The Immunopathology of Pulmonary Rejection after Murine Lung Transplantation.

To improve outcomes following lung transplantation, it is essential to understand the immunological mechanisms that result in chronic graft failure. The associated clinical syndrome is termed chronic lung allograft dysfunction (CLAD), which is known to be induced by alloimmune-dependent (i.e., rejection) and alloimmune-independent factors (e.g., infections, reflux and environmental factors). We aimed to explore the alloimmune-related mechanism, i.e., pulmonary rejection. In this study, we use a murine orthotopic left lung transplant model using isografts and allografts (C57BL/6 or BALB/c as donors to C57BL/6 recipients), with daily immunosuppression (10 mg/kg cyclosporin A and 1.6 mg/kg methylprednisolone). Serial sacrifice was performed at days 1, 7 and 35 post-transplantation (n = 6 at each time point for each group). Left transplanted lungs were harvested, a single-cell suspension was made and absolute numbers of immune cells were quantified using multicolor flow cytometry. The rejection process followed the principles of a classic immune response, including innate but mainly adaptive immune cells. At day 7 following transplantation, the numbers of interstitial macrophages, monocytes, dendritic cells, NK cells, NKT cells, CD4+ T cells and CD8+ T and B cells were increased in allografts compared with isografts. Only dendritic cells and CD4+ T cells remained elevated at day 35 in allografts. Our study provides insights into the immunological mechanisms of true pulmonary rejection after murine lung transplantation. These results might be important in further research on diagnostic evaluation and treatment for CLAD.

The memory of airway epithelium damage in smokers and COPD patients.

Chronic obstructive pulmonary disease (COPD), a devastating and irreversible lung disease, causes structural and functional defects in the bronchial epithelium, the (ir)reversibility of which remains unexplored in vitro. This study aimed to investigate the persistence of COPD-related epithelial defects in long-term airway epithelial cultures derived from non-smokers, smokers, and COPD patients. Barrier function, polarity, cell commitment, epithelial-to-mesenchymal transition, and inflammation were evaluated and compared with native epithelium characteristics. The role of inflammation was explored using cytokines. We show that barrier dysfunction, compromised polarity, and lineage abnormalities observed in smokers and COPD persisted for up to 10 wk. Goblet cell hyperplasia was associated with recent cigarette smoke exposure. Conversely, increased IL-8/CXCL-8 release and abnormal epithelial-to-mesenchymal transition diminished over time. These ex vivo observations matched surgical samples' abnormalities. Cytokine treatment induced COPD-like changes in control cultures and reactivated epithelial-to-mesenchymal transition in COPD cells. In conclusion, these findings suggest that the airway epithelium of smokers and COPD patients retains a multidimensional memory of its original state and previous cigarette smoke-induced injuries, maintaining these abnormalities for extended periods.

The hemodynamic interplay between pulmonary ischemia-reperfusion injury and right ventricular function in lung transplantation: a translational porcine model.

Lung transplantation (LTx) is a challenging procedure. Following the process of ischemia-reperfusion injury, the transplanted pulmonary graft might become severely damaged, resulting in primary graft dysfunction. In addition, during the intraoperative window, the right ventricle (RV) is at risk of acute failure. The interaction of right ventricular function with lung injury is, however, poorly understood. We aimed to address this interaction in a translational porcine model of pulmonary ischemia-reperfusion injury. Advanced pulmonary and hemodynamic assessment was used, including right ventricular pressure-volume loop analysis. The acute model was based on clamping and unclamping of the left lung hilus, respecting the different hemodynamic phases of a clinical lung transplantation. We found that forcing entire right ventricular cardiac output through a lung suffering from ischemia-reperfusion injury increased afterload (pulmonary vascular resistance from baseline to end experiment P < 0.0001) and induced right ventricular failure (RVF) in 5/9 animals. Notably, we identified different compensation patterns in failing versus nonfailing ventricles (arterial elastance P = 0.0008; stroke volume P < 0.0001). Furthermore, increased vascular pressure and flow produced by the right ventricle resulted in higher pulmonary injury, as measured by ex vivo CT density (correlation: pressure r = 0.8; flow r = 0.85). Finally, RV ischemia as measured by troponin-T was negatively correlated with pulmonary injury (r = -0.76); however, troponin-T values did not determine RVF in all animals. In conclusion, we demonstrate a delicate balance between development of pulmonary ischemia-reperfusion injury and right ventricular function during lung transplantation. Furthermore, we provide a physiological basis for potential benefit of extracorporeal life support technology.NEW & NOTEWORTHY In contrast to the abundant literature of mechanical pulmonary artery clamping to increase right ventricular afterload, we developed a model adding a biological factor of pulmonary ischemia-reperfusion injury. We did not only focus on the right ventricular behavior, but also on the interaction with the injured lung. We are the first to describe this interaction while addressing the hemodynamic intraoperative phases of clinical lung transplantation.

Lung Transplant Outcome From Selected Older Donors (≥70 Years) Equals Younger Donors (<70 Years): A Propensity-matched Analysis.

OBJECTIVE: To describe our experience with lung transplantation (LTx) from donors ≥70 years and compare short and long-term outcomes to a propensity-matched cohort of donors <70 years. BACKGROUND: Although extended-criteria donors have been widely used to enlarge the donor pool, the experience with LTx from older donors (≥70 years) remains limited. METHODS: All single-center bilateral LTx between 2010 and 2020 were retrospectively analyzed. Matching (1:1) was performed for the donor (type, sex, smoking history, x-ray abnormalities, partial pressure of oxygen/fraction of inspired oxygen ratio, and time on ventilator) and recipient characteristics (age, sex, LTx indication, perioperative extracorporeal life support, and cytomegalovirus mismatch). Primary graft dysfunction grade-3, 5-year patient, and chronic lung allograft dysfunction-free survival were analyzed. RESULTS: Out of 647 bilateral LTx, 69 were performed from donors ≥70 years. The mean age in the older donor cohort was 74 years (range: 70-84 years) versus 49 years (range: 12-69 years) in the matched younger group. No significant differences were observed in the length of ventilatory support, intensive care unit, or hospital stay. Primary graft dysfunction-3 was 26% in the older group versus 29% in younger donor recipients ( P = 0.85). Reintervention rate was comparable (29% vs 16%; P = 0.10). Follow-up bronchoscopy revealed no difference in bronchial anastomotic complications ( P = 1.00). Five-year patient and chronic lung allograft dysfunction-free survivals were 73.6% versus 73.1% ( P = 0.72) and 51.5% versus 59.2% ( P = 0.41), respectively. CONCLUSIONS: LTx from selected donors ≥70 years is feasible and safe, yielding comparable short and long-term outcomes in a propensity-matched analysis with younger donors (<70 years).

RIPK1 kinase-dependent inflammation and cell death contribute to the pathogenesis of COPD.

BACKGROUND: Receptor-interacting protein kinase 1 (RIPK1) is a key mediator of regulated cell death (including apoptosis and necroptosis) and inflammation, both drivers of COPD pathogenesis. We aimed to define the contribution of RIPK1 kinase-dependent cell death and inflammation in the pathogenesis of COPD. METHODS: We assessed RIPK1 expression in single-cell RNA sequencing (RNA-seq) data from human and mouse lungs, and validated RIPK1 levels in lung tissue of COPD patients via immunohistochemistry. Next, we assessed the consequences of genetic and pharmacological inhibition of RIPK1 kinase activity in experimental COPD, using Ripk1 S25D/S25D kinase-deficient mice and the RIPK1 kinase inhibitor GSK'547. RESULTS: RIPK1 expression increased in alveolar type 1 (AT1), AT2, ciliated and neuroendocrine cells in human COPD. RIPK1 protein levels were significantly increased in airway epithelium of COPD patients compared with never-smokers and smokers without airflow limitation. In mice, exposure to cigarette smoke (CS) increased Ripk1 expression similarly in AT2 cells, and further in alveolar macrophages and T-cells. Genetic and/or pharmacological inhibition of RIPK1 kinase activity significantly attenuated airway inflammation upon acute and subacute CS exposure, as well as airway remodelling, emphysema, and apoptotic and necroptotic cell death upon chronic CS exposure. Similarly, pharmacological RIPK1 kinase inhibition significantly attenuated elastase-induced emphysema and lung function decline. Finally, RNA-seq on lung tissue of CS-exposed mice revealed downregulation of cell death and inflammatory pathways upon pharmacological RIPK1 kinase inhibition. CONCLUSIONS: RIPK1 kinase inhibition is protective in experimental models of COPD and may represent a novel promising therapeutic approach.

Can we attenuate ischaemia-reperfusion injury of allografts in a porcine left lung transplant models by adsorption of cytokines?

OBJECTIVES: Primary graft dysfunction resulting from ischaemia-reperfusion injury remains a major obstacle after lung transplantation (LTx) and is associated with morbidity and mortality. Continuous release of inflammatory cytokines, due to the process of ischaemia and reperfusion, triggers a complex cascade of apoptosis and necrosis resulting in graft dysfunction. Previous studies demonstrated successful graft improvement by cytokine filtration during ex vivo lung perfusion. We hypothesize that plasma cytokine filtration with CytoSorb® during in vivo graft perfusion immediately after implantation may attenuate ischaemia-reperfusion injury after left LTx in a porcine model. METHODS: Left porcine LTx was performed with allografts preserved for 24 h at 4°C. In the treatment group [T] (n = 7), a veno-venous shunt was created to insert the cytokine filter (CytoSorbents, Berlin, Germany). In the sham group [S] (n = 4), the shunt was created without the filter. Haemodynamic parameters, lung mechanics, blood gases and plasma cytokines were assessed during 6 h in vivo reperfusion. RESULTS: During 6 h of reperfusion, significant differences in plasma pro-inflammatory cytokine [interferon (IFN)-α, IFN-γ and interleukin (IL)-6] concentrations were observed between [T] and [S], but surprisingly with higher plasma levels in the [T] group. Plasma concentrations of other pro-inflammatory cytokines (IL-1ß, IL-12p40, IL-4, IL-6, IL-8, IFN-α, IFN-γ and tumour necrosis factor-α) and anti-inflammatory cytokines (IL-10) did not find any evidence for a difference. Furthermore, our study failed to show meaningful difference in haemodynamics and blood gases. Also, no statistically significant differences were found between [T] and [S] in biopsies and wet-to-dry ratio at the end of the experiment. CONCLUSIONS: In our porcine left LTx model cytokine filtration did not achieve the intended effect. This is in contrast to previous studies with CytoSorb use during ex vivo lung perfusion as a surrogate LTx model. Our findings might highlight the fact that the theoretical benefit of inserting an additional cytokine adsorber to improve graft function in clinical practice should be critically evaluated with further studies.

Allogeneic Hematopoietic Stem Cell Transplantation After Prior Lung Transplantation for Hereditary Pulmonary Alveolar Proteinosis: A Case Report.

Pulmonary alveolar proteinosis (PAP) is a rare, diffuse lung disorder characterized by surfactant accumulation in the small airways due to defective clearance by alveolar macrophages, resulting in impaired gas exchange. Whole lung lavage is the current standard of care treatment for PAP. Lung transplantation is an accepted treatment option when whole lung lavage or other experimental treatment options are ineffective, or in case of extensive pulmonary fibrosis secondary to PAP. A disadvantage of lung transplantation is recurrence of PAP in the transplanted lungs, especially in hereditary PAP. The hereditary form of PAP is an ultra-rare condition caused by genetic mutations in genes encoding for the granulocyte macrophage-colony stimulating factor (GM-CSF) receptor, and intrinsically affects bone marrow derived-monocytes, which differentiate into macrophages in the lung. Consequently, these macrophages typically display disrupted GM-CSF receptor-signaling, causing defective surfactant clearance. Bone marrow/hematopoietic stem cell transplantation may potentially reverse the lung disease in hereditary PAP. In patients with hereditary PAP undergoing lung transplantation, post-lung transplant recurrence of PAP may theoretically be averted by subsequent hematopoietic stem cell transplantation, which results in a graft-versus-disease (PAP) effect, and thus could improve long-term outcome. We describe the successful long-term post-transplant outcome of a unique case of end-stage respiratory failure due to hereditary PAP-induced pulmonary fibrosis, successfully treated by bilateral lung transplantation and subsequent allogeneic hematopoietic stem cell transplantation. Our report supports treatment with serial lung and hematopoietic stem cell transplantation to improve quality of life and prolong survival, without PAP recurrence, in selected patients with end-stage hereditary PAP.

The MUC5B Promoter Polymorphism is Not Associated With Non-ILD Chronic Respiratory Diseases or Post-transplant Outcome.

The MUC5B promoter polymorphism (rs35705950) has been associated with interstitial lung disease (ILD) and with prolonged pre-transplant survival in idiopathic pulmonary fibrosis (IPF), but no information is available regarding its prevalence in other respiratory diseases and its influence on post-transplant outcome. We included the Leuven lung transplantation cohort between 1991 and 2015 (n = 801). We assessed the minor allele frequency (MAF) of the MUC5B variant in the entire study cohort and investigated the influence of recipient MUC5B promoter polymorphism on post-transplant outcome in patients who were transplanted after 2004. MUC5B was successfully genotyped in 746 patients. The MAF was significantly higher in ILD (17.6%) compared to chronic obstructive pulmonary disease (COPD)/emphysema (9.3%), cystic fibrosis (CF)/bronchiectasis (BRECT) (7.5%) and pulmonary hypertension (PHT) (7.4%) (p < 0.001). No association was observed between rs35705950 and chronic lung allograft dysfunction (CLAD)/graft loss in the ILD population [CLAD: HR 1.37 95% CI (0.70-2.68); graft loss: HR 1.02 95% CI (0.55-1.89)], nor the entire study cohort [CLAD: HR 0.96 95% CI (0.69-1.34); graft loss: HR 0.97 95% CI (0.70-1.35)]. The MUC5B promoter polymorphism is a very specific predictive factor for the presence of pulmonary fibrosis as it is only associated with pulmonary fibrosis and not with other chronic respiratory diseases. While the MUC5B promoter variant is associated with better pre-transplant survival among IPF patients, recipient MUC5B promoter variant does not play a role in post-transplant outcome.

Pulmonary graft-versus-host disease and chronic lung allograft dysfunction: two sides of the same coin?

Allogeneic haematopoietic stem-cell transplantation (HSCT) and lung transplantation are both established life-saving treatment options for carefully selected patients with various haematological disorders or end-stage lung diseases, respectively. However, long-term survival after allogeneic HSCT is severely limited by chronic graft-versus-host disease (cGVHD)-of which pulmonary cGVHD in particular has a very poor prognosis-and long-term survival after lung transplantation is hampered by chronic lung allograft dysfunction (CLAD). Both pulmonary cGVHD and CLAD are characterised by similar underlying immunopathology, which results in transplant-related pulmonary fibrosis and structural lung remodelling, leading to respiratory dysfunction. Accurate clinical identification and appropriate management is of utmost importance to allow for timely diagnosis, to further optimise current preventive and treatment strategies of pulmonary cGVHD and CLAD, and to ameliorate quality of life and long-term outcomes after allogeneic HSCT and lung transplantation. In this Review, we provide a unique state-of-the-art perspective of both entities for respiratory care practitioners.

A Comprehensive Review on the Surgical Aspect of Lung Transplant Models in Mice and Rats.

Lung transplantation improves the outcome and quality of life of patients with end-stage pulmonary disease. However, the procedure is still hampered by the lack of suitable donors, the complexity of the surgery, and the risk of developing chronic lung allograft dysfunction. Over the past decades, translational experiments in animal models have led to a better understanding of physiology and immunopathology following the lung transplant procedure. Small animal models (e.g., rats and mice) are mostly used in experiments regarding immunology and pathobiology and are preferred over large animal models due to the ethical aspects, the cost-benefit balance, and the high throughput possibility. In this comprehensive review, we summarize the reported surgical techniques for lung transplantation in rodent models and the management of perioperative complications. Furthermore, we propose a guide to help identify the appropriate species for a given experiment and discuss recent experimental findings in small animal lung transplant models.

Increased LGR6 Expression Sustains Long-Term Wnt Activation and Acquisition of Senescence in Epithelial Progenitors in Chronic Lung Diseases.

Chronic lung diseases (CLDs) represent a set of disorders characterized by the progressive loss of proper lung function. Among severe CLDs, the incidence of chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) has grown over the last decades, mainly in the elderly population. Several studies have highlighted an increased expression of senescence-related markers in the resident progenitor cells in COPD and IPF, possibly undermining epithelial integrity and contributing to the progression and the aggravation of both diseases. Recently, the chronic activation of the canonical Wnt/ß-catenin pathway was shown to induce cellular senescence. Here, we investigated the localization and the expression of leucin-rich repeat-containing G-protein-coupled receptor 6 (LGR6), a protein that activates and potentiates the canonical Wnt signalling. Through immunohistochemical analyses, we identified a lesion-associated rise in LGR6 levels in abnormal lung epithelial progenitors in COPD and IPF when compared to histologically normal tissues. Moreover, in areas of aberrant regeneration, chronic damage and fibrosis, LGR6-expressing epithelial progenitors displayed a major increase in the expression of senescence-associated markers. Our study suggests the involvement of LGR6 in the chronic activation of the Wnt/ß-catenin pathway, mediating the impairment and exhaustion of epithelial progenitors in COPD and IPF.

MiR-223 is increased in lungs of patients with COPD and modulates cigarette smoke-induced pulmonary inflammation.

Since microRNA (miR)-223-3p modulates inflammatory responses and chronic obstructive pulmonary disease (COPD) is associated with amplified pulmonary inflammation, we hypothesized that miR-223-3p plays a role in COPD pathogenesis. Expression of miR-223-3p was measured in lung tissue of two independent cohorts with patients with GOLD stage II-IV COPD, never smokers, and smokers without COPD. The functional role of miR-223-3p was studied in deficient mice and on overexpression in airway epithelial cells from COPD and controls. We observed higher miR-223-3p levels in patients with COPD stage II-IV compared with (non)-smoking controls, and levels were associated with higher neutrophil numbers in bronchial biopsies of patients with COPD. MiR-223-3p expression was also increased in lungs and bronchoalveolar lavage of cigarette smoke (CS)-exposed mice. CS-induced neutrophil and monocyte lung infiltration was stronger in miR-223-deficient mice on acute (5 days) exposure, but attenuated on subchronic (4 wk) exposure. Additionally, miR-223 deficiency attenuated acute and subchronic CS-induced lung infiltration of dendritic cells and T lymphocytes. Finally, in vitro overexpression of miR-223-3p in non-COPD airway epithelial cells suppressed C-X-C motif chemokine ligand 8 (CXCL8) and granulocyte monocyte-colony stimulation factor (GM-CSF) secretion and gene expression of the proinflammatory transcription factor TRAF6. Importantly, this suppressive effect of miR-223-3p was compromised in COPD-derived cultures. In conclusion, we demonstrate that miR-223-3p is increased in lungs of patients with COPD and CS-exposed mice and is associated with neutrophilic inflammation. In vivo data indicate that miR-223 acts as negative regulator of acute CS-induced neutrophilic and monocytic inflammation. In vitro data suggest that miR-223-3p does so by suppressing proinflammatory airway epithelial responses, which is less effective in COPD epithelium.

The transition from normal lung anatomy to minimal and established fibrosis in idiopathic pulmonary fibrosis (IPF).

BACKGROUND: The transition from normal lung anatomy to minimal and established fibrosis is an important feature of the pathology of idiopathic pulmonary fibrosis (IPF). The purpose of this report is to examine the molecular and cellular mechanisms associated with this transition. METHODS: Pre-operative thoracic Multidetector Computed Tomography (MDCT) scans of patients with severe IPF (n = 9) were used to identify regions of minimal(n = 27) and established fibrosis(n = 27). MDCT, Micro-CT, quantitative histology, and next-generation sequencing were used to compare 24 samples from donor controls (n = 4) to minimal and established fibrosis samples. FINDINGS: The present results extended earlier reports about the transition from normal lung anatomy to minimal and established fibrosis by showing that there are activations of TGFBI, T cell co-stimulatory genes, and the down-regulation of inhibitory immune-checkpoint genes compared to controls. The expression patterns of these genes indicated activation of a field immune response, which is further supported by the increased infiltration of inflammatory immune cells dominated by lymphocytes that are capable of forming lymphoid follicles. Moreover, fibrosis pathways, mucin secretion, surfactant, TLRs, and cytokine storm-related genes also participate in the transitions from normal lung anatomy to minimal and established fibrosis. INTERPRETATION: The transition from normal lung anatomy to minimal and established fibrosis is associated with genes that are involved in the tissue repair processes, the activation of immune responses as well as the increased infiltration of CD4, CD8, B cell lymphocytes, and macrophages. These molecular and cellular events correlate with the development of structural abnormality of IPF and probably contribute to its pathogenesis.

Once daily tacrolimus conversion in lung transplantation: A prospective study on safety and medication adherence.

BACKGROUND: Lung transplantation (LTx) requires a calcineurin inhibitor-based immunosuppressive regimen. A once daily (QD) tacrolimus regimen was developed to increase medication adherence. However, data concerning its safety and efficacy in LTx are lacking. METHODS: In this prospective study, stable LTx patients were consecutively converted from twice daily (BID) tacrolimus to QD tacrolimus on a 1 mg:1 mg basis. Trough level (Cmin), renal function, cholesterol, fasting glucose, potassium and lung function were monitored six months before and up to one year after conversion. Adherence and its barriers were assessed by self-reported questionnaires (Basel Assessment of Adherence to Immunosuppressive Medications Scale (BAASIS) and Identification of Medication Adherence Barriers questionnaire (IMAB)) and blood-based assays (mean Cmin and coefficient of variation (CV)). RESULTS: We included 372 patients, in whom we observed a decrease in tacrolimus Cmin of 18.5% (p < 0.0001) post-conversion, requiring subsequent daily dose adaptations in both cystic fibrosis (CF) (n = 72) and non-CF patients (n = 300). We observed a small decrease in eGFR one year post-conversion (p = 0.024). No significant changes in blood creatinine, potassium, fasting glucose, cholesterol or rate of lung function decline were observed. In a subgroup of 166 patients, significantly fewer patients missed doses (8.4% vs. 19.3%, p = 0.016) or had irregular intake post-conversion (19.3% vs. 32.5%, p = 0.019). Mean Cmin and CV, as well as the total number of barriers, also decreased significantly post-conversion. CONCLUSIONS: In LTx, conversion from BID to QD tacrolimus (1 mg:1 mg) requires close monitoring of tacrolimus Cmin. QD tacrolimus after transplantation is safe with respect to renal function, metabolic parameters and allograft function and improves LTx recipient adherence.

Genome-wide CRISPR screening identifies TMEM106B as a proviral host factor for SARS-CoV-2.

The ongoing COVID-19 pandemic has caused a global economic and health crisis. To identify host factors essential for coronavirus infection, we performed genome-wide functional genetic screens with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human coronavirus 229E. These screens uncovered virus-specific as well as shared host factors, including TMEM41B and PI3K type 3. We discovered that SARS-CoV-2 requires the lysosomal protein TMEM106B to infect human cell lines and primary lung cells. TMEM106B overexpression enhanced SARS-CoV-2 infection as well as pseudovirus infection, suggesting a role in viral entry. Furthermore, single-cell RNA-sequencing of airway cells from patients with COVID-19 demonstrated that TMEM106B expression correlates with SARS-CoV-2 infection. The present study uncovered a collection of coronavirus host factors that may be exploited to develop drugs against SARS-CoV-2 infection or future zoonotic coronavirus outbreaks.

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.

Diagnostic Yield of 18F-FDG PET After Lung Transplantation: A Single-center, Retrospective Cohort Study.

BACKGROUND: To investigate the diagnostic yield of 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET) in lung transplant recipients. METHODS: A single-center, retrospective cohort study including 234 18F-FDG PET examinations in 199 lung transplant recipients. Indication for PET referral, 18F-FDG PET diagnosis/findings and final clinical diagnosis were classified into 3 groups: malignancy, infection/inflammation not otherwise specified, and chronic lung allograft dysfunction with restrictive allograft syndrome phenotype. Sensitivity/specificity analysis was performed to determine accuracy of 18F-FDG PET in each group. RESULTS: Sensitivity of 18F-FDG PET for malignancy was 91.4% (95% confidence interval, 82.5%-96.0%) and specificity was 82.3% (95% confidence interval, 74.5%-88.1%). Infection/inflammation not otherwise specified and restrictive allograft syndrome as indication for 18F-FDG PET comprised relatively small groups (14 and 31 cases, respectively). In addition, 18F-FDG PET revealed clinically relevant incidental findings in 15% of cases. CONCLUSIONS: Referral for 18F-FDG PET after lung transplantation mainly occurred to confirm or rule out malignancy. In this specific setting, 18F-FDG PET has a high diagnostic yield. Accuracy of 18F-FDG PET for other indications is less clear, given small sample sizes. Clinically relevant diagnoses, unrelated to the primary indication for 18F-FDG PET, are found relatively often in this immunocompromised cohort.

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

BACKGROUND: Physiological lung ageing is associated with a gradual decline in dynamic lung volumes and a progressive increase in residual volume due to diminished elastic recoil of the lung, loss of alveolar tissue, and lower chest wall compliance. However, the effects of ageing on the small airways (ie, airways <2·0 mm in diameter) remain largely unknown. By using a combination of ex-vivo conventional CT (resolution 1 mm), whole lung micro-CT (resolution 150 µm), and micro-CT of extracted cores (resolution 10 µm), we aimed to provide a multiresolution assessment of the small airways in lung ageing in a large cohort of never smokers. METHODS: For this cross-sectional study, we included donor lungs collected from 32 deceased never-smoking donors (age range 16-83 years). Ex-vivo CT and whole lung high-resolution CT (micro-CT) were used to determine total airway numbers, stratified by airway diameter. Micro-CT was used to assess the number, length, and diameter of terminal bronchioles (ie, the last generation of conducting airways); mean linear intercept; and surface density in four lung tissue cores from each lung, extracted using a uniform sampling approach. Regression ß coefficients are calculated using linear regression and polynomial models. FINDINGS: Ex-vivo CT analysis showed an age-dependent decrease in the number of airways of diameter 2·0 mm to less than 2·5 mm (ß coefficient per decade -0·119, 95% CI -0·193 to -0·045; R2=0·29) and especially in airways smaller than 2·0 mm in diameter (-0·158, -0·233 to -0·084; R2=0·47), between 30 and 80 years of age, but not of the larger (≥2·5 mm) diameter airways (-0·00781, -0·04409 to 0·02848; R2=0·0007). In micro-CT analysis of small airways, the total number of terminal bronchioles per lung increased until the age of 30 years, after which an almost linear decline in the number of terminal bronchioles was observed (ß coefficient per decade -2035, 95% CI -2818 to -1252; R2=0·55), accompanied by a non-significant increase in alveolar airspace size (6·44, -0·57 to 13·45, R2=0·10). Moreover, this decrease in terminal bronchioles was associated with the age-related decline of pulmonary function predicted by healthy reference values. INTERPRETATION: Loss of terminal bronchioles is an important structural component of age-related decline in pulmonary function of healthy, non-smoking individuals. FUNDING: Research Foundation-Flanders, KU Leuven, Parker B Francis Foundation, UGent, Canadian Institutes for Health.

Profibrotic epithelial TGF-ß1 signaling involves NOX4-mitochondria cross talk and redox-mediated activation of the tyrosine kinase FYN.

Idiopathic pulmonary fibrosis (IPF) is characterized by a disturbed redox balance and increased production of reactive oxygen species (ROS), which is believed to contribute to epithelial injury and fibrotic lung scarring. The main pulmonary sources of ROS include mitochondria and NADPH oxidases (NOXs), of which the NOX4 isoform has been implicated in IPF. Non-receptor SRC tyrosine kinases (SFK) are important for cellular homeostasis and are often dysregulated in lung diseases. SFK activation by the profibrotic transforming growth factor-ß (TGF-ß) is thought to contribute to pulmonary fibrosis, but the relevant SFK isoform and its relationship to NOX4 and/or mitochondrial ROS in the context of profibrotic TGF-ß signaling is not known. Here, we demonstrate that TGF-ß1 can rapidly activate the SRC kinase FYN in human bronchial epithelial cells, which subsequently induces mitochondrial ROS (mtROS) production, genetic damage shown by the DNA damage marker γH2AX, and increased expression of profibrotic genes. Moreover, TGF-ß1-induced activation of FYN involves initial activation of NOX4 and direct cysteine oxidation of FYN, and both FYN and mtROS contribute to TGF-ß-induced induction of NOX4. NOX4 expression in lung tissues of IPF patients is positively correlated with disease severity, although FYN expression is down-regulated in IPF and does not correlate with disease severity. Collectively, our findings highlight a critical role for FYN in TGF-ß1-induced mtROS production, DNA damage response, and induction of profibrotic genes in bronchial epithelial cells, and suggest that altered expression and activation of NOX4 and FYN may contribute to the pathogenesis of pulmonary fibrosis.

Pathology of Idiopathic Pulmonary Fibrosis Assessed by a Combination of Microcomputed Tomography, Histology, and Immunohistochemistry.

Idiopathic pulmonary fibrosis (IPF) is a fibrotic disease with the histology of usual interstitial pneumonia (UIP). Although the pathologist's visual inspection is central in histologic assessments, three-dimensional microcomputed tomography (microCT) assessment may complement the pathologist's scoring. We examined associations between the histopathologic features of UIP and IPF in explanted lungs and quantitative microCT measurements, including alveolar surface density, total lung volume taken up by tissue (%), and terminal bronchiolar number. Sixty frozen samples from 10 air-inflated explanted lungs with severe IPF and 36 samples from 6 donor control lungs were scanned with microCT and processed for histologic analysis. An experienced pathologist scored three major UIP criteria (patchy fibrosis, honeycomb, and fibroblastic foci), five additional pathologic changes, and immunohistochemical staining for CD68-, CD4-, CD8-, and CD79a-positive cells, graded on a 0 to 3+ scale. The alveolar surface density and terminal bronchiolar number decreased and the tissue percentage increased in lungs with IPF compared with controls. In lungs with IPF, lower alveolar surface density and higher tissue percentage were correlated with greater scores of patchy fibrosis, fibroblastic foci, honeycomb, CD79a-positive cells, and lymphoid follicles. A decreased number of terminal bronchioles was correlated with honeycomb score but not with the other scores. The three-dimensional microCT measurements reflect the pathological UIP and IPF criteria and suggest that the reduction in the terminal bronchioles may be associated with honeycomb cyst formation.