OPTIMAL: An OPTimized Imaging Mass cytometry AnaLysis framework for benchmarking segmentation and data exploration.

Analysis of imaging mass cytometry (IMC) data and other low-resolution multiplexed tissue imaging technologies is often confounded by poor single-cell segmentation and suboptimal approaches for data visualization and exploration. This can lead to inaccurate identification of cell phenotypes, states, or spatial relationships compared to reference data from single-cell suspension technologies. To this end we have developed the "OPTimized Imaging Mass cytometry AnaLysis (OPTIMAL)" framework to benchmark any approaches for cell segmentation, parameter transformation, batch effect correction, data visualization/clustering, and spatial neighborhood analysis. Using a panel of 27 metal-tagged antibodies recognizing well-characterized phenotypic and functional markers to stain the same Formalin-Fixed Paraffin Embedded (FFPE) human tonsil sample tissue microarray over 12 temporally distinct batches we tested several cell segmentation models, a range of different arcsinh cofactor parameter transformation values, 5 different dimensionality reduction algorithms, and 2 clustering methods. Finally, we assessed the optimal approach for performing neighborhood analysis. We found that single-cell segmentation was improved by the use of an Ilastik-derived probability map but that issues with poor segmentation were only really evident after clustering and cell type/state identification and not always evident when using "classical" bivariate data display techniques. The optimal arcsinh cofactor for parameter transformation was 1 as it maximized the statistical separation between negative and positive signal distributions and a simple Z-score normalization step after arcsinh transformation eliminated batch effects. Of the five different dimensionality reduction approaches tested, PacMap gave the best data structure with FLOWSOM clustering out-performing phenograph in terms of cell type identification. We also found that neighborhood analysis was influenced by the method used for finding neighboring cells with a "disc" pixel expansion outperforming a "bounding box" approach combined with the need for filtering objects based on size and image-edge location. Importantly, OPTIMAL can be used to assess and integrate with any existing approach to IMC data analysis and, as it creates .FCS files from the segmentation output and allows for single-cell exploration to be conducted using a wide variety of accessible software and algorithms familiar to conventional flow cytometrists.

Distinct lung cell signatures define the temporal evolution of diffuse alveolar damage in fatal COVID-19.

BACKGROUND: Lung damage in severe COVID-19 is highly heterogeneous however studies with dedicated spatial distinction of discrete temporal phases of diffuse alveolar damage (DAD) and alternate lung injury patterns are lacking. Existing studies have also not accounted for progressive airspace obliteration in cellularity estimates. We used an imaging mass cytometry (IMC) analysis with an airspace correction step to more accurately identify the cellular immune response that underpins the heterogeneity of severe COVID-19 lung disease. METHODS: Lung tissue was obtained at post-mortem from severe COVID-19 deaths. Pathologist-selected regions of interest (ROIs) were chosen by light microscopy representing the patho-evolutionary spectrum of DAD and alternate disease phenotypes were selected for comparison. Architecturally normal SARS-CoV-2-positive lung tissue and tissue from SARS-CoV-2-negative donors served as controls. ROIs were stained for 40 cellular protein markers and ablated using IMC before segmented cells were classified. Cell populations corrected by ROI airspace and their spatial relationships were compared across lung injury patterns. FINDINGS: Forty patients (32M:8F, age: 22-98), 345 ROIs and >900k single cells were analysed. DAD progression was marked by airspace obliteration and significant increases in mononuclear phagocytes (MnPs), T and B lymphocytes and significant decreases in alveolar epithelial and endothelial cells. Neutrophil populations proved stable overall although several interferon-responding subsets demonstrated expansion. Spatial analysis revealed immune cell interactions occur prior to microscopically appreciable tissue injury. INTERPRETATION: The immunopathogenesis of severe DAD in COVID-19 lung disease is characterised by sustained increases in MnPs and lymphocytes with key interactions occurring even prior to lung injury is established. FUNDING: UK Research and Innovation/Medical Research Council through the UK Coronavirus Immunology Consortium, Barbour Foundation, General Sir John Monash Foundation, Newcastle University, JGW Patterson Foundation, Wellcome Trust.

Downregulation of MALAT1 is a hallmark of tissue and peripheral proliferative T cells in COVID-19.

T cells play key protective but also pathogenic roles in COVID-19. We studied the expression of long non-coding RNAs (lncRNAs) in COVID-19 T-cell transcriptomes by integrating previously published single-cell RNA sequencing datasets. The long intergenic non-coding RNA MALAT1 was the most highly transcribed lncRNA in T cells, with Th1 cells demonstrating the lowest and CD8+ resident memory cells the highest MALAT1 expression, amongst CD4+ and CD8+ T-cells populations, respectively. We then identified gene signatures that covaried with MALAT1 in single T cells. A significantly higher number of transcripts correlated negatively with MALAT1 than those that correlated. Enriched functional annotations of the MALAT1- anti-correlating gene signature included processes associated with T-cell activation such as cell division, oxidative phosphorylation, and response to cytokine. The MALAT1 anti-correlating gene signature shared by both CD4+ and CD8+ T-cells marked dividing T cells in both the lung and blood of COVID-19 patients. Focussing on the tissue, we used an independent patient cohort of post-mortem COVID-19 lung samples and demonstrated that MALAT1 suppression was indeed a marker of MKI67+ proliferating CD8+ T cells. Our results reveal MALAT1 suppression and its associated gene signature are a hallmark of human proliferating T cells.

Identification of a protein expression signature distinguishing early from organising diffuse alveolar damage in COVID-19 patients.

Diffuse alveolar damage (DAD) is the histological expression of acute respiratory distress syndrome and characterises lung pathology due to infection with SARS-CoV-2, and other respiratory pathogens of clinical significance. DAD reflects a time-dependent immunopathological process, progressing from an early/exudative stage through to an organising/fibrotic stage, yet within an individual these different stages of DAD may coexist. Understanding the progression of DAD is central to the development of new therapeutics to limit progressive lung damage. Here, we applied highly multiplexed spatial protein profiling to autopsy lung tissues derived from 27 patients who died from COVID-19 and identified a protein signature (ARG1, CD127, GZMB, IDO1, Ki67, phospho-PRAS40 (T246) and VISTA) that distinguishes early DAD from late DAD with good predictive accuracy. These proteins warrant further investigation as potential regulators of DAD progression.

miRNA Expression in Fibroblastic Foci within Idiopathic Pulmonary Fibrosis Lungs Reveals Novel Disease-Relevant Pathways.

miRNAs are 22 nucleotides long and belong to a class of noncoding RNAs that plays an important role in regulating gene expression at a post-transcriptional level. Studies show aberrant levels of miRNAs to be associated with profibrotic processes in idiopathic pulmonary fibrosis (IPF). However, most of these studies used whole IPF tissue or in vitro monocultures in which fibrosis was artificially induced. The current study used laser microdissection to collect fibroblastic foci (FF), the key pathologic lesion in IPF, isolated miRNAs, and compared their expression levels with those found in whole IPF lung tissue and/or in vitro cultured fibroblast from IPF or normal lungs. Sequencing libraries were generated, and data generated were bioinformatically analyzed. A total of 18 miRNAs were significantly overexpressed in FF tissue when compared with whole IPF tissue. Of those, 15 were unique to FF. Comparison of FF with cultured IPF fibroblasts also revealed differences in miRNA composition that impacted several signaling pathways. The miRNA composition of FF is both overlapping and distinct from that of whole IPF tissue or cultured IPF fibroblasts and highlights the importance of characterizing FF biology as a phenotypically and functionally discrete tissue microenvironment.

The trajectory of COVID-19 cardiopulmonary disease: insights from an autopsy study of community-based, pre-hospital deaths.

Background: Post mortem examination of lung and heart tissue has been vital to developing an understanding of COVID-19 pathophysiology; however studies to date have almost uniformly used tissue obtained from hospital-based deaths where individuals have been exposed to major medical and pharmacological interventions. Methods: In this study we investigated patterns of lung and heart injury from 46 community-based, pre-hospital COVID-19-attributable deaths who underwent autopsy. Results: The cohort comprised 22 females and 24 males, median age 64 years (range 19-91) at time of death with illness duration range 0-23 days. Comorbidities associated with poor outcomes in COVID-19 included obesity (body mass index >30 kg·m-2) in 19 out of 46 cases (41.3%). Diffuse alveolar damage in its early exudative phase was the most common pattern of lung injury; however significant heterogeneity was identified with bronchopneumonia, pulmonary oedema consistent with acute cardiac failure, pulmonary thromboembolism and microthrombosis also identified and often in overlapping patterns. Review of clinical records and next of kin accounts suggested a combination of unexpectedly low symptom burden, rapidly progressive disease and psychosocial factors may have contributed to a failure of hospital presentation prior to death. Conclusions: Identifying such advanced acute lung injury in community-based deaths is extremely unusual and raises the question why some with severe COVID-19 pneumonitis were not hospitalised. Multiple factors including low symptom burden, rapidly progressive disease trajectories and psychosocial factors provide possible explanations.

Post-mortem lung tissue: the fossil record of the pathophysiology and immunopathology of severe COVID-19.

The lungs are the main site that is affected in severe COVID-19, and post-mortem lung tissue provides crucial insights into the pathophysiology of severe disease. From basic histology to state-of-the-art multiparameter digital pathology technologies, post-mortem lung tissue provides snapshots of tissue architecture, and resident and inflammatory cell phenotypes and composition at the time of death. Contrary to early assumptions that COVID-19 in the lungs is a uniform disease, post-mortem findings have established a high degree of disease heterogeneity. Classic diffuse alveolar damage represents just one phenotype, with disease divisible by early and late progression as well as by pathophysiological process. A distinct lung tissue state occurs with secondary infection; extrapulmonary causes of death might also originate from a pathological process in the lungs linked to microthrombosis. This heterogeneity of COVID-19 lung disease must be recognised in the management of patients and in the development of novel treatment strategies.

Pathology and natural history of organ fibrosis.

Histopathological assessment of fibrosis focusing on morphological patterns provides important information for the management of patients with chronic diseases of the kidney, liver and the lung. This review summarizes key histopathological features of pulmonary, renal and hepatic fibrosis and discusses advances in the understanding of the pathogenesis of pulmonary fibrosis and pathogenetic insights with translational implications for renal fibrosis. The review also tackles new staging approaches based on liver fibrosis dynamics and evaluation of fibrosis regression, digital pathology and second harmonic generation microscopy methods for hepatic fibrosis assessment and critical appraisal of non-invasive tests for liver and renal fibrosis evaluation.

Giant cell myocarditis presenting with acute heart failure.

Idiopathic giant cell myocarditis (GCM) is a rare and rapidly progressing form of myocarditis predominantly affecting younger people. We report a case of a 23-year-old athletic patient who presented with features of acute heart failure due to GCM and discuss his management that included a left ventricular assist device as a bridge to transplant. He died immediately following the transplant.We also review the literature on this rare disease, highlighting the advances in the management of the disease including immunosuppressive therapy, ventricular assist devices and heart transplantation.

Development of an ex vivo technique to achieve reanimation of hearts sourced from a porcine donation after circulatory death model.

BACKGROUND: This study reports on the development of a novel method for achieving ex vivo reanimation of hearts from a porcine donation after circulatory death (DCD) model without the use of donor pretreatment. METHODS: Porcine hearts (n = 23) were procured 10-29 min after confirmation of asystole. All hearts underwent initial flush with AQIX RS-I solution (London, UK). A 2-h preservation period followed: group 1 hearts (n1-n11) were preserved using static cold storage, group 2 hearts (n12-n17) were preserved using oxygenated, hypothermic machine perfusion (MP), and group 3 hearts (n18-n23) were subjected to retrograde oxygen persufflation. Reperfusion was performed on a Langendorff modification of a Model 33 Functional Circulation circuit. In hearts n16-n23, a dialysis circuit was incorporated into the circuit to facilitate removal of metabolites. The experimental protocol was allowed to follow an evolutionary course, with the aim of achieving greater success with reanimation. RESULTS: In group 1 (static cold storage), 7 of the 11 hearts (63.6%) achieved reanimation on the ex vivo circuit. Two of the six hearts (33.3%) in group 2 (MP) were successfully reanimated. All the six hearts (100%) in group 3 (persufflation) were successfully reanimated. The period of sustained reanimation increased when dialysis was incorporated into the circuit with a maximum of 300 min. CONCLUSIONS: Porcine DCD hearts after 29 min of warm ischemia can be reanimated using the method described. A mechanism of reoxygenation (oxygenated MP or coronary sinus oxygen persufflation) during preservation appears mandatory for hearts from DCDs. Persufflation was associated with a higher probability of successful reanimation. Dialysis in the warm phase was useful in removing metabolites that could interfere with reanimation. The results demonstrate the potential of DCDs to counter the decline affecting heart transplantation.

Platelet thromboembolism after lung transplantation.

Lung transplant patients have an increased risk of pulmonary embolism which is often associated with hypercoagulability disorders. We present a case of sudden death resulting from pulmonary intravascular platelet thromboembolism following a single-lung transplant.

[Diagnoses and diagnostic procedures in 500 consecutive patients with clinical suspicion of interstitial lung disease]. / Diagnósticos y procedimientos en 500 pacientes consecutivos con sospecha clínica de enfermedad pulmonar intersticial.

OBJECTIVE: To determine the diagnostic yield achieved with the application of current recommendations for evaluating patients with suspected interstitial lung disease (ILD) and the procedures that must be applied to reach a definitive diagnosis. PATIENTS AND METHODS: Over a 10-year period, 500 consecutive patients attending an ILD outpatient clinic who showed features of diffuse lung involvement were assessed with a single diagnostic protocol. Results were introduced in a dedicated database and diagnoses for idiopathic interstitial pneumonia were established according to a recent consensus classification. RESULTS: A definitive diagnosis was reached in 427 (85%) patients: in 125 without invasive procedures and in 302 with invasive procedures. In 73 (14.6%) cases a definitive diagnosis was not reached, and patients were placed in the group of unclassifiable interstitial pneumonia. Idiopathic interstitial pneumonia was the predominant group with 193 (39%) patients. The main specific entities included sarcoidosis with 93 (19%) patients, usual interstitial pneumonia with 84 (17%) patients, and hypersensitivity pneumonitis with 75 (15%) patients. Thirty (6%) patients were diagnosed with an illness other than ILD (false ILD). In 332 patients, we performed a total of 433 invasive procedures: transbronchial biopsy in 252 (direct diagnostic yield, 38%, or if used also to exclude other specific diagnosis, 50%), bronchoalveolar lavage in 260 (yield, 5%), and open lung biopsy in 141 (yield, 93%). Hence, following the current diagnostic approach, a definitive diagnosis was established for 85% of patients, for 25% solely on clinical grounds and imaging criteria and for 60% on the basis of invasive procedures. Diagnosis by open lung biopsy was still required for 141 (28%) patients. CONCLUSIONS: The diagnostic yield was high when the recommended study protocol was followed. A quarter of the diagnoses were reached with clinical criteria alone, but another quarter could only be made after open lung biopsy.

Diagnósticos y procedimientos en 500 pacientes consecutivos con sospecha clínica de enfermedad pulmonar intersticial / Diagnoses and diagnostic procedures in 500 consecutive patients with clinical suspicion of interstitial lung disease

Objetivo: Determinar las tasas de diagnósticos alcanzados con el seguimiento de las directrices actuales y los procedimientos que deben utilizarse para establecer el diagnóstico definitivo mediante la aplicación del nuevo protocolo en la evaluación de los pacientes con sospecha de enfermedad pulmonar intersticial (EPI). Pacientes y métodos: Durante un período de 10 años se evaluó, mediante un único protocolo diagnóstico, a 500 pacientes consecutivos atendidos en una consulta ambulatoria de EPI que presentaban las características de esta enfermedad. Los resultados se introdujeron en una base de datos específica y los diagnósticos de neumonía intersticial idiopática (NII) se establecieron siguiendo los criterios del reciente Consenso. Resultados: Se estableció un diagnóstico definitivo en 427 pacientes (85%), en 125 de ellos sin procedimientos invasivos y en 302 con procedimientos invasivos. En 73 casos (14,6%) no se alcanzó un diagnóstico definitivo y en estos pacientes se estableció el diagnóstico de neumonía intersticial no clasificable. La NII constituyó el grupo predominante, con 193 casos (39%). Las entidades específicas principales fueron: sarcoidosis (n = 93; 19%), neumonía intersticial usual (n = 84; 17%) y neumonitis por hipersensibilidad (n = 75; 15%). En 30 pacientes (6%) se estableció el diagnóstico de una enfermedad distinta a la EPI (falsa neumonía intersticial). Se realizó un total de 433 procedimientos invasivos en 332 pacientes (66%): biopsia transbronquial en 252 (rendimiento diagnóstico directo: 38% y rendimiento diagnóstico cuando se utilizó para excluir otros diagnósticos específicos: 50%); lavado broncoalveolar en 260 (rendimiento diagnóstico: 5%), y biopsia pulmonar quirúrgica en 141 (rendimiento diagnóstico: 93%). Por lo tanto, siguiendo el protocolo diagnóstico actual, se estableció un diagnóstico definitivo en el 85% de los pacientes; de ellos, en el 25% el diagnóstico se estableció únicamente en función de los datos clínicos y de los criterios de imagen, mientras que en el 60% se realizó con procedimientos invasivos. En 141 pacientes (28%) fue necesaria la biopsia pulmonar quirúrgica para establecer el diagnóstico. Conclusiones: La tasa de diagnósticos es elevada cuando se utiliza el protocolo de estudio recomendado. La cuarta parte de los diagnósticos se efectúa mediante criterios clínicos como procedimiento único; sin embargo, otra cuarta parte de los diagnósticos requiere la realización de una biopsia pulmonar quirúrgica

Objective: To determine the diagnostic yield achieved with the application of current recommendations for evaluating patients with suspected interstitial lung disease (ILD) and the procedures that must be applied to reach a definitive diagnosis. Patients and methods: Over a 10-year period, 500 consecutive patients attending an ILD outpatient clinic who showed features of diffuse lung involvement were assessed with a single diagnostic protocol. Results were introduced in a dedicated database and diagnoses for idiopathic interstitial pneumonia were established according to a recent consensus classification. Results: A definitive diagnosis was reached in 427 (85%) patients: in 125 without invasive procedures and in 302 with invasive procedures. In 73 (14.6%) cases a definitive diagnosis was not reached, and patients were placed in the group of unclassifiable interstitial pneumonia. Idiopathic interstitial pneumonia was the predominant group with 193 (39%) patients. The main specific entities included sarcoidosis with 93 (19%) patients, usual interstitial pneumonia with 84 (17%) patients, and hypersensitivity pneumonitis with 75 (15%) patients. Thirty (6%) patients were diagnosed with an illness other than ILD (false ILD). In 332 patients, we performed a total of 433 invasive procedures: transbronchial biopsy in 252 (direct diagnostic yield, 38%, or if used also to exclude other specific diagnosis, 50%), bronchoalveolar lavage in 260 (yield, 5%), and open lung biopsy in 141 (yield, 93%). Hence, following the current diagnostic approach, a definitive diagnosis was established for 85% of patients, for 25% solely on clinical grounds and imaging criteria and for 60% on the basis of invasive procedures. Diagnosis by open lung biopsy was still required for 141 (28%) patients. Conclusions: The diagnostic yield was high when the recommended study protocol was followed. A quarter of the diagnoses were reached with clinical criteria alone, but another quarter could only be made after open lung biopsy

De novo nephrotic syndrome following pegylated interferon alfa 2b/ribavirin therapy for chronic hepatitis C infection.

This paper describes the case of a patient with HCV, without previous evidence of nephropathy, who following two well-tolerated cycles of treatment with interferon alone developed nephrotic syndrome after a third attempt with ribavirin associated with peginterferon alfa 2b. The patient exhibited a total remission when the antiviral treatment was discontinued.

Mixoma que abarca desde la vena cava superior hasta la arteria pulmonar derecha / Myxoma extending from the superior vena cava to the right pulmonary artery

No disponible

No disponible

Clinical significance of hypoxia-inducible factor-1a messenger RNA expression in locally advanced non-small-cell lung cancer after platinum agent and gemcitabine chemotherapy followed by surgery.

Hypoxia-inducible factor-1a (HIF-1a) is a key regulator of the angiogenic cascade. This study analyzed HIF-1a messenger RNA expression levels using real-time quantitative polymerase chain reaction (PCR) in paraffin-embedded surgical specimens from 54 stage IIB-III patients with non-small-cell lung cancer (NSCLC) treated with induction platinum/gemcitabine followed by surgery between September 1998 and December 2002. Radiographic response was observed in 61% of patients. Median survival was 37.8 months. Forty-five patients with complete resection attained a 52-month median survival, whereas 8 patients with incomplete resection had a 12-month median survival, and 1 unresectable patient had a survival of 14 months. No significant differences were observed in overall survival (OS) or event-free survival (EFS) according to HIF-1a expression levels. Patients were divided into quartiles according to HIF-1a gene expression levels. Median EFS for the 13 patients in the lowest quartile has not been reached yet, whereas median EFS for the 13 patients in the top quartile was 9 months (P = 0.192). Similarly, median OS for the 13 patients in the lowest quartile has not been reached yet, whereas median OS for the 13 patients in the top quartile was 52 months (P = 0.297). The cisplatin/gemcitabine combination is highly active in neoadjuvant treatment. Hypoxia-inducible factor-1a expression levels analyzed by real-time quantitative PCR in surgery specimens after platinum/gemcitabine therapy do not correlate with the outcome of patients with stage II/III NSCLC.

Tuberculosis in lung transplant recipients.

BACKGROUND: Post-lung transplant infection is one of the leading causes of morbidity and mortality. The cause and incidence are similar in many series; however, infections such as Mycobacterium tuberculosis are influenced by the epidemiologic situation. The authors present a prospective and observational study to define the incidence, clinical presentation, and course of tuberculosis in a cohort of lung transplant patients at a single center in Spain. METHODS: Between 1990 and 2002, cutaneous delayed-type hypersensitivity testing and pathologic and microbiologic study of explanted lungs were conducted in 187 lung transplant patients. Serial bronchoscopies with transbronchial biopsy and bronchioalveolar lavage were performed during follow-up. The diagnosis of tuberculosis was established only when M. tuberculosis was identified in any sample or when histopathologic study was conclusive. RESULTS: Forty-eight patients were classified as anergic (25.6%) and 61 (32.6%) were classified as having a positive tuberculin skin test. Of the 109 patients, 95 received latent tuberculosis infection prophylaxis. Tuberculosis was diagnosed in 12 patients (6.41%); in six of them, diagnosis was determined from the explanted lungs. The remainder were diagnosed during follow-up. Fever and dyspnea were the most common symptoms. Chest radiographic findings presented an alveolar pattern. All patients responded well to antituberculous therapy; no deaths were attributable to tuberculosis. CONCLUSIONS: In the authors' experience, tuberculosis is not rare in lung transplant patients and can be managed successfully with antituberculous therapy without rifampin. A systematic protocol for diagnosing tuberculosis of the explanted lung is useful for reducing tuberculous complications of the implanted lung.