Establishment of the epithelial-specific transcriptome of normal and malignant human breast cells based on MPSS and array expression data.

INTRODUCTION: Diverse microarray and sequencing technologies have been widely used to characterise the molecular changes in malignant epithelial cells in breast cancers. Such gene expression studies to identify markers and targets in tumour cells are, however, compromised by the cellular heterogeneity of solid breast tumours and by the lack of appropriate counterparts representing normal breast epithelial cells. METHODS: Malignant neoplastic epithelial cells from primary breast cancers and luminal and myoepithelial cells isolated from normal human breast tissue were isolated by immunomagnetic separation methods. Pools of RNA from highly enriched preparations of these cell types were subjected to expression profiling using massively parallel signature sequencing (MPSS) and four different genome wide microarray platforms. Functional related transcripts of the differential tumour epithelial transcriptome were used for gene set enrichment analysis to identify enrichment of luminal and myoepithelial type genes. Clinical pathological validation of a small number of genes was performed on tissue microarrays. RESULTS: MPSS identified 6,553 differentially expressed genes between the pool of normal luminal cells and that of primary tumours substantially enriched for epithelial cells, of which 98% were represented and 60% were confirmed by microarray profiling. Significant expression level changes between these two samples detected only by microarray technology were shown by 4,149 transcripts, resulting in a combined differential tumour epithelial transcriptome of 8,051 genes. Microarray gene signatures identified a comprehensive list of 907 and 955 transcripts whose expression differed between luminal epithelial cells and myoepithelial cells, respectively. Functional annotation and gene set enrichment analysis highlighted a group of genes related to skeletal development that were associated with the myoepithelial/basal cells and upregulated in the tumour sample. One of the most highly overexpressed genes in this category, that encoding periostin, was analysed immunohistochemically on breast cancer tissue microarrays and its expression in neoplastic cells correlated with poor outcome in a cohort of poor prognosis estrogen receptor-positive tumours. CONCLUSION: Using highly enriched cell populations in combination with multiplatform gene expression profiling studies, a comprehensive analysis of molecular changes between the normal and malignant breast tissue was established. This study provides a basis for the identification of novel and potentially important targets for diagnosis, prognosis and therapy in breast cancer.

3D dose reconstruction for narrow beams using ion chamber array measurements.

3D dose reconstruction is a verification of the delivered absorbed dose. Our aim was to describe and evaluate a 3D dose reconstruction method applied to phantoms in the context of narrow beams. A solid water phantom and a phantom containing a bone-equivalent material were irradiated on a 6 MV linac. The transmitted dose was measured by using one array of a 2D ion chamber detector. The dose reconstruction was obtained by an iterative algorithm. A phantom set-up error and organ interfraction motion were simulated to test the algorithm sensitivity. In all configurations convergence was obtained within three iterations. A local reconstructed dose agreement of at least 3% / 3mm with respect to the planned dose was obtained, except in a few points of the penumbra. The reconstructed primary fluences were consistent with the planned ones, which validates the whole reconstruction process. The results validate our method in a simple geometry and for narrow beams. The method is sensitive to a set-up error of a heterogeneous phantom and interfraction heterogeneous organ motion.

Investigation of high-resolution functional magnetic resonance imaging by means of surface and array radiofrequency coils at 7 T.

In this investigation, high-resolution, 1x1x1-mm(3) functional magnetic resonance imaging (fMRI) at 7 T is performed using a multichannel array head coil and a surface coil approach. Scan geometry was optimized for each coil separately to exploit the strengths of both coils. Acquisitions with the surface coil focused on partial brain coverage, while whole-brain coverage fMRI experiments were performed with the array head coil. BOLD sensitivity in the occipital lobe was found to be higher with the surface coil than with the head array, suggesting that restriction of signal detection to the area of interest may be beneficial for localized activation studies. Performing independent component analysis (ICA) decomposition of the fMRI data, we consistently detected BOLD signal changes and resting state networks. In the surface coil data, a small negative BOLD response could be detected in these resting state network areas. Also in the data acquired with the surface coil, two distinct components of the positive BOLD signal were consistently observed. These two components were tentatively assigned to tissue and venous signal changes.

Tissue-based immune monitoring I: tumor core needle biopsies allow in-depth interrogation of the tumor microenvironment.

We sought to assess the feasibility and reproducibility of performing tissue-based immune characterization of the tumor microenvironment using CT-compatible needle biopsy material. Three independent biopsies were obtained intraoperatively from one metastatic epithelial ovarian cancer lesion of 7 consecutive patients undergoing surgical cytoreduction using a 16-gauge core biopsy needle. Core specimens were snap-frozen and subjected to immunohistochemistry (IHC) against human CD3, CD4, CD8, and FoxP3. A portion of the cores was used to isolate RNA for 1) real-time quantitative (q)PCR for CD3, CD4, CD8, FoxP3, IL-10 and TGF-beta, 2) multiplexed PCR-based T cell receptor (TCR) CDR3 Vβ region spectratyping, and 3) gene expression profiling. Pearson's correlations were examined for immunohistochemistry and PCR gene expression, as well as for gene expression array data obtained from different tumor biopsies. Needle biopsy yielded sufficient tissue for all assays in all patients. IHC was highly reproducible and informative. Significant correlations were seen between the frequency of CD3+, CD8+ and FoxP3+ T cells by IHC with CD3ε, CD8A, and FoxP3 gene expression, respectively, by qPCR (r=0.61, 0.86, and 0.89; all p< 0.05). CDR3 spectratyping was feasible and highly reproducible in each tumor, and indicated a restricted repertoire for specific TCR Vβ chains in tumor-infiltrating T cells. Microarray gene expression revealed strong correlation between different biopsies collected from the same tumor. Our results demonstrate a feasible and reproducible method of immune monitoring using CT-compatible needle biopsies from tumor tissue, thereby paving the way for sophisticated translational studies during tumor biological therapy.

Efficient targeted transcript discovery via array-based normalization of RACE libraries.

Rapid amplification of cDNA ends (RACE) is a widely used approach for transcript identification. Random clone selection from the RACE mixture, however, is an ineffective sampling strategy if the dynamic range of transcript abundances is large. To improve sampling efficiency of human transcripts, we hybridized the products of the RACE reaction onto tiling arrays and used the detected exons to delineate a series of reverse-transcriptase (RT)-PCRs, through which the original RACE transcript population was segregated into simpler transcript populations. We independently cloned the products and sequenced randomly selected clones. This approach, RACEarray, is superior to direct cloning and sequencing of RACE products because it specifically targets new transcripts and often results in overall normalization of transcript abundance. We show theoretically and experimentally that this strategy leads indeed to efficient sampling of new transcripts, and we investigated multiplexing the strategy by pooling RACE reactions from multiple interrogated loci before hybridization.

Subtelomeric 6p deletion: clinical and array-CGH characterization in two patients

We report on two patients with de novo subtelomeric terminal deletion of chromosome 6p. Patient 1 is an 8-month-old female born with normal growth parameters, typical facial features of 6pter deletion, bilateral corectopia, and protruding tongue. She has severe developmental delay, profound bilateral neurosensory deafness, poor visual contact, and hypsarrhythmia since the age of 6 months. Patient 2 is a 5-year-old male born with normal growth parameters and unilateral hip dysplasia; he has a characteristic facial phenotype, bilateral embryotoxon, and moderate mental retardation. Further characterization of the deletion, using high-resolution array comparative genomic hybridization (array-CGH; Agilent Human Genome kit 244 K), revealed that Patient 1 has a 8.1 Mb 6pter-6p24.3 deletion associated with a contiguous 5.8 Mb 6p24.3-6p24.1 duplication and Patient 2 a 5.7 Mb 6pter-6p25.1 deletion partially overlapping with that of Patient 1. Complementary FISH and array analysis showed that the inv del dup(6) in Patient 1 originated de novo. Our results demonstrate that simple rearrangements are often more complex than defined by standard techniques. We also discuss genotype-phenotype correlations including previously reported cases of deletion 6p.

Nuclear factor I-C links platelet-derived growth factor and transforming growth factor beta1 signaling to skin wound healing progression.

Transforming growth factor beta (TGF-beta) and platelet-derived growth factor A (PDGFAlpha) play a central role in tissue morphogenesis and repair, but their interplay remain poorly understood. The nuclear factor I C (NFI-C) transcription factor has been implicated in TGF-beta signaling, extracellular matrix deposition, and skin appendage pathologies, but a potential role in skin morphogenesis or healing had not been assessed. To evaluate this possibility, we performed a global gene expression analysis in NFI-C(-/-) and wild-type embryonic primary murine fibroblasts. This indicated that NFI-C acts mostly to repress gene expression in response to TGF-beta1. Misregulated genes were prominently overrepresented by regulators of connective tissue inflammation and repair. In vivo skin healing revealed a faster inflammatory stage and wound closure in NFI-C(-/-) mice. Expression of PDGFA and PDGF-receptor alpha were increased in wounds of NFI-C(-/-) mice, explaining the early recruitment of macrophages and fibroblasts. Differentiation of fibroblasts to contractile myofibroblasts was also elevated, providing a rationale for faster wound closure. Taken together with the role of TGF-beta in myofibroblast differentiation, our results imply a central role of NFI-C in the interplay of the two signaling pathways and in regulation of the progression of tissue regeneration.

Olfactory traces and spatial learning in rats.

We conducted an experiment to assess the use of olfactory traces for spatial orientation in an open environment in rats, Rattus norvegicus. We trained rats to locate a food source at a fixed location from different starting points, in the presence or absence of visual information. A single food source was hidden in an array of 19 petri dishes regularly arranged in an open-field arena. Rats were trained to locate the food source either in white light (with full access to distant visuospatial information) or in darkness (without any visual information). In both cases, the goal was in a fixed location relative to the spatial frame of reference. The results of this experiment revealed that the presence of noncontrolled olfactory traces coherent with the spatial frame of reference enables rats to locate a unique position as accurately in darkness as with full access to visuospatial information. We hypothesize that the olfactory traces complement the use of other orientation mechanisms, such as path integration or the reliance on visuospatial information. This experiment demonstrates that rats can rely on olfactory traces for accurate orientation, and raises questions about the establishment of such traces in the absence of any other orientation mechanism. Copyright 1998 The Association for the Study of Animal Behaviour.

A novel gene expression signature in peripheral blood mononuclear cells for early detection of colorectal cancer.

BACKGROUND: Early detection and treatment of colorectal adenomatous polyps (AP) and colorectal cancer (CRC) is associated with decreased mortality for CRC. However, accurate, non-invasive and compliant tests to screen for AP and early stages of CRC are not yet available. A blood-based screening test is highly attractive due to limited invasiveness and high acceptance rate among patients. AIM: To demonstrate whether gene expression signatures in the peripheral blood mononuclear cells (PBMC) were able to detect the presence of AP and early stages CRC. METHODS: A total of 85 PBMC samples derived from colonoscopy-verified subjects without lesion (controls) (n = 41), with AP (n = 21) or with CRC (n = 23) were used as training sets. A 42-gene panel for CRC and AP discrimination, including genes identified by Digital Gene Expression-tag profiling of PBMC, and genes previously characterised and reported in the literature, was validated on the training set by qPCR. Logistic regression analysis followed by bootstrap validation determined CRC- and AP-specific classifiers, which discriminate patients with CRC and AP from controls. RESULTS: The CRC and AP classifiers were able to detect CRC with a sensitivity of 78% and AP with a sensitivity of 46% respectively. Both classifiers had a specificity of 92% with very low false-positive detection when applied on subjects with inflammatory bowel disease (n = 23) or tumours other than CRC (n = 14). CONCLUSION: This pilot study demonstrates the potential of developing a minimally invasive, accurate test to screen patients at average risk for colorectal cancer, based on gene expression analysis of peripheral blood mononuclear cells obtained from a simple blood sample.

Nanosensors for cancer detection.

Cancer is a major burden in today's society and one of the leading causes of death in industrialised countries. Various avenues for the detection of cancer exist, most of which rely on standard methods, such as histology, ELISA, and PCR. Here we put the focus on nanomechanical biosensors derived from atomic force microscopy cantilevers. The versatility of this novel technology has been demonstrated in different applications and in some ways surpasses current technologies, such as microarray, quartz crystal microbalance and surface plasmon resonance. The technology enables label free biomarker detection without the necessity of target amplification in a total cellular background, such as BRAF mutation analysis in malignant melanoma. A unique application of the cantilever array format is the analysis of conformational dynamics of membrane proteins associated to surface stress changes. Another development is characterisation of exhaled breath which allows assessment of a patient's condition in a non-invasive manner.

Profiling of T-cell receptor signaling complex assembly in human CD4 T-lymphocytes using RP protein arrays.

The RP protein (RPP) array approach immobilizes minute amounts of cell lysates or tissue protein extracts as distinct microspots on NC-coated slide. Subsequent detection with specific antibodies allows multiplexed quantification of proteins and their modifications at a scale that is beyond what traditional techniques can achieve. Cellular functions are the result of the coordinated action of signaling proteins assembled in macromolecular complexes. These signaling complexes are highly dynamic structures that change their composition with time and space to adapt to cell environment. Their comprehensive analysis requires until now relatively large amounts of cells (>5 x 10(7)) due to their low abundance and breakdown during isolation procedure. In this study, we combined small scale affinity capture of the T-cell receptor (TCR) and RPP arrays to follow TCR signaling complex assembly in human ex vivo isolated CD4 T-cells. Using this strategy, we report specific recruitment of signaling components to the TCR complex upon T-cell activation in as few as 0.5 million of cells. Second- to fourth-order TCR interacting proteins were accurately quantified, making this strategy specially well-suited to the analysis of membrane-associated signaling complexes in limited amounts of cells or tissues, e.g., ex vivo isolated cells or clinical specimens.

Fungi, bacteria and soil pH: the oxalate-carbonate pathway as a model for metabolic interaction

The oxalatecarbonate pathway involves the oxidation of calcium oxalate to low-magnesium calcite and represents a potential long-term terrestrial sink for atmospheric CO2. In this pathway, bacterial oxalate degradation is associated with a strong local alkalinization and subsequent carbonate precipitation. In order to test whether this process occurs in soil, the role of bacteria, fungi and calcium oxalate amendments was studied using microcosms. In a model system with sterile soil amended with laboratory cultures of oxalotrophic bacteria and fungi, the addition of calcium oxalate induced a distinct pH shift and led to the final precipitation of calcite. However, the simultaneous presence of bacteria and fungi was essential to drive this pH shift. Growth of both oxalotrophic bacteria and fungi was confirmed by qPCR on the frc (oxalotrophic bacteria) and 16S rRNA genes, and the quantification of ergosterol (active fungal biomass) respectively. The experiment was replicated in microcosms with non-sterilized soil. In this case, the bacterial and fungal contribution to oxalate degradation was evaluated by treatments with specific biocides (cycloheximide and bronopol). Results showed that the autochthonous microflora oxidized calcium oxalate and induced a significant soil alkalinization. Moreover, data confirmed the results from the model soil showing that bacteria are essentially responsible for the pH shift, but require the presence of fungi for their oxalotrophic activity. The combined results highlight that the interaction between bacteria and fungi is essential to drive metabolic processes in complex environments such as soil.

Waddlia chondrophila Infects and Multiplies in Ovine Trophoblast Cells Stimulating an Inflammatory Immune Response.

BACKGROUND: Waddlia chondrophila (W. chondrophila) is an emerging abortifacient organism which has been identified in the placentae of humans and cattle. The organism is a member of the order Chlamydiales, and shares many similarities at the genome level and in growth studies with other well-characterised zoonotic chlamydial abortifacients, such as Chlamydia abortus (C. abortus). This study investigates the growth of the organism and its effects upon pro-inflammatory cytokine expression in a ruminant placental cell line which we have previously utilised in a model of C. abortus pathogenicity. METHODOLOGY/PRINCIPAL FINDINGS: Using qPCR, fluorescent immunocytochemistry and electron microscopy, we characterised the infection and growth of W. chondrophila within the ovine trophoblast AH-1 cell line. Inclusions were visible from 6 h post-infection (p.i.) and exponential growth of the organism could be observed over a 60 h time-course, with significant levels of host cell lysis being observed only after 36 h p.i. Expression of CXCL8, TNF-α, IL-1α and IL-1β were determined 24 h p.i. A statistically significant response in the expression of CXCL8, TNF-α and IL-1β could be observed following active infection with W. chondrophila. However a significant increase in IL-1β expression was also observed following the exposure of cells to UV-killed organisms, indicating the stimulation of multiple innate recognition pathways. CONCLUSIONS/SIGNIFICANCE: W. chondrophila infects and grows in the ruminant trophoblast AH-1 cell line exhibiting a complete chlamydial replicative cycle. Infection of the trophoblasts resulted in the expression of pro-inflammatory cytokines in a dose-dependent manner similar to that observed with C. abortus in previous studies, suggesting similarities in the pathogenesis of infection between the two organisms.

Effect of vitamin D on Epstein-Barr (EBV)-specific CD8+T cells in patients with early multiple sclerosis (MS)

Background: Infection with EBV and a lack in vitamin D may be important environmental triggers of MS. 1,25-(OH)2D3 mediates a shift of antigen presenting cells (APC) and CD4+ T cells to a less inflammatory profile. Although CD8+ T cells do express the vitamin D receptor, a direct effect of 1,25(OH)2D3 on these cells has not been demonstrated until now. Since CD8+ T cells are important immune mediators of the inflammatory response in MS, we examined whether vitamin D directly affects the CD8+ T cell response, and more specifically if it modulates the EBV-specific CD8+ T cell response. Material and Methods: To explore whether the vitamin D status may influence the pattern of the EBV-specific CD8+ T cell response, PBMC of 10 patients with early MS and 10 healthy controls (HC) were stimulated with a pool of immunodominant 8-10 mer peptide epitopes known to elicit CD8+ T cell responses. PBMC were stimulated with this EBV CD8 peptide pool, medium (negative control) or anti- CD3/anti-CD28 beads (positive control). The following assays were performed: ELISPOT to assess the secretion of IFN-gamma by T cells in general; cytometric beads array (CBA) and ELISA to determine whichcytokines were released by EBV-specific CD8+ T cells after six days of culture; and intracellular cytokine staining assay to determine by which subtype of T cells secreted given cytokines. To examine whether vitamin D could directly modulate CD8+ T cell immune responses, we depleted CD4+ T cells using negative selection. Results: We found that pre-treatment of vitamin D had an antiinflammatory action on both EBV-specific CD8+ T cells and on CD3/ CD28-stimulated T cells: secretion of pro-inflammatory cytokines (IFNgamma and TNF-alpha) was decreased, whereas secretion of antiinflammatory cytokines (IL-5 and TGF-beta) was increased. At baseline, CD8+ T cells of early MS patients showed a higher secretion of TNFalpha and lower secretion of IL-5. Addition of vitamin D did not restore the same levels of both cytokines as compared to HC. Vitamin D-pretreated CD8+T cells exhibited a decreased secretion of IFN-gamma and TNF-alpha, even after depletion of CD4+ T cells from culture. Conclusion: Vitamin D has a direct anti-inflammatory effect on CD8+ T cells independently from CD4+ T cells. CD8+ T cells of patients with earlyMS are less responsive to the inflammatory effect of vitamin D than HC, pointing toward an intrinsic dysregulation of CD8+ T cells. The modulation of EBV-specific CD8+T cells by vitaminDsuggests that there may be interplay between these twomajor environmental factors of MS. This study was supported by a grant from the Swiss National Foundation (PP00P3-124893), and by an unrestricted research grant from Bayer to RDP.

Genomic, Epigenomic and Transcriptomic Molecular Characterization of Splenic Marginal Zone Lymphoma Reveals Recurrent Abnormalities in miR-182

Splenic marginal zone lymphoma (SMZL) is a low grade B-cell non-Hodgkin's lymphoma. The molecular pathology of this entity remains poorly understood. To characterise this lymphoma at the molecular level, we performed an integrated analysis of 1) genome wide genetic copy number alterations 2) gene expression profiles and 3) epigenetic DNA methylation profiles.We have previously shown that SMZL is characterised by recurrent alterations of chromosomes 7q, 6q, 3q, 9q and 18; however, gene resolution oligonucleotide array comparative genomic hybridisation did not reveal evidence of cryptic amplification or deletion in these regions. The most frequently lost 7q32 region contains a cluster of miRNAs. qRT-PCR revealed that three of these (miR-182/96/183) show underexpression in SMZL, and miR-182 is somatically mutated in >20% of cases of SMZL, as well as in >20% of cases of follicular lymphoma, and between 5-15% of cases of chronic lymphocytic leukaemia, MALT-lymphoma and hairy cell leukaemia. We conclude that miR-182 is a strong candidate novel tumour suppressor miRNA in lymphoma.The overall gene expression signature of SMZL was found to be strongly distinct fromthose of other lymphomas. Functional analysis of gene expression data revealed SMZL to be characterised by abnormalities in B-cell receptor signalling (especially through the CD19/21-PI3K/AKT pathway) and apoptotic pathways. In addition, genes involved in the response to viral infection appeared upregulated. SMZL shows a unique epigenetic profile, but analysis of differentially methylated genes showed few with methylation related transcriptional deregulation, suggesting that DNA methylation abnormalities are not a critical component of the SMZL malignant phenotype.