Graph-based description of tertiary lymphoid organs at single-cell level

Our aim is to complement observer-dependent approaches of immune cell evaluation in microscopy images with reproducible measures for spatial composition of lymphocytic infiltrates. Analyzing such patterns of inflammation is becoming increasingly important for therapeutic decisions, for example in transplantation medicine or cancer immunology. We developed a graph-based assessment of lymphocyte clustering in full whole slide images. Based on cell coordinates detected in the full image, a Delaunay triangulation and distance criteria are used to build neighborhood graphs. The composition of nodes and edges are used for classification, e.g. using a support vector machine. We describe the variability of these infiltrates on CD3/CD20 duplex staining in renal biopsies of long-term functioning allografts, in breast cancer cases, and in lung tissue of cystic fibrosis patients. The assessment includes automated cell detection, identification of regions of interest, and classification of lymphocytic clusters according to their degree of organization. We propose a neighborhood feature which considers the occurrence of edges with a certain type in the graph to distinguish between phenotypically different immune infiltrates. Our work addresses a medical need and provides a scalable framework that can be easily adjusted to the requirements of different research questions

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Comparison of IP-10 production induced by ex-vivo stimulation with Respiratory Syncytial Virus (RSV) and the TLR3 agonist Poly I: C in human and non-human primate lung tissues

Background: Human Respiratory Syncytial virus (HRSV) is a major cause of respiratory disease young children and is known as a risk factor for asthma. HRSV and the viral mimic Poly I:C activate toll‐like receptor 3 (TLR3), initiating an innate response with release of immune mediators, e.g. interferon gamma‐induced protein 10 (IP‐10). In this work, we aimed to perform an ex vivo HRSV infection in precision‐cut lung slices (PCLS) from human, rhesus, and cynomolgus macaques, comparing whenever possible the response with the viral surrogate poly I:C. Method: PCLS containing airways were prepared from lung sections of human, rhesus, and cynomolgus macaques. The slices were inoculated with HRSV‐A2 106 IU/mL, UV‐inactivated HRSV, or vehicle control for 48 hours. Macaque slices were also incubated with Poly I:C 100 μg/mL with and without the immunosuppressive dexamethasone 50 μg/mL. Viral replication, tissue viability, and immune response assays were assessed in supernatants, lysates, or slices. Results: The inoculum infectivity of 106 IU/mL as well the UV‐inactivation were confirmed by plaque‐assay on Hep‐2 cells Immunofluorescence staining using a FITC‐labeled anti‐RSV showed the presence of infected macrophages in PCLS, but not in mock infected samples. HRSV stimulation slightly decreased tissue viability, as seen by Live/DEAD staining and LDH assay. The viral infection increased IP‐10 production in PCLS of human, rhesus, and cynomolgus macaques, reaching respectively 13.3, 3.4, and 1.7 fold‐increase in comparison to the vehicle controls. Poly I:C stimulation caused IP10 response comparable to HRSV in rhesus and cynomolgus PCLS. The IP‐10 production ratio comparing HRSV/Poly I:C was 1.1 in rhesus and 0.9 in cynomolgus PCLS. Conclusion: HRSV infects ex vivo PCLS of human and non‐human primates, inducing the release of the pro‐inflammatory chemokine IP‐10. This response is comparable to the viral surrogate poly I:C. In the future, these systems can be used to further investigate host response to HRSV, especially in the context of asthma development

Cigarette smoke induced pathophysiological changes in the extracellular matrix but not inflammation as early events in fresh human lung tissue

Cigarette smoke (Cs) inhalation is a main reason to develop chronic obstructive pulmonary disease (COPD), characterised by degradation of alveoli, mucus hypersecretion, emphysema development and inflammation [1]. The pathophysiology of COPD is not well understood so the mechanisms that underlie various components of COPD need to be modelled in vitro, specifically using Cs. We assessed the pathophysiological effects of Cs and Cs condensate (Csc) on fresh human lung tissue. Human Precision Cut Lung Slices (PCLS) were exposed to Csc submerged or whole Cs in an Air-Liquid Interface using the in vitro exposure device P.R.I.T.® ExpoCube®. Cytotoxicity, release of cytokines and extracellular matrix (ECM) proteins were measured and gene expression analysis upon RNA isolation from PCLS was performed. PCLS exposed to Csc or Cs showed concentration-dependent loss of tissue viability. Gene expression analysis upon RNA isolated from lung tissue exposed to nontoxic doses of Csc of literature-based COPD-relevant genes indicate damage of epithelium by upregulation of genes involved in tissue injury (e.g. mmp) and specific metabolic activity (e.g. cyp1a1). Samples exposed to control substance LPS did not show increased expression in emphysema associated genes but in genes indicating inflammation (e.g. il-6). These data can be supported by increased production of proteins related to the extracellular matrix (e.g. MMP9, RAGE) and inhibited expression in pro-inflammatory cytokines after Cs exposure in an air-liquid culture. Supportive results were presented in BAL samples from COPD subjects showing markers of apoptosis [2]. These results indicate that early stages of Cs induced lung tissue changes are primarily not provoked by immunological processes but by apoptosis, increased specific metabolic activity and damage of the epithelium. [1] Hodge, S. et al. (2005). ERJ 25 (3), S. 447-454. [2] Shaykhiev, R.; Crystal, R. (2014). AnnalsATS 11 Suppl 5, S252-8

Comparative Analysis of Gene Expression in Fibroblastic Foci in Patients with Idiopathic Pulmonary Fibrosis and Pulmonary Sarcoidosis

Background: Fibroblastic foci (FF) are characteristic features of usual interstitial pneumonia (UIP)/idiopathic pulmonary fibrosis (IPF) and one cardinal feature thought to represent a key mechanism of pathogenesis. Hence, FF have a high impact on UIP/IPF diagnosis in current guidelines. However, although less frequent, these histomorphological hallmarks also occur in other fibrotic pulmonary diseases. Currently, there is therefore a gap in knowledge regarding the underlying molecular similarities and differences of FF in different disease entities. Methods: In this work, we analyzed the compartment-specific gene expression profiles of FF in IPF and sarcoidosis in order to elucidate similarities and differences as well as shared pathomechanisms. For this purpose, we used laser capture microdissection, mRNA and protein expression analysis. Biological pathway analysis was performed using two different gene expression databases. As control samples, we used healthy lung tissue that was donated but not used for lung transplantation. Results: Based on Holm Bonferroni corrected expression data, mRNA expression analysis revealed a significantly altered expression signature for 136 out of 760 genes compared to healthy controls while half of these showed a similar regulation in both groups. Immunostaining of selected markers from each group corroborated these results. However, when comparing all differentially expressed genes with the fdr-based expression data, only 2 of these genes were differentially expressed between sarcoidosis and IPF compared to controls, i.e., calcium transport protein 1 (CAT1) and SMAD specific E3 ubiquitin protein ligase 1 (SMURF1), both in the sarcoidosis group. Direct comparison of sarcoidosis and IPF did not show any differentially regulated genes independent from the statistical methodology. Biological pathway analysis revealed a number of fibrosis-related pathways pronounced in IPF without differences in the regulatory direction. Conclusions: These results demonstrate that FF of end-stage IPF and sarcoidosis lungs, although different in initiation, are similar in gene and protein expression, encouraging further studies on the use of antifibrotic agents in sarcoidosis

From macroscopy to ultrastructure : an integrative approach to pulmonary pathology

Pathology and radiology are complimentary tools, and their joint application is often crucial in obtaining an accurate diagnosis in non-neoplastic pulmonary diseases. However, both come with significant limitations of their own: Computed Tomography (CT) can only visualize larger structures due to its inherent–relatively–poor resolution, while (histo) pathology is often limited due to small sample size and sampling error and only allows for a 2D investigation. An innovative approach of inflating whole lung specimens and subjecting these subsequently to CT and whole lung microCT allows for an accurate matching of CT-imaging and histopathology data of exactly the same areas. Systematic application of this approach allows for a more targeted assessment of localized disease extent and more specifically can be used to investigate early mechanisms of lung diseases on a morphological and molecular level. Therefore, this technique is suitable to selectively investigate changes in the large and small airways, as well as the pulmonary arteries, veins and capillaries in relation to the disease extent in the same lung specimen. In this perspective we provide an overview of the different strategies that are currently being used, as well as how this growing field could further evolve

Beyond bronchiolitis obliterans : in-depth histopathologic characterization of bronchiolitis obliterans syndrome after lung transplantation

Bronchiolitis obliterans syndrome (BOS) is considered an airway-centered disease, with bronchiolitis obliterans (BO) as pathologic hallmark. However, the histologic spectrum of pure clinical BOS remains poorly characterized. We provide the first in-depth histopathologic description of well-characterized BOS patients and patients without chronic lung allograft dysfunction (CLAD), defined according to the recent consensus guidelines. Explant lung tissue from 52 clinically-defined BOS and 26 non-CLAD patients (collected 1993–2018) was analyzed for histologic parameters, including but not limited to airway lesions, vasculopathy and fibrosis. In BOS, BO lesions were evident in 38 (73%) patients and varied from concentric sub-epithelial fibrotic BO to inflammatory BO, while 10/14 patients without BO displayed ‘vanishing airways’, defined by a discordance between arteries and airways. Chronic vascular abnormalities were detected in 22 (42%) patients. Ashcroft fibrosis scores revealed a median of 43% (IQR: 23–69) of normal lung parenchyma per patient; 26% (IQR: 18–37) of minimal alveolar fibrous thickening; and 11% (IQR: 4–18) of moderate alveolar thickening without architectural damage. Patchy areas of definite fibrotic damage to the lung structure (i.e., Ashcroft score ≥5) were present in 28 (54%) patients. Fibrosis was classified as bronchocentric (n = 21/28, 75%), paraseptal (n = 17/28, 61%) and subpleural (n = 15/28, 54%). In non-CLAD patients, BO lesions were absent, chronic vascular abnormalities present in 1 (4%) patient and mean Ashcroft scores were significantly lower compared to BOS (p = 0.0038) with 78% (IQR: 64–88) normally preserved lung parenchyma. BOS explant lungs revealed evidence of various histopathologic findings, including vasculopathy and fibrotic changes, which may contribute to the pathophysiology of BOS

Small airway loss in the physiologically ageing lung : a cross-sectional study in unused donor lungs

Background Physiological lung ageing is associated with a gradual decline in dynamic lung volumes and a progressive increase in residual volume due to diminished elastic recoil of the lung, loss of alveolar tissue, and lower chest wall compliance. However, the effects of ageing on the small airways (ie, airways <2.0 mm in diameter) remain largely unknown. By using a combination of ex-vivo conventional CT (resolution 1 mm), whole lung micro-CT (resolution 150 mu m), and micro-CT of extracted cores (resolution 10 mu m), we aimed to provide a multiresolution assessment of the small airways in lung ageing in a large cohort of never smokers. Methods For this cross-sectional study, we included donor lungs collected from 32 deceased never-smoking donors (age range 16-83 years). Ex-vivo CT and whole lung high-resolution CT (micro-CT) were used to determine total airway numbers, stratified by airway diameter. Micro-CT was used to assess the number, length, and diameter of terminal bronchioles (ie, the last generation of conducting airways); mean linear intercept; and surface density in four lung tissue cores from each lung, extracted using a uniform sampling approach. Regression beta coefficients are calculated using linear regression and polynomial models. Findings Ex-vivo CT analysis showed an age-dependent decrease in the number of airways of diameter 2.0 mm to less than 2.5 mm (beta coefficient per decade -0.119, 95% CI -0.193 to -0.045; R-2=0.29) and especially in airways smaller than 2.0 mm in diameter (-0.158, -0.233 to -0.084; R-2=0.47), between 30 and 80 years of age, but not of the larger (>= 2.5 mm) diameter airways (-0.00781, -0.04409 to 0.02848; R-2=0.0007). In micro-CT analysis of small airways, the total number of terminal bronchioles per lung increased until the age of 30 years, after which an almost linear decline in the number of terminal bronchioles was observed (beta coefficient per decade -2035, 95% CI -2818 to -1252; R-2=0.55), accompanied by a non-significant increase in alveolar airspace size (6.44, -0.57 to 13.45, R-2=0.10). Moreover, this decrease in terminal bronchioles was associated with the age-related decline of pulmonary function predicted by healthy reference values. Interpretation Loss of terminal bronchioles is an important structural component of age-related decline in pulmonary function of healthy, non-smoking individuals