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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.

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.

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.

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.

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.

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.

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 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

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.

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.

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.

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

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.

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.

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 Klebsiella pneumoniae 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 Klebsiella 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, Klebsiella 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.

Cardiac allograft vasculopathy is regarded as a progressive and diffuse intimal hyperplastic lesion of arteries and veins that leads to insidious vessel narrowing and to allograft ischemic disease, such as acute myocardial infarction or sudden cardiac death. The coronary lesions in transplanted hearts are considered as a particular type of arteriosclerosis with many similarities but also significant differences compared to native coronary atherosclerosis. It is particularly difficult for pathologists to systematically classify the lesions and to elucidate their origins, since over time, the allograft immune responses cause vascular pathology characterized by not only the onset of de novo fibrocellular lesions but also remodeling of already-existing native atherosclerotic lesions in the donor heart. Intraplaque hemorrhages, which result from newly formed leaky microvessels, may cause rapid increase of stenosis and generate a substrate for plaque destabilization. Comparing cardiac allograft vasculopathy from explanted hearts at autopsy with native coronary atherosclerosis from hearts removed at transplantation has revealed that ongoing intraplaque hemorrhages are also an important feature of cardiac allograft vasculopathy and may be important factors in the rapid progression of cardiac allograft vasculopathy.

BACKGROUND Streptococcus pneumoniae is the most common causative pathogen in community-acquired pneumonia. Mast cells (MCs) are located mainly at the host-environment interface where they function as sentinels. OBJECTIVE Our goal was to study the role of MCs during pneumonia caused by S. pneumoniae. METHODS Lung tissue of patients who had died from pneumococcal pneumonia or a nonpulmonary cause was stained for MCs and tryptase. Wild-type (WT) and MC-deficient (Kit(W-sh/W-sh)) mice were observed or sacrificed after induction of pneumonia by intranasal inoculation of S. pneumoniae. In separate experiments, WT mice were treated with doxantrazole or cromoglycate, which are MC stabilizing agents. RESULTS The constitutive presence of tryptase-positive MCs was reduced in affected lungs from pneumonia patients. Kit(W-sh/W-sh) mice showed a prolonged survival during the first few days after median lethal dose (LD)100 and LD50 infection, while overall mortality did not differ from that in WT mice. Relative to WT mice, Kit(W-sh/W-sh) mice showed reduced bacterial counts with less bacterial dissemination to distant organs and less inflammation. Neither doxantrazole nor cromoglycate influenced antibacterial defense or inflammatory responses after airway infection with S. pneumoniae. CONCLUSIONS MCs exhibit an unfavorable role in host defense during pneumococcal pneumonia by a mechanism independent of degranulation.

Colon cancer is a clinically diverse disease. This heterogeneity makes it difficult to determine which patients will benefit most from adjuvant therapy and impedes the development of new targeted agents. More insight into the biological diversity of colon cancers, especially in relation to clinical features, is therefore needed. We demonstrate, using an unsupervised classification strategy involving over 1,100 individuals with colon cancer, that three main molecularly distinct subtypes can be recognized. Two subtypes have been previously identified and are well characterized (chromosomal-instable and microsatellite-instable cancers). The third subtype is largely microsatellite stable and contains relatively more CpG island methylator phenotype-positive carcinomas but cannot be identified on the basis of characteristic mutations. We provide evidence that this subtype relates to sessile-serrated adenomas, which show highly similar gene expression profiles, including upregulation of genes involved in matrix remodeling and epithelial-mesenchymal transition. The identification of this subtype is crucial, as it has a very unfavorable prognosis and, moreover, is refractory to epidermal growth factor receptor-targeted therapy.

The complex biology of asthma compels the use of more relevant human allergens, such as house dust mite (HDM), to improve the translation of animal models into human asthma. LPS exposure is associated with aggravations of asthma, but the mechanisms remain unclear. Here, we studied the effects of increasing LPS doses on HDM-evoked allergic lung inflammation. To this end, mice were intranasally sensitized and challenged with HDM with or without increasing doses of LPS (0.001-10 μg). LPS dose-dependently inhibited HDM-induced eosinophil recruitment into the lungs and mucus production in the airways. LPS attenuated the production of Th2 cytokines (IL-4, IL-5, IL-10, and IL-13) in HDM-challenged lungs, while enhancing the HDM-induced release of IL-17, IL-33, IFN-γ, and TNF-α. The shift toward a Th1 inflammatory response was further illustrated by predominant neutrophilic lung inflammation after LPS administration at higher doses. LPS did not influence HDM-induced plasma IgE concentrations. Although LPS did not significantly affect the activation of coagulation or complement in HDM-challenged lungs, it reduced HDM-initiated endothelial cell activation. This study is the first to provide insights into the effects of LPS in an allergic lung inflammation model making use of a clinically relevant allergen without a systemic adjuvant, revealing that LPS dose-dependently inhibits HDM-induced pulmonary Th2 responses.

Objective and design: We examined the role of IL-6 in the temporal development of cardiac ischemia–reperfusion injury employing a closed-chest I/R model. Materials/methods: Infarction, local and systemic inflammation, neutrophil infiltration, coagulation and ST elevation/resolution were compared between wild-type (WT) and IL-6-deficient (IL-6−/−) mice after 1 h ischemia and 0, ½, 3, and 24 h reperfusion. Results: IL-6 deficiency reduced infarct size at 3 h reperfusion (28.8 ± 4.5 % WT vs 17.6 ± 2.5 % IL-6−/−), which reduction persisted and remained similar at 24 h reperfusion (25.1 ± 3.0 % WT vs 14.6 ± 4.4 % IL-6−/−). Serum Amyloid A was reduced at 24 h reperfusion only (57.5 ± 4.9 WT vs 24.8 ± 5.6 ug/ml IL-6−/− mice). Cardiac cytokines (IL-6, IL-1β and TNFα) peaked at 3 h reperfusion, but IL-1β and TNFα levels were unaffected by IL-6 deficiency. Significant neutrophil influx was only detected at 24 h reperfusion and was similar for WT and IL-6−/−. Tissue factor peaked at 24 h reperfusion, whereas fibrin/fibrinogen peaked at 3 h reperfusion and was completely resolved at 24 h reperfusion; both coagulation factors were unaltered by IL-6 deficiency. Prolonged ST elevation was observed during ischemia that completely resolved for both genotypes at early reperfusion. Conclusions: The data suggest that, in the absence of major surgical intervention, IL-6 contributes to the development of infarct size in the early phase of reperfusion; this contribution did not depend on neutrophil influx, IL-1β and TNFα, tissue factor and fibrin.

Objectives: We describe a 62-year-old woman with a 15-year history of a plasma cell dyscrasia (monoclonal IgGκ), manifested by type I cryoglobulinemia and dermal vasculitis. Methods: In addition to the clinical examinations, light microscopy with immunohistochemistry, sequential multicolor immunohistochemistry, and electron microscopy were used to characterize the crystalline deposits. Results: At initial presentation and for a later flare, she was treated with cyclophosphamide and prednisolone with good clinical response. She had renal function decline, microscopic hematuria, and proteinuria. A renal biopsy specimen revealed the presence of glomerular macrophages and duplication of the capillary walls with cellular interposition. Glomerular cells contained abundant needle-shaped eosinophilic crystalline inclusions positive for κ light chain. Electron microscopy confirmed the presence of intracytoplasmatic crystalline structures in endothelial cells, podocytes, and macrophages but not in the tubular epithelium. Rituximab treatment was started. At follow-up (now up to 6 months), renal function remained stable. Conclusions: This patient displays a unique renal manifestation of type I cryoglobulinemia related to a plasma cell dyscrasia.

Background: Areas of microvascular proliferation have been observed in a subpopulation of symptomatic congenital vascular malformations later in life. We investigated whether this angiogenic response is followed by a stage of maturation. Methods: Resections of vascular malformations (n = 15), infantile hemangiomas (IHs) (n = 8) and pyogenic granulomas (PGs) (n = 5) were studied. Histopathologically, all lesions were screened for presence of foci of immature and/or mature microvessels. These areas were further studied immunohistochemically for differential expression of several angiogenic factors, cell cycle-dependent proteins, p53 and active caspase3. Immunostains were scored semiquantitatively. Results: Immature microvessel areas were present in five vascular malformations (all of the arteriovenous type), five IHs and five PGs; these lesions also contained transitions between immature and mature microvessels. Conglomerates of mature microvessels were found in 19 cases (6 vascular malformations, 5 PGs and 8 IHs). Expression of vascular endothelial growth factor-A, angiopoietin-1, Ki-67, p16 and p21/27 ratios were overall significantly lower in mature areas than in immature areas including those in vascular malformations. P53 and caspase3 expression was scarce in all lesions. Conclusions: Microvascular areas in vascular malformations appear to follow the same pattern of vascular proliferation and maturation as seen in other microvascular lesions of skin and soft tissue. Meijer-Jorna LB, van der Loos CM, Teeling P, de Boer OJ, Florquin S, van der Horst CMAM, van der Wal AC. Proliferation and maturation of microvessels in arteriovenous malformations expression patterns of angiogenic and cell cycle-dependent factors

AimsInflammation plays a crucial role in plaque vulnerability. Calcifications can be detected by means of in vivo imaging techniques. The study purpose is to assess a potential association between tissue localization of calcifications and the inflammatory biomarkers, C-reactive protein (CRP), osteopontin and lipoprotein-associated phospholipase A2 (Lp-PLA2), in plaque tissue of patients with acute myocardial infarction (AMI).Methods and resultsThrombectomy materials obtained from patients with electrocardiographically documented ST-segment elevation type of AMI (STEMI) were histologically screened for presence of thrombus, plaque tissues and calcifications. Size of calcifications was measured morphometrically, and their colocalization with the inflammatory biomarkers macrophages, CRP, osteopontin and Lp-PLA2 was assessed with immunostaining. A total of 171 samples containing plaque tissues were obtained from 562 thrombectomy procedures. Calcifications were observed in 67 (39%) plaque fragments, with diameters ranging from 4 to 170μm. Plaque tissues with calcifications contained more frequently extracellular CRP and intracellular CRP in macrophages than those without calcifications (85%, 59% vs. 64%, 32%, P=0.012 and 0.005 respectively). Similar results were obtained with osteopontin immunostaining (98%, 76% vs. 56%, 40%; P

Item does not contain fulltext Patients without a spleen are susceptible for overwhelming sepsis with Streptococcus pneumoniae. We investigated the relative contribution of the pneumococcal capsule in the reduced host defense after splenectomy. Sham-operated or splenectomized mice were inoculated with serotype 2 or 4 S. pneumoniae (D39, TIGR4) or the isogenic nonencapsulated mutants (D39Deltacps, TIGR4Deltacps). After splenectomy, intranasal infection with D39 resulted in increased mortality, increased bacterial dissemination and exaggerated systemic inflammation rather then altering inflammation in the lungs. Intravenous infection also resulted in enhanced mortality, bacterial growth and systemic inflammation after splenectomy. In contrast, the spleen did not contribute to host defense during infection with D39Deltacps. Similar observations were made for TIGR4: increased bacterial growth and inflammation after intravenous infection with wild-type, but not nonencapsulated bacteria in splenectomized mice. These results indicate that the capsule of S. pneumoniae is indeed responsible for increased vulnerability of asplenic mice to invasive pneumococcal disease.

Nine commercially available vascular endothelial growth factor (VEGF) antibodies were investigated for their ability to immunostain vascular malformations (VMs) with or without immature capillary proliferation. First, all antibodies were optimized for their performance in IHC, with placenta and colon adenocarcinoma as positive control tissues. Five antibodies were regarded as unfit for VEGF immunostaining based on poor immunostaining criteria. Subsequently, Western blot analysis using VEGF rabbit polyclonal antibody (Thermo RB-9031) revealed a clear 45-kDa band in tissue extracts from VMs with immature capillary proliferation and a high Ki67-labeling index, whereas tissue extracts from mature VMs without microvascular proliferation and no Ki67-labeling index demonstrated only a very weak 45-kDa band. In contrast, two VEGF antibodies, including the popular Santa Cruz A-20, revealed bands at 45 kDa of similar intensity in tissue extracts from both types of VMs. Staining characteristics of the 45-kDa band were reflected in the results obtained in IHC. (J Histochem Cytochem 58:109–118, 2010)

Background/Aims: Cyclosporin A (CsA) stimulates the development of fibrosis. We investigated whether CsA contributes to peritoneal alterations induced by long-term exposure to dialysis solutions. Methods: Ten rats received peritoneal infusion of dialysis solution and oral CsA for 8 weeks. Eight received only the dialysis solution (controls). Peritoneal function was assessed at 8 weeks followed by sacrifice. The number of vessels was counted, fibrosis was assessed and hydroxyproline was determined. PCR was performed for vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF) and transforming growth factor-β (TGF-β). Results: Histology revealed more fibrosis, hydroxyproline and vessels (thick walled) in CsA-exposed animals. Peritoneal transport was not different. The mRNA content of TGF-β, CTGF and VEGF was higher in CsA. Conclusion: CsA combined with exposure to dialysis solutions was associated with increased peritoneal fibrosis and angiogenesis.

Staphylococcus aureushas evolved as an important cause of pneumonia in both hospital and community settings. Staphylococcal lung infection can lead to overwhelming pulmonary inflammation. During infection, neutrophils release complexes of myeloid-related protein (MRP)8 and MRP14 (MRP8/14). MRP8/14 has been shown to exert pro-inflammatory and chemotactic activity, and to assist in the killing ofS. aureus. In the current study we sought to determine the role of MRP8/14 in the host response duringS. aureuspneumonia.Pneumonia was induced in wildtype and MRP14-deficient mice (mice unable to form MRP8/14) by intranasal inoculation of 1×107 CFU ofS. aureusUSA300. Mice were sacrificed at 6, 24, 48 or 72 h after infection for analyses.S. aureuspneumonia was associated with a strong rise in MRP8/14 in bronchoalveolar lavage fluid and lung tissue. Surprisingly, MRP14 deficiency had a limited effect on bacterial clearance and was associated with increased cytokine levels in bronchoalveolar lavage fluid and aggravated lung histopathology.MRP14 deficiency in addition was associated with a diminished transmigration of neutrophils into bronchoalveolar lavage fluid at late time-points after infection together with reduced release of nucleosomes.MRP8/14 serves in an unexpected protective role for the lung in staphylococcal pneumonia.

Inflammation and thrombosis are key events in the long-lasting sequence of atherosclerotic plaque initiation, plaque growth, and eventual onset of complications leading to clinically manifest disease. Recent cellular and molecular studies have indicated that inside the plaque tissue complex, proinflammatory and prothrombotic mechanisms are intimately associated, and tissue factor (TF) is one of the main proteins that may link both processes. It is therefore not surprising that TF expression appeared to be a prominent feature in various types of vulnerable atherosclerotic plaques (i.e., lesions that specifically predispose to the onset of symptomatic atherosclerotic disease).

A key event in neointima formation and atherogenesis is the migration of vascular smooth muscle cells (VSMCs) into the intima. This is controlled by cytokines and extracellular matix (ECM) components within the microenvironment of the diseased vessel wall. At present, these signals have only been partially identified. In this study, we demonstrate that Met, the receptor tyrosine kinase for hepatocyte growth factor (HGF), is expressed on VSMCs isolated from the intima of atherosclerotic plaques of carotid arteries. Stimulation with HGF led to activation of Met as well as to activation of P13-K, PKB/Akt, MEK, and the MAP kinases Erk1 and -2. Moreover, HGF induced lamellipodia formation, a characteristic feature of motile cells, and promoted VSMC migration across fibronectin-coated filters. The HGF-induced cell migration was mediated by beta1 integrins and required P13-K activation. Our results suggest a role for the HGF-Met signaling pathway in the pathogenesis of atherosclerosis and restenosis. (C) 2002 Elsevier Science (USA). All rights reserved

Background During pneumonia, inflammation and coagulation are activated as part of anti-bacterial host defense. Activated protein C (APC) has anticoagulant and anti-inflammatory properties and until recently was a registered drug for the treatment of severe sepsis. Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia. Methods We aimed to investigate the effect of high APC levels during experimental pneumococcal pneumonia. Wild type (WT) and APC overexpressing (APChigh)-mice were intranasally infected with S. pneumoniae and sacrificed after 6, 24 or 48 hours, or followed in a survival study. Results In comparison to WT mice, APChigh-mice showed decreased bacterial dissemination to liver and spleen, while no differences in bacterial loads were detected at the primary site of infection. Although no differences in the extent of lung histopathology were seen, APChigh-mice showed a significantly decreased recruitment of neutrophils into lung tissue and bronchoalveolar lavage fluid. Activation of coagulation was not altered in APChigh-mice. No differences in survival were observed between WT and APChigh-mice (P =0.06). Conclusion APC overexpression improves host defense during experimental pneumococcal pneumonia. This knowledge may add to a better understanding of the regulation of the inflammatory and procoagulant responses during severe Gram-positive pneumonia. Electronic supplementary material The online version of this article (doi:10.1186/s12879-014-0559-3) contains supplementary material, which is available to authorized users.

Intraplaque hemorrhage (IPH) is a crucial factor in progression and destabilization of an atherosclerotic plaque. Anti-thromboembolic drugs are widely used as prophylactic treatment against arterial and venous thrombotic diseases, but a major complication is bleeding. We investigated the association between exposure to anti-thromboembolic therapy and IPH in postmortem coronary arteries. Coronary arteries with postmortem angiographically confirmed extensive atherosclerosis were obtained at autopsy from patients who had received oral anticoagulants (n = 10), platelet aggregation inhibitors (n = 10), or no anti-thrombotic drugs (n = 10) before death. Coronary arteries were cut at 3-mm interval, and all plaque-containing segments were immunohistochemically screened for IPH and microvessels. These data were related to overall plaque composition and the use of anti-thromboembolic therapies. IPH was found in 483 out of 904 (53 %) coronary segments with advanced atherosclerotic plaques and more frequently in patients on oral anticoagulants (174/284, 61 %) than in patients on anti-platelets (198/376, 53 %) or without therapy (111/244, 46 %) (P = 0.02 and P = 0.001, respectively). Also, intraplaque microvascular leakage was more frequently observed in patients on anticoagulants than in non-treated patients (P = 0.03). Finally, the IPH appeared to be larger in plaques of patients on anticoagulant treatment (P

Accumulating evidence shows that protease-activated receptor-1 (PAR-1) plays an important role in the development of fibrosis, including lung fibrosis. However, whether PAR-1 also plays a role in the development of skin fibrosis remains elusive. The aim of this study was to determine the role of PAR-1 in the development of skin fibrosis. To explore possible mechanisms by which PAR-1 could play a role, human dermal fibroblasts and keratinocytes were stimulated with specific PAR-1 agonists or antagonists. To investigate the role of PAR-1 in skin fibrosis, we subjected wild-type and PAR-1-deficient mice to a model of bleomycin-induced skin fibrosis. PAR-1 activation leads to increased proliferation and extra cellular matrix (ECM) production, but not migration of human dermal fibroblasts (HDF) in vitro. Moreover, transforming growth factor (TGF)-beta production was increased in keratinocytes upon PAR-1 activation, but not in HDF. The loss of PAR-1 in vivo significantly attenuated bleomycin-induced skin fibrosis. The bleomycin-induced increase in dermal thickness and ECM production was reduced significantly in PAR-1-deficient mice compared with wild-type mice. Moreover, TGF-beta expression and the number of proliferating fibroblasts were reduced in PAR-1-deficient mice although the difference did not reach statistical significance. This study demonstrates that PAR-1 contributes to the development of skin fibrosis and we suggest that PAR-1 potentiates the fibrotic response mainly by inducing fibroblast proliferation and ECM production.