The effect of tracheal occlusion in congenital diaphragmatic hernia in the nitrofen rat lung explant model.

PURPOSE: Here, we establish a tracheal occlusion (TO) model with rat lung explants in nitrofen-induced pulmonary hypoplasia in the congenital diaphragmatic hernia (CDH). METHODS: We extracted lungs from rats on an embryonic day 18. We mimicked TO in the lung explants by tying the trachea. We assessed lung weight, morphometry, and abundance of Ki-67, Active caspase-3, and Prosurfactant Protein C (proSP-C) with immunofluorescence. RESULTS: Lung weight was higher in TO + than TO - on day 1. Abundance of Ki-67 was higher in TO + than TO - (0.15 vs. 0.32, p = 0.009 for day 1, 0.07 vs. 0.17, p = 0.004 for day 2, 0.07 vs. 0.12, p = 0.044 for day 3), and Active caspase-3 was higher in TO + than TO - on day 2 and day 3 (0.04 vs. 0.03 p = 0.669 for day 1, 0.03 vs. 0.13 p < 0.001 for day 2, 0.04 vs. 0.17 p = 0.008 for day3). However, proSP-C protein abundance was lower in TO + than TO - (67.9 vs. 59.1 p = 0.033 for day 1, 73.5 vs. 51.6 p = 0.038 for day 2, 83.1 vs. 56.4 p = 0.009 for day 3). CONCLUSIONS: The TO model in lung explants mimics the outcomes of current surgical models of TO and further studies can reveal the cellular and molecular effects of TO in CDH lungs.

Incidentally Detected Malignancies in Lung Explants.

Introduction: Incidentally detected malignancies in lung explants portend risk of early cancer recurrence and metastases with posttransplant immunosuppression. We present a series of lung transplant recipients with previously unverified malignancies in native lung explants. Design: We reviewed the histopathology, radiographic imaging, and management of lung explant malignancies at our institution over 10 years (2011-2020). Endpoints were survival and allograft rejection. Results: An explant malignancy was found in 1.3% (11/855) of lung transplant recipients (6 [55%] men; median age 68 years; 6 [55%] ex-smokers [median pack-years, 25]). Nine (82%) were adenocarcinoma, 1 (9%) was squamous cell carcinoma (SCC), and 1 (9%) was follicular lymphoma. Three patients (27%) had multifocal involvement (≥3 lobes), 4 (36%) had nodal involvement, and the median (range) tumor size was 2.7 (0.4-19) cm. The median interval between last imaging and transplant was 58 (29-144) days. Mycophenolate mofetil was discontinued or reduced in all; everolimus was used in 2 patients, and cisplatin-pemetrexed chemotherapy was used in 2 patients. The prevalence of acute cellular rejection and chronic rejection was 27% and 9%, respectively. Lung recipients with cancer had significantly lower survival than those without (36.4% vs 67.3%, p = 0.002); median survival was 27 (17, 65) months in 4 recipients who were alive and cancer-free at the end of the study period. Conclusions: Unidentified malignancies, commonly adenocarcinoma, can be detected in explanted native lungs. Pneumonectomy may be curative in SCC, lymphoproliferative disorders, and stage I adenocarcinoma. Modulating immunosuppression to prevent allograft rejection and tumor proliferation is warranted.

Silencing Heat Shock Protein 47 (HSP47) in Fibrogenic Precision-Cut Lung Slices: A Surprising Lack of Effects on Fibrogenesis?

Idiopathic pulmonary fibrosis (IPF) is a chronic disease that is characterized by the excessive deposition of scar tissue in the lungs. As currently available treatments are unable to restore lung function in patients, there is an urgent medical need for more effective drugs. Developing such drugs, however, is challenging because IPF has a complex pathogenesis. Emerging evidence indicates that heat shock protein 47 (HSP47), which is encoded by the gene Serpinh1, may be a suitable therapeutic target as it is required for collagen synthesis. Pharmacological inhibition or knockdown of HSP47 could therefore be a promising approach to treat fibrosis. The objective of this study was to assess the therapeutic potential of Serpinh1-targeting small interfering RNA (siRNA) in fibrogenic precision-cut lung slices prepared from murine tissue. To enhance fibrogenesis, slices were cultured for up to 144 h with transforming growth factor ß1. Self-deliverable siRNA was used to knockdown mRNA and protein expression, without affecting the viability and morphology of slices. After silencing HSP47, only the secretion of fibronectin was reduced while other aspects of fibrogenesis remained unaffected (e.g., myofibroblast differentiation as well as collagen secretion and deposition). These observations are surprising as others have shown that Serpinh1-targeting siRNA suppressed collagen deposition in animals. Further studies are therefore warranted to elucidate downstream effects on fibrosis upon silencing HSP47.

Microbial Community Composition in Explanted Cystic Fibrosis and Control Donor Lungs.

To date, investigations of the microbiota in the lungs of people with Cystic Fibrosis (PWCF) have primarily focused on microbial community composition in luminal mucus, with fewer studies observing the microbiota in tissue samples from explanted lung tissue. Here, we analysed both tissue and airway luminal mucus samples extracted from whole explanted lungs of PWCF and unused donor lungs. We determined if the lung microbiota in end-stage CF varied within and between patients, was spatially heterogeneous and related to localized structural damage. Microbial community composition was determined by Illumina MiSeq sequencing and related to the CF-Computed Tomography (CT) score and features of end-stage lung disease on micro-CT. Ninety-eight CF tissue (n=11 patients), 20 CF luminal mucus (n=8 patients) and 33 donor tissue (n=4 patients) samples were analysed. Additionally, we compared 20 paired CF tissue and luminal mucus samples that enabled a direct "geographical" comparison of the microbiota in these two niches. Significant differences in microbial communities were apparent between the 3 groups. However, overlap between the three groups, particularly between CF and donor tissue and CF tissue and CF luminal mucus was also observed. Microbial diversity was lower in CF luminal mucus compared to CF tissue, with dominance higher in luminal mucus. For both CF and donor tissue, intra- and inter-patient variability in ecological parameters was observed. No relationships were observed between ecological parameters and CF-CT score, or features of end-stage lung disease. The end-stage CF lung is characterised by a low diversity microbiota, differing within and between individuals. No clear relationship was observed between regional microbiota variation and structural lung damage.

Chloroquine Inhibits the Release of Inflammatory Cytokines by Human Lung Explants.

On human lung parenchymal explants, chloroquine concentration clinically achievable in the lung (100 µM) inhibited the lipopolysaccharide-induced release of TNF-ɑ (by 76%), IL-6 (by 68%), CCL2 (by 72%), and CCL3 (by 67%). Besides its antiviral activity, chloroquine might also mitigate the cytokine storm associated with severe pneumonia caused by coronaviruses.

Antiviral potential of human IFN-α subtypes against influenza A H3N2 infection in human lung explants reveals subtype-specific activities.

Influenza is an acute respiratory infection causing high morbidity and mortality in annual outbreaks worldwide. Antiviral drugs are limited and pose the risk of resistance development, calling for new treatment options. IFN-α subtypes are immune-stimulatory cytokines with strong antiviral activities against IAV in vitro and in vivo. However, the clinical use of IFN-α2, the only licensed subtype of this multi-gene family, could not prevent or limit IAV infections in humans. However, the other subtypes were not investigated.Therefore, this study evaluated the induction and antiviral potential of all human IFN-α subtypes during H3N2 IAV infection in human lung explants. We found that subtypes with weak antiviral activities were preferentially induced during IAV infection in human lungs. Intriguingly, non-induced subtypes α16, α5 and α4 suppressed viral replication up to 230-fold more efficiently than α2. Furthermore, our results demonstrate that subtypes with stronger antiviral activities induce higher expression of IAV-specific restriction factors and that MxA expression is a determinant of the subtype-specific antiviral activity towards H3N2 IAV. These results corroborate that IFN-α subtypes exhibit differential antiviral activities and emphasize that subtypes α16, α5 and α4 should be further investigated for the prevention and treatment of severe infections with seasonal H3N2 IAV.

High Diagnostic Accuracy of Transbronchial Cryobiopsy in Fibrotic Interstitial Lung Diseases Compared to Final Explant Diagnosis.

BACKGROUND: A diagnostic lung biopsy may be required in some cases of fibrotic interstitial lung diseases (ILD). Transbronchial cryobiopsy has been suggested as a possible alternative to surgical lung biopsy. However, previous estimates of its diagnostic yield were not validated compared to the definitive diagnosis in explanted lungs. OBJECTIVES: We aimed to assess the diagnostic accuracy of cryobiopsy in fibrotic ILD patients who subsequently had lung transplantation. METHODS: All 197 patients who underwent lung transplantation at our Center due to fibrotic ILD from January 2010 to May 2018, were screened for the presence of a pre-transplant cryobiopsy. Fourteen patients who underwent cryobiopsy before transplantation were identified. Two expert lung pathologists blindedto the explant diagnoses, independently examined these cryobiopsy specimens to decide if they match guideline criteria for usual interstitial pneumonia (UIP) pattern or an alternative diagnosis. The primary measure was the diagnostic accuracy of cryobiopsy to detect or refute a UIP pattern, as compared to the final explant diagnosis. RESULTS: Median time between cryobiopsy and transplantation was 1.4 years. All 14 cryobiopsy samples contained adequate alveolar tissue. The explant diagnosis of 13/14 patients was UIP. The two pathologists correctly diagnosed or refuted UIP in the cryobiopsy specimen in 12/14 cases (85.7%) and 11/14 cases (78.6%), respectively. The level of diagnostic agreement between pathologists was good (kappa 0.59, p = 0.016). CONCLUSIONS: Compared to the final explant diagnosis, transbronchial cryobiopsy had high diagnostic accuracy and good inter-observer agreement for UIP pattern. These findings support a potential diagnostic role for cryobiopsy in experienced centers.

Imaging and Analysis of Mouse Embryonic Whole Lung, Isolated Tissue, and Lineage-Labelled Cell Culture.

Research on lung development and disease frequently utilizes mouse models to conduct in vitro experiments. Such experiments involve multiple methodologically distinct stages, from careful consideration of mouse models used to obtain biological samples, to the culturing and imaging of those samples, and finally, to post-imaging analysis. Here, we detail basic protocols to assist with each of these stages. First, we discuss harvesting and preparing biological samples; second, we focus on culturing embryonic whole lung explants and isolated mesenchyme and epithelium; third, we specify the basics of obtaining still and live images; and finally, we bring these methods together by considering and briefly analyzing a lineage-labelling experiment.

Incidental extensive adenocarcinoma in lungs explanted from a transplant recipient with an idiopathic pulmonary fibrosis flare-up: A clinical dilemma.

Patients under consideration for lung transplantation as treatment for end-stage lung diseases such as idiopathic pulmonary fibrosis (IPF) often have risk factors such as a history of smoking or concomitant emphysema, both of which can predispose the patient to lung cancer. In fact, IPF itself increases the risk of lung cancer development by 6.8% to 20%. Solid organ malignancy (non-skin) is an established contraindication for lung transplantation. We encountered a clinical dilemma in a patient who presented with an IPF flare-up and underwent urgent evaluation for lung transplantation. After transplant, the patient's explanted lungs showed extensive adenocarcinoma in situ, with the foci of invasion and metastatic adenocarcinoma in N1-level lymph nodes, as well as usual interstitial pneumonia. Retrospectively, we saw no evidence to suggest malignancy in addition to the IPF flare-up. Clinical diagnostic dilemmas such as this emphasize the need for new noninvasive testing that would facilitate malignancy diagnosis in patients too sick to undergo invasive tissue biopsy for diagnosis. Careful pathological examination of explanted lungs in patients with IPF is critical, as it can majorly influence immunosuppressive regimens, surveillance imaging, and overall prognosis after lung transplant.

Characterization of TLR-induced inflammatory responses in COPD and control lung tissue explants.

PURPOSE: Viruses are a common cause of exacerbations in chronic obstructive pulmonary disease (COPD). They activate toll-like receptors (TLRs) 3, 7, and 8, leading to a pro-inflammatory response. We have characterized the responses of TLR3 and TLR7/8 in lung tissue explants from COPD patients and control smokers. METHODS: We prepared lung whole tissue explants (WTEs) from patients undergoing surgery for confirmed or suspected lung cancer. In order to mimic the conditions of viral infection, we used poly(I:C) for TLR3 stimulation and R848 for TLR7/8 stimulation. These TLR ligands were used alone and in combination. The effects of tumor necrosis factor α (TNFα) neutralization and dexamethasone on TLR responses were examined. Inflammatory cytokine release was measured by enzyme-linked immunosorbent assay and gene expression by quantitative real-time polymerase chain reaction. RESULTS: WTEs from COPD patients released higher levels of pro-inflammatory cytokines compared with WTEs from smokers. Activation of multiple TLRs led to a greater than additive release of TNFα and CCL5. TNFα neutralization and dexamethasone treatment decreased cytokine release. CONCLUSION: This WTE model shows an enhanced response of COPD compared with controls, suggesting an increased response to viral infection. There was amplification of innate immune responses with multiple TLR stimulation.