Gender specific airway gene expression in COPD sub-phenotypes supports a role of mitochondria and of different types of leukocytes.

Chronic obstructive pulmonary disease (COPD) is a destructive inflammatory disease and the genes expressed within the lung are crucial to its pathophysiology. We have determined the RNAseq transcriptome of bronchial brush cells from 312 stringently defined ex-smoker patients. Compared to healthy controls there were for males 40 differentially expressed genes (DEGs) and 73 DEGs for females with only 26 genes shared. The gene ontology (GO) term "response to bacterium" was shared, with several different DEGs contributing in males and females. Strongly upregulated genes TCN1 and CYP1B1 were unique to males and females, respectively. For male emphysema (E)-dominant and airway disease (A)-dominant COPD (defined by computed tomography) the term "response to stress" was found for both sub-phenotypes, but this included distinct up-regulated genes for the E-sub-phenotype (neutrophil-related CSF3R, CXCL1, MNDA) and for the A-sub-phenotype (macrophage-related KLF4, F3, CD36). In E-dominant disease, a cluster of mitochondria-encoded (MT) genes forms a signature, able to identify patients with emphysema features in a confirmation cohort. The MT-CO2 gene is upregulated transcriptionally in bronchial epithelial cells with the copy number essentially unchanged. Both MT-CO2 and the neutrophil chemoattractant CXCL1 are induced by reactive oxygen in bronchial epithelial cells. Of the female DEGs unique for E- and A-dominant COPD, 88% were detected in females only. In E-dominant disease we found a pronounced expression of mast cell-associated DEGs TPSB2, TPSAB1 and CPA3. The differential genes discovered in this study point towards involvement of different types of leukocytes in the E- and A-dominant COPD sub-phenotypes in males and females.

High degree of polyclonality hinders somatic mutation calling in lung brush samples of COPD cases and controls.

Chronic obstructive pulmonary disease (COPD) is induced by cigarette smoking and characterized by inflammation of airway tissue. Since smokers with COPD have a higher risk of developing lung cancer than those without, we hypothesized that they carry more mutations in affected tissue. We called somatic mutations in airway brush samples from medium-coverage whole genome sequencing data from healthy never and ex-smokers (n = 8), as well as from ex-smokers with variable degrees of COPD (n = 4). Owing to the limited concordance of resulting calls between the applied tools we built a consensus, a strategy that was validated with high accuracy for cancer data. However, consensus calls showed little promise of representing true positives due to low mappability of corresponding sequence reads and high overlap with positions harbouring known genetic polymorphisms. A targeted re-sequencing approach suggested that only few mutations would survive stringent verification testing and that our data did not allow the inference of any difference in the mutational load of bronchial brush samples between former smoking COPD cases and controls. High polyclonality in airway brush samples renders medium-depth sequencing insufficient to provide the resolution to detect somatic mutations. Deep sequencing data of airway biopsies are needed to tackle the question.

Methotrexate treatment efficacy in sarcoidosis might be related to TNF-α polymorphism: real life preliminary study.

INTRODUCTION: Methotrexate therapy improves lung function in selected sarcoidosis patients. Variation in TNF gene was associated with response to treatment. Aim: To determine the predictive role of-308 G/A, -857C/T, -863 C/A and -1031 T/C TNF-α polymorphism in the efficacy of MTX for progressive pulmonary sarcoidosis. MATERIAL AND METHODS: Twenty-eight sarcoidosis patients treated with MTX (6-24 months) were genotyped for TNF-α polymorphisms: -1031 T/C, -857C/T, -308 G/A and -863 C/A. Pulmonary function test (PFT) were performed every 6 months to determine treatment response, until the drug withdrawal. RESULTS: No correlation between the initial clinical presentation of sarcoidosis and TNF α polymorphisms was found, neither for every allele nor for combined genotypes distribution. According to PFT evaluation we have discovered 3 types of response to MTX: early (ER), late (LR) and No-response (NR). TNF-α-308 A allele carriers have got significantly higher chance to be LR, p=0.02, RRI:83%. TNF-α-308 GG genotype transferred the 3-fold higher probability of early vs late response to MTX, p=0.02. Combined genotyping allowed to distinguish LR from ER and NR groups. ER and NR patients are genetically similar (-857CC-308GG). LR are "genetically" different group of patients (-857C/T-308GG or -857CC-308A/G) with 5-fold greater probability to be LR than TNF-α-857CC-308GG patients, p=0,005 sensitivity 85%, specificity: 43%, PPV 58%, NPV 75%. TNF-α-308GG-857CC patients have significantly lower chance to be LR comparing to other response type p=0.03 OR=0,075 95% CI=0.07-0.08. CONCLUSION: Two types of positive response to MTX therapy (early and late) in chronic respiratory sarcoidosis are associated with polymorphic changes in TNF gene.

Influence of lung CT changes in chronic obstructive pulmonary disease (COPD) on the human lung microbiome.

BACKGROUND: Changes in microbial community composition in the lung of patients suffering from moderate to severe COPD have been well documented. However, knowledge about specific microbiome structures in the human lung associated with CT defined abnormalities is limited. METHODS: Bacterial community composition derived from brush samples from lungs of 16 patients suffering from different CT defined subtypes of COPD and 9 healthy subjects was analyzed using a cultivation independent barcoding approach applying 454-pyrosequencing of 16S rRNA gene fragment amplicons. RESULTS: We could show that bacterial community composition in patients with changes in CT (either airway or emphysema type changes, designated as severe subtypes) was different from community composition in lungs of patients without visible changes in CT as well as from healthy subjects (designated as mild COPD subtype and control group) (PC1, Padj = 0.002). Higher abundance of Prevotella in samples from patients with mild COPD subtype and from controls and of Streptococcus in the severe subtype cases mainly contributed to the separation of bacterial communities of subjects. No significant effects of treatment with inhaled glucocorticoids on bacterial community composition were detected within COPD cases with and without abnormalities in CT in PCoA. Co-occurrence analysis suggests the presence of networks of co-occurring bacteria. Four communities of positively correlated bacteria were revealed. The microbial communities can clearly be distinguished by their associations with the CT defined disease phenotype. CONCLUSION: Our findings indicate that CT detectable structural changes in the lung of COPD patients, which we termed severe subtypes, are associated with alterations in bacterial communities, which may induce further changes in the interaction between microbes and host cells. This might result in a changed interplay with the host immune system.

The influence of comorbidities on mortality in sarcoidosis: a observational prospective cohort study.

AIM: The aim of this study was to identify the frequency and prevalence of comorbidities in sarcoid patients and to assess their influence on overall mortality in the cohort of patients with sarcoidosis. MATERIALS AND METHODS: A cohort of 557 patients with histologically confirmed sarcoidosis diagnosed between 2007 and 2011 and a group of non-sarcoid controls were observed. All patients were carefully observed for comorbidities and mortality. RESULTS: 291 males (52.2%) and 266 females (47.8%) with mean age 48.4 ± 12.0 years in sarcoidosis group and a group of 100 controls with mean age (49.25 ± 10.3) were observed. The mean number of comorbidities in both groups was similar (0.9 ± 0.99 vs 0.81 ± 0.84 NS). The frequency of thyroid disease was significantly higher in sarcoidosis group comparing to controls at the time of diagnosis (OR = 3.62 P = 0.0144). During the observation period (median 58.0 months), 16 patients died (2.9%). The mean number of comorbidities was significantly higher in the groups of non-survivors as compared to survivors (2.8 ± 1.0, vs 0.8 ± 0.9), P < 0.0001. CONCLUSION: The comorbidity burden has strong impact on mortality in sarcoidosis. Thyroid diseases are more frequent in sarcoidosis than in non-sarcoid controls.

Acceptance of home support and integrated care among advanced COPD patients who live outside large medical centers.

BACKGROUND: Poor self-management constitutes a risk factor for COPD deterioration. Patients from rural areas located at a considerable distance from large medical centers frequently need home-support in advanced stages of the disease. Integrated care has been proposed as a comprehensive model for appropriate treatment, coordination and holistic support. The aim of the study was to assess whether home visits provided by trained assistants are needed and accepted by advanced COPD patients living in rural areas a to evaluate whether an individual short educational program can actually improve such patients' knowledge of COPD and inhaler use. METHODS: Thirty patients with severe or very severe but stable COPD participated in one-month home-assistance interventions twice a week. RESULTS: The total value ≥70 of SGRQ (St George's Respiratory Questionnaire) was recorded in 18 (60%) patients. At the beginning of the study, the patients' knowledge of COPD and inhalation techniques was highly unsatisfactory. Significant improvement in all items (p=0.00) was obtained after the intervention. The risk for poor self-management was high. All patients had at least one 'factor' that indicated the need for home-support. A total of 240 visits (100%) were completed. Patients expressed high acceptance for home-based support delivered by medical assistants twice a week for one month. No patients opposed this kind of care and most of them expressed interest in receiving it in the future. CONCLUSIONS: The results suggest a compelling need for home care and demonstrate full acceptance of this kind of support on the part of advanced COPD patients.

Emphysema- and airway-dominant COPD phenotypes defined by standardised quantitative computed tomography.

EvA (Emphysema versus Airway disease) is a multicentre project to study mechanisms and identify biomarkers of emphysema and airway disease in chronic obstructive pulmonary disease (COPD). The objective of this study was to delineate objectively imaging-based emphysema-dominant and airway disease-dominant phenotypes using quantitative computed tomography (QCT) indices, standardised with a novel phantom-based approach.441 subjects with COPD (Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages 1-3) were assessed in terms of clinical and physiological measurements, laboratory testing and standardised QCT indices of emphysema and airway wall geometry.QCT indices were influenced by scanner non-conformity, but standardisation significantly reduced variability (p<0.001) and led to more robust phenotypes. Four imaging-derived phenotypes were identified, reflecting "emphysema-dominant", "airway disease-dominant", "mixed" disease and "mild" disease. The emphysema-dominant group had significantly higher lung volumes, lower gas transfer coefficient, lower oxygen (PO2 ) and carbon dioxide (PCO2 ) tensions, higher haemoglobin and higher blood leukocyte numbers than the airway disease-dominant group.The utility of QCT for phenotyping in the setting of an international multicentre study is improved by standardisation. QCT indices of emphysema and airway disease can delineate within a population of patients with COPD, phenotypic groups that have typical clinical features known to be associated with emphysema-dominant and airway-dominant disease.

Causes of deaths in COPD patients in primary care setting--a 6-year follow-up.

INTRODUCTION: COPD is one of the most frequent respiratory diseases responsible for patients' disability and mortality. In 2005 a single primary care practice, COPD was diagnosed in 183 out of 1,960 eligible subjects ≥ 40 years (9.3%). The aim of this study was to assess mortality rate and causes of deaths in this group after 6 years. MATERIAL AND METHODS: In 2011 we invited all 183 patients with COPD recognised in 2005. We performed spirometry, physical examination, questionnaire of respiratory symptoms, smoking habits, concomitant diseases and treatment. Information about deaths was taken from primary care register, furthermore, family members were asked to deliver medical documentation or death certificate. RESULTS: In 2011 we studied only 74 subjects (40.4%), 43 subjects died (23.5%) and 66 subjects were lost from the follow-up (36.1%). Cardiovascular diseases were the most frequent causes of deaths - 21 subjects (48.8%) (heart attack - 8 patients and stroke - 8 patients). Respiratory failure in the course of COPD exacerbation was the cause of 10 deaths (23.3%). Neoplastic diseases lead to 9 deaths (20.9%) (lung cancer 7 patients). Renal insufficiency was responsible for one death (2.325%), and the causes of 2 deaths remained unknown (4.65%). Subjects who died (predominantly males) were older, had higher MRC score and lower FEV1. CONCLUSIONS: Study performed six years after COPD diagnosis revealed that 23.5% of subjects died. The main causes of deaths were the following: cardiovascular diseases (mainly heart attack and stroke), COPD exacerbations and lung cancer (more than 75%). Death risk in COPD patients was associated with age, male sex, dyspnoea and severity of the disease.

Methotrexate as a single agent for treating pulmonary sarcoidosis: a single centre real-life prospective study.

INTRODUCTION: The first-line therapy in chronic sarcoidosis, according to WASOG/ATS/ERS recommendations, is GCS. This therapy is associated with significant adverse effects and finally does not alter the natural history of the disease. The objective of our study was to evaluate the efficacy and safety of monotherapy with MTX, as an alternative to GCS, in progressive pulmonary sarcoidosis. MATERIAL AND METHODS: An open prospective real-life, single-centre trial was performed on 50 patients with biopsy proven sarcoidosis, 28M and 22F, mean age 45.55 ± 8.9 years. The average duration of disease before MTX therapy was 12.34 ± 20.49 years, GCS therapy in the past was applied in 41 patients. All patients received MTX (10 mg or 15 mg weekly) between 2004 and 2013 because of chronic progressive pulmonary sarcoidosis. Therapy was planned for 24 months. Patients underwent regular clinical evaluation, pulmonary function assessment, exercise ability testing (6MWT), and chest radiography for therapy effectiveness every six months and side effects monitoring every 4-6 weeks. Forty-nine patients were included for statistical analysis of treatment efficacy. They were retrospectively allocated to "MTX responder" group if an improvement of 10% of FEV1, FVC, TLC, or 15% of DLCO from the initial value was documented for at least one parameter or "non-responders" if the patient did not meet the above-mentioned criteria. RESULTS: Duration of treatment ranged from 6 to 24 months, mean time 60.75 ± 34.1 weeks. For the whole cohort significant improvement after MTX therapy was observed for minimal SaO2 (%) (p = 0.043) and for decrease of DSaO2 (%) (p = 0.048) in six-minute walk test. The results were significantly better for patients treated with 15 mg than for those treated with 10 mg weekly and for those who obtained a greater total amount of MTX during therapy. Significant difference of DLCO%pred was observed after six months of MTX therapy between groups treated 15 mg vs 10 mg weekly (73.27 ± 12.7% vs. 63.15 ± 16.4%, p = 0.03). Twenty-five patients (55%) met the criteria of "MTX responders" group. Patients who responded well to treatment had significantly lower TLC and FVC initial values comparing to "MTX non-responders". After treatment the only significant difference in PFT between groups was noted for DLCO%pred. Eleven patients (22%) stopped the treatment due to adverse events of MTX, mild hepatic abnormalities were observed in ten patients (20%), and concomitant infection was found in four patients. There were no patients with a fatal outcome. CONCLUSIONS: MTX as a single agent in the treatment of sarcoidosis has proved to be a safe and effective steroid alternative. Selected patients with chronic pulmonary sarcoidosis experience definite PFT improvements after MTX treatment. There is need to search for predictors of MTX treatment effectiveness.

Fatal clinical outcome in a patient with sarcoidosis-lymphoma syndrome.

A 62-year-old female suspected of malignant disease underwent a splenectomy that revealed noncaseating granulomas in the histological specimen. Chest X-ray (CXR) and lung CT scans suggested sarcoidosis stage II. TBLB showed noncaseating granulomas. A diagnosis of sarcoidosis was made. Initially no treatment was needed as partial remission on CXR and normal lung function were observed. During the follow up she underwent open lung biopsy and axillary lymph node biopsy because of radiological progression with presence of CXR opacities imitating metastases and recurrent lymphadenopathy. No malignant cells were found. Spontaneous partial resolution of disseminated changes on the CXR was observed. Because of progressive deterioration in lung function and the clinical course of the disease strongly suggesting the progression of systemic sarcoidosis, the patient was given steroid treatment, which initially resulted in partial remission of pulmonary disseminated changes, peripheral lymphadenopathy and improvement in lung function test. Eight months later severe deterioration in general condition, anaemia, leukocytosis, hypoxemia, massive hepatomegaly and recurrence of general lymphadenopathy along with progression of disseminated changes were found. She died before the final diagnosis was established. Post-mortem examination showed a nodal marginal zone B-cell lymphoma with monocytoid B-cells, according to WHO classification. The malignant cells were found in the jugular, mediastinal, paratracheal, paragastric, paraintestinal and retroperitoneal lymph nodes and they infiltrated the lungs, pleura, liver, thyroid gland and pancreas. No sarcoid granulomas were found in the autopsy.

Impact of mild anaemia on dyspnoea during exertion and exercise tolerance in patients with acute exacerbation of chronic obstructive pulmonary disease.

INTRODUCTION: Dyspnoea and decreased exercise tolerance are symptoms of acute exacerbation of chronic obstructive pulmonary disease (AECOPD). Anaemia is a risk factor for reduced functional capacity and dyspnoea in stable COPD. There is limited information about the impact of anaemia on functional capacity and dyspnoea of patients during AECOPD. The aim of this study was to evaluate the impact of decreased blood haemoglobin concentration on the results of six-minute walking test (6MWT) in patients during AECOPD. MATERIAL AND METHODS: A post hoc analysis of data collected from prospective long-term studies on AECOPD. Haemoglobin concentration from the first obtainable hospital measurement were included in the assessment. 6MWT was performed after clinical improvement of the patient. Dyspnoea at baseline and after exercise and oxygen saturation (SpO(2)) during exercise was measured. RESULTS: (presented as means ± SD): 402 patients with exacerbation of COPD (COPD stage 3.5 ± 0.6) were examined. Patients with anaemia (26% of those studied, age 74.5 ± 8.2 years) achieved 258.1 ± 125.1 m during 6MWT, with exertional desaturation of 2.9 ± 2.6%. Patients without anaemia (74% of those studied, age 70.2 ± 8.7 years) achieved 271 ± 136.0 m during 6MWT with exertional desaturation of 3.8 ± 3.7%. The haemoglobin concentration did not correlate with 6MWT, dyspnoea during 6MWT, or exercise oxygenation and blood desaturation during exercise. CONCLUSION: Mildly decreased blood haemoglobin concentration did not influence the results of 6MWT in patients with AECOPD.

[Six-minute walk test in sarcoidosis patients with cardiac involvement]. / Test 6-minutowego chodu u chorych na sarkoidoze pluc z zajeciem serca.

INTRODUCTION: Cardiac involvement in sarcoidosis is of critical importance, due to the poor prognosis if this organ manifestation is left undiagnosed and untreated. The six-minute walk test (6 MWT) is a useful test to evaluate exercise tolerance of sarcoid patients. We aimed to assess the 6 MWT value in diagnosis, course and treatment monitoring of patients with cardiac sarcoidosis. MATERIAL AND METHODS: 47 patients were included: 22 with pulmonary sarcoidosis and cardiac involvement (13 women, 9 men), 25 with pulmonary sarcoidosis, with no changes in the heart (15 women, 10 men), and 18 healthy volunteers as controls (12 women, 6 men). Out of 22 patients with cardiac involvement 11 were treated for heart sarcoidosis with prednisone (9 pts - initial dose 60 mg daily and 2 pts - 40 mg daily). 11 pts in this group were not treated. In all patients sarcoidosis was confirmed histopatologically. Magnetic resonance imaging was used to diagnose involvement of the heart. In the studied groups we assessed: heart rate (HR), oxygen saturation, and distance in 6 MWT and Borg dyspnea score. RESULTS: Patients with cardiac sarcoidosis desaturated more during exercise (DSaO2max = 3.5 ± 3.2 vs. 0.38 ± 0.69; p = 0.004) and had a lower increase of HR in first minute during the 6 MWT (DHR1 = 21.81 ± 11.72 vs. 50.61 ± 12.35; p = 0.0001) when compared to healthy subjects. Significantly lower increase of HR in first minute of 6 MWT was observed in patients with cardiac sarcoidosis when compared to patients with pulmonary sarcoidosis with no cardiac involvement (DHR1 = 21.81 ± 11.72 vs. 38.8 ± 18.17, p = 0.01). After introduction of treatment in sarcoidosis group, significantly higher (p = 0.02) increase of HR in first minute of 6 MWT as compared to baseline test was observed. CONCLUSIONS: The six-minute walk test is useful in diagnosing cardiac involvement in sarcoidosis. The increase in HR during exercise and decrease degree of desaturation were a good predictors of the response to therapy.

The EvA study: aims and strategy.

The EvA study is a European Union-funded project under the Seventh Framework Programme (FP7), which aims at defining new markers for chronic obstructive pulmonary disease (COPD) and its subtypes. The acronym is derived from emphysema versus airway disease, indicating that the project targets these two main phenotypes of the disease. The EvA study is based on the concept that emphysema and airway disease are governed by different pathophysiological processes, are driven by different genes and have differential gene expression in the lung. To define these genes, patients and non-COPD controls are recruited for clinical examination, lung function analysis and computed tomography (CT) of the lung. CT scans are used to define the phenotypes based on lung density and airway wall thickness. This is followed by bronchoscopy in order to obtain samples from the airways and the alveoli. These tissue samples, along with blood samples, are then subjected to genome-wide expression and association analysis and markers linked to the phenotypes are identified. The population of the EvA study is different from other COPD study populations, since patients with current oral glucocorticoids, antibiotics and exacerbations or current smokers are excluded, such that the signals detected in the molecular analysis are due to the distinct inflammatory process of emphysema and airway disease in COPD.