Search

Supplementary Materials and Methods; Supplementary Tables S1-S3.

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. We evaluated the applicability of whole blood–based gene expression profiling for the detection of non–small cell lung cancer (NSCLC).Experimental Design: Expression profiles were generated from PAXgene-stabilized blood samples from three independent groups consisting of NSCLC cases and controls (n = 77, 54, and 102), using the Illumina WG6-VS2 system.Results: Several genes are consistently differentially expressed in whole blood of NSCLC patients and controls. These expression profiles were used to build a diagnostic classifier for NSCLC, which was validated in an independent validation set of NSCLC patients (stages I–IV) and hospital-based controls. The area under the receiver operator curve was calculated to be 0.824 (P < 0.001). In a further independent dataset of stage I NSCLC patients and healthy controls the AUC was 0.977 (P < 0.001). Specificity of the classifier was validated by permutation analysis in both validation cohorts. Genes within the classifier are enriched in immune-associated genes and show specificity for NSCLC.Conclusions: Our results show that gene expression profiles of whole blood allow for detection of manifest NSCLC. These results prompt further development of gene expression–based biomarker tests in peripheral blood for the diagnosis and early detection of NSCLC. Clin Cancer Res; 17(10); 3360–7. ©2011 AACR.

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. Activated GARP-positive IL-8 + Tregs were particularly enriched in livers of patients with chronic hepatitis C in close proximity to areas of fibrosis and their numbers were correlated with the stage of fibrosis. Moreover, Tregs induced upregulation of profibrogenic markers TIMP1, MMP2, TGF-beta1, alpha-SMA, collagen, and CCL2 in primary human hepatic stellate cells (HSC). HSC activation, but not Treg suppressor function, was blocked by adding a neutralizing IL-8 antibody. Conclusions Our studies identified Foxp3 + CD4 + Tregs as an additional intrahepatic source of IL-8 in chronic hepatitis C acting on HSC. Thus, Foxp3 + CD4 + Tregs in chronic hepatitis C have acquired differentiation as regulators of fibrogenesis in addition to suppressing local immune responses.

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. Microglia and ESdM, both cultured in serum-free medium, were closely related to sorted microglia and showed a unique transcriptome profile, clearly distinct to other myeloid cell types, T cells, astrocytes, and neurons. ESdM and primary cultured microglia showed strong overlap in their transcriptome. Only 143 genes were differentially expressed between both cell types, mainly derived from immune-related genes with a higher activation status of proinflammatory and immune defense genes in primary microglia compared to ESdM. Flow cytometry analysis of cell surface markers CD54, CD74, and CD274 selected from the microarray confirmed the close phenotypic relation between ESdM and primary cultured microglia. Thus, assessment of genome-wide transcriptional regulation demonstrates that microglial cells are unique and clearly distinct from other macrophage cell types.

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. We found that vav1 is an important regulator of MHCII expression and transport. Microarray analysis of unstimulated bone marrow-derived macrophages revealed a novel role of vav1 in transcriptional regulation of the MHCII locus, possibly by indirect means. Primary immune cells from vav1-deficient mice had a significantly lower constitutive surface expression of MHCII with the strongest impact observed on splenic and peritoneal B cells. Impaired MHCII expression resulted in a diminished capacity for T-cell activation. Using 6-thio-GTP, a specific inhibitor of the GEF function of vav1, we were able to show that the GEF activity is required for MHCII upregulation in B cells after stimulation with LPS. Furthermore, our data show that vav1 not only affects transcription of the MHCII locus but also is an important regulator of MHCII protein transport to the cell surface.

Background: Psychosocial factors attract interest in investigating the occurrence of cardiovascular disease. Design This study aimed to examine the impact of critical life events on the development of myocardial infarction in smokers and ex-smokers. We hypothesized that critical life events increase the risk of the disease. Methods: Data were taken from the Cologne Smoking Study (CoSmoS), a retrospective multicentre case-control study that examines which psychosocial factors may lead to a higher risk for smokers and ex-smokers of suffering from a myocardial infarction. Our sample consisted of n = 278 myocardial infarction participants and control participants. Both groups had a history of smoking. Logistic regression was used in the analysis. Results: The study results of the smoking and ex-smoking participants showed that sociodemographic data like gender and age have an effect on the development of myocardial infarction. Physical activity seems to offer protection aganist myocardial infarction. Final, the unexpected result that the experience of at least one critical life event seems to have a positive effect on health and so lowers the risk of myocardial infarction. Conclusions: Socio-demographic data and physical activity have an effect on the development of myocardial infarction. Participants with experience of critical life events appear to be strengthened after the events and possess adequate resources to protect their health.

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. Expression profiles obtained from cryopreserved peripheral blood mononuclear cells differed substantially from those of their nonfrozen counterpart samples. Furthermore, expression profiles in cryopreserved peripheral blood mononuclear cell samples were found to undergo significant alterations with increasing storage period, whereas long-term freezing of PAXgene RNA stabilized whole blood samples did not significantly affect stability of gene expression profiles. This report describes important technical aspects contributing toward the establishment of robust and reliable guidance for gene expression studies using peripheral blood and provides a promising strategy for reliable implementation in routine handling for diagnostic purposes.

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. We evaluated the applicability of whole blood–based gene expression profiling for the detection of non–small cell lung cancer (NSCLC). Experimental Design: Expression profiles were generated from PAXgene-stabilized blood samples from three independent groups consisting of NSCLC cases and controls (n = 77, 54, and 102), using the Illumina WG6-VS2 system. Results: Several genes are consistently differentially expressed in whole blood of NSCLC patients and controls. These expression profiles were used to build a diagnostic classifier for NSCLC, which was validated in an independent validation set of NSCLC patients (stages I–IV) and hospital-based controls. The area under the receiver operator curve was calculated to be 0.824 (P < 0.001). In a further independent dataset of stage I NSCLC patients and healthy controls the AUC was 0.977 (P < 0.001). Specificity of the classifier was validated by permutation analysis in both validation cohorts. Genes within the classifier are enriched in immune-associated genes and show specificity for NSCLC. Conclusions: Our results show that gene expression profiles of whole blood allow for detection of manifest NSCLC. These results prompt further development of gene expression–based biomarker tests in peripheral blood for the diagnosis and early detection of NSCLC. Clin Cancer Res; 17(10); 3360–7. ©2011 AACR.

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. Consequently, new gene expression based prognostic and predictive tests are emerging that promise an improvement in predicting survival and therapy response. Initial evidence has emerged that this leads to allocation of fewer patients into high-risk groups allowing a reduction of chemotherapy treatment. Moreover, pattern-based approaches have also been developed to predict response to endocrine therapy or particular chemotherapy regimens. Irrespective of current pitfalls such as lack of validation and standardization, these pattern-based biomarkers will prove useful for clinical decision making in the near future, especially if more patients get access to this form of personalized medicine.

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. Here we evaluate a recently introduced bead array–based technology as a robust method for the generation of blood-based human miRNA expression profiles. Importantly the comparison of different RNA extraction strategies resulted in dissimilar profiles depending on the RNA extraction method as well as on the underlying source. Expression profiles obtained from peripheral mononuclear cells (PBMCs) substantially differed from those of whole blood samples, whereby both sources per se yielded reproducible and reliable results. Expression profiles were also distinct when using either fresh or frozen PBMCs. Moreover RNA size fractioning resulted in discriminative miRNA expression profiles compared with total RNA based profiles. This study outlines important steps toward the establishment of a robust strategy for blood-based miRNA profiling and provides a reliable strategy for its implementation in routine handling for diagnostic purposes.

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.Biomarkers are developed to improve prevention, diagnosis or therapeutic outcome of a given disease. Although each application reveals distinct developmental strategies, evidence-based approval of new biomarkers is important for the success of new drugs, diagnostic tests or recommendations in preventive medicine. Current hurdles to bringing biomarkers into clinical practice are reviewed, thereby focusing on adequate approaches to overcome these limitations in the future.The reader will get an introduction to strategies resolving actual barriers in clinical biomarker development.The identification of evidence-based biomarkers is crucial for the success of individualized therapeutic approaches. Developmental strategies have to be adapted to clinical need, thereby focusing on biomarker validation in clinical settings as well as on the establishment of standardized biomarker test systems for routine application. Consortia have been established bringing together representatives of government, academia and industry to improve future biomarker development.

Tumorerkrankungen stellen in Deutschland nach wie vor die zweithaufigste Tordesursache dar. Neben der Entwicklung wirksamer primarer und sekundarer Praventionsmasnahmen sind vor allem Methoden zur Verbesserung der Diagnose und Subklassifikation sowie zur Prognoseeinschatzung der einzelnen Tumorentitaten dazu geeignet, die krebsbedingte Letalitat langfristig zu senken. Die klassische Diagnose eines Tumors stutzt sich vielfach auf seine histomorphologische Charakterisierung, wohingegen die prognostische Einschatzung zusatzlich durch das Stadium der Erkrankung bei Diagnose bestimmt wird. Nach der Etablierung von Hochdurchsatzmethoden zur Erstellung von Genexpressionsprofilen von Tumorbiopsien wurde im vergangenen Jahrzehnt untersucht, ob die molekulare Charakterisierung von Tumoren die diagnostische Sicherheit zusatzlich erhohen und die Einschatzung der Prognose verbessern kann. Hierzu wurden vor allem Studien beim Mamma-, Lungen- und Kolonkarzinom durchgefuhrt mit dem Ziel, in der adjuvanten Situation bei Vorliegen eines fruhen Stadiums feststellen zu konnen, welche Patientenkollektive tatsachlich von einer Chemotherapie profitieren wurden. Ferner wurden Genexpressionsprofile dazu genutzt, Tumoren mit unklarem Primarius ihrem Ursprungsgewebe zuzuordnen. Daruber hinaus wurde mittels Genexpressionsanalyse eine erganzende molekulare Klassifikation sowohl von B-Zell-Non-Hodgkin-Lymphomen als auch von lymphatischen und myeloischen Leukamien ermoglicht, wodurch moglicherweise sowohl die Diagnostik als auch die Einschatzung der Prognose dieser Erkrankungen langfristig verbessert werden konnen. Obwohl mittlerweile zahlreiche Signaturen und Pradiktoren zu einzelnen Neoplasien veroffentlicht wurden, fand in Deutschland bisher keiner der Tests Eingang in die leitlinienbasierte Diagnostik und Therapie einer malignen Erkrankung. Dies ist vor allem darauf zuruckzufuhren, dass die vielversprechenden Ergebnisse zunachst in grosen prospektiven klinischen Studien evaluiert werden mussen. Erst nach Abschluss dieser Studien wird zu entscheiden sein, ob und welche Testverfahren tatsachlich geeignet sind, den Verlauf einer bestimmten Tumorerkrankung signifikant zu verbessern.

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. Biomarker development for clinical application encompasses phases for their discovery and characterization, assay development and, finally, implementation using automated platforms employed in clinical laboratories. However, translation from bench to bedside outside a research-oriented environment has proven to be more difficult. This is reflected by only few new biomarkers being integrated into clinical application in the last years. This article reviews currently used high-throughput technologies for the identification of biomarkers, as well as present approaches to increase the percentage of biomarkers that pass the barriers for clinical application.

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. HMGB1 expression was analysed in 18 non-Hodgkin lymphomas and two lymphoma cell lines. 11/18 primary lymphomas expressed HMGB1 mRNA at a level exceeding the average of normal lymph nodes. Immunohistochemistry showed that HMGB1 positivity is confined to the lymphoma cells. No correlation between HMGB1 expression and grading was found. However, a high percentage of lymphomas is overexpressing a danger-signalling protein. This protein can support the growth and angiogenesis of lymphoma cells in a paracrine way when released e.g. due to necrosis. Thus it constitutes an interesting therapeutic target as well.

Hodgkin and Reed-Sternberg (H/RS) cells are characterized by chromosomal instability. Nevertheless, neither specific nor consistent chromosomal alterations could be characterized in H/RS cells. Microsatellite instability (MSI) is another form of genomic instability but its role in the pathogenesis of classical Hodgkin's disease (cHD) has not been investigated so far. We analyzed MSI and mismatch repair (MMR) protein expression in H/RS cells of cHD in order to assess genomic instability in these cells. Using a sensitive single cell approach, MSI-low was detected in a portion of single cells of the H/RS cell line L1236. Mutations of genes encoding for hMSH2 and hMLH1 were excluded by RT-PCR in L1236 cells. An analysis of pooled single H/RS cells of seven primary cases of cHD showed loss of heterozygosity for some allelic markers but absence of MSI in all 7 cases. Owing to a tight correlation between MSI-high, inactivating mutations of MMR genes and MMR protein expression in colon cancer, MMR protein expression commonly is used as a marker for MSI. In order to screen additional primary cases of cHD for MSI, we performed immunohistochemistry for hMSH2 and hMLH1 in 6 of the 7 cases analyzed by single cell PCR and 20 additional cases of cHD. H/RS cells from 25 out of 26 cases showed a nuclear staining pattern for hMSH2 and hMLH1 similar to germinal center B cells of non-malignant lymph nodes. These results indicate a proficient MMR system in most H/RS cells. It is concluded that a defect MMR system is unlikely to contribute to the malignant phenotype and genomic instability of H/RS cells in cHD.

In T cell-dependent immune responses, class switch recombination occurs in germinal centres. There is now evidence that Hodgkin/Reed–Sternberg cells are derived from germinal centre B cells. Cytokines specifically determine the direction of class switching, i.e the isotype of the new antibodies. We performed restriction analyses and polymerase chain reaction on the immunoglobulin heavy chain loci for five Hodgkin's disease-derived B-cell lines and one Hodgkin's disease-derived T-cell line in order to analyse class switch recombination. In all the B-cell lines, class switch recombination had been targeted to Cα4 or Cα1/2. This showed that cell-line precursors had undergone class switching, probably under the influence of TH2 or TH3 cell-derived cytokines. Deletions comprising several constant region genes were observed in cell lines L428, L1236, L591 and KMH2. Karyotype analyses of two of these revealed translocational breakpoints within the immunoglobulin heavy chain gene locus. Our data support the view that a chromosomal instability may occur during class switch recombination in Hodgkin/Reed–Sternberg cells causing chromosomal breaks. Thus, as in other germinal centre B cell-derived lymphomas, the immunoglobulin gene locus may be frequently involved in structural chromosomal aberrations in Hodgkin's disease.

Hodgkin-Reed Sternberg cells are derived from germinal centre B-cells in most cases. Somatic mutations affecting their rearranged immunoglobulin genes were detected, rendering potential functional rearrangements non-functional. Under physiological conditions such cells would be designated to undergo apoptosis within the germinal centre. In search for the specific transforming event that prevents Hodgkin-Reed Sternberg cells from programmed cell death, cytogenetic analyses were broadly performed but did not reveal specific chromosomal aberrations. Analysis of these cells on the molecular level is difficult to perform due to the scarcity of the cells in the lymphoma tissue and the given limitations of in situ studies. To overcome these limitations, the cell line L1236, known to be derived from Hodgkin-Reed Sternberg cells in situ, was chosen for allelotype analysis. Using a panel of microsatellite loci assigned to nearly all chromosomal arms, regions of loss of heterozygosity were detected on chromosomal arms 6p, 9q and 17p. The size of lost segments was estimated by amplification of additional microsatellite loci mapped to the respective regions. Further analyses of single Hodgkin-Reed Sternberg cells will reveal whether LOH affecting these regions is a recurrent event in HD and to which extent the smallest commonly affected region can be estimated. © 2001 Cancer Research Campaign http://www.bjcancer.com

Classical Hodgkin's disease (HD) is associated with Epstein-Barr virus (EBV) infection. Although in developing countries EBV can be demonstrated in Hodgkin-Reed-Sternberg (H-RS) cells in up to 95% of HD cases, in industrialized countries only about 50% of HD cases are associated with EBV. An open question remains whether EBV in the EBV-negative cases has escaped detection by standard screening procedures due to deletions in the viral genome associated with integration of viral fragments into the host cell genome. We, among others, recently described this phenomenon in Burkitt's lymphoma cells. To investigate whether H-RS cells in latent membrane protein-1 (LMP-1)-negative HD cases harbor fragments of the EBV genome, we combined fluorescence in situ hybridization (FISH) using a set of six overlapping DNA probes spanning the whole EBV genome with immunophenotyping of fresh frozen lymphoma sections. Results in the eight cases analyzed were as follows: in three LMP-1-positive cases, FISH analysis yielded specific signals for each EBV DNA probe in H-RS cells, which had been identified by morphology and CD30 staining. In contrast, none of the EBV DNA probes hybridized to the H-RS cells in the five LMP-1-negative cases. Thus, there is no evidence for the presence of fragments of the viral genome integrated into the host cell genome in the LMP-1-negative cases. Furthermore, in the LMP-1-positive cases analyzed, no large deletions in the viral genome were detected. These results show that, in classical HD, LMP-1-negative cases do not harbor EBV DNA within the H-RS cells. Whether, in these cases, a still unknown virus contributes to the transformation and maintenance of the malignant phenotype remains to be established.

SummaryDevelopment 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. Such priming generated memory T cells that were spared from deletion by immature DCs. Similar to central memory T cells, liver-primed T cells differentiated into effector CTLs upon antigen re-encounter on matured DCs even after prolonged absence of antigen. Their reactivation required combinatorial signaling through the TCR, CD28, and IL-12R and controlled bacterial and viral infections. Gene expression profiling identified liver-primed T cells as a distinct Neuropilin-1+ memory population. Generation of liver-primed memory T cells may prevent pathogens that avoid DC maturation by innate immune escape from also escaping adaptive immunity through attrition of the T cell repertoire.

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. Using the Kronecker product rule, a NOIA statistical model is built to model gene-environment interactions. By treating the genotypic values as the logarithm of odds, the NOIA statistical models are extended to the analysis of case-control data. Results: Our simulations showed that power for testing associations while allowing for interaction using the NOIA statistical model is much higher than using functional models for most of the scenarios we simulated. When applied to lung cancer data, much smaller p values were obtained using the NOIA statistical model for either the main effects or the SNP-smoking interactions for some of the SNPs tested. Conclusion: The NOIA statistical models are usually more powerful than the functional models in detecting main effects and interaction effects for both quantitative traits and binary traits.

The inflammatory context of antigen-presentation dictates the outcome of CD8+ T cell responses. Pathogen-induced inflammatory signals ensure the generation of an effector CD8+ T cell response and long-lasting T cell memory, whereas antigen-presentation in absence of inflammatory stimuli, e.g. of self-antigens, causes CD8+ T cell-deletion. The systemic distribution of antigens from certain pathogens without inflammatory stimuli therefore imposes the risk of deleting CD8+ T cells needed for anti-infectious defense. We hence wondered whether cross-presentation of exogenous antigens in the liver, the central organ for clearing soluble and gut-derived antigens, might allow for memory T cell generation even in the absence of inflammation. When restricting presentation of soluble ovalbumin to liver sinusoidal endothelial cells (LSEC), the most abundant population of liver-resident antigen-presenting cells, cross-priming by LSEC generated an antigen-experienced CD8+ T cell population that persisted long time and was insensitive to DC-mediated cross-tolerance. As LSEC-primed T cells re-expressed CD62L and relocated to secondary lymphoid organs (SLOs), a hallmark of central memory T cells (TCM), we directly compared the phenotype and in vivo distribution of both T cell populations. Clearly, LSEC-primed OT-I T cells resembled TCM induced by L. monocytogenes-OVA infection in their localization to SLOs and showed high expression of CD127, CD122 and the memory-specific transcription factor Eomes. In response to bacterial and viral infection, LSEC-primed T cells expanded and differentiated into effector T cells similar to TCM. Taken together, these data suggest that antigen-presentation under non-inflammatory conditions, if conducted by LSEC in the liver, allows for memory T cell generation. Those T cells may serve for defense against pathogens that in the first instance avoid causing inflammation.

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. Absence of Krt1 caused a prenatal increase in interleukin-18 (IL-18) and S100A8/A9, accompanied by a barrier defect and perinatal lethality. Depletion of IL-18 partially rescued Krt1−/− mice. IL-18 release was keratinocyte-autonomous, KRT1- and caspase-1-dependent, supporting an upstream role of KRT1 in the pathology. Finally, transcriptome profiling revealed a Krt1-mediated gene expression signature similar to atopic eczema (AE) and psoriasis, but different from Krt5-deficiency and epidermolysis bullosa simplex (EBS). Our data suggest a functional link between KRT1 and human inflammatory skin diseases.

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., miRNeasy Kit and PAXgene Blood miRNA Kit, or alternatively for the enrichment of low-molecular-weight RNA (LMW RNA) fractions including small RNAs, e.g., using the miRNeasy Kit. Furthermore, modifications of classical RNA purification protocols to facilitate the recovery of small RNAs are highlighted.

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., miRNeasy Kit and PAXgene Blood miRNA Kit, or alternatively for the enrichment of low-molecular-weight RNA (LMW RNA) fractions including small RNAs, e.g., using the miRNeasy Kit. Furthermore, modifications of classical RNA purification protocols to facilitate the recovery of small RNAs are highlighted.

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. Downstream of TLR4, signalling through TRIF but not MyD88 impaired the development of specific T-cell immunity against secondary infections. We identified type I interferon (IFN) released from splenic macrophages as the critical factor causing T-cell immune paralysis. Early during sepsis, type I IFN acted selectively on dendritic cells (DCs) by impairing antigen presentation and secretion of pro-inflammatory cytokines. Our results reveal a novel immune regulatory role for type I IFN in the initiation of septic immune paralysis, which is distinct from its well-known immune stimulatory effects. Moreover, we identify potential molecular targets for therapeutic intervention to overcome impairment of T-cell immunity after sepsis.

Hodgkin-Reed Sternberg (H-RS) cells harbour clonal immunoglobulin gene (Ig) rearrangements in almost all cases of classical Hodgkin's disease but lack Ig gene expression. In the H-RS cell line L1236, a somatic mutation of the Ig heavy-chain gene promoter octamer motif has been described as a putative reason for absence of Ig gene expression. We addressed transcriptional activity of this mutated promoter by performing reporter gene studies and gel retardation assays. The results showed that the mutation outside the coding region of the rearranged Ig gene in L1236 cells leads to downregulation of Ig gene expression in H-RS cells.

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. Confirmatory factor analysis (CFA) was conducted to examine the model fit and to retest the single-factor structure of the instrument using the Mplus software. Further construct validity was tested with bivariate analysis using an instrument which measures patients' trust in physicians. Results CFA supported the unidimensional structure of the instrument. The factor loadings exceed the threshold of ≥ 0.50. All indicator reliabilities were higher than 0.30. The composite reliability was 0.86 and the average variance extracted (AVE) resulted in a value of 0.50. The calculation of global fit indices identified a CFI value of 1.00 and for TLI a value of 1.02. The root mean square error of approximation (RMSEA) indicates that 0% of the information is not accounted for by the model. The chi-square value was χ2df = 6 = 4.63 (p = 0.59). Analysis of discriminant validity resulted in a non-significiant correlation of r = 0.092 (p = 0.350). Conclusions Results indicate preliminary evidence for the construct validity of the "SmoCess-GP" instrument which therefore appears to be a promising tool for analyzing the extent of smoking cessation advice offered by GPs from the patients' perspective. Future research should examine the psychometric properties in a population based sample, further improvements of the instrument and should apply other methods of validation.

Contains fulltext : 89395.pdf (Publisher’s version ) (Closed access) 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. Associations between the variants and the risk of lung cancer were estimated by logistic regression models. All statistical tests were two-sided. RESULTS: Associations between 15q25 and the risk of lung cancer were replicated in white ever-smokers (rs16969968: odds ratio [OR] = 1.26, 95% confidence interval [CI] = 1.21 to 1.32, P(trend) = 2 x 10(-26)), and this association was stronger for those diagnosed at younger ages. There was no association in never-smokers or in Asians between either of the 15q25 variants and the risk of lung cancer. For the chromosome 5p15 region, we confirmed statistically significant associations in whites for both rs2736100 (OR = 1.15, 95% CI = 1.10 to 1.20, P(trend) = 1 x 10(-10)) and rs402710 (OR = 1.14, 95% CI = 1.09 to 1.19, P(trend) = 5 x 10(-8)) and identified similar associations in Asians (rs2736100: OR = 1.23, 95% CI = 1.12 to 1.35, P(trend) = 2 x 10(-5); rs402710: OR = 1.15, 95% CI = 1.04 to 1.27, P(trend) = .007). The associations between the 5p15 variants and lung cancer differed by histology; odds ratios for rs2736100 were highest in adenocarcinoma and for rs402710 were highest in adenocarcinoma and squamous cell carcinomas. This pattern was observed in both ethnic groups. Neither of the two variants on chromosome 6p21 was associated with the risk of lung cancer. CONCLUSIONS: In this international genetic association study of lung cancer, previous associations found in white populations were replicated and new associations were identified in Asian populations. Future genetic studies of lung cancer should include detailed stratification by histology.

The pathogenetic events leading to the malignant transformation of Hodgkin–Reed–Sternberg cells are unknown. As Hodgkin–Reed–Sternberg cells are resistant to CD95-mediated apoptosis and chromosomal aberrations involving the 1p22 region harbouring the proapoptotic BCL10 gene represent a recurrent event in Hodgkin's disease-derived cell lines, analysis of the BCL10 gene and its transcripts was performed. As transcription of wild-type BCL10 was detected in all Hodgkin's disease-derived cell lines analysed, alterations of the coding sequence of the BCL10 gene are unlikely to contribute to the malignant transformation of the Hodgkin–Reed–Sternberg cell.

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. Over the last 10 years, numerous studies addressed the use of gene expression profiling of peripheral blood from patients with malignancies, infectious diseases, autoimmunity and even cardiovascular diseases. Despite this great effort, no single test has yet been established using microarray-based transcriptional profiling of peripheral blood. Here we highlight the advances in the field of blood transcriptomics during the last 10 years and also critically discuss the issues that need to be resolved before blood transcriptomics will become part of daily diagnostics in the leukemias, as well as in other diseases showing involvement of peripheral blood.

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. Response was correlated to expression of AKT, p-AKT, EGFR, S6K1, p-S6K1, PTEN and to the mutation status of EGFR and KRAS. As expected, mutation of the EGFR predicted response to EGFR-TKI. The resistance mutation T790M conferred resistance to treatment with gefitinib, but not to the irreversible EGFR inhibitor PD16,8393. In cell lines independent of the EGFR, expression of PTEN correlated with resistance to AKT inhibition, EGFR expression correlated to resistance to 17-AAG and L744832 and S6K1 as well as p-S6K1 expression correlated with sensitivity to rapamycin.

There is overwhelming evidence that at least a substantial number of cases, if not all, of Hodgkin's disease (HD) represent monoclonal B-cell disorders. The treatment of HD is changing strikingly. In early stages of disease, extended field irradiation has been the standard resulting in excellent cure rates. Due to the recognition of fatal long-term effects, however, especially the high rates of second solid tumors, extended field radiotherapy is now being abandoned by most study groups. Instead, mild chemotherapy for control of occult disease is combined with involved field irradiation. In intermediate stage HD, where combined modality treatment is the treatment of choice, extended field irradiation is substituted by involved field irradiation for the same reasons. In advanced stage HD, 8 cycles of polychemotherapy (plus additional radiotherapy for large tumor masses and residual lymphomas) for decades has cured only 50% to 60% of patients. The development of a new dose-intensified regimen (BEACOPP) for the first time has significantly improved that prognosis. In relapsed HD, recently published phase III studies suggest an improvement of relapse-free survival of patients using high-dose chemotherapy followed by autologous stem cell transplantation (ASCT).

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. Eleven cases of in classical Hodgkin lymphoma (cHL) were initially screened with 21 microsatellite markers scattered over the entire genome. Loss of heterozygosity was detected in40% of informative loci in most cases indicating a deletion of a substantial part of the genome of H-RS cells. Allelic losses and imbalances on chromosome 6q were detected in most of these cases. A deletion mapping of 6q was performed in 16 cases of cHL. This detailed analysis of 6q led to the identification of a 3.3-Mbp region around D6S311 flanked by D6S978 and D6S1564 that was altered in 11 of 14 cases of cHL analyzed. In conclusion, allelotyping of single H-RS cells revealed monoallelic chromosomal deletions and genomic imbalances on 6q that might affect genes critically involved in the pathogenesis of H-RS cells.

Summary. In the vast majority of cases, Hodgkin–Reed Sternberg (H-RS) cells, the malignant cells in Hodgkin's lymphoma (HL), are derived from germinal centre B cells. In some cases, somatic mutations within the rearranged immunoglobulin heavy (IgH) chain genes were detected, rendering potentially functional gene rearrangements non-functional. In these H-RS cells the expression of high-affinity B-cell receptors (BCR) was prevented. As in other cases only one non-productive IgH chain gene rearrangement was amplified from H-RS cells, it was speculated whether, in these cases, the functionally rearranged IgH chain genes were lost. An alternative explanation might be that the rearranged genes could not be amplified owing to a high load of somatic mutations within the primer binding sites. Here, we showed that, in the HL-derived Epstein–Barr virus (EBV)-positive cell line L591, only one non-functional somatically mutated IgH gene rearrangement could be detected. The other potentially functional IgH gene rearrangement was lost as a result of an unbalanced translocation affecting the long arm of chromosome 14. Moreover, L591 cells express the EBV latent membrane proteins LMP1 and LMP2A, which might have contributed to the ‘escape’ of these cells from apoptosis within the germinal centre. We conclude that, apart from the introduction of ‘crippling mutations’ into the rearranged VDJ genes rearrangement, deletions of the IGH locus may be regarded as another mechanism to prevent the expression of a BCR in H-RS cells.

Hodgkin and Reed-Sternberg (H/RS) cells are characterized by chromosomal instability. Nevertheless, neither specific nor consistent chromosomal alterations could be characterized in H/RS cells. Microsatellite instability (MSI) is another form of genomic instability but its role in the pathogenesis of classical Hodgkin's disease (cHD) has not been investigated so far. We analyzed MSI and mismatch repair (MMR) protein expression in H/RS cells of cHD in order to assess genomic instability in these cells. Using a sensitive single cell approach, MSI-low was detected in a portion of single cells of the H/RS cell line L1236. Mutations of genes encoding for hMSH2 and hMLH1 were excluded by RT-PCR in L1236 cells. An analysis of pooled single H/RS cells of seven primary cases of cHD showed loss of heterozygosity for some allelic markers but absence of MSI in all 7 cases. Owing to a tight correlation between MSI-high, inactivating mutations of MMR genes and MMR protein expression in colon cancer, MMR protein expression commonly is used as a marker for MSI. In order to screen additional primary cases of cHD for MSI, we performed immunohistochemistry for hMSH2 and hMLH1 in 6 of the 7 cases analyzed by single cell PCR and 20 additional cases of cHD. H/RS cells from 25 out of 26 cases showed a nuclear staining pattern for hMSH2 and hMLH1 similar to germinal center B cells of non-malignant lymph nodes. These results indicate a proficient MMR system in most H/RS cells. It is concluded that a defect MMR system is unlikely to contribute to the malignant phenotype and genomic instability of H/RS cells in cHD.

International audience; Ubiquitous mitochondrial creatine kinase (uMtCK), a key enzyme in energy metabolism, was identified by differential display PCR to be specifically overexpressed in L1236, the first cell line of definite Hodgkin origin. RT-PCR confirmed overexpression of uMtCK in the L1236 cell line and the absence of cytosolic B-CK, which is co-expressed with MtCK physiologically. Cyclocreatine (cCr), whose phosphorylated form is a very poor substrate for CK, inhibited proliferation of the L1236 cell line nearly entirely. This inhibition by cCr was partially reversed by competition with creatine, which by itself had no effect on proliferation of the L1236 cell line. Although these results support a role of CK activity in the inhibitory action of cCr, it remains open whether the cCr effect is due to its inhibition of CK-linked energy metabolism or if alternative mechanisms have to be considered. Because the anti-proliferative effect of cCr was not due to induction of apoptosis, in contrast to most other anticancer agents, treatment with the creatine analogue cCr may represent an advantageous therapeutic approach for cells resistant to programmed cell death.

Members of the nuclear factor (NF)-kappaB family of transcription factors play a crucial role in cellular activation, immune responses, and oncogenesis. In most cells, they are kept inactive in the cytosol by complex formation with members of the inhibitor of NF-kappaB (IkappaB) family, whose degradation activates NF-kappaB in response to diverse stimuli. In Hodgkin's lymphoma (HL), high constitutive nuclear activity of NF-kappaB is characteristic of the malignant Hodgkin and Reed-Sternberg (H/RS) cells, which occur at low number in a background of nonneoplastic inflammatory cells. In single H/RS cells micromanipulated from histological sections of HL, we detect clonal deleterious somatic mutations in the IkappaBalpha gene in two of three Epstein-Barr virus (EBV)-negative cases but not in two EBV-positive cases (in which a viral oncogene may account for NF-kappaB activation). There was no evidence for IkappaBalpha mutations in two non-HL entities or in normal germinal center B cells. This study establishes deleterious IkappaBalpha mutations as the first recurrent genetic defect found in H/RS cells, indicating a role of IkappaBalpha defects in the pathogenesis of HL and implying that IkappaBalpha is a tumor suppressor gene.

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. Our sample consisted of n = 158 participants who had suffered lung cancer (diagnosis in the patient document) and n = 144 control group participants. Both groups had a history of smoking. Data on social networks were collected by asking participants whether they participated in a sports club and about the number of friends and relatives in their social environment. In addition, sociodemographic data (gender, age, education, marital status, residence and religion), physical activity and data on pack years (the cumulative number of cigarettes smoked by an individual, calculated by multiplying the number of cigarettes smoked per day by the number of years the person has smoked divided by 20) were collected to control for potential confounders. Logistic regression was used for the statistical analysis. Results The results reveal that participants who are physically active are at a lower risk of lung cancer than those who are not (adjusted OR = 0.53*; CI = 0.29-0.97). Older age and lower education seem also to be risk factors for the development of lung cancer. The extent of smoking, furthermore, measured by pack years is statistically significant. Active participation in a sports club, number of friends and relatives had no statistically significant influence on the development of the cancer. Conclusions The results of the study suggest that there is a lower risk for physically active participants to develop lung cancer. In the study sample, physical activity seemed to have a greater protective effect than participation in a sports club or social network of friends and relatives. Further studies have to investigate in more detail physical activity and other club participations.

Objective. In most cases of classic Hodgkin's disease (HD), Hodgkin and Reed-Sternberg (H-RS) cells clonally derive from germinal-center B cells. Within their rearranged immunoglobulin genes, somatic mutations rendering potentially functional immunoglobulin gene rearrangements nonfunctional were detected, indicating that H-RS cells do not express a B-cell receptor. Under physiologic conditions, these cells would undergo apoptosis within the germinal center. However, H-RS cells clonally expand, disseminate, and lead to clonal relapse of HD, indicating their resistance to induced programmed cell death. The underlying mechanism remains to be elucidated. Analysis of receptor-ligand interactions in primary H-RS cells is difficult to perform due to their scarcity in vivo and their low proliferation rate in vitro. Materials and Methods. Two B-cellular H-RS cell lines (L1236 and L428) were used to test for the expression of CD95 by flow cytometry and for the induction of apoptosis after incubation with CD95L obtained from retrovirally transduced murine myoblasts. Sequence analysis of CD95 cDNA obtained from these H-RS cell lines was performed. Results. Expression of CD95 on the cell surface was detected in both cell lines. However, after incubation with CD95L, the cells did not undergo apoptosis. To test whether mutations within the CD95 cDNA sequence caused resistance to apoptosis in H-RS cells, sequence analysis of CD95 cDNA obtained from L1236 and L428 was performed. In both cell lines, CD95 was not affected by somatic mutations. Conclusions . Our results indicate that the two H-RS cell lines L1236 and L428 are resistant to CD95-mediated apoptosis induced via CD95L, although wild-type CD95 is expressed. For further characterization of the mechanisms leading to prevention of apoptotic cell death in H-RS cells, it is necessary to determine impairments within the signaling cascade following CD95 activation.

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. Methods We used extended panels of arrayed antigens and determined autoantibody signatures of sera from patients with different kinds of lung cancer, different common non-tumor lung pathologies, and controls without any lung disease by a newly developed computer aided image analysis procedure. The resulting signatures were classified using linear kernel Support Vector Machines and 10-fold cross-validation. Results The novel approach allowed for discriminating lung cancer patients from controls without any lung disease with a specificity of 97.0%, a sensitivity of 97.9%, and an accuracy of 97.6%. The classification of stage IA/IB tumors and controls yielded a specificity of 97.6%, a sensitivity of 75.9%, and an accuracy of 92.9%. The discrimination of lung cancer patients from patients with non-tumor lung pathologies reached an accuracy of 88.5%. Conclusion We were able to separate lung cancer patients from subjects without any lung disease with high accuracy. Furthermore, lung cancer patients could be seprated from patients with other non-tumor lung diseases. These results provide clear evidence that blood-based tests open new avenues for the early diagnosis of lung cancer.