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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.

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.

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.

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.

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

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

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.

Human respiratory syncytial virus (RSV) is the most important cause of severe lower respiratory tract disease (LRTD) in young children worldwide. Extensive neutrophil accumulation in the lungs and occlusion of small airways by DNA-rich mucus plugs are characteristic features of severe RSV-LRTD. Activated neutrophils can release neutrophil extracellular traps (NETs), extracellular networks of DNA covered with antimicrobial proteins, as part of the first-line defence against pathogens. NETs can trap and eliminate microbes; however, abundant NET formation may also contribute to airway occlusion. In this study, we investigated whether NETs are induced by RSV and explored their potential anti-viral effect in vitro. Second, we studied NET formation in vivo during severe RSV-LRTD in infants and bovine RSV-LRTD in calves, by examining bronchoalveolar lavage fluid and lung tissue sections, respectively. NETs were visualized in lung cytology and tissue samples by DNA and immunostaining, using antibodies against citrullinated histone H3, elastase and myeloperoxidase. RSV was able to induce NET formation by human neutrophils in vitro. Furthermore, NETs were able to capture RSV, thereby precluding binding of viral particles to target cells and preventing infection. Evidence for the formation of NETs in the airways and lungs was confirmed in children with severe RSV-LRTD. Detailed histopathological examination of calves with RSV-LRTD showed extensive NET formation in dense plugs occluding the airways, either with or without captured viral antigen. Together, these results suggest that, although NETs trap viral particles, their exaggerated formation during severe RSV-LRTD contributes to airway obstruction.

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.

Streptococcus pneumoniae is a common cause of pneumonia and sepsis. Toll-like receptors (TLRs) play a pivotal role in the host defense against infection. In this study, we sought to determine the role of single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR a.k.a. TIR8), a negative regulator of TLR signaling, in pneumococcal pneumonia and sepsis. Wild-type and SIGIRR-deficient (sigirr-/-) mice were infected intranasally (to induce pneumonia) or intravenously (to induce primary sepsis) with S. pneumoniae and euthanized after 6, 24, or 48 h for analyses. Additionally, survival studies were performed. sigirr-/- mice showed delayed mortality during lethal pneumococcal pneumonia. Accordingly, sigirr-/- mice displayed lower bacterial loads in lungs and less dissemination of the infection 24 h after the induction of pneumonia. SIGIRR deficiency was associated with increased interstitial and perivascular inflammation in lung tissue early after infection, with no impact on neutrophil recruitment or cytokine production. sigirr-/- mice also demonstrated reduced bacterial burdens at multiple body sites during S. pneumoniae sepsis. sigirr-/- alveolar macrophages and neutrophils exhibited an increased capacity to phagocytose viable pneumococci. These results suggest that SIGIRR impairs the antibacterial host defense during pneumonia and sepsis caused by S. pneumoniae.

The interplay between inflammation and blood coagulation is an essential part of host defense during severe pneumosepsis. Melioidosis, instigated by the Gram-negative bacterium Burkholderia pseudomallei, is a frequent cause of pneumosepsis in Southeast Asia. Patients with severe pneumosepsis, including melioidosis, have decreased circulating levels of protein C. Activated protein C has anticoagulant and anti-inflammatory properties. In this study, we aimed to investigate the effect of sustained elevated activated protein C levels on the host response during melioidosis. Animal study. University research laboratory. Wild type and activated protein C overexpressing C57BL/6 mice. Mice were intranasally infected with viable B. pseudomallei and killed after 24, 48, or 72 hours for harvesting of lungs, liver, spleen, and blood. Additionally, survival studies were performed. Plasma activated protein C concentrations in activated protein C overexpressing mice (median 18.1 ng/mL) were in the same range as previously measured in patients treated with recombinant human activated protein C. Activated protein C overexpressing mice demonstrated enhanced susceptibility to B. pseudomallei infection compared with wild type mice as evidenced by a strongly increased mortality accompanied by enhanced bacterial loads in the lungs, blood, and distant organs 48 hours after infection. Additionally, at this time point, activated protein C overexpressing mice showed elevated levels of proinflammatory cytokines in lungs and plasma, together with increased pulmonary histopathology scores and neutrophil influx. At 72 hours postinfection, decreased levels of thrombin-antithrombin complexes, reflecting inhibition of coagulation, were measured in lungs of activated protein C overexpressing mice. Constitutively enhanced expression of activated protein C impairs host defense during severe Gram-negative sepsis caused by B. pseudomallei

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.

SummaryNeutrophils are important cellular sources of interleukin (IL) 17A and –F. Moreover, upon activation neutrophils are able to excrete chromatin embedded with components from their cytoplasmic granules to form ‘neutrophil extracellular traps’ (NETs). Recent studies suggested that NETs contribute to thrombosis by promoting fibrin deposition and platelet aggregation. IL17A may also promote thrombosis by enhancing platelet aggregation. In the present study we investigated the presence of neutrophils, NETs and IL17A and –F in coronary thrombosuction specimens obtained from patients after acute myocardial infarction. Neutrophils and NETs were identified using histochemical (H&E, Feulgen procedure) and immunohistochemical stainings (Histone H1, myeloperoxidase, neutrophil elastase) in 15 fresh, 15 lytic and 15 organised thrombi. The presence and distribution of IL17A and –F was studied using (immuno)histochemical double staining and spectral image analysis, rtPCR and Western blot. High numbers of neutrophils are present (10–30% of the thrombus mass) in fresh and lytic, but not in organized thrombus. NETs were frequently observed in fresh (4/15) and lytic (12/15), but never in organised thrombus specimens. Double staining combining the Feulgen reaction with Histone- H1, MPO or neutrophil elastase confirmed colocalisation with DNA. Cytoplasmatic IL17A/F staining was found in the majority of the neutrophils, extracellularly and in NETs. Western blotting confirmed the presence of IL17A and IL17F in thrombus specimens. In conclusion, a large burden of neutrophils, neutrophil extracellular traps and IL17A and –F are important constituents of fresh and lytic thrombus after acute myocardial infarction. The specific colocalisation of these indicates a role during thrombus stabilisation and growth.

Item does not contain fulltext Polyomavirus-associated nephropathy (PVAN) occurs in a significant percentage of renal transplant recipients, with BK virus reactivation as the main causative agent. PVAN leads to tubular damage and may result in allograft loss. In this study, we analyzed the antiviral immune response in PVAN. Transcription of the proinflammatory cytokine interleukin-18 (IL-18) was significantly higher in PVAN biopsies compared with T cell-mediated rejection (TCMR) (1.42 +/- 0.20 and 0.69 +/- 0.10, respectively; *P = 0.0021). Tubular expression of IL-18 was significantly increased in PVAN compared with TCMR (2.00 +/- 0.24 and 1.333 +/- 0.13, respectively; *P = 0.028). In contrast, in TCMR, IL-18 was expressed predominantly by CD163-positive macrophages. These data suggest that the antiviral immune response in PVAN is partly coordinated by the tubular epithelium, whereas in TCMR, this may be controlled by inflammatory cells.

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.

Item does not contain fulltext Although both polyomavirus infection and T cell-mediated rejection (TCMR) are characterized by tubulointerstitial inflammation in the renal allograft, these conditions are treated with opposing therapeutic regimens. To gain more insight into the differences between antiviral and alloimmune responses, we performed a case-control study, in which we immunophenotyped the inflammatory infiltrates in renal biopsy specimens with BK polyomavirus-associated nephropathy (BKPyVAN) and specimens with TCMR. Compared with TCMR, BKPyVAN was diagnosed later after transplantation; therefore, BKPyVAN specimens showed more chronic damage than TCMR specimens showed. However, TCMR and BKPyVAN specimens had comparable levels of tubulointerstitial inflammation. Adjustment for confounders in various multivariable models revealed more blood dendritic cell antigen-1(+) (BDCA-1(+)) myeloid dendritic cells (mDCs) present during BKPyVAN (odds ratio, 2.31; 95% confidence interval, 1.03 to 5.16; P=0.04) than during TCMR. Double immunostaining for SV40 and BDCA-1 showed that, during BKPyVAN, BDCA-1(+) mDCs localized in proximity to the polyomavirus-infected tubular epithelial cells. We ensured that time of biopsy after transplantation was not a confounding factor by including additional specimens with late TCMR and protocol biopsy specimens matched for biopsy time. These additional specimens showed amounts of BDCA-1(+) mDCs comparable with amounts in the early TCMR specimens. These results suggest that BDCA-1(+) mDCs, known to be involved in the antiviral immune response during various viral infections, might have a pivotal role during BKPyVAN infection in the grafted kidney.

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.

Determination of hepatocyte proliferation activity is hampered by the presence of Ki67-positive non-parenchymal cells. We validated a multicolor immunohistochemical (IHC) approach using multispectral tissue and cell segmentation software. Portal vein branches to the cranial liver lobes of 10 rabbits were embolized, leading to atrophy of the cranial lobes and hyperplasia of the caudal lobes. Slides from cranial and caudal lobes ( n=20) were double-stained (CK8+18 and Ki67) and triple-stained (CK8+18, Ki67, and CD31). The Ki67 proliferation index was calculated using automated tissue and cell segmentation software and compared with manual counting by two independent observers. A substantial variation was seen in the number of Ki67-positive hepatocytes in the different specimens in both double and triple staining (range, 0–50). Correlation coefficients between manual counting and the digital analysis were 0.76 for observer 1 ( p2 = 0.91 for observer 1 and R2 = 0.89 for observer 2, p

Summary Purpose: An International League Against Epilepsy (ILAE) consensus classification system for focal cortical dysplasias (FCDs) has been published in 2011 specifying clinicopathologic FCD variants. The aim of the present work was to microscopically assess interobserver agreement and intraobserver reproducibility for FCD categories among an international group of neuropathologists with different levels of experience and access to epilepsy surgery tissue. Methods: Surgical FCD specimens covering a broad histopathology spectrum were retrieved from 22 patients with epilepsy. Three surgical nonepilepsy specimens served as controls. A total of 188 slides with routine or immunohistochemical stainings were digitalized with a slide scanner to allow Internet-based microscopy review. Nine experienced neuropathologists were invited to review these cases twice at a time gap of 3 months and different orders of case presentation. The 2011 ILAE FCD consensus classification served as instruction. Kappa analysis was calculated to estimate interobserver and intraobserver agreement levels. In a third evaluation round, 21 additional neuropathologists with different experience and access to epilepsy surgery reviewed the same case series. Key Findings: Interobserver agreement was good (I° = 0.6360), with 84% consensus of diagnoses during the first evaluation (21 of 25 cases). Kappa values increased to 0.6532 after reevaluation, and consensus was obtained in 24 (96%) of 25 cases. Overall intraobserver reproducibility was also good (I° = 0.7824, ranging from 0.4991 to 1.000). Fewest changes in the classification were made in the FCD type II group (2.2% of 225 original diagnoses), whereas the majority of changes occurred in FCD type III (13.7% of 225 original diagnoses). In the third evaluation round, interobserver agreement was reflected by the level of experience of each neuropathologist, with I° values ranging from moderate (0.5056; high level of experience >40 cases/year) to low (0.3265; low level of experience

Li X, van der Meer J J, van der Loos C M, Ploegmakers H J P, de Boer O J, de Winter R J & van der Wal A C (2012) Histopathology 61, 88–97 Microvascular endoglin (CD105) expression correlates with tissue markers for atherosclerotic plaque vulnerability in an ageing population with multivessel coronary artery disease Aims: Vulnerable atherosclerotic plaques are lesions with a high propensity to develop plaque disruption and superimposed thrombosis. No systematic studies have been carried out on tissue markers for plaque vulnerability throughout the entire coronary artery system at the end stages of coronary atherosclerosis. Methods and results: Nine autopsied patients (mean age 77 years) with angiographically severe trivascular coronary atherosclerosis were selected for this study. All visible lesions in postmortem coronary angiograms (n = 125) were histologically and immunohistochemically screened for the presence of intraplaque haemorrhages (activated) microvessels and inflammatory infiltrates. Intraplaque haemorrhages were observed in 76/125 plaques (61%). Chronic inflammation was found superficially in 59/125 plaques (47%) and deeply inside the plaque tissue in 103/125 plaques (83%). Microvessels were found in 100/125 lesions (80%), of which 58% showed endothelial expression of the vascular activation marker CD105. Moreover, microvascular CD105 positivity correlated positively with plaque haemorrhage and deeply seated plaque inflammation. Conclusions: Plaque inflammation and haemorrhages can be found at a high frequency throughout the coronary artery system of elderly patients with multivessel coronary atherosclerosis. Microvascular expression of endoglin (CD105), which correlates positively with both of these features of plaque vulnerability, can serve as a marker of the risk of developing coronary thrombotic complications.

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.

Atherosclerosis as well as the subsequent progression towards cardiovascular events are considered to, at least partially, be a consequence of chronic inflammatory activity. Therefore, we decided to evaluate the impact of short-term immunosuppressive treatment on plaque characteristics in patients with symptomatic carotid artery stenosis. Twenty-one patients were randomized to receive either 1000 mg. Mycophenolate mofetil (MMF) BD or placebo for at least 2 weeks prior to undergoing carotid endarterectomy (CEA). The serial sections of the CEA specimens were immunostained for activated T-cells (CD3(+)CD69(+)), regulatory T-cells (CD3(+)FOXP3(+)) and macrophages (CD68). In addition, gene expression pro. ling was performed by Illumina gene-array. Immunostaining revealed a reduction of activated T-cells in nine MMF-treated patients compared to 11 placebo-treated control patients (19.7% vs. 28.1%; p

Background and Purpose— Evidence is accumulating that infection with influenza A virus contributes to atherothrombotic disease. Vaccination against influenza decreases the risk of atherosclerotic syndromes, indicating that inflammatory mechanisms may be involved. We tested the hypothesis that influenza A virus–specific T cells contribute to atherosclerotic plaque inflammation, which mediates the onset of plaque rupture. Methods— T-cell cultures were generated from atherosclerotic segments and peripheral blood of 30 patients with symptomatic carotid artery disease. The response of plaque and peripheral blood T cells to influenza A virus was analyzed and expressed as a stimulation index (SI). Selective outgrowth of intraplaque influenza A–specific T cells was calculated by the ratio of plaque T cell SI and peripheral blood T cell SI for each patient. Accordingly, the patients were categorized as high- (SI ratio ≥5), intermediate- (5 Results— High proliferative responses of plaque-derived T cells to influenza A virus were frequently observed. Among the 30 patients, 5 were categorized as high responders, 10 were intermediate responders, and 15 were nonresponders. Live influenza A virus could not be detected in the atherosclerotic plaques by polymerase chain reaction. Conclusions— Selective outgrowth of influenza A virus–specific T cells occurs within the microenvironment of human atherosclerotic plaques. Influenza virus–derived antigens or alternatively, mimicry antigens, appear to be potential candidates for triggering or sustaining plaque inflammation, which eventually leads to symptomatic plaque complications.

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).