophil extracellular traps (NETs). Uncontrolled activation of PAD4 and subsequent citrullination of proteins is increasingly recognized as a driver of (auto)immune diseases. Currently, our understanding of PAD4 structure-function relationships and activity control in vivo is incomplete.

Aims: To provide the current state-of-the-art on PAD4 structure-activity relationships and involvement of PAD4 in autoimmune disorders as well as in thrombo-inflammatory disease.

Materials & Methods: Literature review and molecular modelling Results: In this review, we used molecular modelling to generate a three-dimensional structure of the complete PAD4 molecule. Using our model, we discuss the catalytic conversion of the arginine substrate to citrulline. Besides mechanistic insight into PAD4 function, we give an overview of biological functions of PAD4 and mechanisms that influence its activation. In addition, we discuss the crucial role of PAD4-mediated citrullination of histones during the formation of NETs. Subsequently, we focus on the role of PAD4-mediated NET formation and its role in pathogenesis of rheumatoid arthritis, sepsis and (immune-)thrombosis. Finally, we summarize current efforts to design different classes of PAD4 inhibitors that are being developed for improved treatment of autoimmune disorders as well as thrombo-inflammatory disease.

Discussion: Advances in PAD4 structure-function are still necessary to gain a complete insight in mechanisms that control PAD4 activity in vivo. The involvement of PAD4 in several diseases signifies the need for a PAD4 inhibitor. Although progress has been made to produce an isotype specific and potent PAD4 inhibitor, currently no PAD4 inhibitor is ready for clinical use.

Conclusion: More research into PAD4 structure and function and into the regulation of its activity is required for the development of PAD4 specific inhibitors that may prove vital to combat and prevent autoimmune disorders and (thrombo)inflammatory disease.
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http://dx.doi.org/10.1111/jth.15313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8360066PMC
July 2021

Structure-based peptide design targeting intrinsically disordered proteins: Novel histone H4 and H2A peptidic inhibitors.

Comput Struct Biotechnol J 2021 21;19:934-948. Epub 2021 Jan 21.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands.

A growing body of research has demonstrated that targeting intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDPRs) is feasible and represents a new trending strategy in drug discovery. However, the number of inhibitors targeting IDPs/IDPRs is increasing slowly due to limitations of the methods that can be used to accelerate the discovery process. We have applied structure-based methods to successfully develop the first peptidic inhibitor ( - istone nhibitory ptide) that targets histone H4 that are released from NETs (Neutrophil Extracellular Traps). HIPe binds stably to the disordered N-terminal tail of histone H4, thereby preventing histone H4-induced cell death. Recently, by utilisation of the same state-of-the-art approaches, we have developed a novel peptidic inhibitor ( - yclical istone H2A nterference eptide) that binds to NET-resident histone H2A, which results in a blockade of monocyte adhesion and consequently reduction in atheroprogression. Here, we present comprehensive details on the computational methods utilised to design and develop HIPe and CHIP. We have exploited protein-protein complexes as starting structures for rational peptide design and then applied binding free energy methods to predict and prioritise binding strength of the designed peptides with histone H4 and H2A. By doing this way, we have modelled only around 20 peptides and from these were able to select 4-5 peptides, from a total of more than a trillion candidate peptides, for functional characterisation in different experiments. The developed computational protocols are generic and can be widely used to design and develop novel inhibitors for other disordered proteins.
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http://dx.doi.org/10.1016/j.csbj.2021.01.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7856395PMC
January 2021

N-glycan-mediated shielding of ADAMTS13 prevents binding of pathogenic autoantibodies in immune-mediated TTP.

Blood 2021 05;137(19):2694-2698

Department of Molecular and Cellular Hemostasis, Sanquin-Academic Medical Center Landsteiner Laboratory, Amsterdam, The Netherlands.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is an autoimmune disorder caused by the development of autoantibodies targeting different domains of ADAMTS13. Profiling studies have shown that residues R568, F592, R660, Y661, and Y665 within exosite-3 of the spacer domain provide an immunodominant region of ADAMTS13 for pathogenic autoantibodies that develop in patients with iTTP. Modification of these 5 core residues with the goal of reducing autoantibody binding revealed a significant tradeoff between autoantibody resistance and proteolytic activity. Here, we employed structural bioinformatics to identify a larger epitope landscape on the ADAMTS13 spacer domain. Models of spacer-antibody complexes predicted that residues R568, L591, F592, K608, M609, R636, L637, R639, R660, Y661, Y665, and L668 contribute to an expanded epitope within the spacer domain. Based on bioinformatics-guided predictions, we designed a panel of N-glycan insertions in this expanded epitope to reduce the binding of spacer domain autoantibodies. One N-glycan variant (NGLY3-ADAMTS13, containing a K608N substitution) showed strongly reduced reactivity with TTP patient sera (28%) as compared with WT-ADAMTS13 (100%). Insertion of an N-glycan at amino acid position 608 did not interfere with processing of von Willebrand factor, positioning the resulting NGLY3-ADAMTS13 variant as a potential novel therapeutic option for treatment of iTTP.
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http://dx.doi.org/10.1182/blood.2020007972DOI Listing
May 2021

Autocitrullination of PAD4 does not alter its enzymatic activity: In vitro and in silico studies.

Int J Biochem Cell Biol 2021 05 30;134:105938. Epub 2021 Jan 30.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 6200 MD, Maastricht, the Netherlands. Electronic address:

Background: Protein arginine deiminase 4 (PAD4) is an enzyme capable of converting arginine (positively charged residue) into citrulline (neutral residue). PAD4 is a promiscuous enzyme since it citrullinates various substrates, including small peptides, large proteins and itself. The effect of autocitrullination on PAD4 activity remains controversial and inconclusive. We hypothesized that PAD4 autocitrullination may influence the activity of PAD4 by indirectly altering its binding to substrate.

Methods: We employed mass spectrometry analysis to study the process of autocitrullination. The kinetics of citrullination of PAD4 and citrullinated PAD4 (citPAD4) towards substrates of different sizes (0.17-15.4 kDa), i.e. free arginine, a peptidyl substrate, and histone H3, were studied by colorimetric assay and Western blotting. Molecular dynamics (MD) simulations were performed to investigate structural dynamic and binding properties of PAD4/citPAD4 in the absence and presence of substrates.

Results: We observed that 23/27 arginine residues in PAD4 (85 %) can be citrullinated, including R372, R374 and R639 located near the substrate binding pocket. PAD4 and citPAD4 expressed comparable enzymatic activities towards different substrates. In agreement with experimental results, MD simulations indicated that autocitrullination does not change the shape of the substrate binding pocket and PAD4/citPAD4 exhibited comparable binding free energy with a H3-derived peptidyl substrate (6-TARKS-10).

Conclusion: While the effect of autocitrullination on PAD4 activity thus far remained unclear and controversial, here we have demonstrated that autocitrullination does not affect the activity of PAD4. Thus, the regulation of PAD4 activity is probably not controlled by autocitrullination but likely by other mechanisms that need further investigation.
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http://dx.doi.org/10.1016/j.biocel.2021.105938DOI Listing
May 2021

Endotoxinemia Accelerates Atherosclerosis Through Electrostatic Charge-Mediated Monocyte Adhesion.

Circulation 2021 01 10;143(3):254-266. Epub 2020 Nov 10.

Institute for Cardiovascular Prevention (IPEK), LMU Munich Hospital, Germany (A.S., A.O.-G., C.W., P. Lemnitzer, J.R.V., C.P., L.P.O., J.W., Y.D., O.S.).

Background: Acute infection is a well-established risk factor of cardiovascular inflammation increasing the risk for a cardiovascular complication within the first weeks after infection. However, the nature of the processes underlying such aggravation remains unclear. Lipopolysaccharide derived from Gram-negative bacteria is a potent activator of circulating immune cells including neutrophils, which foster inflammation through discharge of neutrophil extracellular traps (NETs). Here, we use a model of endotoxinemia to link acute infection and subsequent neutrophil activation with acceleration of vascular inflammation Methods: Acute infection was mimicked by injection of a single dose of lipopolysaccharide into hypercholesterolemic mice. Atherosclerosis burden was studied by histomorphometric analysis of the aortic root. Arterial myeloid cell adhesion was quantified by intravital microscopy.

Results: Lipopolysaccharide treatment rapidly enhanced atherosclerotic lesion size by expansion of the lesional myeloid cell accumulation. Lipopolysaccharide treatment led to the deposition of NETs along the arterial lumen, and inhibition of NET release annulled lesion expansion during endotoxinemia, thus suggesting that NETs regulate myeloid cell recruitment. To study the mechanism of monocyte adhesion to NETs, we used in vitro adhesion assays and biophysical approaches. In these experiments, NET-resident histone H2a attracted monocytes in a receptor-independent, surface charge-dependent fashion. Therapeutic neutralization of histone H2a by antibodies or by in silico designed cyclic peptides enables us to reduce luminal monocyte adhesion and lesion expansion during endotoxinemia.

Conclusions: Our study shows that NET-associated histone H2a mediates charge-dependent monocyte adhesion to NETs and accelerates atherosclerosis during endotoxinemia.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.046677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7914394PMC
January 2021

Modifying ADAMTS13 to modulate binding of pathogenic autoantibodies of patients with acquired thrombotic thrombocytopenic purpura.

Haematologica 2020 11 1;105(11):2619-2630. Epub 2020 Nov 1.

Department of Molecular and Cellular Hemostasis, Sanquin-Academic Medical Center, The Netherlands.

Antibodies that develop in patients with immune thrombotic thrombocytopenic purpura (iTTP) commonly target the spacer epitope R568/F592/R660/Y661/Y665 (RFRYY). In this study we present a detailed contribution of each residue in this epitope for autoantibody binding. Different panels of mutations were introduced here to create a large collection of full-length ADAMTS13 variants comprising conservative (Y←→F), semi-conservative (Y/F→L), non-conservative (Y/F→N) or alanine (Y/F/R→A) substitutions. Previously reported Gain-of-Function (GoF, KYKFF) and truncated 'MDTCS' variants were also included. Sera of 18 patients were screened against all variants. Conservative mutations of the aromatic residues did not reduce the binding of autoantibodies. Moderate resistance was achieved by replacing R568 and R660 by lysines or alanines. Semi-conservative mutations of aromatic residues show a moderate effectiveness in autoantibody resistance. Non-conservative asparagine or alanine mutations of aromatic residues are the most effective. In the mixtures of autoantibodies from the majority (89%) of patients screened, autoantibodies targeting the spacer RFRYY epitope have preponderance compared to other epitopes. Reductions in ADAMTS13 proteolytic activity were observed for all full-length mutant variants, in varying degrees. The greatest activity reductions were observed in the most autoantibody-resistant variants (15-35% residual activity in FRETS-VWF73). Among these, a triple-alanine mutant RARAA showed activity in a VWF multimer assay. This study shows that non-conservative and alanine modifications of residues within the exosite-3 spacer RFRYY epitope in full-length ADAMTS13 resist the binding of autoantibodies from iTTP patients, while retaining residual proteolytic activity. Our study provides a framework for the design of autoantibody-resistant ADAMTS13 variants for further therapeutic development.
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http://dx.doi.org/10.3324/haematol.2019.226068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604655PMC
November 2020

Role of Vitamin K-Dependent Factors Protein S and GAS6 and TAM Receptors in SARS-CoV-2 Infection and COVID-19-Associated Immunothrombosis.

Cells 2020 09 28;9(10). Epub 2020 Sep 28.

Department of Cell Death and Proliferation, IIBB-CSIC, IDIBAPS, 08036 Barcelona, Spain.

The vitamin K-dependent factors protein S (PROS1) and growth-arrest-specific gene 6 (GAS6) and their tyrosine kinase receptors TYRO3, AXL, and MERTK, the TAM subfamily of receptor tyrosine kinases (RTK), are key regulators of inflammation and vascular response to damage. TAM signaling, which has largely studied in the immune system and in cancer, has been involved in coagulation-related pathologies. Because of these established biological functions, the GAS6-PROS1/TAM system is postulated to play an important role in SARS-CoV-2 infection and progression complications. The participation of the TAM system in vascular function and pathology has been previously reported. However, in the context of COVID-19, the role of TAMs could provide new clues in virus-host interplay with important consequences in the way that we understand this pathology. From the viral mimicry used by SARS-CoV-2 to infect cells, to the immunothrombosis that is associated with respiratory failure in COVID-19 patients, TAM signaling seems to be involved at different stages of the disease. TAM targeting is becoming an interesting biomedical strategy, which is useful for COVID-19 treatment now, but also for other viral and inflammatory diseases in the future.
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http://dx.doi.org/10.3390/cells9102186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601762PMC
September 2020

Serial measurements in COVID-19-induced acute respiratory disease to unravel heterogeneity of the disease course: design of the Maastricht Intensive Care COVID cohort (MaastrICCht).

BMJ Open 2020 09 29;10(9):e040175. Epub 2020 Sep 29.

Department of Intensive Care, Maastricht University Medical Center+, Maastricht, The Netherlands.

Introduction: The course of the disease in SARS-CoV-2 infection in mechanically ventilated patients is unknown. To unravel the clinical heterogeneity of the SARS-CoV-2 infection in these patients, we designed the prospective observational Maastricht Intensive Care COVID cohort (MaastrICCht). We incorporated serial measurements that harbour aetiological, diagnostic and predictive information. The study aims to investigate the heterogeneity of the natural course of critically ill patients with a SARS-CoV-2 infection.

Methods And Analysis: Mechanically ventilated patients admitted to the intensive care with a SARS-CoV-2 infection will be included. We will collect clinical variables, vital parameters, laboratory variables, mechanical ventilator settings, chest electrical impedance tomography, ECGs, echocardiography as well as other imaging modalities to assess heterogeneity of the course of a SARS-CoV-2 infection in critically ill patients. The MaastrICCht is also designed to foster various other studies and registries and intends to create an open-source database for investigators. Therefore, a major part of the data collection is aligned with an existing national intensive care data registry and two international COVID-19 data collection initiatives. Additionally, we create a flexible design, so that additional measures can be added during the ongoing study based on new knowledge obtained from the rapidly growing body of evidence. The spread of the COVID-19 pandemic requires the swift implementation of observational research to unravel heterogeneity of the natural course of the disease of SARS-CoV-2 infection in mechanically ventilated patients. Our study design is expected to enhance aetiological, diagnostic and prognostic understanding of the disease. This paper describes the design of the MaastrICCht.

Ethics And Dissemination: Ethical approval has been obtained from the medical ethics committee (Medisch Ethische Toetsingscommissie 2020-1565/3 00 523) of the Maastricht University Medical Centre+ (Maastricht UMC+), which will be performed based on the Declaration of Helsinki. During the pandemic, the board of directors of Maastricht UMC+ adopted a policy to inform patients and ask their consent to use the collected data and to store serum samples for COVID-19 research purposes. All study documentation will be stored securely for fifteen years after recruitment of the last patient. The results will be published in peer-reviewed academic journals, with a preference for open access journals, while particularly considering deposition of the manuscripts on a preprint server early.

Trial Registration Number: The Netherlands Trial Register (NL8613).
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http://dx.doi.org/10.1136/bmjopen-2020-040175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526030PMC
September 2020

The Anticoagulant and Nonanticoagulant Properties of Heparin.

Thromb Haemost 2020 Oct 20;120(10):1371-1383. Epub 2020 Aug 20.

Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands.

Heparins represent one of the most frequently used pharmacotherapeutics. Discovered around 1926, routine clinical anticoagulant use of heparin was initiated only after the publication of several seminal papers in the early 1970s by the group of Kakkar. It was shown that heparin prevents venous thromboembolism and mortality from pulmonary embolism in patients after surgery. With the subsequent development of low-molecular-weight heparins and synthetic heparin derivatives, a family of related drugs was created that continues to prove its clinical value in thromboprophylaxis and in prevention of clotting in extracorporeal devices. Fundamental and applied research has revealed a complex pharmacodynamic profile of heparins that goes beyond its anticoagulant use. Recognition of the complex multifaceted beneficial effects of heparin underscores its therapeutic potential in various clinical situations. In this review we focus on the anticoagulant and nonanticoagulant activities of heparin and, where possible, discuss the underlying molecular mechanisms that explain the diversity of heparin's biological actions.
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http://dx.doi.org/10.1055/s-0040-1715460DOI Listing
October 2020

Variability of Microcirculatory Measurements in Critically Ill Patients.

Shock 2020 07;54(1):9-14

Department of Biochemistry, Maastricht University, Maastricht, the Netherlands.

Introduction: Monitoring the microcirculation may be helpful in guiding resuscitation in patients with circulatory shock. Sublingual side-stream dark field imaging cameras allow for noninvasive, bedside evaluation of the microcirculation, although their use in clinical practice has not yet been validated. The GlycoCheck system automatically analyzes images to determine glycocalyx thickness, red blood cell filling percentage, and vessel density. Although GlycoCheck has been used to study microcirculation in critically ill patients, little is known about the reproducibility of measurements in this population.

Materials And Methods: A total of 60 critically ill patients were studied. Three consecutive microcirculation measurements were performed with the GlycoCheck system in 40 of these patients by one of two experienced observers. Twenty patients were assessed by both observers. Intra- and interobserver variability were assessed using intraclass correlation coefficients (ICCs).

Results: ICCs of single measurements were poor for glycocalyx thickness and good for filling percentage and vessel density. Reproducibility could be substantially increased for all parameters when three consecutive measurements were performed and averaged.

Discussion: GlycoCheck can be used to study microcirculation. However, to obtain reliable results three consecutive measurements should be performed and averaged. The variation of the measurements currently hampers the clinical application in individual patients.
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http://dx.doi.org/10.1097/SHK.0000000000001470DOI Listing
July 2020

A Synthetic Triple Helical Collagen Peptide as a New Agonist for Flow Cytometric Measurement of GPVI-Specific Platelet Activation.

Thromb Haemost 2019 Dec 21;119(12):2005-2013. Epub 2019 Oct 21.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands.

Synthetic cross-linked collagen-related peptide (CRP-XL) is a glycoprotein VI (GPVI) receptor activator for platelet activation. This triple helical peptide, widely used in platelet function tests, is synthesized and cross-linked through cysteine residues at its N-terminus and C-terminus. Currently, there is only one laboratory, which is capable to produce this valuable peptide for clinical applications. In an attempt to provide a standardized alternative for CRP-XL, we developed a synthetic triple helical collagen peptide (STH-CP) with the same primary sequence as CRP-XL (GPC-(GPO)-GPCG-amide), which was both on the C-terminus and on the N-terminus fixed on a scaffold with a binding side for each of the three peptides. The performance of STH-CP on platelet function was studied using flow cytometry and compared with CRP-XL. We found that platelet activation pattern in response to STH-CP and CRP-XL is similar, although the STH-CP requires sixfold higher concentrations to activate platelets to the same state. The intra-assay percent coefficient of variation of STH-CP and CRP-XL were both < 5% and the interindividual variation measured in 118 individuals for both peptides was around 23 and 21% for αIIbβ3 activation and P-selectin expression, respectively. The STH-CP in ready-to-use reaction mix has lower variation than CRP-XL over 1-year storage. In reference values and seasonal variation study, the platelet activation response showed a strong correlation between STH-CP and CRP-XL.Our findings show that this new STH-CP is a stable and potent platelet GPVI agonist which can induce the same reproducible platelet activation as CRP-XL and that STH-CP can be considered as a good alternative for CRP-XL.
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http://dx.doi.org/10.1055/s-0039-1697660DOI Listing
December 2019

Acetaminophen Overdose as a Potential Risk Factor for Parkinson's Disease.

Clin Transl Sci 2019 11 15;12(6):609-616. Epub 2019 Jul 15.

Department of Toxicogenomics, Maastricht University, Maastricht, The Netherlands.

Four complementary approaches were used to investigate acetaminophen overdose as a risk factor for Parkinson's disease (PD). Circulating microRNAs (miRNAs) serum profiles from acetaminophen-overdosed patients were compared with patients with terminal PD, revealing four shared miRNAs. Similarities were found among molecular structures of dopamine (DA), acetaminophen, and two known PD inducers indicating affinity for dopaminergic transport. Potential interactions between acetaminophen and the human DA transporter were confirmed by molecular docking modeling and binding free energy calculations. Thus, it is plausible that acetaminophen is taken up by the dopaminergic transport system into the substantia nigra (SN). A ChEMBL query identified proteins that are similarly targeted by DA and acetaminophen. Here, we highlight CYP3A4, present in the SN, a predominant metabolizer of acetaminophen into its toxic metabolite N-acetyl-p-benzoquinone imine and shown to be regulated in PD. Overall, based on our results, we hypothesize that overdosing of acetaminophen is a potential risk factor for parkinsonism.
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http://dx.doi.org/10.1111/cts.12663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6853143PMC
November 2019

Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death.

Nature 2019 05 1;569(7755):236-240. Epub 2019 May 1.

Department of Pathology, AMC, Amsterdam, The Netherlands.

The perpetuation of inflammation is an important pathophysiological contributor to the global medical burden. Chronic inflammation is promoted by non-programmed cell death; however, how inflammation is instigated, its cellular and molecular mediators, and its therapeutic value are poorly defined. Here we use mouse models of atherosclerosis-a major underlying cause of mortality worldwide-to demonstrate that extracellular histone H4-mediated membrane lysis of smooth muscle cells (SMCs) triggers arterial tissue damage and inflammation. We show that activated lesional SMCs attract neutrophils, triggering the ejection of neutrophil extracellular traps that contain nuclear proteins. Among them, histone H4 binds to and lyses SMCs, leading to the destabilization of plaques; conversely, the neutralization of histone H4 prevents cell death of SMCs and stabilizes atherosclerotic lesions. Our data identify a form of cell death found at the core of chronic vascular disease that is instigated by leukocytes and can be targeted therapeutically.
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http://dx.doi.org/10.1038/s41586-019-1167-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6716525PMC
May 2019

Deficiency of the T cell regulator Casitas B-cell lymphoma-B aggravates atherosclerosis by inducing CD8+ T cell-mediated macrophage death.

Eur Heart J 2019 01;40(4):372-382

Department of Medical Biochemistry, Amsterdam Cardiovascular Sciences (ACS), Amsterdam University Medical Centers, University of Amsterdam, Room K1-110, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

Aims: The E3-ligase CBL-B (Casitas B-cell lymphoma-B) is an important negative regulator of T cell activation that is also expressed in macrophages. T cells and macrophages mediate atherosclerosis, but their regulation in this disease remains largely unknown; thus, we studied the function of CBL-B in atherogenesis.

Methods And Results: The expression of CBL-B in human atherosclerotic plaques was lower in advanced lesions compared with initial lesions and correlated inversely with necrotic core area. Twenty weeks old Cblb-/-Apoe-/- mice showed a significant increase in plaque area in the aortic arch, where initial plaques were present. In the aortic root, a site containing advanced plaques, lesion area rose by 40%, accompanied by a dramatic change in plaque phenotype. Plaques contained fewer macrophages due to increased apoptosis, larger necrotic cores, and more CD8+ T cells. Cblb-/-Apoe-/- macrophages exhibited enhanced migration and increased cytokine production and lipid uptake. Casitas B-cell lymphoma-B deficiency increased CD8+ T cell numbers, which were protected against apoptosis and regulatory T cell-mediated suppression. IFNγ and granzyme B production was enhanced in Cblb-/-Apoe-/- CD8+ T cells, which provoked macrophage killing. Depletion of CD8+ T cells in Cblb-/-Apoe-/- bone marrow chimeras rescued the phenotype, indicating that CBL-B controls atherosclerosis mainly through its function in CD8+ T cells.

Conclusion: Casitas B-cell lymphoma-B expression in human plaques decreases during the progression of atherosclerosis. As an important regulator of immune responses in experimental atherosclerosis, CBL-B hampers macrophage recruitment and activation during initial atherosclerosis and limits CD8+ T cell activation and CD8+ T cell-mediated macrophage death in advanced atherosclerosis, thereby preventing the progression towards high-risk plaques.
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http://dx.doi.org/10.1093/eurheartj/ehy714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340101PMC
January 2019

Inhibiting Inflammation with Myeloid Cell-Specific Nanobiologics Promotes Organ Transplant Acceptance.

Immunity 2018 11 6;49(5):819-828.e6. Epub 2018 Nov 6.

Translational and Molecular Imaging Institute, Department of Radiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Inducing graft acceptance without chronic immunosuppression remains an elusive goal in organ transplantation. Using an experimental transplantation mouse model, we demonstrate that local macrophage activation through dectin-1 and toll-like receptor 4 (TLR4) drives trained immunity-associated cytokine production during allograft rejection. We conducted nanoimmunotherapeutic studies and found that a short-term mTOR-specific high-density lipoprotein (HDL) nanobiologic treatment (mTORi-HDL) averted macrophage aerobic glycolysis and the epigenetic modifications underlying inflammatory cytokine production. The resulting regulatory macrophages prevented alloreactive CD8 T cell-mediated immunity and promoted tolerogenic CD4 regulatory T (Treg) cell expansion. To enhance therapeutic efficacy, we complemented the mTORi-HDL treatment with a CD40-TRAF6-specific nanobiologic (TRAF6i-HDL) that inhibits co-stimulation. This synergistic nanoimmunotherapy resulted in indefinite allograft survival. Together, we show that HDL-based nanoimmunotherapy can be employed to control macrophage function in vivo. Our strategy, focused on preventing inflammatory innate immune responses, provides a framework for developing targeted therapies that promote immunological tolerance.
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http://dx.doi.org/10.1016/j.immuni.2018.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6251711PMC
November 2018

Chrono-pharmacological Targeting of the CCL2-CCR2 Axis Ameliorates Atherosclerosis.

Cell Metab 2018 Jul 31;28(1):175-182.e5. Epub 2018 May 31.

Institute for Cardiovascular Prevention (IPEK), Ludwig Maximilian University, Munich 80336, Germany; German Center for Cardiovascular Research (DZHK), Partner Site Munich Heart Alliance, Munich, Germany; Department of Physiology and Pharmacology (FyFa) and Department of Medicine, Karolinska Institutet, Stockholm 17177, Sweden. Electronic address:

Onset of cardiovascular complications as a consequence of atherosclerosis exhibits a circadian incidence with a peak in the morning hours. Although development of atherosclerosis extends for long periods of time through arterial leukocyte recruitment, we hypothesized that discrete diurnal invasion of the arterial wall could sustain atherogenic growth. Here, we show that myeloid cell recruitment to atherosclerotic lesions oscillates with a peak during the transition from the activity to the resting phase. This diurnal phenotype is regulated by rhythmic release of myeloid cell-derived CCL2, and blockade of its signaling abolished oscillatory leukocyte adhesion. In contrast, we show that myeloid cell adhesion to microvascular beds peaks during the early activity phase. Consequently, timed pharmacological CCR2 neutralization during the activity phase caused inhibition of atherosclerosis without disturbing microvascular recruitment. These findings demonstrate that chronic inflammation of large vessels feeds on rhythmic myeloid cell recruitment, and lay the foundation for chrono-pharmacology-based therapy.
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http://dx.doi.org/10.1016/j.cmet.2018.05.002DOI Listing
July 2018

Child-onset thrombotic thrombocytopenic purpura caused by p.R498C and p.G259PfsX133 mutations in ADAMTS13.

Eur J Haematol 2018 May 15. Epub 2018 May 15.

Department of Haematology, Universitair Ziekenhuis Brussel (UZ Brussel), Brussels, Belgium.

Introduction: Patients suffering from congenital thrombotic thrombocytopenic purpura (cTTP) have a deficiency in ADAMTS13 due to mutations in their ADAMTS13 gene.

Objective: The aim of this study was to determine ADAMTS13 parameters (activity, antigen, and mutations), to investigate if the propositus suffered from child-onset cTTP, and to study the in vitro effect of the ADAMTS13 mutations.

Methods: ADAMTS13 activity and antigen were determined using the FRETS VWF73 assay and ELISA and ADAMTS13 mutations via sequencing of the exons. Mutant proteins were expressed in Chinese hamster ovary cells, and their expression was studied using fluorescence microscopy and ELISA. Molecular modeling was used to evaluate the effect of the mutations on ADAMTS13 structure and stability.

Results: The propositus was diagnosed with cTTP at the age of 20. ADAMTS13 activity was below 10%, and 2 compound heterozygous mutations, the p.R498C point and the p.G259PfsX133 frameshift mutation, were identified. Expression of ADAMTS13 mutants revealed that the p.R498C and the p.G259PfsX133 mutation cause secretion and translation defects in vitro, respectively. Molecular modeling showed that the R498 intra-domain interactions are lacking in the p.R498C mutant, resulting in protein instability.

Conclusion: The ADAMTS13 mutations result in a severe ADAMTS13 deficiency explaining the patient's phenotype.
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http://dx.doi.org/10.1111/ejh.13094DOI Listing
May 2018

Insights into 3D Structure of ADAMTS13: A Stepping Stone towards Novel Therapeutic Treatment of Thrombotic Thrombocytopenic Purpura.

Thromb Haemost 2018 01 5;118(1):28-41. Epub 2018 Jan 5.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands.

ADAMTS13 (A D: isintegrin A: nd M: etalloprotease with a T: hromboS: pondin type-1 motif, member 13: ) and von Willebrand factor (VWF) can be considered as scale weights which control platelet adhesion during primary haemostasis. In a very uncommon condition designated thrombotic thrombocytopenic purpura (TTP), functional absence of ADAMTS13 tips the balance toward VWF-mediated platelet adhesion in the microcirculation. TTP is associated with a high mortality and arises from either a congenital or acquired autoimmune deficiency of the plasma enzyme ADAMTS13. In case of acquired ADAMTS13 deficiency, autoantibodies bind to and inhibit the function of ADAMTS13. Currently available treatments of TTP aim to supply ADAMTS13 through plasma exchange or are aimed at B-cell depletion with rituximab. None of the available therapeutics, however, aims at protection of ADAMTS13 from circulating autoantibodies. In this review, our aim is to describe the structure-function relationship of ADAMTS13 employing homology models and previously published crystal structures. Structural bioinformatics investigation of ADAMTS13 reveals many insights and explains how mutations and autoantibodies may lead to the pathophysiology of TTP. The results of these studies provide a roadmap for the further development of rationally designed therapeutics for the treatment of patients with acquired TTP. In addition, we share our opinion on the state of the art of the open-closed conformations of ADAMTS13 which regulate the activity of this highly specific VWF cleaving protease.
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http://dx.doi.org/10.1160/TH17-06-0404DOI Listing
January 2018

Inhibition of CD40-TRAF6 interactions by the small molecule inhibitor 6877002 reduces neuroinflammation.

J Neuroinflammation 2017 05 12;14(1):105. Epub 2017 May 12.

Department of Medical Biochemistry, Subdivision of Experimental Vascular Biology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands.

Background: The influx of leukocytes into the central nervous system (CNS) is a key hallmark of the chronic neuro-inflammatory disease multiple sclerosis (MS). Strategies that aim to inhibit leukocyte migration across the blood-brain barrier (BBB) are therefore regarded as promising therapeutic approaches to combat MS. As the CD40L-CD40 dyad signals via TNF receptor-associated factor 6 (TRAF6) in myeloid cells to induce inflammation and leukocyte trafficking, we explored the hypothesis that specific inhibition of CD40-TRAF6 interactions can ameliorate neuro-inflammation.

Methods: Human monocytes were treated with a small molecule inhibitor (SMI) of CD40-TRAF6 interactions (6877002), and migration capacity across human brain endothelial cells was measured. To test the therapeutic potential of the CD40-TRAF6-blocking SMI under neuro-inflammatory conditions in vivo, Lewis rats and C57BL/6J mice were subjected to acute experimental autoimmune encephalomyelitis (EAE) and treated with SMI 6877002 for 6 days (rats) or 3 weeks (mice).

Results: We here show that a SMI of CD40-TRAF6 interactions (6877002) strongly and dose-dependently reduces trans-endothelial migration of human monocytes. Moreover, upon SMI treatment, monocytes displayed a decreased production of ROS, tumor necrosis factor (TNF), and interleukin (IL)-6, whereas the production of the anti-inflammatory cytokine IL-10 was increased. Disease severity of EAE was reduced upon SMI treatment in rats, but not in mice. However, a significant reduction in monocyte-derived macrophages, but not in T cells, that had infiltrated the CNS was eminent in both models.

Conclusions: Together, our results indicate that SMI-mediated inhibition of the CD40-TRAF6 pathway skews human monocytes towards anti-inflammatory cells with reduced trans-endothelial migration capacity, and is able to reduce CNS-infiltrated monocyte-derived macrophages during neuro-inflammation, but minimally ameliorates EAE disease severity. We therefore conclude that SMI-mediated inhibition of the CD40-TRAF6 pathway may represent a beneficial treatment strategy to reduce monocyte recruitment and macrophage activation in the CNS and has the potential to be used as a co-treatment to combat MS.
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http://dx.doi.org/10.1186/s12974-017-0875-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5427621PMC
May 2017

Chemokine interactome mapping enables tailored intervention in acute and chronic inflammation.

Sci Transl Med 2017 04;9(384)

Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximilians-Universität München, Munich, Germany.

Chemokines orchestrate leukocyte trafficking and function in health and disease. Heterophilic interactions between chemokines in a given microenvironment may amplify, inhibit, or modulate their activity; however, a systematic evaluation of the chemokine interactome has not been performed. We used immunoligand blotting and surface plasmon resonance to obtain a comprehensive map of chemokine-chemokine interactions and to confirm their specificity. Structure-function analyses revealed that chemokine activity can be enhanced by CC-type heterodimers but inhibited by CXC-type heterodimers. Functional synergism was achieved through receptor heteromerization induced by CCL5-CCL17 or receptor retention at the cell surface via auxiliary proteoglycan binding of CCL5-CXCL4. In contrast, inhibitory activity relied on conformational changes (in CXCL12), affecting receptor signaling. Obligate CC-type heterodimers showed high efficacy and potency and drove acute lung injury and atherosclerosis, processes abrogated by specific CCL5-derived peptide inhibitors or knock-in of an interaction-deficient CXCL4 variant. Atheroprotective effects of CCL17 deficiency were phenocopied by a CCL5-derived peptide disrupting CCL5-CCL17 heterodimers, whereas a CCL5 α-helix peptide mimicked inhibitory effects on CXCL12-driven platelet aggregation. Thus, formation of specific chemokine heterodimers differentially dictates functional activity and can be exploited for therapeutic targeting.
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http://dx.doi.org/10.1126/scitranslmed.aah6650DOI Listing
April 2017

Presence of Cytotoxic Extracellular Histones in Machine Perfusate of Donation After Circulatory Death Kidneys.

Transplantation 2017 04;101(4):e93-e101

1 Department of Surgery, Maastricht University Medical Center, Maastricht, The Netherlands. 2 Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands. 3 Department of Methodology and Statistics, School for Public Health and Primary Care (CAPHRI), Faculty of Health, Medicine and Life Sciences, Maastricht University, The Netherlands. 4 Department of Nephrology, Maastricht University Medical Center, Maastricht, The Netherlands. 5 Department of Surgery, Academic Medical Center Amsterdam, Amsterdam, The Netherlands.

Background: Extracellular histones are cytotoxic molecules that are related to cell stress and death. They have been shown to play a crucial role in multiple pathophysiologic processes like sepsis, inflammation, vascular dysfunction, and thrombosis. Their role in organ donation and graft function and survival is still unknown. The aim of this study was to assess whether an association exists between the presence of extracellular histones in machine perfusates and deceased donor kidney viability.

Methods: Machine perfusates of 390 donations after circulatory death kidneys were analyzed for histone concentration, and corresponding graft function and survival were assessed.

Results: Extracellular histone concentrations were significantly higher in perfusates of kidneys with posttransplant graft dysfunction (primary nonfunction and delayed graft function) and were an independent risk factor for delayed graft function (odds ratio, 2.152; 95% confidence interval [95% CI], 1.199-3.863) and 1 year graft failure (hazard ratio, 1.386; 95% CI, 1.037-1.853), but not for primary nonfunction (odds ratio, 1.342; 95% CI, 0.900-2.002). One year graft survival was 12% higher in the group with low histone concentrations (P = 0.008) as compared with the group that contained higher histone concentrations.

Conclusions: This study warrants future studies to probe for a possible role of cytotoxic extracellular histones in organ viability and suggests that quantitation of extracellular histones might contribute to assessment of posttransplant graft function and survival.
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http://dx.doi.org/10.1097/TP.0000000000001590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228581PMC
April 2017
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