Breast cancer cells rely on environmental pyruvate to shape the metastatic niche.

The extracellular matrix is a major component of the local environment-that is, the niche-that determines cell behaviour1. During metastatic growth, cancer cells shape the extracellular matrix of the metastatic niche by hydroxylating collagen to promote their own metastatic growth2,3. However, only particular nutrients might support the ability of cancer cells to hydroxylate collagen, because nutrients dictate which enzymatic reactions are active in cancer cells4,5. Here we show that breast cancer cells rely on the nutrient pyruvate to drive collagen-based remodelling of the extracellular matrix in the lung metastatic niche. Specifically, we discovered that pyruvate uptake induces the production of α-ketoglutarate. This metabolite in turn activates collagen hydroxylation by increasing the activity of the enzyme collagen prolyl-4-hydroxylase (P4HA). Inhibition of pyruvate metabolism was sufficient to impair collagen hydroxylation and consequently the growth of breast-cancer-derived lung metastases in different mouse models. In summary, we provide a mechanistic understanding of the link between collagen remodelling and the nutrient environment in the metastatic niche.

A Pathology-based Case Series of Influenza- and COVID-19-associated Pulmonary Aspergillosis: The Proof Is in the Tissue.

Rationale: Invasive pulmonary aspergillosis has emerged as a frequent coinfection in severe coronavirus disease (COVID-19), similarly to influenza, yet the clinical invasiveness is more debated. Objectives: We investigated the invasive nature of pulmonary aspergillosis in histology specimens of influenza and COVID-19 ICU fatalities in a tertiary care center. Methods: In this monocentric, descriptive, retrospective case series, we included adult ICU patients with PCR-proven influenza/COVID-19 respiratory failure who underwent postmortem examination and/or tracheobronchial biopsy during ICU admission from September 2009 until June 2021. Diagnosis of probable/proven viral-associated pulmonary aspergillosis (VAPA) was made based on the Intensive Care Medicine influenza-associated pulmonary aspergillosis and the European Confederation of Medical Mycology (ECMM) and the International Society for Human and Animal Mycology (ISHAM) COVID-19-associated pulmonary aspergillosis consensus criteria. All respiratory tissues were independently reviewed by two experienced pathologists. Measurements and Main Results: In the 44 patients of the autopsy-verified cohort, 6 proven influenza-associated and 6 proven COVID-19-associated pulmonary aspergillosis diagnoses were identified. Fungal disease was identified as a missed diagnosis upon autopsy in 8% of proven cases (n = 1/12), yet it was most frequently found as confirmation of a probable antemortem diagnosis (n = 11/21, 52%) despite receiving antifungal treatment. Bronchoalveolar lavage galactomannan testing showed the highest sensitivity for VAPA diagnosis. Among both viral entities, an impeded fungal growth was the predominant histologic pattern of pulmonary aspergillosis. Fungal tracheobronchitis was histologically indistinguishable in influenza (n = 3) and COVID-19 (n = 3) cases, yet macroscopically more extensive at bronchoscopy in influenza setting. Conclusions: A proven invasive pulmonary aspergillosis diagnosis was found regularly and with a similar histological pattern in influenza and in COVID-19 ICU case fatalities. Our findings highlight an important need for VAPA awareness, with an emphasis on mycological bronchoscopic work-up.

Effects of repeated infections with non-typeable Haemophilus influenzae on lung in vitamin D deficient and smoking mice.

BACKGROUND: In chronic obstructive pulmonary disease (COPD), exacerbations cause acute inflammatory flare-ups and increase the risk for hospitalization and mortality. Exacerbations are common in all disease stages and are often caused by bacterial infections e.g., non-typeable Heamophilus influenzae (NTHi). Accumulating evidence also associates vitamin D deficiency with the severity of COPD and exacerbation frequency. However, it is still unclear whether vitamin D deficiency when combined with cigarette smoking would worsen and prolong exacerbations caused by repeated infections with the same bacterial strain. METHODS: Vitamin D sufficient (VDS) and deficient (VDD) mice were exposed to nose-only cigarette smoke (CS) for 14 weeks and oropharyngeally instilled with NTHi at week 6, 10 and 14. Three days after the last instillation, mice were assessed for lung function, tissue remodeling, inflammation and immunity. The impact of VDD and CS on inflammatory cells and immunoglobulin (Ig) production was also assessed in non-infected animals while serum Ig production against NTHi and dsDNA was measured in COPD patients before and 1 year after supplementation with Vitamin D3. RESULTS: VDD enhanced NTHi eradication, independently of CS and complete eradication was reflected by decreased anti-NTHi Ig's within the lung. In addition, VDD led to an increase in total lung capacity (TLC), lung compliance (Cchord), MMP12/TIMP1 ratio with a rise in serum Ig titers and anti-dsDNA Ig's. Interestingly, in non-infected animals, VDD exacerbated the CS-induced anti-NTHi Ig's, anti-dsDNA Ig's and inflammatory cells within the lung. In COPD patients, serum Ig production was not affected by vitamin D status but anti-NTHi IgG increased after vitamin D3 supplementation in patients who were Vitamin D insufficient before treatment. CONCLUSION: During repeated infections, VDD facilitated NTHi eradication and resolution of local lung inflammation through production of anti-NTHi Ig, independently of CS whilst it also promoted autoantibodies. In COPD patients, vitamin D supplementation could be protective against NTHi infections in vitamin D insufficient patients. Future research is needed to decipher the determinants of dual effects of VDD on adaptive immunity. TRAIL REGISTRATION: ClinicalTrials, NCT00666367. Registered 23 April 2008, https://www.clinicaltrials.gov/ct2/show/study/NCT00666367 .

Oxygenated machine perfusion at room temperature as an alternative for static cold storage in porcine donor hearts.

BACKGROUND: There is a continued interest in ex situ heart perfusion as an alternative strategy for donor heart preservation. We hypothesize that oxygenated machine perfusion of donor hearts at a temperature that avoids both normothermia and deep hypothermia offers adequate and safe preservation. METHODS: Cardioplegia-arrested porcine donor hearts were randomly assigned to six hours of preservation using cold storage (CS, n = 5) or machine perfusion using an oxygenated acellular perfusate at 21°C (MP, n = 5). Subsequently, all grafts were evaluated using the Langendorff method for 120 min. Metabolic parameters and histology were analyzed. Systolic function was assessed by contractility and elastance. Diastolic function was assessed by lusitropy and stiffness. RESULTS: For both groups, in vivo baseline and post-Langendorff biopsies were comparable, as were lactate difference and myocardial oxygen consumption. Injury markers gradually increased and were comparable. Significant weight gain was seen in MP (p = 0.008). Diastolic function was not impaired in MP, and lusitropy was superior from 30 min up to 90 min of reperfusion. Contractility was superior in MP during the first hour of evaluation. CONCLUSION: We conclude that the initial functional outcome of MP-preserved hearts was transiently superior compared to CS, with no histological injury post-Langendorff. Our machine perfusion strategy could offer feasible and safe storage of hearts prior to transplantation. Future studies are warranted for further optimization.

A robust human norovirus replication model in zebrafish larvae.

Human noroviruses (HuNoVs) are the most common cause of foodborne illness, with a societal cost of $60 billion and 219,000 deaths/year. The lack of robust small animal models has significantly hindered the understanding of norovirus biology and the development of effective therapeutics. Here we report that HuNoV GI and GII replicate to high titers in zebrafish (Danio rerio) larvae; replication peaks at day 2 post infection and is detectable for at least 6 days. The virus (HuNoV GII.4) could be passaged from larva to larva two consecutive times. HuNoV is detected in cells of the hematopoietic lineage and the intestine, supporting the notion of a dual tropism. Antiviral treatment reduces HuNoV replication by >2 log10, showing that this model is suited for antiviral studies. Zebrafish larvae constitute a simple and robust replication model that will largely facilitate studies of HuNoV biology and the development of antiviral strategies.

Cobalt exposure via skin alters lung immune cells and enhances pulmonary responses to cobalt in mice.

Cobalt has been associated with allergic contact dermatitis and occupational asthma. However, the link between skin exposure and lung responses to cobalt is currently unknown. We investigated the effect of prior dermal sensitization to cobalt on pulmonary physiological and immunological responses after subsequent challenge with cobalt via the airways. BALB/c mice received epicutaneous applications (25 µL/ear) with 5% CoCl2*6H2O (Co) or the vehicle (Veh) dimethyl sulfoxide (DMSO) twice; they then received oropharyngeal challenges with 0.05% CoCl2*6H2O or saline five times, thereby obtaining four groups: Veh/Veh, Co/Veh, Veh/Co, and Co/Co. To detect early respiratory responses noninvasively, we performed sequential in vivo microcomputed tomography (µCT). One day after the last challenge, we assessed airway hyperreactivity (AHR) to methacholine, inflammation in bronchoalveolar lavage (BAL), innate lymphoid cells (ILCs) and dendritic cells (DCs) in the lungs, and serum IgE. Compared with the Veh/Veh group, the Co/Co group showed increased µCT-derived lung response, increased AHR to methacholine, mixed neutrophilic and eosinophilic inflammation, elevated monocyte chemoattractant protein-1 (MCP-1), and elevated keratinocyte chemoattractant (KC) in BAL. Flow cytometry in the Co/Co group demonstrated increased DC, type 1 and type 2 conventional DC (cDC1/cDC2), monocyte-derived DC, increased ILC group 2, and natural cytotoxicity receptor-ILC group 3. The Veh/Co group showed only increased AHR to methacholine and elevated MCP-1 in BAL, whereas the Co/Veh group showed increased cDC1 and ILC2 in lung. We conclude that dermal sensitization to cobalt may increase the susceptibility of the lungs to inhaling cobalt. Mechanistically, this enhanced susceptibility involves changes in pulmonary DCs and ILCs.

Quantitative analysis of airway obstruction in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare, cystic lung disease with progressive pulmonary function loss caused by progressively proliferating LAM cells. The degree of airway obstruction has not been well investigated within the pathogenesis of LAM.Using a combination of ex vivo computed tomography (CT), microCT and histology, the site and nature of airway obstruction in LAM explant lungs was compared with matched control lungs (n=5 each). The total number of airways per generation, total airway counts, terminal bronchioles number and surface density were compared in LAM versus control.Ex vivo CT analysis demonstrated a reduced number of airways from generation 7 on (p<0.0001) in LAM compared with control, whereas whole-lung microCT analysis confirmed the three- to four-fold reduction in the number of airways. Specimen microCT analysis further demonstrated a four-fold decrease in the number of terminal bronchioles (p=0.0079) and a decreased surface density (p=0.0079). Serial microCT and histology images directly showed the loss of functional airways by collapse of airways on the cysts and filling of the airway by exudate.LAM lungs show a three- to four-fold decrease in the number of (small) airways, caused by cystic destruction which is the likely culprit for the progressive loss of pulmonary function.

Programmed death-ligand 1 expression influenced by tissue sample size. Scoring based on tissue microarrays' and cross-validation with resections, in patients with, stage I-III, non-small cell lung carcinoma of the European Thoracic Oncology Platform Lungscape cohort.

PD-L1, as assessed by immunohistochemistry, is a predictive biomarker for immuno-oncology treatment in lung cancer. Different scoring methods have been used to assess its status, resulting in a wide range of positivity rates. We use the European Thoracic Oncology Platform Lungscape non-small cell lung carcinoma cohort to explore this issue. PD-L1 expression was assessed via immunohistochemistry on tissue microarrays (up to four cores per case), using the DAKO 28-8 immunohistochemistry assay, following a two-round external quality assessment procedure. All samples were analyzed under the same protocol. Cross-validation of scoring between tissue microarray and whole sections was performed in 10% randomly selected samples. Cutoff points considered: ≥1, 50 (primarily), and 25%. At the two external quality assessment rounds, tissue microarray scoring agreement rates between pathologists were: 73% and 81%. There were 2008 cases with valid immunohistochemistry tissue microarray results (50% all cores evaluable). Concordant cases at 1, 25, and 50% were: 85, 91, and 93%. Tissue microarray core results were identical for 70% of cases. Sensitivity of the tissue microarray method for 1, 25, and 50% was: 80, 78, and 79% (specificity: 90, 95, 98%). Complete agreement between tissue microarrays and whole sections was achieved for 60% of the cases. Highest sensitivity rates for 1% and 50% cutoffs were detected for higher number of cores. Underestimation of PD-L1 expression on small samples is more common than overestimation. We demonstrated that classification of PD-L1 on small biopsy samples does not represent the overall expression of PD-L1 in all non-small cell cancer carcinoma cases, although the majority of cases are 'correctly' classified. In future studies, sampling more and larger biopsies, recording the biopsy size and tumor load may permit further refinement, increasing predictive accuracy.

Identification and characterization of chronic lung allograft dysfunction patients with mixed phenotype: A single-center study.

RATIONALE: Patients can change chronic lung allograft dysfunction (CLAD) phenotype, especially from BOS to mixed phenotype. Our aim was to further characterize these patients. METHOD: Mixed CLAD was defined as a restrictive physiology with persistent CT opacities, after initial bronchiolitis obliterans syndrome (BOS) diagnosis. The incidence, prognosis, pulmonary function, radiology, pathology, and airway inflammation were compared between patients with restrictive allograft syndrome (RAS) and mixed CLAD. RESULT: A total of 268 (44%) patients developed CLAD of which 47 (18%) were diagnosed with RAS "ab initio," 215 (80%) with BOS, and 6 (2%) an undefined phenotype. Twenty-five patients developed a mixed CLAD phenotype (24 BOS to mixed and 1 RAS to mixed). Survival after mixed phenotype diagnosis was comparable (P = .39) to RAS. More emphysema patients developed a mixed phenotype (P = .020) compared to RAS ab initio, while mixed CLAD patients had a lower FEV1 (P < .0001) and FEV1 /FVC (P = .0002) at diagnosis compared to RAS ab initio. CT scans in patients with the mixed phenotype demonstrated apical predominance of the opacities (P = .0034) with pleuroparenchymal fibroelastosis on histopathology. CONCLUSION: We further characterized patients with a mixed phenotype of CLAD. Although the survival after diagnosis was comparable to RAS ab initio patients, there was a difference in demography, pulmonary function, radiology, and pathology.

Phenotypical diversity of airway morphology in chronic lung graft vs. host disease after stem cell transplantation.

Pulmonary graft vs. host disease is a diverse and underestimated complication following allogenic hematopoietic stem cell transplantation. We aimed to compare the airway architecture with chronic lung allograft dysfunction post lung transplantation. Inflated explant lungs from graft vs. host disease patients were compared with lungs with chronic lung allograft dysfunction following lung transplantation, and control lungs using a combination of CT, microCT, and histology (n = 6 per group) and pathology in the (small) airways was further quantified and analyzed. Following allogenic hematopoietic stem cell transplantation, three patients presented as bronchiolitis obliterans syndrome and three patients showed interstitial changes and restriction. The CT analysis demonstrated a strong similarity between bronchiolitis obliterans syndrome after lung transplantation and post allogenic hematopoietic stem cell transplantation, evidenced by severe ( > 50%) airway obstruction from generation 9, with 70.8% of the airways ending in obstruction. Further analysis indicated that the airways either collapsed or accumulated matrix along a segment of the airway. In patients with restriction and interstitial changes following allogenic hematopoietic stem cell transplantation, the degree of airway obstruction was lower compared with bronchiolitis obliterans syndrome post allogenic hematopoietic stem cell transplantation, but similar to restrictive allograft syndrome post lung transplantation, showing a lower proportion of airway obstruction (20-35%), decreased number of terminal bronchioles per lung (p < 0.01), and parenchymal fibrosis. We observed similarities in the airway and parenchymal morphometric changes in lung graft vs. host disease and with chronic lung allograft dysfunction following lung transplantation, suggesting similar pathophysiological mechanisms.

The histomorphological spectrum of restrictive chronic lung allograft dysfunction and implications for prognosis.

Chronic lung allograft dysfunction continues to be the main contributor to poor long-term allograft survival after lung transplantation. The restrictive phenotype of chronic lung allograft dysfunction carries a particularly poor prognosis. Little is known about the pathogenetic mechanisms involved in restrictive chronic lung allograft dysfunction. In this study, we performed histomorphological and immunohistochemical analysis of restrictive chronic lung allograft dysfunction lungs. Explant lung tissue from 21 restrictive chronic lung allograft dysfunction patients was collected and histopathologic patterns of rejection, fibrosis and vascular changes were scored after routine histochemical stains and additional immunohistochemistry for endothelial markers and C4d. In all, 75% of cases showed evidence of acute cellular rejection; lymphocytic bronchiolitis was absent in most lungs, whereas in 55% there was obliterative bronchiolitis. Almost half of the cases showed a pattern consistent with pleuroparenchymal fibro-elastosis (n=10), and a subset showed nonspecific interstitial pneumonia (n=5) or irregular emphysema (n=5). Fibrinous alveolar exudates were frequently seen in association with fibrosis (n=6), but no diffuse alveolar damage was found. Evidence of microvascular damage was present in most cases. An emphysematous pattern of fibrosis was associated with a better survival (P=0.0030), whereas fibrinous exudates were associated with a worse survival (P=0.0007). In addition to the previously described nonspecific interstitial pneumonia and pleuroparenchymal fibro-elastosis patterns in restrictive chronic lung allograft dysfunction, we are the first to describe a pattern of fibrosis-induced subpleural/paraseptal emphysema. This pattern confers a better survival, whereas fibrinous exudates are associated with a worse survival. We believe that our findings offer a pathogenetic theory for pleuroparenchymal fibro-elastosis in restrictive chronic lung allograft dysfunction, and show that restrictive chronic lung allograft dysfunction is an increasingly heterogeneous disease with presumably different mechanisms of subpattern formation.

Irritant-induced asthma to hypochlorite in mice due to impairment of the airway barrier.

Inhalation of commonly present irritants, such as chlorine and chlorine derivatives, can cause adverse respiratory effects, including irritant-induced asthma (IIA). We hypothesize that due to airway barrier impairment, exposure to hypochlorite (ClO-) can result in airway hypersensitivity. C57Bl/6 mice received an intra-peritoneal (i.p.) injection of the airway damaging agent naphthalene (NA, 200 mg/kg body weight) or vehicle (mineral oil, MO). In vivo micro-computed tomography (CT) images of the lungs were acquired before and at regular time points after the i.p. TREATMENT: After a recovery period of 14 days an intranasal (i.n.) challenge with 0.003% active chlorine (in ClO-) or vehicle (distilled water, H2O) was given, followed by assessment of the breathing frequency. One day later, pulmonary function, along with pulmonary inflammation was determined. Lung permeability was assessed by means of total broncho-alveolar lavage (BAL) protein content and plasma surfactant protein (SP)-D levels. In vivo micro-CT imaging revealed enlargement of the lungs and airways early after NA treatment, with a return to normal at day 14. When challenged i.n. with ClO-, NA-pretreated mice immediately responded with a sensory irritant response. Twenty-four hours later, NA/ClO- mice showed airway hyperreactivity (AHR), accompanied by a neutrophilic and eosinophilic inflammation. NA administration followed by ClO- induced airway barrier impairment, as shown by increased BAL protein and plasma SP-D concentrations; histology revealed epithelial denudation. These data prove that NA-induced lung impairment renders the lungs of mice more sensitive to an airway challenge with ClO-, confirming the hypothesis that incomplete barrier repair, followed by irritant exposure results in airway hypersensitivity.

Forced expiration measurements in mouse models of obstructive and restrictive lung diseases.

BACKGROUND: Pulmonary function measurements are important when studying respiratory disease models. Both resistance and compliance have been used to assess lung function in mice. Yet, it is not always clear how these parameters relate to forced expiration (FE)-related parameters, most commonly used in humans. We aimed to characterize FE measurements in four well-established mouse models of lung diseases. METHOD: Detailed respiratory mechanics and FE measurements were assessed concurrently in Balb/c mice, using the forced oscillation and negative pressure-driven forced expiration techniques, respectively. Measurements were performed at baseline and following increasing methacholine challenges in control Balb/c mice as well as in four disease models: bleomycin-induced fibrosis, elastase-induced emphysema, LPS-induced acute lung injury and house dust mite-induced asthma. RESULTS: Respiratory mechanics parameters (airway resistance, tissue damping and tissue elastance) confirmed disease-specific phenotypes either at baseline or following methacholine challenge. Similarly, lung function defects could be detected in each disease model by at least one FE-related parameter (FEV0.1, FEF0.1, FVC, FEV0.1/FVC ratio and PEF) at baseline or during the methacholine provocation assay. CONCLUSIONS: FE-derived outcomes in four mouse disease models behaved similarly to changes found in human spirometry. Routine combined lung function assessments could increase the translational utility of mouse models.

End-stage cystic fibrosis lung disease is characterised by a diverse inflammatory pattern: an immunohistochemical analysis.

BACKGROUND: Cystic fibrosis (CF) lung disease is characterised by vigorous airway inflammation eventually resulting in severe lung damage. This study aimed to describe the diversity of the inflammatory pattern in end-stage CF lungs by evaluating and quantifying which components of the innate and adaptive immunity are involved, and by assessing whether this is gender-specific. METHODS: CF explant lung tissue (n = 20) collected at time of transplantation and control tissue (n = 22) was sectioned (9 µm) and stained for neutrophils, eosinophils, mast cells, dendritic cells, macrophages, CD4 T cells, cytotoxic T cells and B cells. Quantification with special attention for immune cell location was performed. RESULTS: Neutrophils, mast cells, dendritic cells, macrophages, CD4 T and cytotoxic T cells were significantly increased in CF compared to controls and there was a disproportionate increase of neutrophils around the airways in CF. Large amounts of lymphoid follicles were found in the CF lung and they had a skewed B cell/T cell composition. Upon subdividing the CF patients into a male and female population, eosinophils, mast cells and CD4 T cells were increased specifically in CF females. In this subpopulation, lymphoid follicles had less B cells and more CD8 T cells. CONCLUSION: These data demonstrate a diverse inflammatory response in the CF lung, reflected by an increase of both myeloid and lymphoid immune cells. Inflammation in the CF lung appeared to be gender-specific in our population, as the significant increase of eosinophils, mast cells and CD4 T cells was especially notable in the female subpopulation.

Immunological diversity in phenotypes of chronic lung allograft dysfunction: a comprehensive immunohistochemical analysis.

Chronic rejection after organ transplantation is defined as a humoral- and cell-mediated immune response directed against the allograft. In lung transplantation, chronic rejection is nowadays clinically defined as a cause of chronic lung allograft dysfunction (CLAD), consisting of different clinical phenotypes including restrictive allograft syndrome (RAS) and bronchiolitis obliterans syndrome (BOS). However, the differential role of humoral and cellular immunity is not investigated up to now. Explant lungs of patients with end-stage BOS (n = 19) and RAS (n = 18) were assessed for the presence of lymphoid (B and T cells) and myeloid cells (dendritic cells, eosinophils, mast cells, neutrophils, and macrophages) and compared to nontransplant control lung biopsies (n = 21). All myeloid cells, with exception of dendritic cells, were increased in RAS versus control (neutrophils, eosinophils, and mast cells: all P < 0.05, macrophages: P < 0.001). Regarding lymphoid cells, B cells and cytotoxic T cells were increased remarkably in RAS versus control (P < 0.001) and in BOS versus control (P < 0.01). Interestingly, lymphoid follicles were restricted to RAS (P < 0.001 versus control and P < 0.05 versus BOS). Our data suggest an immunological diversity between BOS and RAS, with a more pronounced involvement of the B-cell response in RAS characterized by a structural organization of lymphoid follicles. This may impact future therapeutic approaches.

Morphometric Analysis of Explant Lungs in Cystic Fibrosis.

RATIONALE: After repeated cycles of lung infection and inflammation, patients with cystic fibrosis (CF) evolve to respiratory insufficiency. Although histology and imaging have provided descriptive information, a thorough morphometric analysis of end-stage CF lung disease is lacking. OBJECTIVES: To quantify the involvement of small and large airways in end-stage CF. METHODS: Multidetector computed tomography (MDCT) and micro-CT were applied to 11 air-inflated CF explanted lungs and 7 control lungs to measure, count, and describe the airway and parenchymal abnormalities in end-stage CF lungs. Selected abnormalities were further investigated with thin section histology. MEASUREMENTS AND MAIN RESULTS: On MDCT, CF explanted lungs showed an increased median (interquartile range) number (631 [511-710] vs. 344 [277-349]; P = 0.003) and size of visible airways (cumulative airway diameter 217 cm [209-250] vs. 91 cm [80-105]; P < 0.001) compared with controls. Airway obstruction was seen, starting from generation 6 and increasing to 40 to 50% of airways from generation 9 onward. Micro-CT showed that the total number of terminal bronchioles was decreased (2.9/ml [2.6-4.4] vs. 5.3/ml [4.8-5.7]; P < 0.001); 49% were obstructed, and the cross-sectional area of the open terminal bronchioles was reduced (0.093 mm(2) [0.084-0.123] vs. 0.179 mm(2) [0.140-0.196]; P < 0.001). On micro-CT, 41% of the obstructed airways reopened more distally. This remodeling was confirmed on histological analysis. Parenchymal changes were also seen, mostly in a patchy and peribronchiolar distribution. CONCLUSIONS: Extensive changes of dilatation and obstruction in nearly all airway generations were observed in end-stage CF lung disease.

Tracheal tissue-engineering: in-vivo biocompatibility of mechanically-stripped allogenic rabbit trachea with autologous epithelial covering.

BACKGROUND: Successful trachea transplantation comprises the use of biocompatible constructs with little immune-reactivity, submucosal revascularization and creation of an epithelial covering. Allogenic chondrocytes might be protected from an overt immune-response due to physical isolation. Our aim was to evaluate in-vivo biocompatibility of allotracheae, stripped of their highly-immunogenic inner lining. Secondly, we established whether these constructs might serve as suitable scaffolds for autologous epithelial grafting. METHODS: Mucosa and submucosa of 12 rabbit donor tracheae were mechanically peeled off. Cartilage was covered with Integra™ regeneration-template. Constructs were implanted within the recipient's lateral thoracic artery flap. Integra of 6 revascularized allotracheae was grafted with autologous buccal mucosa. Macroscopical, histological analysis and immunohistochemistry were performed. RESULTS: Revascularization and buccal grafting was incomplete in the first 2 circular constructs. To enhance blood-vessel outgrowth, the following 10 transplants were opened longitudinally before implantation. Integra revascularized well. Grafted tracheae showed satisfactory mucosa-adherence, albeit with invasion of migrating epithelium within the Integra-scaffold. CONCLUSIONS: Mechanically-stripped allotracheae exhibited beneficial biocompatibility up to two months. This approach might open doors in the treatment of long-segment tracheal pathologies of which immunosuppression is contra-indicated. Thickness of this layered construct limited practical feasibility of orthotopic transfer, though with further refinements, a clinically-useful transplant could be created.

Longitudinal in vivo microcomputed tomography of mouse lungs: No evidence for radiotoxicity.

Before microcomputed tomography (micro-CT) can be exploited to its full potential for longitudinal monitoring of transgenic and experimental mouse models of lung diseases, radiotoxic side effects such as inflammation or fibrosis must be considered. We evaluated dose and potential radiotoxicity to the lungs for long-term respiratory-gated high-resolution micro-CT protocols. Free-breathing C57Bl/6 mice underwent four different retrospectively respiratory gated micro-CT imaging schedules of repeated scans during 5 or 12 wk, followed by ex vivo micro-CT and detailed histological and biochemical assessment of lung damage. Radiation exposure, dose, and absorbed dose were determined by ionization chamber, thermoluminescent dosimeter measurements and Monte Carlo calculations. Despite the relatively large radiation dose delivered per micro-CT acquisition, mice did not show any signs of radiation-induced lung damage or fibrosis when scanned weekly during 5 and up to 12 wk. Doubling the scanning frequency and once tripling the radiation dose as to mimic the instant repetition of a failed scan also stayed without detectable toxicity after 5 wk of scanning. Histological analyses confirmed the absence of radiotoxic damage to the lungs, thereby demonstrating that long-term monitoring of mouse lungs using high-resolution micro-CT is safe. This opens perspectives for longitudinal monitoring of (transgenic) mouse models of lung diseases and therapeutic response on an individual basis with high spatial and temporal resolution, without concerns for radiation toxicity that could potentially influence the readout of micro-CT-derived lung biomarkers. This work further supports the introduction of micro-CT for routine use in the preclinical pulmonary research field where postmortem histological approaches are still the gold standard.

Contribution of inflammation and impaired angiogenesis to the pathobiology of chronic thromboembolic pulmonary hypertension.

Deficient angiogenesis and systemic inflammation could be involved in the pathophysiology of chronic thromboembolic pulmonary hypertension (CTEPH). We aimed to characterise the histopathology of pulmonary vascular lesions from 52 CTEPH patients who underwent a pulmonary endarterectomy (PEA) and investigate a potential link between clinical, biological and morphometric parameters.Collagen, elastin, fibrin, lipid, endothelial, smooth muscle and inflammatory cell content was investigated using immunohistochemistry. Qualitative changes were evaluated using severity scores. Circulating levels of inflammatory mediators were measured using ELISA.Neointima, thrombotic, recanalised and atherosclerotic lesions were found. Accumulation of macrophages, T-lymphocytes and neutrophils was found mainly in atherosclerotic and thrombotic lesions. Angiogenesis was observed in all kinds of lesions; low-scored angiogenesis predicted adverse outcome, including persistent pulmonary hypertension post-PEA, start of medical therapy and poor survival. C-reactive protein (CRP), interleukin-10, monocyte chemotactic protein-1, macrophage inflammatory protein-1α and matrix metalloproteinase (MMP)-9 were significantly elevated in CTEPH patients. Plasma CRP and MMP-9 levels correlated with neutrophil and macrophage accumulation, respectively.Enhanced systemic inflammation parallels local inflammatory cell infiltration in major pulmonary arteries at advanced stages of CTEPH. Impaired neovascularisation is associated with poor survival, start of medical treatment and persistent pulmonary hypertension post-PEA. These findings suggest that inflammation and impaired angiogenesis could contribute to the progression of the disease.

Linking clinical phenotypes of chronic lung allograft dysfunction to changes in lung structure.

Chronic lung allograft dysfunction (CLAD) remains the major barrier to long-term success after lung transplantation. This report compares gross and microscopic features of lungs removed from patients receiving a redo-transplant as treatment for CLAD. Lungs donated by patients with either the bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS) phenotype of CLAD and appropriate control lungs (eight per group) were air-inflated, frozen solid and kept frozen while a multi-detector computed tomography (MDCT) was obtained. The lung was then cut into 2-cm thick transverse slices and sampled for micro-CT and histopathology. The MDCT showed reduced lung volume with increased lung weight and density in RAS versus BOS and control (p<0.05). Although pre-terminal bronchioles were obstructed in both phenotypes, RAS lungs showed a reduction of pre-terminal bronchioles (p<0.01). Micro-CT and matched histopathology showed that RAS was associated with reduced numbers of terminal bronchioles/lung compared to BOS and controls (p<0.01), with expansion of the interstitial compartment and obliteration of the alveolar airspaces by fibrous connective tissue. RAS is associated with greater destruction of both pre-terminal and terminal bronchioles. Additionally, the interstitial compartments are expanded and alveolar airspaces are obliterated by accumulation of fibrous connective tissue.