Publications by authors named "Onno J de Boer"

84 Publications

Artificial Intelligence-Based Segmentation of Residual Tumor in Histopathology of Pancreatic Cancer after Neoadjuvant Treatment.

Cancers (Basel) 2021 Oct 12;13(20). Epub 2021 Oct 12.

Department of Surgery, Amsterdam UMC, Cancer Center Amsterdam, University of Amsterdam, 1081 HV Amsterdam, The Netherlands.

Background: Histologic examination of resected pancreatic cancer after neoadjuvant therapy (NAT) is used to assess the effect of NAT and may guide the choice for adjuvant treatment. However, evaluating residual tumor burden in pancreatic cancer is challenging given tumor response heterogeneity and challenging histomorphology. Artificial intelligence techniques may offer a more reproducible approach.

Methods: From 64 patients, one H&E-stained slide of resected pancreatic cancer after NAT was digitized. Three separate classes were manually outlined in each slide (i.e., tumor, normal ducts, and remaining epithelium). Corresponding segmentation masks and patches were generated and distributed over training, validation, and test sets. Modified U-nets with varying encoders were trained, and F1 scores were obtained to express segmentation accuracy.

Results: The highest mean segmentation accuracy was obtained using modified U-nets with a DenseNet161 encoder. Tumor tissue was segmented with a high mean F1 score of 0.86, while the overall multiclass average F1 score was 0.82.

Conclusions: This study shows that artificial intelligence-based assessment of residual tumor burden is feasible given the promising obtained F1 scores for tumor segmentation. This model could be developed into a tool for the objective evaluation of the response to NAT and may potentially guide the choice for adjuvant treatment.
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http://dx.doi.org/10.3390/cancers13205089DOI Listing
October 2021

Bruton's Tyrosine Kinase-Mediated Signaling in Myeloid Cells Is Required for Protective Innate Immunity During Pneumococcal Pneumonia.

Front Immunol 2021 6;12:723967. Epub 2021 Sep 6.

Center for Experimental and Molecular Medicine (CEMM), Amsterdam University Medical Centers (UMC), Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands.

Bruton's tyrosine kinase (Btk) is a cytoplasmic kinase expressed in B cells and myeloid cells. It is essential for B cell development and natural antibody-mediated host defense against bacteria in humans and mice, but little is known about the role of Btk in innate host defense . Previous studies have indicated that lack of (natural) antibodies is paramount for impaired host defense against in patients and mice with a deficiency in functional Btk. In the present study, we re-examined the role of Btk in B cells and myeloid cells during pneumococcal pneumonia and sepsis in mice. The antibacterial defense of Btk mice was severely impaired during pneumococcal pneumosepsis and restoration of natural antibody production in Btk mice by transgenic expression of Btk specifically in B cells did not suffice to protect against infection. Btk mice with reinforced Btk expression in MhcII cells, including B cells, dendritic cells and macrophages, showed improved antibacterial defense as compared to Btk mice. Bacterial outgrowth in Lysmcre-Btk/Y mice was unaltered despite a reduced capacity of Btk-deficient alveolar macrophages to respond to pneumococci. Mrp8cre-Btk/Y mice with a neutrophil specific paucity in Btk expression, however, demonstrated impaired antibacterial defense. Neutrophils of Mrp8cre-Btk/Y mice displayed reduced release of granule content after pulmonary installation of lipoteichoic acid, a gram-positive bacterial cell wall component relevant for pneumococci. Moreover, Btk deficient neutrophils showed impaired degranulation and phagocytosis upon incubation with pneumococci . Taken together, the results of our study indicate that besides regulating B cell-mediated immunity, Btk is critical for regulation of myeloid cell-mediated, and particularly neutrophil-mediated, innate host defense against .
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http://dx.doi.org/10.3389/fimmu.2021.723967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8450579PMC
September 2021

A literature review of microvascular proliferation in arteriovenous malformations of skin and soft tissue.

J Clin Transl Res 2021 Aug 30;7(4):540-557. Epub 2021 Jul 30.

Department of Pathology, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Background And Aim: Arteriovenous malformations (AVM) are defined as being quiescent vascular masses composed of mature vessels. However, recent studies reported areas of microvascular proliferation (MVP) in AVM, indicating a process of angiogenesis. As this finding questions the previous definition, the primary objective of this review was to evaluate whether angiogenesis occurs in vascular malformations of skin and soft tissue, and second, to identify potential factors involved in MVP.

Method: Due to the multifaceted nature of this subject, a hermeneutic methodology was used to select articles that were likely to provide a deeper understanding of MVP in vascular malformations. Through citation tracking and database searching in PubMed and Web of Science, relevant articles were identified. All study designs concerning occurrence of MVP in AVM of skin and soft tissue in all age groups were included in the study. The Newcastle-Ottawa scale was used for quality assessment.

Results: 16 studies were included in this review which reported occurrence of MVP areas in between the otherwise mature vessels of vascular malformations. In these studies, angiogenesis was reported only in AVM-type of vascular malformations. Increased levels of pro-angiogenic factors were also reported and proliferation was found most prominently during adolescence. Finally, several types of hormone receptors also have been described in tissues of AVM.

Conclusion: Overall, the reviewed data support occurrence of active angiogenesis, highlighted by the presence of MVP in the arteriovenous type of vascular malformations, and a possible concurrent lesion progression towards a higher Schobinger stage of clinical severity. The relative scarcity of data at present implies that further research is required to elucidate the nature of MVP in AVM, which could have implications for developing targeted pharmacotherapy.

Relevance For Patients: Active angiogenesis caused by MVP in AVM patients is known to be correlating to clinical symptoms and contributing to the progression of the disease, recurrence rate, and patient's quality of life.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8445624PMC
August 2021

Macrophage-secreted MMP9 induces mesenchymal transition in pancreatic cancer cells via PAR1 activation.

Cell Oncol (Dordr) 2020 Dec 18;43(6):1161-1174. Epub 2020 Aug 18.

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

Purpose: Targeting tumor-infiltrating macrophages limits progression and improves chemotherapeutic responses in pancreatic ductal adenocarcinoma (PDAC). Protease-activated receptor (PAR)1 drives monocyte/macrophage recruitment, and stromal ablation of PAR1 limits cancer growth and enhances gemcitabine sensitivity in experimental PDAC. However, the functional interplay between PAR1, macrophages and tumor cells remains unexplored. Here we address the PAR1-macrophage-tumor cell crosstalk and assess its contributions to tumor progression.

Methods: PAR1 expression and macrophage infiltration were correlated in primary PDAC biopsies using gene expression datasets and tissue microarrays. Medium transfer experiments were used to evaluate the functional consequences of macrophage-tumor cell crosstalk and to assess the contribution of PAR1 to the observed responses. PAR1 cleavage assays were used to identify a macrophage-secreted PAR1 agonist, and the effects of candidate proteases were assessed in medium transfer experiments with specific inhibitors and/or recombinant agonist.

Results: PAR1 expression correlates with macrophage infiltration in primary PDACs, and macrophages induce mesenchymal transition of PDAC cells through PAR1 activation. Protease profiling identified macrophage-secreted matrix metalloprotease 9 (MMP9) as the relevant PAR1 agonist in PDAC. PAR1 and/or MMP9 inhibition limited macrophage-driven mesenchymal transition. Likewise, preventing mesenchymal transition by silencing ZEB1 or by pharmacological inhibition of the MMP9/PAR1 axis significantly reduced the ability of tumor cells to survive the anti-tumor activities of macrophages.

Conclusion: Macrophages secrete MMP9, which acts upon PDAC cell PAR1 to induce mesenchymal transition. This macrophage-induced mesenchymal transition supports the tumor-promoting role of macrophage influx, explaining the dichotomous contributions of these immune cells to tumor growth.
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http://dx.doi.org/10.1007/s13402-020-00549-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7717035PMC
December 2020

Genetic variants in SUSD2 are associated with the risk of ischemic heart disease.

J Clin Lipidol 2020 Jul - Aug;14(4):470-481. Epub 2020 Jun 3.

Department of Vascular Medicine, Amsterdam University Medical Center, Location AMC, Amsterdam, The Netherlands; Department of Experimental Vascular Medicine, Amsterdam University Medical Center, Location AMC, Amsterdam, The Netherlands. Electronic address:

Background: Genetic factors partly determine the risk for premature myocardial infarction (MI).

Objectives: We report the identification of a novel rare genetic variant in a kindred with an autosomal dominant trait for premature MI and atherosclerosis and explored the association of a common nonsynonymous variant in the same gene with the risk of ischemic heart disease (IHD) in a population-based study.

Methods: Next-generation sequencing was performed in a small pedigree with premature MI or subclinical atherosclerosis. A common variant, rs8141797 A>G (p.Asn466Ser), in sushi domain-containing protein 2 (SUSD2) was studied in the prospective Copenhagen General Population Studies (N = 105,408) for association with IHD.

Results: A novel heterozygous nonsense mutation in SUSD2 (c.G583T; p.Glu195Ter) was associated with the disease phenotype in the pedigree. SUSD2 protein was expressed in aortic specimens in the subendothelial cell layer and around the vasa vasorum. Furthermore, the minor G-allele of rs8141797 was associated with per allele higher levels of SUSD2 mRNA expression in the heart and vasculature. In the Copenhagen General Population Study, hazard ratios for IHD were 0.92 (95% CI: 0.87-0.97) in AG heterozygotes and 0.86 (0.62-1.19) in GG homozygotes vs noncarrriers (P-trend = .002). Finally, in meta-analysis including 73,983 IHD cases and 215,730 controls, the odds ratio for IHD per G-allele vs A-allele was 0.93 (0.90-0.96) (P = 4.6 × 10).

Conclusions: The identification of a truncating mutation in SUSD2, which was associated with premature MI and subclinical atherosclerosis, combined with the finding that a common missense variant in SUSD2 was strongly associated with a lower risk of IHD, suggest that SUSD2 may alter the risk of atherosclerosis.
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http://dx.doi.org/10.1016/j.jacl.2020.05.100DOI Listing
August 2021

Authors' Response to Letter to the Editor on "Unidentified Variables May Account for Variability in Multiplexing Results".

J Histochem Cytochem 2020 05;68(5):355-356

Department of Dermatology, Amsterdam University Medical Centers, location AMC, University of Amsterdam, Amsterdam, The Netherlands.

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http://dx.doi.org/10.1369/0022155420925082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226621PMC
May 2020

Pathological validation and prognostic potential of quantitative MRI in the characterization of pancreas cancer: preliminary experience.

Mol Oncol 2020 09 23;14(9):2176-2189. Epub 2020 Jun 23.

Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC, University of Amsterdam, The Netherlands.

Patient stratification based on biological variation in pancreatic ductal adenocarcinoma (PDAC) subtypes could help to improve clinical outcome. However, noninvasive assessment of the entire tumor microenvironment remains challenging. In this study, we investigate the biological basis of dynamic contrast-enhanced (DCE), intravoxel incoherent motion (IVIM), and R2*-derived magnetic resonance imaging (MRI) parameters for the noninvasive characterization of the PDAC tumor microenvironment and evaluate their prognostic potential in PDAC patients. Patients diagnosed with treatment-naïve resectable PDAC underwent MRI. After resection, a whole-mount tumor slice was analyzed for collagen fraction, vessel density, and hypoxia and matched to the MRI parameter maps. MRI parameters were correlated to immunohistochemistry-derived tissue characteristics and evaluated for prognostic potential. Thirty patients were included of whom 21 underwent resection with whole-mount histology available in 15 patients. DCE K and v , ADC, and IVIM D correlated with collagen fraction. DCE k and IVIM f correlated with vessel density and R2* with tissue hypoxia. Based on MRI, two main PDAC phenotypes could be distinguished; a stroma-high phenotype demonstrating high vessel density and high collagen fraction and a stroma-low phenotype demonstrating low vessel density and low collagen fraction. Patients with the stroma-high phenotype (high k and high IVIM D, n = 8) showed longer overall survival (not reached vs. 14 months, P = 0.001, HR = 9.1, P = 0.004) and disease-free survival (not reached vs. 2 months, P < 0.001, HR 9.3, P = 0.003) compared to the other patients (n = 22). Median follow-up was 41 (95% CI: 36-46) months. MRI was able to accurately characterize tumor collagen fraction, vessel density, and hypoxia in PDAC. Based on imaging parameters, a subgroup of patients with significantly better prognosis could be identified. These first results indicate that stratification-based MRI-derived biomarkers could help to tailor treatment and improve clinical outcome and warrant further research.
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http://dx.doi.org/10.1002/1878-0261.12688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463316PMC
September 2020

Automated Detection and Grading of Non-Muscle-Invasive Urothelial Cell Carcinoma of the Bladder.

Am J Pathol 2020 07 10;190(7):1483-1490. Epub 2020 Apr 10.

Department of Pathology, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands.

Accurate grading of non-muscle-invasive urothelial cell carcinoma is of major importance; however, high interobserver variability exists. A fully automated detection and grading network based on deep learning is proposed to enhance reproducibility. A total of 328 transurethral resection specimens from 232 patients were included, and a consensus reading by three specialized pathologists was used. The slides were digitized, and the urothelium was annotated by expert observers. The U-Net-based segmentation network was trained to automatically detect urothelium. This detection was used as input for the classification network. The classification network aimed to grade the tumors according to the World Health Organization grading system adopted in 2004. The automated grading was compared with the consensus and individual grading. The segmentation network resulted in an accurate detection of urothelium. The automated grading shows moderate agreement (κ = 0.48 ± 0.14 SEM) with the consensus reading. The agreement among pathologists ranges between fair (κ = 0.35 ± 0.13 SEM and κ = 0.38 ± 0.11 SEM) and moderate (κ = 0.52 ± 0.13 SEM). The automated classification correctly graded 76% of the low-grade cancers and 71% of the high-grade cancers according to the consensus reading. These results indicate that deep learning can be used for the fully automated detection and grading of urothelial cell carcinoma.
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http://dx.doi.org/10.1016/j.ajpath.2020.03.013DOI Listing
July 2020

Etosis, rather than apoptosis or cell proliferation, typifies thrombus progression - An immunohistochemical study of coronary aspirates.

Int J Cardiol Heart Vasc 2020 Feb 25;26:100439. Epub 2019 Nov 25.

Department of Pathology, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, Netherlands.

Background: Coronary thrombosis is a process with unpredictable clinical outcome. Changes of thrombus composition overtime influence tissue repair and stabilization. We investigated rates of cell deaths and cell proliferation at different time points after initiation of thrombosis.

Methods: Thrombectomy aspirates of 55 myocardial infarction patients were selected and histomorphologically classified as fresh (25), lytic (25), partially fibrocellular (10), completely fibrocellular (10). Paraffin sections were immunostained with anti-(cleaved) caspase-3/Casp3 (apoptosis), Citrullinated histone/CitH 3 (etosis), C-reactive protein/CRP and Ki67 (proliferation) in combination with either Feulgen counterstaining (DNA) or cell markers for granulocytes, macrophages, SMCs, platelets and endothelium. Rates of apoptosis, etosis and proliferation were measured as a percentage of total number of immunopositive pixels versus total number of DNA positive pixels, while co-localization with cell markers was assessed by digital image analysis.

Results: Positive staining of CitH3 was observed more frequently (93%) than Casp3 (70%), Ki67 (79%) or CRP (59%) (p < 0.05). Moreover, rate of etosis, found in granulocytes and macrophages, differed significantly among thrombi of different age, being higher in lytic (12.82) than in fresh (8.52) and late-organized (2.75) (p < 0.05). Such differences were not observed for the rates of apoptosis or cell proliferation related to thrombus age. CRP staining was present in fresh, lytic and organized thrombi, but did not reliably identify necrotic areas.

Conclusions: Different patterns of cell death and cell proliferation are noticed during progression of coronary thrombus overtime, but with significant differences for only etosis. Etosis could potentially serve as a biomarker for thrombus instability with clinical significance.
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http://dx.doi.org/10.1016/j.ijcha.2019.100439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046519PMC
February 2020

Comparison of Two Different Immunohistochemical Quadruple Staining Approaches to Identify Innate Lymphoid Cells in Formalin-fixed Paraffin-embedded Human Tissue.

J Histochem Cytochem 2020 02 27;68(2):127-138. Epub 2019 Dec 27.

Department of Dermatology, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, The Netherlands.

Lack of specific markers for innate lymphoid cells (ILCs) limit our knowledge on their spatial organization in situ. We compared two quadruple-color staining protocols for detection of the three principal human ILC subsets in formalin-fixed paraffin-embedded specimens. ILC subset-associated archetypical transcription factors (TFs) T-bet, GATA3, and RORγt were used as positive identifiers in combination with lymphoid lineage markers to exclude non-ILCs. One method ("virtual quadruple staining") comprised of iterative single stainings on the same section performing digital scanning and subsequent immunoglobulin and chromogen stripping after each staining round. The second technique ("true-color quadruple staining") comprised sequential double stainings with permanent colors. Both protocols appeared suitable for accurate detection of each ILC subset, and as added result, concomitant visualization of their T cell subset counterpart. Only true-color quadruple staining enabled simultaneous detection of all three ILC subsets within one section. Furthermore, we found that type 3 and type 1 ILCs (ILC1s) represent the major subsets in colon and that part of the ILC1s typically colocalizes with blood vessels. Our data highlight the utility of TFs combined with lineage markers for the identification of ILC subsets and proposed workflow opens the way to gain deeper insight of their anatomical distribution.
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http://dx.doi.org/10.1369/0022155419897257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003497PMC
February 2020

Fibrotic aortic valve disease after radiotherapy: an immunohistochemical study in breast cancer and lymphoma patients.

Cardiovasc Pathol 2020 Mar - Apr;45:107176. Epub 2019 Nov 15.

Department of Cardiothoracic Surgery, Amsterdam University Medical Center, University of Amsterdam, Heart Center, Amsterdam, the Netherlands. Electronic address:

Background: Radiation-associated aortic valve (AV) stenosis is frequently seen as a late sequela after thoracic radiotherapy (RT). Although the clinical relationship between thoracic radiotherapy and valvular dysfunction has been established, the process leading to accelerated aortic valve stenosis remains unclear. The aim of this study was to determine whether increased inflammatory cell infiltration, fibrosis, and calcification is present in aortic valves after radiotherapy at the time of aortic valve replacement.

Methods: Stenotic aortic valve specimens from 43 patients were obtained after surgical aortic valve replacement. A total 28 patients had previously undergone radiotherapy for breast cancer or malignant lymphoma. A total 15 patients were included as control. The valve leaflets were assessed by (immuno)histochemistry for inflammatory cell composition (CD3, CD20, CD68, and CD163) and extracellular matrix changes (collagen and calcification).

Results: Aortic valve cell density after radiotherapy for lymphoma was markedly decreased when compared with other groups. Irradiated aortic valve show similar (low) degrees of late T and B lymphocyte infiltration as control valves, whereas macrophage marker CD68 was decreased after radiotherapy for breast cancer. Collagen content was increased following radiotherapy. Aortic valves of patients with lymphoma contained significantly less calcified tissue when compared with the other groups.

Conclusion: High-dose radiation at a young age (patients with lymphoma) results in cell loss and premature fibrotic aortic valve stenosis as opposed to the degenerative calcific stenosis observed in patients with breast cancer. Our findings suggest a possible dose-dependent effect of radiotherapy on aortic valve fibrosis. The active presence of inflammatory cells may be limited to the acute phase after radiotherapy.
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http://dx.doi.org/10.1016/j.carpath.2019.107176DOI Listing
June 2020

Prekallikrein inhibits innate immune signaling in the lung and impairs host defense during pneumosepsis in mice.

J Pathol 2020 01 25;250(1):95-106. Epub 2019 Nov 25.

Center of Experimental & Molecular Medicine, Amsterdam University Medical Centers, location Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Prekallikrein (PKK, also known as Fletcher factor and encoded by the gene KLKB1 in humans) is a component of the contact system. Activation of the contact system has been implicated in lethality in fulminant sepsis models. Pneumonia is the most frequent cause of sepsis. We sought to determine the role of PKK in host defense during pneumosepsis. To this end, mice were infected with the common human pathogen Klebsiella pneumoniae via the airways, causing an initially localized infection of the lungs with subsequent bacterial dissemination and sepsis. Mice were treated with a selective PKK-directed antisense oligonucleotide (ASO) or a scrambled control ASO for 3 weeks prior to infection. Host response readouts were determined at 12 or 36 h post-infection, including genome-wide messenger RNA profiling of lungs, or mice were followed for survival. PKK ASO treatment inhibited constitutive hepatic Klkb1 mRNA expression by >80% and almost completely abolished plasma PKK activity. Klkb1 mRNA could not be detected in lungs. Pneumonia was associated with a progressive decline in PKK expression in mice treated with control ASO. PKK ASO administration was associated with a delayed mortality, reduced bacterial burdens, and diminished distant organ injury. While PKK depletion did not influence lung pathology or neutrophil recruitment, it was associated with an upregulation of multiple innate immune signaling pathways in the lungs already prior to infection. Activation of the contact system could not be detected, either during infection in vivo or at the surface of Klebsiella in vitro. These data suggest that circulating PKK confines pro-inflammatory signaling in the lung by a mechanism that does not involve contact system activation, which in the case of respiratory tract infection may impede early protective innate immunity. © 2019 Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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http://dx.doi.org/10.1002/path.5354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6972537PMC
January 2020

Deep learning for automatic Gleason pattern classification for grade group determination of prostate biopsies.

Virchows Arch 2019 Jul 16;475(1):77-83. Epub 2019 May 16.

Department of Biomedical Engineering and Physics, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Histopathologic grading of prostate cancer using Gleason patterns (GPs) is subject to a large inter-observer variability, which may result in suboptimal treatment of patients. With the introduction of digitization and whole-slide images of prostate biopsies, computer-aided grading becomes feasible. Computer-aided grading has the potential to improve histopathological grading and treatment selection for prostate cancer. Automated detection of GPs and determination of the grade groups (GG) using a convolutional neural network. In total, 96 prostate biopsies from 38 patients are annotated on pixel-level. Automated detection of GP 3 and GP ≥ 4 in digitized prostate biopsies is performed by re-training the Inception-v3 convolutional neural network (CNN). The outcome of the CNN is subsequently converted into probability maps of GP ≥ 3 and GP ≥ 4, and the GG of the whole biopsy is obtained according to these probability maps. Differentiation between non-atypical and malignant (GP ≥ 3) areas resulted in an accuracy of 92% with a sensitivity and specificity of 90 and 93%, respectively. The differentiation between GP ≥ 4 and GP ≤ 3 was accurate for 90%, with a sensitivity and specificity of 77 and 94%, respectively. Concordance of our automated GG determination method with a genitourinary pathologist was obtained in 65% (κ = 0.70), indicating substantial agreement. A CNN allows for accurate differentiation between non-atypical and malignant areas as defined by GPs, leading to a substantial agreement with the pathologist in defining the GG.
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http://dx.doi.org/10.1007/s00428-019-02577-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6611751PMC
July 2019

Three-dimensional histopathological reconstruction of bladder tumours.

Diagn Pathol 2019 Mar 28;14(1):25. Epub 2019 Mar 28.

Department of Biomedical Engineering and Physics, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Background: Histopathological analysis is the cornerstone in bladder cancer (BCa) diagnosis. These analysis suffer from a moderate observer agreement in the staging of bladder cancer. Three-dimensional reconstructions have the potential to support the pathologists in visualizing spatial arrangements of structures, which may improve the interpretation of specimen. The aim of this study is to present three-dimensional (3D) reconstructions of histology images.

Methods: En-bloc specimens of transurethral bladder tumour resections were formalin fixed and paraffin embedded. Specimens were cut into sections of 4 μm and stained with Hematoxylin and Eosin (H&E). With a Phillips IntelliSite UltraFast scanner, glass slides were digitized at 20x magnification. The digital images were aligned by performing rigid and affine image alignment. The tumour and the muscularis propria (MP) were manually delineated to create 3D segmentations. In conjunction with a 3D display, the results were visualized with the Vesalius3D interactive visualization application for a 3D workstation.

Results: En-bloc resection was performed in 21 BCa patients. Per case, 26-30 sections were included for the reconstruction into a 3D volume. Five cases were excluded due to export problems, size of the dataset or condition of the tissue block. Qualitative evaluation suggested an accurate registration for 13 out of 16 cases. The segmentations allowed full 3D visualization and evaluation of the spatial relationship of the BCa tumour and the MP.

Conclusion: Digital scanning of en-bloc resected specimens allows a full-fledged 3D reconstruction and analysis and has a potential role to support pathologists in the staging of BCa.
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http://dx.doi.org/10.1186/s13000-019-0803-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6440143PMC
March 2019

Platelet Btk is Required for Maintaining Lung Vascular Integrity during Murine Pneumococcal Pneumosepsis.

Thromb Haemost 2019 Jun 14;119(6):930-940. Epub 2019 Mar 14.

Center for Experimental and Molecular Medicine (CEMM), Amsterdam UMC, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Platelet Bruton's tyrosine kinase (Btk) is an essential signalling protein for the collagen receptor glycoprotein VI (GPVI) and podoplanin receptor C-type-lectin-like receptor-2, which are platelet receptors implicated in the maintenance of vascular integrity during inflammation. Moreover, platelets, platelet GPVI and Btk are important for host defence during murine bacterial pneumosepsis. The aim of this study was to determine the role of platelet Btk in vascular integrity and host defence during murine pneumosepsis caused by the common human pathogens and . Using the Cre-loxP system, male platelet-specific Btk-deficient mice (PF4creBtk/Y) were created. Similar to platelets from total Btk-deficient mice, platelets from PF4creBtk/Y mice showed abrogated aggregation and P-selectin expression when stimulated with the GPVI ligand cross-linked collagen-related peptide. Upon infection with , PF4creBtk/Y mice showed increased lung bleeding, but unimpaired anti-bacterial defence. During pneumosepsis evoked by , platelet Btk deficiency was not associated with lung bleeding and did not impact on host defence, even when platelet function was further compromised by blocking secondary platelet activation by the P2Y receptor antagonist clopidogrel. Together, these data indicate that, while platelet Btk is not important for anti-bacterial defence in pneumosepsis, its role in maintaining vascular integrity in the lung depends on the causative pathogen.
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http://dx.doi.org/10.1055/s-0039-1681046DOI Listing
June 2019

Author Correction: Combining streptozotocin and unilateral nephrectomy is an effective method for inducing experimental diabetic nephropathy in the 'resistant' C57Bl/6J mouse strain.

Sci Rep 2019 Feb 27;9(1):3425. Epub 2019 Feb 27.

Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-38075-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391405PMC
February 2019

The role of Mannose Binding Lectin in the immune response against Borrelia burgdorferi sensu lato.

Sci Rep 2019 02 5;9(1):1431. Epub 2019 Feb 5.

Amsterdam UMC, University of Amsterdam, Center for Experimental and Molecular Medicine, Meibergdreef 9, Amsterdam, Netherlands.

The causative agents of Lyme borreliosis, spirochetes belonging to the Borrelia burgdorferi sensu lato group, have developed several ways to protect themselves against killing by the host complement system. In addition, it has been shown that serum sensitive isolates are (partially) protected by the Ixodes Tick Salivary Lectin Pathway Inhibitor (TSLPI) protein; a salivary gland protein that inhibits the function of Mannose Binding Lectin (MBL). MBL is a C-type lectin that recognizes oligosaccharides on pathogens and activates the complement system via the lectin pathway. MBL deficiency has been linked to a more severe course of several infectious diseases and humans with detectable antibodies against B. burgdorferi are significantly more often MBL deficient compared to humans without antibodies against B. burgdorferi. Here we set out to investigate the role of MBL in the immune response against B. burgdorferi in more detail. We demonstrate that B. burgdorferi N40 needle-infected C57BL/6 MBL deficient mice harbored significantly higher B. burgdorferi numbers in skin tissue during the early course of infection. In line with these findings they also developed higher anti-B. burgdorferi IgG serum antibodies compared to WT controls. In contrast, B. burgdorferi loads in distant tissue such as heart, joints or bladder at later time points were similar for both mouse strains. These in vivo findings were corroborated using a B. burgdorferi N40-infected I. scapularis infestation model. We showed that MBL is capable of binding B. burgdorferi through its carbohydrate recognition domains, but in vitro complement killing assays, peritoneal macrophage and whole blood stimulations, phagocytosis assays and an in vivo migration experiment did not reveal the mechanism by which MBL facilitates early clearance of B. burgdorferi. To conclude, we show a protective role of MBL in the early stages of B. burgdorferi infection, yet the underlying mechanism warrants further investigation.
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http://dx.doi.org/10.1038/s41598-018-37922-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363739PMC
February 2019

Btk inhibitor ibrutinib reduces inflammatory myeloid cell responses in the lung during murine pneumococcal pneumonia.

Mol Med 2019 01 15;25(1). Epub 2019 Jan 15.

Center for Experimental and Molecular Medicine (CEMM), Amsterdam UMC, Academic Medical Center, University of Amsterdam, Meibergdreef 9, Room G2-132, 1105, AZ, Amsterdam, the Netherlands.

Background: Streptococcus pneumoniae is a major causative agent in community-acquired pneumonia and sepsis. Overwhelming lung inflammation during pneumococcal pneumonia may hamper lung function. Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (Btk), a key signaling protein controlling the activation of various immune cells, including macrophages and neutrophils. The aim of this study was to determine whether ibrutinib treatment ameliorates acute lung inflammation during pneumococcal pneumonia.

Methods: Mice were treated orally with ibrutinib and the effect on acute pulmonary inflammation elicited by the gram-positive bacterial cell wall component lipoteichoic acid (LTA) and during ceftriaxone-treated pneumococcal pneumonia was assessed.

Results: Treatment with ibrutinib prior to and after intranasal LTA instillation reduced alveolar macrophage activation, neutrophil influx, cytokine release and plasma leakage into the lung. Postponed treatment with ibrutinib supplementing antibiotic therapy during ongoing pneumococcal pneumonia did not impair bacterial killing in lung, blood and spleen. In this setting, ibrutinib reduced alveolar macrophage and systemic neutrophil activation and substantially diminished further monocyte and neutrophil influx in the lung. In vitro, ibrutinib inhibited macrophage TNF secretion and neutrophil activation upon LTA and pneumococcal stimulation.

Conclusions: Taken together, these data indicate that the Btk inhibitor ibrutinib reduces inflammatory myeloid cell responses during acute pulmonary inflammation evoked by LTA and antibiotic-treated pneumococcal pneumonia and suggest that ibrutinib has the potential to inhibit ongoing lung inflammation in an acute infectious setting.
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http://dx.doi.org/10.1186/s10020-018-0069-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6332549PMC
January 2019

Platelet-Dense Granules Worsen Pre-Infection Thrombocytopenia during Gram-Negative Pneumonia-Derived Sepsis.

J Innate Immun 2019 17;11(2):168-180. Epub 2018 Dec 17.

Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Platelet-dense (δ) granules are important for platelet function. Platelets contribute to host defense and vascular integrity during pneumonia and sepsis, and δ granule products, including adenosine diphosphate (ADP), can influence inflammatory responses. We therefore aimed to study the role of platelet δ granules in the host response during sepsis. Hermansky-Pudlak syndrome (Hps)3coa mice (with reduced δ granule content), mice treated with the platelet ADP receptor inhibitor clopidogrel, and appropriate control mice were infected with the human sepsis pathogen Klebsiella pneumoniae via the airways to induce pneumonia and sepsis. In order to override potential redundancy in platelet functions, we also studied Hps3coa and control mice with moderate antibody-induced thrombocytopenia (10%) prior to infection. We found that sepsis-induced thrombocytopenia tended to be less severe in Hps3coa mice, and was significantly ameliorated in Hps3coa mice with low pre-infection platelet counts. Bacterial growth was similar in Hps3coa and control mice in the presence of normal platelet counts prior to infection, but lower in the lungs of Hps3coa mice with low pre-infection platelet counts. Hps3coa mice had unaltered lung pathology and distant organ injury during pneumosepsis, irrespective of pre-infection platelet counts; lung bleeding did not differ between Hps3coa and control mice. Clopidogrel did not influence any host response parameter. These data suggest that platelet δ granules can play a detrimental role in pneumosepsis by aggravating thrombocytopenia and impairing local antibacterial defense, but that these unfavorable effects only become apparent in the presence of low platelet counts.
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http://dx.doi.org/10.1159/000494147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6738263PMC
May 2020

Extracellular traps derived from macrophages, mast cells, eosinophils and neutrophils are generated in a time-dependent manner during atherothrombosis.

J Pathol 2019 04 25;247(4):505-512. Epub 2019 Jan 25.

Department of Pathology, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Extracellular traps generated by neutrophils contribute to thrombus progression in coronary atherosclerotic plaques. It is not known whether other inflammatory cell types in coronary atherosclerotic plaque or thrombus also release extracellular traps. We investigated their formation by macrophages, mast cells, and eosinophils in human coronary atherosclerosis, and in relation to the age of thrombus of myocardial infarction patients. Coronary arteries with thrombosed or intact plaques were retrieved from patients who died from myocardial infarction. In addition, thrombectomy specimens from patients with myocardial infarction were classified histologically as fresh, lytic or organised. Neutrophil and macrophage extracellular traps were identified using sequential triple immunostaining of CD68, myeloperoxidase, and citrullinated histone H3. Eosinophil and mast cell extracellular traps were visualised using double immunostaining for eosinophil major basic protein or tryptase, respectively, and citrullinated histone H3. Single- and double-stained immunopositive cells in the plaque, adjacent adventitia, and thrombus were counted. All types of leucocyte-derived extracellular traps were present in all thrombosed plaques, and in all types of the in vivo-derived thrombi, but only to a much lower extent in intact plaques. Neutrophil traps, followed by macrophage traps, were the most prominent types in the autopsy series of atherothrombotic plaques, including the adventitia adjacent to thrombosed plaques. In contrast, macrophage traps were more numerous than neutrophil traps in intact plaques (lipid cores) and organised thrombi. Mast cell and eosinophil extracellular traps were also present, but sparse in all instances. In conclusion, not only neutrophils but also macrophages, eosinophils, and mast cells are sources of etosis involved in evolving coronary thrombosis. Neutrophil traps dominate numerically in early thrombosis and macrophage traps in late (organising) thrombosis, implying that together they span all the stages of thrombus progression and maturation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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http://dx.doi.org/10.1002/path.5212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6590313PMC
April 2019

Role of Peptidylarginine Deiminase 4 in Neutrophil Extracellular Trap Formation and Host Defense during Induced Pneumonia-Derived Sepsis.

J Immunol 2018 08 9;201(4):1241-1252. Epub 2018 Jul 9.

Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands.

Peptidylarginine deiminase 4 (PAD4) catalyzes citrullination of histones, an important step for neutrophil extracellular trap (NET) formation. We aimed to determine the role of PAD4 during pneumonia. Markers of NET formation were measured in lavage fluid from airways of critically ill patients. NET formation and host defense were studied during pneumonia-derived sepsis caused by in PAD4 and PAD4 mice. Patients with pneumosepsis, compared with those with nonpulmonary disease, showed increased citrullinated histone 3 (CitH3) levels in their airways and a trend toward elevated levels of NET markers cell-free DNA and nucleosomes. During murine pneumosepsis, CitH3 levels were increased in the lungs of PAD4 but not of PAD4 mice. Combined light and electron microscopy showed NET-like structures surrounding in areas of CitH3 staining in the lung; however, these were also seen in PAD4 mice with absent CitH3 lung staining. Moreover, cell-free DNA and nucleosome levels were mostly similar in both groups. Moreover, and LPS could still induce NETosis in PAD4 neutrophils. Both groups showed largely similar bacterial growth, lung inflammation, and organ injury. In conclusion, these data argue against a major role for PAD4 in NET formation, host defense, or organ injury during pneumonia-derived sepsis.
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http://dx.doi.org/10.4049/jimmunol.1800314DOI Listing
August 2018

Neutrophil Extracellular Traps Participate in All Different Types of Thrombotic and Haemorrhagic Complications of Coronary Atherosclerosis.

Thromb Haemost 2018 06 19;118(6):1078-1087. Epub 2018 Apr 19.

Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands.

Acute coronary syndromes can be initiated by either atherosclerotic fibrous cap ruptures, superficial plaque erosions or intraplaque haemorrhages (IPHs). Since neutrophil extracellular traps (NETs) display pro-inflammatory and pro-thrombotic properties, we investigated the presence, extent and distribution of neutrophils and NETs in different types of plaque complications in relation to the age of overlying thrombus mass or haemorrhage. Sixty-four paraffin-embedded coronary plaque segments of 30 acute myocardial infarction patients were retrieved from the autopsy archives, which contained 44 complicated plaques (17 IPHs, 9 erosions and 18 ruptures) and 20 intact plaques. Complicated plaques were further categorized according to the histological age of thrombus or haemorrhage. Immunohistochemistry was performed to visualize neutrophils (anti-myeloperoxidase, anti-elastase and anti-CD177) and NETs (anti-citrullinated histone-3 and anti-peptidyl-arginine-deiminase-4). The results were scored semi-quantitatively. Neutrophils and NETs were abundantly present in all types of complicated, but not in intact, plaques ( < 0.05). They were found in thrombus, haemorrhages and at the thrombus-plaque interface, with no significant differences in extent between ruptures, erosions and IPHs. Interestingly, adjacent perivascular tissue of complicated, but not of intact plaques, also contained high numbers of neutrophils and NETs ( < 0.05). In thrombus and haemorrhage of different age, neutrophils and NETs were more frequently present in non-organized (fresh) thrombi and in on-going IPHs. In conclusion, netosis is a prominent pro-thrombotic participant in all distinct types of atherothrombosis, which may facilitate the progression of thrombotic or haemorrhagic complications and thus the onset of ensuing clinical coronary ischemic syndromes.
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http://dx.doi.org/10.1055/s-0038-1641749DOI Listing
June 2018

Combining streptozotocin and unilateral nephrectomy is an effective method for inducing experimental diabetic nephropathy in the 'resistant' C57Bl/6J mouse strain.

Sci Rep 2018 04 3;8(1):5542. Epub 2018 Apr 3.

Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Diabetic nephropathy (DN) is the leading cause of chronic kidney disease. Animal models are essential tools for designing new strategies to prevent DN. C57Bl/6 (B6) mice are widely used for transgenic mouse models, but are relatively resistant to DN. This study aims to identify the most effective method to induce DN in a type 1 (T1D) and a type 2 diabetes (T2D) model in B6 mice. For T1D-induced DN, mice were fed a control diet, and randomised to streptozotocin (STZ) alone, STZ+unilateral nephrectomy (UNx), or vehicle/sham. For T2D-induced DN, mice were fed a western (high fat) diet, and randomised to either STZ alone, STZ+UNx, UNx alone, or vehicle/sham. Mice subjected to a control diet with STZ +UNx developed albuminuria, glomerular lesions, thickening of the glomerular basement membrane, and tubular injury. Mice on control diet and STZ developed only mild renal lesions. Furthermore, kidneys from mice on a western diet were hardly affected by diabetes, UNx or the combination. We conclude that STZ combined with UNx is the most effective model to induce T1D-induced DN in B6 mice. In our hands, combining western diet and STZ treatment with or without UNx did not result in a T2D-induced DN model in B6 mice.
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http://dx.doi.org/10.1038/s41598-018-23839-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5882654PMC
April 2018

Immunophenotypic analysis of the chronological events of tissue repair in aortic medial dissections.

Cardiovasc Pathol 2018 May - Jun;34:9-14. Epub 2018 Feb 10.

Dept. of Pathology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105, AZ, Amsterdam, the Netherlands. Electronic address:

Acute medial dissection of aorta can occur in the context of a sudden and unexpected death. For medico-legal reasons it is important to estimate as accurately the histological age of dissections. We evaluated the additional value of a systematic application of immunohistochemistry, compared with conventional histology only, in determining chronological steps of injury and repair. Thirty two paraffin embedded specimens of aortic dissection were retrospectively allocated to one of four defined stages: acute (I), subacute (II), early organizing (III) and scarring (IV) using Hematoxylin and Eosin and Elastica van Gieson stained sections. Subsequent immunohistochemically staining was performed with the following markers: (myeloperoxidase (neutrophils), citrullinated-Histone 3 (neutrophil extracellular traps), CD68 (macrophages), CD3 (T-cells), CD31 and CD34 (endothelial cells), and smooth muscle actin. Immune stained sections were scored semi-quantitatively. Histologically, five cases were identified as stage I, 16 as II, 7 as III and 4 as IV. Additional immunostaining for smooth muscle cells and endothelial cells altered the classification in 25% of cases (all in groups II and III). Immunostaining and semi-quantitative grading of involvement of neutrophils, macrophages and NETs also provided specific distribution patterns over the 4 age categories, including unexpected involvement of the peri adventitial fat tissue. In conclusion, it appears that semi-quantitative immunohistochemistry of resident vascular wall cells, inflammatory cells and NETS represents a useful adjunct in detailed histopathological grading of the chronological age of aortic dissections.
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http://dx.doi.org/10.1016/j.carpath.2018.01.009DOI Listing
October 2018

Platelet glycoprotein VI aids in local immunity during pneumonia-derived sepsis caused by gram-negative bacteria.

Blood 2018 02 29;131(8):864-876. Epub 2017 Nov 29.

Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Platelet collagen receptor glycoprotein VI (GPVI) and podoplanin receptor C-type lectin-like receptor 2 (CLEC2) are receptors implicated in platelet activation that both signal via an immunoreceptor tyrosine-based activation motif. Platelets are necessary for host defense and prevention of hemorrhage during sepsis, but the role of platelet GPVI and CLEC2 herein is unknown. To investigate this, we infected mice depleted of platelet GPVI or CLEC2 by antibody treatment or GPVI mice with the common human sepsis pathogen via the airways to induce pneumonia-derived sepsis. The GPVI ligand collagen and the CLEC2 ligand podoplanin were constitutively present in the lung, whereas the GPVI ligands fibrin and histone were induced during pneumonia. During late-stage infection, both mice depleted of GPVI and GPVI mice showed increased bacterial growth in lungs, and GPVI mice also showed increased bacterial growth in distant body sites. Despite higher bacterial loads, GPVI-depleted mice showed reduced platelet numbers, platelet activation, and platelet-leukocyte complex formation in the bronchoalveolar space. Consistently, in human whole blood, GPVI stimulation of platelets increased platelet-leukocyte complex formation and leukocyte activation, which was accompanied by enhanced phagocytosis of GPVI-depleted mice showed increased lung hemorrhage during infection, but not to the extent observed in platelet-depleted mice, and lung bleeding was not significantly different between GPVI and wild-type mice. CLEC2 depletion did not affect any of the responses during pneumonia. These results suggest that platelet GPVI, but not CLEC2, contributes to local host defense during pneumonia-derived sepsis by enhancing leukocyte function.
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http://dx.doi.org/10.1182/blood-2017-06-788067DOI Listing
February 2018

ASC and NLRP3 impair host defense during lethal pneumonia caused by serotype 3 Streptococcus pneumoniae in mice.

Eur J Immunol 2018 01 3;48(1):66-79. Epub 2017 Nov 3.

Center of Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, The Netherlands.

Streptococcus (S.) pneumoniae is the most common cause of community-acquired pneumonia. The Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, consisting of NLRP3, ASC (the adaptor apoptosis-associated speck-like protein containing a CARD) and caspase-1, has been implicated in protective immunity during pneumonia induced by high doses of S. pneumoniae serotype 2. Here we investigated the role of the NLRP3 inflammasome in the host response during lethal airway infection with a low dose of serotype 3 S. pneumoniae. Mice were euthanized at predefined endpoints for analysis or observed in survival studies. In additional studies, Tlr2 /Tlr4 mice and Myd88 mice incapable of Toll-like receptor signaling were studied. In stark contrast with existing literature, both Nlrp3 and Asc mice showed a strongly improved host defense, as reflected by a markedly reduced mortality rate accompanied by diminished bacterial growth and dissemination. Host defense was unaltered in Tlr2 /Tlr4 mice and Myd88 mice. These results show that the NLRP3 inflammasome impairs host defense during lethal pneumonia caused by serotype 3 S. pneumoniae. Our findings challenge the current paradigm that proximal innate detection systems are indispensable for an adequate host immune response against bacteria.
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http://dx.doi.org/10.1002/eji.201646554DOI Listing
January 2018

Composition of the cellular infiltrate in patients with simple and complex appendicitis.

J Surg Res 2017 06 6;214:190-196. Epub 2017 Mar 6.

Department of Paediatric Surgery, Paediatric Surgical Centre of Amsterdam, Emma Children's Hospital AMC and VU University Medical Centre, Amsterdam, The Netherlands.

Background: It is now well established that there are two types of appendicitis: simple (nonperforating) and complex (perforating). This study evaluates differences in the composition of the immune cellular infiltrate in children with simple and complex appendicitis.

Materials And Methods: A total of 47 consecutive children undergoing appendectomy for acute appendicitis between January 2011 and December 2012 were included. Intraoperative criteria were used to identify patients with either simple or complex appendicitis and were confirmed histopathologically. Immune histochemical techniques were used to identify immune cell markers in the appendiceal specimens. Digital imaging analysis was performed using Image J.

Results: In the specimens of patients with complex appendicitis, significantly more myeloperoxidase positive cells (neutrophils) (8.7% versus 1.2%, P < 0.001) were detected compared to patients with a simple appendicitis. In contrast, fewer CD8+ T cells (0.4% versus 1.3%, P = 0.016), CD20 + cells (2.9% versus 9.0%, P = 0.027), and CD21 + cells (0.2% versus 0.6%, P = 0.028) were present in tissue from patients with complex compared to simple appendicitis.

Conclusions: The increase in proinflammatory innate cells and decrease of adaptive cells in patients with complex appendicitis suggest potential aggravating processes in complex appendicitis. Further research into the underlying mechanisms may identify novel biomarkers to be able to differentiate simple and complex appendicitis.
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http://dx.doi.org/10.1016/j.jss.2017.02.062DOI Listing
June 2017

The change in circulating galectin-3 predicts absence of atrial fibrillation after thoracoscopic surgical ablation.

Europace 2018 05;20(5):764-771

Department of Cardiology, Experimental Cardiology and Cardiothoracic Surgery, Heart Center, Academic Medical Center, Meibergdreef 9, 1100 DD, Amsterdam, The Netherlands.

Aims: Galectin-3 (Gal-3) is an important mediator of cardiac fibrosis, particularly in heart failure. Increased Gal-3 concentration (Gal-3), associated with increased risk of developing atrial fibrillation (AF), may reflect atrial fibrotic remodelling underlying AF progression. We aimed to investigate whether the change in serum Gal-3 reflects alterations of the arrhythmogenic atrial substrate following thoracoscopic AF surgery, and predicts absence of AF.

Methods And Results: Consecutive patients undergoing thoracoscopic AF surgery were included. Left atrial appendages (LAAs) and serum were collected during surgery and serum again 6 months thereafter. Gal-3 was determined in tissue and serum. Interstitial collagen in the LAA was quantified using Picrosirius red staining. Ninety-eight patients (76% male, mean age 60 ± 9 years) underwent thoracoscopic surgery for advanced AF. Patients with increased Gal-3 after ablation compared to baseline had a higher recurrence rate compared to patients with decreased or unchanged Gal-3 (HR 2.91, P = 0.014). These patients more frequently had persistent AF, longer AF duration and thick atrial collagen strands (P = 0.049). At baseline, Gal-3 was similar between patients with and without AF recurrence: 14.8 ± 3.9 µg/L vs. 13.7 ± 3.7 µg/L, respectively in serum (P = 0.16); 94.5 ± 19.4 µg/L vs. 93.3 ± 30.8µg/L, respectively in atrial myocardium (P = 0.83). There was no correlation between serum Gal-3 and left atrial Gal-3 (P = 0.20), nor between serum Gal-3 and the percentage of fibrosis in LAA (P = 0.18).

Conclusion: The change of circulating Gal-3, rather than its baseline value, predicts AF recurrence after thoracoscopic ablation. Patients in whom Gal-3 increases after ablation have a high recurrence rate reflecting ongoing profibrotic signalling, irrespective of arrhythmia continuation.
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http://dx.doi.org/10.1093/europace/eux090DOI Listing
May 2018

Coagulation factor XI improves host defence during murine pneumonia-derived sepsis independent of factor XII activation.

Thromb Haemost 2017 07 11;117(8):1601-1614. Epub 2017 May 11.

Ingrid Stroo, Center for Experimental and Molecular Medicine, Academic Medical Center, Meibergdreef 9, G2-1051105 AZ Amsterdam, the Netherlands, Tel.: +31 20 5666034, E-mail:

Bacterial pneumonia, the most common cause of sepsis, is associated with activation of coagulation. Factor XI (FXI), the key component of the intrinsic pathway, can be activated via factor XII (FXII), part of the contact system, or via thrombin. To determine whether intrinsic coagulation is involved in host defence during pneumonia and whether this is dependent on FXII activation, we infected in parallel wild-type (WT), FXI knockout (KO) and FXII KO mice with two different clinically relevant pathogens, the Gram-positive bacterium Streptococcus pneumoniae and the Gram-negative bacterium Klebsiella pneumoniae, via the airways. FXI deficiency worsened survival and enhanced bacterial outgrowth in both pneumonia models. This was accompanied with enhanced inflammatory responses in FXI KO mice. FXII KO mice were comparable with WT mice in Streptococcus pneumoniae pneumonia. On the contrary, FXII deficiency improved survival and reduced bacterial outgrowth following infection with Klebsiella pneumoniae. In both pneumonia models, local coagulation was not impaired in either FXI KO or FXII KO mice. The capacity to phagocytose bacteria was impaired in FXI KO neutrophils and in human neutrophils where activation of FXI was inhibited. Deficiency for FXII or blocking activation of FXI via FXIIa had no effect on phagocytosis. Taken together, these data suggest that FXI protects against sepsis derived from Streptococcus pneumoniae or Klebsiella pneumoniae pneumonia at least in part by enhancing the phagocytic capacity of neutrophils by a mechanism that is independent of activation via FXIIa.
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http://dx.doi.org/10.1160/TH16-12-0920DOI Listing
July 2017

Therapeutic Administration of a Monoclonal Anti-Il-1β Antibody Protects Against Experimental Melioidosis.

Shock 2016 11;46(5):566-574

*Center for Infection and Immunity Amsterdam (CINIMA)/Center for Experimental and Molecular Medicine (CEMM), Academic Medical Center, Amsterdam, The Netherlands †Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands ‡Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand §Centre for Tropical Medicine and Global Health, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK ||Novartis Institutes for BioMedical Research, Basel, Switzerland ¶Department of Internal medicine, Division of Infectious Diseases, Academic Medical Center, Amsterdam, The Netherlands.

Background: Melioidosis, caused by the gram-negative bacterium Burkholderia pseudomallei, is a common cause of community-acquired sepsis in Southeast Asia and Northern Australia. The NLRP3 inflammasome and its downstream product interleukin-1 beta (IL-1β) have been proposed to play crucial roles in melioidosis. In this study, we characterized the role of IL-1β more closely and we assessed its therapeutic potential.

Methods: mRNA expression of inflammasome components was determined in isolated leukocytes of 32 healthy controls and 34 patients with sepsis caused by B pseudomallei.Wild-type (WT), NLRP3-deficient (Nlrp3), and Asc mice were infected with B pseudomallei. In additional experiments, infected WT mice were treated with an anti-IL-1β antibody. After 24, 48, and 72 hours (h) mice were sacrificed and organs were harvested. Furthermore, survival studies were performed.

Results: Patients with melioidosis exhibited lower mRNA levels of caspase-1, NLRP3, and ASC. Bacterial dissemination and organ damage were increased in B pseudomallei-infected Nlrp3 and Asc mice, together with a reduced pulmonary cell influx. Anti-IL-1β treatment of B pseudomallei challenged mice resulted in strongly reduced bacterial counts, organ damage, and pulmonary granulocyte influx together with reduced mortality. Postponement of anti-IL-1β treatment for 24 h postinfection still protected mice during melioidosis.

Conclusion: Expression of caspase-1, NLRP3, and ASC is altered in melioidosis patients. In mice, both NLRP3 and ASC contribute to the host defense against melioidosis. Anti-IL-1β treatment protects mice against B pseudomallei infection and might be a novel treatment strategy in melioidosis.
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http://dx.doi.org/10.1097/SHK.0000000000000625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5058638PMC
November 2016
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