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BACKGROUND: Compelling evidence has shown cardiac involvement in COVID-19 patients. However, the overall majority of these studies use data obtained during the first wave of the pandemic, while recently differences have been reported in disease course and mortality between first- and second wave COVID-19 patients. The aim of this study was to analyze and compare cardiac pathology between first- and second wave COVID-19 patients.METHODS: Autopsied hearts from first- (n = 15) and second wave (n = 10) COVID-19 patients and from 18 non-COVID-19 control patients were (immuno)histochemically analyzed. CD45+ leukocyte, CD68+ macrophage and CD3+ T lymphocyte infiltration, cardiomyocyte necrosis and microvascular thrombosis were quantified. In addition, the procoagulant factors Tissue Factor (TF), Factor VII (FVII), Factor XII (FXII), the anticoagulant protein Dipeptidyl Peptidase 4 (DPP4) and the advanced glycation end-product N(ε)-Carboxymethyllysine (CML), as markers of microvascular thrombogenicity and dysfunction, were quantified.RESULTS: Cardiac inflammation was significantly decreased in second wave compared to first wave COVID-19 patients, predominantly related to a decrease in infiltrated lymphocytes and the occurrence of lymphocytic myocarditis. This was accompanied by significant decreases in cardiomyocyte injury and microvascular thrombosis. Moreover, microvascular deposits of FVII and CML were significantly lower in second wave compared to first wave COVID-19 patients.CONCLUSIONS: These results show that in our cohort of fatal COVID-19 cases cardiac inflammation, cardiomyocyte injury and microvascular thrombogenicity were markedly decreased in second wave compared to first wave patients. This may reflect advances in COVID-19 treatment related to an increased use of steroids in the second COVID-19 wave.

OBJECTIVE: To perform a scoping review of imaging-based machine-learning models to predict clinical outcomes and identify biomarkers in patients with PDAC. SUMMARY OF BACKGROUND DATA: Patients with PDAC could benefit from better selection for systemic and surgical therapy. Imaging-based machine-learning models may improve treatment selection. METHODS: A scoping review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses-scoping review guidelines in the PubMed and Embase databases (inception-October 2020). The review protocol was prospectively registered (open science framework registration: m4cyx). Included were studies on imaging-based machine-learning models for predicting clinical outcomes and identifying biomarkers for PDAC. The primary outcome was model performance. An area under the curve (AUC) of ≥0.75, or a P-value of ≤0.05, was considered adequate model performance. Methodological study quality was assessed using the modified radiomics quality score. RESULTS: After screening 1619 studies, 25 studies with 2305 patients fulfilled the eligibility criteria. All but 1 study was published in 2019 and 2020. Overall, 23/25 studies created models using radiomics features, 1 study quantified vascular invasion on computed tomography, and one used histopathological data. Nine models predicted clinical outcomes with AUC measures of 0.78-0.95, and C-indices of 0.65-0.76. Seventeen models identified biomarkers with AUC measures of 0.68-0.95. Adequate model performance was reported in 23/25 studies. The methodological quality of the included studies was suboptimal, with a median modified radiomics quality score score of 7/36. CONCLUSIONS: The use of imaging-based machine-learning models to predict clinical outcomes and identify biomarkers in patients with PDAC is increasingly rapidly. Although these models mostly have good performance scores, their methodological quality should be improved.

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 × 10−7). 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.

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 trans and v e, ADC, and IVIM D correlated with collagen fraction. DCE k ep 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 ep 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, Patient stratification based on biological variation in pancreatic ductal adenocarcinoma subtypes could help to improve clinical outcome. In this study, quantitative magnetic resonance imaging (MRI) parameters were directly correlated to immunohistochemistry‐derived tissue characteristics. Furthermore, based on MRI parameters a subgroup of patients with significantly better prognosis could be identified, indicating that patient stratification based on MRI parameters is feasible.

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&amp, 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.

Humans with dysfunctional Bruton's tyrosine kinase (Btk) are highly susceptible to bacterial infections. Compelling evidence indicates that Btk is essential for B cell-mediated immunity, whereas its role in myeloid cell-mediated immunity against infections is controversial. In this study, we determined the contribution of Btk in B cells and neutrophils to host defense against the extracellular bacterial pathogen Klebsiella pneumoniae, a common cause of pulmonary infections and sepsis. Btk-/- mice were highly susceptible to Klebsiella infection, which was not reversed by Btk re-expression in B cells and restoration of natural antibody levels. Neutrophil-specific Btk deficiency impaired host defense against Klebsiella to a similar extent as complete Btk deficiency. Neutrophil-specific Btk deficiency abolished extracellular reactive oxygen species production in response to Klebsiella. These data indicate that expression of Btk in neutrophils is crucial, while in B cells, it is dispensable for in vivo host defense against K. pneumoniae.

The subthalamic nucleus (STN) is successfully used as a surgical target for deep brain stimulation in the treatment of movement disorders. Interestingly, the internal structure of the STN is still incompletely understood. The objective of the present study was to investigate three-dimensional (3D) immunoreactivity patterns for 12 individual protein markers for GABA-ergic, serotonergic, dopaminergic as well as glutamatergic signaling. We analyzed the immunoreactivity using optical densities and created a 3D reconstruction of seven postmortem human STNs. Quantitative modeling of the reconstructed 3D immunoreactivity patterns revealed that the applied protein markers show a gradient distribution in the STN. These gradients were predominantly organized along the ventromedial to dorsolateral axis of the STN. The results are of particular interest in view of the theoretical underpinning for surgical targeting, which is based on a tripartite distribution of cognitive, limbic and motor function in the STN. Electronic supplementary material The online version of this article (10.1007/s00429-019-01960-3) contains supplementary material, which is available to authorized users.

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.

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. Electronic supplementary material The online version of this article (10.1186/s10020-018-0069-7) contains supplementary material, which is available to authorized users.

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 Streptococcus pneumoniae and Klebsiella pneumoniae. Using the Cre-loxP system, male platelet-specific Btk-deficient mice (PF4creBtkfl/Y) were created. Similar to platelets from total Btk-deficient mice, platelets from PF4creBtkfl/Y mice showed abrogated aggregation and P-selectin expression when stimulated with the GPVI ligand cross-linked collagen-related peptide. Upon infection with S. pneumoniae, PF4creBtkfl/Y mice showed increased lung bleeding, but unimpaired anti-bacterial defence. During pneumosepsis evoked by K. pneumoniae, 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 P2Y12 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.

Immunotherapy is a new anti-cancer treatment option, showing promising results in clinical trials. To investigate potential immune biomarkers in esophageal adenocarcinoma (EAC), we explored immune landscape patterns in the tumor microenvironment before and after neoadjuvant chemoradiation (nCRT). Sections from matched pretreatment biopsies and post-nCRT resection specimens (n = 188) were stained for (1) programmed death-ligand 1 (PD-L1, CD274); (2) programmed cell death protein 1 (PD-1, CD279), forkhead box P3 (FOXP3), CD8, pan-cytokeratin multiplex; and (3) an MHC class I, II duplex. The densities of tumor-associated immune cells (TAICs) were calculated using digital image analyses and correlated to histopathological nCRT response [tumor regression grade (TRG)], survival, and post-nCRT immune patterns. PD-L1 positivity defined by a combined positive score of >1 was associated with a better response post-nCRT (TRG 1–3 versus 4, 5, p = 0.010). In addition, high combined mean densities of CD8 +, FOXP3 +, and PD-1 + TAICs in the tumor epithelium and stroma of biopsies were associated with a better response (TRG 1–3 versus 4, 5, p = 0.025 and p = 0.044, respectively). Heterogeneous TAIC density patterns were observed post-nCRT, with significantly higher CD8 + and PD-1 + TAIC mean densities compared with biopsies (both p = 0.000). Three immune landscape patterns were defined post-nCRT: ‘inflamed’, ‘invasive margin’, and ‘desert’, of which ‘inflamed’ was the most frequent (57%). Compared with matched biopsies, resection specimens with ‘inflamed’ tumors showed a significantly higher increase in CD8 + density compared with non-inflamed tumors post-nCRT (p = 0.000). In this cohort of EAC patients, higher TAIC densities in pretreatment biopsies were associated with response to nCRT. This warrants future research into the potential of the tumor-immune landscape for patient stratification and novel (immune) therapeutic strategies.

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.

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.

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.

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.

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 Klebsiella pneumoniae 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 Klebsiella 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.

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

Background: Barrett's esophagus (BE) is a precursor lesion of esophageal adenocarcinoma and may progress from non-dysplastic through low-grade dysplasia (LGD) to high-grade dysplasia (HGD) and cancer. Grading BE is of crucial prognostic value and is currently based on the subjective evaluation of biopsies. This study aims to investigate the potential of machine learning (ML) using spatially resolved molecular data from mass spectrometry imaging (MSI) and histological data from microscopic hematoxylin and eosin (H&E)-stained imaging for computer-aided diagnosis and prognosis of BE. Methods: Biopsies from 57 patients were considered, divided into non-dysplastic (n = 15), LGD non-progressive (n = 14), LGD progressive (n = 14), and HGD (n = 14). MSI experiments were conducted at 50 x 50 mu m spatial resolution per pixel corresponding to a tile size of 96x96 pixels in the co-registered H&E images, making a total of 144,823 tiles for the whole dataset. Results: ML models were trained to distinguish epithelial tissue from stroma with area-under-the-curve (AUC) values of 0.89 (MSI) and 0.95 (H&E)) and dysplastic grade (AUC of 0.97 (MSI) and 0.85 (H&E)) on a tile level, and low-grade progressors from non-progressors on a patient level (accuracies of 0.72 (MSI) and 0.48 (H&E)). Conclusions: In summary, while the H&E-based classifier was best at distinguishing tissue types, the MSI-based model was more accurate at distinguishing dysplastic grades and patients at progression risk, which demonstrates the complementarity of both approaches. Data are available via ProteomeXchange with identifier PXD028949.

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

SummaryBacterial 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.Supplementary Material to this article is available online at www.thrombosis-online.com.

Highlights • Aspirates allow to study cell death and proliferation in relation to thrombus age. • Apoptosis, etosis and proliferation are present in all stages of thrombus evolution. • Etosis is the only marker that associates significantly with older age of thrombus. • Etosis could serve potentially as tissue marker for thrombus aging and progression., 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

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. Electronic supplementary material The online version of this article (10.1007/s00428-019-02577-x) contains supplementary material, which is available to authorized users.

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., peer-reviewed

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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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.

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.

Supplemental Digital Content is available in the text, 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.

Pseudomonas aeruginosa is a flagellated pathogen frequently causing pneumonia in hospitalized patients and sufferers of chronic lung disease. Here we investigated the role of the common Toll-like receptor (TLR) adaptor myeloid differentiation factor (MyD)88 in myeloid vs. lung epithelial cells in clearance of P. aeruginosa from the airways. Mice deficient for MyD88 in lung epithelial cells (Sftpccre -MyD88-lox mice) or myeloid cells (LysMcre- MyD88-lox mice) and bone marrow chimeric mice deficient for TLR5 (the receptor recognizing Pseudomonas flagellin) in either parenchymal or hematopoietic cells were infected with P. aeruginosa via the airways. Sftpccre -MyD88-lox mice demonstrated a reduced influx of neutrophils into the bronchoalveolar space and an impaired early antibacterial defense after infection with P. aeruginosa, whereas the response of LysMcre- MyD88-lox mice did not differ from control mice. The immune-enhancing role of epithelial MyD88 was dependent on recognition of pathogen-derived flagellin by epithelial TLR5, as demonstrated by an unaltered clearance of mutant P. aeruginosa lacking flagellin from the lungs of Sftpccre -MyD88-lox mice and an impaired bacterial clearance in bone marrow chimeric mice lacking TLR5 in parenchymal cells. These data indicate that early clearance of P. aeruginosa from the airways is dependent on flagellin-TLR5-MyD88-dependent signaling in respiratory epithelial cells.

Background Remodeling of extracellular matrix is a key process during wound healing, which is strictly regulated by matrix metalloproteinases (MMPs) and their tissue inhibitors [tissue inhibitors of metalloproteinases (TIMPs)]. In this study, we evaluated intrathrombotic MMPs and TIMPs and their cellular origin during thrombus evolution after disruption of coronary atherosclerotic plaque. Materials and methods Thrombectomy materials ( N =120) obtained from patients with acute myocardial infarction were histologically classified in three groups based on thrombus age: fresh ( 5days) thrombi; materials showing a heterogeneous composition were classified according to oldest part. Presence and cellular origin of MMPs (MMP-1, MMP-2, MMP-8, MMP-9, and MMP-14) and TIMPs (TIMP-1, TIMP-2, and TIMP-3) was evaluated with immunostains (double) and with polymerase chain reaction. Results and conclusion MMPs and TIMPs were present in all the thrombectomy samples. A distinct temporal change in extent and cellular origin of MMPs and TIMPs during thrombus evolution was observed. In the early (fresh and lytic) stages of thrombus, high numbers of neutrophilic granulocytes occupy the thrombus mass and produce large amounts of MMPs and TIMPs. However, with progression of thrombus evolution (organizing stage) and diminishment of neutrophil granulocytes, there is disappearance of MMP-8 and MMP-9, steep decline of MMP-1 and TIMP-2, and progressive decrease of TIMP-3. In contrast, intrathrombotic MMP-2 and MMP-14 are present at a constant high level during the entire process of thrombus evolution. These temporal changes indicate a complex time-dependent function of MMPs, which are largely granulocyte derived, in the healing process of thrombus after plaque disruption.

Background and aimsIntracranial atherosclerosis is one of the main causes of ischemic stroke. However, the characteristics of intracranial arteries and atherosclerosis have rarely been studied. Therefore, we systematically investigated atherosclerotic changes in all arteries of the Circle of Willis (CoW).MethodsSixty-seven CoWs obtained at autopsy from randomly chosen hospital patients (mean age, 67.3 ± 12.5 years), of which a total of 1220 segments were collected from 22 sites. Atherosclerotic plaques were classified according to the revised American Heart Association classification and were related to local vessel characteristics, such as the presence of an external and internal elastic lamina and the elastic fibre density of the media.Results181 out of the 1220 segments had advanced plaques (15%), which were mainly observed in large arteries such as the internal carotid, middle cerebral, basilar and vertebral artery. Only 11 out of 1220 segments (1%) showed complicated plaques (p

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.

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 (p

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

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.