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IntroductionEndobronchial foreign body aspiration is not common in adults, but it is a life-threatening event. Recurrent pneumonias by chronic retention of foreign body often lead to initial medical presentation of the patient. However, lymphoplasmacellular bronchitis with adenomatous hyperplasia and squamous epithelium metaplasia with complete or partial blockage of lobar bronchus mimicking lung tumor is rare in literature, and this particular condition is often misdiagnosed.Case presentationwe report our experience in the diagnostic and management of two elderly patients with recurrent pneumonia, admitted in hospital for further examination. In both patients, with no history of aspiration, the cherry pit was detected during bronchoscopy and recanalization with flexible cryoprobe, surrounded by purulent secretion, occluding completely the right upper lobe in the first case, and partially the left lower lobe associated with persistent actinomycosis in the second case, with signs of local inflammation, bronchial adenomatous hyperplasia mimicking lung tumor at initial bronchoscopic examination. Histology showed a lymphoplasmacellullar bronchitis with adenomatous hyperplasia and squamous epithelium metaplasia because of chronic retention of foreign body.ConclusionBronchoscopy examination should be considered in cases where there is an unresolved chronic cough with recurrent pneumonia or persistent actinomycosis in patients with high risk. Cryoprobe is a safe and feasible approach for treatment of airway obstructions due to chronic foreign body retention. Furthermore, relevant findings are discussed here, along with a review of the pathologic alterations and treatment modalities seen in chronic retention of foreign body and airway injury.

Recent breakthroughs in single-cell sequencing, advancements in cellular and tissue imaging techniques, innovations in cell lineage tracing, and insights into the epigenome collectively illuminate the enigmatic landscape of alveolar macrophages in the lung under homeostasis and disease conditions. Our current knowledge reveals the cellular and functional diversity of alveolar macrophages within the respiratory system, emphasising their remarkable adaptability. By synthesising insights from classical cell and developmental biology studies, we provide a comprehensive perspective on alveolar macrophage functional plasticity. This includes an examination of their ontology-related features, their role in maintaining tissue homeostasis under steady-state conditions and the distinct contribution of bone marrow-derived macrophages (BMDMs) in promoting tissue regeneration and restoring respiratory system homeostasis in response to injuries. Elucidating the signalling pathways within inflammatory conditions, the impact of various triggers on tissue-resident alveolar macrophages (TR-AMs), as well as the recruitment and polarisation of macrophages originating from the bone marrow, presents an opportunity to propose innovative therapeutic approaches aimed at modulating the equilibrium between phenotypes to induce programmes associated with a pro-regenerative or homeostasis phenotype of BMDMs or TR-AMs. This, in turn, can lead to the amelioration of disease outcomes and the attenuation of detrimental inflammation. This review comprehensively addresses the pivotal role of macrophages in the orchestration of inflammation and resolution phases after lung injury, as well as ageing-related shifts and the influence of clonal haematopoiesis of indeterminate potential mutations on alveolar macrophages, exploring altered signalling pathways and transcriptional profiles, with implications for respiratory homeostasis.

Abstract Influenza A virus (IAV) infection mobilizes bone marrow-derived macrophages (BMDM) that gradually undergo transition to tissue-resident alveolar macrophages (TR-AM) in the inflamed lung. Combining high-dimensional single-cell transcriptomics with complex lung organoid modeling, in vivo adoptive cell transfer, and BMDM-specific gene targeting, we found that transitioning (“regenerative”) BMDM and TR-AM highly express Placenta-expressed transcript 1 (Plet1). We reveal that Plet1 is released from alveolar macrophages, and acts as important mediator of macrophage-epithelial cross-talk during lung repair by inducing proliferation of alveolar epithelial cells and re-sealing of the epithelial barrier. Intratracheal administration of recombinant Plet1 early in the disease course attenuated viral lung injury and rescued mice from otherwise fatal disease, highlighting its therapeutic potential.

Organoid models have become an integral part of the research methodology in the lung field. These systems allow for the study of progenitor and stem cell self-renewal, self-organization, and differentiation. Distinct models of lung organoids mimicking various anatomical regions of mature lungs have emerged in parallel to the increased gain of knowledge regarding epithelial stem and progenitor cell populations and the corresponding mesenchymal cells that populate the in vivo niche. In the distal lung, type 2 alveolar epithelial cells (AEC2s) represent a stem cell population that is engaged in regenerative mechanisms in response to various insults. These cells self-renew and give rise to AEC1s that carry out gas exchange. Multiple experimental protocols allowing the generation of alveolar organoids, or alveolospheres, from murine lungs have been described. Among the drawbacks have been the requirement of transgenic mice allowing the isolation of AEC2s with high viability and purity, and the occasional emergence of bronchiolar and bronchioalveolar organoids. Here, we provide a refined gating strategy and an optimized protocol for the generation of alveolospheres from wild-type mice. Our approach not only overcomes the need for transgenic mice to generate such organoids, but also yields a pure culture of alveolospheres that is devoid of bronchiolar and bronchioalveolar organoids. Our protocol contributes to the standardization of this important research tool.

Alveolar macrophages (AMs) are the sentinel cells of the alveolar space, maintaining homeostasis, fending off pathogens, and controlling lung inflammation. During acute lung injury, AMs orchestrate the initiation and resolution of inflammation in order to ultimately restore homeostasis. This central role in acute lung inflammation makes AMs attractive targets for therapeutic interventions. Single-cell RNA-Seq and spatial omics approaches, together with methodological advances such as the generation of human macrophages from pluripotent stem cells, have increased understanding of the ontogeny, function, and plasticity of AMs during infectious and sterile lung inflammation, which could move the field closer to clinical application. However, proresolution phenotypes might conflict with proinflammatory and antibacterial responses. Therefore, therapeutic targeting of AMs at vulnerable time points over the course of infectious lung injury might harbor the risk of serious side effects, such as loss of antibacterial host defense capacity. Thus, the identification of key signaling hubs that determine functional fate decisions in AMs is of the utmost importance to harness their therapeutic potential.

IntroductionAllogeneic stem cell transplantation is used to cure hematologic malignancies or deficiencies of the hematopoietic system. It is associated with severe immunodeficiency of the host early after transplant and therefore early reactivation of latent herpesviruses such as CMV and EBV within the first 100 days are frequent. Small studies and case series indicated that application of herpes virus specific T cells can control and prevent disease in this patient population.MethodsWe report the results of a randomized controlled multi centre phase I/IIa study (MULTIVIR-01) using a newly developed T cell product with specificity for CMV and EBV derived from the allogeneic stem cell grafts used for transplantation. The study aimed at prevention and preemptive treatment of both viruses in patients after allogeneic stem cell transplantation targeting first infusion on day +30. Primary endpoints were acute transfusion reaction and acute-graft versus-host-disease after infusion of activated T cells.ResultsThirty-three patients were screened and 9 patients were treated with a total of 25 doses of the T cell product. We show that central manufacturing can be achieved successfully under study conditions and the product can be applied without major side effects. Overall survival, transplant related mortality, cumulative incidence of graft versus host disease and number of severe adverse events were not different between treatment and control groups. Expansion of CMV/EBV specific T cells was observed in a fraction of patients, but overall there was no difference in virus reactivation. DiscussionOur study results indicate peptide stimulated epitope specific T cells derived from stem cell grafts can be administered safely for prevention and preemptive treatment of reactivation without evidence for induction of acute graft versus host disease.Clinical trial registrationhttps://clinicaltrials.gov, identifier NCT02227641.

IntroductionAcute respiratory distress syndrome (ARDS) is a common complication of influenza virus (IV) infection. During ARDS, alveolar protein concentrations often reach 40-90% of plasma levels, causing severe impairment of gas exchange and promoting deleterious alveolar remodeling. Protein clearance from the alveolar space is at least in part facilitated by the multi-ligand receptor megalin through clathrin-mediated endocytosis.MethodsTo investigate whether IV infection impairs alveolar protein clearance, we examined albumin uptake and megalin expression in MLE-12 cells and alveolar epithelial cells (AEC) from murine precision-cut lung slices (PCLS) and in vivo, under IV infection conditions by flow cytometry and western blot. Transcriptional levels from AEC and broncho-alveolar lavage (BAL) cells were analyzed in an in-vivo mouse model by RNAseq.ResultsIV significantly downregulated albumin uptake, independently of activation of the TGF-β1/GSK3β axis that has been previously implicated in the regulation of megalin function. Decreased plasma membrane abundance, total protein levels, and mRNA expression of megalin were associated with this phenotype. In IV-infected mice, we identified a significant upregulation of matrix metalloproteinase (MMP)-14 in BAL fluid cells. Furthermore, the inhibition of this protease partially recovered total megalin levels and albumin uptake.DiscussionOur results suggest that the previously described MMP-driven shedding mechanisms are potentially involved in downregulation of megalin cell surface abundance and clearance of excess alveolar protein. As lower alveolar edema protein concentrations are associated with better outcomes in respiratory failure, our findings highlight the therapeutic potential of a timely MMP inhibition in the treatment of IV-induced ARDS.

Abstract Background Persistent symptoms after initial COVID-19 infection are common and are frequently referred to by the umbrella terms “post-COVID syndrome” and “long COVID”. The sheer number of affected patients pose an increasing challenge to healthcare systems worldwide. To date, our understanding of the pathophysiology of the post-COVID syndrome remains poor and the extent to which persistent cardiopulmonary abnormalities contribute to the symptom complex is unclear. We sought to determine the presence and impact of cardiopulmonary sequelae after COVID-19 in longitudinal assessment. Methods We report on 71 patients who underwent comprehensive, longitudinal testing in regular intervals for up to 12 months after their initial COVID-19 diagnosis. Testing included pulmonary function testing, cardiopulmonary exercise testing, dedicated left and right heart echocardiography, lung ultrasonography, and cardiac MRI. Results Our results demonstrate that subjective quality of life after COVID-19 (EQ-5D visual acuity scale, VAS, 67.4 for patients treated as outpatient, 79.2 for patients admitted to the general floor, 71.8 for patients treated in an ICU) is not related to the severity of the initial infection. Maximal exercise capacity is also reduced (VO2max 79% predicted, SD ± 19%); however, this is driven in large parts by patients who had initially required ICU-level of care. The degree of objective reduction in exertion did not correlate with quality of life scores. Pulmonary function testing revealed mild and persistent reduction in DLCO over the first 12 months without significant restrictive or obstructive lung disease. Left and right heart function was intact with good RV function and intact RV/PA coupling, imaging findings suggestive of myocarditis were uncommon (7% of patients). Conclusion A reduction in exercise capacity after COVID-19 is common, but is most prominent in patients previously treated in the ICU and more likely related to deconditioning or fatigue than to cardiopulmonary impairment. Subjective quality of life scores are independent of the severity of initial infection and do not correlate with objective measures of cardiopulmonary function. In our cohort, persistent cardiopulmonary impairment after COVID-19 was uncommon. The post-COVID syndrome is unlikely to be the result of cardiopulmonary sequalae and may reflect a post-ICU syndrome in some. Trial registration Registered on clinicaltrials.gov (NCT04442789), Date: June 23, 2020

Mycobacterium genavense infection, a rare nontuberculous mycobacteria infection, occurs in heavily immunocompromised patients (i.e., those with advanced HIV disease, genetic disorders, or acquired immunologic disorders and those undergoing immunosuppressive therapy). We report a case of disseminated M. genavense infection preceding Hodgkin lymphoma in a patient without obvious risk factors for this infection.

The widespread presence of autoantibodies in acute infection with SARS-CoV-2 is increasingly recognized, but the prevalence of autoantibodies in non–SARS-CoV-2 infections and critical illness has not yet been reported. We profiled IgG autoantibodies in 267 patients from 5 independent cohorts with non–SARS-CoV-2 viral, bacterial, and noninfectious critical illness. Serum samples were screened using Luminex arrays that included 58 cytokines and 55 autoantigens, many of which are associated with connective tissue diseases (CTDs). Samples positive for anti-cytokine antibodies were tested for receptor blocking activity using cell-based functional assays. Anti-cytokine antibodies were identified in > 50% of patients across all 5 acutely ill cohorts. In critically ill patients, anti-cytokine antibodies were far more common in infected versus uninfected patients. In cell-based functional assays, 11 of 39 samples positive for select anti-cytokine antibodies displayed receptor blocking activity against surface receptors for Type I IFN, GM-CSF, and IL-6. Autoantibodies against CTD-associated autoantigens were also commonly observed, including newly detected antibodies that emerged in longitudinal samples. These findings demonstrate that anti-cytokine and autoantibodies are common across different viral and nonviral infections and range in severity of illness.

Here, Shaban et al. show that coronaviruses modulate ER stress and the unfolded protein response. The ER stress inducer thapsigargin exerts potent antiviral effects, partially reverses the virus-induced translational shut-down, reprograms the host proteome and suppresses autophagic flux, thereby inhibiting coronavirus replication at multiple levels.

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Abstract Background The technology of single cell RNA sequencing (scRNA-seq) has gained massively in popularity as it allows unprecedented insights into cellular heterogeneity as well as identification and characterization of (sub-)cellular populations. Furthermore, scRNA-seq is almost ubiquitously applicable in medical and biological research. However, these new opportunities are accompanied by additional challenges for researchers regarding data analysis, as advanced technical expertise is required in using bioinformatic software. Results Here we present WASP, a software for the processing of Drop-Seq-based scRNA-Seq data. Our software facilitates the initial processing of raw reads generated with the ddSEQ or 10x protocol and generates demultiplexed gene expression matrices including quality metrics. The processing pipeline is realized as a Snakemake workflow, while an R Shiny application is provided for interactive result visualization. WASP supports comprehensive analysis of gene expression matrices, including detection of differentially expressed genes, clustering of cellular populations and interactive graphical visualization of the results. The R Shiny application can be used with gene expression matrices generated by the WASP pipeline, as well as with externally provided data from other sources. Conclusions With WASP we provide an intuitive and easy-to-use tool to process and explore scRNA-seq data. To the best of our knowledge, it is currently the only freely available software package that combines pre- and post-processing of ddSEQ- and 10x-based data. Due to its modular design, it is possible to use any gene expression matrix with WASP’s post-processing R Shiny application. To simplify usage, WASP is provided as a Docker container. Alternatively, pre-processing can be accomplished via Conda, and a standalone version for Windows is available for post-processing, requiring only a web browser.

Acute respiratory distress syndrome (ARDS) results in catastrophic lung failure and has an urgent, unmet need for improved early recognition and therapeutic development. Neutrophil influx is a hallmark of ARDS and is associated with the release of tissue-destructive immune effectors, such as matrix metalloproteinases (MMPs) and membrane-anchored metalloproteinase disintegrins (ADAMs). Here, we observed using intravital microscopy that Adam8–/– mice had impaired neutrophil transmigration. In mouse pneumonia models, both genetic deletion and pharmacologic inhibition of ADAM8 attenuated neutrophil infiltration and lung injury while improving bacterial containment. Unexpectedly, the alterations of neutrophil function were not attributable to impaired proteolysis but resulted from reduced intracellular interactions of ADAM8 with the actin-based motor molecule Myosin1f that suppressed neutrophil motility. In 2 ARDS cohorts, we analyzed lung fluid proteolytic signatures and identified that ADAM8 activity was positively correlated with disease severity. We propose that in acute inflammatory lung diseases such as pneumonia and ARDS, ADAM8 inhibition might allow fine-tuning of neutrophil responses for therapeutic gain.

A hallmark of acute respiratory distress syndrome (ARDS) is an accumulation of protein-rich alveolar edema that impairs gas exchange and leads to worse outcomes. Thus, understanding the mechanisms of alveolar albumin clearance is of high clinical relevance. Here, we investigated the mechanisms of the cellular albumin uptake in a three-dimensional culture of precision-cut lung slices (PCLS). We found that up to 60% of PCLS cells incorporated labeled albumin in a time- and concentration-dependent manner, whereas virtually no uptake of labeled dextran was observed. Of note, at a low temperature (4 °C), saturating albumin receptors with unlabeled albumin and an inhibition of clathrin-mediated endocytosis markedly decreased the endocytic uptake of the labeled protein, implicating a receptor-driven internalization process. Importantly, uptake rates of albumin were comparable in alveolar epithelial type I (ATI) and type II (ATII) cells, as assessed in PCLS from a SftpcCreERT2/+: tdTomatoflox/flox mouse strain (defined as EpCAM+CD31−CD45−tdTomatoSPC−T1α+ for ATI and EpCAM+CD31−CD45−tdTomatoSPC+T1α− for ATII cells). Once internalized, albumin was found in the early and recycling endosomes of the alveolar epithelium as well as in endothelial, mesenchymal, and hematopoietic cell populations, which might indicate transcytosis of the protein. In summary, we characterize albumin uptake in alveolar epithelial cells in the complex setting of PCLS. These findings may open new possibilities for pulmonary drug delivery that may improve the outcomes for patients with respiratory failure.

Several acute and chronic lung diseases are associated with alveolar hypoventilation leading to accumulation of CO2 (hypercapnia). The β-subunit of the Na,K-ATPase plays a pivotal role in maintaining epithelial integrity by functioning as a cell adhesion molecule and regulating cell surface stability of the catalytic α-subunit of the transporter, thereby, maintaining optimal alveolar fluid balance. Here, we identified the E3 ubiquitin ligase for the Na,K-ATPase β-subunit, which promoted polyubiquitination, subsequent endocytosis and proteasomal degradation of the protein upon exposure of alveolar epithelial cells to elevated CO2 levels, thus impairing alveolar integrity. Ubiquitination of the Na,K-ATPase β-subunit required lysine 5 and 7 and mutating these residues (but not other lysines) prevented trafficking of Na,K-ATPase from the plasma membrane and stabilized the protein upon hypercapnia. Furthermore, ubiquitination of the Na,K-ATPase β-subunit was dependent on prior phosphorylation at serine 11 by protein kinase C (PKC)-ζ. Using a protein microarray, we identified the tumor necrosis factor receptor-associated factor 2 (TRAF2) as the E3 ligase driving ubiquitination of the Na,K-ATPase β-subunit upon hypercapnia. Of note, prevention of Na,K-ATPase β-subunit ubiquitination was necessary and sufficient to restore the formation of cell-cell junctions under hypercapnic conditions. These results suggest that a hypercapnic environment in the lung may lead to persistent epithelial dysfunction in affected patients. As such, the identification of the E3 ligase for the Na,K-ATPase may provide a novel therapeutic target, to be employed in patients with acute or chronic hypercapnic respiratory failure, aiming to restore alveolar epithelial integrity.

Summary: Organoid models have been shown to be valuable tools for studying epithelial-mesenchymal crosstalk during biological and pathological settings. Our data identified ACTA2+ PDGFRα+ repair-supportive mesenchymal cells as an important component of the conducting airway niche. Here, we provide a detailed protocol for culturing airway organoids, or bronchiolospheres, which provide an assessment of the ability of mesenchymal cells to support club-cell growth.For complete details on the use and execution of this protocol, please refer to Moiseenko et al. (2020).

Fgf10 is a key gene during development, homeostasis and repair after injury. We previously reported a knock-in Fgf10Cre–ERT2 line (with the Cre-ERT2 cassette inserted in frame with the start codon of exon 1), called thereafter Fgf10Ki–v1, to target FGF10Pos cells. While this line allowed fairly efficient and specific labeling of FGF10Pos cells during the embryonic stage, it failed to target these cells after birth, particularly in the postnatal lung, which has been the focus of our research. We report here the generation and validation of a new knock-in Fgf10Cre–ERT2 line (called thereafter Fgf10Ki–v2) with the insertion of the expression cassette in frame with the stop codon of exon 3. Fgf10Ki−v2/+ heterozygous mice exhibited comparable Fgf10 expression levels to wild type animals. However, a mismatch between Fgf10 and Cre expression levels was observed in Fgf10Ki–v2/+ lungs. In addition, lung and limb agenesis were observed in homozygous embryos suggesting a loss of Fgf10 functional allele in Fgf10Ki–v2 mice. Bioinformatic analysis shows that the 3′UTR, where the Cre-ERT2 cassette is inserted, contains numerous putative transcription factor binding sites. By crossing this line with tdTomato reporter line, we demonstrated that tdTomato expression faithfully recapitulated Fgf10 expression during development. Importantly, Fgf10Ki–v2 mouse is capable of significantly targeting FGF10Pos cells in the adult lung. Therefore, despite the aforementioned limitations, this new Fgf10Ki–v2 line opens the way for future mechanistic experiments involving the postnatal lung.

Idiopathic pulmonary fibrosis is associated with myofibroblast activation in the lungs and metabolic alterations. Here, the authors show that the antidiabetic drug metformin has antifibrotic effects in human-derived samples and mouse models, by modulating a number of metabolic pathways to induce lipogenic transdifferentiation of myofibroblasts.

Insulin-like growth factor (IGF) signaling controls the development and growth of many organs, including the lung. Loss of function of Igf1 or its receptor Igf1r impairs lung development and leads to neonatal respiratory distress in mice. Although many components of the IGF signaling pathway have shown to be dysregulated in idiopathic pulmonary fibrosis (IPF), the expression pattern of such components in different cellular compartments of the developing and/or fibrotic lung has been elusive. In this study, we provide a comprehensive transcriptional profile for such signaling components during embryonic lung development in mice, bleomycin-induced pulmonary fibrosis in mice and in human IPF lung explants. During late gestation, we found that Igf1 is upregulated in parallel to Igf1r downregulation in the lung mesenchyme. Lung tissues derived from bleomycin-treated mice and explanted IPF lungs revealed upregulation of IGF1 in parallel to downregulation of IGF1R, in addition to upregulation of several IGF binding proteins (IGFBPs) in lung fibrosis. Finally, treatment of IPF lung fibroblasts with recombinant IGF1 led to myogenic differentiation. Our data serve as a resource for the transcriptional profile of IGF signaling components and warrant further research on the involvement of this pathway in both lung development and pulmonary disease.

Idiopathic pulmonary fibrosis (IPF) is a progressive and often lethal interstitial lung disease of unknown aetiology. IPF is characterised by myofibroblast activation, tissue stiffening, and alveolar epithelium injury. As current IPF treatments fail to halt disease progression or induce regeneration, there is a pressing need for the development of novel therapeutic targets. In this regard, tri-dimensional (3D) models have rapidly emerged as powerful platforms for disease modelling, drug screening and discovery. In this review, we will touch on how 3D in vitro models such as hydrogels, precision-cut lung slices, and, more recently, lung organoids and lung-on-chip devices have been generated and/or modified to reveal distinct cellular and molecular signalling pathways activated during fibrotic processes. Markedly, we will address how these platforms could provide a better understanding of fibrosis pathophysiology and uncover effective treatment strategies for IPF patients.

Three-dimensional (3D) organoid culture systems have rapidly emerged as powerful tools to study organ development and disease. The lung is a complex and highly specialized organ that comprises more than 40 cell types that offer several region-specific roles. During organogenesis, the lung goes through sequential and morphologically distinctive stages to assume its mature form, both structurally and functionally. As branching takes place, multipotent epithelial progenitors at the distal tips of the growing/bifurcating epithelial tubes progressively become lineage-restricted, giving rise to more differentiated and specialized cell types. Although many cellular and molecular mechanisms leading to branching morphogenesis have been explored, deeper understanding of biological processes governing cell-fate decisions and lung patterning is still needed. Given that these distinct processes cannot be easily analyzed in vivo, 3D culture systems have become a valuable platform to study organogenesis in vitro. This minireview focuses on the current lung organoid systems that recapitulate developmental events occurring before and during branching morphogenesis. In addition, we also discuss their limitations and future directions.

Coronavirus disease 2019 (COVID-19)-associated acute respiratory distress syndrome (ARDS) is associated with high mortality. Lung-protective ventilation is the current standard of care in patients with ARDS, but it might lead to hypercapnia, which is independently associated with worse outcomes. Extracorporeal carbon dioxide removal (ECCO2R) has been proposed as an adjuvant therapy to avoid progression of clinical severity and limit further ventilator-induced lung injury, but its use in COVID-19 has not been described yet. Acute kidney injury requiring renal replacement therapy (RRT) is common among critically ill COVID-19 patients. In centers with available dialysis, low-flow ECCO2R (

Abstract Background Sclerosing angiomatoid nodular transformation is a benign disorder of splenic tissue and is often mistaken as a potentially malignant entity in the diagnostic process. To the best of our knowledge, this is the first report of sclerosing angiomatoid nodular transformation mimicking metastasis of melanoma in the literature. Case presentation A 43-year-old white man presented with a newly found splenic mass 4 years ago to our Department of Gastroenterology. He was diagnosed as having a superficial spreading malignant melanoma localized at his left instep 7 years ago and was successfully treated with radical local resection. Several diagnostic procedures were conducted. Ultrasound showed a hypoechoic lesion in the inferior pole of his spleen with a diameter of 2 cm, blurred boundaries, and inhomogeneous interior pattern. Contrast-enhanced ultrasound was inconclusive and showed only discrete contrast enhancement of the lesion with accentuated nodule-like enrichment of the boundaries in the arterial phase. Computed tomography and magnetic resonance imaging scans showed two splenic lesions which were highly suspicious of metastasis. Magnetic resonance imaging of his head was inconspicuous. Bone scintigraphy showed no abnormal results. Fine-needle aspiration indicated metastasis of the above-mentioned malignant melanoma. We conducted a laparoscopic splenectomy. His intraoperative and postoperative course were uneventful. In contrast to the result of the fine-needle aspiration, the presence of metastasis of melanoma could not be confirmed. Histological analysis revealed nodule-like arrangement of fibroblasts with low cell density and a predominance of dilated capillaries, indicating sclerosing angiomatoid nodular transformation of the spleen. Conclusions There are no preoperative diagnostic imaging procedures which can definitely differentiate sclerosing angiomatoid nodular transformation from malignancies in cases of morphological and immunophenotypic variations of the specimen. Morphological and immunophenotypic variations of the specimen represent a diagnostic challenge and can mimic malignoma. As reported in our case, the specimen obtained by ultrasound-guided fine-needle aspiration led to the diagnosis of metastasis of melanoma. Splenectomy is often conducted due to a splenic mass suspicious of malignoma as described in our case or with unknown valency in different diagnostic imaging procedures.

Progress in developing new therapies for bronchopulmonary dysplasia (BPD) is sometimes complicated by the lack of a standardised animal model. Our objective was to develop a robust hyperoxia-based mouse model of BPD that recapitulated the pathological perturbations to lung structure noted in infants with BPD. Newborn mouse pups were exposed to a varying fraction of oxygen in the inspired air (FiO2) and a varying window of hyperoxia exposure, after which lung structure was assessed by design-based stereology with systemic uniform random sampling. The efficacy of a candidate therapeutic intervention using parenteral nutrition was evaluated to demonstrate the utility of the standardised BPD model for drug discovery. An FiO2 of 0.85 for the first 14 days of life decreased total alveoli number and concomitantly increased alveolar septal wall thickness, which are two key histopathological characteristics of BPD. A reduction in FiO2 to 0.60 or 0.40 also caused a decrease in the total alveoli number, but the septal wall thickness was not impacted. Neither a decreasing oxygen gradient (from FiO2 0.85 to 0.21 over the first 14 days of life) nor an oscillation in FiO2 (between 0.85 and 0.40 on a 24 h:24 h cycle) had an appreciable impact on lung development. The risk of missing beneficial effects of therapeutic interventions at FiO2 0.85, using parenteral nutrition as an intervention in the model, was also noted, highlighting the utility of lower FiO2 in selected studies, and underscoring the need to tailor the model employed to the experimental intervention. Thus, a state-of-the-art BPD animal model that recapitulates the two histopathological hallmark perturbations to lung architecture associated with BPD is described. The model presented here, where injurious stimuli have been systematically evaluated, provides a most promising approach for the development of new strategies to drive postnatal lung maturation in affected infants.

It has been shown previously that increased circulating endothelial cells-derived extracellular vesicles represent an important pathological attribute of pulmonary hypertension. Although it is a well-known fact that inflammatory cells may also release extracellular vesicles, and pulmonary hypertension is a disease associated with abnormal inflammation, there is no profound knowledge with regard to the role of inflammatory cells-derived extracellular vesicles. Therefore, our study demonstrated that circulating levels of extracellular vesicles derived from T-cells are enhanced in various pulmonary hypertension forms and that endothelial cells-derived extracellular vesicles may have distinctive profiles in different clinical subgroups of pulmonary hypertension, which still remains as a poorly treatable and life-threatening disorder.

Abstract Bronchopulmonary dysplasia (BPD) is a common complication of preterm birth characterized by arrested lung alveolarization, which generates lungs that are incompetent for effective gas exchange. We report here deregulated expression of miR‐34a in a hyperoxia‐based mouse model of BPD, where miR‐34a expression was markedly increased in platelet‐derived growth factor receptor (PDGFR)α‐expressing myofibroblasts, a cell type critical for proper lung alveolarization. Global deletion of miR‐34a; and inducible, conditional deletion of miR‐34a in PDGFRα+ cells afforded partial protection to the developing lung against hyperoxia‐induced perturbations to lung architecture. Pdgfra mRNA was identified as the relevant miR‐34a target, and using a target site blocker in vivo, the miR‐34a/Pdgfra interaction was validated as a causal actor in arrested lung development. An antimiR directed against miR‐34a partially restored PDGFRα+ myofibroblast abundance and improved lung alveolarization in newborn mice in an experimental BPD model. We present here the first identification of a pathology‐relevant microRNA/mRNA target interaction in aberrant lung alveolarization and highlight the translational potential of targeting the miR‐34a/Pdgfra interaction to manage arrested lung development associated with preterm birth.

Apicomplexan parasites are obligatory intracellular protozoa. In the case of Toxoplasma gondii, Neospora caninum or Besnoitia besnoiti, to ensure proper tachyzoite production, they need nutrients and cell building blocks. However, apicomplexans are auxotrophic for cholesterol, which is required for membrane biosynthesis. P-glycoprotein (P-gp) is a transmembrane transporter involved in xenobiotic efflux. However, the physiological role of P-gp in cholesterol metabolism is unclear. Here, we analyzed its impact on parasite proliferation in T. gondii-, N. caninum- and B. besnoiti-infected primary endothelial cells by applying different generations of P-gp inhibitors. Host cell treatment with verapamil and valspodar significantly diminished tachyzoite production in all three parasite species, whereas tariquidar treatment affected proliferation only in B. besnoiti. 3D-holotomographic analyses illustrated impaired meront development driven by valspodar treatment being accompanied by swollen parasitophorous vacuoles in the case of T. gondii. Tachyzoite and host cell pre-treatment with valspodar affected infection rates in all parasites. Flow cytometric analyses revealed verapamil treatment to induce neutral lipid accumulation. The absence of a pronounced anti-parasitic impact of tariquidar, which represents here the most selective P-gp inhibitor, suggests that the observed effects of verapamil and valspodar are associated with mechanisms independent of P-gp. Out of the three species tested here, this compound affected only B. besnoiti proliferation and its effect was much milder as compared to verapamil and valspodar.

Branching morphogenesis is the basic developmental mode common to organs such as the lungs that undergo a process of ramification from a rudimentary tree. However, the precise molecular and cellular bases underlying the formation of branching organs are still unclear. As inactivation of fibroblast growth factor receptor 2b (Fgfr2b) signaling during early development leads to lung agenesis, thereby preventing the analysis of this pathway at later developmental stages, we used transgenic mice to induce expression of a soluble form of Fgfr2b to inactivate Fgfr2b ligands at embryonic day (E) 14.5, corresponding to the mid-pseudoglandular stage of lung development. We identified an Fgfr2b signaling signature comprised of 46 genes enriched in the epithelium, some of which were common to, but most of them distinct from, the previously identified Fgfr2b signaling signature at E12.5. Our results indicate that Fgfr2b signaling at E14.5 controls mostly proliferation and alveolar type 2 cell (AT2) differentiation. In addition, inhibition of Fgfr2b signaling at E14.5 leads to morphological and cellular impairment at E18.5, with defective alveolar lineage formation. Further studies will have to be conducted to elucidate the role of Fgfr2b signaling at successive stages (canalicular/saccular/alveolar) of lung development as well as during homeostasis and regeneration and repair after injury.

Alveolar edema, impaired alveolar fluid clearance, and elevated CO2 levels (hypercapnia) are hallmarks of the acute respiratory distress syndrome (ARDS). This study investigated how hypercapnia affects maturation of the Na,K-ATPase (NKA), a key membrane transporter, and a cell adhesion molecule involved in the resolution of alveolar edema in the endoplasmic reticulum (ER). Exposure of human alveolar epithelial cells to elevated CO2 concentrations caused a significant retention of NKA-β in the ER and, thus, decreased levels of the transporter in the Golgi apparatus. These effects were associated with a marked reduction of the plasma membrane (PM) abundance of the NKA-α/β complex as well as a decreased total and ouabain-sensitive ATPase activity. Furthermore, our study revealed that the ER-retained NKA-β subunits were only partially assembled with NKA α-subunits, which suggests that hypercapnia modifies the ER folding environment. Moreover, we observed that elevated CO2 levels decreased intracellular ATP production and increased ER protein and, particularly, NKA-β oxidation. Treatment with α-ketoglutaric acid (α-KG), which is a metabolite that has been shown to increase ATP levels and rescue mitochondrial function in hypercapnia-exposed cells, attenuated the deleterious effects of elevated CO2 concentrations and restored NKA PM abundance and function. Taken together, our findings provide new insights into the regulation of NKA in alveolar epithelial cells by elevated CO2 levels, which may lead to the development of new therapeutic approaches for patients with ARDS and hypercapnia.

Acute lung injury is associated with formation of pulmonary edema leading to impaired gas exchange. Patients with acute respiratory distress syndrome (ARDS) require mechanical ventilation to improve oxygenation; however, the use of relatively low tidal volumes (to minimize further injury of the lung) often leads to further accumulation of carbon dioxide (hypercapnia). Hypercapnia has been shown to impair alveolar fluid clearance (AFC), thereby causing retention of pulmonary edema, and may lead to worse outcomes; however, the underlying molecular mechanisms remain incompletely understood. AFC is critically dependent on the epithelial sodium channel (ENaC), which drives the vectorial transport of Na+ across the alveolar epithelium. Thus, in the current study, we investigated the mechanisms by which hypercapnia effects ENaC cell surface stability in alveolar epithelial cells (AECs). Elevated CO2 levels led to polyubiquitination of β-ENaC and subsequent endocytosis of the α/β-ENaC complex in AECs, which were prevented by silencing the E3 ubiquitin ligase, Nedd4-2. Hypercapnia-induced ubiquitination and cell surface retrieval of ENaC were critically dependent on phosphorylation of the Thr615 residue of β-ENaC, which was mediated by the extracellular signal-regulated kinase (ERK)1/2. Furthermore, activation of ERK1/2 led to subsequent activation of AMP-activated protein kinase (AMPK) and c-Jun N-terminal kinase (JNK)1/2 that in turn phosphorylated Nedd4-2 at the Thr899 residue. Importantly, mutation of Thr899 to Ala markedly inhibited the CO2-induced polyubiquitination of β-ENaC and restored cell surface stability of the ENaC complex, highlighting the critical role of Nedd4-2 phosphorylation status in targeting ENaC. Collectively, our data suggest that elevated CO2 levels promote activation of the ERK/AMPK/JNK axis in a human AEC line, in which ERK1/2 phosphorylates β-ENaC whereas JNK mediates phosphorylation of Nedd4-2, thereby facilitating the channel–ligase interaction. The hypercapnia-induced ENaC dysfunction may contribute to impaired alveolar edema clearance and thus, interfering with these molecular mechanisms may improve alveolar fluid balance and lead to better outcomes in patients with ARDS.

Pneumonia may be caused by a wide range of pathogens and is considered the most common infectious cause of death in humans. Murine acute lung infection models mirror human pathologies in many aspects and contribute to our understanding of the disease and the development of novel treatment strategies. Despite progress in other fields of tissue imaging, histopathology remains the most conclusive and practical read out tool for the descriptive and semiquantitative evaluation of mouse pneumonia and therapeutic interventions. Here, we systematically describe and compare the distinctive histopathological features of established models of acute pneumonia in mice induced by Streptococcus (S.) pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Legionella pneumophila, Escherichia coli, Middle East respiratory syndrome (MERS) coronavirus, influenza A virus (IAV) and superinfection of IAV-incuced pneumonia with S. pneumoniae. Systematic comparisons of the models revealed striking differences in the distribution of lesions, the characteristics of pneumonia induced, principal inflammatory cell types, lesions in adjacent tissues, and the detectability of the pathogens in histological sections. We therefore identified core criteria for each model suitable for practical semiquantitative scoring systems that take into account the pathogen- and model-specific patterns of pneumonia. Other critical factors that affect experimental pathologies are discussed, including infectious dose, time kinetics, and the genetic background of the mouse strain. The substantial differences between the model-specific pathologies underscore the necessity of pathogen- and model-adapted criteria for the comparative quantification of experimental outcomes. These criteria also allow for the standardized validation and comparison of treatment strategies in preclinical models.

From June of 2009 to August of 2010 the influenza subtype H1N1pdm09 caused a worldwide pandemic. The impact on populations and health care systems around the globe evolved differently. Substantial data come from the German national surveillance network in an outpatient and private practice setting, while information on hospitalized patients in Germany is rather limited.Data from the Federal Statistics Office comprising health insurance claims of the entire nationwide inpatient sample from 2005 to 2012 were used to identify patients who were hospitalized for laboratory-confirmed influenza and to analyse demographical aspects, comorbidities, hospitalization duration, outcomes and ventilator use during the pandemic and seasonal waves of influenza.A number of 34,493 admissions for laboratory-confirmed influenza occurred during waves between 2005 and 2012. During the pandemic seasonal waves, the number of hospitalizations vastly surpassed the level that was seen in any of the seasonal waves. A major demographic shift was seen with respect to patient age, as younger patients (< 60 years old) were more frequently hospitalized. Mean length of stay was shorter (149 vs. 193 hours), mean time on ventilation tended to be shorter (261 vs. 305 hours) in young children (< 4 years old) and longer (393 vs. 339 hours) in the elderly (> 60 years old). Time to ventilation was shorter in non-fatal cases (328 vs. 349 hours) and longer in fatal cases (419 vs. 358 hours). Logistic regression was used to show the impact of comorbidities and co-diagnoses on mortality and the need for ventilation, as well as differences between pandemic and seasonal influenza.Inpatient data suggest differences in patient populations during pandemic and seasonal influenza. Younger patients were more frequently hospitalized. Differences with respect to the presence of certain comorbidities and co-diagnoses, length of stay, time to ventilation and ventilation time could be identified.

Influenza Virus (IV) pneumonia is associated with severe damage of the lung epithelium and respiratory failure. Apart from efficient host defense, structural repair of the injured epithelium is crucial for survival of severe pneumonia. The molecular mechanisms underlying stem/progenitor cell mediated regenerative responses are not well characterized. In particular, the impact of IV infection on lung stem cells and their regenerative responses remains elusive. Our study demonstrates that a highly pathogenic IV infects various cell populations in the murine lung, but displays a strong tropism to an epithelial cell subset with high proliferative capacity, defined by the signature EpCamhighCD24lowintegrin(α6)high. This cell fraction expressed the stem cell antigen-1, highly enriched lung stem/progenitor cells previously characterized by the signature integrin(β4)+CD200+, and upregulated the p63/krt5 regeneration program after IV-induced injury. Using 3-dimensional organoid cultures derived from these epithelial stem/progenitor cells (EpiSPC), and in vivo infection models including transgenic mice, we reveal that their expansion, barrier renewal and outcome after IV-induced injury critically depended on Fgfr2b signaling. Importantly, IV infected EpiSPC exhibited severely impaired renewal capacity due to IV-induced blockade of β-catenin-dependent Fgfr2b signaling, evidenced by loss of alveolar tissue repair capacity after intrapulmonary EpiSPC transplantation in vivo. Intratracheal application of exogenous Fgf10, however, resulted in increased engagement of non-infected EpiSPC for tissue regeneration, demonstrated by improved proliferative potential, restoration of alveolar barrier function and increased survival following IV pneumonia. Together, these data suggest that tropism of IV to distal lung stem cell niches represents an important factor of pathogenicity and highlight impaired Fgfr2b signaling as underlying mechanism. Furthermore, increase of alveolar Fgf10 levels may represent a putative therapy to overcome regeneration failure after IV-induced lung injury.

[This corrects the article DOI: 10.1371/journal.ppat.1003188.].

Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria.

In addition to their well-known antibacterial activity some antimicrobial peptides and proteins (AMPs) display also antiviral effects. A 27 aa peptide from the N-terminal part of human bactericidal/permeability-increasing protein (BPI) previously shown to harbour antibacterial activity inhibits the infectivity of multiple Influenza A virus strains (H1N1, H3N2 and H5N1) the causing agent of the Influenza pneumonia. In contrast, the homologous murine BPI-peptide did not show activity against Influenza A virus. In addition human BPI-peptide inhibits the activation of immune cells mediated by Influenza A virus. By changing the human BPI-peptide to the sequence of the mouse homologous peptide the antiviral activity was completely abolished. Furthermore, the human BPI-peptide also inhibited the pathogenicity of the Vesicular Stomatitis Virus but failed to interfere with HIV and measles virus. Electron microscopy indicate that the human BPI-peptide interferes with the virus envelope and at high concentrations was able to destroy the particles completely.

Influenza viruses (IV) cause pneumonia in humans with progression to lung failure and fatal outcome. Dysregulated release of cytokines including type I interferons (IFNs) has been attributed a crucial role in immune-mediated pulmonary injury during severe IV infection. Using ex vivo and in vivo IV infection models, we demonstrate that alveolar macrophage (AM)-expressed IFN-β significantly contributes to IV-induced alveolar epithelial cell (AEC) injury by autocrine induction of the pro-apoptotic factor TNF-related apoptosis-inducing ligand (TRAIL). Of note, TRAIL was highly upregulated in and released from AM of patients with pandemic H1N1 IV-induced acute lung injury. Elucidating the cell-specific underlying signalling pathways revealed that IV infection induced IFN-β release in AM in a protein kinase R- (PKR-) and NF-κB-dependent way. Bone marrow chimeric mice lacking these signalling mediators in resident and lung-recruited AM and mice subjected to alveolar neutralization of IFN-β and TRAIL displayed reduced alveolar epithelial cell apoptosis and attenuated lung injury during severe IV pneumonia. Together, we demonstrate that macrophage-released type I IFNs, apart from their well-known anti-viral properties, contribute to IV-induced AEC damage and lung injury by autocrine induction of the pro-apoptotic factor TRAIL. Our data suggest that therapeutic targeting of the macrophage IFN-β-TRAIL axis might represent a promising strategy to attenuate IV-induced acute lung injury.