Unique transcriptome signature of mouse microglia.

Microglial cells can be derived directly from the dissociated brain tissue by sorting procedures, from postnatal glial cultures by mechanic isolation or from pluripotent stem cells by differentiation. The detailed molecular phenotype of microglia from different sources is still unclear. Here, we performed a whole transcriptome analysis of flow cytometry-sorted microglia, primary postnatal cultured microglia, embryonic stem cell derived microglia (ESdM), and other cell types.

Intrahepatic IL-8 producing Foxp3⁺CD4⁺ regulatory T cells and fibrogenesis in chronic hepatitis C.

Background & Aims: Regulatory CD4(+) T cells (Tregs) are considered to affect outcomes of HCV infection, because they increase in number during chronic hepatitis C and can suppress T-cell functions.

Methods: Using microarray analysis, in situ immunofluorescence, ELISA, and flowcytometry, we characterised functional differentiation and localisation of adaptive Tregs in patients with chronic hepatitis C.

Results: We found substantial upregulation of IL-8 in Foxp3(+)CD4(+) Tregs from chronic hepatitis C.

Liver-primed memory T cells generated under noninflammatory conditions provide anti-infectious immunity.

Development of CD8(+) T cell (CTL) immunity or tolerance is linked to the conditions during T cell priming. Dendritic cells (DCs) matured during inflammation generate effector/memory T cells, whereas immature DCs cause T cell deletion/anergy. We identify a third outcome of T cell priming in absence of inflammation enabled by cross-presenting liver sinusoidal endothelial cells.

Vav1 regulates MHCII expression in murine resting and activated B cells.

Vav1 is a guanine nucleotide exchange factor (GEF) for Rho GTPases, which is exclusively expressed in cells of the hematopoietic system. In addition to its well-documented GEF activity, it was suggested to have other functions due to the presence of multiple domains and nuclear localization signals in its protein structure. Although GEF-dependent and GEF-independent functions of vav have been implicated in T-cell development and T-cell receptor signaling, the role of vav1 in antigen-presenting cells is poorly understood.

Keratin 1 maintains skin integrity and participates in an inflammatory network in skin through interleukin-18.

Keratin 1 (KRT1) and its heterodimer partner keratin 10 (KRT10) are major constituents of the intermediate filament cytoskeleton in suprabasal epidermis. KRT1 mutations cause epidermolytic ichthyosis in humans, characterized by loss of barrier integrity and recurrent erythema. In search of the largely unknown pathomechanisms and the role of keratins in barrier formation and inflammation control, we show here that Krt1 is crucial for maintenance of skin integrity and participates in an inflammatory network in murine keratinocytes.

High-resolution transcriptome of human macrophages.

Macrophages are dynamic cells integrating signals from their microenvironment to develop specific functional responses. Although, microarray-based transcriptional profiling has established transcriptional reprogramming as an important mechanism for signal integration and cell function of macrophages, current knowledge on transcriptional regulation of human macrophages is far from complete. To discover novel marker genes, an area of great need particularly in human macrophage biology but also to generate a much more thorough transcriptome of human M1- and M1-like macrophages, we performed RNA sequencing (RNA-seq) of human macrophages.

Natural and orthogonal interaction framework for modeling gene-environment interactions with application to lung cancer.

Objectives: We aimed at extending the Natural and Orthogonal Interaction (NOIA) framework, developed for modeling gene-gene interactions in the analysis of quantitative traits, to allow for reduced genetic models, dichotomous traits, and gene-environment interactions. We evaluate the performance of the NOIA statistical models using simulated data and lung cancer data.

Methods: The NOIA statistical models are developed for additive, dominant, and recessive genetic models as well as for a binary environmental exposure.

The association between active participation in a sports club, physical activity and social network on the development of lung cancer in smokers: a case-control study.

Background: This study analyses the effect of active participation in a sports club, physical activity and social networks on the development of lung cancer in patients who smoke. Our hypothesis is that study participants who lack social networks and do not actively participate in a sports club are at a greater risk for lung cancer than those who do.

Methods: Data for the study were taken from the Cologne Smoking Study (CoSmoS), a retrospective case-control study examining potential psychosocial risk factors for the development of lung cancer.

Blood-based miRNA preparation for noninvasive biomarker development.

This chapter describes several methods for the isolation of miRNAs from peripheral whole blood samples or constituent fractions thereof, such as peripheral blood mononuclear cells, plasma, and serum. The methods described here are recently introduced protocols dedicated to the isolation of total RNAs including small RNAs, e.g.

Expression of type I interferon by splenic macrophages suppresses adaptive immunity during sepsis.

Early during Gram-negative sepsis, excessive release of pro-inflammatory cytokines can cause septic shock that is often followed by a state of immune paralysis characterized by the failure to mount adaptive immunity towards secondary microbial infections. Especially, the early mechanisms responsible for such immune hypo-responsiveness are unclear. Here, we show that TLR4 is the key immune sensing receptor to initiate paralysis of T-cell immunity after bacterial sepsis.

RNA-stabilized whole blood samples but not peripheral blood mononuclear cells can be stored for prolonged time periods prior to transcriptome analysis.

Microarray-based transcriptome analysis of peripheral blood as surrogate tissue has become an important approach in clinical implementations. However, application of gene expression profiling in routine clinical settings requires careful consideration of the influence of sample handling and RNA isolation methods on gene expression profile outcome. We evaluated the effect of different sample preservation strategies (eg, cryopreservation of peripheral blood mononuclear cells or freezing of PAXgene-stabilized whole blood samples) on gene expression profiles.

Blood-based gene expression signatures in non-small cell lung cancer.

Purpose: Blood-based surrogate markers would be attractive biomarkers for early detection, diagnosis, prognosis, and prediction of therapeutic outcome in cancer. Disease-associated gene expression signatures in peripheral blood mononuclear cells (PBMC) have been described for several cancer types. However, RNA-stabilized whole blood-based technologies would be clinically more applicable and robust.

Prediction and prognosis: impact of gene expression profiling in personalized treatment of breast cancer patients.

Breast cancer is a complex disease, whose heterogeneity is increasingly recognized. Despite considerable improvement in breast cancer treatment and survival, a significant proportion of patients seems to be over- or undertreated. To date, single clinicopathological parameters show limited success in predicting the likelihood of survival or response to endocrine therapy and chemotherapy.

Replication of lung cancer susceptibility loci at chromosomes 15q25, 5p15, and 6p21: a pooled analysis from the International Lung Cancer Consortium.

Background: Genome-wide association studies have identified three chromosomal regions at 15q25, 5p15, and 6p21 as being associated with the risk of lung cancer. To confirm these associations in independent studies and investigate heterogeneity of these associations within specific subgroups, we conducted a coordinated genotyping study within the International Lung Cancer Consortium based on independent studies that were not included in previous genome-wide association studies.

Methods: Genotype data for single-nucleotide polymorphisms at chromosomes 15q25 (rs16969968, rs8034191), 5p15 (rs2736100, rs402710), and 6p21 (rs2256543, rs4324798) from 21 case-control studies for 11 645 lung cancer case patients and 14 954 control subjects, of whom 85% were white and 15% were Asian, were pooled.

The influence of occupational stress factors on the nicotine dependence: a cross sectional study.

Objective: This study analyses the association between occupational stress factors and nicotine dependence. Our hypothesis is that occupational stress factors increase nicotine dependence.

Methods: Data were taken from the Cologne Smoking Study, a case-control study that examines which genetic/psychosocial factors lead to a higher risk for smokers to suffer from cardiac infarction, lung cancer and/or to become addicted to nicotine.

Bead array-based microrna expression profiling of peripheral blood and the impact of different RNA isolation approaches.

Blood-based mRNA expression profiling has already become an important issue in clinical applications. More recently, the characterization of the small RNA transcriptome offers additional avenues for diagnostic approaches. However, when applying miRNA expression profiling in routine clinical settings, the method of RNA preservation and the manner of RNA extraction as well as the reliability of the miRNA profiling procedure have to be carefully considered.

Re-overcoming barriers in translating biomarkers to clinical practice.

Importance Of The Field: Recently, there has been growing evidence for the concept of personalized medicine as the implementation of genomic and molecular information in the delivery of healthcare. In parallel, the identification of biomarkers has become of enormous significance as a prerequisite for individualized intervention regimens.

Areas Covered In This Review: Biomarkers are developed to improve prevention, diagnosis or therapeutic outcome of a given disease.

Identification of lung cancer with high sensitivity and specificity by blood testing.

Background: Lung cancer is a very frequent and lethal tumor with an identifiable risk population. Cytological analysis and chest X-ray failed to reduce mortality, and CT screenings are still controversially discussed. Recent studies provided first evidence for the potential usefulness of autoantigens as markers for lung cancer.

Validation of the "SmoCess-GP" instrument - a short patient questionnaire for assessing the smoking cessation activities of general practitioners: a cross-sectional study.

Background: We developed an instrument assessing the extent of smoking cessation activities by general practitioners (GPs) within the Cologne Smoking Study (CoSmoS). The objective of the present study was to examine further psychometric quality of the "SmoCess-GP" instrument (Smoking Cessation by General Practitioners).

Methods: 127 current smokers who had participated in the Cologne Smoking Study (CoSmoS) were included in our analyses.

Application of T cell-based transcriptomics to identify three candidate biomarkers for monitoring anti-TGFbetaR therapy.

Objectives: The development of targeted drugs would greatly benefit from the simultaneous identification of biomarkers to determine the aspects of bioactivity, drug safety and efficacy, particularly when affecting receptor-signaling pathways. However, the establishment of appropriate systems to monitor drug-induced events requires an accessible surrogate tissue for functional read out.

Methods: Therefore we present a universal platform based upon T cell-based gene expression profiling for the identification of biomarkers using the antitransforming growth factor beta receptor inhibitor LY2109761 as an example.

Blood-based transcriptomics: leukemias and beyond.

In 1999, Golub et al. proposed for the first time microarray-based transcriptional profiling to be used as a new technology for the differential diagnosis of acute myeloid leukemias and acute lymphocytic leukemias. This very preliminary study sparked great enthusiasm beyond the leukemias.

Use of genome-wide high-throughput technologies in biomarker development.

In recent years, the usage of high-throughput technologies in the fields of genomics, transcriptomics, proteomics and metabolomics for biomarker discovery has expanded enormously. Biomarkers can be applied for many purposes, including diagnosis, prognosis, staging and selecting appropriate patient therapy. In addition, biomarkers can provide information on disease mechanism or progression.

Non-Hodgkin lymphoma expressing high levels of the danger-signalling protein HMGB1.

HMGB1 is a high mobility group protein that can act either as a DNA binding protein or extracellularly as a cytokine-like danger signal. Extracellular HMGB1, either actively secreted or passively released by necrotic cells, is linked to inflammation and cancer. Herein, the results of a study to quantify the expression of HMGB1 in lymphomas by quantitative real-time RT-PCR are presented.

Expression of signaling mediators downstream of EGF-receptor predict sensitivity to small molecule inhibitors directed against the EGF-receptor pathway.

The EGF-receptor (EGFR) and downstream signaling molecules have emerged as promising targets for inhibition by small molecules in the treatment of nonsmall cell lung cancer (NSCLC). In this study expression of pivotal signaling molecules in the EGFR pathway were used to predict response to inhibitors of the EGFR signaling cascade. NSCLC cell lines were treated with the EGFR tyrosine kinase inhibitor (TKI) gefitinib and PD16,8393, the AKT inhibitor SH-6 and LY294002, the farnesyltransferase inhibitor L744832, and the mTOR inhibitor rapamycin.

Allelic losses on chromosome 6q25 in Hodgkin and Reed Sternberg cells.

We established a molecular cytogenetic approach to identify consistent genetic aberrations in classical Hodgkin lymphoma. Single laser-micromanipulated Hodgkin and Reed Sternberg (H-RS) cells and the respective germ line tissue were PCR-amplified using highly polymorphic microsatellite probes. Loss of heterozygosity and genomic imbalances of the fluorochrome-labeled microsatellites were determined by fragment length analysis.