Human sample, from patients or healthy donors, are a valuable link between basic research and clinic. Especially in translational research, they play an essential role in understanding development ...and progression of diseases as well as in developing new diagnostic and therapeutic tools. Stored in biobanks, fast access to appropriate material becomes possible. However, biobanking in a clinical context faces several challenges. In practice, collecting samples during clinical routine does not allow to strictly adhere to protocols of sample collection in all aspects. This may influence sample quality to variable degrees. Time from sample draw to asservation is a variable factor, and influences of prolonged storage at ambient temperature of tissues are not well understood. We investigated whether delays between 5 minutes and 3 hours, and the use of RNAlater RNA-preserving reagent would lead to a relevant drop in sample quality, measured by quantitative mRNA expression analysis. Our findings suggest that even under ambient conditions, delays up to 3 hours do not have a major impact on sample quality as long as the tissue remains intact.
Idiopathic pulmonary fibrosis (IPF) is characterized by excessive deposition of extracellular matrix, in particular, collagens. Two IPF therapeutics, nintedanib and pirfenidone, decelerate lung ...function decline, but their underlying mechanisms of action are poorly understood. In this study, we sought to analyze their effects on collagen synthesis and maturation at important regulatory levels. Primary human fibroblasts from patients with IPF and healthy donors were treated with nintedanib (0.01-1.0 μM) or pirfenidone (100-1,000 μM) in the absence or presence of transforming growth factor-β1. Effects on collagen, fibronectin, FKBP10, and HSP47 expression, and collagen I and III secretion, were analyzed by quantitative polymerase chain reaction and Western blot. The appearance of collagen fibrils was monitored by scanning electron microscopy, and the kinetics of collagen fibril assembly was assessed using a light-scattering approach. In IPF fibroblasts, nintedanib reduced the expression of collagen I and V, fibronectin, and FKBP10 and attenuated the secretion of collagen I and III. Pirfenidone also down-regulated collagen V but otherwise showed fewer and less pronounced effects. By and large, the effects were similar in donor fibroblasts. For both drugs, electron microscopy of IPF fibroblast cultures revealed fewer and thinner collagen fibrils compared with untreated controls. Finally, both drugs dose-dependently delayed fibril formation of purified collagen I. In summary, both drugs act on important regulatory levels in collagen synthesis and processing. Nintedanib was more effective in down-regulating profibrotic gene expression and collagen secretion. Importantly, both drugs inhibited collagen I fibril formation and caused a reduction in and an altered appearance of collagen fibril bundles, representing a completely novel mechanism of action for both drugs.
There are various reconstructive methods after total sternectomy. Reproducibility is scarce due to overall small patient numbers. Therefore we present a standardized, interdisciplinary approach for ...thoracic and plastic surgery.
Four patients underwent interdisciplinary chest wall reconstruction with STRATOS® titanium bars and myocutaneous vastus lateralis muscle free flap in our center.
All patients reported chest wall stability after reconstruction. They reported good quality of life, no dyspnea, prolonged pain or impairment in lung function from rigid reconstruction. FEV1/FVC was overall better after surgery. Secondary wound healing was not impaired and there was no implant defect in follow up.
We recommend an interdisciplinary surgical approach in chest wall reconstruction after total sternectomy. The combination of rigid reconstruction with titanium bars and a myocutaneous vastus lateralis muscle free flap renders excellent results in patient satisfaction and is objectifiable via spirometry.
Analyzing the molecular heterogeneity of different forms of organ fibrosis may reveal common and specific factors and thus identify potential future therapeutic targets.
We sought to use ...proteome-wide profiling of human tissue fibrosis to (1) identify common and specific signatures across end-stage interstitial lung disease (ILD) cases, (2) characterize ILD subgroups in an unbiased fashion, and (3) identify common and specific features of lung and skin fibrosis.
We collected samples of ILD tissue (n = 45) and healthy donor control samples (n = 10), as well as fibrotic skin lesions from localized scleroderma and uninvolved skin (n = 6). Samples were profiled by quantitative label-free mass spectrometry, Western blotting, or confocal imaging.
We determined the abundance of more than 7,900 proteins and stratified these proteins according to their detergent solubility profiles. Common protein regulations across all ILD cases, as well as distinct ILD subsets, were observed. Proteomic comparison of lung and skin fibrosis identified a common upregulation of marginal zone B- and B1-cell-specific protein (MZB1), the expression of which identified MZB1
/CD38
/CD138
/CD27
/CD45
/CD20
plasma B cells in fibrotic lung and skin tissue. MZB1 levels correlated positively with tissue IgG and negatively with diffusing capacity of the lung for carbon monoxide.
Despite the presumably high molecular and cellular heterogeneity of ILD, common protein regulations are observed, even across organ boundaries. The surprisingly high prevalence of MZB1-positive plasma B cells in tissue fibrosis warrants future investigations regarding the causative role of antibody-mediated autoimmunity in idiopathic cases of organ fibrosis, such as idiopathic pulmonary fibrosis.
Poor oral hygiene can cause infections and inflammatory diseases. Data on its impact on outcome after lung transplantation (LuTX) is scarce. Most transplant centers have individual standards ...regarding dental care as there is no clinical guideline. This study's objective was to assess LuTX-listed patient's dental status and determine its effect on postoperative outcome.
Two hundred patients having undergone LuTX from 2014 to 2019 were selected. Collected data comprised LuTX-indication, periodontal status, and number of carious teeth/fillings. A preoperative panoramic dental X-ray and a dentist's consultative clarification were mandatory.
63.5% had carious dental status, differing significantly regarding TX-indication (p < 0.001; ILD: 41.7% vs. CF: 3.1% of all patients with carious teeth). Mean age at the time of LuTX differed significantly within these groups. Neither preoperative carious dental status nor periodontitis or bone loss deteriorated post-LuTX survival significantly. No evidence was found that either resulted in a greater number of deaths related to an infectious etiology.
This study shows that carious dental status, periodontitis, and bone loss do not affect post-TX survival. However, literature indicates that they can cause systemic/pulmonary infections that deteriorate post-LuTX survival. Regarding the absence of standardized guidelines regarding dental care and LuTX, we strongly recommend emphasizing research in this field.
Abstract Background The aim of this single-center study was to analyze factors predicting long-term outcomes following surgical resection of pulmonary metastases in patients with renal cell ...carcinoma. Methods Two hundred two consecutive patients entered the study. Overall survival was analyzed by the Kaplan-Meier method. Multivariate analysis was performed using Cox regression models. Results In 175 cases (87%), curative resection of the pulmonary metastases was achievable, with median survival of 43 months. Multivariate analysis revealed complete metastasectomy (R0), metastasis size >3 cm, positive nodal status of the primary tumor, synchronous metastases, pleural infiltration, and tumor-infiltrated hilar or mediastinal lymph nodes as independent prognostic factors for survival. On the basis of these findings, a new scoring system (the Munich score) was established to predict survival, which discriminates 3 groups with low, intermediate, and high risk for poor outcomes (median survival, 90, 31, and 14 months, respectively, P < .001). Conclusions The aim of the Munich score is to define patients with low, intermediate, and high risk for poor survival and will help identify patients who may benefit from further adjuvant therapy.
Interstitial lung disease (ILD) is a heterogenous group of lung disorders where destruction and incomplete regeneration of the lung parenchyma often results in persistent architectural distortion of ...the pulmonary scaffold. Continuous mesenchyme-centered, disease-relevant signaling likely initiates and perpetuates the fibrotic remodeling process, specifically targeting the epithelial cell compartment, thereby destroying the gas exchange area.
With the aim of identifying functional mediators of the lung mesenchymal-epithelial crosstalk with potential as new targets for therapeutic strategies, we developed a 3D organoid co-culture model based on human induced pluripotent stem cell-derived alveolar epithelial type 2 cells that form alveolar organoids in presence of lung fibroblasts from fibrotic-ILD patients, in our study referring to cases of pulmonary fibrosis, as well as control cell line (IMR-90).
While organoid formation capacity and size was comparable in the presence of fibrotic-ILD or control lung fibroblasts, metabolic activity was significantly increased in fibrotic-ILD co-cultures. Alveolar organoids cultured with fibrotic-ILD fibroblasts further demonstrated reduced stem cell function as reflected by reduced Surfactant Protein C gene expression together with an aberrant basaloid-prone differentiation program indicated by elevated Cadherin 2, Bone Morphogenic Protein 4 and Vimentin transcription. To screen for key mediators of the misguided mesenchymal-to-epithelial crosstalk with a focus on disease-relevant inflammatory processes, we used mass spectrometry and characterized the secretome of end stage fibrotic-ILD lung fibroblasts in comparison to non-chronic lung disease (CLD) patient fibroblasts. Out of the over 2000 proteins detected by this experimental approach, 47 proteins were differentially abundant comparing fibrotic-ILD and non-CLD fibroblast secretome. The fibrotic-ILD secretome profile was dominated by chemokines, including CXCL1, CXCL3, and CXCL8, interfering with growth factor signaling orchestrated by Interleukin 11 (IL11), steering fibrogenic cell-cell communication, and proteins regulating extracellular matrix remodeling including epithelial-to-mesenchymal transition. When in turn treating alveolar organoids with IL11, we recapitulated the co-culture results obtained with primary fibrotic-ILD fibroblasts including changes in metabolic activity.
We identified mediators likely contributing to the disease-perpetuating mesenchymal-to-epithelial crosstalk in ILD. In our alveolar organoid co-cultures, we were able to highlight the importance of fibroblast-initiated aberrant epithelial differentiation and confirmed IL11 as a key player in fibrotic-ILD pathogenesis by unbiased fibroblast secretome analysis.
IntroductionEnvironmental pollutants injure the mucociliary elevator, thereby provoking disease progression in chronic obstructive pulmonary disease (COPD). Epithelial resilience mechanisms to ...environmental nanoparticles in health and disease are poorly characterised.MethodsWe delineated the impact of prevalent pollutants such as carbon and zinc oxide nanoparticles, on cellular function and progeny in primary human bronchial epithelial cells (pHBECs) from end-stage COPD (COPD-IV, n=4), early disease (COPD-II, n=3) and pulmonary healthy individuals (n=4). After nanoparticle exposure of pHBECs at air–liquid interface, cell cultures were characterised by functional assays, transcriptome and protein analysis, complemented by single-cell analysis in serial samples of pHBEC cultures focusing on basal cell differentiation.ResultsCOPD-IV was characterised by a prosecretory phenotype (twofold increase in MUC5AC+) at the expense of the multiciliated epithelium (threefold reduction in Ac-Tub+), resulting in an increased resilience towards particle-induced cell damage (fivefold reduction in transepithelial electrical resistance), as exemplified by environmentally abundant doses of zinc oxide nanoparticles. Exposure of COPD-II cultures to cigarette smoke extract provoked the COPD-IV characteristic, prosecretory phenotype. Time-resolved single-cell transcriptomics revealed an underlying COPD-IV unique basal cell state characterised by a twofold increase in KRT5+ (P=0.018) and LAMB3+ (P=0.050) expression, as well as a significant activation of Wnt-specific (P=0.014) and Notch-specific (P=0.021) genes, especially in precursors of suprabasal and secretory cells.ConclusionWe identified COPD stage-specific gene alterations in basal cells that affect the cellular composition of the bronchial elevator and may control disease-specific epithelial resilience mechanisms in response to environmental nanoparticles. The identified phenomena likely inform treatment and prevention strategies.
Prognostic phenotypes of early-stage lung adenocarcinoma Lamort, Anne-Sophie; Kaiser, Jan Christian; Pepe, Mario A.A. ...
European respiratory journal/The European respiratory journal,
07/2022, Volume:
60, Issue:
1
Journal Article
Peer reviewed
Open access
Background
Survival after curative resection of early-stage lung adenocarcinoma (LUAD) varies and prognostic biomarkers are urgently needed.
Methods
Large-format tissue samples from a prospective ...cohort of 200 patients with resected LUAD were immunophenotyped for cancer hallmarks TP53, NF1, CD45, PD-1, PCNA, TUNEL and FVIII, and were followed for a median of 2.34 (95% CI 1.71–3.49) years.
Results
Unsupervised hierarchical clustering revealed two patient subgroups with similar clinicopathological features and genotype, but with markedly different survival: “proliferative” patients (60%) with elevated TP53, NF1, CD45 and PCNA expression had 50% 5-year overall survival, while “apoptotic” patients (40%) with high TUNEL had 70% 5-year survival (hazard ratio 2.23, 95% CI 1.33–3.80; p=0.0069). Cox regression and machine learning algorithms including random forests built clinically useful models: a score to predict overall survival and a formula and nomogram to predict tumour phenotype. The distinct LUAD phenotypes were validated in The Cancer Genome Atlas and KMplotter data, and showed prognostic power supplementary to International Association for the Study of Lung Cancer tumour–node–metastasis stage and World Health Organization histologic classification.
Conclusions
Two molecular subtypes of LUAD exist and their identification provides important prognostic information.
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