Long-term expanding human airway organoids for disease modeling.

Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non-structural viral NS2 protein, and preferentially recruits neutrophils upon co-culturing. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.

Increase of mast cells in COVID-19 pneumonia may contribute to pulmonary fibrosis and thrombosis.

AIMS: Lung tissue from COVID-19 patients shares similar histomorphological features with chronic lung allograft disease, also suggesting activation of autoimmune-related pathways in COVID-19. To more clearly understand the underlying spectrum of pathophysiology in COVID-19 pneumonia, we analysed mRNA expression of autoimmune-related genes in post-mortem lung tissue from COVID-19 patients. METHODS AND RESULTS: Formalin-fixed, paraffin-embedded lung tissue samples of 18 COVID-19 patients and eight influenza patients were used for targeted gene expression profiling using NanoString technology. Multiplex immunofluorescence for tryptase and chymase was applied for validation. Genes related to mast cells were significantly increased in COVID-19. This finding was strengthened by multiplex immunofluorescence also showing a significant increase of tryptase- and chymase-positive cells in COVID-19. Furthermore, receptors for advanced glycation end-products (RAGE) and pro-platelet basic protein (PPBP) were up-regulated in COVID-19 compared to influenza. Genes associated with Type I interferon signalling showed a significant correlation to detected SARS-CoV2 pathway-related genes. The comparison of lung tissue samples from both groups based on the presence of histomorphological features indicative of acute respiratory distress syndrome did not result in finding any specific gene or pathways. CONCLUSION: Two separate means of measuring show a significant increase of mast cells in SARS-CoV-2-infected lung tissue compared to influenza. Additionally, several genes involved in fibrosis and thrombosis, among which are RAGE and PPBP, are up-regulated in COVID-19. As mast cells are able to induce thrombosis and fibrosis, they may play an important role in the pathogenesis of COVID-19.

Unfavourable outcome of glucocorticoid treatment in suspected idiopathic pulmonary fibrosis.

BACKGROUND AND OBJECTIVE: The diagnostic classification of 'possible idiopathic pulmonary fibrosis (posIPF)' is characterized by a radiological pattern of inconsistent usual interstitial pneumonia (UIP) on high-resolution computed tomography (HRCT) scan and a UIP pattern in surgical lung biopsy (SLB). The evidence base to guide treatment for patients with posIPF is lacking; the clinician must choose between observation, treatment with immunomodulatory agents or anti-fibrotic agents. METHODS: To evaluate outcomes of immunomodulatory treatment, a multicentre cohort of 59 posIPF patients treated with prednisone was analysed retrospectively. Prednisone starting dose was 0.5 mg/kg/day and tapered to 0.15 mg/day/kg over 6 months. Outcome measures were forced vital capacity (FVC) and serious adverse events (SAE), defined as death or hospital admissions. RESULTS: The majority of prednisone-treated posIPF patients were non-responders (68%) with a decrease in FVC >5% or death within 6 months from baseline; 90% of patients with radiographical presence of honeycombing were non-responders. In contrast, six out of seven patients with focal desquamative interstitial pneumonia-like reaction in the SLB who had stopped smoking for <5 years ago were responders to prednisone, demonstrating <5% FVC decline. The mean decline of FVC was 8.7% (95% CI: 3.1-14.3%) before treatment and 20% (95% CI: 9.4-31.1%) after treatment (P = 0.018) in the 32 patients with available FVC data. Twelve SAE occurred within the first 3 months on prednisone (at dosage >0.3 mg/kg/day), including five deaths. CONCLUSION: Patients with posIPF demonstrated an accelerated FVC decline and a substantial number of SAE on steroid therapy.

Evaluation of multicontrast MRI including fat suppression and inversion recovery spin echo for identification of intra-plaque hemorrhage and lipid core in human carotid plaque using the mahalanobis distance measure.

Intra-plaque hemorrhage (IPH) and lipid core, characteristics of rupture prone carotid plaques, are often visualized in vivo with MRI using T1 weighted gradient and spin echo, respectively. Increasing magnetic field strength may help to identify IPH and lipid core better. As a proof of concept, automatic segmentation of plaque components was performed with the Mahalanobis distance (MD) measure derived from image contrast from multicontrast MR images including inversion recovery spin echo and T1 weighted gradient echo with fat suppression. After MRI of nine formaldehyde-fixated autopsy specimens, the MDs and Euclidean Distances between plaque component intensities were calculated for each MR weighting. The distances from the carotid bifurcation and the size and shape of calcification spots were used as landmarks for coregistration of MRI and histology. MD between collagen/cell-rich area and IPH was largest with inversion recovery spin echo (4.2/9.3, respectively), between collagen/cell-rich area/foam cells and lipid core with T1 weighted gradient echo with fat suppression (26.9/38.2/4.6, respectively). The accuracy of detection of IPH, cell-rich area, and collagen increased when the MD classifier was used compared with the Euclidean Distance classifier. The enhanced conspicuity of lipid core and IPH in human carotid artery plaque, using ex vivo T1 weighted gradient echo with fat suppression and inversion recovery spin echo MRI and MD classifiers, demands further in vivo evaluation in patients.

Surfactant protein C mutations are the basis of a significant portion of adult familial pulmonary fibrosis in a dutch cohort.

RATIONALE: Familial clustering of adult idiopathic interstitial pneumonias (IIP) suggests that genetic factors might play an important role in disease development. Mutations in the gene encoding surfactant protein C (SFTPC) have been found in children and families with idiopathic pneumonias, whereas cocarriage of a mutation in ATP-binding cassette subfamily A member 3 (ABCA3) was postulated to have a disease-modifying effect. OBJECTIVES: To investigate the contribution of SFTPC mutations to adult familial pulmonary fibrosis (FPF) and the disease-modifying effect of mutations in ABCA3 within their families. METHODS: Twenty-two unrelated patients with FPF (10%) were identified within our single-center cohort of 229 patients with IIP. SFTPC was sequenced in 20 patients with FPF and 20 patients with sporadic IIP. In patients with an SFTPC mutation, sequencing of ABCA3 was performed. Discovered variants were typed in more than 100 control subjects and 121 additional patients with sporadic IIP. MEASUREMENTS AND MAIN RESULTS: In 5/20 unrelated patients with FPF (25%; confidence interval, 10-49) a mutation in SFTPC was detected: M71V, IVS4+2, and three times I73T. No mutations were detected in the sporadic or control cohort. Patients with SFTPC mutations presented with a histopathological pattern of usual interstitial pneumonia and nodular septa thickening and multiple lung cysts in combination with ground glass or diffuse lung involvement on chest high-resolution computed tomography. Two variants in ABCA3 were found in adult patients with FPF but not in affected children. CONCLUSIONS: Mutations in SFTPC are a frequent cause of FPF in adult patients in our cohort. Nonclassifiable radiological patterns with cystic changes and histopathological patterns of usual interstitial pneumonia are characteristics of adult SFTPC mutation carriers.

Telomere shortening and DNA damage in culprit cells of different types of progressive fibrosing interstitial lung disease.

Pulmonary fibrosis is strongly associated with telomere shortening and increased DNA damage. Key cells in the pathogenesis involve alveolar type 2 (AT2) cells, club cells and myofibroblasts; however, to what extent these cells are affected by telomere shortening and DNA damage is not yet known. We sought to determine the degree of, and correlation between, telomere shortening and DNA damage in different cell types involved in the pathogenesis of progressive fibrosing interstitial lung disease. Telomere length and DNA damage were quantified, using combined fluorescence in situ hybridisation and immunofluorescence staining techniques, in AT2 cells, club cells and myofibroblasts of controls and patients with pulmonary fibrosis and a telomerase reverse transcriptase mutation (TERT-PF), idiopathic pulmonary fibrosis (IPF) and fibrotic hypersensitivity pneumonitis (fHP). In IPF and TERT-PF lungs, AT2 cells contained shorter telomeres and expressed higher DNA damage signals than club cells and myofibroblasts. In fHP lungs, club cells contained highly elevated levels of DNA damage, while telomeres were not obviously short. In vitro, we found significantly shorter telomeres and higher DNA damage levels only in AT2 surrogate cell lines treated with telomerase inhibitor BIBR1532. Our study demonstrated that in IPF and TERT-PF lungs, telomere shortening and accumulation of DNA damage primarily affects AT2 cells, further supporting the importance of AT2 cells in these diseases, while in fHP the particularly high telomere-independent DNA damage signals in club cells underscores its bronchiolocentric pathogenesis. These findings suggest that cell type-specific telomere shortening and DNA damage may help to discriminate between different drivers of fibrogenesis.

The Extent of Inflammatory Cell Infiltrate and Fibrosis in Lungs of Telomere- and Surfactant-Related Familial Pulmonary Fibrosis.

Familial pulmonary fibrosis (FPF) is a monogenic disease most commonly involving telomere- (TERT) or surfactant- (SFTP) related mutations. These mutations have been shown to alter lymphocytic inflammatory responses, and FPF biopsies with histological lymphocytic infiltrates have been reported. Recently, a model of a surfactant mutation in mice showed that the disease initially started with an inflammatory response followed by fibrogenesis. Since inflammation and fibrogenesis are targeted by different drugs, we investigated whether the degree of these two features co-localize or occur independently in different entities of FPF, and whether they influence survival. We quantified the number of lymphocyte aggregates per surface area, the extent of diffuse lymphocyte cell infiltrate, the number of fibroblast foci per surface area, and the percentage of fibrotic lung surface area in digitally scanned hematoxylin and eosin (H&E) sections of diagnostic surgical biopsies of patients with TERT-related FPF (TERT-PF; n = 17), SFTP-related FPF (SFTP-PF; n = 7), and sporadic idiopathic pulmonary fibrosis (sIPF; n = 10). For comparison, we included biopsies of patients with cellular non-specific interstitial pneumonia (cNSIP; n = 10), an inflammatory interstitial lung disease with high lymphocyte influx and usually responsive to immunosuppressive therapy. The degree of inflammatory cell infiltrate and fibrosis in TERT-PF and SFTP-PF was not significantly different from that in sIPF. In comparison with cNSIP, the extent of lymphocyte infiltrates was significantly lower in sIPF and TERT-PF, but not in SFTP-PF. However, in contrast with cNSIP, in sIPF, TERT-PF, and SFTP-PF, diffuse lymphocyte cell infiltrates were predominantly present and lymphocyte aggregates were only present in fibrotic areas (p < 0.0001). Furthermore, fibroblast foci and percentage of fibrotic lung surface were associated with survival (p = 0.022 and p = 0.018, respectively), while this association was not observed for lymphocyte aggregates or diffuse lymphocytic infiltration. Inflammatory cells in diagnostic lung biopsies of TERT-PF, SFTP-PF, and sIPF were largely confined to fibrotic areas. However, based on inflammation and fibrosis, no differences were found between FPF and sIPF, substantiating the histological similarities between monogenic familial and sporadic disease. Furthermore, the degree of fibrosis, rather than inflammation, correlates with survival, supporting that fibrogenesis is the key feature for therapeutic targeting of FPF.

Small cell carcinoma of the lung and large cell neuroendocrine carcinoma interobserver variability.

AIMS: To test the hypothesis that the published morphological criteria permit reliable segregation of small cell carcinoma of the lung (SCLC) and large cell neuroendocrine carcinoma (LCNEC) cases by determining the interobserver variation. METHODS AND RESULTS: One hundred and seventy cases of SCLC, LCNEC and cases diagnosed as neuroendocrine lung carcinoma before LCNEC had been established as a diagnostic category were retrieved from the archives of the assessor's institutes. A representative haematoxylin and eosin section from each case was selected for review. Batches of cases were circulated among nine pathologists with a special interest in pulmonary pathology. Participants were asked to classify the cases histologically according to the 2004 World Health Organization (WHO) criteria. The diagnoses were collected and kappa values calculated. Unanimity of diagnosis was achieved for only 20 cases; a majority diagnosis was reached for 115 cases. In 35 cases no consensus diagnosis could be reached. There was striking variability amongst assessors in diagnosing SCLC and LCNEC. The overall level of agreement for all cases included in this study was fair (kappa=0.40). CONCLUSIONS: Using non-preselected cases, the morphological WHO criteria for diagnosing SCLC and LCNEC leave room for subjective pathological interpretation, which results in imprecise categorization of SCLC and LCNEC cases.

Neonatal dexamethasone treatment in the rat leads to kidney damage in adulthood.

UNLABELLED: Recently, concern has been raised that corticosteroid treatment of preterm neonates might be associated with adverse effects later in life, including early development of hypertension. Here, we investigate the impact of neonatal dexamethasone (Dex) treatment on early renal cell proliferation and nephron number. We analyzed mitotic activity in renal cortex of rat pups neonatally treated with Dex. Nephron number was measured and possible renal damage was quantified by counting inflammatory foci, ED-1 positive cells (macrophages), and the desmin score (activated podocytes). Mitotic activity was 34 and 29% lower on d 2 and 4 in Dex-treated rats compared with saline-treated controls. The number of glomeruli was lower at 4 wk, but nephron size was unchanged after Dex treatment, as calculated from glomerular density and (lower) body- and kidney weight. At wk 50, the glomerular number was significantly lower in Dex-treated rats, whereas body and kidney weight were the same as in Sal controls. Dex rats also showed more kidney damage, manifested by a approximately 3.5-fold increase in inflammation foci/mm and in ED-1 positive cells/mm and a approximately 4.3-fold increased desmin score. Temporary suppression of mitotic activity during neonatal Dex treatment leads to reduction of nephron number and more kidney damage later in life. ABBREVIATIONS: :

From organ to cell: Multi-level telomere length assessment in patients with idiopathic pulmonary fibrosis.

RATIONALE: A subset of patients with idiopathic pulmonary fibrosis (IPF) contains short leukocyte telomeres or telomere related mutations. We previously showed that alveolar type 2 cells have short telomeres in fibrotic lesions. Our objectives were to better understand how telomere shortening associates with fibrosis in IPF lung and identify a subset of patients with telomere-related disease. METHODS: Average telomere length was determined in multiple organs, basal and apical lung, and diagnostic and end-stage fibrotic lung biopsies. Alveolar type 2 cells telomere length was determined in different areas of IPF lungs. RESULTS: In IPF but not in controls, telomere length in lung was shorter than in other organs, providing rationale to focus on telomere length in lung. Telomere length did not correlate with age and no difference in telomere length was found between diagnostic and explant lung or between basal and apical lung, irrespective of the presence of a radiological apicobasal gradient or fibrosis. Fifteen out of 28 IPF patients had average lung telomere length in the range of patients with a telomerase (TERT) mutation, and formed the IPFshort group. Only in this IPFshort and TERT group telomeres of alveolar type 2 cells were extremely short in fibrotic areas. Additionally, whole exome sequencing of IPF patients revealed two genetic variations in RTEL1 and one in PARN in the IPFshort group. CONCLUSIONS: Average lung tissue telomere shortening does not associated with fibrotic patterns in IPF, however, approximately half of IPF patients show excessive lung telomere shortening that is associated with pulmonary fibrosis driven by telomere attrition.

Characterization of in vitro and in vivo lesions made by a novel multichannel ablation generator and a circumlinear decapolar ablation catheter.

INTRODUCTION: The aim of this study is to characterize ablation lesions using varying ratios of bipolar:unipolar energy and to show the feasibility of a circular decapolar pulmonary vein ablation catheter (PVAC) to create transmural lesions in an in vivo porcine superior vena cava (SVC) model. METHODS AND RESULTS: Ablations were performed on (1) isolated blocks of bovine myocardium, (2) thigh muscle preparations (4 pigs), and (3) the junction between SVC and right atrium (6 pigs). Radiofrequency (RF) energy was delivered simultaneously to all electrodes (target temperature 60 degrees C, maximum power 10 W/electrode) with various ratios of bipolar:unipolar energy. (1) In vitro PVAC resulted in circumscript lesions. Changing RF energy mode from unipolar only to bipolar:unipolar 1:1, 2:1 and 4:1 and bipolar only decreased lesion depth significantly (6.7 +/- 0.2 vs. 5.0 +/- 0.2, 4.1 +/- 0.3, 3.6 +/- 0.1 and 3.1 +/- 0.1 mm, P < 0.001). (2) Similar results were obtained in vivo (thigh muscle) showing less deep lesions with more bipolar energy modes (e.g., 5.4 +/- 1.3 for unipolar only vs. 4.1 +/- 0.8 mm for bipolar only, P < 0.05). (3) Ablation at the SVC did not result in device-related complications. All animals survived and were sacrificed after 7 +/- 1 days except for one pig, which died immediately postoperatively from an unknown cause. Macroscopy and microscopy showed circumferential transmural SVC lesions. CONCLUSIONS: Temperature-controlled, power-limited RF energy with the PVAC is feasible for creating circumferential transmural lesions (SVC) that sometimes extended to neighboring structures. Lesion depth can be titrated by varying the bipolar:unipolar energy ratio. This novel ablation system paves the way for single-catheter ablation of AF.

Histopathological changes of the heart after neonatal dexamethasone treatment: studies in 4-, 8-, and 50-week-old rats.

Dexamethasone (Dex), for prevention of chronic lung disease in preterm infants, showed potential negative long-term effects. Studies regarding long-term cardiovascular effects are lacking. We investigated possible histopathological myocardial changes after neonatal Dex in the young and adult rat heart. Rats were treated with Dex on d 1, 2, and 3 (0.5, 0.3, and 0.1 mg/kg) of life. Control-pups received saline. At 4, 8, and 50 wk after birth rats were killed and anatomic data collected. Heart tissue was stained with hematoxylin and eosin, Cadherin-periodic acid schiff, and sirius red for cardiomyocyte morphometry and collagen determination. Presence of macrophages and mast cells was analyzed. Cardiomyocyte length of the Dex-treated rats was increased in all three age groups, whereas ventricular weight was reduced. Cardiomyocyte volumes were increased at 50 wk indicating cellular hypertrophy. Collagen content gradually increased with age and was 62% higher in Dex rats at 50 wk. Macrophage focus score and mast cell count were also higher. Neonatal Dex affects normal heart growth resulting in cellular hypertrophy and increased collagen deposition in the adult rat heart. Because previous studies in rats showed premature death, suggesting cardiac failure, cardiovascular follow-up of preterm infants treated with glucocorticoids should be considered.

Contractile and morphological properties of hamster retractor muscle following 16 h of cold preservation.

INTRODUCTION: Cold hypoxia is a common factor in cold tissue preservation and mammalian hibernation. The purpose of this study was to determine the effects of cold preservation on the function of the retractor (RET) muscle of the hamster in the non-hibernating state and compare these with previously published data (van der Heijden et al., 2000) on the rat cutaneus trunci (CT) muscle. MATERIALS AND METHODS: After cold storage (16 h at 4 degrees C), muscles were stimulated electrically to measure maximum tetanus tension (P(0)) and histologically analyzed. The protective effects of addition of the antioxidants trolox and deferiprone and the calcium release inhibitor BDM to the storage fluid were determined. RESULTS: After storage, the twitch threshold current was increased (from 60 to 500 microA) and P(0) was decreased to 27% of control. RET morphology remained unaffected. RET muscle function was protected by trolox and deferiprone (P(0), resp., 43% and 59% of control). Addition of BDM had no effect on the RET. CONCLUSIONS: The observed effects of cold preservation and of trolox and deferiprone on the RET were comparable to those on CT muscle function, as reported in a previously published study (van der Heijden et al., 2000). Both hamster RET and rat CT muscles show considerable functional damage due to actions of reactive oxygen species. In contrast to the CT, in the RET cold preservation-induced functional injury could not be prevented by BDM and was not accompanied by morphological damage such as necrosis and edema. This suggests that the RET myocytes possess a specific adaptation to withstand the Ca(2+) overload induced by cold ischemia.

Implementation of Novel Molecular Biomarkers for Non-small Cell Lung Cancer in the Netherlands: How to Deal With Increasing Complexity.

The diagnostic landscape of non-small cell lung cancer (NSCLC) is changing rapidly with the availability of novel treatments. Despite high-level healthcare in the Netherlands, not all patients with NSCLC are tested with the currently relevant predictive tumor markers that are necessary for optimal decision-making for today's available targeted or immunotherapy. An expert workshop on the molecular diagnosis of NSCLC involving pulmonary oncologists, clinical chemists, pathologists, and clinical scientists in molecular pathology was held in the Netherlands on December 10, 2018. The aims of the workshop were to facilitate cross-disciplinary discussions regarding standards of practice, and address recent developments and associated challenges that impact future practice. This paper presents a summary of the discussions and consensus opinions of the workshop participants on the initial challenges of harmonization of the detection and clinical use of predictive markers of NSCLC. A key theme identified was the need for broader and active participation of all stakeholders involved in molecular diagnostic services for NSCLC, including healthcare professionals across all disciplines, the hospitals and clinics involved in service delivery, healthcare insurers, and industry groups involved in diagnostic and treatment innovations. Such collaboration is essential to integrate different technologies into molecular diagnostics practice, to increase nationwide patient access to novel technologies, and to ensure consensus-preferred biomarkers are tested.

Dominant arrhythmia vulnerability of the right ventricle in senescent mice.

BACKGROUND: Several cardiac disorders affect the right ventricle (RV) and left ventricle (LV) equally, but nevertheless, RV vulnerability to conduction slowing and arrhythmias exceeds that of the LV. OBJECTIVE: This study sought to assess the mechanism of dominant RV arrhythmia vulnerability in senescent mice as a model of general reduced myocardial integrity. METHODS: Epicardial ventricular activation mapping was performed on senescent (22 months) and adult (3 months) Langendorff perfused mouse hearts. Arrhythmia inducibility was tested by programmed stimulation. Conduction velocity longitudinal and transversal (CVT) to fiber orientation, conduction heterogeneity, and effective refractory period were determined. Subsequently, hearts were processed for immunohistochemistry, Western blotting, and Sirius red staining. RESULTS: In senescent RV, but not LV, CVT was reduced and wavelength decreased, whereas anisotropic ratio and conduction heterogeneity increased. Arrhythmias, based on anisotropic reentry, were induced in 55% of senescent hearts only and predominantly in RV. In senescent mice, Connexin 43 (Cx43) and Cardiac Sodium Channel (Nav1.5) were decreased and interstitial fibrosis increased comparably in RV and LV. However, in senescent mice, heterogeneously distributed patches of replacement fibrosis were present throughout the entire RV myocardium, but only in midendocardium and subendocardium of LV. Cx43 expression in these areas was disrupted. CONCLUSION: Widespread presence of replacement fibrosis in senescent RV compared with LV, combined with Cx43 and Nav1.5 disruption, potentiate shorter wavelength, conduction slowing, and conduction heterogeneity in RV, resulting in greater vulnerability of senescent RV to arrhythmias.

Long-term cardiovascular effects of neonatal dexamethasone treatment: hemodynamic follow-up by left ventricular pressure-volume loops in rats.

Dexamethasone is clinically applied in preterm infants to treat or prevent chronic lung disease. However, concern has emerged about adverse side effects. The cardiovascular short-term side effects of neonatal dexamethasone treatment are well documented, but long-term consequences are unknown. Previous studies showed suppressed mitosis during dexamethasone treatment, leading to reduced ventricular weight, depressed systolic function, and compensatory dilatation in prepubertal rats. In addition, recent data indicated a reduced life expectancy. Therefore, we investigated the long-term effects of neonatal dexamethasone treatment on cardiovascular function. Neonatal rats were treated with dexamethasone or received saline. Cardiac function was determined in 8-, 50-, and 80-wk-old animals, representing young adult, middle-aged, and elderly stages. A pressure-conductance catheter was introduced into the left ventricle to measure pressure-volume loops. Subsequently, the hearts were collected for histological examination. Our results showed reduced ventricular and body weights in dexamethasone-treated rats at 8 and 80 wk, but not at 50 wk. Cardiac output and diastolic function were unchanged, but systolic function was depressed at 50 and 80 wk, evidenced by reduced ejection fractions and rightward shifts of the end-systolic pressure-volume relationships. We concluded that previously demonstrated early adverse effects of neonatal dexamethasone treatment are transient but that reduced ventricular weight and systolic dysfunction become manifest again in elderly rats. Presumably, cellular hypertrophy initially compensates for the dexamethasone treatment-induced lower number of cardiomyocytes, but this mechanism falls short at a later stage, leading to systolic dysfunction. If applicable to humans, cardiac screening of a relatively large patient group to enable secondary prevention may be indicated.

Telomerase Reverse Transcriptase Polymorphism rs2736100: A Balancing Act between Cancer and Non-Cancer Disease, a Meta-Analysis.

The enzyme telomerase reverse transcriptase (TERT) is essential for telomere maintenance. In replicating cells, maintenance of telomere length is important for the preservation of vital genetic information and prevention of genomic instability. A common genetic variant in TERT, rs2736100 C/A, is associated with both telomere length and multiple diseases. Carriage of the C allele is associated with longer telomere length, while carriage of the A allele is associated with shorter telomere length. Furthermore, some diseases have a positive association with the C and some with the A allele. In this study, meta-analyses were performed for two groups of diseases, cancerous diseases, e.g., lung cancer and non-cancerous diseases, e.g., pulmonary fibrosis, using data from genome-wide association studies and case-control studies. In the meta-analysis it was found that cancer positively associated with the C allele (pooled OR 1.16 [95% CI 1.09-1.23]) and non-cancerous diseases negatively associated with the C allele (pooled OR 0.81 [95% CI 0.65-0.99]). This observation illustrates that the ambiguous role of telomere maintenance in disease hinges, at least in part, on a single locus in telomerase genes. The dual role of this single nucleotide polymorphism also emphasizes that therapeutic agents aimed at influencing telomere maintenance should be used with caution.

Cell Type-Specific Quantification of Telomere Length and DNA Double-strand Breaks in Individual Lung Cells by Fluorescence In Situ Hybridization and Fluorescent Immunohistochemistry.

Telomeres are small repetitive DNA sequences at the ends of chromosomes which act as a buffer in age-dependent DNA shortening. Insufficient telomere repeats will be recognized as double-strand breaks. Presently, it is becoming more evident that telomere attrition, whether or not caused by mutations in telomere maintenance genes, plays an important role in many inflammatory and age-associated diseases. In this report, a method to (semi)quantitatively assess telomere length and DNA double-strand breaks in formalin-fixed paraffin-embedded (FFPE) tissue is described. Therefore, a novel combination of quantitative fluorescence in situ hybridization, tissue elution, and immunofluorescence staining techniques was developed. Caveolin-1 (type 1 pneumocytes), pro-surfactant protein C (type 2 pneumocytes), club cell-10 (club cells), and alpha smooth muscle actin (smooth muscle cells) markers were used to identify cell types. To visualize all the different probes, restaining the tissue by heat-mediated slide elution is essential. Fluorescent signals of telomeres and DNA double-strand breaks were quantified using the Telometer plugin of ImageJ. As example, we analyzed lung tissue from a familial pulmonary fibrosis patient with a mutation in the telomere-associated gene poly(A)-specific ribonuclease ( PARN). The protocol displays a novel opportunity to directly quantitatively link DNA double-strand breaks to telomere length in specific FFPE cells.

The chemokine and chemokine receptor profile of infiltrating cells in the wall of arteries with cardiac allograft vasculopathy is indicative of a memory T-helper 1 response.

BACKGROUND: Despite improvement in short-term patient survival after heart transplantation (HTx), long-term survival rates have not improved much, mainly because of cardiac allograft vasculopathy (CAV). Cytokines and chemokines are considered to play an important role in CAV development. METHODS AND RESULTS: We focused on coronary arteries of HTx patients and made an inventory of the infiltrating cells and the expression of cytokines as well as chemokines and chemokine receptors (C+CR) in the different layers of the vessel wall with CAV. Tissue slides were stained for a variety of cell markers (CD3, CD4, CD8, CD20, CD68, CD79a), chemokines (monokine induced by interferon [MIG], interferon-inducible protein 10 [IP-10], interferon-inducible T cell-alpha chemoattractant [ITAC], RANTES [regulated on activation normal T cell expressed and secreted], and fractalkine), and chemokine receptors (CXCR3, CCR5, and CX3CR1). In reference coronary arteries (not transplanted), almost no infiltrating cells were found, and in transplanted hearts with CAV (HTx+CAV), a large number of T cells were observed (CD4:CD8=2:1), mainly localized in the neointima and adventitia. Most of these T cells appeared to be activated (human leukocyte antigen DR positive). Coronary arteries from transplanted hearts without CAV (HTx-CAV), HTx+CAV, and references were also analyzed for cytokine and C+CR mRNA expression with the use of quantitative polymerase chain reaction. Interferon-gamma was highly expressed in HTx+CAV compared with HTx-CAV. Interleukin-4 and interleukin-10 were expressed at the same level in both HTx groups and references. In HTx+CAV, all C+CR, but especially the T-helper 1 (TH1) C+CR, were more abundant than in the HTx-CAV and references. However, TH2 CCR4 expression did not differ significantly between both HTx groups. CONCLUSIONS: In coronary arteries with CAV, most T cells are CD4+ and express human leukocyte antigen DR. These activated TH cells are mainly memory TH1 cells on the basis of their C+CR profile and cytokine expression.