Airway Elastin is increased in severe asthma and relates to proximal wall area: histological and computed tomography findings from the U-BIOPRED severe asthma study.

BACKGROUND: Airway remodelling, which may include goblet cell hyperplasia / hypertrophy, changes in epithelial integrity, accumulation of extracellular matrix components, smooth muscle hypertrophy and thickening of the lamina reticularis, is a feature of severe asthma and contributes to the clinical phenotype. OBJECTIVE: Within the U-BIOPRED severe asthma study, we have assessed histological elements of airway remodelling and their relationship to computed tomography (CT) measures of proximal airway dimensions. METHODS: Bronchial biopsies were collected from two severe asthma groups, one non-smoker (SAn, n = 28) and one current/ex-smoker (SAs/ex, n = 13), and a mild-moderate asthma group (MMA, n = 28) classified and treated according to GINA guidelines, plus a healthy control group (HC, n = 33). Movat's pentachrome technique was used to identify mucin, elastin and total collagen in these biopsies. The number of goblet cells (mucin+) was counted as a percentage of the total number of epithelial cells and the percentage mucin epithelial area measured. The percentage area of elastic fibres and total collagen within the submucosa was also measured, and the morphology of the elastic fibres classified. Participants in the asthma groups also had a CT scan to assess large airway morphometry. RESULTS: The submucosal tissue elastin percentage was higher in both severe asthma groups (16.1% SAn, 18.9% SAs/ex) compared with the HC (9.7%) but did not differ between asthma groups. There was a positive relationship between elastin and airway wall area measured by CT (n = 18-20, rho=0.544, p = 0.024), which also related to an increase in elastic fibres with a thickened lamellar morphological appearance. Mucin epithelial area and total collagen were not different between the four groups. Due to small numbers of suitable CT scans, it was not feasible to compare airway morphometry between the asthma groups. CONCLUSION: These findings identify a link between extent of elastin deposition and airway wall thickening in severe asthma.

Epithelial IL-6 trans-signaling defines a new asthma phenotype with increased airway inflammation.

BACKGROUND: Although several studies link high levels of IL-6 and soluble IL-6 receptor (sIL-6R) to asthma severity and decreased lung function, the role of IL-6 trans-signaling (IL-6TS) in asthmatic patients is unclear. OBJECTIVE: We sought to explore the association between epithelial IL-6TS pathway activation and molecular and clinical phenotypes in asthmatic patients. METHODS: An IL-6TS gene signature obtained from air-liquid interface cultures of human bronchial epithelial cells stimulated with IL-6 and sIL-6R was used to stratify lung epithelial transcriptomic data (Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes [U-BIOPRED] cohorts) by means of hierarchical clustering. IL-6TS-specific protein markers were used to stratify sputum biomarker data (Wessex cohort). Molecular phenotyping was based on transcriptional profiling of epithelial brushings, pathway analysis, and immunohistochemical analysis of bronchial biopsy specimens. RESULTS: Activation of IL-6TS in air-liquid interface cultures reduced epithelial integrity and induced a specific gene signature enriched in genes associated with airway remodeling. The IL-6TS signature identified a subset of patients with IL-6TS-high asthma with increased epithelial expression of IL-6TS-inducible genes in the absence of systemic inflammation. The IL-6TS-high subset had an overrepresentation of frequent exacerbators, blood eosinophilia, and submucosal infiltration of T cells and macrophages. In bronchial brushings Toll-like receptor pathway genes were upregulated, whereas expression of cell junction genes was reduced. Sputum sIL-6R and IL-6 levels correlated with sputum markers of remodeling and innate immune activation, in particular YKL-40, matrix metalloproteinase 3, macrophage inflammatory protein 1ß, IL-8, and IL-1ß. CONCLUSIONS: Local lung epithelial IL-6TS activation in the absence of type 2 airway inflammation defines a novel subset of asthmatic patients and might drive airway inflammation and epithelial dysfunction in these patients.

Contribution of airway eosinophils in airway wall remodeling in asthma: Role of MMP-10 and MET.

BACKGROUND: Eosinophils play an important role in the pathophysiology of asthma being implicated in airway epithelial damage and airway wall remodeling. We determined the genes associated with airway remodeling and eosinophilic inflammation in patients with asthma. METHODS: We analyzed the transcriptomic data from bronchial biopsies of 81 patients with moderate-to-severe asthma of the U-BIOPRED cohort. Expression profiling was performed using Affymetrix arrays on total RNA. Transcription binding site analysis used the PRIMA algorithm. Localization of proteins was by immunohistochemistry. RESULTS: Using stringent false discovery rate analysis, MMP-10 and MET were significantly overexpressed in biopsies with high mucosal eosinophils (HE) compared to low mucosal eosinophil (LE) numbers. Immunohistochemical analysis confirmed increased expression of MMP-10 and MET in bronchial epithelial cells and in subepithelial inflammatory and resident cells in asthmatic biopsies. Using less-stringent conditions (raw P-value < 0.05, log2 fold change > 0.5), we defined a 73-gene set characteristic of the HE compared to the LE group. Thirty-three of 73 genes drove the pathway annotation that included extracellular matrix (ECM) organization, mast cell activation, CC-chemokine receptor binding, circulating immunoglobulin complex, serine protease inhibitors, and microtubule bundle formation pathways. Genes including MET and MMP10 involved in ECM organization correlated positively with submucosal thickness. Transcription factor binding site analysis identified two transcription factors, ETS-1 and SOX family proteins, that showed positive correlation with MMP10 and MET expression. CONCLUSION: Pathways of airway remodeling and cellular inflammation are associated with submucosal eosinophilia. MET and MMP-10 likely play an important role in these processes.

A Transcriptome-driven Analysis of Epithelial Brushings and Bronchial Biopsies to Define Asthma Phenotypes in U-BIOPRED.

RATIONALE: Asthma is a heterogeneous disease driven by diverse immunologic and inflammatory mechanisms. OBJECTIVES: Using transcriptomic profiling of airway tissues, we sought to define the molecular phenotypes of severe asthma. METHODS: The transcriptome derived from bronchial biopsies and epithelial brushings of 107 subjects with moderate to severe asthma were annotated by gene set variation analysis using 42 gene signatures relevant to asthma, inflammation, and immune function. Topological data analysis of clinical and histologic data was performed to derive clusters, and the nearest shrunken centroid algorithm was used for signature refinement. MEASUREMENTS AND MAIN RESULTS: Nine gene set variation analysis signatures expressed in bronchial biopsies and airway epithelial brushings distinguished two distinct asthma subtypes associated with high expression of T-helper cell type 2 cytokines and lack of corticosteroid response (group 1 and group 3). Group 1 had the highest submucosal eosinophils, as well as high fractional exhaled nitric oxide levels, exacerbation rates, and oral corticosteroid use, whereas group 3 patients showed the highest levels of sputum eosinophils and had a high body mass index. In contrast, group 2 and group 4 patients had an 86% and 64% probability, respectively, of having noneosinophilic inflammation. Using machine learning tools, we describe an inference scheme using the currently available inflammatory biomarkers sputum eosinophilia and fractional exhaled nitric oxide levels, along with oral corticosteroid use, that could predict the subtypes of gene expression within bronchial biopsies and epithelial cells with good sensitivity and specificity. CONCLUSIONS: This analysis demonstrates the usefulness of a transcriptomics-driven approach to phenotyping that segments patients who may benefit the most from specific agents that target T-helper cell type 2-mediated inflammation and/or corticosteroid insensitivity.

Severe asthma exists despite suppressed tissue inflammation: findings of the U-BIOPRED study.

The U-BIOPRED study is a multicentre European study aimed at a better understanding of severe asthma. It included three steroid-treated adult asthma groups (severe nonsmokers (SAn group), severe current/ex-smokers (SAs/ex group) and those with mild-moderate disease (MMA group)) and healthy controls (HC group). The aim of this cross-sectional, bronchoscopy substudy was to compare bronchial immunopathology between these groups.In 158 participants, bronchial biopsies and bronchial epithelial brushings were collected for immunopathologic and transcriptomic analysis. Immunohistochemical analysis of glycol methacrylate resin-embedded biopsies showed there were more mast cells in submucosa of the HC group (33.6 mm-2) compared with both severe asthma groups (SAn: 17.4 mm-2, p<0.001; SAs/ex: 22.2 mm-2, p=0.01) and with the MMA group (21.2 mm-2, p=0.01). The number of CD4+ lymphocytes was decreased in the SAs/ex group (4.7 mm-2) compared with the SAn (11.6 mm-2, p=0.002), MMA (10.1 mm-2, p=0.008) and HC (10.6 mm-2, p<0.001) groups. No other differences were observed.Affymetrix microarray analysis identified seven probe sets in the bronchial brushing samples that had a positive relationship with submucosal eosinophils. These mapped to COX-2 (cyclo-oxygenase-2), ADAM-7 (disintegrin and metalloproteinase domain-containing protein 7), SLCO1A2 (solute carrier organic anion transporter family member 1A2), TMEFF2 (transmembrane protein with epidermal growth factor like and two follistatin like domains 2) and TRPM-1 (transient receptor potential cation channel subfamily M member 1); the remaining two are unnamed.We conclude that in nonsmoking and smoking patients on currently recommended therapy, severe asthma exists despite suppressed tissue inflammation within the proximal airway wall.

Morphometric analysis of inflammation in bronchial biopsies following exposure to inhaled diesel exhaust and allergen challenge in atopic subjects.

BACKGROUND: Allergen exposure and air pollution are two risk factors for asthma development and airway inflammation that have been examined extensively in isolation. The impact of combined allergen and diesel exhaust exposure has received considerably less attention. Diesel exhaust (DE) is a major contributor to ambient particulate matter (PM) air pollution, which can act as an adjuvant to immune responses and augment allergic inflammation. We aimed to clarify whether DE increases allergen-induced inflammation and cellular immune response in the airways of atopic human subjects. METHODS: Twelve atopic subjects were exposed to DE 300 µg.m(-3) or filtered air for 2 h in a blinded crossover study design with a four-week washout period between arms. One hour following either filtered air or DE exposure, subjects were exposed to allergen or saline (vehicle control) via segmental challenge. Forty-eight hours post-allergen or control exposure, bronchial biopsies were collected. The study design generated 4 different conditions: filtered air + saline (FAS), DE + saline (DES), filtered air + allergen (FAA) and DE + allergen (DEA). Biopsies sections were immunostained for tryptase, eosinophil cationic protein (ECP), neutrophil elastase (NE), CD138, CD4 and interleukin (IL)-4. The percent positivity of positive cells were quantified in the bronchial submucosa. RESULTS: The percent positivity for tryptase expression and ECP expression remained unchanged in the bronchial submucosa in all conditions. CD4 % positive staining in DEA (0.311 ± 0.060) was elevated relative to FAS (0.087 ± 0.018; p = 0.035). IL-4% positive staining in DEA (0.548 ± 0.143) was elevated relative to FAS (0.127 ± 0.062; p = 0.034). CD138 % positive staining in DEA (0.120 ± 0.031) was elevated relative to FAS (0.017 ± 0.006; p = 0.015), DES (0.044 ± 0.024; p = 0.040), and FAA (0.044 ± 0.008; p = 0.037). CD138% positive staining in FAA (0.044 ± 0.008) was elevated relative to FAS (0.017 ± 0.006; p = 0.049). NE percent positive staining in DEA (0.224 ± 0.047) was elevated relative to FAS (0.045 ± 0.014; p = 0.031). CONCLUSIONS: In vivo allergen and DE co-exposure results in elevated CD4, IL-4, CD138 and NE in the respiratory submucosa of atopic subjects, while eosinophils and mast cells are not changed. TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01792232.

Effects of controlled diesel exhaust exposure on apoptosis and proliferation markers in bronchial epithelium - an in vivo bronchoscopy study on asthmatics, rhinitics and healthy subjects.

BACKGROUND: Epidemiological evidence demonstrates that exposure to traffic-derived pollution worsens respiratory symptoms in asthmatics, but controlled human exposure studies have failed to provide a mechanism for this effect. Here we investigated whether diesel exhaust (DE) would induce apoptosis or proliferation in the bronchial epithelium in vivo and thus contribute to respiratory symptoms. METHODS: Moderate (n = 16) and mild (n = 16) asthmatics, atopic non-asthmatic controls (rhinitics) (n = 13) and healthy controls (n = 21) were exposed to filtered air or DE (100 µg/m(3)) for 2 h, on two separate occasions. Bronchial biopsies were taken 18 h post-exposure and immunohistochemically analysed for pro-apoptotic and anti-apoptotic proteins (Bad, Bak, p85 PARP, Fas, Bcl-2) and a marker of proliferation (Ki67). Positive staining was assessed within the epithelium using computerized image analysis. RESULTS: No evidence of epithelial apoptosis or proliferation was observed in healthy, allergic or asthmatic airways following DE challenge. CONCLUSION: In the present study, we investigated whether DE exposure would affect markers of proliferation and apoptosis in the bronchial epithelium of asthmatics, rhinitics and healthy controls, providing a mechanistic basis for the reported increased airway sensitivity in asthmatics to air pollutants. In this first in vivo exposure investigation, we found no evidence of diesel exhaust-induced effects on these processes in the subject groups investigated.

Increased expression of the 5-lipoxygenase pathway and its cellular localization in Barrett's adenocarcinoma.

AIMS: Up-regulation of the 5-lipoxygenase (5-LOX) leukotriene pathway is evident in numerous tumour types, and has been linked to the promotion of cancer cell growth. The aim of this study was to evaluate the immunohistochemical expression of 5-LOX pathway proteins in oesophageal adenocarcinoma and its premalignant lesion, Barrett's metaplasia. METHODS AND RESULTS: Tissue samples were collected at endoscopy from 16 patients with Barrett's metaplasia and from seven with oesophageal adenocarcinoma; five proximal squamous oesophagus samples were used as controls. Immunohistochemical analyses were performed on stromal and epithelial areas with optimized concentrations of primary antibodies for 5-LOX, 5-LOX-activating protein (FLAP), and the distal enzymes leukotriene (LT) A(4) hydrolase (LTA(4) H) and LTC(4) synthase (LTC(4) S). the diagnosis was histologically confirmed from adjacent sections by a gastrointestinal pathologist. Striking increases in the stromal immunoexpression of 5-LOX (P = 0.041), FLAP (P = 0.038), LTA(4) H (P = 0.0008) and LTC(4) S (P = 0.036) were seen in adenocarcinoma tissue. Stromal FLAP and LTA(4) H immunostaining correlated with elevated neutrophil counts (P < 0.001). LTC(4) S was also notably overexpressed within epithelial cells in both Barrett's metaplasia (P < 0.001) and adenocarcinoma (P < 0.01) tissue. CONCLUSIONS: Key biosynthetic enzymes of the LTB(4) and LTC(4) biosynthetic pathways are incrementally expressed across the spectrum of squamous, Barrett's metaplasia and oesophageal adenocarcinoma tissues, suggesting, for the first time, a role for both LT subfamilies in disease progression.

Tumor necrosis factor alpha (TNF-α) autoregulates its expression and induces adhesion molecule expression in asthma.

Subjects with mild asthma underwent repeated low-dose allergen exposure and bronchial biopsies were examined for the expression of TNF-α and adhesion molecules. Bronchial biopsies from moderately severe asthmatics were then tested in an explant culture system to assess the effect of Der p and CDP-870, a TNF-α blocking pegylated-antibody Fab, on expression of TNF-α and adhesion molecules. Low-dose allergen challenge significantly upregulated sub-mucosal mast cells, TNF-α(+) cells, and VCAM. When bronchial explants were exposed to Der p and CDP 870 for 24h, CDP 870 caused a significant reduction in TNF-α release both at baseline and following stimulation with Der p allergen. The bronchial biopsies showed significant upregulation of TNF-α positive cells and ICAM-1 following exposure to Der p (p=0.03) and this was reduced in the presence of CDP-870. So, allergen exposure up-regulates TNF-α expression in asthma and down-stream targets, including adhesion molecules that contribute to airway inflammation.

Proinflammatory doses of diesel exhaust in healthy subjects fail to elicit equivalent or augmented airway inflammation in subjects with asthma.

BACKGROUND: Exposure to traffic-derived air pollutants, particularly diesel emissions, has been associated with adverse health effects, predominantly in individuals with pre-existing respiratory disease. Here the hypothesis that this heightened sensitivity reflects an augmentation of the transient inflammatory response previously reported in healthy adults exposed to diesel exhaust is examined. METHODS: 32 subjects with asthma (mild to moderate severity) and 23 healthy controls were exposed in a double-blinded crossover control fashion to both filtered air and diesel exhaust (100 µg/m(3) PM(10)) for 2 h. Airway inflammation was assessed by bronchoscopy 18 h postexposure. In addition, lung function, fraction of exhaled nitric oxide and bronchial reactivity to metacholine were examined in the subjects with asthma. RESULTS: In healthy control subjects a significant increase in submucosal neutrophils (p=0.004) was observed following the diesel challenge. Significant increases in neutrophil numbers (p=0.01), and in the concentrations of interleukin 6 (p=0.03) and myeloperoxidase (p=0.04), were also seen in bronchial wash after diesel, relative to the control air challenge. No evidence of enhanced airway inflammation was observed in the subjects with asthma following the diesel exposure. CONCLUSIONS: Exposure to diesel exhaust at concentrations consistent with roadside levels elicited an acute and active neutrophilic inflammation in the airways of healthy subjects. This response was absent in subjects with asthma, as was evidence supporting a worsening of allergic airway inflammation.

Prosurvival activity for airway neutrophils in severe asthma.

BACKGROUND: Airway neutrophilia is a recognised feature of chronic severe asthma, but the mechanisms that underlie this phenomenon are unknown. Evidence for factors present in airway secretions that prolong neutrophil survival has been sought and it has been hypothesised that these might be augmented in neutrophilic asthma. METHODS: Non-smoking subjects with severe asthma (SA) or mild asthma (MA) and healthy control subjects (HC) underwent sputum induction. The SA group was subdivided into subjects with neutrophil counts above (SA-high) and those within the normal range (SA-low). Apoptotic neutrophils were enumerated in the cellular phase while the fluid phase was assessed for its ability to prolong the in vitro survival of blood-derived neutrophils using morphometric and flow cytometric analyses. RESULTS: There was a significant difference between all four subject groups with respect to the percentage of apoptotic sputum neutrophils (Kruskal-Wallis, p=0.042). Cuzick test showed a highly significant (p=0.008) trend towards decreasing numbers of apoptotic neutrophils across the four groups with increasing asthma severity and neutrophil count. The sputum antiapoptotic activity was also different between the groups (p=0.039), with a highly significant (p=0.005) decreasing trend across the four groups. The survival effect could not be inhibited by blocking selective chemotaxin receptors, neutralising neutrophil survival factors, inhibiting phosphatidylinositol-3-kinase (using LY294002) or with pertussis toxin pretreatment. Similarly, it could not be explained by lipopolysaccharide contamination or by the presence of inhaled corticosteroids in sputum. CONCLUSIONS: These data demonstrate the capacity of as yet unidentified factor(s) in the airways of subjects with asthma to delay human neutrophil apoptosis and extend their lifespan as a potential mechanism contributing to unresolving airways neutrophilia in severe asthma.

Diesel exhaust increases EGFR and phosphorylated C-terminal Tyr 1173 in the bronchial epithelium.

BACKGROUND: Epidemiological studies have demonstrated adverse health effects of environmental pollution. Diesel exhaust (DE) is a major contributor to particulate matter pollution. DE exposure has been shown to induce a pronounced inflammatory response in the airways, together with an enhanced epithelial expression of cytokines such as IL-8, Gro-alpha, IL-13 and activation of redox sensitive transcription factors (NFkappaB, AP-1), and MAP kinases (p38, JNK). The aim of the present investigation was to elucidate the involvement of the epidermal growth factor receptor (EGFR) signalling pathway in the epithelial response to DE in-vivo. RESULTS: Immunohistochemical staining was used to quantify the expression of the EGFR, phosphorylated Tyrosine residues, MEK and ERK in the bronchial epithelium of archived biopsies from 15 healthy subjects following exposure to DE (PM10, 300 mug/m3) and air. DE induced a significant increases in the expression of EGFR (p = 0.004) and phosphorylated C-terminal Tyr 1173 (p = 0.02). Other investigated EGFR tyrosine residues, Src related tyrosine (Tyr 416), MEK and ERK pathway were not changed significantly by DE. CONCLUSION: Exposure to DE (PM10, 300 mug/m3) caused enhanced EGFR expression and phosphorylation of the tyrosine residue (Tyr 1173) which is in accordance with the previously demonstrated activation of the JNK, AP-1, p38 MAPK and NFkB pathways and associated downstream signalling and cytokine production. No effects were seen on the MEK and ERK pathway suggesting that at the investigated time point (6 hours post exposure) there was no proliferative/differentiation signalling in the bronchial epithelium. The present findings suggest a key role for EGFR in the bronchial response to diesel exhaust.

Using Baclofen to Explore GABA-B Receptor Function in Alcohol Dependence: Insights From Pharmacokinetic and Pharmacodynamic Measures.

Background: The role of GABA-B neurotransmission in addiction has recently received increased attention, with clinical trials indicating that baclofen, a GABA-B receptor agonist, may reduce alcohol consumption, craving and promote abstinence. However, the optimal dose to treat alcohol dependence is unclear with patients requesting and tolerating much higher doses of baclofen, compared with other clinical uses. We assessed the pharmacokinetics and pharmacodynamics (PK/PD) of baclofen to provide insight into GABA-B sensitivity in this patient group, relative to controls. Methods: Male healthy volunteers (controls, n = 12) and abstinent alcohol dependent individuals (AD, n = 8) received single oral doses of baclofen or placebo in a 3-way crossover design. Controls received placebo/10 mg/60 mg baclofen in a randomized, double-blind design, AD received placebo/60 mg/90 mg baclofen in a single-blind design. PK/PD measures were recorded at baseline and multiple time-points up to 6 h post-dosing, including plasma baclofen, plasma growth hormone (GH), Subjective High Assessment Scale (SHAS) and biphasic alcohol effects scale (BAES). Repeated measures ANOVA analysis explored "change from baseline" dose, time, group, and interaction effects, t-tests compared peak effects. Results: Dose-dependent effects of baclofen on PK and PD measures were observed in both control and AD groups. Whilst there were no significant group differences in any baclofen PK parameters (t 1/2, t max , C max , AUC), marked differences in PD effects were clearly evident. In controls, 60 mg baclofen significantly increased total SHAS and BAES scores, and significantly increased plasma GH levels compared with placebo, with peak effects at 60-120 min, in line with its PK profile. In AD, 60 mg baclofen had limited effects on these parameters; SHAS scores, BAES scores and plasma GH levels were significantly blunted compared with controls (significant group*time interactions P = 0.0014, 0.0015 and P < 0.0001, respectively). Conclusions: Our study shows blunted sensitivity to baclofen in AD relative to controls, with no difference in PK suggesting a lower GABA-B receptor sensitivity. This may explain why higher baclofen doses are requested and tolerated in the treatment of alcohol dependence. Our data has implications for choice of dose in future clinical trials in AD and possibly other substances of dependence.

Blockade of alcohol's amnestic activity in humans by an alpha5 subtype benzodiazepine receptor inverse agonist.

Alcohol produces many subjective and objective effects in man including pleasure, sedation, anxiolysis, plus impaired eye movements and memory. In human volunteers we have used a newly available GABA-A/benzodiazepine receptor inverse agonist that is selective for the alpha5 subtype (a5IA) to evaluate the role of this subtype in mediating these effects of alcohol on the brain. After pre-treatment with a5IA, we found almost complete blockade of the marked impairment caused by alcohol (mean breath concentration 150mg/100ml) of word list learning and partial but non-significant reversal of subjective sedation without effects on other measures such as intoxication, liking, and slowing of eye movements. This action was not due to alterations in alcohol kinetics and so provides the first proof of concept that selectively decreasing GABA-A receptor function at a specific receptor subtype can offset some actions of alcohol in humans. It also supports growing evidence for a key role of the alpha5 subtype in memory. Inverse agonists at other GABA-A receptor subtypes may prove able to reverse other actions of alcohol, and so offer a new approach to understanding the actions of alcohol in the human brain and in the treatment of alcohol related disorders in humans.

Squamous Metaplasia Is Increased in the Bronchial Epithelium of Smokers with Chronic Obstructive Pulmonary Disease.

AIMS: To quantify the extent of squamous metaplasia in bronchial biopsies and relate it to the presence of chronic obstructive pulmonary disease (COPD), a smoking-related pathology. METHODS: Bronchial biopsies (n = 15 in each group) from smokers with COPD GOLD stage1 and GOLD stage2, smokers without COPD and healthy non-smokers were stained immunohistochemically with a panel of antibodies that facilitated the identification of pseudostratified epithelium and distinction of squamous metaplasia and squamous epithelium from tangentially cut epithelium. The percentage length of each of these epithelial phenotypes was measured as a percent of total epithelial length using computerised image analysis. Sections were also stained for carcinoembryonic antigen and p53, early markers of carcinogenesis, and Ki67, and the percentage epithelial expression measured. RESULTS: The extent of squamous metaplasia was significantly increased in both COPD1 and COPD2 compared to healthy smokers and healthy non-smokers. The amount of fully differentiated squamous epithelium was also increased in COPD1 and COPD2 compared to healthy non-smokers, as was the expression of carcinoembryonic antigen. These features correlated with one other. CONCLUSION: In subjects with COPD there is a loss of pseudostratified epithelium accompanied by an increase in squamous metaplasia with transition into a fully squamous epithelium and expression of early markers of carcinogenesis.

Increased sympathetic response to standing in panic disorder.

Although autonomic function has been investigated in panic disorder (PD), previous studies have not used non-invasive beat by beat blood pressure (BP) monitoring to assess the rapid dynamics of BP during autonomic reflex tests. The hypothesis of the current study was that patients with PD would show increased cardiovascular sympathetic reactivity compared with healthy or anxious controls, as assessed by the initial overshoot of diastolic BP during the immediate response to standing. Patients with PD (n=56), social phobia (n=28) and healthy volunteers (n=56) were tested using finger photoplethysmography during an orthostatic challenge. Panic disorder patients showed an increased BP overshoot compared with both control groups. Moreover, in a preliminary assessment of selective serotonin reuptake inhibitor treatment effects, the BP overshoot was significantly reduced towards normal values. These findings are consistent with recent evidence for increased sympathetic baroreflex function in PD and may be relevant to the pathophysiology of the disorder.

Correlation of subjective and objective sleep measurements at different stages of the treatment of depression.

Studies of the correlation of subjective and objective sleep measures in depressed patients have produced mixed results so far. Further, they were carried out in sleep laboratories and tended to obtain one-off assessments, thus not taking into account the effect of treatment. We investigated forty (40) patients over the course of 8-week treatment of depression with either paroxetine or nefazodone. We used home polysomnography at baseline, nights 3 and 10, and week 8 of treatment, with extensive assessments of subjective sleep, the morning after each sleep recording. The patients were able to judge accurately their total sleep time and sleep onset latency, both before and during treatment. However, they were inaccurate in estimating the number of times they woke up during the night. Sleep satisfaction correlated negatively with Stage 1 sleep at baseline. Sleep quality was represented by a combination of subjective parameters measuring the ease of initiation and maintenance of sleep, and it appeared to derive from slow wave sleep and sleep continuity as seen in polysomnography. The partial discrepancy between subjective and objective measures suggests that a cognitive element is combined with the biological element to produce the sleep problems reported by depressed patients.