Quantitative Image Analysis of Fibrillar Collagens Reveals Novel Diagnostic and Prognostic Biomarkers and Histotype-Dependent Aberrant Mechanobiology in Lung Cancer.

Fibrillar collagens are the most abundant extracellular matrix components in non-small cell lung cancer (NSCLC). However, the potential of collagen fiber descriptors as a source of clinically relevant biomarkers in NSCLC is largely unknown. Similarly, our understanding of the aberrant collagen organization and associated tumor-promoting effects is very scarce. To address these limitations, we identified a digital pathology approach that can be easily implemented in pathology units based on CT-FIRE software (version 2; https://loci.wisc.edu/software/ctfire) analysis of Picrosirius red (PSR) stains of fibrillar collagens imaged with polarized light (PL). CT-FIRE settings were pre-optimized to assess a panel of collagen fiber descriptors in PSR-PL images of tissue microarrays from surgical NSCLC patients (106 adenocarcinomas [ADC] and 89 squamous cell carcinomas [SCC]). Using this approach, we identified straightness as the single high-accuracy diagnostic collagen fiber descriptor (average area under the curve = 0.92) and fiber density as the single descriptor consistently associated with a poor prognosis in both ADC and SCC independently of the gold standard based on the TNM staging (hazard ratio, 2.69; 95% CI, 1.55-4.66; P < .001). Moreover, we found that collagen fibers were markedly straighter, longer, and more aligned in tumor samples compared to paired samples from uninvolved pulmonary tissue, particularly in ADC, which is indicative of increased tumor stiffening. Consistently, we observed an increase in a panel of stiffness-associated processes in the high collagen fiber density patient group selectively in ADC, including venous/lymphatic invasion, fibroblast activation (α-smooth muscle actin), and immune evasion (programmed death-ligand 1). Similarly, a transcriptional correlation analysis supported the potential involvement of the major YAP/TAZ pathway in ADC. Our results provide a proof-of-principle to use CT-FIRE analysis of PSR-PL images to assess new collagen fiber-based diagnostic and prognostic biomarkers in pathology units, which may improve the clinical management of patients with surgical NSCLC. Our findings also unveil an aberrant stiff microenvironment in lung ADC that may foster immune evasion and dissemination, encouraging future work to identify therapeutic opportunities.

Reduced airway levels of fatty-acid binding protein 4 in COPD: relationship with airway infection and disease severity.

BACKGROUND: For still unclear reasons, chronic airway infection often occurs in patients with Chronic Obstructive Pulmonary Disease (COPD), particularly in those with more severe airflow limitation. Fatty-acid binding protein 4 (FABP4) is an adipokine involved in the innate immune response against infection produced by alveolar macrophages (Mɸ). We hypothesized that airway levels of FABP4 may be altered in COPD patients with chronic airway infection. METHODS: In this prospective and controlled study we: (1) compared airway FABP4 levels (ELISA) in induced sputum, bronchoalveolar lavage fluid (BALF) and plasma samples in 52 clinically stable COPD patients (65.2 ± 7.9 years, FEV1 59 ± 16% predicted) and 29 healthy volunteers (55.0 ± 12.3 years, FEV1 97 ± 16% predicted); (2) explored their relationship with the presence of bacterial airway infection, defined by the presence of potentially pathogenic bacteria (PPB) at ≥103 colony-forming units/ml in BALF; (3) investigated their relationship with the quantity and proportion of Mɸ in BALF (flow cytometry); and, (4) studied their relationship with the severity of airflow limitation (FEV1), GOLD grade and level of symptoms (CAT questionnaire). RESULTS: We found that: (1) airway levels of FABP4 (but not plasma ones) were reduced in COPD patients vs. controls [219.2 (96.0-319.6) vs. 273.4 (203.1-426.7) (pg/ml)/protein, p = 0.03 in BALF]; (2) COPD patients with airway infection had lower sputum FABP4 levels [0.73 (0.35-15.3) vs. 15.6 (2.0-29.4) ng/ml, p = 0.02]; (3) in COPD patients, the number and proportion of Mɸ were positively related with FABP4 levels in BALF; (4) BALF and sputum FABP4 levels were positively related with FEV1, negatively with the CAT score, and lowest in GOLD grade D patients. CONCLUSIONS: Airway FABP4 levels are reduced in COPD patients, especially in those with airway infection and more severe disease. The relationship observed between Mɸ and airway FABP4 levels supports a role for FABP4 in the pathogenesis of airway infection and disease severity in COPD.

Determinants of empirical antipseudomonal antibiotic prescription for adults with pneumonia in the emergency department.

BACKGROUND: Antipseudomonal antibiotics should be restricted to patients at risk of Pseudomonas aeruginosa infection. However, the indications in different guidelines on community-acquired pneumonia (CAP) are discordant. Our objectives were to assess the prevalence of antipseudomonal antibiotic prescriptions and to identify determinants of empirical antibiotic choices in the emergency department. METHODS: Observational, retrospective, one-year cohort study in hospitalized adults with pneumonia. Antibiotic choices and clinical and demographic data were recorded on a standardized form. Antibiotics with antipseudomonal activity were classified into two groups: a) ß-lactam antipseudomonals (ß-APS), including carbapenems, piperacillin / tazobactam or cefepime (in monotherapy or combination) and b) monotherapy with antipseudomonal quinolones. RESULTS: Data were recorded from 549 adults with pneumonia, with Pseudomonas aeruginosa being isolated in only nine (1.6%). Most (85%) prescriptions were compliant with SEPAR guidelines and 207 (37%) patients received antibiotics with antipseudomonal activity (14% ß-APS and 23% levofloxacin). The use of ß-APS was independently associated with ICU admission (OR 8.16 95% CI 3.69-18.06) and prior hospitalization (OR 6.76 95% CI 3.02-15.15), while levofloxacin was associated with negative pneumococcal urine antigen tests (OR 3.41 95% CI 1.70-6.85) but negatively associated with ICU admission (OR 0.26 95% CI 0.08-0.86). None of these factors were associated with P aeruginosa episodes. In univariate analysis, prior P aeruginosa infection/colonization (2/9 vs 6/372, p = 0.013), severe COPD (3/9 vs 26/372, p = 0.024), multilobar involvement (7/9 vs 119/372, p = 0.007) and prior antibiotic (6/9 vs 109/372, p = 0.025) were significantly associated with P aeruginosa episodes. CONCLUSIONS: Antipseudomonal prescriptions were common, in spite of the very low incidence of Pseudomonas aeruginosa. The rationale for prescription was influenced by both severity-of-illness and pneumococcal urine antigen test (levofloxacin) and prior hospitalization and ICU admission (ß-APS). However, these factors were not associated with P aeruginosa episodes. Only prior P aeruginosa infection/colonization and severe COPD seem to be reliable indicators in clinical practice.

5 protein-based signature for resectable lung squamous cell carcinoma improves the prognostic performance of the TNM staging.

INTRODUCTION: Prognostic biomarkers have been very elusive in the lung squamous cell carcinoma (SCC) and none is currently being used in the clinical setting. We aimed to identify and validate the clinical utility of a protein-based prognostic signature to stratify patients with early lung SCC according to their risk of recurrence or death. METHODS: Patients were staged following the new International Association for the Study of Lung Cancer (IASLC) staging criteria (eighth edition, 2018). Three independent retrospective cohorts of 117, 96 and 105 patients with lung SCC were analysed to develop and validate a prognostic signature based on immunohistochemistry for five proteins. RESULTS: We identified a five protein-based signature whose prognostic index (PI) was an independent and significant predictor of disease-free survival (DFS) (p<0.001; HR=4.06, 95% CI 2.18 to 7.56) and overall survival (OS) (p=0.004; HR=2.38, 95% CI 1.32 to 4.31). The prognostic capability of PI was confirmed in an external multi-institutional cohort for DFS (p=0.042; HR=2.01, 95% CI 1.03 to 3.94) and for OS (p=0.031; HR=2.29, 95% CI 1.08 to 4.86). Moreover, PI added complementary information to the newly established IASLC TNM 8th edition staging system. A combined prognostic model including both molecular and anatomical (TNM) criteria improved the risk stratification in both cohorts (p<0.05). CONCLUSION: We have identified and validated a clinically feasible protein-based prognostic model that complements the updated TNM system allowing more accurate risk stratification. This signature may be used as an advantageous tool to improve the clinical management of the patients, allowing the reduction of lung SCC mortality through a more accurate knowledge of the patient's potential outcome.

A novel protein-based prognostic signature improves risk stratification to guide clinical management in early-stage lung adenocarcinoma patients.

Each of the pathological stages (I-IIIa) of surgically resected non-small-cell lung cancer has hidden biological heterogeneity, manifested as heterogeneous outcomes within each stage. Thus, the finding of robust and precise molecular classifiers with which to assess individual patient risk is an unmet medical need. Here, we identified and validated the clinical utility of a new prognostic signature based on three proteins (BRCA1, QKI, and SLC2A1) to stratify early-stage lung adenocarcinoma patients according to their risk of recurrence or death. Patients were staged according to the new International Association for the Study of Lung Cancer (IASLC) staging criteria (8th edition, 2018). A test cohort (n = 239) was used to assess the value of this new prognostic index (PI) based on the three proteins. The prognostic signature was developed by Cox regression with the use of stringent statistical criteria (TRIPOD: Transparent reporting of a multivariable prediction model for individual prognosis or diagnosis). The model resulted in a highly significant predictor of 5-year outcome for disease-free survival (p < 0.001) and overall survival (p < 0.001). The prognostic ability of the model was externally validated in an independent multi-institutional cohort of patients (n = 114, p = 0.021). We also demonstrated that this molecular classifier adds relevant information to the gold standard TNM-based pathological staging, with a highly significant improvement of the likelihood ratio. We subsequently developed a combined PI including both the molecular and the pathological data that improved the risk stratification in both cohorts (p ≤ 0.001). Moreover, the signature may help to select stage I-IIA patients who might benefit from adjuvant chemotherapy. In summary, this protein-based signature accurately identifies those patients with a high risk of recurrence and death, and adds further prognostic information to the TNM-based clinical staging, even when the new IASLC 8th edition staging criteria are applied. More importantly, it may be a valuable tool for selecting patients for adjuvant therapy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Relationship between the respiratory microbiome and the severity of airflow limitation, history of exacerbations and circulating eosinophils in COPD patients.

BACKGROUND: The respiratory microbiome is altered in COPD patients but its relationship with core components of the disease, such as the severity of airflow limitation, the frequency of exacerbations or the circulating levels of eosinophils, is unclear. METHODS: Cross-sectional study comprising 72 clinically stable COPD patients (mean age 68 [SD 7.9] years; FEV1 48.7 [SD 20.1]% of reference) who provided spontaneous sputum samples for 16S rRNA gene amplification and sequencing. The microbiome composition was analysed with QIIME. RESULTS: We observed that: (1) more severe airflow limitation was associated with reduced relative abundance (RA) of Treponema and an increase in Pseudomonas; (2) patients with ≥2 exacerbations the previous year showed a significantly different bacterial community with respect to non-exacerbators (p = 0.014), with changes in 13 genera, including an increase of Pseudomonas, and finally, (3) peripheral eosinophils levels ≥2% were associated with more diverse microbiome [Chao1 224.51 (74.88) vs 277.39 (78.92) p = 0.006; Shannon 3.94 (1.05) vs 4.54 (1.06) p = 0.020], and a significant increase in the RAs of 20 genera. CONCLUSION: The respiratory microbiome in clinically stable COPD patients varies significantly according to the severity of airflow limitation, previous history of exacerbations and circulating eosinophils levels.

Multi-level differential network analysis of COPD exacerbations.

Patients with chronic obstructive pulmonary disease (COPD) often suffer episodes of exacerbation (ECOPD) that impact negatively the course of their disease. ECOPD are heterogeneous events of unclear pathobiology and non-specific diagnosis. Network analysis is a novel research approach that can help unravelling complex biological systems. We hypothesised that the comparison of multi-level (i.e., clinical, physiological, biological, imaging and microbiological) correlation networks determined during ECOPD and convalescence can yield novel patho-biologic information.In this proof-of-concept study we included 86 patients hospitalised because of ECOPD in a multicentre study in Spain. Patients were extensively characterised both during the first 72 h of hospitalisation and during clinical stability, at least 3 months after hospital discharge.We found that 1) episodes of ECOPD are characterised by disruption of the network correlation observed during convalescence; and 2) a panel of biomarkers that include increased levels of dyspnoea, circulating neutrophils and C-reactive protein (CRP) has a high predictive value for ECOPD diagnosis (AUC 0.97).We conclude that ECOPD 1) are characterised by disruption of network homeokinesis that exists during convalescence; and 2) can be identified objectively by using a panel of three biomarkers (dyspnoea, circulating neutrophils and CRP levels) frequently determined in clinical practice.

The microbiome in respiratory medicine: current challenges and future perspectives.

The healthy lung has previously been considered to be a sterile organ because standard microbiological culture techniques consistently yield negative results. However, culture-independent techniques report that large numbers of microorganisms coexist in the lung. There are many unknown aspects in the field, but available reports show that the lower respiratory tract microbiota: 1) is similar in healthy subjects to the oropharyngeal microbiota and dominated by members of the Firmicutes, Bacteroidetes and Proteobacteria phyla; 2) shows changes in smokers and well-defined differences in chronic respiratory diseases, although the temporal and spatial kinetics of these changes are only partially known; and 3) shows relatively abundant non-cultivable bacteria in chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, cystic fibrosis and bronchiectasis, with specific patterns for each disease. In all of these diseases, a loss of diversity, paralleled by an over-representation of Proteobacteria (dysbiosis), has been related to disease severity and exacerbations. However, it is unknown whether dysbiosis is a cause or a consequence of the damage to bronchoalveolar surfaces.Finally, little is known about bacterial functionality and the interactions between viruses, fungi and bacteria. It is expected that future research in bacterial gene expressions, metagenomics longitudinal analysis and host-microbiome animal models will help to move towards targeted microbiome interventions in respiratory diseases.

The respiratory microbiome in bronchial mucosa and secretions from severe IgE-mediated asthma patients.

BACKGROUND: The bronchial microbiome in chronic lung diseases presents an abnormal pattern, but its microbial composition and regional differences in severe asthma have not been sufficiently addressed. The aim of the study was to describe the bacterial community in bronchial mucosa and secretions of patients with severe chronic asthma chronically treated with corticosteroids in addition to usual care according to Global Initiative for Asthma. Bacterial community composition was obtained by 16S rRNA gene amplification and sequencing, and functional capabilities through PICRUSt. RESULTS: Thirteen patients with severe asthma were included and provided 11 bronchial biopsies (BB) and 12 bronchial aspirates (BA) suitable for sequence analyses. Bacteroidetes, Firmicutes, Proteobacteria and Actinobacteria showed relative abundances (RAs) over 5% in BB, a cutoff that was reached by Streptococcus and Prevotella at genus level. Legionella genus attained a median RA of 2.7 (interquartile range 1.1-4.7) in BB samples. In BA a higher RA of Fusobacteria was found, when compared with BB [8.7 (5.9-11.4) vs 4.2 (0.8-7.5), p = 0.037], while the RA of Proteobacteria was lower in BA [4.3 (3.7-6.5) vs 17.1 (11.2-33.4), p = 0.005]. RA of the Legionella genus was also significantly lower in BA [0.004 (0.001-0.02) vs. 2.7 (1.1-4.7), p = 0.005]. Beta-diversity analysis confirmed the differences between the microbial communities in BA and BB (R2 = 0.20, p = 0.001, Adonis test), and functional analysis revealed also statistically significant differences between both types of sample on Metabolism, Cellular processes, Human diseases, Organismal systems and Genetic information processing pathways. CONCLUSIONS: The microbiota in the bronchial mucosa of severe asthma has a specific pattern that is not accurately represented in bronchial secretions, which must be considered a different niche of bacteria growth.

Personalized respiratory medicine: exploring the horizon, addressing the issues. Summary of a BRN-AJRCCM workshop held in Barcelona on June 12, 2014.

This Pulmonary Perspective summarizes the content and main conclusions of an international workshop on personalized respiratory medicine coorganized by the Barcelona Respiratory Network ( www.brn.cat ) and the AJRCCM in June 2014. It discusses (1) its definition and historical, social, legal, and ethical aspects; (2) the view from different disciplines, including basic science, epidemiology, bioinformatics, and network/systems medicine; (3) the bottlenecks and opportunities identified by some currently ongoing projects; and (4) the implications for the individual, the healthcare system and the pharmaceutical industry. The authors hope that, although it is not a systematic review on the subject, this document can be a useful reference for researchers, clinicians, healthcare managers, policy-makers, and industry parties interested in personalized respiratory medicine.

Severity-related changes of bronchial microbiome in chronic obstructive pulmonary disease.

Bronchial colonization by potentially pathogenic microorganisms (PPMs) is often demonstrated in chronic obstructive pulmonary disease (COPD), but culture-based techniques identify only a portion of the bacteria in mucosal surfaces. The aim of the study was to determine changes in the bronchial microbiome of COPD associated with the severity of the disease. The bronchial microbiome of COPD patients was analyzed by 16S rRNA gene amplification and pyrosequencing in sputum samples obtained during stable disease. Seventeen COPD patients were studied (forced expiratory volume in the first second expressed as a percentage of the forced vital capacity [FEV1%] median, 35.0%; interquartile range [IQR], 31.5 to 52.0), providing a mean of 4,493 (standard deviation [SD], 2,598) sequences corresponding to 47 operational taxonomic units (OTUs) (SD, 17) at a 97% identity level. Patients were dichotomized according to their lung function as moderate to severe when their FEV1% values were over the median and as advanced when FEV1% values were lower. The most prevalent phyla in sputum were Proteobacteria (44%) and Firmicutes (16%), followed by Actinobacteria (13%). A greater microbial diversity was found in patients with moderate-to-severe disease, and alpha diversity showed a statistically significant decrease in patients with advanced disease when assessed by Shannon (ρ = 0.528; P = 0.029, Spearman correlation coefficient) and Chao1 (ρ = 0.53; P = 0.028, Spearman correlation coefficient) alpha-diversity indexes. The higher severity that characterizes advanced COPD is paralleled by a decrease in the diversity of the bronchial microbiome, with a loss of part of the resident flora that is replaced by a more restricted microbiota that includes PPMs.

Pseudomonas aeruginosa isolates in severe chronic obstructive pulmonary disease: characterization and risk factors.

BACKGROUND: Patients with severe chronic obstructive pulmonary disease (COPD) are at increased risk of infection by P. aeruginosa. The specific role of bronchiectasis in both infection and chronic colonization by this microorganism in COPD, however, remains ill defined.To evaluate the prevalence and risk factors for P. aeruginosa recovery from sputum in outpatients with severe COPD, characterizing P. aeruginosa isolates by pulsed-field gel electrophoresis (PFGE) and focusing on the influence of bronchiectasis on chronic colonization in these patients. METHODS: A case-cohort study of 118 patients with severe COPD attended at a Respiratory Day Unit for an acute infectious exacerbation and followed up over one year. High-resolution CT scans were performed during stability for bronchiectasis assessment and sputum cultures were obtained during exacerbation and stability in all patients. P. aeruginosa isolates were genotyped by PFGE. Determinants of the recovery of P. aeruginosa in sputum and chronic colonization by this microorganism were assessed by multivariate analysis. RESULTS: P. aeruginosa was isolated from 41 of the 118 patients studied (34.7%). Five of these 41 patients (12.2%) with P. aeruginosa recovery fulfilled criteria for chronic colonization. In the multivariate analysis, the extent of bronchiectasis (OR 9.8, 95% CI: 1.7 to 54.8) and the number of antibiotic courses (OR 1.7, 95% CI: 1.1 to 2.5) were independently associated with an increased risk of P. aeruginosa isolation. Chronic colonization was unrelated to the presence of bronchiectasis (p=0.75). In patients with chronic colonization the isolates of P. aeruginosa retrieved corresponded to the same clones during the follow-up, and most of the multidrug resistant isolates (19/21) were harbored by these patients. CONCLUSIONS: The main risk factors for P. aeruginosa isolation in severe COPD were the extent of bronchiectasis and exposure to antibiotics. Over 10% of these patients fulfilled criteria for chronic colonization by P. aeruginosa and showed clonal persistence, independently of the presence of bronchiectasis.

COPD: systemic proteomic profiles in frequent and infrequent exacerbators.

Background: Some patients with COPD suffer frequent exacerbations (FE). We hypothesised that their systemic proteomic profile would be different from that of non-frequent exacerbators (NFE). The objective of the present study was to contrast the systemic proteomic profile in FE versus NFE. As a reference, we also determined the systemic proteomic profile of healthy controls (HC) and COPD patients during an actual episode of exacerbation (AE). Methods: In the analysis we included 40 clinically stable COPD patients (20 FE and 20 NFE), and 20 HC and 10 AE patients. Their plasma samples were analysed by combining two complementary proteomic approaches: label-free liquid chromatography-tandem mass spectrometry and multiplex immunoassays. Gene Ontology annotation, pathway enrichment and network analyses were used to investigate molecular pathways associated with differentially abundant proteins/peptides (DAPs). Results: Compared with HC, we identified 40 DAPs in FE, 10 in NFE and 63 in AE. Also compared to HC, pathway functional and protein-protein network analyses revealed dysregulation of inflammatory responses involving innate and antibody-mediated immunity in COPD, particularly in the FE group, as well as during an AE episode. Besides, we only identified alterations in the complement and coagulation cascades in AE. Conclusion: There are specific plasma proteome profiles associated with FE, which are partially shared with findings observed during AE, albeit others are uniquely present during the actual episode of AE.

Effects of immunocompromise and comorbidities on pneumococcal serotypes causing invasive respiratory infection in adults: implications for vaccine strategies.

BACKGROUND: The 13-valent pneumococcal conjugate vaccine (PCV13) has recently been approved for use in immunocompromised adults. However, it is unclear whether there is an association between specific underlying conditions and infection by individual serotypes. The objective was to determine the prevalence of serotypes covered by PCV13 in a cohort of patients with invasive pneumococcal disease of respiratory origin and to determine whether there are specific risk factors for each serotype. METHODS: An observational study of adults hospitalized with invasive pneumococcal disease in 2 Spanish hospitals was conducted during the period 1996-2011. A multinomial regression analysis was performed to identify conditions associated with infection by specific serotypes (grouped according their formulation in vaccines and individually). RESULTS: A total of 1094 patients were enrolled; the infecting serotype was determined in 993. In immunocompromised patients, 64% of infecting serotypes were covered by PCV13. After adjusting for age, smoking, alcohol abuse, and nonimmunocompromising comorbidities, the group of serotypes not included in either PCV13 or PPV23 were more frequently isolated in patients with immunocompromising conditions and cardiopulmonary comorbidities. Regarding individual serotypes, 6A, 23F, 11A, and 33F were isolated more frequently in patients with immunocompromise and specifically in some of their subgroups. The subgroup analysis showed that serotype10A was also associated with HIV infection. CONCLUSIONS: Specific factors related to immunocompromise seem to determine the appearance of invasive infection by specific pneumococcal serotypes. Although the coverage of serotypes in the 13-valent conjugate pneumococcal vaccine (PCV13) was high, some non-PCV13-emergent serotypes are more prevalent in immunocompromised patients.

Effect of dynamic random leaks on the monitoring accuracy of home mechanical ventilators: a bench study.

BACKGROUND: So far, the accuracy of tidal volume (VT) and leak measures provided by the built-in software of commercial home ventilators has only been tested using bench linear models with fixed calibrated and continuous leaks. The objective was to assess the reliability of the estimation of tidal volume (VT) and unintentional leaks in a single tubing bench model which introduces random dynamic leaks during inspiratory or expiratory phases. METHODS: The built-in software of four commercial home ventilators and a fifth ventilator-independent ad hoc designed external software tool were tested with two levels of leaks and two different models with excess leaks (inspiration or expiration). The external software analyzed separately the inspiratory and expiratory unintentional leaks. RESULTS: In basal condition, all ventilators but one underestimated tidal volume with values ranging between -1.5 ± 3.3% to -8.7% ± 3.27%. In the model with excess of inspiratory leaks, VT was overestimated by all four commercial software tools, with values ranging from 18.27 ± 7.05% to 35.92 ± 17.7%, whereas the ventilator independent-software gave a smaller difference (3.03 ± 2.6%). Leaks were underestimated by two applications with values of -11.47 ± 6.32 and -5.9 ± 0.52 L/min. With expiratory leaks, VT was overestimated by the software of one ventilator and the ventilator-independent software and significantly underestimated by the other three, with deviations ranging from +10.94 ± 7.1 to -48 ± 23.08%. The four commercial tools tested overestimated unintentional leaks, with values between 2.19 ± 0.85 to 3.08 ± 0.43 L/min. CONCLUSIONS: In a bench model, the presence of unintentional random leaks may be a source of error in the measurement of VT and leaks provided by the software of home ventilators. Analyzing leaks during inspiration and expiration separately may reduce this source of error.

Computer-assisted diagnosis for an early identification of lung cancer in chest X rays.

Computer-assisted diagnosis (CAD) algorithms have shown its usefulness for the identification of pulmonary nodules in chest x-rays, but its capability to diagnose lung cancer (LC) is unknown. A CAD algorithm for the identification of pulmonary nodules was created and used on a retrospective cohort of patients with x-rays performed in 2008 and not examined by a radiologist when obtained. X-rays were sorted according to the probability of pulmonary nodule, read by a radiologist and the evolution for the following three years was assessed. The CAD algorithm sorted 20,303 x-rays and defined four subgroups with 250 images each (percentiles ≥ 98, 66, 33 and 0). Fifty-eight pulmonary nodules were identified in the ≥ 98 percentile (23,2%), while only 64 were found in lower percentiles (8,5%) (p < 0.001). A pulmonary nodule was confirmed by the radiologist in 39 out of 173 patients in the high-probability group who had follow-up information (22.5%), and in 5 of them a LC was diagnosed with a delay of 11 months (12.8%). In one quarter of the chest x-rays considered as high-probability for pulmonary nodule by a CAD algorithm, the finding is confirmed and corresponds to an undiagnosed LC in one tenth of the cases.

Microbiome diversity in the bronchial tracts of patients with chronic obstructive pulmonary disease.

Culture of bacteria from bronchial secretions in respiratory patients has low sensitivity and does not allow for complete assessment of microbial diversity across different bronchial compartments. In addition, a significant number of clinical studies are based on sputum samples, and it is not known to what extent they describe the real diversity of the mucosa. In order to identify previously unrecognized lower airway bacteria and to investigate the complexity and distribution of microbiota in patients with chronic obstructive pulmonary disease (COPD), we performed PCR amplification and pyrosequencing of the 16S rRNA gene in patients not showing signs or symptoms of infection. Four types of respiratory samples (sputum, bronchial aspirate, bronchoalveolar lavage, and bronchial mucosa) were taken from each individual, obtaining on average >1,000 16S rRNA sequences per sample. The total number of genera per patient was >100, showing a high diversity, with Streptococcus, Prevotella, Moraxella, Haemophilus, Acinetobacter, Fusobacterium, and Neisseria being the most commonly identified. Sputum samples showed significantly lower diversity than the other three sample types. Lower-bronchial-tree samples, i.e., bronchoalveolar lavage and bronchial mucosa, showed a very similar bacterial compositions in contrast to sputum and bronchial aspirate samples. Thus, sputum and bronchial aspirate samples are upper bronchial tree samples that are not representative of the lower bronchial mucosa flora, and bronchoalveolar lavage samples showed the results closest to those for the bronchial mucosa. Our data confirm that the bronchial tree is not sterile in COPD patients and support the existence a different microbiota in the upper and lower compartments.

Specific IgA and metalloproteinase activity in bronchial secretions from stable chronic obstructive pulmonary disease patients colonized by Haemophilus influenzae.

BACKGROUND: Haemophilus influenzae is the most common colonizing bacteria of the bronchial tree in chronic obstructive pulmonary disease (COPD), and positive cultures for this potentially pathogenic microorganism (PPM) has been associated with local inflammation changes that may influence the relationships between H. influenzae and the bronchial mucosa. METHODS: A cross-sectional analysis of stable COPD patients enrolled in the Phenotype and Course of Chronic Obstructive Pulmonary Disease (PAC-COPD) Study, focusing on bronchial colonization by H. influenzae, was performed. Specific IgA against the PPM was measured by optical density, and metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) using ELISA in sputum samples. Levels in patients colonized by H. influenzae and non-colonized patients were compared. RESULTS: Sputum supernatant for the measurement of specific IgA against H. influenzae was available from 54 stable COPD patients, who showed levels of specific IgA significantly lower in colonized (n=21) than in non-colonized patients (n=33) (15 [4-37] versus 31 [10-75], p=0.033, Mann-Whitney U test). Proenzyme MMP-9 was measured in 44 patients, and it was higher in colonized (n=12, 1903 [1488-6699] ng/ml) than in non-colonized patients (n=32, 639 [373-972] ng/ml) (p<0.001, Mann-Whitney U test). Active form of MMP-9 was also higher in colonized (126 [25-277] ng/ml) than in non-colonized patients (39 [14-68] ng/ml) (p=0.021, Mann-Whitney U test), and the molar ratio between proenzyme MMP-9 and TIMP-1 was above 1 (2.1 [0.1-12.5]) in colonized patients, significantly higher than the ratio found in non-colonized patients (0.2 [0.08-0.5]) (p=0.030, Mann-Whitney U test). CONCLUSIONS: Clinically stable COPD patients colonized by H. influenzae had lower levels of specific IgA against the microorganism and higher values of the active form of MMP-9 in their sputum supernatant than non-colonized patients. Bronchial colonization by H. influenzae may cause structural changes in the extracellular matrix through a defective defense and the production of active metalloproteinases.

Contribution of cell blocks obtained through endobronchial ultrasound-guided transbronchial needle aspiration to the diagnosis of lung cancer.

BACKGROUND: Conventional smears of samples obtained by endobronchial ultrasound with real-time transbronchial needle aspiration (EBUS-TBNA) have proven useful in lung cancer staging, but the value of additional information from cell-block processing of EBUS-TBNA samples has only been marginally investigated. This study focussed on the contribution of cell block analysis to the diagnostic yield in lung cancer. METHODS: Patients referred for lung cancer diagnosis and/or staging by means of EBUS-TBNA were enrolled, the adequacy of the obtained samples for preparing cell blocks was assessed, and the additional pathologic or genetic information provided from cell block analysis was examined. RESULTS: In 270 lung cancer patients referred for EBUS-TBNA (mean age, 63.3 SD 10.4 years) 697 aspirations were performed. Cell blocks could be obtained from 334 aspirates (47.9%) and contained diagnostic material in 262 (37.6%) aspirates, providing information that was additional to conventional smears in 50 of the 189 samples with smears that were non-diagnostic, corresponding 21 of these blocks to malignant nodes, and allowing lung cancer subtyping of 4 samples. Overall, cell blocks improved the pathologic diagnosis attained with conventional smears in 54 of the 697 samples obtained with EBUS-TBNA (7.7%). Cell blocks obtained during EBUS-TBNA also made epithelial growth factor receptor mutation analysis possible in 39 of the 64 patients with TBNA samples showing metastatic adenocarcinoma (60.1%). Overall, cell blocks provided clinically significant information for 83 of the 270 patients participating in the study (30.7%). CONCLUSIONS: Cell-block preparation from EBUS-TBNA samples is a simple way to provide additional information in lung cancer diagnosis. Analysis of cell blocks increases the diagnostic yield of the procedure by nearly seven per cent and allows for genetic analysis in a sixty per cent of the patients with metastatic adenocarcinoma.

Anti-tissue antibodies are related to lung function in chronic obstructive pulmonary disease.

RATIONALE: Chronic obstructive pulmonary disease (COPD) is a multicomponent disease. Autoimmunity can contribute to the pathogenesis of COPD. OBJECTIVES: This study investigates the prevalence of circulating antinuclear antibodies (ANA) and anti-tissue (AT) antibodies, two common markers of autoimmunity, in COPD and their relationship with several components of the disease. METHODS: We determined lung function, the serum titers of ANA and AT by immunofluorescence, and the serum levels of C-reactive protein (CRP) by high sensitivity nephelometry in 328 patients with clinically stable COPD and in 67 healthy controls recruited in the PAC-COPD study. Multiple linear and logistic regression analysis was used to analyze results. MEASUREMENTS AND MAIN RESULTS: The prevalence of abnormal ANA and AT titers was 34% and 26% in patients and 3% and 6% in controls, respectively. Levels of AT greater than or equal to 1:320 were seen in 21% of patients with COPD and were independently associated with the severity of airflow limitation and gas transfer impairment (P < 0.05). Neither ANA or AT titers was related to body mass index, current smoking status, use of inhaled steroids, the Charlson index, or serum C-reactive protein values. CONCLUSIONS: Between a quarter and a third of patients with clinically stable COPD present abnormal titers of circulating ANA and AT. The observed relationship between AT and lung function supports a role for autoimmunity in the pathogenesis of COPD.