Author: H. Fehrenbach

Authors: J. R. Nyengaard; H. J. G. Gundersen

The present article reviews the relevant stereological estimators for obtaining reliable quantitative structural data from the lungs. Stereological sampling achieves reliable, quantitative information either about the whole lung or complete lobes, whilst minimising the workload. Studies have used systematic random sampling, which has fixed and constant sampling probabilities on all blocks, sections and fields of view. For an estimation of total lung or lobe volume, the Cavalieri principle can be used, but it is not useful in estimating individual cell volume due to various effects from over- or underprojection. If the number of certain structures is required, two methods can be used: the disector and the fractionator.

The disector method is a three-dimensional stereological probe for sampling objects according to their number. However, it may be affected on tissue deformation and, therefore, the fractionator method is often the preferred sampling principle. In this method, a known and predetermined fraction of an object is sampled in one or more steps, with the final step estimating the number. Both methods can be performed in a physical and optical manner, therefore enabling cells and larger lung structure numbers (e.g. number of alveoli) to be estimated. Some estimators also require randomisation of orientation, so that all directions have an equal chance of being chosen. Using such isotropic sections, surface area, length, and diameter can be estimated on a Cavalieri set of sections.

Stereology can also illustrate the potential for transport between two compartments by analysing the barrier width. Estimating the individual volume of cells can be achieved by local stereology using a two-step procedure that first samples lung cells using the disector and then introduces individual volume estimation of the sampled cells.

The coefficient of error of most unbiased stereological estimators is a combination of variance from blocks, sections, fields of view, and noise due to random positioning of the probes. This can be decreased by increasing the number of units from the element causing the most variance. Overall, stereology provides lung scientists with efficient tools for estimating structural components correctly.

Author: M. Ochs

Acute lung injury is associated with a variety of histopathological alterations, such as oedema formation, damage to the components of the blood–air barrier and impairment of the surfactant system. Stereological methods are indispensable tools with which to properly quantitate these structural alterations at the light and electron microscopic level. The stereological parameters that are relevant for the analysis of acute lung injury are reviewed in the present article.

Authors: D. M. Hyde; L. A. Miller; E. S. Schelegle; M. V. Fanucchi; L. S. Van Winkle; N. K. Tyler; M. V. Avdalovic; M. J. Evans; R. Kajekar; A. R. Buckpitt; K. E. Pinkerton; J. P. Joad; L. J. Gershwin; R. Wu; C. G. Plopper

Asthma is a worldwide health problem that affects 300 million people, as estimated by the World Health Organization. A key question in light of this statistic is: “what is the most appropriate laboratory animal model for human asthma?”

The present authors used stereological methods to assess airways in adults and during post-natal development, and their response to inhaled allergens to compare rodents and nonhuman primates to responses in humans.

An epithelial–mesenchymal trophic unit was defined in which all of the compartments interact with each other. Asthma manifests itself by altering not only the epithelial compartment but also other compartments (e.g. interstitial, vascular, immunological and nervous). All of these compartments show significant alteration in an airway generation-specific manner in rhesus monkeys but are limited to the proximal airways in mice. The rhesus monkey model shares many of the key features of human allergic asthma including the following: 1) allergen-specific immunoglobulin (Ig)E and skin-test positivity; 2) eosinophils and IgE+ cells in airways; 3) a T-helper type 2 cytokine profile in airways; 4) mucus cell hyperplasia; 5) subepithelial fibrosis; 6) basement membrane thickening; and 7) persistent baseline hyperreactivity to histamine or methacholine.

In conclusion, the unique responses to inhaled allergens shown in rhesus monkeys make it the most appropriate animal model of human asthma.

Author: H. Fehrenbach

A variety of animal models have been suggested as models of pulmonary emphysema; these are critically discussed in the present article from a stereologist's perspective. In addition, a stereological design for the quantification of experimentally induced emphysema is proposed.

On the basis of the widely accepted definition of pulmonary emphysema being an “abnormal permanent enlargement of the airspaces distal to the terminal bronchioles, accompanied by destruction of their walls,” quantitative morphology is the only method with which to reliably assess the presence of emphysema. Recognising this, careful inspection of animal models that are based on instillation of elastase, genetic alterations, inhalation of cigarette smoke or induction of apoptosis, reveals that both criteria of emphysema definition were demonstrated in surprisingly few of them.

Several aspects are suggested to be critical for the understanding of animal models of human emphysema. For example, genetic models that rely on the inhibition of the formation of alveoli during post-natal alveolarisation should clearly be distinguished from models that rely on the loss of mature alveoli after alveolarisation is complete. Furthermore, inhalation models that are characterised by exposed animals exhibiting a severe loss of body weight should carefully examine the relative contribution of intervention and weight loss, respectively. Models that rely on the exposure of juvenile animals for several weeks or even months should take into account the effects of normal lung growth and ageing.

Stereology offers appropriate tools with which to quantify the parameters relevant to assess development and the regeneration of emphysema. Stereologists continue to develop tools that will help ascertain the reliability of established and new models. If inappropriate parameters continue to be used for the evaluation of animal models of emphysema, thinking and resources are likely to be misdirected and the models may limit rather than expand the understanding of human emphysema and the development of new therapies.

Author: C. C. W. Hsia

Compensatory lung growth is defined by the absolute increases in the quantity of functioning lung tissue in response to injury and/or disease, leading to a positive impact on functional outcome. The pneumonectomy model is used as an example to illustrate the salient features of compensatory growth and the specific considerations in its morphological quantification, including techniques of sampling and analysis, resolution of ultrastructural details, selection of markers for measurement, and interpretation of results in the context of organ architecture and physiology. The potential for structure–function dissociation is described in the present article. The current paper will discuss the application of high-resolution computed tomography to noninvasively quantify regional anatomy as well as temporal evolution of lung growth.

Authors: P. G. Woodruff; A. L. Innes

Bronchoscopy with bronchial biopsies allows sampling of airway tissue in prospective clinical studies and is increasingly applied for the study of airway inflammation and remodelling.

It is important to recognise that there are limitations inherent to the use of bronchial biopsies, including the inability to randomly sample the airway and susceptibility to the “reference trap”. Nonetheless, certain stereological principles can be applied to the analysis of these specimens, which may improve the validity of the results obtained. These principles relate to choice of reference space, adequacy of sampling and treatment of tissue orientation in the estimation of thickness and surface area.

With attention to these principles, the present authors have developed protocols for the assessment of multiple measures of airway remodelling in bronchial biopsies, including airway epithelial mucin content, epithelial goblet cell size, reticular basement membrane thickness and smooth muscle morphological outcomes. They have also developed protocols for the enumeration of inflammatory cells important in the study of asthma and chronic obstructive pulmonary disease, including eosinophils and neutrophils.

In the present article, relevant stereological principles and the details of these specific protocols will be reviewed.

Authors: P. S. Burge; V. C. Moore

Authors: StephenA. Renshaw; CatherineA. Loynes; DanielM. Trushell; PhilipW. Ingham; Moira K. B. Whyte

Although we are separated from zebrafish by 160 million years of evolution, we share many features of the innate and adaptive immune systems. In addition, we can manipulate the genome of zebrafish, and observe the effects on inflammation in vivo as they are transparent in their larval stages. This has exciting implications for the study of inflammatory diseases.

We have established a model of inflammation in the zebrafish tail, in which caspase dependent cell death is required for resolution. For example, addition of the pan-caspase inhibitor zVD added at 4 hours after tailfin injury increases the number of neutrophils present from 6.0+/−1.0 to 28.9+/− 3.3 (mean +/− s.e.m. p<0.001 n = 3).

The transparency of the larvae makes these an ideal model for the study of in vivo inflammation, and we have generated fluorescent systems for the easy visualisation of neutrophilic inflammation and resolution in vivo.

We are also performing an unbiased forward genetic screen for mutants with defective resolution of inflammation, and to date have identified 38 putative mutants. These techniques allow new approaches to understanding the molecular controls of inflammation resolution.

Authors: EmmaH. Baker; Andrew Irwin; AmandaL. Brennan; Brian Trost; Alan Britten; Sue Heenan; Barry Peterson

Measurement of pulmonary leukocyte margination could be a useful biomarker of lung inflammation in COPD, but analysis is complicated by recirculation of labeled leukocytes.

15 minutes of planar nuclear images were obtained after injecting autologous ^99mTc-labeled leukocytes in 4 never-smoked controls, 6 stable mild/moderate and 2 exacerbating COPD patients. COPD patients were also imaged for 10 minutes after in vivo red blood cell (RBC) labeling with ^99mTc and were re-imaged 2 weeks later to determine reproducibility. Activity as a function of time was measured in regions of interest over lungs and heart.

A multi-compartment mathematical model was used to correct for recirculation but failed to provide a biomarker that clearly separated controls from COPD. A simpler model for activity in lungs and heart as a function of time (A(t)) was applied only to time points before recirculation: [A(t) = X1 x (1-exp(-X2 x t)) x exp(-X3 x t), where Xi are adjusted to match the data]. The ratio (R) of X3 (downslope of curve) in the lungs to X3 in the heart was investigated as a biomarker of margination. Values of R were reasonable (1.06±0.08 (SE)) for RBC (i.e. no margination) and 0.76±0.10 for controls (∼25% margination). In stable COPD patients R was significantly smaller (0.19±0.09, p<0.01) than controls and was reproducible (0.25±0.10). R during exacerbation was surprisingly large (0.88±0.22), possibly due to steroid treatment, but R was similar to stable COPD patients 2 weeks later (0.09±0.04).

R requires only 2–3 minutes of imaging and may be a useful biomarker of margination. However it remains to be shown whether R truly reflects inflammation in COPD.

Authors: Hajime Yoshisue; Stephen T. Holgate; Donna E. Davies; AnthonyP. Sampson

We have reported that cysteinyl-leukotrienes (cys-LTs) synergise not only with epidermal growth factor (EGF) but also with platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) to induce mitogenesis in human bronchial fibroblasts. We now describe the molecular mechanisms underlying this synergism. Mitogenesis was assessed by incorporation of [³H]thymidine into DNA and changes in protein phosphorylation by Western blotting. Surprisingly, no CysLT receptor antagonists (MK-571, montelukast, BAY u9773) prevented the synergistic mitogenesis. LTD4 did not cause phosphorylation of EGFR nor did it augment EGF-induced phosphorylation of EGFR, and the synergy between LTD4 and EGF was not blocked by the metalloproteinase inhibitor GM6001 or by an HB-EGF neutralising antibody. The EGFR-selective kinase inhibitor, AG1478, suppressed the synergy by LTD4 and EGF, but had no effect on the synergy with PDGF and FGF. While inhibitors of mitogen-activated protein kinase, phosphatidylinositol 3-kinase and protein kinase C (PKC) prevented the synergy, these drugs also inhibited mitogenesis elicited by EGF alone. In contrast, pertussis toxin (PTX) efficiently inhibited the potentiating effect of LTD4 on EGF-induced mitogenesis, as well as that provoked by PDGF or FGF, but had no effect on mitogenesis elicited by the growth factors alone. Whereas LTD4 alone did not augment phosphorylation of extracellular signal-regulated kinase (Erk)-1/2 and Akt, it increased phosphorylation of PKC in a Gαi-dependent manner. Addition of LTD4 prolonged the duration of EGF-induced phosphorylation of Erk-1/2 and Akt, both of which were sensitive to PTX. The effect of cys-LTs involves a PTX-sensitive and PKC-mediated intracellular pathway leading to sustained growth factor-dependent phosphorylation of Erk-1/2 and Akt.

Authors: C. Pilette; B. Detry; A. Guisset; Y. Sibille

A decreased bronchial expression of secretory component (SC) was demonstrated in severe COPD, and correlated with neutrophils. Mechanisms of epithelial cell/neutrophils interactions remain however poorly understood.

Calu-3 (human bronchial epithelial) cells were incubated after confluence (in triplicate conditions) with various ratios of activated neutrophils (0.5:1 to 15:1, neutrophils: Calu-3 cells). After 48hrs of co-culture supernatants were assayed for SC by ELISA.

SC production by Calu-3 cells increased at intermediate neutrophil numbers (316±32 versus 193±19ng·ml⁻¹, ratio of 5:1 versus control, mean±SEM of 3 experiments, p = 0.05). In contrast, a trend for decrease in SC was observed with high neutrophil numbers (111±19 versus 193±19ng·ml⁻¹, ratio of 15:1 versus control, p = 0.06). The addition of secretory leukocyte protease inhibitor further increased SC upregulation at intermediate ratios, and inhibited the SC decrease at high neutrophil numbers. The mechanism of SC up-regulation by neutrophils did not implicate TNF-α or IL-1β.

This study provides direct evidence of a dual effect of neutrophils on epithelial SC. Our data suggest that neutrophils could differently affect epithelial immune secretory function according to the extent of neutrophil influx and/or to the reactivity of airway epithelial cells.

Authors: Stefan Pabst; Vildan Yenice; Martina Lennarz; Georg Baumgarten; Pascal Knuefermann; Bettina Hecht; Adrian Gillissen; Hans Vetter; Christian Grohe

The etiology of chronic obstructive lung disease (COPD) is unclear. It is supposed to be the product of an exogenous antigenic stimulus, such as tobacco smoke, and an endogenous genetic susceptibility. Toll-like receptors (TLR) are signal molecules, essential for the cellular response to bacterial cell wall components. Lipopolysaccharide (LPS) binds to TLR4 and two different polymorphisms for the TLR4 gene (Asp299Gly and Thr399Ile) have recently been described. TLR5 is the receptor for flagellin, aconstituent of Gram-positive and -negative bacterial flagella. A functional relevant TLR5 polymorphism (TLR5392STOP) has already been identified. The coactivation of both TLR4 and 5 seems to play an important role in the mediation of host-defense mechanisms. We genotyped 138 Caucasian patients with COPD and 135 healthy controls for the TLR5 polymorphism TLR5392STOP respectively for Asp299Gly and Thr399Ile polymorphisms in theTLR4gene. Among COPD patients the prevalence for the TLR5 mutant allele was 9.42% (26/276). The prevalence for each Asp299Gly and Thr399Ile mutant allele was 4.71% (13/276). In the control group the TLR5 mutation prevalence was 5.19% (14/270) (P = 0.138), the prevalence of each TLR4 polymorphism was 2.96% (8/270) (p = 0.279). In the subgroup of 54 patients with a stable course of COPD, defined as less than three hospitalizations over the last three years due to COPD, we found a significant association with the TLR5392STOP gene polymorphism (P = 0.026). These data suggest that the TLR5392STOP polymorphism is associated with a stable course of COPD, whereas TLR4 polymorphisms have no impact neither on the onset nor on the course of COPD.

Authors: Melanie Königshoff; Jochen Wilhelm; Andreas Jahn; Oana Amarie; Kamila Kitowska; Anke Wilhelm; Rainer M. Bohle; Werner Seeger; Frank Rose; Ludger Fink; Andreas Guenther; Oliver Eickelberg

Fibrotic lung disease is characterized by distorted lung architecture and severe loss of respiratory function secondary to alveolar epithelial cell (AEC) hyperplasia, enhanced extracellular matrix (ECM) deposition and fibroblast proliferation. Repetitive epithelial injuries with impaired alveolar wound healing and altered AEC gene expression represent a trigger mechanism for development of fibrosis. To reveal gene regulatory networks in lung fibrosis, we compared gene expression profiles of freshly isolated AEC obtained from mice 14 days after saline or bleomycin (BM) instillation using whole genome microarray analysis. Several genes of the Wnt signaling pathway, in particular WISP-1, a member of the CCN family, were highly regulated. WISP-1 protein expression was demonstrated in proliferating AEC in BM-treated lungs by immunofluorescence. When analyzing all six CCN family members, WISP-1 was upregulated the most 14 days after BM challenge, as analyzed by qRT-PCR. To elucidate WISP-1 function, cultured primary mouse AEC were stimulated with WISP-1 and demonstrated a 230% increase in proliferation, analyzed by 3H-thymidine incorporation. This was mediated through enhanced phosphorylation, but not expression of protein kinase B (PKB/Akt), as detected by immunoblot. Finally, increased expression of WISP-1 was detected in lung homogenates and isolated AEC from IPF patients, using qRT-PCR. Immunohistochemical analysis of WISP-1 and Ki67 verified the existence of hyperplastic and proliferative AEC expressing WISP-1 in vivo. Our study thus identifies WISP-1 as a novel regulator of AEC injury and repair, and suggests that WISP-1 is a key mediator in pulmonary fibrosis.

Authors: Suzanne Zuyderduyn; DennisK. Ninaber; Pieter S. Hiemstra; Klaus F. Rabe

Inflammatory cells that infiltrate and surround the airway smooth muscle (ASM) layer express antimicrobial peptides including the cathelicidin LL-37. LL-37 has been shown to activate epithelial cells by transactivation of the epidermal growth factor receptor (EGFR). Previously, we have shown that LL-37-induced IL-8 release by ASM cells was not dependent on either formyl peptide receptors or the EGFR (ATS 2005). In monocytes LL-37 induces processing of IL-1β through activation of the purinergic P2X7 receptor. Therefore, the aim of our study was to evaluate the role of purinergic receptors in LL-37-induced activation of ASM cells, and to explore the involvement of several intracellular signalling pathways.

ASM cells were cultured and serum-deprived 24 hours before stimulation with LL-37 (10 μg·ml⁻¹). The purinergic receptor antagonist suramin and inhibitors of ERK1/2, p38, Src and PI3K were preincubated for one hour. ERK1/2 phosphorylation was assessed by Western Blot, and IL-8 release was determined in supernatants using a sandwich ELISA. RT-PCR was performed for P2X7 on untreated ASM cells. LL-37 induced ERK1/2 phosphorylation and IL-8 release; both were inhibited by suramin (IL-8: 86%). Inhibitors of ERK1/2, p38 and Src signalling also reduced LL-37-induced IL-8 release (by 67%, 63% and 76%, respectively), suggesting a role for these pathways. P2X7 mRNA was expressed in ASM cells.

These data show that LL-37-induced IL-8 release is mediated via purinergic receptors, ERK1/2 activation, p38 and Src signalling. Our PCR data are in line with the hypothesis that also in ASM P2X7 is the purinergic receptor involved in LL-37 signalling, although this needs further investigation.

Authors: P. Marno; C. Bryden; W. Bird; H. A. Watkin

Many COPD sufferers find that their symptoms become worse during colder weather, which can lead to an exacerbation resulting in hospital admission. This study investigates different measures of cold, assessing which most strongly relate to COPD admissions and whether they can be used to forecast risk of exacerbation.

COPD admissions (ICD10 J40–J44) for the five Strategic Health Authorities (SHAs) in London and corresponding meteorological data were extracted for October–March 1997–2003. Correlations and regressions were used to compare the effects on admissions of:

<li><p>daily mean, maximum and minimum temperature;

significant drops in temperature;

weekly average maximum temperature;

“cumulative cold”, summing the number of degrees the daily maximum temperature was below a threshold across a week;

different windchill indices.

Authors: Sarah J. Deacon; Emma E. Vincent; Sally J. Singh; Michael C. Steiner; Paul Greenhaff; Michael D. Morgan

We conducted a randomised, placebo-controlled trial to examine whether creatine supplementation augments the benefits of pulmonary rehabilitation (PR), containing aerobic exercise and resistance training (RT).

80 subjects with COPD (GOLD stages I–IV) [mean (SD) age 68 (7.8) yrs, FEV1 44.1 (20.3) % predicted] completed 21 sessions of PR, with enhanced RT, following baseline measurements and randomisation to take creatine (Cr) or placebo (Pl) supplement. A subgroup had muscle biopsies.

Groups were well matched at baseline except for gender (M:F Cr 19:19, Pl 31:11, Chi² p = 0.03). Mean change in functional performance & muscle strength after PR are shown. Health status (CRQ-SR) improved significantly after PR but did not differ between groups. Muscle biopsies showed evidence of creatine uptake.

This adequately powered study showed significant improvements in all outcomes following PR. Creatine supplementation did not enhance these benefits.

Authors: Sundeep Kaul; IanM. Stell; S. Chinn; M. Polkey; J. Moxham

Supplemental O2 is frequently added to bi-level non-invasive ventilation circuits to maintain Sa,O2 >90%. Oxygen can be added at several points & in the presence of different inspiratory pressures. The effect of varying entrainment sites and inspiratory pressures (IPAP) on PO2, PCO2, Fio2, inspiratory triggering and expiratory triggering in COPD patients is unknown.

18 patients with stable COPD (mean FEV1 47%) participated in the study. Oxygen was added at 4 sites in the ventilatory circuit (site 1: between mask and exhalation port; site 2: just distal to exhalation port; site 3: at ventilator outlet; site 4: directly into the mask via an inlet). The effect of varying entrainment sites and inspiratory pressures on arterial PO2, PCO2, FIO2, was recorded at 3 mins. The same full face mask (Respironics, Image 3) & ventilator (Respironics, BIPAP ST 30) was used.

Results for PO2 are shown at IPAP 10/EPAP 4 [table 1].

Anova was used to analyse the data.

Site 4 (via mask) was associated with a significantly higher PO2 at all flow rates compared with sites 1, 2 and 3 (P<0.001).

Site 3 (at ventilator outlet) was associated with the lowest PO2 at all flow rates, particularly at 15 L min (P<0.001).

Adding oxygen to the mask at lower IPAPs result in higher oxygen delivery.

Authors: JocimarA. Martins; ArmèleD. Andrade; RodrigoS. Assis; Rovilson Lara; VerônicaF. Parreira

Excessive tracheobronchial secretion is a common complication of chronic obstruction pulmonary disease (COPD) and its presence is associated with lower survival, greater hospitalization and death. Slow expiration with the glottis open in a lateral posture (ELTGOL) is a chest physiotherapy procedure for the removal of lung secretions. Studies which have assessed ELTGOL through pulmonary scintigraphy were not found [1]. The aim this study was to evaluate the effects of ELTGOL on the mucociliary clearance in patients with COPD.

Twelve patients with COPD aged 45 to 75 years were studied. After inhaling 20 mCi of 99mTc-DTPA [2], six posterior static scintigraphy images were taken in intervals of 0, 20, 40, 60, 80 and 120 minutes (figure 1).

The study was carried out in two stages (control and experimental) in a randomized order with a minimum interval of one week between the stages. In the experimental stage, ELTGOL was carried out after the first image. The right lung was studied, comparing the percentage of retention [3] of radioactive aerosol obtained during the control stage with the experimental condition, for each interval studied. The study was approved by the local ethics committee and all patients gave their written consent participate. The t test was used for data analysis with a significance level of α<0.05.

As shown in figure 2, significant differences between groups were found between all time intervals.

The present results showed the ELTGOL significantly increased the mucociliary clearance suggesting that ELTGOL is an effective technique to improve secretion removal in patients with COPD.

Authors: Stephan Budweiser; RudolfA. Jörres; Theresa Riedl; Frank Heinemann; Michael Pfeifer

We studied the role of base excess (BE) as marker of chronic hypercapnia and survival in patients with chronic obstructive pulmonary disease (COPD) and chronic hypercapnic respiratory failure (CHRF). Moreover, it was investigated whether the effects of non-invasive positive pressure ventilation (NPPV) on CHRF were reflected in BE and survival.

In 240 (160 without exacerbation) patients with COPD (mean±sd FEV1 30.7±9.7 %pred; PaCO2 56.9±9.9 mmHg) body-mass index (BMI), lung function, respiratory muscle function, blood gases and 6-minute walking distance (6-MWD) were assessed prior to initiation of NPPV. In addition, the changes of risk factors 6.3±2.9 months after initiation of NPPV were evaluated.

Overall mortality during the follow-up time (26.0±24.5 months) was 34.6%. Deaths resulted predominantly from respiratory causes (65.1%); among those, respiratory failure was most frequent (85.2%). Univariate analysis revealed BMI, FEV1, maximal inspiratory pressure (PImax), inspiratory load (P0.1), haemoglobin, 6-MWD, hyperinflation (IC/TLC, RV/TLC), blood gases and BE to be associated (p<0.05 each) with prognosis. In multivariate analyses, however, only BMI, RV/TLC and BE turned out to be independent cross-sectional predictors (p<0.05). Kaplan-Meier analyses showed that BE had predictive value particularly in patients with BMI≥25 kg·m⁻², RV/TLC≥70 % and PaCO2≥57 mmHg. Furthermore, changes of BMI, RV/TLC and BE (p<0.01) were associated with improved prognosis in severe hypercapnic COPD.

In patients with COPD and CHRF, BE was a prognostic marker for mortality, that was independent from other factors, particularly PaCO2. In addition, reversal of CHRF was reflected in BE and appeared to have an impact on prognosis.

Authors: Masaru Hasegawa; Yasuyuki Nasuhara; Yuya Onodera; Hironi Makita; Tomoko Betsuyaku; Masaharu Nishimura

We have recently developed new software to obtain longitudinal images and accurate short axis images of airways with an inner diameter > 2 mm located anywhere in the lung, using curved multiplanar reconstruction. Using this software, we demonstrated in patients with COPD that FEV1 (%predicted) was highly correlated with airway dimensions and the correlation coefficients improved as the airway became smaller in size (3). In this study, our aims are to further confirm the significant relationship between airway dimensions and airflow limitation in larger number of subjects, and to examine the relationship of airway dimensions with lung volumes in 95 patients with COPD (stage 0, 10; stage I, 23; stage II, 35; stage III, 24; stage IV, 3). We analyzed the airway dimensions from the 3^rd to the 6^th generations of the apical bronchus (B1) of the right upper lobe and the anterior basal bronchus (B8) of the right lower lobe. Lung volumes were measured by the helium closed circuit method. Both airway luminal area (Ai) and wall area percent (WA%) of all the generations, except a few, from the two bronchi were significantly correlated with RV and RV/TLC, but not with TLC or FRC. More importantly, the correlation coefficients (r) between airway dimensions and RV/TLC improved as the airways became smaller in size from the 3^rd to 6^th generations in both bronchi (r =  −0.483, −0482, −0.553, −0.624 for Ai of B8; r = 0.316, 0.380, 0.499, 0.551 for WA% of B8). These findings provide further evidence that distal (small) airways rather than proximal (large) airways are the determinants for airflow limitation in COPD.

Authors: R. P. Bowler; J. Hokanson; M. Taylor; S. Levy; E. M. Canaham; E. Regan; C. Wheeler; M. Nicks; E. Chan; J. D. Crapo

Tobacco smoke contains a high concentration of oxidants and is the primary cause of chronic obstructive pulmonary disease (COPD). Extracellular superoxide dismutase (EC-SOD) is the major antioxidant enzyme in the extracellular space of the lung and is part of the lung defense against these oxidants. We hypothesized that EC-SOD is a risk factor for COPD. We found that EC-SOD plasma levels were significantly higher (p<0.001) in 337 patients with COPD (147±7 ng·ml⁻¹) versus 343 controls (96±9 ng·ml⁻¹) and that lower FEV1s were associated with lower EC-SOD levels. To identify whether the EC-SOD gene was associated with COPD, we resequenced a subset of 188 subjects and identified 33 novel SNPs. Two of these SNPs (rs8192287 and rs8192288) were associated with a reduced odds of having COPD (OR 0.05 and 0.34; P<0.05). Haplotype analysis using a total of 5 EC-SOD SNPs (Table 1) further identified a protective haplotype (TTCGC) that was found in 11.4% of controls, but only 2.1% of subjects with COPD (P<0.001). These data indicate that EC-SOD genotype may partially predict whether smokers are resistant to the effects smoking.

Author: S. Carraro

At the European Respiratory Society (ERS) Annual Congress 2006, I received the ERS Annual Award for Pediatric Respiratory Research in Europe for my research work on noninvasive markers of airway inflammation. Most of the studies in which I have been involved were conducted at the University of Padova, Padova, Italy. We have studied asthmatic children, and have demonstrated that some markers of inflammation (e.g cysteinyl leukotrienes) and oxidative stress (e.g. 8-isoprostane and malondialdehyde) are increased in their exhaled breath condensate, while antioxidant products, such as glutathione, are reduced. In addition, while I was working in the laboratory of Dr Benjamin Gaston at the University of Virginia, Charlottesville, VA, USA, I conducted some basic research studies. A first group of experiments was aimed at investigating the role of rhinovirus infection in airway acidification. A second group of experiments was conducted to investigate the mechanisms involved in the regulation of expression S-nitrosoglutathione reductase (GSNOR), an enzyme that has a role in asthma pathogenesis, breaking down the endogenous bronchodilator GSNO.

Authors: Cleo C. van Diemen; Dirkje S. Postma; JudithM. Vonk; Marcel Bruinenberg; Jan P. Schouten; H. Marike Boezen

ADAM33 (A Disintegrin and Metalloprotease 33) has been identified as a susceptibility gene for asthma and single nucleotide polymorphisms (SNPs) in this gene have been associated with excess decline of lung function in asthmatics.

To assess whether SNPs in ADAM33 are associated with accelerated lung function loss in the general population and with chronic obstructive pulmonary disease (COPD).

We have collected DNA from subjects of the Vlagtwedde/Vlaardingen cohort participating in the last survey in 1989/1990 after a follow up of 25 years. Information was collected every 3 years, including lung function measurements. We defined COPD as GOLD stage 2 or higher at the last survey. 1390 subjects from the cohort were genotyped for the following SNPs in ADAM33: F+1, Q−1, S_1, S_2, T_1, T_2, V_4, ST+5. Differences in prevalence of SNPs were analyzed with chi-square tests. Linear mixed effects models were used to analyze FEV1 decline according to genotype.

In the whole population mean adjusted decline was 18.7 and 12.7 ml·y⁻¹ in females and males respectively. Individuals homozygous for minor alleles of SNPs S_2 and Q−1 and heterozygous for SNP S_1 had a significantly accelerated decline in FEV1 of respectively 4.9, 9.6 and 3.6 ml·y⁻¹ compared with wild type. We found a significantly higher prevalence of SNPs F+1, S_1, S_2 and T_2 in subjects with COPD.

We demonstrated that SNPs in ADAM33 are associated with accelerated lung function decline in the general population. These SNPs are also risk factors for COPD.

Authors: J. H. J. Vernooy; R. H. E. Cloots; M. A. Dentener; E. F. M. Wouters

Leptin, originally described as an adipocyte-derived hormone regulating energy expenditure, is now classified as a type I cytokine. We and others recently demonstrated that the lung is an additional source of leptin. The aim of the present study was to investigate the effect of acute and chronic inflammation on pulmonary expression of leptin in male Swiss mice. Acute lung inflammation was induced by a single intratracheal (IT) dose of 5 μg LPS (E.coli O55:B5), and mice were killed 4h, 24h and 72h postexposure. Chronic lung inflammation was induced by repeated LPS exposure (twice a week for 12 wks) and mice were killed after a 1-wk or 8-wks recovery period. Lungs were removed to assess cellular influx, leptin protein (immunohistochemistry) and mRNA (RT-PCR). Acute LPS exposure induced a transient neutrophilia (Vernooy, J. et al. AJRCMB 2001; 24:569–576). Chronic LPS exposure resulted in persistent accumulation of macrophages and CD8+ T-cells and emphysema (Vernooy, J. et al. AJRCMB 2002; 26:152–159). Control mice displayed positive leptin staining in bronchiolar epithelium and alveolar macrophages. Both acute (4h, 24h, 72h) and chronic (1-wk) inflammation was associated with complete suppression of pulmonary leptin expression at protein and mRNA level. Leptin expression was fully restored after 8-wks of recovery. Lungs of mice displayed constitutive leptin expression in bronchiolar epithelium and alveolar macrophages. Acute and chronic lung inflammation resulted in complete suppression of pulmonary leptin expression. Our data suggest that downregulation of leptin expression in lung may contribute to chronic pulmonary inflammation.

Authors: M. Díez; V. I. Peinado; E. Ferrer; J. Ramírez; J. Roca; R. Rodriguez-Roisin; J. A. Barberà

Vascular progenitor cells (VPC) have shown in vitro and in vivo their ability to differentiate into endothelial cells (EC). Some evidence suggests that the plasticity of these cells to differentiate into other cell types might contribute not only to angiogenesis but also to perpetuate vascular lesions. Studies done in pulmonary arteries (PA) of patients with COPD have demonstrated the presence of VPC infiltrating the intima. Since intimal thickening is mainly constituted by smooth muscle cells (SMC), we asked whether VPC could play a role in wall thickening. Accordingly, the objective was to evaluate in vitro the plasticity of VPC to differentiate into SMC and EC of human PA. G-CSF-mobilized peripheral blood CD133+ cells from a commercial primary line were expanded and labelled with acetylated-LDL-DiI for tracking cell purposes. Then, cells were co-cultured with commercial primary lines of human PA EC or SMC (n = 3). As control, CD133+ cells were cultured alone, with minimal medium with or without VEGF (50ng·ml⁻¹). After 6 and 12 days of growth, the phenotype of cultures was characterized by immunofluorescence with: lectin, α-actin and CD31. Cells were also evaluated morphologically. After 6 days, VPC acquired the morphology and the phenotype of the cells with which they were co-cultured, EC (lectin+, CD31+, α-actin-) or SMC (α-actin+, lectin-, CD31-). VPC cultured 12 days alone or with VEGF did not acquire typical morphology and markers of mature EC or SMC of PA. We conclude that VPC have the potential to differentiate in vitro into SMC, and that this plasticity could contribute to perpetuate pulmonary vascular remodelling in COPD.

Authors: E. M. Drost; C. A. Poland; K. Donaldson; W. MacNee

Persistent inflammation is the main pathological process that underlies COPD. Understanding this inflammatory response is a key focus of COPD research with the aim of discovering new therapeutic targets. The nuclear hormone receptor, PPAR is now a recognised modulator of inflammation in various chronic inflammatory conditions, but its role in the persistent airways inflammation in COPD has not been examined. Control of the inflammatory response by PPARα has been shown by antagonising inflammatory signalling pathways, such as NF-κB and AP-1.

PPARα protein levels in lung tissue from patients with COPD were assessed by Western blot. In vitro assays using the human type II alveolar epithelial cell line were performed to assess the effect of PPARα agonist treatment on inflammatory cytokine generation.

An increase in PPARα protein levels was seen for healthy smokers compared with non-smokers (Ratio to β-actin loading control, non-smokers 0.61±0.1, n = 10; healthy smokers 0.97±0.3, n = 11, p>0.05). No increase was seen for current smoker or ex-smoker COPD patients (0.36±0.08, n = 12; 0.49±0.1, n = 8 respectively). In vitro experiments with a human type II alveolar epithelial cell line demonstrated a diminished inflammatory response to TNFα, as measured by the generation of the pro-inflammatory cytokine IL-8, following pre-treatment with the PPARα agonist, WY-14643 (IL-8 generation, control 823±22 pg·ml⁻¹, TNFα 7491±530 pg·ml⁻¹ p<0.001, WY-14643 2559±46 pg·ml⁻¹ p<0.05, n = 3).

We propose PPAR agonists as a potential therapy for reducing the NF-κB-regulated inflammation in COPD airways.

Supported by GlaxoSmithKline

Authors: Kay Roy; Zoe Borrill; Andrew Hazel; Jorgen Vestbo; Dave Singh

Multiple flow rates FeNO data two-compartment mathematical modelling can estimate NO airway wall concentration (CawNO), airway wall diffusing capacity (DawNO), alveolar concentration (CalvNO) and maximal NO flux (JawNO). To compare modelling based on linear, non linear and mixed linear and non linear analyses in COPD. FeNO was measured using the Niox analyser (Aerocrine) at flow rates: 10, 30, 50, 100 and 200ml/s in 50 COPD patients and the data applied to 4 different methods. All methods showed that current smoking reduced CawNO. JawNO data differed between methods (Table 1). All the methods showed that current smoking did not affect CalvNO or DawNO. Comparison of the methods showed that CalvNO and DawNO data were significantly different between all methods, JawNO was different for most between method comparisons, while there was agreement between all the methods for CawNO. Smoking in COPD reduces CawNO , but not CalvNO and DawNO. JawNO, CalvNO and DawNO data are model dependent parameters. CawNO findings were model independent, and hence the most robust modelled parameter.

Author: Mariëlle Pijnenburg

The work described herein was presented as an oral, invited presentation at the European Respiratory Society (ERS) Congress 2006 and was awarded the European Respiratory Society Annual Award for Paediatric Respiratory Research in Europe; the work consists of two parts. The first part is on methodological issues of measuring exhaled nitric oxide fraction (Fe,NO) in children. The second part includes four studies on clinical applications of Fe,NO measurements in asthmatic children.

Authors: M. Pallayova; V. Donic; V. Donicova; Z. Tomori; S. Gresova

Sleep-disordered breathing (SDB) is often associated with impaired glucose metabolism. The study aimed at assessing immediate effect of CPAP on glucose excursions in type 2 diabetic patients with SDB measured with 72-hour continuous glucose monitoring system (CGMS).

8 type 2 diabetic patients with SDB (men, age 48,13±4,91 years, BMI 34,06±7,41 kg·m⁻², HbA1c 7,3±1,4%) underwent 2 overnight polysomnographic examinations including diagnostic night and CPAP night. CGMS was applied on both occasions. Statistical analyses included paired Student's t-test.

CPAP decreased apnoea-hypopnoea index (AHI) from 57,64±9,64·h⁻¹ to 8,05±4,42·h⁻¹ (p<0,0001) with significant improvement of saturation. Frequent episodes of sleep apnoea/hypopnoea and severe oxygen desaturation were followed by significant rise in blood glucose of up to 12,3 mmol·l⁻¹. Duration of post-hypoxic hyperglycemia was 50±10,79 min and its climax tended to be appeared up to 45min post-hypoxia. Nocturnal hyperglycemia strongly correlated with severe oxygen desaturation. Nocturnal glucose values were significantly higher during diagnostic night than during CPAP night (8,19±0,99 mmol·l⁻¹ versus 6,77±1,47 mmol·l⁻¹; p<0,0001). CGMS also showed improved preprandial and 1,5-hour postprandial glucose levels for breakfast after CPAP night. The improvement in overall glucose levels was much greater in patients with BMI<30 kg·m⁻² than in more obese patients.

The results suggest that nocturnal hyperglycemia is closely related to desaturation and CPAP treatment may have an immediate decreasing effect on blood glucose in type 2 diabetic patients with SDB.

Authors: Silke Ryan; Cormac T. Taylor; Walter T. McNicholas

Circulating levels of TNF-α are elevated in Obstructive Sleep Apnoea Syndrome (OSAS) and likely contribute to associated cardiovascular diseases. Furthermore, TNF-α has been suggested as a mediator of excessive daytime sleepiness (EDS). We investigated the predictors of TNF-α in OSAS in large, well-selected patient and control cohorts.

We undertook a prospective study of 30 non-OSAS (including 22 non-sleepy controls and 8 sleepy non-apnoeic snorers), 36 mild-moderate OSAS and 31 severe OSAS male subjects. All groups were closely matched in age, BMI, smoking status, blood pressure and lipid profile. All subjects were free of other disease and were not taking regular medication. Serum for TNF-α assay was drawn following polysomnography (PSG) in all subjects. 49 patients were commenced on CPAP therapy within one week following PSG; sleep studies and TNF-α measurements were repeated 6 weeks later.

TNF-α was higher in OSAS patients than controls (p<0.001). In multivariate analysis, TNF-α was independently associated with the desaturation index (r = 0.399, p<0.001), the Epworth Sleepiness Score (ESS) (r = 0.243, p = 0.005) and total cholesterol (r = 0.216, p = 0.018). Furthermore, levels were higher in sleepy non-OSAS patients than in controls (4.49[3.35,6.94] pg·ml⁻¹ vs 2.46[1.66,3.40]; p = 0.002) but lower than in severe OSAS patients (6.32[5.70,8.17]; p = 0.03). CPAP significantly lowered TNF-α (from 5.56±2.10 to 4.13±2.99 pg·ml⁻¹; p = 0.004).

The severity of intermittent hypoxia is the strongest predictor of TNF-α level supporting a key role of inflammation in the cardiovascular pathophysiology of OSAS. Furthermore, TNF-α is independently associated with EDS.

Authors: IlonkaH. van Veen; Pieter S. Hiemstra; Anneke ten Brinke; Anja Roos; RenateM. Verhoosel; JaapK. Sont; Peter J. Sterk; Klaus F. Rabe; ElisabethH. Bel

There is increasing evidence that activation of the complement system in asthma contributes to ongoing inflammation, tissue damage and airway remodeling. Mannose binding lectin (MBL) is a pattern recognition molecule that serves as the key mediator of the lectin pathway of complement activation. MBL levels are genetically determined and vary widely amongst individuals. In the present study we hypothesized that high MBL levels in asthma are associated with increased loss of lung function over time, as a consequence of inflammatory tissue damage. We measured serum MBL levels by ELISA in 68 patients with severe asthma and prospectively determined the change in post-bronchodilator (pb) FEV1 over a mean period of 5.7 years. The relationship between MBL and change in pbFEV1 (ΔFEV1) was analysed using (multiple) regression analysis and corrected for possible confounders (age, sex, age of onset, asthma duration, and pbFEV1). The median (range) MBL level was 332 (10.8-3587) ng·ml⁻¹. MBL was significantly associated with ΔFEV1 (p<0.04). Patients with a high MBL level (≥332 ng·ml⁻¹) had an increased risk of lung function decline compared to those with low MBL levels (OR (CI): 3.16 (1.14-8.79), p = 0.027); the excess decline being 42 ml·yr⁻¹ (p = 0.01). We conclude that a high MBL level is associated with an increased decline in lung function in patients with severe asthma. MBL might provide a clue towards better understanding of the pathophysiology of ongoing inflammation and subsequent decline in lung function of patients with severe asthma.

Authors: T. Duelien; T. M. L. Eagan; G. E. Eide; A. Gulsvik; P. S. Bakke

The effects of pre- or postnatal passive smoking on the adult incidence of asthma have not been reported previously.

Between 1985 and 1996/1997, we conducted an 11-year community cohort study on the incidence of asthma and respiratory symptoms in Western Norway. The cohort included 3,786 subjects aged 15 to 70 years, of which 2,819 were responders at both baseline and follow-up. The incidence of asthma and five respiratory symptoms by self-reported exposure to maternal smoking in utero and in childhood, as well as smoking by other household members in childhood, was examined.

After adjustment for sex, age, education, hay fever, personal smoking, and occupational exposure, maternal smoking was associated with asthma, phlegm cough, chronic cough, dyspnoea grade 2, attacks of dyspnoea, and wheezing, with odds ratios (95% confidence intervals [CI]) of 3.0 (1.6, 5.6), 1.7 (1.1, 2.6), 1.9 (1.2, 3.0), 1.9 (1.2, 3.0), 2.0 (1.3, 3.0), and 1.4 (0.9, 2.2), respectively. The adjusted attributable fractions (95% CI) of the adult incidence of asthma were 17.3% (5.2, 27.9) caused by maternal smoking and 9.3% (23.2, 33.2) caused by smoking by other household members.

Exposure to pre- and postnatal smoking carries a substantial risk for developing adult asthma and respiratory symptoms.

Authors: Daiana Stolz; Andreas Stulz; Beat Mueller; Michael Tamm

We evaluated the diagnostic accuracy of laboratory biomarkers and BAL differential cell count for the diagnosis of bacterial infection in severe immunosuppressed patients. One-hundred and seven consecutive patients undergoing bronchoscopy for suspected pulmonary infection were included in this study. Assessment included history, clinical examination, chest image studies, CRP, procalcitonin (ProCT), leukocyte counts, and BAL results. Patients were classified as having proven, possible, and non-bacterial infection.

Authors: D. G. Yanbaeva; G. F. Korytina; L. Z. Akhmadishina; S. Z. Zagidullin; T. V. Victorova

Chronic obstructive pulmonary disease (COPD) is a complex heterogeneous respiratory disease. COPD is characterized by a progressive irreversible airflow limitation that is due to a loss of lung elasticity resulting from peripheral airflow obstruction (chronic bronchitis) and parenchymal destruction (emphysema). Matrix metalloproteinases (MMP) are a major group of proteases known to regulate extracellular matrix turnover. They have been suggested to be important in the process of lung diseases associated with tissue remodeling. Polymorphisms in MMPs which known to upregulate their activity may result in the degradation of a lung matrix.

A case-control study was performed to investigate the association of polymorphisms of MMP type 1 (-1607G/GG), 9 (-1562C/T) and 12 (-82A/G) genes with COPD and disease severity. A total of 309 COPD patients admitted to departments of respiratory medicine have been recruited in Ufa city hospitals (## 13, 21, and 22). COPD patients have been undergone a spirometry and a physical examination by a chest physician to refer the GOLD II-IV stages. The control group comprised of 305 healthy subjects without evidence of chronic diseases (Table Basic characteristic of study groups).