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Introduction: Cancer cachexia (CC) is often experienced by lung cancer patients (LCP). It is associated with shrinkage of skeletal muscle mass, immobility, reduced quality of life, and shorter patient survival. Brown (BAT) and white adipose tissue (WAT), plus abdominal muscle mass play a role in the development of CC. Aims: We aimed to find out whether and how adipose and muscle tissue interacts with LC in CC. Methods: Retrospectively, 200 LCP and 30 healthy controls (HC) were analysed for BAT activation via fluorine-18 deoxyglucose positron emission tomography/computed tomography. Mean standardized uptake values (SUVmean) of predefined regions of interest (ROI) in the retroclavicular fat were measured, normalized to liver uptake and reported as SUV ratio (SUVR). ROIs from transversal CT images were used to quantify visceral adipose (VAT) and abdominal muscle mass. Prospectively, 50 LCP were likewise analysed for BAT activation and additionally underwent bioelectrical impedance analysis to assess body composition and analysis of circulating adipokines via enzyme-linked immunosorbent assay. Results: LCP showed higher SUVR in BAT than the HC. Higher SUVR in BAT was associated with lower BMI (r=-0.38) and reduced VAT (r=-0.44). LCP with higher SUVR in BAT were associated with significant weight loss (odds ratio 2.3, P Conclusions: LCP express higher BAT activity than HC, which is correlated with CC. Adiponectin is associated with BAT activation and leaner body composition.

Objective Neuregulin 4 (NRG4) has recently been introduced as a novel brown adipose tissue (BAT)-secreted adipokine with beneficial metabolic effects in mice. However, regulation of Nrg4 in end-stage kidney disease (ESKD) and type 2 diabetes mellitus (T2DM) has not been elucidated, so far. Design/methods Serum NRG4 levels were quantified by ELISA in 60 subjects with ESKD on chronic hemodialysis as compared to 60 subjects with an estimated glomerular filtration rate >50 mL/min/1.73 m2 in a cross-sectional cohort. Within both groups, about half of the patients had a T2DM. Furthermore, mRNA expression of Nrg4 was determined in two mouse models of diabetic kidney disease (DKD) as compared to two different groups of non-diabetic control mice. Moreover, mRNA expression of Nrg4 was investigated in cultured, differentiated mouse brown and white adipocytes, as well as hepatocytes, after treatment with the uremic toxin indoxyl sulfate. Results Median serum NRG4 was significantly lower in patients with ESKD compared to controls and the adipokine was independently associated with a beneficial renal, glucose and lipid profile. In mice with DKD, Nrg4 mRNA expression was decreased in all adipose tissue depots compared to control mice. The uremic toxin indoxyl sulfate did not significantly alter Nrg4 mRNA expression in adipocytes and hepatocytes, in vitro. Conclusions Circulating NRG4 is independently associated with a preserved renal function and mRNA expression of -Nrg4 is reduced in adipose tissue depots of mice with DKD. The BAT-secreted adipokine is further associated with a beneficial glucose and lipid profile supporting NRG4 as potential treatment target in metabolic and renal disease states.

Introduction: Brown adipose tissue (BAT) increases energy expenditure and might be involved in the development of cancer cachexia. Non-small cell lung cancer (NSCLC) cells might induce BAT activation via an adipokine signalling promoting cachexia. Objectives: We aimed to find out whether NSCLC cells influence the expression of adipokines in preadipocytes and BAT involved in the development of BAT activation. Methods: Preadipocytes and differentiated BAT were cultured with conditioned media of six NSCLC cell lines (H1299, A549, H322, PC-9, H1650, H460), respectively. Protein levels of the key-adipokines adiponectin, fibroblast growth factor 21 (FGF21), and irisin involved in the activation of BAT were investigated in both supernatants and cell lysates of preadipocytes and BAT by enzyme-linked immunosorbent assay. Results: Undifferentiated preadipocytes showed higher FGF21 protein expression (p.e.) when cultured with conditioned medium from PC-9 cells. Higher irisin p.e. was observed in H1650 and H322 conditioned media compared with control medium. In differentiated BAT, FGF21 was increased when incubated with conditioned media from all NSCLC cell lines. BAT showed higher irisin p.e. in the media of H1650 and H322 cells but lower p.e. when cultured in H460 medium. Adiponectin p.e. in BAT were stimulated by the media of H1299, A549, H322, H460. Conclusions: PC-9 and H1650 media might potentially induce preadipocyte activation through FGF21 and irisin signalling, respectively. In differentiated BAT, NSCLC cell-derived media increase protein levels of all adipokines investigated. Therefore, BAT activation might play a role in the development of cancer cachexia.

Estimating the degree of pulmonary hyperinflation in chronic obstructive pulmonary disease (COPD) is not always straight forward. Standard pulmonary function tests provide only a crude estimate of this important aspect of COPD. In addition, good patient cooperation cannot always be achieved and therefore adds to the uncertainties with regard to the extent of hyperinflation of the lung. The aim of this investigation was to characterize exhaled breath condensate nitrite in volunteers, healthy smokers, and stable COPD (GOLD-stages 0-4) and to compare this parameter with inflammatory markers in exhaled breath condensate and with lung function in order to test the hypothesis that elevated exhaled breath condensate nitrite reflects hyperinflation in COPD. We found a logarithmic correlation of exhaled breath condensate nitrite to residual volume (r=0.75, p

Early diagnosis of lung carcinoma is greatly desired. A potential source of early information regarding the process of cancerisation in the airways is exhaled breath condensate (EBC). The direct approach to detecting cancerisation is examining DNA from the area of chronic damage, i.e. airways and lung parenchyma. We therefore investigated DNA in EBC of patients with NSCLC and healthy volunteers. Human DNA was amplified by PCR in exhaled breath condensate and used to detect p53 mutations. A PCR of the β-actin gene fragment was used to detect human DNA in each of the EBC samples. In 65.7% of the samples, the β-actin gene was found. Extracted DNA as well as native EBC were equally suited as starting material for amplification. Mutations of the p53 gene were investigated in all EBC samples of NSCLC patients. p53 exons 5–8 were amplified using nested PCR and subsequently sequenced. Mutations were found in four of the patients ( n =11; 36.4%) while no mutation was found in volunteers ( n =10). Mutations detected in EBC were also compared with those of corresponding tumor tissue. Different point mutations in EBC and tumor tissue were revealed in all cases. Our findings demonstrate that exhaled breath condensate may be used for analysis of somatic gene mutations in an area of direct tobacco-related DNA damage.

Lung injury in ventilated lungs may occur due to local or systemic disease and is usually caused by or accompanied by inflammatory processes. Recently, acidification of exhaled breath condensate pH (EBC-pH) has been suggested as marker of inflammation in airway disease. We investigated pH, ammonia, Lactate, pCO2, HCO3-, IL-6 and IL-8 in EBC of 35 ventilated patients (AECC-classification: ARDS: 15, ALI: 12, no lung injury: 8). EBC-pH was decreased in ventilated patients compared to volunteers (5.85 +/- 0.32 vs. 7.46 +/- 0.48; P0.0001). NH4+, lactate, HCO3-, pCO2, IL-6 and IL-8 were analyzed in EBC and correlated with EBC-pH. We observed correlations of EBC-pH with markers of local (EBC IL-6: r = -0.71, P0.0001, EBC IL-8: r = -0.68, P0.0001) but not of systemic inflammation (serum IL-6, serum IL-8) and with indices of severity of lung injury (Murray's Lung Injury Severity Score; r = -0.73, P0.0001, paO2/FiO2; r = 0.54, P0.001). Among factors potentially contributing to pH of EBC, EBC-lactate and EBC-NH4+ were found to correlate with EBC-pH. Inflammation-induced disturbances of regulatory mechanisms, such as glutaminase systems may result in EBC acidification. EBC-pH is suggested to represent a marker of acute lung injury caused by or accompanied by pulmonary inflammation.

Clinical studies suggest prognostic relevance of p16INK4A in nonsmall cell lung cancer (NSCLC) while conflicting results for p53 have been published. However, the importance of the apoptosis regulating gene BAX, a downstream regulator of p53, on the prognosis of NSCLC is unknown. The present study investigated the prognostic relevance of BAX with respect to the status of p53 and P16INK4A in 61 patients with advanced NSCLC. Protein expression of BAX, p53 and p16INK4A was investigated retrospectively by immunohistochemistry. Tumour deoxyribonucleic acid (DNA) was screened for p53 mutations by single strand-conformation polymorphism polymerase chain reaction (PCR) and BAX frameshift mutations by fragment length analysis. Patients with positive BAX protein expression had a significantly longer median survival (14 months) than those patients without BAX expression (6 months, p=0.0004). In contrast, p53 status did not influence prognosis. Patients with p161NK4A negative tumours had a significantly shorter survival (4 months) than those with p16INK41 protein expression (15 months, p=0.0001). Furthermore, the loss of p16INK4A protein expression correlated strongly with the pressure of distant and advanced lymph-node metastases. The best survival was seen in a subgroup of 20 patients with positive p16INK4A expression and intact BAX (p=0.0002). The results of the present study suggest that the loss of BAX and p16INK4A expression are independent markers for poor prognosis in nonsmall cell lung cancer. The study suggests that multimarker analysis of genes involved in apoptosis may be useful for determining individual therapy and for identifying targets for gene-replacement therapy. This should be assessed in a prospective study with a larger cohort of patients.

The objective of this work was to study in more detail the human/murine SCID arthritis model with special emphasis on characteristic features initiated by rheumatoid arthritis (RA) synovial membrane (SM) as compared to appropriate control tissues. Small tissue samples from RA-SM, healthy lymph node, healthy SM, and granulomatous tissue of human origin were implanted into the left knee joint of mice with severe combined immunodeficiency (SCID), and the joints were analysed histologically after 7 days. In addition, a time course study, including non-invasive monitoring by serological parameters (human IgM, IgG, and IL-6) and Tc-99m-scintigraphy, was performed for up to 4 weeks on RA-SM recipients. All tissue implants induced transient exudative joint inflammation while RA-SM initiated a characteristic arthritis with pannus tissue of high cellular density, erosion, multinuclear giant cells, lining cell hyperplasia, fibroblast-like cell layers, chondroideal metaplasia, and fibrin deposits. Significantly elevated levels of human immunoglobulin and characteristic signs of chronic inflammation persisted for more than 4 weeks. We conclude that the hu/mu SCID arthritis with RA-SM implants comprises features of non-specific inflammation which is also transiently seen with control tissues but develops characteristic features of chronic RA-like synovitis thereafter.

Early recognition of lung cancer is a prerequisite for any strategy to improve lung cancer treatment outcome. Here we report a cross-sectional study intended as a proof of principle investigation using breath based detection (exhaled breath condensate, EBC) of angiogenic markers (VEGF, bFGF, angiogenin), TNFand IL-8 to discriminate 74 individuals, with confirmed presence or absence (X-ray, CT) of non-small lung cancer (NSCLC). Levels of angiogenic markers bFGF, angiogenin and VEGF in EBC significantly discriminated between 17 individuals with newly detected NSCLC versus stable and exacerbated chronic obstructive pulmonary disease (COPD) patients as well as healthy volunteers. Levels of IL-8 and TNF- in EBC indicated acute inflammation, e.g. in acute exacerbated COPD (AECOPD) and were not indicative of lung cancer. In a different group of patients that were already treated with two cycles of chemotherapy and who responded with at least a 25% reduction in primary tumor diameter, levels of angiogenic markers were lower compared to patients with newly diagnosed NSCLC. We suggest that breath based detection of angiogenic markers may help in the early detection of lung cancer. © 2009 Published by Elsevier Ireland Ltd.

Tumor vascularisation, the formation of blood vessels is a central process to allow tumor growth beyond limited sizes and to facilitate metastasis formation. Angiogenesis is regulated by a balance of stimulatory and inhibitory factors. Angiogenic factors have been the focus of intense research since the prospects of new therapeutic approaches seemed enormous. Vascular endothelial growth factor (VEGF) has emerged as the most potent and most specific growth factor for endothelial cells and therefore a relevant target for novel anticancer therapy. A wide range of agents have been designed for their ability to interfere the VEGF signalling pathway. In addition, several drugs are currently in advanced clinical development. This review describes the current experimental strategies to inhibit VEGF and will also summarize and discuss the results of recent clinical trials involving anti-VEGF compounds either as standalone therapy or in combination with chemotherapy in lung cancer.

Background: To improve monitoring of lung diseases, we analyzed cytokines in exhaled breath condensate (EBC). The main challenge in measurement of cytokines in EBC is the low protein content, which requires concentration steps that conflict with the need for excessive fluid required by most commonly used kits. Methods: Here, a multiplex bead array for the detection of interleukins (IL) -1b, -6, -8, -10, TNF-a, and IL-12p70 was modified and validated for analysis in EBC samples. Furthermore, 33 healthy volunteers and 11 patients with acute lung injury were investigated. Results: In patients with inflammatory lung diseases, cytokine levels for all investigated cytokines were higher in comparison to healthy smokers or healthy volunteers. Discussion: Multiplexed immunoassays in highly sensitive approaches allow for cytokine detection in EBC. We found significant differences between patients and controls for all investigated cytokines. q 2006 International Society for Analytical Cytology

The majority of NSCLC cell lines produce elevated levels of bFGF but do not secrete considerable amounts. We therefore investigated the influence of cell death inducing mechanisms on bFGF release. In addition we studied the biological consequences on chemosensitivity of NSCLC cell lines to cisplatin. In our experiment we induced i) apoptosis with cisplatin and ii) necrosis with heat treatment in NSCLC cell lines. Supernatants of apoptotic/necrotic NSCLC cell lines exhibited significantly increased levels of bFGF independent of the cell death inducing mechanism. The incubation of three NSCLC cell lines with these supernatants resulted in enhanced cisplatin-induced apoptosis/necrosis in one of the cell lines (H1299) only. Addition of an anti-bFGF antibody to conditioned supernatant of H1299 cells abolished this effect partially. These results suggest that the release of bFGF following cell death contributes to enhanced chemosensitivity in NSCLC cell lines.

The human Y-box binding protein, YB-1, is a multifunctional protein that regulates gene expression. Nuclear expression of YB-1 has been associated with chemoresistance and poor prognosis of tumour patients. Representative samples from autopsied material of primary tumours from 77 patients with NSCLC were investigated by immunohistochemistry for subcellular distribution of YB-1 and p53, in order to evaluate the prognostic role of nuclear expression of YB-1. Cytoplasmic YB-1 expression was found in all tumour samples, whereas nuclear expression was only observed in 48%. There was no correlation with histological classification, clinical parameters or tumour size, stage and metastasis status. However, patients with positive nuclear YB-1 expression in tumours showed reduced survival times when compared with patients without nuclear expression. Including information about the histology and mutational status for p53 increased the prognostic value of nuclear YB-1. Patients with nuclear YB-1 expression and p53 mutations had the worst prognosis (median survival 3 months), while best outcome was found in patients with no nuclear YB-1 and wildtype p53 (median survival 15 months). This suggests that the combined analysis of both markers allows a better identification of subgroups with varying prognosis. Nuclear expression of Y-box binding protien seems to be an independent prognostic marker.

Mechanical ventilation may damage the lung. Low tidal volume (VT) is protective, but VT is scaled to body weight (BW) and may be high in functionally small ARDS lungs. We hypothesized that exhaled breath condensate (EBC) nitrite (NO(2)(-)) concentration may increase with lung distension.Prospective, noncontrolled study.University hospital and medical ICU.Thirty-five ICU patients requiring mechanical ventilation (severe pneumonia, n = 31; exacerbated COPD, n = 4). Patients were scored according to American and European Consensus Conference on ARDS criteria (AECC) [no lung injury, n = 7; acute lung injury, n = 13; ARDS, n = 15], as well as the Murray lung injury severity score (LISS) [score 0, n = 3; score 0.1 to 2.5, n = 19; score2.5, n = 13].EBC was collected and analyzed for NO(2)(-), interleukin (IL)-6, and IL-8. Serum was analyzed for IL-6, IL-8, and procalcitonin.and measurements: EBC NO(2)(-) correlated well with VT (milliliters per kilogram of BW; r = 0.79, p0.0001) and expiratory minute volume (r = 0.60, p0.0001) but not with other ventilatory parameters or parameters of pulmonary (EBC IL-6, EBC IL-8) or systemic (serum IL-6, IL-8, and procalcitonin) inflammation. The ratio of EBC NO(2)(-) and the size of the VT correlated directly with lung injury (AECC, r = 0.66, p0.0001; LISS, r = 0.84, p0.0001).EBC NO(2)(-) increased linearly with VT. The ratio of EBC NO(2)(-) to VT is assumed to reflect NO(2)(-) release at a given VT. An increase in this ratio indicates an inappropriate increase of NO(2)(-) production most likely due to mechanical stress of the remaining open lung units in injured lungs. We conclude that the EBC NO(2)(-)/VT ratio may help to identify situations of critical mechanical stress.

Summary There is only limited information about the prognostic value of p53 immunostaining in cervical cancer. The purpose of this study was to assess the clinical significance of p53 and prognosis in operatively treated cervical carcinoma. A hundred and fourteen primary surgically treated cervical carcinomas (CX) were obtained from the so called Wertheim Archive in the Department of Obstetrics and Gynecology at the University of Leipzig. These included 105 squamous cell cancer (SCC) and nine adenocarcinomas (AC). No cases received neoadjuvant therapy. For immunohistochemical analysis, the cases were tested with the monoclonal antibody DO-7 (DAKO Diagnostics, Denmark). Two hundred tumor cell nuclei were counted for positive nuclear immunostaining, regardless of staining intensity. Cases were stated as positive when a minimum of 10% nuclei showed positive staining. Fresh frozen tissue was available from 21 CX for p53-mutation analysis (exons 4-9) using PCR-based amplification and SSCP-analysis. Of the squamous cell cancers (SCC), 63.8% showed positive nuclear p53-immunostaining; adenocarcinomas (AC) were completely negative (P = 0.0000, Chi2-test). Stage-by-stage analysis revealed no differences in p53-expression. However, combining pT1b- and pT2-cases, the difference in positive immunostaining reached statistical significance (44.4% vs. 71.7%; P = 0.007). There were no differences in p53-reactivity regarding the presence of pelvic lymph node metastases, tumor grading, relapse-free survival and tumor recurrence. In addition, only 5% of CX with positive p53-immunostaining showed genomic alterations in mutational analysis. p53-immunoreactivity showed significant correlation with local tumor progression but not with lymphatic spread, lacking any prognostic impact in surgically treated cervical cancer. There is no correlation of p53-immunostaining with the occurrence of p53-gene mutations in cervical cancer.

Differences in cytokine patterns in stable chronic obstructive pulmonary disease (COPD), exacerbated COPD, smokers without apparent COPD, and healthy volunteers should be of interest for pathophysiological and therapeutic reasons. Methods including lavage, biopsy and sputum have been employed to investigate cytokines in the lung. For asystematic comparison, exhaled breath condensate (EBC) appears to be well suited. We investigated healthy volunteers, smokers without apparent COPD, stable and exacerbated COPD patients (+/- inhalative steroids) and finally those whose exacerbation made mechanical ventilation inevitable, for a more complete picture of inflammatory cytokines in COPD. We chose EBC because it is non-invasive and can be used repeatedly in spontaneous breathing individuals and during mechanical ventilation. EBC cytokines (IL-1 beta, IL-6, IL-8, IL-10, IL-12 p 70, TNF-alpha) were assayed from a single sample using a multiplex array test kit. We observed a significant increase of all cytokines in acute exacerbation compared to stable COPD, smokers, and volunteers. Stable COPD and volunteers exhibited only small differences in cytokine pattern with respect to IL-1 beta and IL-12 (P