Publications by authors named "Ghislain Opdenakker"

264 Publications

CD146/Soluble CD146 Pathway Is a Novel Biomarker of Angiogenesis and Inflammation in Proliferative Diabetic Retinopathy.

Invest Ophthalmol Vis Sci 2021 07;62(9):32

University Hospitals, UZ Gasthuisberg, Leuven, Belgium.

Purpose: Inflammation, angiogenesis and fibrosis are pathological hallmarks of proliferative diabetic retinopathy (PDR). The CD146/sCD146 pathway displays proinflammatory and proangiogenic properties. We investigated the role of this pathway in the pathophysiology of PDR.

Methods: Vitreous samples from 41 PDR and 27 nondiabetic patients, epiretinal fibrovascular membranes from 18 PDR patients, rat retinas, human retinal microvascular endothelial cells (HRMECs) and human retinal Müller glial cells were studied by ELISA, Western blot analysis, immunohistochemistry and immunofluorescence microscopy analysis. Blood-retinal barrier breakdown was assessed with fluorescein isothiocyanate-conjugated dextran.

Results: sCD146 and VEGF levels were significantly higher in vitreous samples from PDR patients than in nondiabetic patients. In epiretinal membranes, immunohistochemical analysis revealed CD146 expression in leukocytes, vascular endothelial cells and myofibroblasts. Significant positive correlations were detected between numbers of blood vessels expressing CD31, reflecting angiogenic activity of PDR, and numbers of blood vessels and stromal cells expressing CD146. Western blot analysis showed significant increase of CD146 in diabetic rat retinas. sCD146 induced upregulation of phospho-ERK1/2, NF-κB , VEGF and MMP-9 in Müller cells. The hypoxia mimetic agent cobalt chloride, VEGF and TNF-α induced upregulation of sCD146 in HRMECs. The MMP inhibitor ONO-4817 attenuated TNF-α-induced upregulation of sCD146 in HRMECs. Intravitreal administration of sCD146 in normal rats significantly increased retinal vascular permeability and induced significant upregulation of phospho-ERK1/2, intercellular adhesion molecule-1 and VEGF in the retina. sCD146 induced migration of HRMECs.

Conclusions: These results suggest that the CD146/sCD146 pathway is involved in the initiation and progression of PDR.
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http://dx.doi.org/10.1167/iovs.62.9.32DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8300056PMC
July 2021

Internal Disulfide Bonding and Glycosylation of Interleukin-7 Protect Against Proteolytic Inactivation by Neutrophil Metalloproteinases and Serine Proteases.

Front Immunol 2021 30;12:701739. Epub 2021 Jun 30.

Laboratory of Immunobiology, Rega Institute for Medical Research/KU Leuven, Department of Microbiology, Immunology and Transplantation, Leuven, Belgium.

Interleukin 7 (IL-7) is a cell growth factor with a central role in normal T cell development, survival and differentiation. The lack of IL-7-IL-7 receptor(R)-mediated signaling compromises lymphoid development, whereas increased signaling activity contributes to the development of chronic inflammation, cancer and autoimmunity. Gain-of-function alterations of the IL-7R and the signaling through Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) are enriched in T cell acute lymphoblastic leukemia (T-ALL) and autocrine production of IL-7 by T-ALL cells is involved in the phenotypes of leukemic initiation and oncogenic spreading. Several IL-7-associated pathologies are also characterized by increased presence of matrix metalloproteinase-9 (MMP-9), due to neutrophil degranulation and its regulated production by other cell types. Since proteases secreted by neutrophils are known to modulate the activity of many cytokines, we investigated the interactions between IL-7, MMP-9 and several other neutrophil-derived proteases. We demonstrated that MMP-9 efficiently cleaved human IL-7 in the exposed loop between the α-helices C and D and that this process is delayed by IL-7 N-linked glycosylation. Functionally, the proteolytic cleavage of IL-7 did not influence IL-7Rα binding and internalization nor the direct pro-proliferative effects of IL-7 on a T-ALL cell line (HPB-ALL) or in primary CD8 human peripheral blood mononuclear cells. A comparable effect was observed for the neutrophil serine proteases neutrophil elastase, proteinase 3 and combinations of neutrophil proteases. Hence, glycosylation and disulfide bonding as two posttranslational modifications influence IL-7 bioavailability in the human species: glycosylation protects against proteolysis, whereas internal cysteine bridging under physiological redox state keeps the IL-7 conformations as active proteoforms. Finally, we showed that mouse IL-7 does not contain the protease-sensitive loop and, consequently, was not cleaved by MMP-9. With the latter finding we discovered differences in IL-7 biology between the human and mouse species.
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http://dx.doi.org/10.3389/fimmu.2021.701739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8278288PMC
June 2021

Kinetics of peripheral blood neutrophils in severe coronavirus disease 2019.

Clin Transl Immunology 2021 29;10(4):e1271. Epub 2021 Apr 29.

Laboratory of Molecular Immunology Department of Microbiology, Immunology and Transplantation Rega Institute, KU Leuven Leuven Belgium.

Objectives: Emerging evidence of dysregulation of the myeloid cell compartment urges investigations on neutrophil characteristics in coronavirus disease 2019 (COVID-19). We isolated neutrophils from the blood of COVID-19 patients receiving general ward care and from patients hospitalised at intensive care units (ICUs) to explore the kinetics of circulating neutrophils and factors important for neutrophil migration and activation.

Methods: Multicolour flow cytometry was exploited for the analysis of neutrophil differentiation and activation markers. Multiplex and ELISA technologies were used for the quantification of protease, protease inhibitor, chemokine and cytokine concentrations in plasma. Neutrophil polarisation responses were evaluated microscopically. Gelatinolytic and metalloproteinase activity in plasma was determined using a fluorogenic substrate. Co-culturing healthy donor neutrophils with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) allowed us to investigate viral replication in neutrophils.

Results: Upon ICU admission, patients displayed high plasma concentrations of granulocyte-colony-stimulating factor (G-CSF) and the chemokine CXCL8, accompanied by emergency myelopoiesis as illustrated by high levels of circulating CD10, immature neutrophils with reduced CXCR2 and C5aR expression. Neutrophil elastase and non-metalloproteinase-derived gelatinolytic activity were increased in plasma from ICU patients. Significantly higher levels of circulating tissue inhibitor of metalloproteinase 1 (TIMP-1) in patients at ICU admission yielded decreased total MMP proteolytic activity in blood. COVID-19 neutrophils were hyper-responsive to CXCL8 and CXCL12 in shape change assays. Finally, SARS-CoV-2 failed to replicate inside human neutrophils.

Conclusion: Our study provides detailed insights into the kinetics of neutrophil phenotype and function in severe COVID-19 patients, and supports the concept of an increased neutrophil activation state in the circulation.
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http://dx.doi.org/10.1002/cti2.1271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8082714PMC
April 2021

From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8.

Front Immunol 2021 11;12:644725. Epub 2021 Mar 11.

Laboratory of Molecular Immunology, Department of Microbiology, Immunology and Transplantation, Rega Institute, Katholieke Universiteit Leuven, Leuven, Belgium.

With ELISAs one detects the ensemble of immunoreactive molecules in biological samples. For biomolecules undergoing proteolysis for activation, potentiation or inhibition, other techniques are necessary to study biology. Here we develop methodology that combines immunosorbent sample preparation and nano-scale liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) for proteoform analysis (ISTAMPA) and apply this to the aglycosyl chemokine CXCL8. CXCL8, the most powerful human chemokine with neutrophil chemotactic and -activating properties, occurs in different NH-terminal proteoforms due to its susceptibility to site-specific proteolytic modification. Specific proteoforms display up to 30-fold enhanced activity. The immunosorbent ion trap top-down mass spectrometry-based approach for proteoform analysis allows for simultaneous detection and quantification of full-length CXCL8(1-77), elongated CXCL8(-2-77) and all naturally occurring truncated CXCL8 forms in biological samples. For the first time we demonstrate site-specific proteolytic activation of CXCL8 in synovial fluids from patients with chronic joint inflammation and address the importance of sample collection and processing.
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http://dx.doi.org/10.3389/fimmu.2021.644725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7991300PMC
March 2021

Chlorite oxidized oxyamylose differentially influences the microstructure of fibrin and self assembling peptide hydrogels as well as dental pulp stem cell behavior.

Sci Rep 2021 Mar 11;11(1):5687. Epub 2021 Mar 11.

OMFS IMPATH Research Group, Faculty of Medicine, Department of Imaging and Pathology, KU Leuven and Oral and Maxillofacial Surgery, University Hospitals Leuven, Kapucijnenvoer 33, 3000, Leuven, Belgium.

Tailored hydrogels mimicking the native extracellular environment could help overcome the high variability in outcomes within regenerative endodontics. This study aimed to evaluate the effect of the chemokine-binding and antimicrobial polymer, chlorite-oxidized oxyamylose (COAM), on the microstructural properties of fibrin and self-assembling peptide (SAP) hydrogels. A further goal was to assess the influence of the microstructural differences between the hydrogels on the in vitro behavior of human dental pulp stem cells (hDPSCs). Structural and mechanical characterization of the hydrogels with and without COAM was performed by atomic force microscopy and scanning electron microscopy to characterize their microstructure (roughness and fiber length, diameter, straightness, and alignment) and by nanoindentation to measure their stiffness (elastic modulus). Then, hDPSCs were encapsulated in hydrogels with and without COAM. Cell viability and circularity were determined using confocal microscopy, and proliferation was determined using DNA quantification. Inclusion of COAM did not alter the microstructure of the fibrin hydrogels at the fiber level while affecting the SAP hydrogel microstructure (homogeneity), leading to fiber aggregation. The stiffness of the SAP hydrogels was sevenfold higher than the fibrin hydrogels. The viability and attachment of hDPSCs were significantly higher in fibrin hydrogels than in SAP hydrogels. The DNA content was significantly affected by the hydrogel type and the presence of COAM. The microstructural stability after COAM inclusion and the favorable hDPSCs' response observed in fibrin hydrogels suggest this system as a promising carrier for COAM and application in endodontic regeneration.
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http://dx.doi.org/10.1038/s41598-021-84405-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7952722PMC
March 2021

CCR2 Is Dispensable for Disease Resolution but Required for the Restoration of Leukocyte Homeostasis Upon Experimental Malaria-Associated Acute Respiratory Distress Syndrome.

Front Immunol 2020 16;11:628643. Epub 2021 Feb 16.

Laboratory of Immunoparasitology, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, KU Leuven, University of Leuven, Leuven, Belgium.

Malaria complications are often lethal, despite efficient killing of parasites with antimalarial drugs. This indicates the need to study the resolution and healing mechanisms involved in the recovery from these complications. NK65-infected C57BL/6 mice develop malaria-associated acute respiratory distress syndrome (MA-ARDS) at 8 days post infection. Antimalarial treatment was started on this day and resulted in the recovery, as measured by the disappearance of the signs of pathology, in >80% of the mice. Therefore, this optimized model represents an asset in the study of mechanisms and leukocyte populations involved in the resolution of MA-ARDS. C-C chemokine receptor type 2 (CCR2) knock-out mice were used to investigate the role of monocytes and macrophages, since these cells are described to play an important role during the resolution of other inflammatory diseases. CCR2 deficiency was associated with significantly lower numbers of inflammatory monocytes in the lungs during infection and resolution and abolished the increase in non-classical monocytes during resolution. Surprisingly, CCR2 was dispensable for the development and the resolution of MA-ARDS, since no effect of the CCR2 knock-out was observed on any of the disease parameters. In contrast, the reappearance of eosinophils and interstitial macrophages during resolution was mitigated in the lungs of CCR2 knock-out mice. In conclusion, CCR2 is required for re-establishing the homeostasis of pulmonary leukocytes during recovery. Furthermore, the resolution of malaria-induced lung pathology is mediated by unknown CCR2-independent mechanisms.
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http://dx.doi.org/10.3389/fimmu.2020.628643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921736PMC
June 2021

Blood-brain barrier resealing in neuromyelitis optica occurs independently of astrocyte regeneration.

J Clin Invest 2021 03;131(5)

Institute of Neuropathology and.

Approximately 80% of neuromyelitis optica spectrum disorder (NMOSD) patients harbor serum anti-aquaporin-4 autoantibodies targeting astrocytes in the CNS. Crucial for NMOSD lesion initiation is disruption of the blood-brain barrier (BBB), which allows the entrance of Abs and serum complement into the CNS and which is a target for new NMOSD therapies. Astrocytes have important functions in BBB maintenance; however, the influence of their loss and the role of immune cell infiltration on BBB permeability in NMOSD have not yet been investigated. Using an experimental model of targeted NMOSD lesions in rats, we demonstrate that astrocyte destruction coincides with a transient disruption of the BBB and a selective loss of occludin from tight junctions. It is noteworthy that BBB integrity is reestablished before astrocytes repopulate. Rather than persistent astrocyte loss, polymorphonuclear leukocytes (PMNs) are the main mediators of BBB disruption, and their depletion preserves BBB integrity and prevents astrocyte loss. Inhibition of PMN chemoattraction, activation, and proteolytic function reduces lesion size. In summary, our data support a crucial role for PMNs in BBB disruption and NMOSD lesion development, rendering their recruitment and activation promising therapeutic targets.
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http://dx.doi.org/10.1172/JCI141694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919716PMC
March 2021

Interferons and other cytokines, genetics and beyond in COVID-19 and autoimmunity.

Cytokine Growth Factor Rev 2021 04 29;58:134-140. Epub 2021 Jan 29.

Laboratory of Immunobiology and Laboratory of Molecular Immunology, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, University of Leuven, KU Leuven, Belgium.

Interferons are the best antiviral agents in vitro against SARS-CoV-2 so far and genetic defects in their signaling cascade or neutralization of alfa-interferons by autoantibodies come with more severe COVID-19. However, there is more, as the SARS-CoV-2 dysregulates not only innate immune mechanisms but also T and B cell repertoires. Most genetic, hematological and immunological studies in COVID-19 are at present phenomenological. However, these and antecedent studies contain the seed grains to resolve many unanswered questions and a whole range of testable hypotheses. What are the links, if existing, between genetics and the occurrence of interferon-neutralizing antibodies? Are NAGGED (neutralizing and generated by gene defect) antibodies involved or not? Is the autoimmune process cause or consequence of virus infection? What are the roles played by cytokine posttranslational modifications, such as proteolysis, glycosylation, citrullination and others? How is systemic autoimmunity linked with type 1 interferons? These questions place cytokines and growth factors at pole positions as keys to unlock basic mechanisms of infection and (auto)immunity. Related to cytokine research, (1) COVID-19 patients develop neutralizing autoantibodies, mainly against alpha interferons and it is not yet established whether this is the consequence or cause of virus replication. (2) The glycosylation of recombinant interferon-beta protects against breaking tolerance and the development of neutralizing antibodies. (3) SARS-CoV-2 induces severe inflammation and release of extracellular proteases leading to remnant epitopes, e.g. of cytokines. (4) In the rare event of homozygous cytokine gene segment deletions, observed neutralizing antibodies may be named NAGGED antibodies. (5) Severe cytolysis releases intracellular content into the extracellular milieu and leads to regulated degradation of intracellular proteins and selection of antibody repertoires, similar to those observed in patients with systemic lupus erythematosus. (6) Systematic studies of novel autoimmune diseases on single cytokines will complement the present picture about interferons. (7) Interferon neutralization in COVID-19 constitutes a preamble of more studies about cytokine-regulated proteolysis in the control of autoimmunity. Here we reformulate these seven conjectures into testable questions for future research.
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http://dx.doi.org/10.1016/j.cytogfr.2021.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845543PMC
April 2021

Evaluation of Proteoforms of the Transmembrane Chemokines CXCL16 and CX3CL1, Their Receptors, and Their Processing Metalloproteinases ADAM10 and ADAM17 in Proliferative Diabetic Retinopathy.

Front Immunol 2020 20;11:601639. Epub 2021 Jan 20.

Laboratory of Molecular Immunology, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium.

The transmembrane chemokine pathways CXCL16/CXCR6 and CX3CL1/CX3CR1 are strongly implicated in inflammation and angiogenesis. We investigated the involvement of these chemokine pathways and their processing metalloproteinases ADAM10 and ADAM17 in the pathophysiology of proliferative diabetic retinopathy (PDR). Vitreous samples from 32 PDR and 24 non-diabetic patients, epiretinal membranes from 18 patients with PDR, rat retinas, human retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied by enzyme-linked immunosorbent assay, immunohistochemistry and Western blot analysis. angiogenesis assays were performed and the adherence of leukocytes to CXCL16-stimulated HRMECs was assessed. CXCL16, CX3CL1, ADAM10, ADAM17 and vascular endothelial growth factor (VEGF) levels were significantly increased in vitreous samples from PDR patients. The levels of CXCL16 were 417-fold higher than those of CX3CL1 in PDR vitreous samples. Significant positive correlations were found between the levels of VEGF and the levels of CXCL16, CX3CL1, ADAM10 and ADAM17. Significant positive correlations were detected between the numbers of blood vessels expressing CD31, reflecting the angiogenic activity of PDR epiretinal membranes, and the numbers of blood vessels and stromal cells expressing CXCL16, CXCR6, ADAM10 and ADAM17. CXCL16 induced upregulation of phospho-ERK1/2, p65 subunit of NF-κB and VEGF in cultured Müller cells and tumor necrosis factor-α induced upregulation of soluble CXCL16 and ADAM17 in Müller cells. Treatment of HRMECs with CXCL16 resulted in increased expression of intercellular adhesion molecule-1 (ICAM-1) and increased leukocyte adhesion to HRMECs. CXCL16 induced HRMEC proliferation, formation of sprouts from HRMEC spheroids and phosphorylation of ERK1/2. Intravitreal administration of CXCL16 in normal rats induced significant upregulation of the p65 subunit of NF-κB, VEGF and ICAM-1 in the retina. Our findings suggest that the chemokine axis CXCL16/CXCR6 and the processing metalloproteinases ADAM10 and ADAM17 might serve a role in the initiation and progression of PDR.
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http://dx.doi.org/10.3389/fimmu.2020.601639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854927PMC
June 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

Hemozoin in Malarial Complications: More Questions Than Answers.

Trends Parasitol 2021 03 22;37(3):226-239. Epub 2020 Oct 22.

Laboratory of Immunoparasitology, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, KU Leuven, 3000 Leuven, Belgium. Electronic address:

Plasmodium parasites contain various virulence factors that modulate the host immune response. Malarial pigment, or hemozoin (Hz), is an undegradable crystalline product of the hemoglobin degradation pathway in the parasite and possesses immunomodulatory properties. An association has been found between Hz accumulation and severe malaria, suggesting that the effects of Hz on the host immune response may contribute to the development of malarial complications. Although the immunomodulatory roles of Hz have been widely investigated, many conflicting data exist, likely due to the variability between experimental set-ups and technical limitations of Hz generation and isolation methods. Here, we critically assess the potential immunomodulatory effects of Hz, its role in malarial complications, and its potential effects after parasite clearance.
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http://dx.doi.org/10.1016/j.pt.2020.09.016DOI Listing
March 2021

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

Citrullination as a novel posttranslational modification of matrix metalloproteinases.

Matrix Biol 2021 01 4;95:68-83. Epub 2020 Nov 4.

Rega Institute for Medical Research, Laboratory of Immunobiology, Department of Microbiology, Immunology and Transplantation, KU Leuven, Herestraat 49 box 1044, Leuven 3000, Belgium. Electronic address:

Matrix metalloproteinases (MMPs) are enzymes with critical roles in biology and pathology. Glycosylation, nitrosylation and proteolysis are known posttranslational modifications (PTMs) regulating intrinsically the activities of MMPs. We discovered MMP citrullination by peptidyl arginine deiminases (PADs) as a new PTM. Upon hypercitrullination, MMP-9 acquired a higher affinity for gelatin than control MMP-9. Furthermore, hypercitrullinated proMMP-9 was more efficiently activated by MMP-3 compared to control MMP-9. JNJ0966, a specific therapeutic inhibitor of MMP-9 activation, inhibited the activation of hypercitrullinated proMMP-9 by MMP-3 significantly less in comparison with control proMMP-9. The presence of citrullinated/homocitrullinated MMP-9 was detected in vivo in neutrophil-rich sputum samples of cystic fibrosis patients. In addition to citrullination of MMP-9, we report efficient citrullination of MMP-1 and lower citrullination levels of MMP-3 and MMP-13 by PAD2 in vitro. In conclusion, citrullination of MMPs is a new PTM worthy of additional biochemical and biological studies.
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http://dx.doi.org/10.1016/j.matbio.2020.10.005DOI Listing
January 2021

Efficacy of B Cell Depletion Therapy with Rituximab in Refractory Chronic Recurrent Uveitis Associated with Vogt-Koyanagi-Harada Disease.

Ocul Immunol Inflamm 2020 Sep 29:1-8. Epub 2020 Sep 29.

Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, KU Leuven, Leuven, Belgium.

Purpose: To evaluate the efficacy of B cell depletion therapy with the chimeric mouse/human anti-CD20 monoclonal antibody rituximab for refractory chronic recurrent granulomatous uveitis associated with Vogt-Koyanagi-Harada (VKH) disease.

Methods: Retrospective study of 9 patients (18 eyes) who failed to respond to conventional combination immunosuppressive therapy.

Results: All the patients received 3 rituximab infusions. The follow-up period after initiation of rituximab therapy ranged from 9 to 36 months (mean ±SD, 19.2 ± 10.1). All patients achieved remission and visual acuity significantly improved ( < .001). Rituximab provided corticosteroid-sparing effect along with control of inflammation. No rituximab-related complications were observed.

Conclusions: Rituximab is effective for the treatment of refractory chronic recurrent granulomatous uveitis associated with VKH disease.
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http://dx.doi.org/10.1080/09273948.2020.1820531DOI Listing
September 2020

Bivalent Inhibitor with Selectivity for Trimeric MMP-9 Amplifies Neutrophil Chemotaxis and Enables Functional Studies on MMP-9 Proteoforms.

Cells 2020 07 7;9(7). Epub 2020 Jul 7.

Laboratory of Immunobiology, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, University of Leuven, KU Leuven, Herestraat 49-bus 1044, B-3000 Leuven, Belgium.

A fundamental part of the immune response to infection or injury is leukocyte migration. Matrix metalloproteinases (MMPs) are a class of secreted or cell-bound endopeptidases, implicated in every step of the process of inflammatory cell migration. Hence, specific inhibition of MMPs is an interesting approach to control inflammation. We evaluated the potential of a bivalent carboxylate inhibitor to selectively inhibit the trimeric proteoform of MMP-9 and compared this with a corresponding monovalent inhibitor. The bivalent inhibitor efficiently inhibited trimeric MMP-9 (IC = 0.1 nM), with at least 500-fold selectivity for MMP-9 trimers over monomers. Surprisingly, in a mouse model for chemotaxis, the bivalent inhibitor amplified leukocyte influxes towards lipopolysaccharide-induced inflammation. We verified by microscopic and flow cytometry analysis increased amounts of neutrophils. In a mouse model for endotoxin shock, mice treated with the bivalent inhibitor had significantly increased levels of MMP-9 in plasma and lungs, indicative for increased inflammation. In conclusion, we propose a new role for MMP-9 trimers in tempering excessive neutrophil migration. In addition, we have identified a small molecule inhibitor with a high selectivity for the trimeric proteoform of MMP-9, which will allow further research on the functions of MMP-9 proteoforms.
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http://dx.doi.org/10.3390/cells9071634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408547PMC
July 2020

Neutrophils: Underestimated Players in the Pathogenesis of Multiple Sclerosis (MS).

Int J Mol Sci 2020 Jun 26;21(12). Epub 2020 Jun 26.

Laboratory of Molecular Immunology, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, KU Leuven, Herestraat 49-Box 1042, 3000 Leuven, Belgium.

Neutrophils are the most abundant circulating and first-responding innate myeloid cells and have so far been underestimated in the context of multiple sclerosis (MS). MS is the most frequent, immune-mediated, inflammatory disease of the central nervous system. MS is treatable but not curable and its cause(s) and pathogenesis remain elusive. The involvement of neutrophils in MS pathogenesis has been suggested by the use of preclinical animal disease models, as well as on the basis of patient sample analysis. In this review, we provide an overview of the possible mechanisms and functions by which neutrophils may contribute to the development and pathology of MS. Neutrophils display a broad variety of effector functions enabling disease pathogenesis, including (1) the release of inflammatory mediators and enzymes, such as interleukin-1β, myeloperoxidase and various proteinases, (2) destruction and phagocytosis of myelin (as debris), (3) release of neutrophil extracellular traps, (4) production of reactive oxygen species, (5) breakdown of the blood-brain barrier and (6) generation and presentation of autoantigens. An important question relates to the issue of whether neutrophils exhibit a predominantly proinflammatory function or are also implicated in the resolution of chronic inflammatory responses in MS.
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http://dx.doi.org/10.3390/ijms21124558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349048PMC
June 2020

Recombinant Protein-Based Nanoparticles: Elucidating their Inflammatory Effects In Vivo and their Potential as a New Therapeutic Format.

Pharmaceutics 2020 May 13;12(5). Epub 2020 May 13.

Department of Ruminant Production, Institut de Recerca i Tecnologia Agroalimentàries (IRTA), 08140 Caldes de Montbui, Spain.

Bacterial inclusion bodies (IBs) are protein-based nanoparticles of a few hundred nanometers formed during recombinant protein production processes in different bacterial hosts. IBs contain active protein in a mechanically stable nanostructured format that has been broadly characterized, showing promising potential in different fields such as tissue engineering, protein replacement therapies, cancer, and biotechnology. For immunomodulatory purposes, however, the interference of the format immunogenic properties-intrinsic to IBs-with the specific effects of the therapeutic protein is still an uncovered gap. For that, active and inactive forms of the catalytic domain of a matrix metalloproteinase-9 (MMP-9 and mutMMP-9, respectively) have been produced as IBs and compared with the soluble form for dermal inflammatory effects in knock-out mice. After protein injections in air-pouches in the mouse model, MMP-9 IBs induce local neutrophil recruitment and increase pro-inflammatory chemokine levels, lasting for at least two days, whereas the effects triggered by the soluble MMP-9 format fade out after 3 h. Interestingly, the IB intrinsic effects (mutMMP-9 IBs) do not last more than 24 h. Therefore, it may be concluded that IBs could be used for the delivery of therapeutic proteins, such as immunomodulating proteins while preserving their stability in the specific tissue and without triggering important unspecific inflammatory responses due to the protein format.
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http://dx.doi.org/10.3390/pharmaceutics12050450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284881PMC
May 2020

Remnant Epitopes Generating Autoimmunity: From Model to Useful Paradigm.

Trends Immunol 2020 May 13;41(5):367-378. Epub 2020 Apr 13.

Laboratory of Molecular Immunology, Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven, University of Leuven, Leuven, Belgium.

Autoimmune diseases are defined as pathologies of adaptive immunity by the presence of autoantibodies or MHC-restricted autoantigen-reactive T cells. Because autoreactivity is a normal process based on mechanisms producing repertoires of antibodies and T cell receptors, crucial questions about disease mechanisms and key steps for interference have been outstanding. We defined 25 years ago the 'remnant epitopes generate autoimmunity' (REGA)-model in which extracellular proteases from innate immune cells generate autoantigens. Here, we refine the REGA-model, tested in diseases ranging from organ-specific autoimmune diseases to systemic lupus erythematosus. It now constitutes a paradigm in which remnant epitopes generate, maintain, and regulate autoimmunity; are dependent on genetic and epigenetic influences; are produced in a disease phase-specific manner; and have therapeutic implications when targeted.
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http://dx.doi.org/10.1016/j.it.2020.03.004DOI Listing
May 2020

Protease propeptide structures, mechanisms of activation, and functions.

Crit Rev Biochem Mol Biol 2020 04 14;55(2):111-165. Epub 2020 Apr 14.

Rega Institute for Medical Research, Department of Microbiology, Immunology and Transplantation, Laboratory of Immunobiology, KU Leuven, Leuven, Belgium.

Proteases are a diverse group of hydrolytic enzymes, ranging from single-domain catalytic molecules to sophisticated multi-functional macromolecules. Human proteases are divided into five mechanistic classes: aspartate, cysteine, metallo, serine and threonine proteases, based on the catalytic mechanism of hydrolysis. As a protective mechanism against uncontrolled proteolysis, proteases are often produced and secreted as inactive precursors, called zymogens, containing inhibitory N-terminal propeptides. Protease propeptide structures vary considerably in length, ranging from dipeptides and propeptides of about 10 amino acids to complex multifunctional prodomains with hundreds of residues. Interestingly, sequence analysis of the different protease domains has demonstrated that propeptide sequences present higher heterogeneity compared with their catalytic domains. Therefore, we suggest that protease inhibition targeting propeptides might be more specific and have less off-target effects than classical inhibitors. The roles of propeptides, besides keeping protease latency, include correct folding of proteases, compartmentalization, liganding, and functional modulation. Changes in the propeptide sequence, thus, have a tremendous impact on the cognate enzymes. Small modifications of the propeptide sequences modulate the activity of the enzymes, which may be useful as a therapeutic strategy. This review provides an overview of known human proteases, with a focus on the role of their propeptides. We review propeptide functions, activation mechanisms, and possible therapeutic applications.
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http://dx.doi.org/10.1080/10409238.2020.1742090DOI Listing
April 2020

The Biological Potential Hidden in Inclusion Bodies.

Pharmaceutics 2020 Feb 15;12(2). Epub 2020 Feb 15.

Department of Ruminant Production, Institut de Recerca i Tecnologia Agroalimentàries (IRTA), 08140 Caldes de Montbui, Spain.

Inclusion bodies (IBs) are protein nanoclusters obtained during recombinant protein production processes, and several studies have demonstrated their potential as biomaterials for therapeutic protein delivery. Nevertheless, IBs have been, so far, exclusively sifted by their biological activity in vitro to be considered in further protein-based treatments in vivo. Matrix metalloproteinase-9 (MMP-9) protein, which has an important role facilitating the migration of immune cells, was used as model protein. The MMP-9 IBs were compared with their soluble counterpart and with MMP-9 encapsulated in polymeric-based micelles (PM) through ionic and covalent binding. The soluble MMP-9 and the MMP-9-ionic PM showed the highest activity values in vitro. IBs showed the lowest activity values in vitro, but the specific activity evolution in 50% bovine serum at room temperature proved that they were the most stable format. The data obtained with the use of an air-pouch mouse model showed that MMP-9 IBs presented the highest in vivo activity compared to the soluble MMP-9, which was associated only to a low and a transitory peak of activity. These results demonstrated that the in vivo performance is the addition of many parameters that did not always correlate with the in vitro behavior of the protein of interest, becoming especially relevant at evaluating the potential of IBs as a protein-based nanomaterial for therapeutic purposes.
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http://dx.doi.org/10.3390/pharmaceutics12020157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076398PMC
February 2020

Oncostatin M-induced astrocytic tissue inhibitor of metalloproteinases-1 drives remyelination.

Proc Natl Acad Sci U S A 2020 03 18;117(9):5028-5038. Epub 2020 Feb 18.

Department of Immunology, Biomedical Research Institute, Hasselt University, 3590 Diepenbeek, Belgium;

The brain's endogenous capacity to restore damaged myelin deteriorates during the course of demyelinating disorders. Currently, no treatment options are available to establish remyelination. Chronic demyelination leads to damaged axons and irreversible destruction of the central nervous system (CNS). We identified two promising therapeutic candidates which enhance remyelination: oncostatin M (OSM), a member of the interleukin-6 family, and downstream mediator tissue inhibitor of metalloproteinases-1 (TIMP-1). While remyelination was completely abrogated in OSMRβ knockout (KO) mice, OSM overexpression in the chronically demyelinated CNS established remyelination. Astrocytic TIMP-1 was demonstrated to play a pivotal role in OSM-mediated remyelination. Astrocyte-derived TIMP-1 drove differentiation of oligodendrocyte precursor cells into mature oligodendrocytes in vitro. In vivo, TIMP-1 deficiency completely abolished spontaneous remyelination, phenocopying OSMRβ KO mice. Finally, TIMP-1 was expressed by human astrocytes in demyelinated multiple sclerosis lesions, confirming the human value of our findings. Taken together, OSM and its downstream mediator TIMP-1 have the therapeutic potential to boost remyelination in demyelinating disorders.
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http://dx.doi.org/10.1073/pnas.1912910117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060743PMC
March 2020

The Proinflammatory and Proangiogenic Macrophage Migration Inhibitory Factor Is a Potential Regulator in Proliferative Diabetic Retinopathy.

Front Immunol 2019 4;10:2752. Epub 2019 Dec 4.

Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium.

The macrophage migration inhibitory factor (MIF)/CD74 signaling pathway is strongly implicated in inflammation and angiogenesis. We investigated the expression of MIF and its receptor CD74 in proliferative diabetic retinopathy (PDR) to reveal a possible role of this pathway in the pathogenesis of PDR. Levels of MIF, soluble (s)CD74, soluble intercellular adhesion molecule-1 (sICAM-1) and vascular endothelial growth factor (VEGF) were significantly increased in the vitreous from patients with PDR compared to nondiabetic control samples. We detected significant positive correlations between the levels of MIF and the levels of sICAM-1 ( = 0.43; = 0.001) and VEGF ( = 0.7; < 0.001). Through immunohistochemical analysis of PDR epiretinal membranes, significant positive correlations were also found between microvessel density (CD31 expression) and the numbers of blood vessels expressing MIF ( = 0.56; = 0.045) and stromal cells expressing MIF ( = 0.79; = 0.001) and CD74 ( = 0.59; = 0.045). Similar to VEGF, MIF was induced in Müller cells cultured under hypoxic conditions and MIF induced phosphorylation of ERK1/2 and VEGF production in Müller cells. Intravitreal administration of MIF in normal rats induced increased retinal vascular permeability and significant upregulation of phospho-ERK1/2, NF-κB, ICAM-1 and vascular cell adhesion molecule-1 expression in the retina. MIF induced migration and proliferation of human retinal microvascular endothelial cells. These results suggest that MIF/CD74 signaling is involved in PDR angiogenesis.
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http://dx.doi.org/10.3389/fimmu.2019.02752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904364PMC
October 2020

Soluble cytokine receptor levels in aqueous humour of patients with specific autoimmune uveitic entities: sCD30 is a biomarker of granulomatous uveitis.

Eye (Lond) 2020 09 5;34(9):1614-1623. Epub 2019 Dec 5.

Department of Microbiology and Immunology, Rega Institute for Medical Research, University of Leuven, KU Leuven, Leuven, Belgium.

Purpose: Soluble cytokine receptors are potential biomarkers for immune activation and have a promising potential as immunotherapeutic agents. We investigated the levels of soluble cytokine receptors in aqueous humour (AH) samples from patients with specific autoimmune uveitic entities.

Methods: Patients with active uveitis associated with Behçet's disease (BD) (n = 13), sarcoidosis (n = 8), HLA-B27-related inflammation (n = 12), Vogt-Koyanagi-Harada (VKH) disease (n = 12) and control subjects (n = 9) were included. AH samples were analyzed with the use of multiplex assays for the proinflammatory cytokine tumour necrosis factor (TNF)-α and the soluble cytokine receptors sCD30, sCD163, sgp130, sIL-6 receptor-α (sIL-6R), sTNFRI and sTNFRII.

Results: TNF-α and soluble cytokine receptor AH levels were significantly higher in uveitis patients (n = 45) compared with controls (n = 9). When nongranulomatous uveitis (BD and HLA-B27-associated uveitis) was compared with granulomatous uveitis (sarcoidosis and VKH disease), the levels of sCD30 and sTNFRI/TNF-α and sTNFRII/TNF-α ratios were significantly enhanced in granulomatous uveitis. Finally, when comparing the profile in the specific uveitis entities, sCD30 levels were highest in patients with VKH disease. sgp130, sCD163, sIL-6R, sTNFRI and sTNFRII did not differ significantly between the four different clinical uveitic subgroups.

Conclusions: Soluble cytokine receptors are significantly upregulated in autoimmune uveitis. CD30 T cells might contribute to the inflammatory process in granulomatous uveitis, particularly in VKH disease. Granulomatous uveitis is also characterized by significantly higher sTNFRs/TNF-α ratios than nongranulomatous uveitis.
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http://dx.doi.org/10.1038/s41433-019-0693-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7608430PMC
September 2020

Homotrimeric MMP-9 is an active hitchhiker on alpha-2-macroglobulin partially escaping protease inhibition and internalization through LRP-1.

Cell Mol Life Sci 2020 Aug 23;77(15):3013-3026. Epub 2019 Oct 23.

Laboratory of Immunobiology, Department of Microbiology and Immunology, Rega Institute for Medical Research, University of Leuven, KU Leuven, Herestraat 49, Bus 1044, 3000, Leuven, Belgium.

Proteolysis is a crucial process in life, tightly controlled by numerous natural protease inhibitors. In human blood, alpha-2-macroglobulin is an emergency protease inhibitor preventing coagulation and damage to endothelia and leukocytes. With the use of a unique protease trapping mechanism, alpha-2-macroglobulin lures active proteases into its snap-trap, shields these from potential substrates and 'flags' their complex for elimination by receptor-mediated endocytosis. Matrix metalloprotease-9/gelatinase B is a secreted protease increased in blood of patients with inflammations, vascular disorders and cancers. Matrix metalloprotease-9 occurs as monomers and stable homotrimers, but the reason for their co-existence remains obscure. We discovered that matrix metalloprotease-9 homotrimers undergo reduced anti-proteolytic regulation by alpha-2-macroglobulin and are able to travel as a proteolytically active hitchhiker on alpha-2-macroglobulin. As a comparison, we revealed that monomeric active matrix metalloprotease-9 is efficiently trapped by human plasma alpha-2-macroglobulin and this masks the detection of activated matrix metalloprotease-9 with standard analysis techniques. In addition, we show that alpha-2-macroglobulin/trimer complexes escape clearance through the receptor low-density lipoprotein receptor-related protein 1, also known as the alpha-2-macroglobulin receptor. Thus, the biochemistry and biology of matrix metalloprotease-9 monomers and trimers are completely different as multimerization enables active matrix metalloprotease-9 to partially avoid alpha-2-macroglobulin regulation both by direct protease inhibition and by removal from the extracellular space by receptor-mediated endocytosis. Finally, for the biomarker field, the analysis of alpha-2-macroglobulin/protease complexes with upgraded technology is advocated as a quotum for protease activation in human plasma samples.
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http://dx.doi.org/10.1007/s00018-019-03338-4DOI Listing
August 2020

The Ulcerative Colitis Response Index for Detection of Mucosal Healing in Patients Treated With Anti-tumour Necrosis Factor.

J Crohns Colitis 2020 Feb;14(2):176-184

Translational Research Centre for GastroIntestinal Disorders (TARGID), Department of Chronic Diseases, Metabolism and Ageing (CHROMETA), KU Leuven, Leuven, Belgium.

Background: Surrogate markers that accurately detect mucosal healing [MH] in patients with ulcerative colitis [UC] are urgently needed. Several stool neutrophil-related proteins are currently used as biomarkers for MH. However, the sensitivity and specificity are not sufficient to avoid unnecessary endoscopic evaluations.

Methods: Novel serum neutrophil-related markers (neutrophil gelatinase B-associated lipocalin and matrix metalloproteinase-9 [NGAL-MMP-9 complex], cathelicidin LL-37 and chitinase 3-like 1 [CHI3L1]), together with C-reactive protein [CRP] and neutrophil counts were studied. Serum samples were obtained from 176 anti-tumour necrosis factor [anti-TNF]-treated UC patients (145 infliximab [IFX] and 31 adalimumab [ADM]) at baseline and after a median of 9.5 weeks. All patients had active disease prior to treatment (Mayo endoscopic subscore [MES] ≥ 2), and MH was defined as MES ≤ 1. Serum was also obtained from 75 healthy controls. Binary logistic regression analysis was used to generate the Ulcerative Colitis Response Index [UCRI]. The performance of individual markers and UCRI was tested with receiver operating characteristic analysis.

Results: All neutrophil-related markers were significantly higher in active UC patients compared to healthy controls. In the IFX cohort, CRP, NGAL-MMP-9, CHI3L1 and neutrophil count decreased significantly after treatment and all marker levels were significantly lower in healers compared to non-healers following IFX. In the ADM cohort, CRP, NGAL-MMP-9, CHI3L1 and neutrophil count decreased significantly only in healers. UCRI [including CRP, CHI3L1, neutrophil count and LL-37] accurately detected MH in both IFX-treated (area under the curve [AUC] = 0.83) and ADM-treated [AUC = 0.79] patients.

Conclusions: The new UCRI index accurately detects MH after treatment with IFX and ADM. This panel is useful for monitoring MH in UC patients under anti-TNF treatment.

Podcast: This article has an associated podcast which can be accessed at https://academic.oup.com/ecco-jcc/pages/podcast.
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http://dx.doi.org/10.1093/ecco-jcc/jjz125DOI Listing
February 2020

Critical Roles of Endogenous Glucocorticoids for Disease Tolerance in Malaria.

Trends Parasitol 2019 11 9;35(11):918-930. Epub 2019 Oct 9.

Laboratory of Immunoparasitology, Department of Microbiology, Immunology, and Transplantation, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium. Electronic address:

During malaria, the hypothalamic-pituitary-adrenal (HPA) axis is activated and glucocorticoid (GC) levels are increased, but their essential roles have been largely overlooked. GCs are decisive for systemic regulation of vital processes such as immune responses, vascular function, and metabolism, which are crucial in malaria. Here, we introduce GCs in general, followed by their versatile roles for disease tolerance in malaria. A complementary comparison is provided with their role in sepsis. Finally, potential translational implications are considered. The failed clinical trials of dexamethasone against cerebral malaria in the past have diminished the interest in GCs in malaria. However, the issue of relative corticosteroid insufficiency has barely been explored in malaria patients, but may hold promise for a better understanding and treatment of specific malaria complications.
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http://dx.doi.org/10.1016/j.pt.2019.08.007DOI Listing
November 2019

Matrix metalloproteinase-9 induces a pro-angiogenic profile in chronic lymphocytic leukemia cells.

Biochem Biophys Res Commun 2019 11 1;520(1):198-204. Epub 2019 Oct 1.

Department of Molecular Biomedicine, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain. Electronic address:

Increased angiogenesis is commonly observed in chronic lymphocytic leukemia (CLL) tissues in correlation with advanced disease. CLL cells express pro- and anti-angiogenic genes and acquire a pro-angiogenic pattern upon interaction with the microenvironment. Because MMP-9 (a microenvironment component) plays important roles in solid tumor angiogenesis, we have studied whether MMP-9 influenced the angiogenic pattern in CLL cells. Immunofluorescence analyses confirmed the presence of MMP-9 in CLL tissues. MMP-9 interaction with CLL cells increased their MMP-9 expression and secretion into the medium. Accordingly, the conditioned media of MMP-9-primed CLL cells significantly enhanced endothelial cell proliferation, compared to control cells. MMP-9 also increased VEGF and decreased TSP-1 and Ang-2 expression, all at the gene and protein level, inducing a pro-angiogenic pattern in CLL cells. Mechanistic analyses demonstrated that downregulation of the selected gene TSP-1 by MMP-9 involved α4β1 integrin, Src kinase family activity and the STAT3 transcription factor. Regulation of angiogenic genes is a novel contribution of MMP-9 to CLL pathology.
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http://dx.doi.org/10.1016/j.bbrc.2019.09.127DOI Listing
November 2019

Translational glycobiology: from bench to bedside.

J R Soc Med 2019 10 17;112(10):424-427. Epub 2019 Sep 17.

National Institute for Bioprocessing Research and Training, Dublin A94 X099, Ireland.

The importance of sugars to protein function is real and is of significant clinical relevance. Technology advances enable large population studies to be carried out, shedding light on individual sugar variation and variations with time. Three-dimensional mass spectroscopy on solid pathological specimens is going to open up a whole new world of pathology visualisation. The door is now open to exploit carbohydrate recognition in new therapeutics by identifying novel biomarkers in cancer to aid diagnosis, and also providing therapeutic targets for treatment. Glycan age correlates with biological age. This means we can map the reversal of biological age with exercise and diet.
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http://dx.doi.org/10.1177/0141076819865863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794674PMC
October 2019

Interleukin-11 Overexpression and M2 Macrophage Density are Associated with Angiogenic Activity in Proliferative Diabetic Retinopathy.

Ocul Immunol Inflamm 2020 May 12;28(4):575-588. Epub 2019 Aug 12.

Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven , Leuven, Belgium.

Purpose: To investigate the expression of IL-11 and its receptor IL-11Rα and to quantify density of CD163 M2 macrophages in proliferative diabetic retinopathy (PDR).

Methods: Vitreous samples from 29 PDR and 19 nondiabetic patients, epiretinal fibrovascular membranes from 15 patients with PDR and Müller cells were studied by enzyme-linked immunosorbent assay, immunohistochemistry and Western blot analysis.

Results: We showed a significant increase in expression of IL-11, soluble(s) IL-11Rα, sCD163 and VEGF in vitreous samples from PDR patients compared to nondiabetic controls. Significant positive correlations were found between levels of VEGF and levels of IL-11 and sCD163. Significant positive correlations were found between microvessel density and number of blood vessels and stromal cells expressing IL-11, IL-11Rα and CD163 in PDR epiretinal membranes. The hypoxia mimetic agent cobalt chloride induced upregulation of IL-11 and IL-11Ra in cultured Müller cells.

Conclusions: IL-11/IL-11Rα signaling and CD163 M2 macrophages might be involved in PDR angiogenesis.
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http://dx.doi.org/10.1080/09273948.2019.1616772DOI Listing
May 2020

Galectin-1 studies in proliferative diabetic retinopathy.

Acta Ophthalmol 2020 Feb 18;98(1):e1-e12. Epub 2019 Jul 18.

Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, KU Leuven, Leuven, Belgium.

Purpose: Galectin-1 regulates endothelial cell function and promotes angiogenesis. We investigated the hypothesis that galectin-1 may be involved in the pathogenesis of proliferative diabetic retinopathy (PDR).

Methods: Vitreous samples from 36 PDR and 20 nondiabetic patients, epiretinal fibrovascular membranes from 13 patients with PDR, rat retinas and human retinal Müller glial cells were studied by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry and Western blot analysis. In vitro angiogenesis assays were performed and the adherence of leukocytes to galectin-1-stimulated human retinal microvascular endothelial cells (HRMECs) was assessed.

Results: The ELISA analysis revealed that galectin-1 and vascular endothelial growth factor (VEGF) levels were significantly higher in vitreous samples from PDR patients than in those from nondiabetics (p < 0.001 for both comparisons). A significant positive correlation was found between the levels of galectin-1 and VEGF (r = 0.354; p = 0.022). In epiretinal membranes, immunohistochemical analysis showed that galectin-1 was expressed in vascular endothelial cells expressing CD31, myofibroblasts expressing α-smooth muscle actin and leukocytes expressing CD45. The galectin-1 receptor neuropilin-1 was expressed on vascular endothelial cells. CD31 staining was used as a marker to assess microvessel density (MVD). Significant positive correlation was detected between MVD in epiretinal membranes and the number of blood vessels expressing galectin-1 (r = 0.848; p < 0.001). Western blot analysis demonstrated significant increase of galectin-1 protein in rat retinas after induction of diabetes. ELISA analysis revealed that hydrogen peroxide and cobalt chloride (CoCl ) induced upregulation of galectin-1 in Müller cells. Treatment with galectin-1 induced upregulation of VEGF in Müller cells and increased leukocyte adhesion to HRMECs. The galectin-1 inhibitor OTX008 attenuated VEGF-induced HRMECs migration and CoCl -induced upregulation of NF-κB, galectin-1 and VEGF in Müller cells.

Conclusions: These results suggest that galectin-1is involved in the pathogenesis of PDR.
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http://dx.doi.org/10.1111/aos.14191DOI Listing
February 2020
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