Exploring protocol bias in airway microbiome studies: one versus two PCR steps and 16S rRNA gene region V3 V4 versus V4.

BACKGROUND: Studies on the airway microbiome have been performed using a wide range of laboratory protocols for high-throughput sequencing of the bacterial 16S ribosomal RNA (16S rRNA) gene. We sought to determine the impact of number of polymerase chain reaction (PCR) steps (1- or 2- steps) and choice of target marker gene region (V3 V4 and V4) on the presentation of the upper and lower airway microbiome. Our analyses included lllumina MiSeq sequencing following three setups: Setup 1 (2-step PCR; V3 V4 region), Setup 2 (2-step PCR; V4 region), Setup 3 (1-step PCR; V4 region). Samples included oral wash, protected specimen brushes and protected bronchoalveolar lavage (healthy and obstructive lung disease), and negative controls. RESULTS: The number of sequences and amplicon sequence variants (ASV) decreased in order setup1 > setup2 > setup3. This trend appeared to be associated with an increased taxonomic resolution when sequencing the V3 V4 region (setup 1) and an increased number of small ASVs in setups 1 and 2. The latter was considered a result of contamination in the two-step PCR protocols as well as sequencing across multiple runs (setup 1). Although genera Streptococcus, Prevotella, Veillonella and Rothia dominated, differences in relative abundance were observed across all setups. Analyses of beta-diversity revealed that while oral wash samples (high biomass) clustered together regardless of number of PCR steps, samples from the lungs (low biomass) separated. The removal of contaminants identified using the Decontam package in R, did not resolve differences in results between sequencing setups. CONCLUSIONS: Differences in number of PCR steps will have an impact of final bacterial community descriptions, and more so for samples of low bacterial load. Our findings could not be explained by differences in contamination levels alone, and more research is needed to understand how variations in PCR-setups and reagents may be contributing to the observed protocol bias.

Coagulation markers as predictors for clinical events in COPD.

BACKGROUND AND OBJECTIVE: Activation of the blood coagulation system is a common observation in inflammatory diseases. The role of coagulation in COPD is underexplored. METHODS: The study included 413 COPD patients and 49 controls from the 3-year Bergen COPD Cohort Study (BCCS). One hundred and forty-eight COPD patients were also examined during AECOPD. The plasma markers of coagulation activation, TAT complex, APC-PCI complex and D-dimer, were measured at baseline and during exacerbations by enzyme immunoassays. Differences in levels of the markers between stable COPD patients and controls, and between stable COPD and AECOPD were examined. The associations between coagulation markers and later AECOPD and mortality were examined by negative binomial and Cox regression analyses. RESULTS: TAT was significantly lower in stable COPD (1.03 ng/mL (0.76-1.44)) than in controls (1.28 (1.04-1.49), P = 0.002). During AECOPD, all markers were higher than in the stable state: TAT 2.56 versus 1.43 ng/mL, APC-PCI 489.3 versus 416.4 ng/mL and D-dimer 763.5 versus 479.7 ng/mL (P < 0.001 for all). Higher D-dimer in stable COPD predicted a higher mortality (HR: 1.60 (1.24-2.05), P < 0.001). Higher TAT was associated with both an increased risk of later exacerbations, with a yearly incidence rate ratio of 1.19 (1.04-1.37), and a faster time to the first exacerbation (HR: 1.25 (1.10-1.42), P = 0.001, all after adjustment). CONCLUSION: Activation of the coagulation system is increased during COPD exacerbations. Coagulation markers are potential predictors of later COPD exacerbations and mortality.

Repeated bronchoscopy in health and obstructive lung disease: is the airway microbiome stable?

OBJECTIVE: Little is known concerning the stability of the lower airway microbiome. We have compared the microbiota identified by repeated bronchoscopy in healthy subjects and patients with ostructive lung diseaseases (OLD). METHODS: 21 healthy controls and 41 patients with OLD completed two bronchoscopies. In addition to negative controls (NCS) and oral wash (OW) samples, we gathered protected bronchoalveolar lavage in two fractions (PBAL1 and PBAL2) and protected specimen brushes (PSB). After DNA extraction, we amplified the V3V4 region of the 16S rRNA gene, and performed paired-end sequencing (Illumina MiSeq). Initial bioinformatic processing was carried out in the QIIME-2 pipeline, identifying amplicon sequence variants (ASVs) with the DADA2 algorithm. Potentially contaminating ASVs were identified and removed using the decontam package in R and the sequenced NCS. RESULTS: A final table of 551 ASVs consisted of 19 × 106 sequences. Alpha diversity was lower in the second exam for OW samples, and borderline lower for PBAL1, with larger differences in subjects not having received intercurrent antibiotics. Permutational tests of beta diversity indicated that within-individual changes were significantly lower than between-individual changes. A non-parametric trend test showed that differences in composition between the two exams (beta diversity) were largest in the PSBs, and that these differences followed a pattern of PSB > PBAL2 > PBAL1 > OW. Time between procedures was not associated with increased diversity. CONCLUSION: The airways microbiota varied between examinations. However, there is compositional microbiota stability within a person, beyond that of chance, supporting the notion of a transient airways microbiota with a possibly more stable individual core microbiome.

Laboratory contamination in airway microbiome studies.

BACKGROUND: The low bacterial load in samples acquired from the lungs, have made studies on the airway microbiome vulnerable to contamination from bacterial DNA introduced during sampling and laboratory processing. We have examined the impact of laboratory contamination on samples collected from the lower airways by protected (through a sterile catheter) bronchoscopy and explored various in silico approaches to dealing with the contamination post-sequencing. Our analyses included quantitative PCR and targeted amplicon sequencing of the bacterial 16S rRNA gene. RESULTS: The mean bacterial load varied by sample type for the 23 study subjects (oral wash>1st fraction of protected bronchoalveolar lavage>protected specimen brush>2nd fraction of protected bronchoalveolar lavage; p < 0.001). By comparison to a dilution series of know bacterial composition and load, an estimated 10-50% of the bacterial community profiles for lower airway samples could be traced back to contaminating bacterial DNA introduced from the laboratory. We determined the main source of laboratory contaminants to be the DNA extraction kit (FastDNA Spin Kit). The removal of contaminants identified using tools within the Decontam R package appeared to provide a balance between keeping and removing taxa found in both negative controls and study samples. CONCLUSIONS: The influence of laboratory contamination will vary across airway microbiome studies. By reporting estimates of contaminant levels and taking use of contaminant identification tools (e.g. the Decontam R package) based on statistical models that limit the subjectivity of the researcher, the accuracy of inter-study comparisons can be improved.

Lung hyperinflation and functional exercise capacity in patients with COPD - a three-year longitudinal study.

BACKGROUND: Lung hyperinflation contributes to dyspnea, morbidity and mortality in chronic obstructive pulmonary disease (COPD). The inspiratory-to-total lung capacity (IC/TLC) ratio is a measure of lung hyperinflation and is associated with exercise intolerance. However, knowledge of its effect on longitudinal change in the 6-min walk distance (6MWD) in patients with COPD is scarce. We aimed to study whether the IC/TLC ratio predicts longitudinal change in 6MWD in patients with COPD. METHODS: This prospective cohort study included 389 patients aged 40-75 years with clinically stable COPD in Global Initiative for Chronic Obstructive Lung Disease stages II-IV. The 6MWD was measured at baseline, and after one and 3 years. We performed generalized estimating equation regression analyses to examine predictors for longitudinal change in 6MWD. Predictors at baseline were: IC/TLC ratio, age, gender, pack years, fat mass index (FMI), fat-free mass index (FFMI), number of exacerbations within 12 months prior to inclusion, Charlson index for comorbidities, forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), and light and hard self-reported physical activity. RESULTS: Reduced IC/TLC ratio (p < 0.001) was a statistically significant predictor for decline in 6MWD. With a 0.1-unit decrease in baseline IC/TLC ratio, the annual decline in 6MWD was 12.7 m (p < 0.001). Study participants with an IC/TLC ratio in the upper quartiles maintained their 6MWD from baseline to year 3, while it was significantly reduced for the patients with an IC/TLC ratio in the lower quartiles. Absence of light and hard physical activity, increased age and FMI, decreased FEV1 and FVC, more frequent exacerbations and higher Charlson comorbidity index were also predictors for lower 6MWD at any given time, but did not predict higher rate of decline over the timespan of the study. CONCLUSION: Our findings demonstrated that patients with less lung hyperinflation at baseline maintained their functional exercise capacity during the follow-up period, and that it was significantly reduced for patients with increased lung hyperinflation.

Growth differentiation factor-15 is a predictor of important disease outcomes in patients with COPD.

Increased levels of growth differentiation factor-15 (GDF15) are associated with cachexia, cardiovascular disease and all-cause mortality. The role of GDF15 in chronic obstructive pulmonary disease (COPD) is unknown.The study included 413 patients with COPD from the Bergen COPD Cohort Study. All patients had a forced expiratory volume in 1 s (FEV1) <80% predicted, a FEV1 to forced vital capacity (FVC) ratio <0.7 and a history of smoking. Spirometry, fat free mass index, blood gases and plasma GDF15 were measured at baseline. Patients were followed for 3 years regarding exacerbations and changes in lung function, and 9 years for mortality. Yearly exacerbation rate, survival and yearly change in FEV1/FVC were evaluated with regression models.Median plasma GDF15 was 0.86 ng·mL-1 (interquartile range 0.64-1.12 ng·mL-1). The distribution was not normal and GDF15 was analysed as a categorical variable. High levels of GDF15 were associated with a higher exacerbation rate (incidence rate ratio 1.39, 95% CI 1.1-1.74, p=0.006, adjusted values). Furthermore, high levels of GDF15 were associated with higher mortality (hazard ratio 2.07, 95% CI 1.4-3.1, p<0.001) and an increased decline in both FEV1 (4.29% versus 3.25%) and FVC (2.63% versus 1.44%) in comparison to low levels (p<0.01 for both).In patients with COPD, high levels of GDF15 were independently associated with a higher yearly rate of exacerbations, higher mortality and increased decline in both FEV1 and FVC.

Antimicrobial peptide levels are linked to airway inflammation, bacterial colonisation and exacerbations in chronic obstructive pulmonary disease.

Antimicrobial peptides (AMPs) are effectors of host defence against infection, inflammation and wound repair. We aimed to study AMP levels in stable chronic obstructive pulmonary disease (COPD) and during acute exacerbations of COPD (AECOPD), and to examine their relation to clinical parameters and inflammatory markers.The 3-year Bergen COPD Cohort Study included 433 COPD patients and 325 controls. Induced sputum was obtained and analysed for levels of the AMPs human cathelicidin (hCAP18/LL-37) and secretory leukocyte protease inhibitor (SLPI), and for the inflammatory markers interleukin (IL)-8, IL-6 and tumour necrosis factor-α (TNF-α) using immunoassays. Systemic hCAP18/LL-37 and vitamin D levels were also studied. Treating AMPs as response variables, non-parametric tests were applied for univariate comparison, and linear regression to obtain adjusted estimates. The risk of AECOPD was assessed by Cox proportional-hazard regression.Sputum AMP levels were higher in patients with stable COPD (n=215) compared to controls (n=45), and further changed during AECOPD (n=56), with increased hCAP18/LL-37 and decreased SLPI levels. Plasma hCAP18/LL-37 levels showed a similar pattern. In stable COPD, high sputum hCAP18/LL-37 levels were associated with increased risk of AECOPD, non-typeable Haemophilus influenzae colonisation, higher age, ex-smoking and higher levels of inflammatory markers.Altered levels of selected AMPs are linked to airway inflammation, infection and AECOPD, suggesting a role for these peptides in airway defence mechanisms in COPD.

Comparing microbiota profiles in induced and spontaneous sputum samples in COPD patients.

BACKGROUND: Induced and spontaneous sputum are used to evaluate the airways microbiota. Whether the sputum types can be used interchangeably in microbiota research is unknown. Our aim was to compare microbiota in induced and spontaneous sputum from COPD patients sampled during the same consultation. METHODS: COPD patients from Bergen, Norway, were followed between 2006/2010, examined during the stable state and exacerbations. 30 patients delivered 36 sample pairs. DNA was extracted by enzymatic and mechanical lysis methods. The V3-V4 region of the 16S rRNA gene was PCR-amplified and prepared for paired-end sequencing. Illumina Miseq System was used for sequencing, and Quantitative Insights Into Microbial Ecology (QIIME) and Stata were used for bioinformatics and statistical analyses. RESULTS: Approximately 4 million sequences were sorted into 1004 different OTUs and further assigned to 106 different taxa. Pair-wise comparison of both taxonomic composition and beta-diversity revealed significant differences in one or both parameters in 1/3 of sample pairs. Alpha-diversity did not differ. Comparing abundances for each taxa identified, showed statistically significant differences between the mean abundances in induced versus spontaneous samples for 15 taxa when disease state was considered. This included potential pathogens like Haemophilus and Moraxella. CONCLUSION: When studying microbiota in sputum samples one should take into consideration how samples are collected and avoid the usage of both induced and spontaneous sputum in the same study.

Macrophage migration inhibitory factor, a role in COPD.

Macrophage migration inhibitor factor (MIF) is a pluripotent cytokine associated with several different inflammatory conditions, but its role within lung inflammation and chronic obstructive pulmonary disease (COPD) is unclear. This study aimed to examine MIF in both stable COPD and during acute exacerbations (AECOPD). The study included 433 patients with COPD aged 41-76 and 325 individuals from the Bergen COPD cohort study who served as controls. All patients had an FEV1 of <80% predicted, FEV1/FVC ratio of <0.7, and a smoking history >10 pack-years. Serum levels of MIF were compared between the two groups at baseline, and for 149 patients, measurements were also carried out during AECOPD. Linear regression models were fitted with MIF as the outcome variable and adjusted for sex, age, body composition, smoking, and Charlson Comorbidity Score (CCS). Median MIF (interquartile range) in patients with COPD was 20.1 ng/ml (13.5-30.9) compared with 14.9 ng/ml (11.1-21.6) in controls (P < 0.01). MIF was bivariately associated with sex, body composition, and CCS (P < 0.05 for all). In the regression analyses, MIF was significantly higher in patients with COPD, coefficient 1.32 (P < 0.01) and 1.30 (P < 0.01) unadjusted and adjusted, respectively. In addition, in 149 patients during episodes of AECOPD, MIF was significantly elevated, with a median of 23.2 ng/ml (14.1-42.3) compared with measurements at stable disease of 19.3 ng/ml (12.4-31.3, P < 0.01). Serum levels of MIF were significantly higher in patients with COPD compared with controls. We also identified an additional increase in MIF levels during episodes of AECOPD.

Variability of within-breath reactance in COPD patients and its association with dyspnoea.

The forced oscillation technique can identify expiratory flow limitation (EFL) when a large difference in inspiratory and expiratory reactance (ΔXrs) occurs. However, flow limitation can vary from breath to breath, and so we compared a multiple-breath ΔXrs approach to the traditional breath-by-breath assessment of EFL. We investigated the within- and between-day reproducibility and the factors that affect the size of ΔXrs when used as a continuous measurement over multiple breaths. In addition, we examined how multiple-breath ΔXrs relates to the sensation of breathlessness. 425 moderate to very severe chronic obstructive pulmonary disease (COPD) patients and 229 controls were included. Spirometry and impedance measurements were performed on a MasterScope CT Impulse Oscillation System. Median ΔXrs approached zero in healthy controls with little variation between measurements. COPD patients generally had higher ΔXrs and higher variability. The COPD patients with ΔXrs >0.1 kPa · L(-1) · s(-1) were prone to be more breathless and had a higher modified Medical Research Council dyspnoea scale score. In controls, the 95th percentile of ΔXrs was as low as 0.07 kPa · L(-1) · s(-1). We describe a new method to assess EFL at a patient level and propose a cut-off, mean ΔXrs >0.1 kPa · L(-1) · s(-1), as a way to identify COPD patients who are more likely to report dyspnoea.

Peak oxygen uptake and breathing pattern in COPD patients--a four-year longitudinal study.

BACKGROUND: Activities of daily living in patients with chronic obstructive pulmonary disease (COPD) are limited by exertional dyspnea and reduced exercise capacity. The aims of the study were to examine longitudinal changes in peak oxygen uptake (V̇O2peak), peak minute ventilation (V̇Epeak) and breathing pattern over four years in a group of COPD patients, and to examine potential explanatory variables of change. METHODS: This longitudinal study included 63 COPD patients, aged 44-75 years, with a mean forced expiratory volume in one second (FEV1) at baseline of 51 % of predicted (SD = 14). The patients performed two cardiopulmonary exercise tests (CPETs) on treadmill 4.5 years apart. The relationship between changes in V̇O2peak and V̇Epeak and possible explanatory variables, including dynamic lung volumes and inspiratory capacity (IC), were analysed by multivariate linear regression analysis. The breathing pattern in terms of the relationship between minute ventilation (V̇E) and tidal volume (VT) was described by a quadratic equation, VT = a + b∙V̇E + c∙V̇E (2), for each test. The VTmax was calculated from the individual quadratic relationships, and was the point where the first derivative of the quadratic equation was zero. The mean changes in the curve parameters (CPET2 minus CPET1) and VTmax were analysed by bivariate and multivariate linear regression analyses with age, sex, height, changes in weight, lung function, IC and inspiratory reserve volume as possible explanatory variables. RESULTS: Significant reductions in V̇O2peak (p < 0.001) and V̇Epeak (p < 0.001) were related to a decrease in resting IC and in FEV1. Persistent smoking contributed to the reduction in V̇O2peak. The breathing pattern changed towards a lower VT at a given V̇E and was related to the reduction in FEV1. CONCLUSION: Increasing static hyperinflation and increasing airway obstruction were related to a reduction in exercise capacity. The breathing pattern changed towards more shallow breathing, and was related to increasing airway obstruction.

Serial Measurements of Arterial Oxygen Tension are Associated with Mortality in COPD.

BACKGROUND: Knowledge on factors associated with mortality can help identify patients with COPD that might benefit from close monitoring and intervention. Arterial blood gases (ABGs) are related to mortality, but both arterial tension of oxygen (PaO2) and arterial tension of carbon dioxide (PaCO2) vary over time. The aim of our study was to investigate the association between repeatedly measured ABGs and mortality in men and women with COPD. METHODS: A cohort of 419 Norwegian subjects with COPD, GOLD stage II-IV, aged 40-75, was followed up with up to seven ABGs, measured during stable phase for three years. Cox proportional hazard models were used to quantify the relationship between both single and repeatedly measured ABGs and all-cause mortality after five years, adjusting for age, sex, and the updated BODE index. RESULTS: A total of 64 subjects died during follow-up. Mean initial arterial oxygen tension (standard deviation) was significantly higher in survivors compared to deceased, with PaO2 (in kPa) 9.4 (1.1) versus 8.8 (1.2), p<0.001. Corresponding numbers for PaCO2 were 5.3 (0.5) and 5.5 (0.7), p < 0.001. In analyses adjusting for age, sex, and the updated BODE index hazard ratios - HR(95% confidence intervals) - for all-cause mortality were 0.73 (0.55, 0.97) and 1.58 (0.90, 2.76) for repeated measures of PaO2 and PaCO2, respectively. CONCLUSION: Both arterial oxygen and carbon dioxide tension were related to mortality in this study, and arterial oxygen tension added prognostic information to the updated BODE index in COPD.

Comparison of inflammatory markers in induced and spontaneous sputum in a cohort of COPD patients.

BACKGROUND: Sputum induction is a non-invasive method for obtaining measurements of inflammation in the airways. Whether spontaneously sampled sputum can be a valid surrogate is unknown. The aim of this study was to compare levels of six inflammatory markers in sputum pairs consisting of induced and spontaneous sputum sampled on the same consultation either in a stable state or during exacerbations of chronic obstructive pulmonary disease (COPD). METHODS: 433 COPD patients aged 40-76, Global initiative for chronic Obstructive Lung Disease (GOLD) stage II-IV were enrolled in 2006/07 and followed every six months for three years. 356 patients were followed for potential exacerbations. Interleukin-6, interleukin-8, interleukin-18, interferon gamma-inducible protein-10, monokine induced by gamma interferon and tumor necrosis factor-alpha (IL-6, IL-8, IL-18, IP-10, MIG and TNF-α) were measured by bead based multiplex immunoassay in 60 paired sputum samples from 45 patients. Albumin was measured by enzyme immunoassay, for concentration correction. Culturing for bacterial growth was performed on 24 samples. Bland-Altman plots were used to assess agreement. The paired non-parametric Wilcoxon signed-rank test, the non-parametric Spearman's rank correlation test and Kruskal-Wallis test were used for statistical analyses. For all analyses, a p-value < 0.05 was considered significant. RESULTS: Agreement between the two measurements was generally low for all six markers. TNF-α was significantly higher in spontaneous sputum at exacerbations (p = 0.002) and trending higher at the steady state (p = 0.06). Correlation coefficients between the levels of markers in induced and spontaneous sputum varied between 0.58 (IL-18) to 0.83 (IP-10). In spontaneous sputum IL-18 and MIG were higher in ex-smokers (p < 0.05). The levels of all markers were higher in GOLD stage III & IV except for IL-6 in spontaneous sputum and IL-18 in induced sputum, compared with GOLD stage II, although not statistically significant. In spontaneous sputum the levels of IL-6 were significantly higher if Haemophilus influenzae (HI) was not cultured. CONCLUSION: We observed a low agreement and significant differences in inflammatory markers between induced and spontaneous sputum, both at steady state and exacerbations. We recommend considering sampling method when reporting on inflammatory markers in sputum.

Paraneoplastic Hu and CRMP5 antibodies are present in smokers without cancer or neurological disease.

BACKGROUND AND OBJECTIVE: We investigated if the paraneoplastic Hu and collapsin response mediator protein 5 (CRMP5) antibodies could be used as early markers for lung cancer in smokers with or without chronic obstructive pulmonary disease (COPD). METHODS: Hu and CRMP5 antibodies were measured by radioimmunoprecipitation assay (RIPA) in sera from 552 smokers; 379 with and 173 without COPD. Three hundred blood donors served as controls. The positive sera were also tested by indirect immunofluorescence and line blot with recombinant proteins. The 552 smokers were matched with data from the Cancer Registry of Norway, and the hospital medical records from the subjects positive for Hu and CRMP5 antibodies were reviewed. The mean follow-up time was 4.4 years (range 2.5-5.7 years). RESULTS: The RIPA showed that 5/379 (1.3%) smokers with COPD had Hu antibodies and 1/379 (0.3%) smokers with COPD had CRMP5 antibodies. Only the smoker with the highest RIPA index had Hu antibodies also detected by immunofluorescence and line blot. One of 173 (0.6%) smokers without COPD had Hu antibodies, but none had CRMP5 antibodies. None of the 300 controls had Hu antibodies, but 2/300 (0.7%) had CRMP5 antibodies. Hu antibodies remained positive for more than 5 years. No cancer or neurological disease was recorded in the Hu or CRMP5 positive patients. The total cancer frequency in the smokers with and without COPD was 70/552 (13%). CONCLUSIONS: Hu and CRMP5 antibodies were not associated with cancer or neurological disease in a large cohort of smokers and are therefore not always paraneoplastic.

Inflammatory cytokine response to exercise in alpha-1-antitrypsin deficient COPD patients 'on' or 'off' augmentation therapy.

BACKGROUND: There is still limited information on systemic inflammation in alpha-1-antitrypsin-deficient (AATD) COPD patients and what effect alpha-1-antitrypsin augmentation therapy and/or exercise might have on circulating inflammatory cytokines. We hypothesized that AATD COPD patients on augmentation therapy (AATD + AUG) would have lower circulating and skeletal muscle inflammatory cytokines compared to AATD COPD patients not receiving augmentation therapy (AATD-AUG) and/or the typical non-AATD (COPD) patient. We also hypothesized that cytokine response to exercise would be lower in AATD + AUG compared to AATD-AUG or COPD subjects. METHODS: Arterial and femoral venous concentration and skeletal muscle expression of TNFα, IL-6, IL-1ß and CRP were measured at rest, during and up to 4-hours after 50% maximal 1-hour knee extensor exercise in all COPD patient groups, including 2 additional groups (i.e. AATD with normal lung function, and healthy age-/activity-matched controls). RESULTS: Circulating CRP was higher in AATD + AUG (4.7 ± 1.6 mg/dL) and AATD-AUG (3.3 ± 1.2 mg/dL) compared to healthy controls (1.5 ± 0.3 mg/dL, p < 0.05), but lower in AATD compared to non-AATD-COPD patients (6.1 ± 2.6 mg/dL, p < 0.05). TNFα, IL-6 and IL-1ß were significantly increased by 1.7-, 1.7-, and 4.7-fold, respectively, in non-AATD COPD compared to AATD COPD (p < 0.05), and 1.3-, 1.7-, and 2.2-fold, respectively, compared to healthy subjects (p < 0.05). Skeletal muscle TNFα was on average 3-4 fold greater in AATD-AUG compared to the other groups (p < 0.05). Exercise showed no effect on these cytokines in any of our patient groups. CONCLUSION: These data show that AATD COPD patients do not experience the same chronic systemic inflammation and exhibit reduced inflammation compared to non-AATD COPD patients. Augmentation therapy may help to improve muscle efflux of TNFα and reduce muscle TNFα concentration, but showed no effect on IL-6, IL-1ß or CRP.

Malnutrition as a prognostic factor for 2-year mortality in hospitalized patients in Norway: A matched cohort study.

BACKGROUND: Risk of malnutrition and malnutrition have been previously associated with increased risk of mortality. It remains unclear, however, whether the severity of malnutrition differentiates in association with all-cause mortality. The aim was to assess the association between being at risk of malnutrition or being diagnosed with malnutrition according to the diagnostic assessment of the Global Leadership Initiative on Malnutrition (GLIM) with all-cause mortality during a 2-year follow-up in hospitalized patients. METHODS: A matched cohort study was conducted in hospitalized patients (excluding cancer, intensive care, and transmissible infections) at a university hospital in Bergen, Norway. All patients underwent nutrition screening with the Nutritional Risk Screening 2002 and a further nutrition assessment using the GLIM criteria. All-cause mortality was estimated from the Norwegian death registry after 2 years, and risk factors were calculated by Cox regression analysis. RESULTS: Among 326 patients included, 55 patients died within 2 years (17% mortality rate). Risk of malnutrition was associated with increased all-cause mortality, which disappeared after adjustment for age and sex. Malnutrition was associated with an increased risk of all-cause mortality at 2 years also after adjustment for age and sex and, additionally, for further comorbidities (hazard ratio = 2.50; 95% CI, 1.41-4.42). When analyzed separately only severe malnutrition was associated with mortality (hazard ratio = 2.73; 95% CI, 1.44-5.15). CONCLUSION: The findings highlight a strong association between inpatients with severe malnutrition, defined by the GLIM criteria, and an increased risk of all-cause mortality within a 2-year follow-up.

TNF-α is associated with loss of lean body mass only in already cachectic COPD patients.

BACKGROUND: Systemic inflammation may contribute to cachexia in patients with chronic obstructive pulmonary disease (COPD). In this longitudinal study we assessed the association between circulating C-reactive protein (CRP), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 levels and subsequent loss of fat free mass and fat mass in more than 400 COPD patients over three years. METHODS: The patients, aged 40-76, GOLD stage II-IV, were enrolled in 2006/07, and followed annually. Fat free mass and fat mass indexes (FFMI & FMI) were calculated using bioelectrical impedance, and CRP, TNF-α, IL-1ß, and IL-6 were measured using enzyme immunoassays. Associations with mean change in FFMI and FMI of the four inflammatory plasma markers, sex, age, smoking, FEV1, inhaled steroids, arterial hypoxemia, and Charlson comorbidity score were analyzed with linear mixed models. RESULTS: At baseline, only CRP was significantly (but weakly) associated with FFMI (r = 0.18, p < 0.01) and FMI (r = 0.27, p < 0.01). Univariately, higher age, lower FEV1, and use of beta2-agonists were the only significant predictors of decline in FFMI, whereas smoking, hypoxemia, Charlson score, and use of inhaled steroids predicted increased loss in FMI. Multivariately, high levels of TNF-α (but not CRP, IL-1ß or IL-6) significantly predicted loss of FFMI, however only in patients with established cachexia at entry. CONCLUSION: This study does not support the hypothesis that systemic inflammation is the cause of accelerated loss of fat free mass in COPD patients, but suggests a role for TNF-α in already cachectic COPD patients.

Association of exposure to environmental tobacco smoke in childhood with chronic obstructive pulmonary disease and respiratory symptoms in adults.

BACKGROUND AND OBJECTIVE: Exposure to environmental tobacco smoke (ETS) is associated with impaired lung function in childhood, which in turn, is associated with chronic obstructive pulmonary disease (COPD) in adulthood. However, little is known regarding the direct association between childhood exposure to ETS and the development of COPD. The main objective of the present study was to examine the associations between childhood ETS exposure and adult COPD and respiratory symptoms. METHODS: Patients with COPD (n = 433) and control subjects (n = 325) participated in the Bergen COPD Cohort Study during 2006-2009. Participants performed spirometry and answered extensive questionnaires. The risk factors for COPD, morning cough, cough with phlegm, chronic cough and dyspnoea were examined using logistic regression analysis. Analyses were stratified by gender. RESULTS: The prevalence of childhood exposure to ETS was 61%. After adjustment, women who were exposed to ETS during childhood had a higher risk of COPD than those who were not exposed: odds ratio 1.9, 95% confidence interval 1.0, 3.7. Other important predictors for COPD and respiratory symptoms among women were occupational dust exposure (COPD), family history of COPD (COPD, all symptoms), current exposure to ETS in the home (morning cough) and education (COPD, dyspnoea). ETS exposure during childhood was associated with respiratory symptoms among males (odds ratios 1.5-1.7). Risk factors for COPD among men were occupational dust exposure, family history of COPD and level of education. Occupational dust exposure and family history of COPD also predicted dyspnoea among males. CONCLUSIONS: Exposure to ETS during childhood was associated with COPD and respiratory symptoms in adulthood. Although active smoking is still the most important risk factor for COPD, reduction of childhood ETS exposure could contribute to the prevention of COPD and respiratory symptoms.

Different COPD disease characteristics are related to different outcomes in the 6-minute walk test.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) can lead to severe disability as the disease advances. The 6-minute walk test (6MWT) is commonly used to measure functional capacity in COPD patients and has three potential outcomes; walking distance, oxygen desaturation, and self-perceived dyspnea assessed by the Borg scale, all reflecting different aspects of COPD. The aim of this study was to identify predictors of all 3 outcomes of 6MWT in patients with COPD. METHODS: 370 COPD patients, aged 40-75 yrs, were included from the first phase of the Bergen COPD cohort study. They were examined with spirometry, bioelectrical impedance measurements, 6MWT, Center for Epidemiologic Studies of Depression (CES-D) Scale, Medical Research Council (MRC) dyspnea scale, Charlson index for co-morbidities, self-reported physical activity questionnaire, plasma levels of C-reactive protein (CRP) and arterial blood gases. RESULTS: Significant predictors in the multivariate analyses were sex, age, FEV(1) in % predicted, symptoms of dyspnea (MRC), co-morbidities (Charlson Index) and self-reported physical activity for walking distance, FEV(1) in % predicted and PaO(2) for oxygen desaturation, and body composition, smoking and co-morbidities for self-perceived dyspnea assessed by the Borg scale. CONCLUSION: Several COPD characteristics have predictive value for the 6MWT, and some COPD characteristics are more strongly related to specific 6MWT outcomes than others.