A proof-of principal study using phase-contrast imaging for the detection of large airway pathologies after lung transplantation.

In this study we aim to evaluate the assessment of bronchial pathologies in a murine model of lung transplantation with grating-based X-ray interferometry in vivo. Imaging was performed using a dedicated grating-based small-animal X-ray dark-field and phase-contrast scanner. While the contrast modality of the dark-field signal already showed several promising applications for diagnosing various types of pulmonary diseases, the phase-shifting contrast mechanism of the phase contrast has not yet been evaluated in vivo. For this purpose, qualitative analysis of phase-contrast images was performed and revealed pathologies due to previous lung transplantation, such as unilateral bronchial stenosis or bronchial truncation. Dependent lung parenchyma showed a strong loss in dark-field and absorption signal intensity, possibly caused by several post transplantational pathologies such as atelectasis, pleural effusion, or pulmonary infiltrates. With this study, we are able to show that bronchial pathologies can be visualized in vivo using conventional X-ray imaging when phase-contrast information is analysed. Absorption and dark-field images can be used to quantify the severity of lack of ventilation in the affected lung.

Antibiotic use during pregnancy increases offspring asthma severity in a dose-dependent manner.

BACKGROUND: The use of antibiotics during pregnancy is associated with increased allergic asthma risk in the offspring, and given that approximately 25% of pregnant women are prescribed antibiotics, it is important to understand the mechanisms contributing to this phenomenon. Currently, there are no studies that directly test this association experimentally. Our objective was to develop a mouse model in which antibiotic treatment during pregnancy results in increased offspring asthma susceptibility. METHODS: Pregnant mice were treated daily from gestation day 8-17 with an oral solution of the antibiotic vancomycin, and three concentrations were tested. At weaning, offspring were subjected to an adjuvant-free experimental asthma protocol using ovalbumin as an allergen. The composition of the gut microbiome was determined in mothers and offspring with samples collected from five different time points; short-chain fatty acids were also analyzed in allergic offspring. RESULTS: We found that maternal antibiotic treatment during pregnancy was associated with increased offspring asthma severity in a dose-dependent manner. Furthermore, maternal vancomycin treatment during pregnancy caused marked changes in the gut microbiome composition in both mothers and pups at several different time points. The increased asthma severity and intestinal microbiome changes in pups were also associated with significantly decreased cecal short-chain fatty acid concentrations. CONCLUSION: Consistent with the "Developmental Origins Hypothesis," our results confirm that exposure to antibiotics during pregnancy shapes the neonatal intestinal environment and increases offspring allergic lung inflammation.

Maternal asthma is associated with persistent changes in allergic offspring antibody glycosylation.

BACKGROUND: Maternal asthma during pregnancy is considered an environmental risk factor for asthma development in children. Immunoglobulin G (IgG) antibodies that are transferred from the mother to the fetus are known to act in a pro- or anti-inflammatory manner depending on their glycosylation status. OBJECTIVE: Using a mouse model, we examined how maternal allergic airway inflammation during pregnancy influenced offspring experimental asthma severity, as well as maternal and offspring serum IgG antibody glycosylation patterns. Additionally, the effects of maternal and offspring exposure to the same or different allergens were investigated. METHODS: Female mice were either sham sensitized or sensitized to casein (CAS) or ovalbumin (OVA) before mating. Subsequently, allergic lung inflammation was induced in pregnant dams via aerosol allergen challenge (sham, CAS or OVA). After weaning, pups were subjected to an experimental asthma protocol using OVA. Asn-297 IgG glycosylation was analysed in maternal and offspring serum. RESULTS: When mothers and offspring were sensitized to the same allergen (OVA-OVA), offspring had more severe experimental asthma. This was evidenced by altered antibody concentrations, increased bronchoalveolar lavage inflammatory cell influx and decreased lung tissue and lung draining lymph node regulatory T cell percentages. When mothers and offspring were sensitized to different allergens (CAS-OVA), this phenotype was no longer observed. Additionally, maternal serum from allergic mothers had significantly higher levels of pro-inflammatory IgG1, shown by decreased galactosylation and sialylation at the Asn-297 glycosylation site. Similar glycosylation patterns were observed in the serum of adult allergic offspring from allergic mothers. CONCLUSIONS AND CLINICAL RELEVANCE: We observed a strong association between maternal experimental asthma during pregnancy, increased offspring airway inflammation and pro-inflammatory IgG glycosylation patterns in mothers and offspring. IgG glycosylation is not a standard measurement in the clinical setting, and we argue that it may be an important parameter to include in future clinical studies.

Inhibition of B cell-dependent lymphoid follicle formation prevents lymphocytic bronchiolitis after lung transplantation.

Lung transplantation (LTx) is the only therapeutic option for many patients with chronic lung disease. However, long-term survival after LTx is severely compromised by chronic rejection (chronic lung allograft dysfunction [CLAD]), which affects 50% of recipients after 5 years. The underlying mechanisms for CLAD are poorly understood, largely due to a lack of clinically relevant animal models, but lymphocytic bronchiolitis is an early sign of CLAD. Here, we report that lymphocytic bronchiolitis occurs early in a long-term murine orthotopic LTx model, based on a single mismatch (grafts from HLA-A2:B6-knockin donors transplanted into B6 recipients). Lymphocytic bronchiolitis is followed by formation of B cell-dependent lymphoid follicles that induce adjacent bronchial epithelial cell dysfunction in a spatiotemporal fashion. B cell deficiency using recipient µMT-/- mice prevented intrapulmonary lymphoid follicle formation and lymphocytic bronchiolitis. Importantly, selective inhibition of the follicle-organizing receptor EBI2, using genetic deletion or pharmacologic inhibition, prevented functional and histological deterioration of mismatched lung grafts. In sum, we provided what we believe to be a mouse model of chronic rejection and lymphocytic bronchiolitis after LTx and identified intrapulmonary lymphoid follicle formation as a target for pharmacological intervention of long-term allograft dysfunction after LTx.

X-Ray Dark-field Imaging to Depict Acute Lung Inflammation in Mice.

The aim of this study was to evaluate the feasibility of early stage imaging of acute lung inflammation in mice using grating-based X-ray dark-field imaging in vivo. Acute lung inflammation was induced in mice by orotracheal instillation of porcine pancreatic elastase. Control mice received orotracheal instillation of PBS. Mice were imaged immediately before and 1 day after the application of elastase or PBS to assess acute changes in pulmonary structure due to lung inflammation. Subsequently, 6 mice from each group were sacrificed and their lungs were lavaged and explanted for histological analysis. A further 7, 14 and 21 days later the remaining mice were imaged again. All images were acquired with a prototype grating-based small-animal scanner to generate dark-field and transmission radiographs. Lavage confirmed that mice in the experimental group had developed acute lung inflammation one day after administration of elastase. Acute lung inflammation was visible as a striking decrease in signal intensity of the pulmonary parenchyma on dark-field images at day 1. Quantitative analysis confirmed that dark-field signal intensity at day 1 was significantly lower than signal intensities measured at the remaining timepoints, confirming that acute lung inflammation can be depicted in vivo with dark-field radiography.

Female mice lacking Pald1 exhibit endothelial cell apoptosis and emphysema.

Paladin (Pald1, mKIAA1274 or x99384) was identified in screens for vascular-specific genes and is a putative phosphatase. Paladin has also been proposed to be involved in various biological processes such as insulin signaling, innate immunity and neural crest migration. To determine the role of paladin we have now characterized the Pald1 knock-out mouse in a broad array of behavioral, physiological and biochemical tests. Here, we show that female, but not male, Pald1 heterozygous and homozygous knock-out mice display an emphysema-like histology with increased alveolar air spaces and impaired lung function with an obstructive phenotype. In contrast to many other tissues where Pald1 is restricted to the vascular compartment, Pald1 is expressed in both the epithelial and mesenchymal compartments of the postnatal lung. However, in Pald1 knock-out females, there is a specific increase in apoptosis and proliferation of endothelial cells, but not in non-endothelial cells. This results in a transient reduction of endothelial cells in the maturing lung. Our data suggests that Pald1 is required during lung vascular development and for normal function of the developing and adult lung in a sex-specific manner. To our knowledge, this is the first report of a sex-specific effect on endothelial cell apoptosis.

Reduced Frizzled Receptor 4 Expression Prevents WNT/ß-Catenin-driven Alveolar Lung Repair in Chronic Obstructive Pulmonary Disease.

RATIONALE: Chronic obstructive pulmonary disease (COPD), in particular emphysema, is characterized by loss of parenchymal alveolar tissue and impaired tissue repair. Wingless and INT-1 (WNT)/ß-catenin signaling is reduced in COPD; however, the mechanisms thereof, specifically the role of the frizzled (FZD) family of WNT receptors, remain unexplored. OBJECTIVES: To identify and functionally characterize specific FZD receptors that control downstream WNT signaling in impaired lung repair in COPD. METHODS: FZD expression was analyzed in lung homogenates and alveolar epithelial type II (ATII) cells of never-smokers, smokers, patients with COPD, and two experimental COPD models by quantitative reverse transcriptase-polymerase chain reaction, immunoblotting, and immunofluorescence. The functional effects of cigarette smoke on FZD4, WNT/ß-catenin signaling, and elastogenic components were investigated in primary ATII cells in vitro and in three-dimensional lung tissue cultures ex vivo. Gain- and loss-of-function approaches were applied to determine the effects of FZD4 signaling on alveolar epithelial cell wound healing and repair, as well as on expression of elastogenic components. MEASUREMENTS AND MAIN RESULTS: FZD4 expression was reduced in human and experimental COPD lung tissues as well as in primary human ATII cells from patients with COPD. Cigarette smoke exposure down-regulated FZD4 expression in vitro and in vivo, along with reduced WNT/ß-catenin activity. Inhibition of FZD4 decreased WNT/ß-catenin-driven epithelial cell proliferation and wound closure, and it interfered with ATII-to-ATI cell transdifferentiation and organoid formation, which were augmented by FZD4 overexpression. Moreover, FZD4 restoration by overexpression or pharmacological induction led to induction of WNT/ß-catenin signaling and expression of elastogenic components in three-dimensional lung tissue cultures ex vivo. CONCLUSIONS: Reduced FZD4 expression in COPD contributes to impaired alveolar repair capacity.

X-ray dark-field radiography facilitates the diagnosis of pulmonary fibrosis in a mouse model.

The aim of this study was to evaluate whether diagnosing pulmonary fibrosis with projection radiography can be improved by using X-ray dark-field radiograms. Pulmonary X-ray transmission and dark-field images of C57Bl/6N mice, either treated with bleomycin to induce pulmonary fibrosis or PBS to serve as controls, were acquired with a prototype grating-based small-animal scanner. Two blinded readers, both experienced radiologists and familiar with dark-field imaging, had to assess dark-field and transmission images for the absence or presence of fibrosis. Furthermore readers were asked to grade their stage of diagnostic confidence. Histological evaluation of the lungs served as the standard of reference in this study. Both readers showed a notably higher diagnostic confidence when analyzing the dark-field radiographs (p < 0.001). Diagnostic accuracy improved significantly when evaluating the lungs in dark-field images alone (p = 0.02) or in combination with transmission images (p = 0.01) compared to sole analysis of absorption images. Interreader agreement improved from good when assessing only transmission images to excellent when analyzing dark-field images alone or in combination with transmission images. Adding dark-field images to conventional transmission images in a murine model of pulmonary fibrosis leads to an improved diagnosis of this disease on chest radiographs.

Noncanonical WNT-5A signaling impairs endogenous lung repair in COPD.

Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide. One main pathological feature of COPD is the loss of functional alveolar tissue without adequate repair (emphysema), yet the underlying mechanisms are poorly defined. Reduced WNT-ß-catenin signaling is linked to impaired lung repair in COPD; however, the factors responsible for attenuating this pathway remain to be elucidated. Here, we identify a canonical to noncanonical WNT signaling shift contributing to COPD pathogenesis. We demonstrate enhanced expression of noncanonical WNT-5A in two experimental models of COPD and increased posttranslationally modified WNT-5A in human COPD tissue specimens. WNT-5A was increased in primary lung fibroblasts from COPD patients and induced by COPD-related stimuli, such as TGF-ß, cigarette smoke (CS), and cellular senescence. Functionally, mature WNT-5A attenuated canonical WNT-driven alveolar epithelial cell wound healing and transdifferentiation in vitro. Lung-specific WNT-5A overexpression exacerbated airspace enlargement in elastase-induced emphysema in vivo. Accordingly, inhibition of WNT-5A in vivo attenuated lung tissue destruction, improved lung function, and restored expression of ß-catenin-driven target genes and alveolar epithelial cell markers in the elastase, as well as in CS-induced models of COPD. We thus identify a novel essential mechanism involved in impaired mesenchymal-epithelial cross talk in COPD pathogenesis, which is amenable to therapy.

Cigarette smoke alters the secretome of lung epithelial cells.

Cigarette smoke is the most relevant risk factor for the development of lung cancer and chronic obstructive pulmonary disease. Many of its more than 4500 chemicals are highly reactive, thereby altering protein structure and function. Here, we used subcellular fractionation coupled to label-free quantitative MS to globally assess alterations in the proteome of different compartments of lung epithelial cells upon exposure to cigarette smoke extract. Proteomic profiling of the human alveolar derived cell line A549 revealed the most pronounced changes within the cellular secretome with preferential downregulation of proteins involved in wound healing and extracellular matrix organization. In particular, secretion of secreted protein acidic and rich in cysteine, a matricellular protein that functions in tissue response to injury, was consistently diminished by cigarette smoke extract in various pulmonary epithelial cell lines and primary cells of human and mouse origin as well as in mouse ex vivo lung tissue cultures. Our study reveals a previously unrecognized acute response of lung epithelial cells to cigarette smoke that includes altered secretion of proteins involved in extracellular matrix organization and wound healing. This may contribute to sustained alterations in tissue remodeling as observed in lung cancer and chronic obstructive pulmonary disease.

Viable Ednra Y129F mice feature human mandibulofacial dysostosis with alopecia (MFDA) syndrome due to the homologue mutation.

Animal models resembling human mutations are valuable tools to research the features of complex human craniofacial syndromes. This is the first report on a viable dominant mouse model carrying a non-synonymous sequence variation within the endothelin receptor type A gene (Ednra c.386A>T, p.Tyr129Phe) derived by an ENU mutagenesis program. The identical amino acid substitution was reported recently as disease causing in three individuals with the mandibulofacial dysostosis with alopecia (MFDA, OMIM 616367) syndrome. We performed standardized phenotyping of wild-type, heterozygous, and homozygous Ednra Y129F mice within the German Mouse Clinic. Mutant mice mimic the craniofacial phenotypes of jaw dysplasia, micrognathia, dysplastic temporomandibular joints, auricular dysmorphism, and missing of the squamosal zygomatic process as described for MFDA-affected individuals. As observed in MFDA-affected individuals, mutant Ednra Y129F mice exhibit hearing impairment in line with strong abnormalities of the ossicles and further, reduction of some lung volumetric parameters. In general, heterozygous and homozygous mice demonstrated inter-individual diversity of expression of the craniofacial phenotypes as observed in MFDA patients but without showing any cleft palates, eyelid defects, or alopecia. Mutant Ednra Y129F mice represent a valuable viable model for complex human syndromes of the first and second pharyngeal arches and for further studies and analysis of impaired endothelin 1 (EDN1)-endothelin receptor type A (EDNRA) signaling. Above all, Ednra Y129F mice model the recently published human MFDA syndrome and may be helpful for further disease understanding and development of therapeutic interventions.

Impairment of Immunoproteasome Function by Cigarette Smoke and in Chronic Obstructive Pulmonary Disease.

RATIONALE: Patients with chronic obstructive pulmonary disease (COPD) and in particular smokers are more susceptible to respiratory infections contributing to acute exacerbations of disease. The immunoproteasome is a specialized type of proteasome destined to improve major histocompatibility complex (MHC) class I-mediated antigen presentation for the resolution of intracellular infections. OBJECTIVES: To characterize immunoproteasome function in COPD and its regulation by cigarette smoke. METHODS: Immunoproteasome expression and activity were determined in bronchoalveolar lavage (BAL) and lungs of human donors and patients with COPD or idiopathic pulmonary fibrosis (IPF), as well as in cigarette smoke-exposed mice. Smoke-mediated alterations of immunoproteasome activity and MHC I surface expression were analyzed in human blood-derived macrophages. Immunoproteasome-specific MHC I antigen presentation was evaluated in spleen and lung immune cells that had been smoke-exposed in vitro or in vivo. MEASUREMENTS AND MAIN RESULTS: Immunoproteasome and MHC I mRNA expression was reduced in BAL cells of patients with COPD and in isolated alveolar macrophages of patients with COPD or IPF. Exposure of immune cells to cigarette smoke extract in vitro reduced immunoproteasome activity and impaired immunoproteasome-specific MHC I antigen presentation. In vivo, acute cigarette smoke exposure dynamically regulated immunoproteasome function and MHC I antigen presentation in mouse BAL cells. End-stage COPD lungs showed markedly impaired immunoproteasome activities. CONCLUSIONS: We here show that the activity of the immunoproteasome is impaired by cigarette smoke resulting in reduced MHC I antigen presentation. Regulation of immunoproteasome function by cigarette smoke may thus alter adaptive immune responses and add to prolonged infections and exacerbations in COPD and IPF.

Proteasome function is not impaired in healthy aging of the lung.

Aging is the progressive loss of cellular function which inevitably leads to death. Failure of proteostasis including the decrease in proteasome function is one hallmark of aging. In the lung, proteasome activity was shown to be impaired in age-related diseases such as chronic obstructive pulmonary disease. However, little is known on proteasome function during healthy aging. Here, we comprehensively analyzed healthy lung aging and proteasome function in wildtype, proteasome reporter and immunoproteasome knockout mice. Wildtype mice spontaneously developed senile lung emphysema while expression and activity of proteasome complexes and turnover of ubiquitinated substrates was not grossly altered in lungs of aged mice. Immunoproteasome subunits were specifically upregulated in the aged lung and the caspase-like proteasome activity concomitantly decreased. Aged knockout mice for the LMP2 or LMP7 immunoproteasome subunits showed no alteration in proteasome activities but exhibited typical lung aging phenotypes suggesting that immunoproteasome function is dispensable for physiological lung aging in mice. Our results indicate that healthy aging of the lung does not involve impairment of proteasome function. Apparently, the reserve capacity of the proteostasis systems in the lung is sufficient to avoid severe proteostasis imbalance during healthy aging.

Coactivator-Associated Arginine Methyltransferase-1 Function in Alveolar Epithelial Senescence and Elastase-Induced Emphysema Susceptibility.

Chronic obstructive pulmonary disease (COPD) is characterized by an irreversible loss of lung function and is one of the most prevalent and severe diseases worldwide. A major feature of COPD is emphysema, which is the progressive loss of alveolar tissue. Coactivator-associated arginine methyltransferase-1 (CARM1) regulates histone methylation and the transcription of genes involved in senescence, proliferation, and differentiation. Complete loss of CARM1 leads to disrupted differentiation and maturation of alveolar epithelial type II (ATII) cells. We thus hypothesized that CARM1 regulates the development and progression of emphysema. To address this, we investigated the contribution of CARM1 to alveolar rarefication using the mouse model of elastase-induced emphysema in vivo and small interfering (si)RNA-mediated knockdown in ATII-like LA4 cells in vitro. We demonstrate that emphysema progression in vivo is associated with a time-dependent down-regulation of CARM1. Importantly, elastase-treated CARM1 haploinsufficient mice show significantly increased airspace enlargement (52.5 ± 9.6 µm versus 38.8 ± 5.5 µm; P < 0.01) and lung compliance (2.8 ± 0.32 µl/cm H2O versus 2.4 ± 0.4 µl/cm H2O; P < 0.04) compared with controls. The knockdown of CARM1 in LA4 cells led to decreased sirtuin 1 expression (0.034 ± 0.003 versus 0.022 ± 0.001; P < 0.05) but increased expression of p16 (0.27 ± 0.013 versus 0.31 ± 0.010; P < 0.5) and p21 (0.81 ± 0.088 versus 1.28 ± 0.063; P < 0.01) and higher ß-galactosidase-positive senescent cells (50.57 ± 7.36% versus 2.21 ± 0.34%; P < 0.001) compared with scrambled siRNA. We further demonstrated that CARM1 haploinsufficiency impairs transdifferentiation and wound healing (32.18 ± 0.9512% versus 8.769 ± 1.967%; P < 0.001) of alveolar epithelial cells. Overall, these results reveal a novel function of CARM1 in regulating emphysema development and premature lung aging via alveolar senescence as well as impaired regeneration, repair, and differentiation of ATII cells.

In Vivo Dark-Field Radiography for Early Diagnosis and Staging of Pulmonary Emphysema.

OBJECTIVES: The aim of this study was to evaluate the suitability of in vivo x-ray dark-field radiography for early-stage diagnosis of pulmonary emphysema in mice. Furthermore, we aimed to analyze how the dark-field signal correlates with morphological changes of lung architecture at distinct stages of emphysema. MATERIALS AND METHODS: Female 8- to 10-week-old C57Bl/6N mice were used throughout all experiments. Pulmonary emphysema was induced by orotracheal injection of porcine pancreatic elastase (80-U/kg body weight) (n = 30). Control mice (n = 11) received orotracheal injection of phosphate-buffered saline. To monitor the temporal patterns of emphysema development over time, the mice were imaged 7, 14, or 21 days after the application of elastase or phosphate-buffered saline. X-ray transmission and dark-field images were acquired with a prototype grating-based small-animal scanner. In vivo pulmonary function tests were performed before killing the animals. In addition, lungs were obtained for detailed histopathological analysis, including mean cord length (MCL) quantification as a parameter for the assessment of emphysema. Three blinded readers, all of them experienced radiologists and familiar with dark-field imaging, were asked to grade the severity of emphysema for both dark-field and transmission images. RESULTS: Histopathology and MCL quantification confirmed the introduction of different stages of emphysema, which could be clearly visualized and differentiated on the dark-field radiograms, whereas early stages were not detected on transmission images. The correlation between MCL and dark-field signal intensities (r = 0.85) was significantly higher than the correlation between MCL and transmission signal intensities (r = 0.37). The readers' visual ratings for dark-field images correlated significantly better with MCL (r = 0.85) than visual ratings for transmission images (r = 0.36). Interreader agreement and the diagnostic accuracy of both quantitative and visual assessment were significantly higher for dark-field imaging than those for conventional transmission images. CONCLUSIONS: X-ray dark-field radiography can reliably visualize different stages of emphysema in vivo and demonstrates significantly higher diagnostic accuracy for early stages of emphysema than conventional attenuation-based radiography.

Quantitative detection of drug dose and spatial distribution in the lung revealed by Cryoslicing Imaging.

Administration of drugs via inhalation is an attractive route for pulmonary and systemic drug delivery. The therapeutic outcome of inhalation therapy depends not only on the dose of the lung-delivered drug, but also on its bioactivity and regional distribution. Fluorescence imaging has the potential to monitor these aspects already during preclinical development of inhaled drugs, but quantitative methods of analysis are lacking. In this proof-of-concept study, we demonstrate that Cryoslicing Imaging allows for 3D quantitative fluorescence imaging on ex vivo murine lungs. Known amounts of fluorescent substance (nanoparticles or fluorophore-drug conjugate) were instilled in the lungs of mice. The excised lungs were measured by Cryoslicing Imaging. Herein, white light and fluorescence images are obtained from the face of a gradually sliced frozen organ block. A quantitative representation of the fluorescence intensity throughout the lung was inferred from the images by accounting for instrument noise, tissue autofluorescence and out-of-plane fluorescence. Importantly, the out-of-plane fluorescence correction is based on the experimentally determined effective light attenuation coefficient of frozen murine lung tissue (10.0 ± 0.6 cm(-1) at 716 nm). The linear correlation between pulmonary total fluorescence intensity and pulmonary fluorophore dose indicates the validity of this method and allows direct fluorophore dose assessment. The pulmonary dose of a fluorescence-labeled drug (FcγR-Alexa750) could be assessed with an estimated accuracy of 9% and the limit of detection in ng regime. Hence, Cryoslicing Imaging can be used for quantitative assessment of dose and 3D distribution of fluorescence-labeled drugs or drug carriers in the lungs of mice.

Endothelial function in patients with familial Mediterranean fever-related amyloidosis and association with cardiovascular events.

OBJECTIVES: Secondary amyloidosis is the most important complication of FMF and endothelial function is more severely impaired. Elevated asymmetric dimethyl arginine (ADMA) may mediate the excess cardiovascular disease (CVD) risk of this group. We aimed to compare endothelial function characteristics, including ADMA, in patients with FMF-related amyloidosis and primary glomerulopathies and to define risk factors for a CVD event. METHODS: We undertook a cross-sectional study with prospective follow-up including consecutive patients with FMF-related amyloidosis (n = 98) or other non-diabetic glomerulopathies (n = 102). All patients had nephrotic-range proteinuria and normal glomerular filtration rate. Flow-mediated dilatation (FMD) was assessed and ADMA levels, CRP and pentraxin 3 (PTX3) were determined. Patients were followed for cardiovascular events. RESULTS: Amyloidosis patients secondary to FMF showed higher levels of ADMA, CRP and PTX3 and lower FMD as compared with patients with other glomerulopathies. Cardiovascular events (n = 54) were registered during 3 years of follow-up. Increased ADMA levels and lower FMD were observed in patients with cardiovascular risk in both groups, but especially in individuals with amyloidosis. CONCLUSION: Patients with FMF-related amyloidosis have increased CVD event risk, probably related to the high ADMA levels, elevated inflammatory markers and decreased FMD measures observed in these patients.

Improved diagnosis of pulmonary emphysema using in vivo dark-field radiography.

OBJECTIVES: The purpose of this study was to assess whether the recently developed method of grating-based x-ray dark-field radiography can improve the diagnosis of pulmonary emphysema in vivo. MATERIALS AND METHODS: Pulmonary emphysema was induced in female C57BL/6N mice using endotracheal instillation of porcine pancreatic elastase and confirmed by in vivo pulmonary function tests, histopathology, and quantitative morphometry. The mice were anesthetized but breathing freely during imaging. Experiments were performed using a prototype small-animal x-ray dark-field scanner that was operated at 35 kilovolt (peak) with an exposure time of 5 seconds for each of the 10 grating steps. Images were compared visually. For quantitative comparison of signal characteristics, regions of interest were placed in the upper, middle, and lower zones of each lung. Receiver-operating-characteristic statistics were performed to compare the effectiveness of transmission and dark-field signal intensities and the combined parameter "normalized scatter" to differentiate between healthy and emphysematous lungs. RESULTS: A clear visual difference between healthy and emphysematous mice was found for the dark-field images. Quantitative measurements of x-ray dark-field signal and normalized scatter were significantly different between the mice with pulmonary emphysema and the control mice and showed good agreement with pulmonary function tests and quantitative histology. The normalized scatter showed a significantly higher discriminatory power (area under the receiver-operating-characteristic curve [AUC], 0.99) than dark-field (AUC, 0.90; P = 0.01) or transmission signal (AUC, 0.69; P < 0.001) alone did, allowing for an excellent discrimination of healthy and emphysematous lung regions. CONCLUSIONS: In a murine model, x-ray dark-field radiography is technically feasible in vivo and represents a substantial improvement over conventional transmission-based x-ray imaging for the diagnosis of pulmonary emphysema.