Publications by authors named "Ingrid de Meester"

103 Publications

The Effect of a Novel Serine Protease Inhibitor on Inflammation and Intestinal Permeability in a Murine Colitis Transfer Model.

Front Pharmacol 2021 24;12:682065. Epub 2021 Jun 24.

Laboratory of Experimental Medicine and Pediatrics, University of Antwerp, Antwerp, Belgium.

A protease/antiprotease disbalance is observed in inflammatory bowel diseases (IBD). We therefore studied the effect of the novel serine protease inhibitor UAMC-00050 on intestinal inflammation and permeability in a chronic colitis T cell transfer mouse model to get further insight into the regulation of T cell-mediated immunopathology. Colitis was induced in severe combined immunodeficient (SCID) mice, by the adoptive transfer of CD4CD25CD62L T cells. Animals were treated intraperitoneally (i.p.) 2x/day with vehicle or UAMC-00050 (5 mg/kg) from week 2 onwards. Colonic inflammation was assessed by clinical parameters, colonoscopy, macroscopy, microscopy, myeloperoxidase activity and cytokine expression levels. At week 4, 4 kDa FITC-dextran intestinal permeability was evaluated and T helper transcription factors, protease-activated receptors and junctional proteins were quantified by RT-qPCR. Adoptive transfer of CD4CD25CD62L T cells resulted in colonic inflammation and an altered intestinal permeability. The serine protease inhibitor UAMC-00050 ameliorated both the inflammatory parameters and the intestinal barrier function. Furthermore, a decrease in colonic mRNA expression of Tbet and PAR4 was observed in colitis mice after UAMC-00050 treatment. The beneficial effect of UAMC-00050 on inflammation was apparent via a reduction of Tbet, IFN-γ, TNF-α, IL-1β and IL-6. Based on these results, we hypothesize a pivotal effect of serine protease inhibition on the Th1 inflammatory profile potentially mediated via PAR4.
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http://dx.doi.org/10.3389/fphar.2021.682065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8264366PMC
June 2021

Prolyl Carboxypeptidase Mediates the C-Terminal Cleavage of (Pyr)-Apelin-13 in Human Umbilical Vein and Aortic Endothelial Cells.

Int J Mol Sci 2021 Jun 22;22(13). Epub 2021 Jun 22.

Laboratory of Medical Biochemistry, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, 2610 Wilrijk, Belgium.

The aim of this study was to investigate the C-terminal cleavage of (pyr)-apelin-13 in human endothelial cells with respect to the role and subcellular location of prolyl carboxypeptidase (PRCP). Human umbilical vein and aortic endothelial cells, pre-treated with prolyl carboxypeptidase-inhibitor compound 8o and/or angiotensin converting enzyme 2 (ACE2)-inhibitor DX600, were incubated with (pyr)-apelin-13 for different time periods. Cleavage products of (pyr)-apelin-13 in the supernatant were identified by mass spectrometry. The subcellular location of PRCP was examined via immunocytochemistry. In addition, PRCP activity was measured in supernatants and cell lysates of LPS-, TNFα-, and IL-1β-stimulated cells. PRCP cleaved (pyr)-apelin-13 in human umbilical vein and aortic endothelial cells, while ACE2 only contributed to this cleavage in aortic endothelial cells. PRCP was found in endothelial cell lysosomes. Pro-inflammatory stimulation induced the secretion of PRCP in the extracellular environment of endothelial cells, while its intracellular level remained intact. In conclusion, PRCP, observed in endothelial lysosomes, is responsible for the C-terminal cleavage of (pyr)-apelin-13 in human umbilical vein endothelial cells, while in aortic endothelial cells ACE2 also contributes to this cleavage. These results pave the way to further elucidate the relevance of the C-terminal Phe of (pyr)-apelin-13.
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http://dx.doi.org/10.3390/ijms22136698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8268575PMC
June 2021

In Vitro Evaluation of the Squaramide-Conjugated Fibroblast Activation Protein Inhibitor-Based Agents AAZTA.SA.FAPi and DOTA.SA.FAPi.

Molecules 2021 Jun 8;26(12). Epub 2021 Jun 8.

Department of Chemistry-TRIGA, Johannes Gutenberg University Mainz, 55128 Mainz, Germany.

Recently, the first squaramide-(SA) containing FAP inhibitor-derived radiotracers were introduced. DATA.SA.FAPi and DOTA.SA.FAPi with their non-radioactive complexes showed high affinity and selectivity for FAP. After a successful preclinical study with [Ga]Ga-DOTA.SA.FAPi, the first patient studies were realized for both compounds. Here, we present a new squaramide-containing compound targeting FAP, based on the AAZTA chelator 1,4-bis-(carboxylmethyl)-6-[bis-(carboxymethyl)-amino-6-pentanoic-acid]-perhydro-1,4-diazepine. For this molecule (AAZTA.SA.FAPi), complexation with radionuclides such as gallium-68, scandium-44, and lutetium-177 was investigated, and the in vitro properties of the complexes were characterized and compared with those of DOTA.SA.FAPi. AAZTA.SA.FAPi and its derivatives labelled with non-radioactive isotopes demonstrated similar excellent inhibitory potencies compared to the previously published SA.FAPi ligands, i.e., sub-nanomolar IC values for FAP and high selectivity indices over the serine proteases PREP and DPPs. Labeling with all three radiometals was easier and faster with AAZTA.SA.FAPi compared to the corresponding DOTA analogue at ambient temperature. Especially, scandium-44 labeling with the AAZTA derivative resulted in higher specific activities. Both DOTA.SA.FAPi and AAZTA.SA.FAPi showed sufficiently high stability in different media. Therefore, these FAP inhibitor agents could be promising for theranostic approaches targeting FAP.
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http://dx.doi.org/10.3390/molecules26123482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8226449PMC
June 2021

Local Colonic Administration of a Serine Protease Inhibitor Improves Post-Inflammatory Visceral Hypersensitivity in Rats.

Pharmaceutics 2021 May 29;13(6). Epub 2021 May 29.

Laboratory of Experimental Medicine and Pediatrics (LEMP), University of Antwerp, 2610 Wilrijk, Belgium.

Dysregulation of the protease-antiprotease balance in the gastrointestinal tract has been suggested as a mechanism underlying visceral hypersensitivity in conditions such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). We aimed to study the potential therapeutic role of an intracolonically administered serine protease inhibitor for the treatment of abdominal pain in a post-inflammatory rat model for IBS. An enema containing 2,4,6-trinitrobenzene sulfonic acid (TNBS) was used to induce colitis in male Sprague-Dawley rats, whereas controls received a saline solution. Colonoscopies were performed to confirm colitis and follow-up mucosal healing. In the post-inflammatory phase, the serine protease inhibitor UAMC-00050 (0.1-5 mg/kg) or its vehicle alone (5% DMSO in HO) was administered in the colon. Thirty minutes later, visceral mechanosensitivity to colorectal distensions was quantified by visceromotor responses (VMRs) and local effects on colonic compliance and inflammatory parameters were assessed. Specific proteolytic activities in fecal and colonic samples were measured using fluorogenic substrates. Pharmacokinetic parameters were evaluated using bioanalytical measurements with liquid chromatography-tandem mass spectrometry. Post-inflammatory rats had increased trypsin-like activity in colonic tissue and elevated elastase-like activity in fecal samples compared to controls. Treatment with UAMC-00050 decreased trypsin-like activity in colonic tissue of post-colitis animals. Pharmacokinetic experiments revealed that UAMC-00050 acted locally, being taken up in the bloodstream only minimally after administration. Local administration of UAMC-00050 normalized visceral hypersensitivity. These results support the role of serine proteases in the pathophysiology of visceral pain and the potential of locally administered serine protease inhibitors as clinically relevant therapeutics for the treatment of IBS patients with abdominal pain.
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http://dx.doi.org/10.3390/pharmaceutics13060811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8229129PMC
May 2021

and Activity-Based Labeling of Fibroblast Activation Protein with UAMC1110-Derived Probes.

Front Chem 2021 14;9:640566. Epub 2021 Apr 14.

Laboratory of Medicinal Chemistry, Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium.

Fibroblast activation protein (FAP) is a proline-selective protease that belongs to the S9 family of serine proteases. It is typically highly expressed in the tumor microenvironment (TME) and especially in cancer-associated fibroblasts, the main cell components of the tumor stroma. The exact role of its enzymatic activity in the TME remains largely unknown. Hence, tools that enable selective, activity-based visualization of FAP within the TME can help to unravel FAP's function. We describe the synthesis, biochemical characterization, and application of three different activity-based probes (biotin-, Cy3-, and Cy5-labeled) based on the FAP-inhibitor UAMC1110, an in-house developed molecule considered to be the most potent and selective FAP inhibitor available. We demonstrate that the three probes have subnanomolar FAP affinity and pronounced selectivity with respect to the related S9 family members. Furthermore, we report that the fluorescent Cy3- and Cy5-labeled probes are capable of selectively detecting FAP in a cellular context, making these chemical probes highly suitable for further biological studies. Moreover, proof of concept is provided for FAP activity staining in patient-derived cryosections of urothelial tumors.
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http://dx.doi.org/10.3389/fchem.2021.640566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8114891PMC
April 2021

Effect of Statin Therapy on the Carboxypeptidase U (CPU, TAFIa, CPB2) System in Patients With Hyperlipidemia: A Proof-of-Concept Observational Study.

Clin Ther 2021 Apr 25. Epub 2021 Apr 25.

Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium. Electronic address:

Purpose: Statins are commonly used in patients with hypercholesterolemia to lower their cholesterol levels and to reduce their cardiovascular risk. There is also considerable evidence that statins possess a range of cholesterol-independent effects, including profibrinolytic properties. This pilot study aimed to explore the influence of statins on procarboxypeptidase U (proCPU) biology and to search for possible effects and associations that can be followed up in a larger study.

Methods: Blood was collected from 16 patients with hyperlipidemia, before and after 3 months of statin therapy (simvastatin 20 mg or atorvastatin 20 mg). Fifteen age-matched normolipemic persons served as control subjects. Lipid parameters and markers of inflammation and fibrinolysis (proCPU levels and clot lysis times) were determined in all samples.

Findings: Mean (SD) proCPU levels were significantly higher in patients with hypercholesterolemia compared to control subjects (1186 [189] U/L vs 1061 [60] U/L). Treatment of these patients with a statin led to a significant average decrease of 11.6% in proCPU levels and brought the proCPU concentrations to the same level as in the control subjects. On a functional level, enhancement in plasma fibrinolytic potential was observed in the statin group, with the largest improvement in fibrinolysis seen in patients with the highest baseline proCPU levels and largest proCPU decrease upon statin treatment.

Implications: Increased proCPU levels are present in patients with hyperlipidemia. Statin treatment significantly decreased proCPU levels and improved plasma fibrinolysis in these patients. Moreover, our study indicates that patients with high baseline proCPU levels are most likely to benefit from statin therapy. The latter should be examined further in a large cohort.
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http://dx.doi.org/10.1016/j.clinthera.2021.03.011DOI Listing
April 2021

A novel serine protease inhibitor as potential treatment for dry eye syndrome and ocular inflammation.

Sci Rep 2020 10 14;10(1):17268. Epub 2020 Oct 14.

Laboratory of Microbiology, Parasitology and Hygiene (LMPH), Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610, Wilrijk, Belgium.

Dry eye syndrome (DES), a multifactorial disorder which leads to ocular discomfort, visual disturbance and tear film instability, has a rising prevalence and limited treatment options. In this study, a newly developed trypsin-like serine protease inhibitor (UAMC-00050) in a tear drop formulation was evaluated to treat ocular inflammation. A surgical animal model of dry eye was employed to investigate the potential of UAMC-00050 on dry eye pathology. Animals treated with UAMC-00050 displayed a significant reduction in ocular surface damage after evaluation with sodium fluorescein, compared to untreated, vehicle treated and cyclosporine-treated animals. The concentrations of IL-1α and TNF-α were also significantly reduced in tear fluid from UAMC-00050-treated rats. Additionally, inflammatory cell infiltration in the palpebral conjunctiva (CD3 and CD45), was substantially reduced. An accumulation of pro-MMP-9 and a decrease in active MMP-9 were found in tear fluid from animals treated with UAMC-00050, suggesting that trypsin-like serine proteases play a role in activating MMP-9 in ocular inflammation in this animal model. Comparative qRT-PCR analyses on ocular tissue indicated the upregulation of tryptase, urokinase plasminogen activator receptor (uPAR) and protease-activated receptor 2 (PAR2). The developed UAMC-00050 formulation was stable up to 6 months at room temperature in the absence of light, non-irritating and sterile with compatible pH and osmolarity. These results provide a proof-of-concept for the in vivo modifying potential of UAMC-00050 on dry eye pathology and suggest a central role of trypsin-like serine proteases and PAR2 in dry eye derived ocular inflammation.
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http://dx.doi.org/10.1038/s41598-020-74159-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7560718PMC
October 2020

Targeting fibroblast activation protein (FAP): next generation PET radiotracers using squaramide coupled bifunctional DOTA and DATA chelators.

EJNMMI Radiopharm Chem 2020 Jul 29;5(1):19. Epub 2020 Jul 29.

Department of Chemistry - TRIGA Site, Johannes Gutenberg University Mainz, 55128, Mainz, Germany.

Background: Fibroblast activation protein (FAP) is a proline selective serine protease that is overexpressed in tumor stroma and in lesions of many other diseases that are characterized by tissue remodeling. In 2014, a most potent FAP-inhibitor (referred to as UAMC1110) with low nanomolar FAP-affinity and high selectivity toward related enzymes such as prolyl oligopeptidase (PREP) and the dipeptidyl-peptidases (DPPs): DPP4, DPP8/9 and DPP2 were developed. This inhibitor has been adopted recently by other groups to create radiopharmaceuticals by coupling bifunctional chelator-linker systems. Here, we report squaric acid (SA) containing bifunctional DATA and DOTA chelators based on UAMC1110 as pharmacophor. The novel radiopharmaceuticals DOTA.SA.FAPi and DATA.SA.FAPi with their non-radioactive derivatives were characterized for in vitro inhibitory efficiency to FAP and PREP, respectively and radiochemical investigated with gallium-68. Further, first proof-of-concept in vivo animal study followed by ex vivo biodistribution were determined with [Ga]Ga-DOTA.SA.FAPi.

Results: [Ga]Ga-DOTA.SA.FAPi and [Ga]Ga-DATA.SA.FAPi showed high complexation > 97% radiochemical yields after already 10 min and high stability over a period of 2 h. Affinity to FAP of DOTA.SA.FAPi and DATA.SA.FAPi and its Ga and Lu-labeled derivatives were excellent resulting in low nanomolar IC values of 0.7-1.4 nM. Additionally, all five compounds showed low affinity for the related protease PREP (high IC with 1.7-8.7 μM). First proof-of-principle in vivo PET-imaging animal studies of the [Ga]Ga-DOTA.SA.FAPi precursor in a HT-29 human colorectal cancer xenograft mouse model indicated promising results with high accumulation in tumor (SUV of 0.75) and low background signal. Ex vivo biodistribution showed highest uptake in tumor (5.2%ID/g) at 60 min post injection with overall low uptake in healthy tissues.

Conclusion: In this work, novel PET radiotracers targeting fibroblast activation protein were synthesized and biochemically investigated. Critical substructures of the novel compounds are a squaramide linker unit derived from the basic motif of squaric acid, DOTA and DATA bifunctional chelators and a FAP-targeting moiety. In conclusion, these new FAP-ligands appear promising, both for further research and development as well as for first human application.
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http://dx.doi.org/10.1186/s41181-020-00102-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7391456PMC
July 2020

Dysregulated activities of proline-specific enzymes in septic shock patients (sepsis-2).

PLoS One 2020 21;15(4):e0231555. Epub 2020 Apr 21.

Laboratory of Medical Biochemistry, University of Antwerp, Antwerp, Belgium.

The proline-specific enzymes dipeptidyl peptidase 4 (DPP4), prolylcarboxypeptidase (PRCP), fibroblast activation protein α (FAP) and prolyl oligopeptidase (PREP) are known for their involvement in the immune system and blood pressure regulation. Only very limited information is currently available on their enzymatic activity and possible involvement in patients with sepsis and septic-shock. The activity of the enzymes was measured in EDTA-plasma of patients admitted to the intensive care unit (ICU): 40 septic shock patients (sepsis-2) and 22 ICU control patients after major intracranial surgery. These data were used to generate receiver operating characteristic (ROC) curves. A survival analysis (at 90 days) and an association study with other parameters was performed. PRCP (day 1) and PREP (all days) enzymatic activities were higher in septic shock patients compared to controls. In contrast, FAP and DPP4 were lower in these patients on all studied time points. Since large differences were found, ROC curves were generated and these yielded area under the curve (AUC) values for PREP, FAP and DPP4 of 0.88 (CI: 0.80-0.96), 0.94 (CI: 0.89-0.99) and 0.86 (CI: 0.77-0.95), respectively. PRCP had a lower predicting value with an AUC of 0.71 (CI: 0.58-0.83). A nominally significant association was observed between survival and the DPP4 enzymatic activity at day 1 (p<0.05), with a higher DPP4 activity being associated with an increase in survival. All four enzymes were dysregulated in septic shock patients. DPP4, FAP and PREP are good in discriminating between septic shock patients and ICU controls and should be further explored to see whether they are already dysregulated in earlier stages, opening perspectives for their further investigation as biomarkers in sepsis. DPP4 also shows potential as a prognostic biomarker. Additionally, the associations found warrant further research.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0231555PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7173796PMC
July 2020

Native, Intact Glucagon-Like Peptide 1 Is a Natural Suppressor of Thrombus Growth Under Physiological Flow Conditions.

Arterioscler Thromb Vasc Biol 2020 03 2;40(3):e65-e77. Epub 2020 Jan 2.

From the Institute for Molecular Cardiovascular Research (IMCAR) (M.S., C.C.F.M.J.B., J.W., W.T., B.W., J.J., H.N.), University Clinic Aachen, Germany.

Objective: In patients with diabetes mellitus, increased platelet reactivity predicts cardiac events. Limited evidence suggests that DPP-4 (dipeptidyl peptidase 4) influences platelets via GLP-1 (glucagon-like peptide 1)-dependent effects. Because DPP-4 inhibitors are frequently used in diabetes mellitus to improve the GLP-1-regulated glucose metabolism, we characterized the role of DPP-4 inhibition and of native intact versus DPP-4-cleaved GLP-1 on flow-dependent thrombus formation in mouse and human blood. Approach and Results: An ex vivo whole blood microfluidics model was applied to approach in vivo thrombosis and study collagen-dependent platelet adhesion, activation, and thrombus formation under shear-flow conditions by multiparameter analyses. In mice, in vivo inhibition or genetic deficiency of DPP-4 (), but not of GLP-1-receptors (), suppressed flow-dependent platelet aggregation. In human blood, GLP-1(7-36), but not DPP-4-cleaved GLP-1(9-36), reduced thrombus volume by 32% and impaired whole blood thrombus formation at both low/venous and high/arterial wall-shear rates. These effects were enforced upon ADP costimulation and occurred independently of plasma factors and leukocytes. Human platelets did not contain detectable levels of GLP-1-receptor transcripts. Also, GLP-1(7-36) did not inhibit collagen-induced aggregation under conditions of stirring or stasis of platelets, pointing to a marked flow-dependent role.

Conclusions: Native, intact GLP-1 is a natural suppressor of thrombus growth under physiological flow conditions, with DPP-4 inhibition and increased intact GLP-1 suppressing platelet aggregation under flow without a main relevance of GLP-1-receptor on platelets.
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http://dx.doi.org/10.1161/ATVBAHA.119.313645DOI Listing
March 2020

Novel Small Molecule-Derived, Highly Selective Substrates for Fibroblast Activation Protein (FAP).

ACS Med Chem Lett 2019 Aug 9;10(8):1173-1179. Epub 2019 Jul 9.

Laboratory of Medicinal Chemistry (UAMC), Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk (Antwerp), Belgium.

Fibroblast activation protein (FAP) is a proline-selective serine protease. It is hardly expressed in healthy adult tissue but upregulated in tissue remodeling sites associated with several diseases including epithelial cancer types, atherosclerosis, arthritis and fibrosis. Ongoing research aims at clinical implementation of FAP as a biomarker for these diseases. Several immunochemical methods that quantify FAP expression have been reported. An alternative/complementary approach focuses on quantification of FAP's enzymatic activity. Developing an activity-based assay for FAP has nonetheless proven challenging because of selectivity issues with respect to prolyl oligopeptidase (PREP). Here, we present substrate-type FAP probes that are structurally derived from a FAP-inhibitor (UAMC1110) that we published earlier. Both cleavage efficiency and FAP-selectivity of the best compounds in the series equal or surpass the most advanced peptide-based FAP substrates reported to date. Finally, proof-of-concept is provided that 4-aminonaphthol containing probes can spatially localize FAP activity in biological samples.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6691567PMC
August 2019

The development and validation of a combined kinetic fluorometric activity assay for fibroblast activation protein alpha and prolyl oligopeptidase in plasma.

Clin Chim Acta 2019 Aug 11;495:154-160. Epub 2019 Apr 11.

Laboratory of Medical Biochemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium. Electronic address:

Background: Fibroblast activiation protein alpha (FAP) is considered a diagnostic and prognostic biomarker for various types of cancer. FAP shares substrate specificity with prolyl oligopeptidase (PREP), studied in (neuro)inflammation and neurodegeneration as well as cancer. Current assays inadequately discriminate between FAP and PREP and there is need for an assay that reliably quantitates the FAP/PREP activity ratio in plasma.

Methods: FAP and PREP activities were measured in human EDTA-plasma in presence of well characterized PREP and FAP inhibitors.

Results: A combined kinetic assay was developed in conditions to optimally measure FAP as well as PREP activity with Z-Gly-Pro-AMC as substrate. Limit of detection was 0.009 U/L and limit of quantitation was 0.027 U/L for the combined FAP-PREP assay. Within-run coefficient of variation was 3% and 4% and between-run precision was 7% and 12% for PREP and FAP, respectively. Accuracy was demonstrated by comparison with established end-point assays. Hemolysis interferes with the assay with 1.5 g/L hemoglobin as cut-off value. PREP (but not FAP) activity can increase upon lysis of platelets and red blood cells during sample preparation.

Conclusion: With this new assay, on average 67% of the Z-Gly-Pro-AMC converting activity in plasma can be attributed to FAP.
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http://dx.doi.org/10.1016/j.cca.2019.04.063DOI Listing
August 2019

CD26/DPP4 - a potential biomarker and target for cancer therapy.

Pharmacol Ther 2019 06 26;198:135-159. Epub 2019 Feb 26.

Department of Thoracic Surgery, University Hospital Rostock, Schillingallee 35, 18057 Rostock, Germany. Electronic address:

CD26/dipeptidyl peptidase (DPP)4 is a membrane-bound protein found in many cell types of the body, and a soluble form is present in body fluids. There is longstanding evidence that various primary tumors and also metastases express CD26/DPP4 to a variable extent. By cleaving dipeptides from peptides with a proline or alanine in the penultimate position at the N-terminus, it regulates the activity of incretin hormones, chemokines and many other peptides. Due to these effects and interactions with other molecules, a tumor promoting or suppressing role can be attributed to CD26/DPP4. In this review, we discuss the existing evidence on the expression of soluble or membrane-bound CD26/DPP4 in malignant diseases, along with the most recent findings on CD26/DPP4 as a therapeutic target in specific malignancies. The expression and possible involvement of the related DPP8 and DPP9 in cancer are also reviewed. A higher expression of CD26/DPP4 is found in a wide variety of tumor entities, however more research on CD26/DPP4 in the tumor microenvironment is needed to fully explore its use as a tumor biomarker. Circulating soluble CD26/DPP4 has also been studied as a cancer biomarker, however, the observed decrease in most cancer patients does not seem to be cancer specific. Encouraging results from experimental work and a recently reported first phase clinical trial targeting CD26/DPP4 in mesothelioma, renal and urological tumors pave the way for follow-up clinical studies, also in other tumor entities, possibly leading to the development of more effective complementary therapies against cancer.
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http://dx.doi.org/10.1016/j.pharmthera.2019.02.015DOI Listing
June 2019

DPP8/DPP9 inhibition elicits canonical Nlrp1b inflammasome hallmarks in murine macrophages.

Life Sci Alliance 2019 02 4;2(1). Epub 2019 Feb 4.

Department of Internal Medicine, Ghent University, Ghent, Belgium

Activating germline mutations in the human inflammasome sensor NLRP1 causes palmoplantar dyskeratosis and susceptibility to Mendelian autoinflammatory diseases. Recent studies have shown that the cytosolic serine dipeptidyl peptidases DPP8 and DPP9 suppress inflammasome activation upstream of NLRP1 and CARD8 in human keratinocytes and peripheral blood mononuclear cells. Moreover, pharmacological inhibition of DPP8/DPP9 protease activity was shown to induce pyroptosis in murine C57BL/6 macrophages without eliciting other inflammasome hallmark responses. Here, we show that DPP8/DPP9 inhibition in macrophages that express a lethal toxin (LeTx)-sensitive allele triggered significantly accelerated pyroptosis concomitant with caspase-1 maturation, ASC speck assembly, and secretion of mature IL-1β and IL-18. Genetic ablation of ASC prevented DPP8/DPP9 inhibition-induced caspase-1 maturation and partially hampered pyroptosis and inflammasome-dependent cytokine release, whereas deletion of caspase-1 or gasdermin D triggered apoptosis in the absence of IL-1β and IL-18 secretion. In conclusion, blockade of DPP8/DPP9 protease activity triggers rapid pyroptosis and canonical inflammasome hallmarks in primary macrophages that express a LeTx-responsive allele.
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http://dx.doi.org/10.26508/lsa.201900313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6362307PMC
February 2019

The CD26/DPP4-inhibitor vildagliptin suppresses lung cancer growth via macrophage-mediated NK cell activity.

Carcinogenesis 2019 04;40(2):324-334

Department of Thoracic Surgery, University Hospital Zurich, Zurich, Switzerland.

CD26/dipeptidyl peptidase 4 (DPP4) is a transmembrane protein which is expressed by various malignant cells. We found that the expression of CD26/DPP4 was significantly higher in lung adenocarcinoma samples in our own patient cohort compared to normal lung tissue. We therefore hypothesize that the inhibition of CD26/DPP4 can potentially suppress lung cancer growth. The CD26/DPP4 inhibitor vildagliptin was employed on Lewis Lung Carcinoma (LLC) cell line and a human lung adenocarcinoma (H460) cell line. Two weeks after subcutaneous injection of tumor cells into C57BL/6 and CD1/nude mice, the size of LLC and H460 tumors was significantly reduced by vildagliptin. Immunohistochemically, the number of macrophages (F4/80+) and NK cells (NKp46+) was significantly increased in vildagliptin-treated tumor samples. Mechanistically, we found in vitro that lung cancer cell lines expressed increased levels of surfactant protein upon vildagliptin treatment thereby promoting the pro-inflammatory activity of macrophages. By the depletion of macrophages with clodronate and by using NK cell deficient (IL-15-/-) mice, tumors reversed to the size of controls, suggesting that indeed macrophages and NK cells were responsible for the observed tumor-suppressing effect upon vildagliptin treatment. FACS analysis showed tumor-infiltrating NK cells to express tumor necrosis-related apoptosis-inducing ligand (TRAIL) which induced the intra-cellular stress marker γH2AX. Accordingly, we found upregulated γH2AX in vildagliptin-treated tumors and TRAIL-treated cell lines. Moreover, the effect of vildagliptin-mediated enhanced NK cell cytotoxicity could be reversed by antagonizing the TRAIL receptor. Our data provide evidence that the CD26/DPP4-inhibitor vildagliptin reduces lung cancer growth. We could demonstrate that this effect is exerted by surfactant-activated macrophages and NK cells that act against the tumor via TRAIL-mediated cytotoxicity.
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http://dx.doi.org/10.1093/carcin/bgz009DOI Listing
April 2019

Peroxynitrite Exposure of CXCL12 Impairs Monocyte, Lymphocyte and Endothelial Cell Chemotaxis, Lymphocyte Extravasation and Anti-HIV-1 Activity.

Front Immunol 2018 28;9:1933. Epub 2018 Aug 28.

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

CXCL12 is a chemotactic cytokine that attracts many different cell types for homeostasis and during inflammation. Under stress conditions, macrophages and granulocytes produce factors such as peroxynitrite as a consequence of their oxidative response. After short incubations of CXCL12 with peroxynitrite, the gradual nitration of Tyr7, Tyr61, or both Tyr7 and Tyr61 was demonstrated with the use of mass spectrometry, whereas longer incubations caused CXCL12 degradation. Native CXCL12 and the nitrated forms, [3-NT]CXCL12 and [3-NT]CXCL12, were chemically synthesized to evaluate the effects of Tyr nitration on the biological activity of CXCL12. All CXCL12 forms had a similar binding affinity for heparin, the G protein-coupled chemokine receptor CXCR4 and the atypical chemokine receptor ACKR3. However, nitration significantly enhanced the affinity of CXCL12 for chondroitin sulfate. Internalization of CXCR4 and β-arrestin 2 recruitment to CXCR4 was significantly reduced for [3-NT]CXCL12 compared to CXCL12, whereas β-arrestin 2 recruitment to ACKR3 was similar for all CXCL12 variants. [3-NT]CXCL12 was weaker in calcium signaling assays and in i chemotaxis assays with monocytes, lymphocytes and endothelial cells. Surprisingly, nitration of Tyr61, but not Tyr7, partially protected CXCL12 against cleavage by the specific serine protease CD26. , the effects were more pronounced compared to native CXCL12. Nitration of any Tyr residue drastically lowered lymphocyte extravasation to joints compared to native CXCL12. Finally, the anti-HIV-1 activity of [3-NT]CXCL12 and [3-NT]CXCL12 was reduced, whereas CXCL12 and [3-NT]CXCL12 were equally potent. In conclusion, nitration of CXCL12 occurs readily upon contact with peroxynitrite and specifically nitration of Tyr7 fully reduces its and biological activities.
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http://dx.doi.org/10.3389/fimmu.2018.01933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6127631PMC
September 2019

Newly developed serine protease inhibitors decrease visceral hypersensitivity in a post-inflammatory rat model for irritable bowel syndrome.

Br J Pharmacol 2018 09 23;175(17):3516-3533. Epub 2018 Jul 23.

Laboratory of Experimental Medicine and Pediatrics, Division of Gastroenterology, University of Antwerp, Antwerp, Belgium.

Background And Purpose: Serine proteases have been re suggested as important mediators of visceral pain. We investigated their effect by using newly developed serine protease inhibitors with a well-characterized inhibitory profile in a rat model of post-inflammatory irritable bowel syndrome (IBS).

Experimental Approach: Colitis was induced in rats receiving intrarectal trinitrobenzenesulphonic acid; controls received 0.9% NaCl. Colonoscopies were performed on day 3, to confirm colitis, and later until mucosal healing. Visceral hypersensitivity was quantified by visceromotor responses (VMRs) to colorectal distension, 30 min after i.p. injection of the serine protease inhibitors nafamostat, UAMC-00050 or UAMC-01162. Serine proteases, protease-activated receptors (PARs) and TRP channels were quantified by qPCR and immunohistochemistry. Proteolytic activity was characterized using fluorogenic substrates.

Key Results: VMR was significantly elevated in post-colitis rats. Nafamostat normalized VMRs at the lowest dose tested. UAMC-00050 and UAMC-01162 significantly decreased VMR dose-dependently. Expression of mRNA for tryptase-αβ-1and PAR4, and tryptase immunoreactivity was significantly increased in the colon of post-colitis animals. Trypsin-like activity was also significantly increased in the colon but not in the faeces. PAR2 and TRPA1 immunoreactivity co-localized with CGRP-positive nerve fibres in control and post-colitis animals.

Conclusions And Implications: Increased expression of serine proteases and activity together with increased expression of downstream molecules at the colonic and DRG level and in CGRP-positive sensory nerve fibres imply a role for serine proteases in post-inflammatory visceral hypersensitivity. Our results support further investigation of serine protease inhibitors as an interesting treatment strategy for IBS-related visceral pain.
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http://dx.doi.org/10.1111/bph.14396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6086981PMC
September 2018

Prolyl carboxypeptidase activity in the circulation and its correlation with body weight and adipose tissue in lean and obese subjects.

PLoS One 2018 17;13(5):e0197603. Epub 2018 May 17.

Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium.

Background: Prolyl carboxypeptidase (PRCP) is involved in the regulation of body weight, likely by hydrolysing alpha-melanocyte-stimulating hormone and apelin in the hypothalamus and in the periphery. A link between PRCP protein concentrations in plasma and metabolic disorders has been reported. In this study, we investigated the distribution of circulating PRCP activity and assessed its relation with body weight and adipose tissue in obese patients and patients who significantly lost weight.

Methods: PRCP activity was measured using reversed-phase high-performance liquid chromatography in different isolated blood fractions and primary human cells to investigate the distribution of circulating PRCP. PRCP activity was measured in serum of individuals (n = 75) categorized based on their body mass index (BMI < 25.0; 25.0-29.9; 30.0-39.9; ≥ 40.0 kg/m2) and the diagnosis of metabolic syndrome. Differences in serum PRCP activity were determined before and six months after weight loss, either by diet (n = 45) or by bariatric surgery (n = 24). Potential correlations between serum PRCP activity and several metabolic and biochemical parameters were assessed. Additionally, plasma PRCP concentrations were quantified using a sensitive ELISA in the bariatric surgery group.

Results: White blood cells and plasma contributed the most to circulating PRCP activity. Serum PRCP activity in lean subjects was 0.83 ± 0.04 U/L and increased significantly with a rising BMI (p<0.001) and decreased upon weight loss (diet, p<0.05; bariatric surgery, p<0.001). The serum PRCP activity alteration reflected body weight changes and was found to be positively correlated with several metabolic parameters, including: total, abdominal and visceral adipose tissue. Plasma PRCP concentration was found to be significantly correlated to serum PRCP activity (0.865; p<0.001). Additionally, a significant decrease (p<0.001) in plasma PRCP protein concentration (mean ± SD) before (18.2 ± 3.7 ng/mL) and 6 months after bariatric surgery (15.7 ± 2.7 ng/mL) was found.

Conclusion: Our novel findings demonstrate that white blood cells and plasma contributed the most to circulating PRCP activity. Additionally, we have shown that there were significant correlations between serum PRCP activity and various metabolic parameters, and that plasma PRCP concentration was significantly correlated to serum PRCP activity. These novel findings on PRCP activity in serum support further investigation of its in vivo role and involvement in several metabolic diseases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197603PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5957431PMC
December 2018

Optimal Evaluation of Programmed Death Ligand-1 on Tumor Cells Versus Immune Cells Requires Different Detection Methods.

Arch Pathol Lab Med 2018 08 2;142(8):982-991. Epub 2018 Apr 2.

From the Departments of Immunohistochemistry (Drs Schats, Van Vré, Boeckx, and Schrijvers and Ms De Bie) and Molecular Pathology (Dr Kockx), HistoGeneX, Antwerp, Belgium; the Department of Medical Biochemistry, University of Antwerp, Antwerp, Belgium (Drs Schats and De Meester); and the Department of Medical Oncology, Universitair Ziekenhuis Brussel, Belgium (Dr Neyns).

Context: - The benefit of programmed death ligand-1 (PD-L1) immunohistochemistry (IHC) as a method to select patients who may benefit from programmed death receptor-1 (PD-1)/PD-L1 immunotherapies remains uncertain in many tumor indications.

Objectives: - To compare the commercially available, approved PD-L1 IHC assays (22C3, 28-8, SP142, SP263), specifically identifying the changes in staining output created by altering the detection method.

Design: - This pilot study investigates the respective PD-L1 kit assay staining patterns and related scoring of tumor cells and immune cells on lung carcinoma and melanoma. Furthermore, the influence of the detection method (platform and related reagents) on PD-L1 antibody performance is studied.

Results: - The SP142 kit reveals more immune cell staining but less tumor cell staining than the other PD-L1 kits. Alternatively, the 22C3 and 28-8 kits show good tumor cell sensitivity, but less pronounced immune cell staining, even in tonsil. Tumor cell staining by the SP263 kit is comparable to that of 22C3 and 28-8 kits, while immune cell staining is better. Strikingly, the selection of the detection method has a major impact on the sensitivity of the assay for PD-L1 detection per cell type. Switching the detection method of the kits could largely circumvent the observed staining differences.

Conclusions: - The diverse sensitivities caused by the choice of the detection method should be taken into consideration when selecting PD-L1 kits or developing PD-L1 IHC laboratory-developed tests. When using alternative kits or laboratory-developed tests, it is strongly recommended to reestablish their clinical utility per therapeutic agent or compare them with the original kit.
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http://dx.doi.org/10.5858/arpa.2017-0159-OADOI Listing
August 2018

Crystal structure of Porphyromonas gingivalis dipeptidyl peptidase 4 and structure-activity relationships based on inhibitor profiling.

Eur J Med Chem 2017 Oct 10;139:482-491. Epub 2017 Aug 10.

Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium. Electronic address:

The Gram-negative anaerobe Porphyromonas gingivalis is associated with chronic periodontitis. Clinical isolates of P. gingivalis strains with high dipeptidyl peptidase 4 (DPP4) expression also had a high capacity for biofilm formation and were more infective. The X-ray crystal structure of P. gingivalis DPP4 was solved at 2.2 Å resolution. Despite a sequence identity of 32%, the overall structure of the dimer was conserved between P. gingivalis DPP4 and mammalian orthologues. The structures of the substrate binding sites were also conserved, except for the region called S2-extensive, which is exploited by specific human DPP4 inhibitors currently used as antidiabetic drugs. Screening of a collection of 450 compounds as inhibitors revealed a structure-activity relationship that mimics in part that of mammalian DPP9. The functional similarity between human and bacterial DPP4 was confirmed using 124 potential peptide substrates.
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http://dx.doi.org/10.1016/j.ejmech.2017.08.024DOI Listing
October 2017

Glycosaminoglycans Regulate CXCR3 Ligands at Distinct Levels: Protection against Processing by Dipeptidyl Peptidase IV/CD26 and Interference with Receptor Signaling.

Int J Mol Sci 2017 Jul 13;18(7). Epub 2017 Jul 13.

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

CXC chemokine ligand (CXCL)9, CXCL10 and CXCL11 direct chemotaxis of mainly T cells and NK cells through activation of their common CXC chemokine receptor (CXCR)3. They are inactivated upon NH₂-terminal cleavage by dipeptidyl peptidase IV/CD26. In the present study, we found that different glycosaminoglycans (GAGs) protect the CXCR3 ligands against proteolytic processing by CD26 without directly affecting the enzymatic activity of CD26. In addition, GAGs were shown to interfere with chemokine-induced CXCR3 signaling. The observation that heparan sulfate did not, and heparin only moderately, altered CXCL10-induced T cell chemotaxis in vitro may be explained by a combination of protection against proteolytic inactivation and altered receptor interaction as observed in calcium assays. No effect of CD26 inhibition was found on CXCL10-induced chemotaxis in vitro. However, treatment of mice with the CD26 inhibitor sitagliptin resulted in an enhanced CXCL10-induced lymphocyte influx into the joint. This study reveals a dual role for GAGs in modulating the biological activity of CXCR3 ligands. GAGs protect the chemokines from proteolytic cleavage but also directly interfere with chemokine-CXCR3 signaling. These data support the hypothesis that both GAGs and CD26 affect the in vivo chemokine function.
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http://dx.doi.org/10.3390/ijms18071513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5536003PMC
July 2017

The expression of proline-specific enzymes in the human lung.

Ann Transl Med 2017 Mar;5(6):130

Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, 2610 Wilrijk, Belgium.

The pathophysiology of lung diseases is very complex and proteolytic enzymes may play a role or could be used as biomarkers. In this review, the literature was searched to make an overview of what is known on the expression of the proline-specific peptidases dipeptidyl peptidase (DPP) 4, 8, 9, prolyl oligopeptidase (PREP) and fibroblast activation protein α (FAP) in the healthy and diseased lung. Search terms included asthma, chronic obstructive pulmonary disease (COPD), lung cancer, fibrosis, ischemia reperfusion injury and pneumonia. Knowledge on the loss or gain of protein expression and activity during disease might tie these enzymes to certain cell types, substrates or interaction partners that are involved in the pathophysiology of the disease, ultimately leading to the elucidation of their functional roles and a potential therapeutic target. Most data could be found on DPP4, while the other enzymes are less explored. Published data however often appear to be conflicting, the applied methods divers and the specificity of the assays used questionable. In conclusion, information on the expression of the proline-specific peptidases in the healthy and diseased lung is lacking, begging for further well-designed research.
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http://dx.doi.org/10.21037/atm.2017.03.36DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5395489PMC
March 2017

Regulation of intestinal permeability: The role of proteases.

World J Gastroenterol 2017 Mar;23(12):2106-2123

Hanne Van Spaendonk, Hannah Ceuleers, Leonie Witters, Eveline Patteet, Joris G De Man, Benedicte Y De Winter, Laboratory of Experimental Medicine and Pediatrics, Division of Gastroenterology, University of Antwerp, 2610 Antwerp, Belgium.

The gastrointestinal barrier is - with approximately 400 m - the human body's largest surface separating the external environment from the internal milieu. This barrier serves a dual function: permitting the absorption of nutrients, water and electrolytes on the one hand, while limiting host contact with noxious luminal antigens on the other hand. To maintain this selective barrier, junction protein complexes seal the intercellular space between adjacent epithelial cells and regulate the paracellular transport. Increased intestinal permeability is associated with and suggested as a player in the pathophysiology of various gastrointestinal and extra-intestinal diseases such as inflammatory bowel disease, celiac disease and type 1 diabetes. The gastrointestinal tract is exposed to high levels of endogenous and exogenous proteases, both in the lumen and in the mucosa. There is increasing evidence to suggest that a dysregulation of the protease/antiprotease balance in the gut contributes to epithelial damage and increased permeability. Excessive proteolysis leads to direct cleavage of intercellular junction proteins, or to opening of the junction proteins activation of protease activated receptors. In addition, proteases regulate the activity and availability of cytokines and growth factors, which are also known modulators of intestinal permeability. This review aims at outlining the mechanisms by which proteases alter the intestinal permeability. More knowledge on the role of proteases in mucosal homeostasis and gastrointestinal barrier function will definitely contribute to the identification of new therapeutic targets for permeability-related diseases.
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http://dx.doi.org/10.3748/wjg.v23.i12.2106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5374123PMC
March 2017

Visceral hypersensitivity in inflammatory bowel diseases and irritable bowel syndrome: The role of proteases.

World J Gastroenterol 2016 Dec;22(47):10275-10286

Hannah Ceuleers, Hanne Van Spaendonk, Nikita Hanning, Jelena Heirbaut, Joris G De Man, Benedicte Y De Winter, Laboratory of Experimental Medicine and Pediatrics, Division of Gastroenterology, University of Antwerp, 2610 Antwerp, Belgium.

Proteases, enzymes catalyzing the hydrolysis of peptide bonds, are present at high concentrations in the gastrointestinal tract. Besides their well-known role in the digestive process, they also function as signaling molecules through the activation of protease-activated receptors (PARs). Based on their chemical mechanism for catalysis, proteases can be classified into several classes: serine, cysteine, aspartic, metallo- and threonine proteases represent the mammalian protease families. In particular, the class of serine proteases will play a significant role in this review. In the last decades, proteases have been suggested to play a key role in the pathogenesis of visceral hypersensitivity, which is a major factor contributing to abdominal pain in patients with inflammatory bowel diseases and/or irritable bowel syndrome. So far, only a few preclinical animal studies have investigated the effect of protease inhibitors specifically on visceral sensitivity while their effect on inflammation is described in more detail. In our accompanying review we describe their effect on gastrointestinal permeability. On account of their promising results in the field of visceral hypersensitivity, further research is warranted. The aim of this review is to give an overview on the concept of visceral hypersensitivity as well as on the physiological and pathophysiological functions of proteases herein.
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http://dx.doi.org/10.3748/wjg.v22.i47.10275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5175241PMC
December 2016

Anti-inflammatory effects on ischemia/reperfusion-injured lung transplants by the cluster of differentiation 26/dipeptidylpeptidase 4 (CD26/DPP4) inhibitor vildagliptin.

J Thorac Cardiovasc Surg 2017 03 15;153(3):713-724.e4. Epub 2016 Nov 15.

Division of Thoracic Surgery, University Hospital Zurich, Zurich, Switzerland. Electronic address:

Objectives: We showed previously that stromal cell-derived factor 1 (SDF-1) is a substrate of cluster of differentiation 26/dipeptidylpeptidase 4 (CD26/DPP4) and exerts regenerative properties on acute lung ischemia-reperfusion injury on CD26/DPP4 inhibition. Here, we extend our studies to test whether an intermediate recovery of lung transplants from ischemia/reperfusion injury by CD26/DPP4 inhibition can be achieved for up to 14 days.

Methods: Syngeneic mouse lung transplantation (Tx) was performed in C57BL/6 and in CD26-/- mice by applying 18 hours of cold ischemia. Donor lungs were preconditioned with saline or the CD26/DPP4 inhibitor vildagliptin (1 μg/mL [3 μM]). In vitro, the influence of vildagliptin and SDF-1 on the macrophage cell line RAW 264.7 was tested. Transplants were analyzed up to 14 days after Tx for the expression of SDF-1, tumor necrosis factor-α (TNF-α), interleukin-10, intercellular adhesion molecule-1 (ICAM-1), immune cell infiltration, and oxygenation.

Results: Cold ischemic time of 18 hours with vildagliptin preconditioning elevated lung SDF-1 levels (P = .0011) and increased interleukin-10-producing macrophages (P = .0165) compared with the control. SDF-1 reduced macrophage-derived TNF-α (P = .0248) in vitro. Five hours after Tx, vildagliptin significantly reduced macrophages and neutrophils (P = .0306), decreased ICAM-1 expression (P = .002), and improved transplant oxygenation (P = .0181). Seven days after Tx, grafts were preserved on CD26/DPP4-inhibition: perivascular macrophages (P = .0046) and TNF-α (P = .0013) were reduced as well as T and B cells. ICAM-1 was absent in CD26/DPP4-inhibited grafts at all time points.

Conclusions: This study proves an intermediate improvement of ischemia/reperfusion-injured lung transplants by the CD26/DPP4-inhibitor vildagliptin up to 14 days. Enhanced levels of SDF-1 induced an anti-inflammatory effect on a cellular and protein level, and render CD26/DPP4 inhibition preconditioning effective for the protection from lung ischemia/reperfusion injury.
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http://dx.doi.org/10.1016/j.jtcvs.2016.10.080DOI Listing
March 2017

Acute Ischemic Stroke Severity, Progression, and Outcome Relate to Changes in Dipeptidyl Peptidase IV and Fibroblast Activation Protein Activity.

Transl Stroke Res 2017 04 26;8(2):157-164. Epub 2016 Aug 26.

Laboratory of Medical Biochemistry, University of Antwerp, Universiteitsplein 1, Building S, room D.S.513, 2610, Wilrijk-Antwerp, Belgium.

Dipeptidyl peptidase IV (DPPIV) inhibition may be a promising therapeutic strategy for acute stroke treatment, given its potential to prolong the biological half-life of neuroprotective substrates. A related protease, fibroblast activation protein (FAP), was recently shown to inactivate the same substrates. Therefore, it should also be investigated as a potential target in stroke. The study aimed to investigate whether stroke severity and outcome correlate with DPPIV and FAP activities and their kinetics shortly after acute ischemic stroke. DPPIV and FAP activities were analyzed in the serum of 50 hyperacute stroke patients at admission, 1 day, 3 days, and 7 days after stroke onset and in 50 age-matched healthy controls. This was done as part of the Middelheim's Interdisciplinary Stroke Study. DPPIV activity tended to increase shortly after stroke compared to the control population. DPPIV and FAP activities steadily decreased in the first week after stroke onset. Higher infarct volumes (≥5 ml) and a more severe stroke (NIHSS >7) at admission were correlated with a stronger decrease in the activities of both enzymes. Moreover, these patients more often developed a progressive stroke, were more often institutionalized. Patients with a stronger increase in DPPIV activity at admission and decrease in the activity of both DPPIV and FAP during the first week after stroke onset had a more severe stroke and worse short-term outcomes.
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http://dx.doi.org/10.1007/s12975-016-0493-3DOI Listing
April 2017

Validated programmed cell death ligand 1 immunohistochemistry assays (E1L3N and SP142) reveal similar immune cell staining patterns in melanoma when using the same sensitive detection system.

Histopathology 2017 Jan 10;70(2):253-263. Epub 2016 Oct 10.

Department of Molecular Pathology, HistoGeneX, Antwerp, Belgium.

Aims: Tumour cell and/or immune cell programmed cell death ligand 1 (PD-L1) expression is considered as a potential biomarker for anti-PD1 and anti-PD-L1 immunotherapy. Currently, different PD-L1 assays are used. This study aims to compare the staining patterns of two PD-L1 antibody clones in melanoma metastases and correlate them with PD-L1 mRNA expression.

Methods And Results: The immunohistochemistry assays were optimized and validated independently on a Ventana Benchmark Ultra (Ventana Medical Systems Inc., Tucson, AZ, USA) (E1L3N) and XT (SP142), using the same detection system. In total, 46 melanoma metastases were stained with both validated immunohistochemistry assays. Stained slides were digitized for qualitative and semi-quantitative evaluation; done by pathologist and semi-automated software analysis. A subset of 21 melanoma metastases was selected for quantification of the PD-L1 mRNA expression. Accuracy and precision criteria were met for both assays. PD-L1 protein and mRNA expression showed remarkably good Spearman's coefficients of 0.90 (E1L3N) and 0.87 (SP142). Despite the remarkable correlation between both PD-L1 assays in expression patterns and quantification values (ρ > 0.90), E1L3N showed significantly more tumour cell staining than SP142.

Conclusions: E1L3N and SP142 IHC assays were optimized and validated successfully and independently for sensitive and accurate PD-L1 detection. Concordance was best for immune cell scoring, while E1L3N tended to detect more tumour cells. Determination of the clinically relevant cut-off values for immune cell versus tumour cell detection requires further research.
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http://dx.doi.org/10.1111/his.13056DOI Listing
January 2017

Prolyl carboxypeptidase purified from human placenta: its characterization and identification as an apelin-cleaving enzyme.

Biochim Biophys Acta 2016 11 20;1864(11):1481-8. Epub 2016 Jul 20.

Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium.

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http://dx.doi.org/10.1016/j.bbapap.2016.07.004DOI Listing
November 2016

Optimization and validation of an existing, surgical and robust dry eye rat model for the evaluation of therapeutic compounds.

Exp Eye Res 2016 05 17;146:172-178. Epub 2016 Mar 17.

Faculty of Pharmaceutical, Biomedical and Veterinary Sciences: Laboratory of Microbiology, Parasitology and Hygiene (LMPH); University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium. Electronic address:

The aim of this research was to optimize and validate an animal model for dry eye, adopting clinically relevant evaluation parameters. Dry eye was induced in female Wistar rats by surgical removal of the exorbital lacrimal gland. The clinical manifestations of dry eye were evaluated by tear volume measurements, corneal fluorescein staining, cytokine measurements in tear fluid, MMP-9 mRNA expression and CD3(+) cell infiltration in the conjunctiva. The animal model was validated by treatment with Restasis(®) (4 weeks) and commercial dexamethasone eye drops (2 weeks). Removal of the exorbital lacrimal gland resulted in 50% decrease in tear volume and a gradual increase in corneal fluorescein staining. Elevated levels of TNF-α and IL-1α have been registered in tear fluid together with an increase in CD3(+) cells in the palpebral conjunctiva when compared to control animals. Additionally, an increase in MMP-9 mRNA expression was recorded in conjunctival tissue. Reference treatment with Restasis(®) and dexamethasone eye drops had a positive effect on all evaluation parameters, except on tear volume. This rat dry eye model was validated extensively and judged appropriate for the evaluation of novel compounds and therapeutic preparations for dry eye disease.
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http://dx.doi.org/10.1016/j.exer.2016.03.006DOI Listing
May 2016
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