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Background: Severe alpha1-antitrypsin (AAT) deficiency associated with low AAT blood concentrations is an established genetic COPD risk factor. Less is known about the respiratory health impact of variation in AAT serum concentrations in the general population. We cross-sectionally investigated correlates of circulating AAT concentrations and its association with FEV1.Methods: In 5187 adults (2669 females) with high-sensitive c-reactive protein (CRP) levels < or = 10 mg/l from the population-based Swiss SAPALDIA cohort, blood was collected at the time of follow-up examination for measuring serum AAT and CRP.Results: Female gender, hormone intake, systolic blood pressure, age in men and in postmenopausal women, as well as active and passive smoking were positively, whereas alcohol intake and BMI inversely correlated with serum AAT levels, independent of CRP adjustment. We observed an inverse association of AAT with FEV1 in the total study population (p < 0.001), that disappeared after adjustment for CRP (p = 0.28). In addition, the AAT and FEV1 association was modified by gender, menopausal status in women, and smoking.Conclusion: The results of this population-based study reflect a complex interrelationship between tobacco exposure, gender related factors, circulating AAT, systemic inflammatory status and lung function. [ABSTRACT FROM AUTHOR]

Sarcoidosis is a highly variable, systemic granulomatous disease of hitherto unknown aetiology. The GenPhenReSa (Genotype-Phenotype Relationship in Sarcoidosis) project represents a European multicentre study to investigate the influence of genotype on disease phenotypes in sarcoidosis.The baseline phenotype module of GenPhenReSa comprised 2163 Caucasian patients with sarcoidosis who were phenotyped at 31 study centres according to a standardised protocol.From this module, we found that patients with acute onset were mainly female, young and of Scadding type I or II. Female patients showed a significantly higher frequency of eye and skin involvement, and complained more of fatigue. Based on multidimensional correspondence analysis and subsequent cluster analysis, patients could be clearly stratified into five distinct, yet undescribed, subgroups according to predominant organ involvement: 1) abdominal organ involvement, 2) ocular-cardiac-cutaneous-central nervous system disease involvement, 3) musculoskeletal-cutaneous involvement, 4) pulmonary and intrathoracic lymph node involvement, and 5) extrapulmonary involvement.These five new clinical phenotypes will be useful to recruit homogenous cohorts in future biomedical studies., Competing Interests: Conflict of interest: Disclosures can be found alongside this article at erj.ersjournals.com, (Copyright ©ERS 2018.)

Time to first investigator-reported acute exacerbation was a key secondary end-point in the INPULSIS trials of nintedanib in patients with idiopathic pulmonary fibrosis (IPF).We used the INPULSIS trial data to investigate risk factors for acute exacerbation of IPF and to explore the impact of nintedanib on risk and outcome of investigator-reported and adjudicated confirmed/suspected acute exacerbations. Mortality following these events and events adjudicated as not acute exacerbations was analysed using the log rank test.Risk of acute exacerbations was most strongly associated with the following variables: baseline forced vital capacity (higher risk with lower value), baseline supplemental oxygen (higher risk with use), baseline antacid medication (higher risk with use), treatment (higher risk with placebo), and for confirmed/suspected acute exacerbations, cigarette smoking. Mortality was similar following investigator-reported and adjudicated confirmed/suspected acute exacerbations. Nintedanib had no significant effect on risk of mortality post-exacerbation.Investigator-reported acute exacerbations of IPF are associated with similar risk factors and outcomes as adjudicated confirmed/suspected acute exacerbations., Competing Interests: Conflict of interest: Disclosures can be found alongside this article at erj.ersjournals.com, (Copyright ©ERS 2017.)

Background: Whole lung lavage (WLL) is the current standard of care treatment for patients affected by pulmonary alveolar proteinosis (PAP). However, WLL is not standardized and international consensus documents are lacking. Our aim was to obtain a factual portrayal of WLL as currently practiced with respect to the procedure, indications for its use, evaluation of therapeutic benefit and complication rate., Methods: A clinical practice survey was conducted globally by means of a questionnaire and included 27 centers performing WLL in pediatric and/or adult PAP patients., Results: We collected completed questionnaires from 20 centres in 14 countries, practicing WLL in adults and 10 centers in 6 countries, practicing WLL in pediatric patients. WLL is almost universally performed under general anesthesia, with a double-lumen endobronchial tube in two consecutive sessions, with an interval of 1-2 weeks between sessions in approximately 50 % of centres. The use of saline warmed to 37 °C, drainage of lung lavage fluid by gravity and indications for WLL therapy in PAP were homogenous across centres. There was great variation in the choice of the first lung to be lavaged: 50 % of centres based the choice on imaging, whereas 50 % always started with the left lung. The choice of position was also widely discordant; the supine position was chosen by 50 % of centres. Other aspects varied significantly among centres including contraindications, methods and timing of follow up, use of chest percussion, timing of extubation following WLL and lung isolation and lavage methods for small children. The amount of fluid used to perform the WLL is a critical aspect. Whilst a general consensus exists on the single aliquot of fluid for lavage (around 800 ml of warm saline, in adults) great variability exists in the total volume instilled per lung, ranging from 5 to 40 liters, with an average of 15.4 liters/lung., Conclusions: This international survey found that WLL is safe and effective as therapy for PAP. However these results also indicate that standardization of the procedure is required; the present survey represents the a first step toward building such a document.

The Z deficiency in α1-antitrypsin (A1ATD) is an under-recognized condition. Alpha1-antitrypsin (A1AT) is the main protein in the α1-globulin fraction of serum protein electrophoresis (SPE); however, evaluation of the α1-globulin protein fraction has received very little attention. Serum Z-type A1AT manifests in polymeric forms, but their interference with quantitative immunoassays has not been reported. Here, 214 894 samples were evaluated by SPE at the G. Fracastoro Hospital of Verona, Italy. Patients with an A1AT level ≤ 0.92 g/L were recalled to complete A1ATD diagnosis. In parallel, to qualitatively and quantitatively characterize A1AT, sera samples from 10 PiZZ and 10 PiMM subjects obtained at the National Institute of Tuberculosis and Lung Diseases in Warsaw, Poland, were subjected to non-denaturing 7.5% PAGE and 7.5% SDS-PAGE followed by Western blot. Moreover, purified A1AT was heated at 60°C and analyzed by a non-denaturing PAGE and 4-15% gradient SDS-PAGE followed by Western blot as well as by isolelectrofocusing and nephelometry. A total of 966 samples manifested percentages ≤ 2.8 or a double band in the alpha1-zone. According to the nephelometry data, 23 samples were classified as severe (A1AT ≤ 0.49 g/L) and 462 as intermediate (A1AT >0.49≤ 1.0 g/L) A1ATD. Twenty subjects agreed to complete the diagnosis and an additional 21 subjects agreed to family screening. We detected 9 cases with severe and 26 with intermediate A1ATD. Parallel experiments revealed that polymerization of M-type A1AT, when measured by nephelometry or isolelectrofocusing, yields inaccurate results, leading to the erroneous impression that it was Z type and not M-type A1AT. We illustrate the need for confirmation of Z A1AT values by "state of the art" method. Clinicians should consider a more in-depth investigation of A1ATD in patients when they exhibit serum polymers and low α1-globulin protein levels by SPE.

Background: The majority of cases with severe pulmonary alveolar proteinosis (PAP) are caused by auto-antibodies against GM-CSF. A multitude of genetic and exogenous causes are responsible for few other cases. Goal of this study was to determine the prevalence of GATA2 deficiency in children and adults with PAP and hematologic disorders., Methods: Of 21 patients with GM-CSF-autoantibody negative PAP, 13 had no other organ involvement and 8 had some form of hematologic disorder. The latter were sequenced for GATA2., Results: Age at start of PAP ranged from 0.3 to 64 years, 4 patients were children. In half of the subjects GATA2-sequence variations were found, two of which were considered disease causing. Those two patients had the typical phenotype of GATA2 deficiency, one of whom additionally showed a previously undescribed feature - a cholesterol pneumonia. Hematologic disorders included chronic myeloic leukemia, juvenile myelo-monocytic leukemia, lymphoblastic leukemia, sideroblastic anemia and two cases of myelodysplastic syndrome (MDS). A 4 year old child with MDS and DiGeorge Syndrome Type 2 was rescued with repetitive whole lung lavages and her PAP was cured with heterologous stem cell transplant., Conclusions: In children and adults with severe GM-CSF negative PAP a close cooperation between pneumologists and hemato-oncologists is needed to diagnose the underlying diseases, some of which are caused by mutations of transcription factor GATA2. Treatment with whole lung lavages as well as stem cell transplant may be successful.

Pulmonary alveolar proteinosis (PAP) is a severe autoimmune disease caused by autoantibodies that neutralize GM-CSF resulting in impaired function of alveolar macrophages. In this study, we characterize 21 GM-CSF autoantibodies from PAP patients and find that somatic mutations critically determine their specificity for the self-antigen. Individual antibodies only partially neutralize GM-CSF activity using an in vitro bioassay, depending on the experimental conditions, while, when injected in mice together with human GM-CSF, they lead to the accumulation of a large pool of circulating GM-CSF that remains partially bioavailable. In contrast, a combination of three non-cross-competing antibodies completely neutralizes GM-CSF activity in vitro by sequestering the cytokine in high-molecular-weight complexes, and in vivo promotes the rapid degradation of GM-CSF-containing immune complexes in an Fc-dependent manner. Taken together, these findings provide a plausible explanation for the severe phenotype of PAP patients and for the safety of treatments based on single anti-GM-CSF monoclonal antibodies.

Background: Malignant Pleural Mesothelioma (MPM) is an aggressive tumor arising from mesothelial cells lining the pleural cavities characterized by resistance to standard therapies. Most of the molecular steps responsible for pleural transformation remain unclear; however, several growth factor signaling cascades are known to be altered during MPM onset and progression. Transducers of these pathways, such as PIK3CA-mTOR-AKT, MAPK, and ezrin/radixin/moesin (ERM) could therefore be exploited as possible targets for pharmacological intervention. This study aimed to identify 'druggable' pathways in MPM and to formulate a targeted approach based on the use of commercially available molecules, such as the multikinase inhibitor sorafenib and the mTOR inhibitor everolimus., Methods: We planned a triple approach based on: i) analysis of immunophenotypes and mutational profiles in a cohort of thoracoscopic MPM samples, ii) in vitro pharmacological assays, ii) in vivo therapeutic approaches on MPM xenografts. No mutations were found in 'hot spot' regions of the mTOR upstream genes (e.g. EGFR, KRAS and PIK3CA)., Results: Phosphorylated mTOR and ERM were specifically overexpressed in the analyzed MPM samples. Sorafenib and everolimus combination was effective in mTOR and ERM blockade; exerted synergistic effects on the inhibition of MPM cell proliferation; triggered ROS production and consequent AMPK-p38 mediated-apoptosis. The antitumor activity was displayed when orally administered to MPM-bearing NOD/SCID mice., Conclusions: ERM and mTOR pathways are activated in MPM and 'druggable' by a combination of sorafenib and everolimus. Combination therapy is a promising therapeutic strategy against MPM.

Alpha1-antitrypsin Deficiency (AATD) is a rare hereditary disorder with an estimated prevalence of about 1/5000 individuals in Italy. Deficient patients are at a higher risk of developing lung emphysema and chronic liver disease. The low estimated prevalence of AATD prompted the establishment of a registry with the aim of learning more about the natural history and the quality of care of these patients. The Italian registry for AATD was established in 1996. In this study, genetic and clinical findings of Italian AATD patients are presented. Moreover, we also evaluated the changes in health-related quality of life (HRQoL) in patients with COPD and AAT deficiency over a three-year period, in relation to augmentation therapy. In a period spanning 18 years (1996-2014) a total of 422 adult subjects with severe AATD were enrolled, namely 258 PI*ZZ, 74 PI*SZ, 4 PI*SS and 86 patients with at least one rare deficient allele. The 21.3% frequency for AATD patients with at least one deficient rare variant is the highest so far recorded in national registries of AATD. The registry data allow a detailed characterization of the natural course of the disease and the level of patient care, as well as confirm the usefulness of early AATD detection.

Rationale: α1-Antitrypsin (AAT) is a potent protease inhibitor, deficiency of which is associated with the presence of emphysema. An imbalance of elastase and antielastase, along with innate inflammation in the lung, is believed to cause lung destruction in α1-antitrypsin deficiency (AATD). It is now apparent that AAT has important immune-regulatory roles that would be lost in AATD, yet adaptive immune responses in the lung have not been investigated in patients with AATD., Objectives: To assess the adaptive immune response in severe AATD emphysema and compare it with that present in "usual" chronic obstructive pulmonary disease (COPD)., Methods: The immune inflammatory response in explanted lungs from 10 subjects with AATD was characterized and quantified, and the results were compared with those of 26 subjects with usual COPD and those of 17 smoking and 11 nonsmoking control subjects with normal lung function., Measurements and Main Results: Lymphoid follicles (LFs) in AATD and usual COPD were markedly increased when compared with control groups. Molecular analysis of B lymphocytes in LFs showed predominantly mono/oligoclonality. LF number correlated negatively with FEV1/FVC. B lymphocytes and CD4(+) and CD8(+) T lymphocytes were significantly increased in AATD and usual COPD when compared with control groups. IL-32, an important cytokine in induction of autoimmunity, was markedly up-regulated in AATD and usual COPD., Conclusions: An important adaptive immune inflammation, comprising B, CD4(+), and CD8(+) lymphocytes, and LFs, is a prominent feature in AATD. These results change the paradigm of the mechanism of AATD-induced emphysema from a pure elastase-antielastase imbalance to a much more complex one involving the adaptive immune system, similarly to what occurs in usual COPD.

Background: Alpha-1 antitrypsin (AAT) is the most abundant circulating antiprotease and is a member of the serine protease inhibitor (SERPIN) superfamily. The gene encoding AAT is the highly polymorphic SERPINA1 gene, found at 14q32.1. Mutations in the SERPINA1 gene can lead to AAT deficiency (AATD) which is associated with a substantially increased risk of lung and liver disease. The most common pathogenic AAT variant is Z (Glu342Lys) which causes AAT to misfold and polymerise within hepatocytes and other AAT-producing cells. A group of rare mutations causing AATD, termed Null or Q0, are characterised by a complete absence of AAT in the plasma. While ultra rare, these mutations confer a particularly high risk of emphysema., Methods: We performed the determination of AAT serum levels by a rate immune nephelometric method or by immune turbidimetry. The phenotype was determined by isoelectric focusing analysis on agarose gel with specific immunological detection. DNA was isolated from whole peripheral blood or dried blood spot (DBS) samples using a commercial extraction kit. The new mutations were identified by sequencing all coding exons (II-V) of the SERPINA1 gene., Results: We have found eight previously unidentified SERPINA1 Null mutations, named: Q0cork, Q0perugia, Q0brescia, Q0torino, Q0cosenza, Q0pordenone, Q0lampedusa, and Q0dublin . Analysis of clinical characteristics revealed evidence of the recurrence of lung symptoms (dyspnoea, cough) and lung diseases (emphysema, asthma, chronic bronchitis) in M/Null subjects, over 45 years-old, irrespective of smoking., Conclusions: We have added eight more mutations to the list of SERPINA1 Null alleles. This study underlines that the laboratory diagnosis of AATD is not just a matter of degree, because the precise determination of the deficiency and Null alleles carried by an AATD individual may help to evaluate the risk for the lung disease.

Idiopathic pulmonary fibrosis (IPF) is defined as a specific form of chronic, progressive fibrosing interstitial pneumonia of unknown cause. The most recent hypotheses on IPF pathogenesis suggest a central role of epithelial cell damage, followed by a dysregulated molecular cross talk between epithelial cells and fibroblasts. Thus, IPF progression has often been assimilated to that of cancer, and several signaling patterns appear to be disrupted in both diseases. Here, we analyze the expression in an IPF series of a panel of molecules, which are known to play a role in tumorigenic process. Our findings, although preliminary, reveal that IPF landscape is enriched in neoplastic potential expressed in a context of complex genomic polyclonality and cellular heterogeneity. These results provide a rationale for further investigations aimed to exploit-in a similar fashion to cancer-targeted therapies for a "precision medicine" approach to IPF., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Alpha-1 antitrypsin is the main inhibitor of neutrophil elastase in the lung. Although it is principally synthesized by hepatocytes, alpha-1 antitrypsin is also secreted by bronchial epithelial cells. Gene mutations can lead to alpha-1 antitrypsin deficiency, with the Z variant being the most clinically relevant due to its propensity to polymerize. The ability of bronchial epithelial cells to produce Z-variant protein and its polymers is unknown., Methods: Experiments using a conformation-specific antibody were carried out on M- and Z-variant-transfected 16HBE cells and on bronchial biopsies and ex vivo bronchial epithelial cells from Z and M homozygous patients. In addition, the effect of an inflammatory stimulus on Z-variant polymer formation, elicited by Oncostatin M, was investigated. Comparisons of groups were performed using t-test or ANOVA. Non-normally distributed data were assessed by Mann-Whitney U test or the Kruskal-Wallis test, where appropriate. A P value of < 0.05 was considered to be significant., Results: Alpha-1 antitrypsin polymers were found at a higher concentration in the culture medium of ex vivo bronchial epithelial cells from Z-variant homozygotes, compared with M-variant homozygotes (P < 0.01), and detected in the bronchial epithelial cells and submucosa of patient biopsies. Oncostatin M significantly increased the expression of alpha-1 antitrypsin mRNA and protein (P < 0.05), and the presence of Z-variant polymers in ex vivo cells (P < 0.01)., Conclusions: Polymers of Z-alpha-1 antitrypsin form in bronchial epithelial cells, suggesting that these cells may be involved in the pathogenesis of lung emphysema and in bronchial epithelial cell dysfunction.

Background: Interstitial lung disease occurring in children is a condition characterized by high frequency of cases due to genetic aberrations of pulmonary surfactant homeostasis, that are also believed to be responsible of a fraction of familial pulmonary fibrosis. To our knowledge, ABCA3 gene was not previously reported as causative agent of fibrosis affecting both children and adults in the same kindred., Methods: We investigated a large kindred in which two members, a girl whose interstitial lung disease was first recognized at age of 13, and an adult, showed a diffuse pulmonary fibrosis with marked differences in terms of morphology and imaging. An additional, asymptomatic family member was detected by genetic analysis. Surfactant abnormalities were investigated at biochemical, and genetic level, as well as by cell transfection experiments., Results: Bronchoalveolar lavage fluid analysis of the patients revealed absence of surfactant protein C, whereas the gene sequence was normal. By contrast, sequence of the ABCA3 gene showed a novel homozygous G > A transition at nucleotide 2891, localized within exon 21, resulting in a glycine to aspartic acid change at codon 964. Interestingly, the lung specimens from the girl displayed a morphologic usual interstitial pneumonitis-like pattern, whereas the specimens from one of the two adult patients showed rather a non specific interstitial pneumonitis-like pattern., Conclusions: We have detected a large kindred with a novel ABCA3 mutation likely causing interstitial lung fibrosis affecting either young and adult family members. We suggest that ABCA3 gene should be considered in genetic testing in the occurrence of familial pulmonary fibrosis.

To improve the knowledge on a variety of severe disorders, research has moved from the analysis of individual proteins to the investigation of all proteins expressed by a tissue/organism. This global proteomic approach could prove very useful: (i) for investigating the biochemical pathways involved in disease; (ii) for generating hypotheses; or (iii) as a tool for the identification of proteins differentially expressed in response to the disease state. Proteomics has not been used yet in the field of respiratory research as extensively as in other fields, only a few reproducible and clinically applicable molecular markers, which can assist in diagnosis, having been currently identified. The continuous advances in both instrumentation and methodology, which enable sensitive and quantitative proteomic analyses in much smaller amounts of biological material than before, will hopefully promote the identification of new candidate biomarkers in this area. The aim of this report is to critically review the application over the decade 2004-2013 of very sophisticated technologies to the study of respiratory disorders. The observed changes in protein expression profiles from tissues/fluids of patients affected by pulmonary disorders opens the route for the identification of novel pathological mediators of these disorders.