Publications by authors named "Jean-Pierre Timmermans"

208 Publications

Correction to: The Pulmonary Neuroepithelial Body Microenvironment.

Adv Anat Embryol Cell Biol 2021 ;233:C1

Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerpen (Wilrijk), Belgium.

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http://dx.doi.org/10.1007/978-3-030-65817-5_6DOI Listing
January 2021

Miltefosine enhances infectivity of a miltefosine-resistant Leishmania infantum strain by attenuating its innate immune recognition.

PLoS Negl Trop Dis 2021 Jul 22;15(7):e0009622. Epub 2021 Jul 22.

University of Antwerp, Department of Biomedical Sciences, Laboratory of Microbiology, Parasitology and Hygiene (LMPH), Wilrijk, Belgium.

Background: Miltefosine (MIL) is currently the only oral drug available to treat visceral leishmaniasis but its use as first-line monotherapy has been compromised by an increasing treatment failure. Despite the scarce number of resistant clinical isolates, MIL-resistance by mutations in a single aminophospholipid transporter gene can easily be selected in a laboratory environment. These mutations result in a reduced survival in the mammalian host, which can partially be restored by exposure to MIL, suggesting a kind of drug-dependency.

Methodology/principal Findings: To enable a combined study of the infection dynamics and underlying immunological events for differential in vivo survival, firefly luciferase (PpyRE9) / red fluorescent protein (DsRed) double-reporter strains were generated of MIL-resistant (MIL-R) and syngeneic MIL-sensitive (MIL-S) Leishmania infantum. Results in C57Bl/6 and BALB/c mice show that MIL-R parasites induce an increased innate immune response that is characterized by enhanced influx and infection of neutrophils, monocytes and dendritic cells in the liver and elevated serum IFN-γ levels, finally resulting in a less efficient establishment in liver macrophages. The elevated IFN-γ levels were shown to originate from an increased response of hepatic NK and NKT cells to the MIL-R parasites. In addition, we demonstrated that MIL could increase the in vivo fitness of MIL-R parasites by lowering NK and NKT cell activation, leading to a reduced IFN-γ production.

Conclusions/significance: Differential induction of innate immune responses in the liver was found to underlie the attenuated phenotype of a MIL-R parasite and its peculiar feature of drug-dependency. The impact of MIL on hepatic NK and NKT activation and IFN-γ production following recognition of a MIL-R strain indicates that this mechanism may sustain infections with resistant parasites and contribute to treatment failure.
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http://dx.doi.org/10.1371/journal.pntd.0009622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8330912PMC
July 2021

Parkinson mice show functional and molecular changes in the gut long before motoric disease onset.

Mol Neurodegener 2021 06 2;16(1):34. Epub 2021 Jun 2.

Department of Informatics and Microsystems and Technology, University of Applied Science Kaiserslautern, Working Group Enteric Nervous System, 66482, Zweibrücken, Germany.

Background: There is increasing evidence that Parkinson's disease (PD) might start in the gut, thus involving and compromising also the enteric nervous system (ENS). At the clinical onset of the disease the majority of dopaminergic neurons in the midbrain is already destroyed, so that the lack of early biomarkers for the disease represents a major challenge for developing timely treatment interventions. Here, we use a transgenic A30P-α-synuclein-overexpressing PD mouse model to identify appropriate candidate markers in the gut before hallmark symptoms begin to manifest.

Methods: Based on a gait analysis and striatal dopamine levels, we defined 2-month-old A30P mice as pre-symptomatic (psA30P), since they are not showing any motoric impairments of the skeletal neuromuscular system and no reduced dopamine levels, but an intestinal α-synuclein pathology. Mice at this particular age were further used to analyze functional and molecular alterations in both, the gastrointestinal tract and the ENS, to identify early pathological changes. We examined the gastrointestinal motility, the molecular composition of the ENS, as well as the expression of regulating miRNAs. Moreover, we applied A30P-α-synuclein challenges in vitro to simulate PD in the ENS.

Results: A retarded gut motility and early molecular dysregulations were found in the myenteric plexus of psA30P mice. We found that i.e. neurofilament light chain, vesicle-associated membrane protein 2 and calbindin 2, together with the miRNAs that regulate them, are significantly altered in the psA30P, thus representing potential biomarkers for early PD. Many of the dysregulated miRNAs found in the psA30P mice are reported to be changed in PD patients as well, either in blood, cerebrospinal fluid or brain tissue. Interestingly, the in vitro approaches delivered similar changes in the ENS cultures as seen in the transgenic animals, thus confirming the data from the mouse model.

Conclusions: These findings provide an interesting and novel approach for the identification of appropriate biomarkers in men.
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http://dx.doi.org/10.1186/s13024-021-00439-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8170976PMC
June 2021

Concluding Remarks and Future Perspectives.

Adv Anat Embryol Cell Biol 2021 ;233:69-70

Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerpen (Wilrijk), Belgium.

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http://dx.doi.org/10.1007/978-3-030-65817-5_5DOI Listing
January 2021

Functional Exploration of the Pulmonary NEB ME.

Adv Anat Embryol Cell Biol 2021 ;233:31-67

Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerpen (Wilrijk), Belgium.

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http://dx.doi.org/10.1007/978-3-030-65817-5_4DOI Listing
January 2021

Studying the Pulmonary NEB ME: A Multidisciplinary Approach.

Adv Anat Embryol Cell Biol 2021 ;233:19-29

Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerpen (Wilrijk), Belgium.

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http://dx.doi.org/10.1007/978-3-030-65817-5_3DOI Listing
January 2021

The Pulmonary NEB ME Is a Complex Intraepithelial Unit.

Adv Anat Embryol Cell Biol 2021 ;233:7-18

Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerpen (Wilrijk), Belgium.

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http://dx.doi.org/10.1007/978-3-030-65817-5_2DOI Listing
January 2021

Pulmonary Sensory Receptors.

Adv Anat Embryol Cell Biol 2021 ;233:1-65

Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerpen (Wilrijk), Belgium.

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http://dx.doi.org/10.1007/978-3-030-65817-5_1DOI Listing
January 2021

Basophil and mast cell activation tests by flow cytometry in immediate drug hypersensitivity: Diagnosis and beyond.

J Immunol Methods 2021 Aug 29;495:113050. Epub 2021 Apr 29.

Department of Immunology, Allergology, Rheumatology and the Infla-Med Centre of Excellence, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp (Belgium) and Immunology, Allergology, Rheumatology, Antwerp University Hospital, Antwerp, Belgium; Department of Immunology, AZ Jan Palfijn Hospital Gent, Ghent, Belgium. Electronic address:

Immediate drug hypersensitivity reactions (IDHRs) constitute a significant health issue with serious consequences of diagnostic error. The primary diagnostics to document IDHRs usually consists of quantification of drug-specific IgE (sIgE) antibodies and skin tests. Unfortunately, the positive predictive value (PPV) and negative predictive value (NPV) of these tests are not absolutely, which leaves room for new tests. Over the last two decades, the basophil activation test (BAT), in which ex vivo activation of individual basophils is quantified by flow cytometry, has emerged as a reliable complementary diagnostic to document IDHRs, to explore allergenic recognition, to study cross-reactivity and to monitor therapy. However, the BAT is technically challenging requiring specialized personnel and equipment, fresh samples and the technique is lost as a diagnostic in patients showing a non-responder status of their cells. By consequence, the BAT has still not entered mainstream application. In contrast, mast cell activation tests (MATs) use serum samples that can be frozen, stored, and shipped to a recognized reference centre experienced in mast cell (MC) lines and/or cultures and capable of offering batch testing with necessary quality controls. This review does not only highlight the use of the BAT and MAT as diagnostics in IDHRs, but also outlines the potential of both techniques in further exploring and unveiling the mechanisms that govern drug-induced basophil and MC activation and degranulation.
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http://dx.doi.org/10.1016/j.jim.2021.113050DOI Listing
August 2021

Microglial derived extracellular vesicles activate autophagy and mediate multi-target signaling to maintain cellular homeostasis.

J Extracell Vesicles 2020 11 25;10(1):e12022. Epub 2020 Nov 25.

Biomedical Research Institute UHasselt Hasselt University Hasselt Belgium.

Microglia, the immunocompetent cells of the central nervous system (CNS), play an important role in maintaining cellular homeostasis in the CNS. These cells secrete immunomodulatory factors including nanovesicles and participate in the removal of cellular debris by phagocytosis or autophagy. Accumulating evidence indicates that specifically the cellular exchange of small extracellular vesicles (EVs), participates in physiology and disease through intercellular communication. However, the contribution of microglial-derived extracellular vesicles (M-EVs) to the maintenance of microglia homeostasis and how M-EVs could influence the phenotype and gene function of other microglia subtypes is unclear. In addition, knowledge of canonical signalling pathways of inflammation and immunity gene expression patterns in human microglia exposed to M-EVs is limited. Here, we analysed the effects of M-EVs produced in vitro by either tumour necrosis factor alpha (TNFα) activated or non-activated microglia BV2 cells. We showed that M-EVs are internalized by both mouse and human C20 microglia cells and that the uptake of M-EVs in microglia induced autophagic vesicles at various stages of degradation including autophagosomes and autolysosomes. Consistently, stimulation of microglia with M-EVs increased the protein expression of the autophagy marker, microtubule-associated proteins 1A/1B light chain 3B isoform II (LC3B-II), and promoted autophagic flux in live cells. To elucidate the biological activities occurring at the transcriptional level in C20 microglia stimulated with M-EVs, the gene expression profiles, potential upstream regulators, and enrichment pathways were characterized using targeted RNA sequencing. Inflammation and immunity transcriptome gene panel sequencing of both activated and normal microglia stimulated with M-EVs showed involvement of several canonical pathways and reduced expression of key genes involved in neuroinflammation, inflammasome and apoptosis signalling pathways compared to control cells. In this study, we provide the perspective that a beneficial activity of in vitro cell culture produced EVs could be the modulation of autophagy during cellular stress. Therefore, we use a monoculture system to study microglia-microglia crosstalk which is important in the prevention and propagation of inflammation in the brain. We demonstrate that in vitro produced microglial EVs are able to influence multiple biological pathways and promote activation of autophagy in order to maintain microglia survival and homeostasis.
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http://dx.doi.org/10.1002/jev2.12022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7890546PMC
November 2020

Unsupervised Machine Learning-Based Clustering of Nanosized Fluorescent Extracellular Vesicles.

Small 2021 02 15;17(5):e2006786. Epub 2021 Jan 15.

Biomedical Research Institute (BIOMED), Hasselt University, Martelarenlaan 42, Hasselt, 3500, Belgium.

Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, multicolor single-molecule burst analysis microscopy is used to detect multiple biomarkers present on single EV. The authors classify the recorded signals and apply the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, the authors use the method to assess both the purity and the inflammatory status of EV, and compare cell culture and plasma-derived EV isolated via different purification methods. This methodology is then applied to identify intercellular adhesion molecule-1 specific EV subgroups released by inflamed endothelial cells, and to prove that apolipoprotein-a1 is an excellent marker to identify the typical lipoprotein contamination in plasma. This methodology can be widely applied on standard confocal microscopes, thereby allowing both standardized quality assessment of patient plasma EV preparations, and diagnostic profiling of multiple EV biomarkers in health and disease.
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http://dx.doi.org/10.1002/smll.202006786DOI Listing
February 2021

Systematic Quantification of Synapses in Primary Neuronal Culture.

iScience 2020 Sep 7;23(9):101542. Epub 2020 Sep 7.

Laboratory of Cell Biology and Histology, University of Antwerp, Wilrijk, Antwerp 2610, Belgium.

Most neurological disorders display impaired synaptic connectivity. Hence, modulation of synapse formation may have therapeutic relevance. However, the high density and small size of synapses complicate their quantification. To improve synapse-oriented screens, we analyzed the labeling performance of synapse-targeting antibodies on neuronal cell cultures using segmentation-independent image analysis based on sliding window correlation. When assessing pairwise colocalization, a common readout for mature synapses, overlap was incomplete and confounded by spurious signals. To circumvent this, we implemented a proximity ligation-based approach that only leads to a signal when two markers are sufficiently close. We applied this approach to different marker combinations and demonstrate its utility for detecting synapse density changes in healthy and compromised cultures. Thus, segmentation-independent analysis and exploitation of resident protein proximity increases the sensitivity of synapse quantifications in neuronal cultures and represents a valuable extension to the analytical toolset for synapse screens.
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http://dx.doi.org/10.1016/j.isci.2020.101542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7516133PMC
September 2020

Effect of TNBS-induced colitis on enteric neuronal subpopulations in adult zebrafish.

Eur J Histochem 2020 Aug 28;64(3). Epub 2020 Aug 28.

Laboratory of Human Anatomy, Faculty of Medicine and Health Sciences, University of Antwerp.

Inflammatory bowel disease (IBD) includes inflammation of the gastrointestinal (GI) tract and is characterized by periods of acute inflammation and remission. Therapeutic management of IBD is still problematic, because of incomplete understanding its pathogenesis. This study focuses on the effect of 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis on changes in enteric neuronal subpopulations in adult zebrafish. These changes are suggested to be related to the altered neuro-immune interactions and GI motility, and in IBD pathogenesis. New insights into neuroplasticity will be instrumental in finding appropriate therapeutic treatments. TNBS was intraluminally administered in the distal intestine (DI) of anesthetized adult zebrafish. A histological time course of the intestinal inflammatory response was created to establish optimal TNBS concentration and acute inflammation phase. Using double immunolabelling on whole mounts, the effect of inflammation on neuronal populations was analyzed. Based on intestinal wall thickening, epithelial fold disruption, reduced goblet cell number, and eosinophil infiltration, our analysis indicated that the optimal TNBS concentration (320 mM in 25% ethanol) inducing non-lethal inflammation reached a peak at 6 hours post-induction. The inflammatory response returned to baseline values at 3 days post-induction. At the acute inflammation phase, no influence on the distribution or proportion of nitrergic neurons was observed, while only the proportion of cholinergic neurons was significantly reduced in the DI. The proportion of serotonergic neurons was significantly increased in the entire intestine during inflammation. This study describes a method of TNBS-induced colitis in the adult zebrafish. Given that the acute inflammation phase is accompanied by neuroplasticity comparable to changes observed in IBD patients, and the unique and versatile characteristics of the zebrafish, allows this model to be used alongside IBD animal models to unravel IBD pathology and to test new IBD therapies.
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http://dx.doi.org/10.4081/ejh.2020.3161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459238PMC
August 2020

Progressive tau aggregation does not alter functional brain network connectivity in seeded hTau.P301L mice.

Neurobiol Dis 2020 09 10;143:105011. Epub 2020 Jul 10.

Laboratory of Cell Biology and Histology, University of Antwerp, Belgium. Electronic address:

Progressive accumulation of hyperphosphorylated tau is a hallmark of various neurodegenerative disorders including Alzheimer's disease. However, to date, the functional effects of tau pathology on brain network connectivity remain poorly understood. To directly interrogate the impact of tau pathology on functional brain connectivity, we conducted a longitudinal experiment in which we monitored a fibril-seeded hTau.P301L mouse model using correlative whole-brain microscopy and resting-state functional MRI. Despite a progressive aggravation of tau pathology across the brain, the major resting-state networks appeared unaffected up to 15 weeks after seeding. Targeted analyses also showed that the connectivity of regions with high levels of hyperphosphorylated tau was comparable to that observed in controls. In line with the ostensible retention of connectivity, no behavioural changes were detected between seeded and control hTau.P301L mice as determined by three different paradigms. Our data indicate that seeded tau pathology, with accumulation of tau aggregates throughout different regions of the brain, does not alter functional connectivity or behaviour in this mouse model. Additional correlative functional studies on different mouse models should help determine whether this is a generalizable trait of tauopathies.
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http://dx.doi.org/10.1016/j.nbd.2020.105011DOI Listing
September 2020

Three-Dimensional Imaging of Intraplaque Neovascularization in a Mouse Model of Advanced Atherosclerosis.

J Vasc Res 2020 1;57(6):348-354. Epub 2020 Jul 1.

Laboratory of Physiopharmacology, University of Antwerp, Antwerp, Belgium,

Multiple lines of evidence suggest that intraplaque (IP) neovascularization promotes atherosclerotic plaque growth, destabilization, and rupture. However, pharmacological inhibition of IP neovascularization remains largely unexplored due to the limited number of animal models that develop IP neovessels and the lack of reliable methods for visualizing IP angiogenesis. Here, we applied 3D confocal microscopy with an optimized tissue-clearing process, immunolabeling-enabled three-dimensional imaging of solvent-cleared organs, to visualize IP neovessels in apolipoprotein E-deficient (ApoE-/-) mice carrying a heterozygous mutation (C1039+/-) in the fibrillin-1 gene. Unlike regular ApoE-/- mice, this mouse model is characterized by the presence of advanced plaques with evident IP neovascularization. Plaques were stained with antibodies against endothelial marker CD31 for 3 days, followed by incubation with fluorescently labeled secondary antibodies. Subsequent tissue clearing with dichloromethane (DCM)/methanol, DCM, and dibenzyl ether allowed easy visualization and 3D reconstruction of the IP vascular network while plaque morphology remained intact.
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http://dx.doi.org/10.1159/000508449DOI Listing
January 2021

Lactobacilli Have a Niche in the Human Nose.

Cell Rep 2020 05;31(8):107674

Department of Bioscience Engineering, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium. Electronic address:

Although an increasing number of beneficial microbiome members are characterized for the human gut and vagina, beneficial microbes are underexplored for the human upper respiratory tract (URT). In this study, we demonstrate that taxa from the beneficial Lactobacillus genus complex are more prevalent in the healthy URT than in patients with chronic rhinosinusitis (CRS). Several URT-specific isolates are cultured, characterized, and further explored for their genetic and functional properties related to adaptation to the URT. Catalase genes are found in the identified lactobacilli, which is a unique feature within this mostly facultative anaerobic genus. Moreover, one of our isolated strains, Lactobacillus casei AMBR2, contains fimbriae that enable strong adherence to URT epithelium, inhibit the growth and virulence of several URT pathogens, and successfully colonize nasal epithelium of healthy volunteers. This study thus demonstrates that specific lactobacilli are adapted to the URT and could have a beneficial keystone function in this habitat.
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http://dx.doi.org/10.1016/j.celrep.2020.107674DOI Listing
May 2020

Flow cytometric basophil activation tests: Staining of exteriorized basophil granule matrix by fluorescent avidin versus appearance of CD63.

Cytometry B Clin Cytom 2020 11 3;98(6):483-490. Epub 2020 Feb 3.

Department of Immunology, Allergology, Rheumatology and the Infla-Med Research Consortium of Excellence, University of Antwerp and Antwerp University Hospital, Antwerp, Belgium.

Background: Staining of exteriorized basophil granule matrix by fluorescent avidin might be a reliable technique to monitor basophil degranulation. This study compares the avidin-based technique with the upregulation of CD203c and appearance of CD63 in response to various stimuli.

Methods: Fourteen individuals responsive to anti-IgE, nine healthy controls, and five birch pollen-allergic patients, and five nonresponders were studied. Activation experiments included anti-IgE, fMLP, interleukin-(IL)-3, and birch pollen allergen. Basophil activation/degranulation was analyzed by flow cytometry and microscopy using anti-CD63, anti-CD203c, and avidin.

Results: Stimulation with anti-IgE, fMLP, and relevant allergen results in upregulation of CD203c, CD63 appearance, and an increase in avidin binding. In response to anti-IgE and allergen, upregulation of CD203c peaks within 10 min, CD63 and avidin binding reach a plateau after 10-20 min. CD63 staining leads to a bimodal distribution, avidin staining causes a unimodal shift with a less clear discrimination between degranulating and nondegranulating cells. In response to fMLP, upregulation of CD203c and CD63 and avidin binding are maximal after 2.5 min. Following incubation with anti-IgE and fMLP, percentages of CD203c+ cells are higher than those of CD63+ and avidin+ cells, pointing to a dissociation between activation and degranulation. Percentages of CD63+ cells are systemically higher than those of avidin+ cells. Incubated with IL-3 only upregulates CD203c, while no CD63 or avidin binding is observed.

Conclusions: Staining of exteriorized proteoglycans by avidin is a reliable technique to quantify basophil degranulation but offers no added value when compared to traditional assays that use CD63 as a readout.
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http://dx.doi.org/10.1002/cyto.b.21868DOI Listing
November 2020

In-Depth Study of Transmembrane Mucins in Association with Intestinal Barrier Dysfunction During the Course of T Cell Transfer and DSS-Induced Colitis.

J Crohns Colitis 2020 Jul;14(7):974-994

Laboratory of Experimental Medicine and Pediatrics, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium.

Background And Aims: There is evidence for a disturbed intestinal barrier function in inflammatory bowel diseases [IBD] but the underlying mechanisms are unclear. Because mucins represent the major components of the mucus barrier and disturbed mucin expression is reported in the colon of IBD patients, we studied the association between mucin expression, inflammation and intestinal permeability in experimental colitis.

Methods: We quantified 4-kDa FITC-dextran intestinal permeability and the expression of cytokines, mucins, junctional and polarity proteins at dedicated time points in the adoptive T cell transfer and dextran sodium sulfate [DSS]-induced colitis models. Mucin expression was also validated in biopsies from IBD patients.

Results: In both animal models, the course of colitis was associated with increased interleukin-1β [IL-1β] and tumour necrosis factor-α [TNF-α] expression and increased Muc1 and Muc13 expression. In the T cell transfer model, a gradually increasing Muc1 expression coincided with gradually increasing 4-kDa FITC-dextran intestinal permeability and correlated with enhanced IL-1β expression. In the DSS model, Muc13 expression coincided with rapidly increased 4-kDa FITC-dextran intestinal permeability and correlated with TNF-α and Muc1 overexpression. Moreover, a significant association was observed between Muc1, Cldn1, Ocln, Par3 and aPKCζ expression in the T cell transfer model and between Muc13, Cldn1, Jam2, Tjp2, aPkcζ, Crb3 and Scrib expression in the DSS model. Additionally, MUC1 and MUC13 expression was upregulated in inflamed mucosa of IBD patients.

Conclusions: Aberrantly expressed MUC1 and MUC13 might be involved in intestinal barrier dysfunction upon inflammation by affecting junctional and cell polarity proteins, indicating their potential as therapeutic targets in IBD.
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http://dx.doi.org/10.1093/ecco-jcc/jjaa015DOI Listing
July 2020

Effects of intestinal alkaline phosphatase on intestinal barrier function in a cecal ligation and puncture (CLP)-induced mouse model for sepsis.

Neurogastroenterol Motil 2020 03 21;32(3):e13754. Epub 2019 Nov 21.

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

Background: Sepsis is a severe pathological condition associated with systemic inflammation, intestinal inflammation, and gastrointestinal barrier dysfunction. Intestinal alkaline phosphatase (IAP) has been demonstrated to detoxify lipopolysaccharide, an important mediator in the pathophysiology of sepsis. We investigated the effect of treatment with IAP on intestinal permeability, intestinal inflammation, and bacterial translocation.

Methods: OF-1 mice were divided into 4 groups (n = 12/group), undergoing either a sham or cecal ligation and puncture (CLP) procedure to induce sepsis. Mice received IAP or a vehicle intraperitoneally 5 minutes prior to the onset of the CLP or sham procedure, which was repeated every 12 hours for two consecutive days. After two days, in vivo intestinal permeability, intestinal inflammation, and bacterial translocation were determined.

Key Results: CLP-induced sepsis resulted in significantly more weight loss, worse clinical disease scores, bacterial translocation, and elevated inflammatory cytokines. Intestinal permeability was increased up to 5-fold (P < .001). IAP activity was significantly increased in septic animals. Treatment with IAP had no effect on clinical outcomes but reduced the increased permeability of the small intestine by 50% (P = .005). This reduction in permeability was accompanied by a modified gene expression of claudin-1 (P = .025), claudin-14 (P = .035), and interleukin 12 (P = .015). A discriminant analysis showed that treatment with IAP is linked to modified mRNA levels of several tight junction proteins and cytokines.

Conclusions And Inferences: Treatment with IAP diminished CLP-induced intestinal barrier disruption, associated with modified expression of several cytokines and claudins. Nevertheless, this effect did not translate into better clinical outcomes in our experimental setup.
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http://dx.doi.org/10.1111/nmo.13754DOI Listing
March 2020

Presence of MrgprD within the gastrointestinal wall: reality or fake?

Cell Tissue Res 2019 Dec 31;378(3):555-558. Epub 2019 Aug 31.

Laboratory of Cell Biology & Histology, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, BE-2610, Wilrijk, Antwerp, Belgium.

Due to their pivotal role in nociception and mast cell biology, the family of Mas-related G protein-coupled receptors (Mrgprs) has recently gained attention for their possible expression and role(s) in the gastrointestinal tract. In this context, based on immunocytochemical stainings using a commercial antibody, a recent study by Zhou et al. reported that the murine Mrgprd member is expressed in mouse gut lamina propria immune cells and in the outer smooth muscle layers pointing to a potential role for MrgprD in inflammatory responses and intestinal immunity. Immunohistochemical staining for G protein-coupled receptors (GPCRs), however, remains challenging and should be cautiously interpreted using appropriate specificity controls. Using the same antibody with an identical dilution, we did observe a similar staining in the same wild-type mouse strain, but an identical staining pattern was also found in mice lacking the MrgprD receptor, indicating that this antibody recognizes epitopes other than those of MrgprD. Moreover, in situ hybridization for MrgprD further indicated the absence of receptor mRNA expression in lamina propria immune cells and in the outer smooth muscle layers. Therefore, the results and conclusions regarding the presence of MrgprD at protein level within the GI wall as described in the study of Zhou and collaborators should be interpreted with strong caution and should be reconsidered in the light of the emerging possible roles of MrgprD and therapeutic perspectives in gastrointestinal pathophysiology.
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http://dx.doi.org/10.1007/s00441-019-03097-5DOI Listing
December 2019

Impact of spray-drying on the pili of Lactobacillus rhamnosus GG.

Microb Biotechnol 2019 09 21;12(5):849-855. Epub 2019 Jun 21.

Department of Bioscience Engineering, Research Group Environmental Ecology and Applied Microbiology, University of Antwerp, Groenenborgerlaan 171, B-2020, Antwerp, Belgium.

The preservation of the viability of microorganisms in probiotic formulations is the most important parameter ensuring the adequate concentration of live microorganisms at the time of administration. The formulation and processing techniques used to produce these probiotic formulations can influence the preservation of the microbial viability. However, it is also required that the bacteria maintain their key probiotic capacities during processing, formulation and shelf life. In this study, we investigated the impact of spray-drying on different cell wall properties of the model probiotic strain Lactobacillus rhamnosus GG, including its adherence to intestinal epithelial cells. The dltD gene knock-out mutant, L. rhamnosus GG CMPG5540, displaying modified cell wall lipoteichoic acids, showed significantly increased colony-forming units after spray-drying and subsequent storage under standard conditions compared to wild-type L. rhamnosus GG. In contrast, disruption of the biosynthesis of exopolysaccharides or pili expression did not impact survival. However, spray-drying did significantly affect the adherence capacity of L. rhamnosus GG. Scanning electron microscopy confirmed that the pili, key surface factors for adherence to intestinal cells and mucus, were sheared off during the spray-drying process. These data thus highlight that both the functionality and viability of probiotics should be assessed during the spray-drying process and subsequent storage.
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http://dx.doi.org/10.1111/1751-7915.13426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6680608PMC
September 2019

Identification of Macrophage Genotype and Key Biological Pathways in Circulating Angiogenic Cell Transcriptome.

Stem Cells Int 2019 2;2019:9545261. Epub 2019 May 2.

Laboratory of Cellular and Molecular Cardiology, Antwerp University Hospital, Edegem, Belgium.

Background: Circulating angiogenic cells (CAC) have been identified as important regulators of vascular biology. However, there is still considerable debate about the genotype and function of CAC.

Methods And Results: Data from publicly available gene expression data sets were used to analyse the transcriptome of in vitro cultured CAC (CAC). Genes and pathways of interest were further evaluated using qPCR comparing CAC versus CD14 monocytic cells. The CAC transcriptome strongly related to tissue macrophages, and more specifically to regulatory M2c macrophages. The cytokine expression profile of CAC was predominantly immune modulatory and resembled the cytokine expression of tumor-associated macrophages (TAM). Pathway analysis revealed previously unrecognized biological processes in CAC, such as riboflavin metabolism and liver X receptor (LXR)/retinoid X receptor (RXR) and farnesoid X receptor (FXR)/retinoid X receptor (RXR) pathways. Analysis of endothelial-specific genes did not show evidence for endothelial transdifferentiation.

Conclusions: CAC are genotypically similar to regulatory M2c macrophages and lack signs of endothelial differentiation.
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http://dx.doi.org/10.1155/2019/9545261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525806PMC
May 2019

Mas-related G protein-coupled receptor C11 (Mrgprc11) induces visceral hypersensitivity in the mouse colon: A novel target in gut nociception?

Neurogastroenterol Motil 2019 08 22;31(8):e13623. Epub 2019 May 22.

Laboratory of Cell Biology and Histology, University of Antwerp, Antwerp, Belgium.

Background: Visceral hypersensitivity, an important cause of abdominal pain in disorders such as IBD and IBS, presents with a poorly understood pathophysiology and limited treatment options. Several members of the Mas-related G protein-coupled receptor family (Mrgprs) have become promising targets in pain research. The potential link between the murine Mrgpr C11 (Mrgprc11) and gut nociception is currently uninvestigated. Therefore, we explored the expression and functional role of Mrgprc11 in the gut nociceptive innervation.

Methods: Mrgprc11 expression was evaluated in DRG neurons innervating the mouse colon using in situ hybridization and immunohistochemistry. Visceromotor responses to colorectal distension (CRD) assessed the effect of the Mrgprc11 agonist, BAM(8-22), on colonic pain sensitivity in healthy mice. Moreover, we determined pERK1/2-immunoreactivity in the thoracolumbar spinal cord after noxious CRD. Finally, from a translational point of view, we looked for expression of the human counterpart of Mrgprc11, MRGPRX1, in human thoracolumbar DRGs.

Key Results: In situ hybridization and immunohistochemistry revealed Mrgprc11 expression in colonic DRG neurons. Intracolonic administration of BAM(8-22) significantly increased colonic pain sensitivity in an Mrgprc11-dependent manner, and led to a significantly increased degree of neuronal activation in the splanchnic spinal cord upon noxious stimulation. Furthermore, MRGPRX1 expression was also detected in human thoracolumbar DRG neurons. CONCLUSIONS & INFERENCES: Our findings established a novel function for Mrgprc11 in the gut nociceptive innervation and propose the receptor as a new player in visceral hypersensitivity. Given the presence of MRGPRX1 in human DRG neurons, our study warrants future research on its therapeutic potential in abdominal pain disorders.
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http://dx.doi.org/10.1111/nmo.13623DOI Listing
August 2019

Regional vulnerability and spreading of hyperphosphorylated tau in seeded mouse brain.

Neurobiol Dis 2019 07 14;127:398-409. Epub 2019 Mar 14.

Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium; Department of Molecular Biotechnology, Ghent University, Coupure Links 653, 9000 Ghent, Belgium.

We have exploited whole brain microscopy to map the progressive deposition of hyperphosphorylated tau in intact, cleared mouse brain. We found that the three-dimensional spreading pattern of hyperphosphorylated tau in the brain of an aging Tau.P301L mouse model did not resemble that observed in AD patients. Injection of synthetic or patient-derived tau fibrils in the CA1 region resulted in a more faithful spreading pattern. Atlas-guided volumetric analysis showed a connectome-dependent spreading from the injection site and also revealed hyperphosphorylated tau deposits beyond the direct anatomical connections. In fibril-injected brains, we also detected a persistent subpopulation of rod-like and swollen microglia. Furthermore, we showed that the hyperphosphorylated tau load could be reduced by intracranial co-administration of, and to a lesser extent, by repeated systemic dosing with an antibody targeting the microtubule-binding domain of tau. Thus, the combination of targeted seeding and in toto staging of tau pathology allowed assessing regional vulnerability in a comprehensive manner, and holds potential as a preclinical drug validation tool.
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http://dx.doi.org/10.1016/j.nbd.2019.03.010DOI Listing
July 2019

Comparative analysis reveals Ce3D as optimal clearing method for in toto imaging of the mouse intestine.

Neurogastroenterol Motil 2019 05 13;31(5):e13560. Epub 2019 Feb 13.

Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerp, Belgium.

Background: The intestinal wall has a complex topographical architecture. The multi-layered network of the enteric nervous system and its intercellular interactions are difficult to map using traditional section-based or whole-mount histology. With the advent of optical clearing techniques, it has become feasible to visualize intact tissue and organs in 3D. However, as yet, a gap still needs to be filled in that no in-depth analysis has been performed yet on the potential of different clearing techniques for the small intestine.

Aim: The goal of this study was to identify an optimal clearing protocol for in toto imaging of mouse intestinal tissue.

Methods: Five aqueous-based clearing protocols (SeeDB2, CUBIC, ScaleS, Ce3D, and UbasM) and four organic reagent-based clearing protocols (3DISCO, iDISCO+, uDISCO, and Visikol ) were assessed in segments of small intestine from CX3CR1 and wild-type mice. Following clearing, optical transparency, tissue morphology, green fluorescent protein (GFP) fluorescence retention, and compatibility with (immuno-)labeling were analyzed.

Key Results: All organic reagent-based clearing protocols-except for Visikol-rendered tissue highly transparent but led to substantial tissue shrinkage and deformation. Of the aqueous-based protocols, only Ce3D yielded full-thickness tissue transparency. In addition, Ce3D displayed excellent GFP retention and preservation of tissue morphology.

Conclusions: Ce3D emerged as a most efficient protocol for enabling rapid full-thickness 3D mapping of the mouse intestinal wall.
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http://dx.doi.org/10.1111/nmo.13560DOI Listing
May 2019

Neuropathy-causing mutations in HSPB1 impair autophagy by disturbing the formation of SQSTM1/p62 bodies.

Autophagy 2019 06 31;15(6):1051-1068. Epub 2019 Jan 31.

a Peripheral Neuropathy Research Group , Institute Born Bunge and University of Antwerp , Antwerp , Belgium.

HSPB1 (heat shock protein family B [small] member 1) is a ubiquitously expressed molecular chaperone. Most mutations in HSPB1 cause axonal Charcot-Marie-Tooth neuropathy and/or distal hereditary motor neuropathy. In this study we show that mutations in HSPB1 lead to impairment of macroautophagic/autophagic flux. In HSPB1 knockout cells, we demonstrate that HSPB1 is necessary for autophagosome formation, which was rescued upon re-expression of HSPB1. Employing a label-free LC-MS/MS analysis on the various HSPB1 variants (wild type and mutants), we identified autophagy-specific interactors. We reveal that the wild-type HSPB1 protein binds to the autophagy receptor SQSTM1/p62 and that the PB1 domain of SQSTM1 is essential for this interaction. Mutations in HSPB1 lead to a decrease in the formation of SQSTM1/p62 bodies, and subsequent impairment of phagophore formation, suggesting a regulatory role for HSPB1 in autophagy via interaction with SQSTM1. Remarkably, autophagy deficits could also be confirmed in patient-derived motor neurons thereby indicating that the impairment of autophagy might be one of the pathomechanisms by which mutations in HSPB1 lead to peripheral neuropathy. Abbreviations: ACD: alpha-crystallin domain; ALS: amyotrophic lateral sclerosis; ATG14: autophagy related 14; BAG1/3: BCL2 associated athanogene 1/3; CMT: Charcot-Marie-Tooth; dHMN: distal hereditary motor neuropathy; GFP: green fluorescent protein; HSPA8: heat shock protein family A (Hsp70) member 8; HSPB1/6/8: heat shock protein family B (small) member 1/6/8; LIR: LC3-interacting region; LC3B: microtubule associated protein 1 light chain 3 beta; PB1: Phox and Bem1; SQSTM1: sequestosome 1; STUB1/CHIP: STIP1 homology and U-box containing protein 1; UBA: ubiquitin-associated; WIPI1: WD repeat domain, phosphoinositide interacting 1; WT: wild-type.
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http://dx.doi.org/10.1080/15548627.2019.1569930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6526868PMC
June 2019

Selective activation and proliferation of a quiescent stem cell population in the neuroepithelial body microenvironment.

Respir Res 2018 Oct 26;19(1):207. Epub 2018 Oct 26.

Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610, Wilrijk, Antwerpen, Belgium.

Background: The microenvironment (ME) of neuroepithelial bodies (NEBs) harbors densely innervated groups of pulmonary neuroendocrine cells that are covered by Clara-like cells (CLCs) and is believed to be important during development and for adult airway epithelial repair after severe injury. Yet, little is known about its potential stem cell characteristics in healthy postnatal lungs.

Methods: Transient mild lung inflammation was induced in mice via a single low-dose intratracheal instillation of lipopolysaccharide (LPS). Bronchoalveolar lavage fluid (BALF), collected 16 h after LPS instillation, was used to challenge the NEB ME in ex vivo lung slices of control mice. Proliferating cells in the NEB ME were identified and quantified following simultaneous LPS instillation and BrdU injection.

Results: The applied LPS protocol induced very mild and transient lung injury. Challenge of lung slices with BALF of LPS-treated mice resulted in selective Ca-mediated activation of CLCs in the NEB ME of control mice. Forty-eight hours after LPS challenge, a remarkably selective and significant increase in the number of divided (BrdU-labeled) cells surrounding NEBs was observed in lung sections of LPS-challenged mice. Proliferating cells were identified as CLCs.

Conclusions: A highly reproducible and minimally invasive lung inflammation model was validated for inducing selective activation of a quiescent stem cell population in the NEB ME. The model creates new opportunities for unraveling the cellular mechanisms/pathways regulating silencing, activation, proliferation and differentiation of this unique postnatal airway epithelial stem cell population.
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http://dx.doi.org/10.1186/s12931-018-0915-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6203996PMC
October 2018

Experimentally Induced Biliary Atresia by Means of Rotavirus-Infection Is Directly Linked to Severe Damage of the Microvasculature in the Extrahepatic Bile Duct.

Anat Rec (Hoboken) 2019 05 22;302(5):818-824. Epub 2018 Nov 22.

Department of Pediatric Surgery, Medizinische Hochschule Hannover, Hannover, Germany.

Vascular damage has been reported to contribute to atresia formation in several diseases including biliary atresia. This study focused on the extrahepatic biliary plexus in experimental biliary atresia. Newborn BALB/cAnNCrl-pups were infected with rhesus rotavirus within 24 hr after birth to induce experimental biliary atresia. The extrahepatic biliary plexus was examined by confocal microscopy on whole-mount preparations, scored by three independent researchers, and further evaluated at the subcellular level with transmission electron microscopy. Imaging results revealed a progressive destruction of the extrahepatic biliary vascular plexus in the course of experimental biliary atresia induced by rotavirus infection. Endothelial cell damage was already visible as cell swelling and necrosis in the first days after infection and a damaged microcirculation that rapidly deteriorated with progression of obliterative cholangiopathy, was observed in the infected mice as early as 72 hr after birth. In experimental biliary atresia, the destruction of the extrahepatic biliary vascular plexus starts already in the first days postinfection and clearly precedes the morphological symptoms of atresia. The deterioration of the vascular bed architecture continues with disease progression. Therefore, we conclude that the (ultra)structural changes in the extrahepatic biliary microvasculature occurring before the visible onset of atresia has a predictive diagnostic value and this impairment in blood supply to the extrahepatic bile duct may be an important contributing factor to the pathogenesis of acquired biliary atresia. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc. Anat Rec, 302:818-824, 2019. © 2018 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/ar.23974DOI Listing
May 2019

Human amniotic membrane dressing for the treatment of an infected wound due to an entero-cutaneous fistula: Case report.

Int J Surg Case Rep 2018 13;51:11-13. Epub 2018 Aug 13.

Department of Urology, Lukas Hospital Neuss, Germany; University of Duisburg-Essen, Essen, Germany.

Introduction: Infected wounds are difficult to treat and there are no standardized protocols.

Presentation Of Case: We report a case of infected postoperative wound and entero-cutaneous fistula in a 83 years-old woman. An innovative treatment protocol for Human amniotic membrane (HAM)-assisted dressing of infected wound as the Idea Stage following the IDEAL recommendations is presented. The development of amnion preparation and the involved treatment steps are described. No adverse events and no graft rejection have been detected.

Discussion: Favorable results confirm the technical simplicity, safety and efficacy of this procedure. HAM has been shown to promote wound healing and to have antibacterial characteristics, which was supported by the presented case.

Conclusion: We are able to report a successful treatment of an infected wound caused by entero-cutaneous fistula with HAM dressing. Following the IDEAL recommendations, consecutive prospective cohort trials are justified.
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http://dx.doi.org/10.1016/j.ijscr.2018.08.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6104582PMC
August 2018
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