We have developed a high-throughput method for analyzing the methylation status of hundreds of preselected genes simultaneously and have applied it to the discovery of methylation signatures that distinguish normal from cancer tissue samples. Through an adaptation of the GoldenGate genotyping assay implemented on a BeadArray platform, the methylation state of 1536 specific CpG sites in 371 genes (one to nine CpG sites per gene) was measured in a single reaction by multiplexed genotyping of 200 ng of bisulfite-treated genomic DNA. The assay was used to obtain a quantitative measure of the methylation level at each CpG site. After validating the assay in cell lines and normal tissues, we analyzed a panel of lung cancer biopsy samples (N = 22) and identified a panel of methylation markers that distinguished lung adenocarcinomas from normal lung tissues with high specificity. These markers were validated in a second sample set (N = 24). These results demonstrate the effectiveness of the method for reliably profiling many CpG sites in parallel for the discovery of informative methylation markers. The technology should prove useful for DNA methylation analyses in large populations, with potential application to the classification and diagnosis of a broad range of cancers and other diseases.

No abstract

Background: Cigarette smoke (CS) is the predominant pathogenetic factor in the development of chronic bronchitis and chronic obstructive pulmonary disease. The knowledge about the cellular and molecular mechanisms underlying the smoke-induced inflammation in epithelial cells is limited. Objectives: The aim of this study was to develop an in vitro model to monitor the effects of volatile CS on differentiated airway epithelial cells. Methods: The airway epithelial cell line MM-39 and primary human bronchial epithelial cells were cultivated as air-liquid interface cultures and exposed directly to volatile CS. We used two types of exposure models, one using ambient air, the other using humidified and warm air. Cytokine levels were measured by quantitative PCR and ELISA. Phosphorylation of p38 MAP kinase was assessed by Western blot analysis. To reduce the smoke-induced inflammation, antisense oligonucleotides directed against the p65 subunit of NF-ĸB were applied. Results: Exposure of epithelia to cold and dry air resulted in a significant inflammatory response. In contrast, exposure to humidified warm air did not elicit a cellular response. Stimulation with CS resulted in upregulation of mRNA for IL-6 and IL-8 and protein release. Exposure to CS combined with heat-inactivated bacteria synergistically increased levels of the cytokines. Reactions of differentiated epithelial cells to smoke are mediated by the MAP kinase p38 and the transcription factor NF-ĸB. Conclusions: We developed an exposure model to examine the consequences of direct exposure of differentiated airway epithelial cells to volatile CS. The model enables to measure the cellular reactions to smoke exposure and to determine the outcome of therapeutic interventions.

Chronic obstructive pulmonary disease (COPD) is a major health problem. The disease is driven by abnormal inflammatory reactions in response to inhaled particles and fumes. Therefore, inflammatory mediators are postulated to be of distinct importance. In the present case-control study, we investigated interleukin (IL)-promoter polymorphisms known to correlate with altered transcription levels of their gene products in patients with COPD. We analyzed tumor necrosis factor-alpha (TNF-alpha)-308, TNF-beta-intron1-252, IL-6-174, IL-10-819, and IL-10-1082 polymorphisms in 469 individuals using restriction fragment length polymorphism-based converted polymerase chain reaction. The study population consisted of 113 patients with COPD based on chronic bronchitis, divided into subgroups by severity (I degrees -III degrees ), 113 matched hospitalized individuals suffering from severe coronary heart disease without pulmonary disease (age-, sex-, and smoking-matched control group), and 243 healthy individuals (population control group). The matched analysis showed no significant differences in genotype distribution of all tested polymorphisms between the matched controls and the COPD patients. However, comparison with the population controls revealed significant differences in IL-10-1082 A/G genotype frequencies (P = 0.0247 for the whole COPD group, P = 0.009 for smokers only), with the genotypes carrying the G allele more common in the COPD cases [odds ratio (OR) = 1.66, 95% confidence interval (CI) 1.01-2.75; P = 0.046]. Interestingly, this shift toward more G alleles was even more pronounced in the matched control group (OR = 2.55, 95% CI 1.47-4.41; P = 0.0007), suggesting both presented groups share corresponding underlying mechanisms. The IL-10-1082_G allele is known to correlate with altered IL-10 levels. Therefore, it might be associated with altered or abnormal inflammatory response, a mechanism that could be postulated to be important in both chronic bronchitis and coronary heart disease.

Apart from cigarette smoking, genetic factors seem to be of importance in the development of lung cancer. The present case-control study investigated frequencies of five inflammatory response gene polymorphisms (TNF-α-308, TNF-β-Intron1-252, IL-6-174, IL-10-819 and IL-10-1082) in patients with lung cancer and controls. The study population consisted of 117 patients with lung cancer (77 patients with NSCLC, including 40 Squamous Cell Carcinoma and 26 Adenocarcinoma, and 40 patients with SCLC), 117 matched controls without pulmonary disease and 243 healthy individuals (population control). Genotype analyses revealed no difference in genotype frequencies using matched-pair analysis. However, in comparison to the population control, the IL-10-1082 genotypes carrying the G allele appeared with higher frequency in the SCLC group (p = 0.006) [SCLC: 84.6%, population controls: 64.6%]. This yields an odds ratio of 3.01 for SCLC (95% CI = [1.21, 7.48]). No associations were seen for all other polymorphisms analysed. The study raises the possibility of a correlation between the IL-10-1082_G allele and the presence of SCLC in a German population. The functional IL-10-1082 polymorphism correlates with altered IL-10 levels and might influence lung cancer susceptibility by altered inflammatory responses in the airways.

SUMMARYObjective: To examine the attitudes toward counseling about sudden unexpected death in epilepsy (SUDEP) and other epilepsy risk factors among Austrian, German, and Swiss neurologists and neuropediatricians, and to determine factors associated with not discussing SUDEP. Methods: Questionnaires were sent to approximately 5,000 neurologists and neuropediatricians in 2014 regarding respondents' demographics, their working environments, and how often they discussed SUDEP, suicidal ideations on anticonvulsive medication, driving restrictions, and risks in daily life activities. Results: In total, 519 surveys were completed (respondents' mean age: 45.5 years, 41.6% female, 66.9% adult neurologists, 31.0% neuropediatricians). A minority of 2.7% reported that they counseled all of their patients on SUDEP, 8.7% counseled most of the time (50-90%), 20.8% sometimes (10-49%), 44.5% rarely (1-9%), and 23.3% reported not counseling about SUDEP at all. In contrast, 92.9% reported that they counseled all patients about driving restrictions and 81.5% about risks in daily life activities. Suicidal ideations were discussed in 59.0% for some and in 3.3% for all patients, whereas 35.1% of respondents reported never discussing suicidal ideations. Independent predictors of not discussing SUDEP were no additional epilepsy training, no or uncertain SUDEP cases in the past, <10 years in practice, <25 epilepsy patients seen per quarter, and the opinion of a lack of consequences in SUDEP prevention. The opinion that SUDEP is a risk factor in particular patient groups and the attitude that all risks should be discussed predicted counseling on SUDEP. Significance: Our findings show a discrepancy between guidelines and practice regarding the discussion of premature mortality due to SUDEP or suicidality. Both are not

Our data demonstrate that therapeutic application of DeltaN23-KGF has the potential to induce alveolar maintenance programs in emphysematous lungs and suggest that the regenerative effect on interstitial tissue is linked to AE2 cell-derived TGF-beta1.

Purpose Patients with advanced cancer often receive suboptimal end-of-life (EOL) care. Particularly males with advanced cancer are more likely to receive EOL care that is more aggressive, even if death is imminent. Critical factors determining EOL care are EOL conversations or advance care planning. However, information about gender-related factors influencing EOL conversations is lacking. Therefore, the current study investigates gender differences concerning the content, the desired time point, and the mode of initiation of EOL conversations in cancer patients. Methods In a cross-sectional study, 186 female and male cancer patients were asked about their preferences for EOL discussions using a semi-structured interview, focusing on (a) the importance of six different topics (medical and nursing care, organizational, emotional, social, and spiritual/religious aspects), (b) the desired time point, and (c) the mode of discussion initiation. Results The importance of EOL topics differs significantly regarding issue (p = 0.002, η 2 = 0.02) and gender (p < 0.001, η 2 = 0.11). Males wish to avoid the engagement in discussions about death and dying particularly if they are anxious about their endof-life period. They wish to be addressed regarding the "hard facts" nursing and medical care only. In contrast, females prefer to speak more about "soft facts" and to be addressed about each EOL topic. Independent of gender, the majority of patients prefer to talk rather late: when the disease is getting worse (58%), at the end of their therapy, or when loosing self-sufficiency (27.5%). Conclusion The tendency of patients to talk late about EOL issues increases the risk of delayed or missed EOL conversations, which may be due to a knowledge gap regarding the possibility of disease-associated incapability. Furthermore, there are significant gender differences influencing the access to EOL conversations. Therefore, for daily clinical routine, we suggest an early two-step, gender-sensitive approach to end-of-life conversations.