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Purpose Fractional exhaled nitric oxide (FENO50) level and peripheral blood eosinophil count may serve as indicators of airway eosinophilia. The aim of this study was to estimate the diagnostic value of these markers for detecting airway eosinophilia in patients with stable chronic obstructive pulmonary disease (COPD) and those experiencing an acute exacerbation (AECOPD). Patients and methods FENO50 levels, sputum and blood eosinophil counts were assessed in 53 clinically stable ex-smoker COPD patients and 67 ex-smoker COPD patients experiencing a severe exacerbation. In AECOPD, clinical variables were measured at the time of hospital admission and discharge following treatment. Results In stable COPD, blood eosinophil count but not FENO50 level was found to be a good predictor of airway eosinophilia (area under the receiver operating characteristic curve [ROC AUC]: ≥0.82). The sensitivity and the specificity of the test ranged between 75% and 98%, the negative predictive value (NPV) was high (>90%). In AECOPD, FENO50 was predictive for airway eosinophilia (ROC AUC: >0.8) with high NPV (>88%), but with lower sensitivity and specificity (64-70%). In contrast, the predictive accuracy of blood eosinophil count for airway eosinophilia in AECOPD was modest (ROC AUC: 0.54-0.63). The combined use of the two markers provided only limited additional benefit. Correlation analyses supported ROC curve findings. Conclusion In stable COPD the peripheral blood eosinophil count, while in AECOPD the FENO50 level is a good surrogate marker of airway eosinophilia.

Imre Barta,1,* Csilla Paska,1,* Balazs Antus1,2 1Department of Pathophysiology, National Koranyi Institute of Pulmonology, Budapest, Hungary; 2Department of Pulmonology, National Koranyi Institute of Pulmonology, Budapest, Hungary*These authors contributed equally to this workCorrespondence: Balazs Antus, Department of Pathophysiology, National Koranyi Institute of Pulmonology, Koranyi Frigyes ut 1, Tel +36 1 391 3309, Fax +36 1 200 7060, Email antusb@hotmail.comPurpose: Cytokines are extracellular signaling proteins that have been widely implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Here, we investigated cytokine expression both at the mRNA and protein level in the sputum of healthy individuals, stable COPD patients, and those experiencing a severe acute exacerbation (AECOPD) requiring hospitalization.Patients and Methods: Sputum was collected in 19 healthy controls, 25 clinically stable COPD patients, and 31 patients with AECOPD. In AECOPD patients sample collection was performed both at the time of hospital admission and at discharge following treatment. Sputum supernatant was analyzed by an antibody microarray detecting 120 cytokines simultaneously, while the mRNA expression of 14 selected cytokines in sputum cells was investigated by real-time PCR (qPCR).Results: Proteomic analysis identified interleukin (IL)-6 and growth-regulated oncogene (GRO)α as the only sputum cytokines that were differentially expressed between stable COPD patients and healthy controls. At the onset of AECOPD, several cytokines exhibited altered sputum expression compared to stable COPD. Recovery from AECOPD induced significant changes in the sputum cytokine protein profile; however, the length of hospitalization was insufficient for most cytokines to return to stable levels. With regard to gene expression analysis by qPCR, we found that bone morphogenetic protein (BMP)-4 was up-regulated, while IL-1α, monokine-induced by interferon-γ (MIG), and BMP-6 were down-regulated at the mRNA level in patients with AECOPD compared to stable disease.Conclusion: The sputum cytokine signature of AECOPD differs from that of stable COPD. Protein level changes are asynchronous with changes in gene expression at the mRNA level in AECOPD. The observation that the levels of most cytokines do not stabilize with acute treatment of AECOPD suggests a prolonged effect of exacerbation on the status of COPD patients.Keywords: airway, biomarker, gene, inflammation, microarray, PCR

Purpose Fractional exhaled nitric oxide (FENO50) level and peripheral blood eosinophil count may serve as indicators of airway eosinophilia. The aim of this study was to estimate the diagnostic value of these markers for detecting airway eosinophilia in patients with stable chronic obstructive pulmonary disease (COPD) and those experiencing an acute exacerbation (AECOPD). Patients and Methods FENO50 levels, sputum and blood eosinophil counts were assessed in 53 clinically stable ex-smoker COPD patients and 67 ex-smoker COPD patients experiencing a severe exacerbation. In AECOPD, clinical variables were measured at the time of hospital admission and discharge following treatment. Results In stable COPD, blood eosinophil count but not FENO50 level was found to be a good predictor of airway eosinophilia (area under the receiver operating characteristic curve [ROC AUC]: ≥0.82). The sensitivity and the specificity of the test ranged between 75% and 98%, the negative predictive value (NPV) was high (>90%). In AECOPD, FENO50 was predictive for airway eosinophilia (ROC AUC: >0.8) with high NPV (>88%), but with lower sensitivity and specificity (64–70%). In contrast, the predictive accuracy of blood eosinophil count for airway eosinophilia in AECOPD was modest (ROC AUC: 0.54–0.63). The combined use of the two markers provided only limited additional benefit. Correlation analyses supported ROC curve findings. Conclusion In stable COPD the peripheral blood eosinophil count, while in AECOPD the FENO50 level is a good surrogate marker of airway eosinophilia.

Continuous positive airway pressure (CPAP) treatment results in nearly complete remission of symptoms of obstructive sleep apnoea (OSA); however, its effect on OSA comorbidities including cardiovascular diseases remains contradictory. Here we investigated the short- and long-term effect of CPAP treatment on matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in patients with severe OSA. Serum levels of 7 MMPs and 3 TIMPs were followed in OSA patients (n = 28) with an apnoea-hypopnoea index of ≥30 events/h at the time of diagnosis and at control visits (2 months, 6 months and 5 years) after initiation of fixed-pressure CPAP treatment. The first few months of CPAP therapy resulted in significant decrease of MMP-8 and MMP-9 levels (MMP-8: 146 (79–237) vs. 287 (170–560) pg/mL; MMP-9: 10.1 (7.1–14.1) vs. 12.7 (10.4–15.6) ng/mL, p

The anti-aging factor, klotho has been identified as a tumor suppressor in various human cancers, including lung cancer. In vitro studies provided evidence that klotho expression influences the characteristics of lung cancer cells, however, in vivo results are lacking. The aim of our study was to evaluate whether circulating klotho protein might serve as a potential biomarker of lung cancer. Blood samples were taken from 45 newly diagnosed lung cancer patients (31 NSCLC, 14 SCLC) and 43 control subjects. Plasma klotho concentration was measured using ELISA. No difference in plasma klotho values was detected between patients and control subjects (366.3 (257.9–486.8) vs. 383.5 (304.6–489.7) pg/ml respectively (median (IQR)); p > 0.05). Plasma klotho levels in patients with distant metastasis did not differ from less advanced stage disease (354.2 (306.9–433.3 vs. 328.5 (242.5–419.7) pg/ml, p > 0.05). In contrast, analyzed with one-way ANOVA, significant difference (p = 0.04) was found between the examined histological types of lung cancer: adenocarcinoma (353 (329.4–438.5) pg/ml), squamous cell carcinoma (308 (209.6–348.1) pg/ml) and small cell lung cancer (388.8 (289.9–495.4) pg/ml). However, Tukey’s post hoc test did not reveal significant difference between any pairs of histological groups. There was no difference between any histological subtype and health either. Our results suggest that circulating klotho protein cannot be considered as a biomarker for lung cancer. Further studies are warranted in order to examine the relationship between klotho expression in lung tissue and circulating levels of the protein, and to explore its mechanism of action in lung cancer.

Chronic obstructive pulmonary disease (COPD) is associated with the accelerated aging of the lung. The protein klotho has been implicated in longevity, and there is some evidence that it might be involved in the pathomechanism of chronic respiratory diseases. Therefore, we aimed to examine whether the clinical condition of COPD patients is reflected in plasma klotho concentration. As plasma concentration of the protein is modulated by physiological factors that are generally improved during pulmonary rehabilitation, we hypothesized that a complex rehabilitation program may alter plasma klotho concentration. Blood samples were taken from 31 stable COPD patients. Clinical parameters such as respiratory function, 6-minute walking distance (6MWD), impact of disease (CAT), dyspnea, grip strength, chest expansion and breath holding time, smoking history, and body mass index (BMI) were evaluated. 19 patients who participated in a 3-week inpatient rehabilitation program had blood sample collection on the first, third, and last days of the program and had the above functional measurements before and after rehabilitation. Plasma klotho concentration was assessed by enzyme-linked immunosorbent assay. Klotho levels showed no correlation with clinical parameters (FEV1%, 6MWD, grip strength, CAT, smoking history, p0.05). Coefficient of variation of klotho measurements was 4.5% between Day 1 and Day 3. Although the rehabilitation resulted in significant improvements in 6MWD, CAT, grip strength, and chest expansion, klotho levels did not change significantly (510.1 ± 149.9 vs. 504.2 ± 139.8 pg/ml, p0.05). Plasma klotho concentration can be reliably measured in stable COPD; however, its levels are not correlated with clinical parameters of patients. Despite functional improvement, klotho level remains unchanged during the rehabilitation program.

Background: Oxidative stress may play a pivotal role in the development of acute exacerbation of chronic obstructive pulmonary disease (AECOPD). In this study we assessed simultaneously the degree of DNA damage and the activities of two major antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) in AECOPD. Methods: SOD and CAT activities and the level of 8-hydroxy-2’-deoxyguanosine (8-OHdG), a product of oxidatively damaged DNA were measured in the sputum and serum of 13 stable COPD patients and 19 AECOPD patients, first, at the time of hospital admission, and again at discharge following treatment. Measurements were performed using colorimetric and enzyme immunoassays. Data are presented as median with IQR. Results: Sputum 8-OHdG levels were increased in AECOPD compared to stable disease (2648 [683-7736] vs. 510 [425-991] pg/mL, p 0.05). Conclusions: DNA is oxidatively damaged in AECOPD. The parallel increase in SOD and CAT activities may represent a protective regulatory response locally in the airways.

Aims: Previously, antibody microarrays analysis revealed significant differences in the levels of 12 cytokines between stable COPD and acute exacerbation (AECOPD). Here we compared the expression of these cytokines at the mRNA level between stable COPD, AECOPD and various stages during the course of recovery from exacerbation. Patients and Methods: Sputum samples were collected from ex-smoker COPD patients in stable state (n=26) and from those in acute exacerbation (n=31) at hospital admission and discharge; after 6 weeks of early pulmonary rehabilitation and after 3 month of home recovery. Levels of BMP4, BMP6, IL1α, IL1R, IL6, IL8, M-CSF, MIG, Gro-α, CCL23, Rantes and Osteoprotegerin (OPG) mRNA were determined by real-time PCR using PPIA and GNB2L1 as housekeeping genes. Relative gene expressions were calculated using the pairwise fixed reallocation randomization test. Results: Compared to stable COPD, in AECOPD BMP4 was significantly up-regulated (2.7-fold), while BMP6, IL1α and MIG were down-regulated (3.1-, 2.4- and 4.9-fold; respectively). Increased expression (p Conclusion: Changes at the protein level are paralleled by changes in gene expression in AECOPD, however on a different time and amplitude scale. The level of several cytokines is not stabilized even after 5 month of recovery, indicating a long lasting effect of exacerbation on the status of COPD patients.

Continuous positive airway pressure (CPAP) provides a well-documented symptomatic relief for most patients with obstructive sleep apnea (OSA); however, its effect on dyslipidaemia remains contradictory. The aim of this longitudinal pilot study was to investigate the effect of long-term CPAP treatment on the lipid profile of patients with severe OSA. Fasting serum levels of total cholesterol (TC), low- and high-density lipoprotein cholesterol (LDL-C and HDL-C) and triglyceride (TG) were longitudinally measured in 33 OSA patients with an apnea-hypopnea index (AHI) of ≥30 events/hr, at the time of diagnosis (baseline) and at control visits following fixed-pressure CPAP treatment. Compared to baseline values, even as short as a 2-month CPAP therapy resulted in a significant decrease of both TC and LDL-C levels (TC, 5.62 ± 0.22 vs. 5.18 ± 0.21 mmol/L; LDL-C, 3.52 ± 0.19 vs. 3.19 ± 0.2 mmol/L; p 0.05 for each). These lipid fractions exhibited similar improvements at 6 months and after 5 years of CPAP treatment (TC, 5.1 ± 0.17 mmol/L; LDL-C, 2.86 ± 0.16 mmol/L; p 0.01 for each). The reduction in lipid levels was greater in younger patients and/or in those who had higher body mass index (BMI) (p 0.05). There were no significant correlations between AHI and lipid levels (p 0.05); BMI showed a weak negative association with HDL-C fraction (BMI, r = -0.263, p 0.05). CPAP therapy had neither short- nor long-term effects on TG and HDL-C levels (p 0.05). CPAP therapy has a rapid and long-lasting beneficial effect on the lipid profile of patients with severe OSA.

Sputum often contains large amounts of contaminating bacterial DNA that, if not eliminated during RNA isolation, may interfere with gene expression studies. During RNA isolation only repeated DNase treatment can effectively remove contaminating bacterial DNA from samples, but this compromises RNA quality. In this study we tested alternative methods to facilitate the removal of DNA and improve the quality of RNA obtained. Sputum samples obtained from patients with chronic obstructive pulmonary disease were processed with dithiothreitol and subjected to various RNA isolation methods, yet with modified protocols. Modifications included prolonged DNase treatment or vortexing of sputum cells in the presence of beads prior to RNA isolation. Bacterial DNA contamination was tested by PCR using universal bacterial primers, while RNA quality was assessed by real-time PCR using GAPDH primers for amplicons of different length. We found that the RNeasy Plus Mini kit equipped with the gDNA eliminator spin column was able to completely eliminate bacterial DNA, if sputum cells were lysed in the presence of bashing beads. Notably, compared with the standard protocol, the modified procedure yielded better quality RNA as well, as indicated by improved threshold profiles of qPCR. Bead vortexing of cells was less effective when combined with other RNA isolation methods, and the repeated DNase treatment needed to completely remove contaminating DNA from the samples reduced the quality of RNA markedly. Bead vortexing in combination with certain RNA extraction methods greatly facilitates the isolation of sputum RNA that is free of contaminating bacterial DNA, and is suitable for downstream applications.

Introduction Exhaled carbon monoxide (eCO) has been widely implicated as a pulmonary biomarker in respiratory diseases. The aim of this study was to investigate whether the treatment of patients with severe acute exacerbation of chronic obstructive pulmonary disease (AECOPD) could be aided by monitoring the changes in eCO. Methods The levels of eCO along with routine clinical parameters were analyzed in 29 current smoker and 33 ex-smoker COPD patients, first at the time of hospital admission, and again at discharge following the standard treatment. Patients with AECOPD were also stratified according to sputum bacteria. Results At exacerbation, the levels of eCO were increased in current smokers compared to ex-smokers (6.0 [2.0-9.5] versus 1.0 [1.0-2.0] ppm, p 0.001). Similarly, eCO levels were higher in smokers after treatment (7.0 [2.0-12.5] versus 1.0 [1.0-2.0] ppm, p 0.001). Treatment of AECOPD did not affect eCO concentrations. The levels of eCO were not statistically different between bacterial and non-bacterial AECOPD either. Investigating a subgroup of current smoker patients (n = 15), there was a significant correlation between the levels of eCO and blood carboxyhemoglobin concentrations both at exacerbation and discharge. No associations were found between eCO and lung function or blood gas parameters. Conclusion Our results suggest that monitoring eCO during the treatment of AECOPD is of limited clinical value.

Background: Superoxide dismutase (SOD) and catalase (CAT) have been implicated as major antioxidant enzymes of the human lungs. In this study we investigated whether activities of these enzymes are altered in patients hospitalized with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). Methods: SOD and CAT activities were measured in the sputum and serum of 36 COPD patients, first, at the time of hospital admission, and again at the time of hospital discharge following treatment of AECOPD. Antioxidants were also assessed in 24 stable COPD patients and 23 healthy controls. Measurements were performed using colorimetric assays. Data are presented as mean±SEM. Results: Sputum SOD and CAT activities were significantly increased in patients with AECOPD compared to those with stable disease (SOD: 0.142 [0.053-0.81] vs. 0.038 [0.002-0.146] U/mL, p 0.05). Both SOD and CAT activity significantly correlated with sputum neutrophil (SOD: r=0.557, p Conclusion: Our data demonstrate that sputum antioxidant activity is elevated in AECOPD and suggest that SOD and CAT is an integral part of the human defense mechanism against the increased oxidant production associated with AECOPD.

Superoxide dismutases (SODs) and catalase (CAT) have been implicated as major antioxidant enzymes of the human lungs. In this study, we investigated whether activities of these enzymes are altered in the airways of patients hospitalized with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). SOD and CAT activities were measured in the sputum, exhaled breath condensate, and serum of 36 COPD patients experiencing a severe exacerbation. Measurements were performed using colorimetric assays in samples collected at the time of hospital admission and at the time of hospital discharge following treatment of AECOPD. For comparison, antioxidants were also assessed in 24 stable COPD patients and 23 healthy control subjects. SOD and CAT activities in sputum were significantly increased in patients with AECOPD compared to those with stable disease (SOD: 0.142 [0.053-0.81] vs. 0.038 [0.002-0.146] U/mL, p 0.01; CAT: 48.7 [18.7-72.6] vs. 10.2 [2.9-40.6] nmol/min/mL, p 0.05), while treatment of exacerbation led to a decrease in enzyme activities (SOD: 0.094 [0.046-0.45] U/mL, p 0.05; CAT: 28.0 [7.3-60.4] nmol/min/mL, p 0.005). No changes were observed in the serum (p 0.05). Both SOD and CAT activities significantly correlated with sputum neutrophil and lymphocyte cell counts in patients with AECOPD. Moreover, SOD and CAT values correlated with each other and also with sputum malondialdehyde, an established marker for oxidative stress. Our data demonstrate that sputum antioxidant activity is elevated during COPD exacerbation and suggest that activation of SODs and CAT is an integral part of the human defense mechanism against the increased oxidant production associated with AECOPD.

Chronic obstructive pulmonary disease (COPD) is a major and rapidly increasing health problem associated with a chronic inflammatory response, predominantly in small airways and lung parenchyma. Oxidative stress induced by reactive oxygen and nitrogen species (ROS and RNS) plays a central role in the pathophysiology of COPD. There is evidence that several molecules formed during oxidative processes may have the potential to serve as biomarkers of oxidative stress in the airways of patients with COPD. Among these molecules carbon monoxide, ethane and pentane can be measured in the exhaled air, while 8-isoprostane, malondialdehyde, 4- hydroxyhexenal, 4-hyroxynonenal, acrolein, hydrogen peroxide, nitrogen oxides and 3-nitrotyrosine can be detected in exhaled breath condensate and/or sputum supernatant. In this review the molecular background of these processes including the formation of ROS and RNS, the biosynthesis of essential ω-3 and ω-6 polyunsaturated fatty acids as building blocks of lipids in the cellular membranes and their enzymatic and non-enzymatic metabolism to eicosanoids and related compounds have been summarized. Moreover, the formation of oxidative stress markers studied most commonly in the context of COPD has been briefly discussed. The associations between biomarkers and clinical variables have also been highlighted in an attempt to illustrate the potential clinical applicability of these biomarker measurements.

Background: The oxidative stress endured by cells in COPD damages macromolecules such as lipids, proteins and nucleic acids. Aims: We wanted to see if the increased oxidative stress during COPD exacerbations (AECOPD) is detectable at DNA/RNA level, and if so, whether the extent of nucleic acid damage correlates with the pulmonary status of the patient. Methods: 19 patients hospitalized for AECOPD were enrolled, along with 13 stable COPD patients and 13 healthy subjects as controls. Sputum and blood samples were collected. AECOPD patients provided one set of samples before treatment with steroids and antibiotics commenced and another set at discharge from hospital. The degree of DNA/RNA oxidative damage was determined by measuring 8-hydroxy-29-deoxyguanosine (8-OH(d)G). Results: The status of AECOPD patients has significantly improved upon treatment as evidenced by lung function tests (FEV1: 46.6±4.9% vs. 38.0±5.2%, p Conclusions: The increased oxidative stress in AECOPD induces local but not systemic damage to nucleic acids. The improvement in lung function upon treatment is mirrored by a reduction in nucleic acid damage, thus making sputum 8-OH(d)G a potentially suitable biomarker for monitoring the disease.

Breath tests cover the fraction of nitric oxide in expired gas (FENO), volatile organic compounds (VOCs), variables in exhaled breath condensate (EBC) and other measurements. For EBC and for FENO, official recommendations for standardised procedures are more than 10 years old and there is none for exhaled VOCs and particles. The aim of this document is to provide technical standards and recommendations for sample collection and analytic approaches and to highlight future research priorities in the field. For EBC and FENO, new developments and advances in technology have been evaluated in the current document. This report is not intended to provide clinical guidance on disease diagnosis and management.Clinicians and researchers with expertise in exhaled biomarkers were invited to participate. Published studies regarding methodology of breath tests were selected, discussed and evaluated in a consensus-based manner by the Task Force members.Recommendations for standardisation of sampling, analysing and reporting of data and suggestions for research to cover gaps in the evidence have been created and summarised.Application of breath biomarker measurement in a standardised manner will provide comparable results, thereby facilitating the potential use of these biomarkers in clinical practice.

Background and objective Exercise-induced bronchoconstriction (EIB) is the temporary narrowing of the airways caused by physical exercise. Its exact pathophysiology is unclear; however, acute changes in airways pH may play a role. Exhaled breath condensate (EBC) pH was suggested as a surrogate indicator for airway acid–base status, but its value is also affected by volatile molecules and respiratory droplet dilution. The aim of the study was to assess changes in EBC pH during EIB. Methods Twenty-two asthmatics who reported breathlessness following exercise and 16 healthy individuals participated in the study. Lung function test was performed and exhaled breath samples were collected for pH, dilution factor and volatile compound pattern measurements (Cyranose 320) pre-exercise and at 0, 10, 20 and 30 min after physical exercise challenge. Fractional exhaled nitric oxide was measured before exercise. Results EIB developed in 13 asthmatic subjects. In these participants, but not in the EIB-negative asthmatics (P = 0.51), EBC pH reduced significantly during exercise (P = 0.01). In addition, changes in EBC pH were related to the degree of bronchospasm in the EIB-positive group (P = 0.01, r = 0.68). Exhaled volatile pattern became altered (P 0.05). Conclusions The development of EIB was related to acute changes of EBC pH, which suggest the role of airway pH decrease in the pathophysiology of EIB. Exercise-induced changes in exhaled biomarkers suggest methodological precautions to avoid physical exercise before performing exhaled breath tests.

Background: Eicosanoids are small lipid molecules with diverse biological functions in the airways. Objectives: The aim of this study was to investigate changes in leukotriene B4 (LTB4), 8-isoprostane, prostaglandin E2 (PGE2) and cysteinyl-leukotriene (cys-LT) levels in the sputum of patients with chronic obstructive pulmonary disease (COPD) at the onset of a severe exacerbation and during the course of recovery. Methods: Thirty-seven ex-smoker COPD patients suffering an episode of acute exacerbation were enrolled. Samples were taken (i) on hospital admission and (ii) after regular treatment. Twenty-five stable ex-smoker COPD patients served as controls. Eicosanoids were determined by enzyme immunoassay. Results: Sputum PGE2 [39.8 (13.3-103.3) vs. 5.05 (2.3-12.1) pg/ml, p < 0.001], 8-isoprostane [89.5 (36.9-184.7) vs. 29.7 (13.8-68.8) pg/ml, p < 0.01] and LTB4 [587.7 (252.9-774.8) vs. 276.1 (105.4-594.7) pg/ml, p < 0.05] levels were increased in patients with exacerbation compared to stable subjects. After treatment only PGE2 levels decreased significantly [at discharge: 19.6 (4.6-52.5) pg/ml, p < 0.01], the levels of other eicosanoids remained elevated (p = NS). Sputum cys-LT levels were similar in stable patients and in those with exacerbation and treatment did not influence cys-LTs either. There was a significant correlation between PGE2 and sputum neutrophil and lymphocyte cell counts in patients with exacerbation. Conclusions: Our results suggest that 8-isoprostane, LTB4 and PGE2 but not cys-LTs may be involved in exacerbation-associated inflammatory processes in the airways of patients with COPD. Validation of PGE2 for use as a biomarker of recovery from an exacerbation requires further studies.

Background and objective Oxidative stress plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). In this longitudinal study changes in the level of malondialdehyde (MDA), an end product of polyunsaturated fatty acid peroxidation, were investigated in the airways of patients with acute exacerbation of COPD (AECOPD). Methods Levels of MDA were measured in sputum and exhaled breath condensate (EBC) of 34 COPD patients at the time of hospital admission due to an acute exacerbation of the disease, and again following treatment at the time of hospital discharge. MDA was also assessed in 21 stable patients with COPD and 20 healthy controls. Measurements were performed using high-performance liquid chromatography. Results Sputum MDA levels were significantly increased in AECOPD (220.0 ± 17.5 nmol/L) compared with stable disease (144.6 ± 14.3 nmol/L, P

Fractional exhaled nitric oxide (FENO) may be a pulmonary biomarker in chronic obstructive pulmonary disease (COPD). In this prospective study, the relationship between FENO and airway inflammation was assessed in COPD exacerbations. FENO and lung function were measured, and sputum was collected from 49 ex-smoking COPD patients, first at the time of hospital admission and again at discharge following treatment. There was a significant positive correlation between the percentage of sputum eosinophils and FENO concentrations, both at exacerbation (r = 0.593, p 0.001) and discharge (r = 0.337, p = 0.044). The increase in forced expiratory volume in one second (FEV(1)) after treatment was greater in patients with sputum eosinophilia (ΔFEV(1) 0.35 ± 0.12 vs. 0.13 ± 0.04 L, p = 0.046), and FENO was a strong predictor of sputum eosinophilia (area under the receiver operating characteristic curve, 0.89). The optimum cut point was 19 parts per billion (sensitivity: 90 %; specificity: 74 %). Our data suggest that FENO is a good surrogate marker of eosinophilic inflammation in COPD patients with exacerbations.

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of mortality and morbidity worldwide. In addition to generating high healthcare costs, COPD imposes a significant burden in terms of disability and impaired quality of life. Unlike many leading causes of death and disability, COPD is projected to increase in many regions of the world as the frequency of smoking is rising and the population is aging. The pharmacological treatment of COPD includes bronchodilators to relax smooth muscle, such as β2-agonists (salbutamol, terbutaline, and fenoterol, short-acting β2-agonists as well as salmeterol, formoterol, and indacaterol, and long-acting β2-agonists) and anticholinergics, such as ipratropium, oxitropium (short-acting anticholinergic), and tiotropium (long-acting anticholinergic). Although airway inflammation in COPD poorly responds to steroids, several inhaled corticosteroids (fluticasone, budesonide, and beclomethasone) are in use in combination with long-acting β2-agonists. Other medications include theophylline (both a bronchodilator and a phosphodiesterase inhibitor) and the phosphodiesterase-4 antagonists, such as roflumilast. Finally, a number of novel long-acting anticholinergics and β2-agonists with once- or twice-daily profiles are in development and clinical testing.

Background Despite accumulating evidence about its adverse health effects, water-pipe tobacco smoking has become very popular among youth. The aim of this study was to compare smoke exposure and the kinetics of exhaled carbon monoxide (eCO) between water-pipe and cigarette smokers under different conditions. Methods Using a cross-over study design, changes in eCO and urinary cotinine levels were measured in a cohort of 32 healthy university students after sessions of water-pipe smoking indoors and outdoors. An indoor cigarette smoking session with equal amounts of tobacco was conducted for reference purposes. Both active and passive smokers participated in all sessions. Results In indoor sessions, we found that among active participants, eCO levels were approximately 7.5-fold higher in water-pipe users than cigarette smokers. eCO levels remained significantly elevated even 10 h after discontinuing water-pipe smoking. Notably, eCO levels in passive water-pipe smokers were in the same range as in active cigarette smokers. Compared with indoor sessions, eCO levels in active water-pipe users were reduced in outdoor environments. Nonetheless, levels were still higher in these subjects than those in active cigarette smokers measured in indoor sessions. Urinary cotinine levels were comparable in active water-pipe and cigarette smokers. Conclusions Our results suggest that water-pipe smoking is associated with significantly higher toxicant exposure than cigarette smoking even in outdoor environments. Furthermore, even passive, indoor water-pipe smoke exposure may have significant health hazards compared with those of active cigarette smoking.

Background: Although increased oxidative stress has been widely implicated in pathogenesis of acute exacerbation of chronic obstructive pulmonary disease (AECOPD), the role of antioxidants in the process is poorly understood. In this study changes in the activity of catalase (CAT), an ubiquitous antioxidant enzyme, were investigated in patients with AECOPD. Methods: CAT activity was measured in sputum and plasma of 14 healthy controls, 14 stable COPD patients and 20 COPD patients (males: 13, age: 71.8±1.8 years, pack-years: 45.8±7.3) at the time of hospital admission due to an exacerbation, and again at discharge following treatment. Measurement was performed by enzyme immunoassay. Data are presented as mean±SEM. Results: Sputum CAT activity was significantly increased in AECOPD compared with stable disease and healthy controls (ex: 62.3±9.9 vs. stable: 32.3±10.8 and control: 5.3±1.8 nmol/min/ml, p 0.05). No correlations were observed between CAT values and fractional exhaled nitric oxide or lung function parameters (p>0.05). By contrast, we found significant associations between CAT activity and sputum neutrophil cell counts (stable: r=0.799, p Conclusions: AECOPD is accompanied with increased antioxidant activity in the airways, which could play a regulatory role in the redox balance upon recovery from AECOPD.

Introduction: Oxidative stress plays an important role in the pathogenesis of COPD and in acute exacerbation of the disease (AECOPD). Pulmonary rehabilitation (PR) has maintenance effect on exercise physiologic parameters in COPD. Aims: The aim of our study was to investigate the effect of early PR on lung function, exercise physiologic variables and oxidative stress after AECOPD. Methods: 18 AECOPD patients (FEV 1 : 45±17, TLCO: 41±16 %pred) were enrolled, 10 patients completed a 6 week standardized PR and 8 patients served as controls (C). Body plethysmography, diffusion capacity and cardio-pulmonary exercise test were carried out and MDA levels in sputum and plasma were measured in a longitudional manner (at the onset of AECOPD (V1), before PR (V2), after PR (V3), at 3 months after PR(V4)). Results: Lung function parameters did not change significantly in PR and C group. The effect of PR was manifested in WR (V2:52±12 vs. V3:67±5* vs. V4:62±22), VO2/kg (V2:12,1±2,4 vs. V3: 14,1±1,1* vs. V4:13,7±4,5 mL/kg/min., *p Conclusion: Early pulmonary rehabilitation after AECOPD results in favourable exercise physiologic effects that are sustained for at least 3 months after completion of the program. Effective PR may stabilize oxidative stress at a lower level following treatment of AECOPD.

Background: By-products of lipid peroxidation including malondialdehyde (MDA) have been implicated in the pathophysiology of chronic obstructive pulmonary disease (COPD). The aim of this longitudinal study was to investigate changes in airway and systemic MDA levels during recovery from severe acute exacerbations in COPD patients. Methods: 18 AECOPD patients (age: 69.4±1.3 years, 45.5±7.1 pack-years, GOLD: I-IV) were followed. Clinical assessments and sample collections took place at hospital admission, at discharge after treatment, and finally at a follow-up visit when patients were clinically stable. MDA was determined by high-performance liquid chromatography. Data are presented as mean±SEM. Results: MDA levels in sputum were significantly decreased at discharge compared to those at admission (138.8±16.4 vs. 103.7±11.7 nmol/l, p 0.05). At stable disease, sputum (113.9±25.5 nmol/l) and plasma (109.5±13.9 nmol/l) MDA values were comparable (p>0.05), and were not different from those observed at hospital discharge either (p>0.05). There were significant positive correlations between sputum but not plasma MDA levels and arterial blood carbon-dioxide tension (r=0.684, p 1 % pred) (r=0.562, p 0.05). Conclusions: Recovery from AECOPD is reflected by changes in airway but not systemic MDA levels. Sputum MDA could be used as a marker for monitoring oxidative stress in AECOPD.

Although oxidative stress is thought to play a pivotal role in the pathogenesis of inflammatory airway diseases, its assessment in clinical practice remains elusive. In recent years, it has been conceptualized that oxidative stress markers in sputum should be employed to monitor oxidative processes in patients with asthma, chronic obstructive pulmonary disease (COPD), or cystic fibrosis (CF). In this review, the use of sputum-based oxidative markers was explored and potential clinical applications were considered. Among lipid peroxidation-derived products, 8-isoprostane and malondialdehyde have been the most frequently investigated, while nitrosothiols and nitrotyrosine may serve as markers of nitrosative stress. Several studies have showed higher levels of these products in patients with asthma, COPD, or CF compared to healthy subjects. Marker concentrations could be further increased during exacerbations and decreased along with recovery of these diseases. Measurement of oxidized guanine species and antioxidant enzymes in the sputum could be other approaches for assessing oxidative stress in pulmonary patients. Collectively, even though there are promising findings in this field, further clinical studies using more established detection techniques are needed to clearly show the benefit of these measurements in the follow-up of patients with inflammatory airway diseases.

In recent years induced sputum analysis has become a non-invasive method for the assessment of airway inflammation in obstructive airway diseases. Sputum induction is safe and well tolerated by the patients. The method has been standardized, and this has markedly improved the quality and reproducibility of sputum samples. Identification of sputum eosinophilia has the greatest clinical relevance as it predicts a favorable response to corticosteroids. Treatment strategy aiming normalisation of sputum eosinophil cell count may reduce the rate of exacerbations in asthma. Profiling inflammatory mediators in sputum supernatant provides new insights into the pathogenesis of asthma and chronic obstructive pulmonary disease. Cell type analysis in spontaneous sputum may also provide much information about inflammatory processes in the airways. Based on the results of clinical studies sputum analysis should be more often used in clinical settings in the future. Orv. Hetil., 2012, 153, 1847–1854.

Exhaled breath condensate (EBC) pH has been proposed as a useful, non-invasive marker of airway inflammation in pulmonary diseases. In this study we tested whether cystic fibrosis (CF) is associated with acidification of EBC, when pH is assessed by the CO2 gas standardization method. EBC was collected using two different devices (EcoScreen and R-Tube) in 46 stable CF patients during routine clinical visits and in 28 healthy controls. Mean EBC pH in CF patients and in healthy controls was similar (EcoScreen: CF patients: 6.38 ± 0.03 versus controls: 6.39 ± 0.03, p = 0.699; R-tube: CF patients: 5.94 ± 0.04 versus controls: 6.02 ± 0.03, p = 0.159). Inflammatory cell counts in spontaneously expectorated sputum obtained in a subset of patients (n = 20) showed no correlation with pH values. EBC samples collected with the R-tube were more acidic than those collected with the EcoScreen device (p

Airway inflammation plays a central role in the pathophysiology of chronic obstructive pulmonary disease. Exposure to cigarette smoke induces the recruitment of inflammatory cells in the airways, which in turn produces various cytokines, chemokines, proteases and pro-inflammatory mediators leading ultimately to increased oxidative stress, a protease/anti-protease imbalance and progressive lung tissue injury. Biomarkers may be useful in monitoring airway inflammation and oxidative stress, defining different phenotypes of the disease and evaluating the response of therapies. Exhaled breath condensate collection is a simple and completely non-invasive method of sampling the lower respiratory tract in humans. Exhaled breath condensate may be a rich source of pulmonary biomarkers including hydrogen peroxide, cytokines, metabolites of the arachidonic acid, nitric oxides and the pH. However, the concentration of these biomarkers is often very low, which may cause several problems in their detection. The clinical applicability of exhaled breath condensate biomarkers cannot be assessed until methods of sample collection and analysis have been standardized. Orv. Hetil., 2012, 153, 843–851.

Hydrogen peroxide (H2O2) is detectable in exhaled breath condensate (EBC) and has been proposed to be a surrogate marker of oxidative stress in the airways. In this study we tested whether the breathing pattern during EBC collection influences the concentration of exhaled H2O2.EBC was collected during (1) tidal breathing and (2) breathing with increased tidal volume for 10 min from 16 healthy volunteers. On-line H2O2 measurement was performed by the EcoCheck™ biosensor system. Repeated measurements were also conducted to assess intrasubject reproducibility.Minute ventilation, tidal volume, expiratory flow rate were all increased significantly when subjects were asked to perform breathing with increased tidal volume. In parallel, EBC volume increased (1413±59 vs. 1959±71 μL, p0.001), whereas exhaled H2O2 levels decreased significantly (1400±170 vs. 840±130 nmol/L, p0.001). H2O2 levels did not correlate with any individual breathing parameters (p0.05). Assessment of intersubject variability of H2O2 measurements during the two types of breathing revealed a coefficient of variation of 49 and 54%, respectively. The EBC H2O2 measurement was highly reproducible (888±176 vs. 874±156 nmol/L) as tested during normal breathing.These data demonstrate that the concentration of H2O2 in EBC depends on the ventilatory pattern during sample collection that has to be taken into consideration in all EBC H2O2 assays.

Exercise-caused metabolic changes can be followed by monitoring exhaled volatiles; however it has not been previously reported if a spectrum of exhaled gases is modified after physical challenge. We have hypothesized that changes in volatile molecules assessed by an electronic nose may be the reason for the alkalization of the exhaled breath condensate (EBC) fluid following physical exercise.Ten healthy young subjects performed a 6-minute running test. Exhaled breath samples pre-exercise and post-exercise (0 min, 15 min, 30 min and 60 min) were collected for volatile pattern ("smellprint") determination and pH measurements (at 5.33 kPa CO2), respectively. Exhaled breath smellprints were analyzed using principal component analysis and were related to EBC pH.Smellprints (p=0.04) and EBC pH (p=0.01) were altered during exercise challenge. Compared to pre-exercise values, smellprints and pH differed at 15 min, 30 min and 60 min following exercise (p0.05), while no difference was found at 0 min post-exercise. In addition, a significant correlation was found between volatile pattern of exhaled breath and EBC pH (p=0.01, r=-0.34).Physical exercise changes the pattern of exhaled volatiles together with an increase in pH of breath. Changes in volatiles may be responsible for increase in EBC pH.

Exhaled breath condensate pH has been proposed as a noninvasive marker of airway inflammation. However, due to standardization difficulties in pH measurement techniques, different pH readings were obtained in previous studies.In this longitudinal study we assessed condensate pH in patients with an exacerbation of asthma or chronic obstructive airway disease using the very precise carbon dioxide standardization method that negates the effect of this gas on condensate acidity.Condensate pH, fractional exhaled nitric oxide, lung function, and blood gases were measured in 20 nonsmoking patients with asthma and 21 smoking and 17 ex-smoking patients with chronic obstructive airway disease first at hospital admission due to an acute exacerbation of the disease and again at discharge after treatment. Condensate pH was also assessed in 18 smoking and 18 nonsmoking healthy control subjects.In patients with asthma, condensate pH was significantly decreased at the time of exacerbation compared with nonsmoking control subjects and increased with treatment. In patients with chronic obstructive airway disease, condensate pH remained unchanged during exacerbation, both in smokers and ex-smokers. Nevertheless, condensates collected from smokers were more acidic than those of ex-smokers. A similar difference was observed between smoker and nonsmoker healthy control subjects. No correlations were found between condensate pH and fractional exhaled nitric oxide or lung function variables measured either at admission or discharge.Our data suggest that exacerbation of asthma, but not chronic obstructive airway disease, is associated with acidification of breath condensate.

A kilégzett levegőben mérhető nitrogén-monoxid a legszélesebb körben vizsgált légúti biomarker. A stabil állapotú krónikus obstruktív tüdőbetegségben a kilégzett nitrogén-monoxid-szint hasonló vagy csak kismértékben emelkedett az egészségesekhez képest. Mivel a nitrogén-monoxid-szint szoros összefüggést mutat a légúti eosinophilia mértékével, és mivel az eosinophil típusú légúti gyulladás szteroidokra érzékenyebb, az emelkedett nitrogén-monoxid-szinttel rendelkező betegek jobb válaszkészséget mutatnak az inhalációs vagy szisztémás kortikoszteroidkezelésre. A krónikus obstruktív tüdőbetegség akut exacerbatiója során a kilégzett nitrogén-monoxid szintje megemelkedik, majd ennek kezelése után csökken. Mivel a nitrogén-monoxid-szint és a kezelés során elért légzésfunkciós javulás szoros korrelációt mutat egymással, a nitrogén-monoxid-méréssel a terápiás válasz megjósolható. Összefoglalva: a nitrogén-monoxid-méréssel a krónikus obstruktív tüdőbetegségben szenvedő betegek olyan alcsoportját lehet elkülöníteni, amelynek szteroidérzékenysége nagyobb. Orv. Hetil., 2010, 151, 2083–2088.

Background and objective: Fractional exhaled nitric oxide (FENO) has been implicated as a pulmonary biomarker in various respiratory diseases, including COPD. In this longitudinal study, the benefit of measuring FENO in a routine clinical setting was assessed in COPD patients hospitalized with an exacerbation of the disease. Methods: FENO, lung function and blood gases were measured in 58 COPD patients at hospital admission due to an exacerbation, and at discharge following treatment with corticosteroids and bronchodilators. Results: FENO levels were significantly decreased at discharge, compared with those at admission (geometric mean 25.3 ppb (95% CI: 21.2–30.1) vs 19.7 ppb (95% CI: 17.2–22.6); P = 0.002). There was a significant positive correlation between FENO concentrations at admission and the increase in FEV1 after treatment (r = 0.441, P

Exhaled breath condensate (EBC) analysis is a promising method for investigating airway pathology. In this pilot study we tested the cytokine pattern of EBC of lung transplant patients with and without clinical evidence of bronchiolitis obliterans syndrome (BOS). Breath condensates collected from eight BOS patients and eight stable BOS-free lung transplant recipients in three consecutive visits were pooled in order to increase protein concentration and were then used for antibody microarray analysis detecting 120 cytokines simultaneously. Nine cytokines exhibited more than twofold increase and four exhibited more than twofold decrease in BOS patients as compared to stable subjects. We conclude that inflammatory cytokines are present in EBC of lung transplant recipients, however the potential benefit of detecting the EBC proteome warrants further studies.

Exhaled breath condensate analysis is an attractive but still not fully standardised method for investigating airway pathology. Adherence of biomarkers to various condensing surfaces and changes in condensing temperature has been considered to be responsible for the variability of the results. Our aims were to compare the efficacy of different types of condensers and to test the influence of condensing temperature on condensate composition.Breath condensates from 12 healthy persons were collected in two settings: (1) by using three condensers of different type (EcoScreen, R-Tube, Anacon) and (2) by using R-Tube condenser either cooled to -20 or -70 degrees C. Condensate pH at standardised CO(2) level was determined; protein content was measured by the Bradford method and leukotrienes by EIA.Breath condensates collected using EcoScreen were more alkaline (6.45+/-0.20 vs. 6.19+/-0.23, p0.05 and 6.10+/-0.26, p0.001) and contained more protein (3.89+/-2.03 vs. 2.65+/-1.98, n.s. and 1.88+/-1.99 microg/ml, p0.004) as compared to the other devices. Only parameters obtained with R-Tube and Anacon correlated. Condensing temperature affected condensate pH (5.99+/-0.20 at -20 degrees C and 5.82+/-0.07 at -70 degrees C, p0.05) but not protein content. Leukotriene B(4) was not found in any sample and cysteinyl-leukotriene was not found in condensates collected with R-Tube or Anacon.Condenser type influences sample pH, total protein content and cysteinyl-leukotriene concentration. Condensing temperature influences condensate pH but not total protein content. These results suggest that adherence of the biomarkers to condenser surface and condensing temperature may play a role but does not fully explain the variability of EBC biomarker levels.

Lung transplantation has become an accepted therapeutic modality for end-stage diseases of the lungs and the pulmonary circulation. In the past two decades more than 20,000 lung transplantations were performed all over the world. Due to improvements in immunosuppressive regimens the mortality rate of severe acute rejections has decreased up to 2% in the first post-transplant year. By contrast, infections became the most common cause of morbidity and mortality after lung transplantation. It was reported that 21.2 and 40% of annual deaths are due to infections in the first 30 days and one year, respectively. In the first month 35-70% of transplant recipients develop bacterial pneumonia caused often by Gram-negative organisms especially by Pseudomonas species. All patients should receive prophylactic antibiotics after the operation, which are to be modified according to the resistance patterns of pathogens isolated from the donor lungs. In the early post-operative period, the frequency of invasive fungal (Aspergillus and Candida) and cytomegalovirus (CMV) infections appears to be less then 10% due to prophylactic amphotericin inhalation and systemic valganciclovir administration for 100 days. After withdrawing these drugs, these infections became more common. In the late post-transplant period, the development of bronchiolitis obliterans syndrome (BOS) may predispose to infections. BOS may be manifested in approximately 50% of patients 5 years post-transplant. Routinely or urgently performed screening tests (laboratory and radiological investigations, lung function tests, sputum culture, bronchoscopy) and specific treatments are of central importance in the management of infections. In this review we discuss the clinical manifestation, the diagnosis and the treatment possibilities of the most common pulmonary infections in lung transplant recipients.

Background: Measurement of pH in exhaled breath condensate (EBC) may represent a novel method for investigating airway pathology. Objectives: The aim of this longitudinal study was to assess the variability of EBC pH in stable lung transplant recipients (LTR). Methods: During routine clinical visits 74 EBC pH measurements were performed in 17 LTR. EBC pH was also measured in 19 healthy volunteers on four separate occasions. EBC pH was determined at standard CO2 partial pressure by a blood gas analyzer. Results: Mean EBC pH in clinically stable LTR and in controls was similar (6.38 ± 0.09 vs. 6.44 ± 0.16; p =nonsignificant). Coefficient of variation for pH in LTR and controls was 2.1 and 2.3%, respectively. The limits of agreement for between-visit variability determined by the Bland-Altman test in LTR and healthy volunteers were also comparable (–0.29 and 0.46 vs. –0.53 and 0.44). Conclusions: Our data suggest that the variability of EBC pH in stable LTR is relatively small, and it is similar to that in healthy nontransplant subjects.

Background Blockade of the renin–angiotensin and the endothelin (ET) systems ameliorates fibrous airway obliteration in rat tracheal allografts. The aim of this study was to test which of these pharmacologic interventions attenuates the process more effectively, and whether combination therapy confers additional benefit. Methods Rat tracheas were heterotopically transplanted from Brown-Norway donors into Lewis recipients. Recipients received the dual endothelin-1 (ET-1) receptor blocker, bosentan (100 mg/kg), the angiotensin-converting enzyme (ACE) inhibitor, ramipril (5 mg/kg), bosentan plus ramipril (doses as just noted) or vehicle. Grafts were harvested at Days 7 and 21 for morphometric studies and molecular analysis by real-time polymerase chain reaction (PCR). Results At Day 21, bosentan and ramipril treatment reduced the percentage of luminal occlusion compared with vehicle to a similar extent. In animals that received the combined treatment, luminal obliteration was further reduced compared with the respective monotherapies. Furthermore, rats treated with bosentan plus ramipril showed a lower percent of epithelial necrosis. The beneficial effects of bosentan and ramipril, alone or in combination, were associated with reduced mRNA levels of transforming growth factor (TGF)-β1 and platelet-derived growth factor (PDGF)-A in the tracheal allografts. Conclusions Our data suggest that ET receptor blockade prevents fibrous airway obliteration to a similar extent as ACE inhibition in this model. Interruption of both pathways provides superior graft protection as compared with single-system blockade.

The aim of this longitudinal study was to test whether pulmonary infections influence fractional exhaled nitric oxide levels (FENO) in otherwise clinically-stable lung transplant recipients. Levels of FENO were measured at least on 11 occasions in nine lung transplant recipients who attended for routine or urgent clinical review over 27.0 +/- 3.2 months period. Diagnosis of infection was based on clinical symptoms, functional measurements and radiological findings. Concentrations of FENO were also determined in 12 healthy volunteers. During follow-up, six patients had one, two had three, and one had four episodes of pulmonary infections. Overall, six upper and 10 lower respiratory tract infections were noted. Recipients with active infections developed increased FENO levels as compared with their own baseline levels measured in the clinically well period (10.8 +/- 1.3 vs. 7.6 +/- 1.1 ppb, p < 0.05). After antibiotic treatment, elevated FENO concentrations returned to baseline in association with full clinical recovery. Baseline FENO levels in lung transplant recipients and in healthy volunteers (6.0 +/- 0.5 ppb) were similar. The sensitivity and specificity of FENO measurement in detecting pulmonary infections were 57 and 96%, respectively. Our data suggest that pulmonary infections are associated with increased FENO levels in patients with lung allografts. Nevertheless, the measurement of FENO by itself as a screening tool for infections seems to be limited by its low sensitivity.

BACKGROUND: We recently demonstrated that oestrogens ameliorate the progression of chronic allograft nephropathy (CAN). In our present study, we investigated the role of progesterone and selective oestrogen receptor modulators (SERMs) in this process. METHODS: Female Fisher (F344) kidneys were orthotopically transplanted into intact or ovariectomized female Lewis recipients. Ovariectomized recipients were divided into four groups and were treated with either progesterone alone or in combination with oestradiol, oestradiol alone or vehicle. Intact recipients were divided into three groups and were treated with SERMs such as tamoxifen and one of its new derivatives, droloxifene or vehicle. Animals were harvested 24 weeks after transplantation for histological and immunohistological studies as well as for molecular analysis. RESULTS: Administration of progesterone resulted in increased urinary protein excretion as well as profound glomerulosclerosis and mononuclear cell infiltration. The combined treatment had similar detrimental effects on the development of CAN. In contrast, oestradiol treatment alone improved graft function, reduced glomerulosclerosis and diminished cellular infiltration. SERMs again impaired allograft function and promoted the development of CAN. Renal allograft damage paralleled intragraft mRNA expression of transforming growth factor-beta1 in all groups. CONCLUSIONS: Our results suggest that addition of progesterone diminishes the beneficial effects of oestrogens on the development of CAN in rats. Similarly to progesterone, SERMs worsened long-term renal allograft outcome.

Background Chronic allograft nephropathy (CAN) is responsible for most cases of late kidney allograft loss. However, no effective treatment is available so far. Everolimus (RAD) (40-O [2-hydroxyethyl] rapamycin) is a new immunosuppressive agent with antiproliferative and apoptosis-enhancing effects. We asked whether everolimus can ameliorate CAN even at advanced stages, whether everolimus treatment affects the level of growth factor mRNA, and whether everolimus treatment affects the number of apoptotic cells in the graft. Methods We transplanted kidneys from Fisher rats into Lewis rats and treated recipients with everolimus over different time periods. Grafts were analyzed 20 or 28 weeks after transplantation. Results Everolimus delayed the progression of CAN when started at an early stage. Surprisingly, everolimus even ameliorated CAN when initiated at an advanced stage. Interestingly, apoptosis was more prevalent in treated animals, particularly in those with delayed treatment as compared with controls. Conclusions Everolimus ameliorates CAN as a result of antiproliferative or apoptosis-enhancing effects.