Comparison of Whole Blood and Peripheral Blood Mononuclear Cell Gene Expression for Evaluation of the Perioperative Inflammatory Response in Patients with Advanced Heart Failure

Background: Heart failure (HF) prevalence is increasing in the United States. Mechanical Circulatory Support (MCS) therapy is an option for Advanced HF (AdHF) patients. Perioperatively, multiorgan dysfunction (MOD) is linked to the effects of device implantation, augmented by preexisting HF. Early recognition of MOD allows for better diagnosis, treatment, and risk prediction. Gene expression profiling (GEP) was used to evaluate clinical phenotypes of peripheral blood mononuclear cells (PBMC) transcriptomes obtained from patients’ blood samples. Whole blood (WB) samples are clinically more feasible, but their performance in comparison to PBMC samples has not been determined. Methods: We collected blood samples from 31 HF patients (57¡15 years old) undergoing cardiothoracic surgery and 7 healthy age-matched controls, between 2010 and 2011, at a single institution. WB and PBMC samples were collected at a single timepoint postoperatively (median day 8 postoperatively) (25–75% IQR 7–14 days) and subjected to Illumina single color Human BeadChip HT12 v4 whole genome expression array analysis. The Sequential Organ Failure Assessment (SOFA) score was used to characterize the severity of MOD into low (# 4 points), intermediate (5–11), and high ($ 12) risk categories correlating with GEP. Results: Results indicate that the direction of change in GEP of individuals with MOD as compared to controls is similar when determined from PBMC versus WB. The main enriched terms by Gene Ontology (GO) analysis included those involved in the inflammatory response, apoptosis, and other stress response related pathways. The data revealed 35 significant GO categories and 26 pathways overlapping between PBMC and WB. Additionally, class prediction using machine learning tools demonstrated that the subset of significant genes shared by PBMC and WB are sufficient to train as a predictor separating the SOFA groups. Conclusion: GEP analysis of WB has the potential to become a clinical tool for immune-monitoring in patients with MO

Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation

Regulatory T cells (T(reg) cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (T(eff) cell) function and gain of suppressive activity by T(reg) cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T(reg) cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3' untranslated region. Release of SATB1 from the control of Foxp3 in T(reg) cells caused loss of suppressive function, establishment of transcriptional T(eff) cell programs and induction of T(eff) cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining T(reg) cell functionality.Marc Beyer... Timothy Sadlon...Simon C Barry... et al

Cancer vaccine enhanced, non-tumor-reactive CD8(+) T cells exhibit a distinct molecular program associated with "division arrest anergy"

Immune-mediated tumor rejection relies on fully functional T-cell responses and neutralization of an adverse tumor microenvironment. In clinical trials, we detected peptide-specific but non-tumor-reactive and therefore not fully functional CD8(+) T cells post-vaccination against tumor antigens. Understanding the molecular mechanisms behind nontumor reactivity will be a prerequisite to overcome this CD8(+) T-cell deviation. We report that these non-tumor-reactive CD8(+) T cells are characterized by a molecular program associated with hallmarks of "division arrest anergy." Non-tumor-reactive CD8(+) T cells are characterized by coexpression of CD7, CD25, and CD69 as well as elevated levels of lck(p505) and p27(kip1). In vivo quantification revealed high prevalence of non-tumor-reactive CD8(+) T cells with increased levels during cancer vaccination. Furthermore, their presence was associated with a trend toward shorter survival. Dynamics and frequencies of non-target-reactive CD8(+) T cells need to be further addressed in context of therapeutic vaccine development in cancer, chronic infections, and autoimmune diseases

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

©2011 AAC

Crosstalk between keratinocytes and adaptive immune cells in an IkappaBalpha protein-mediated inflammatory disease of the skin.

Inflammatory diseases at epithelial borders develop from aberrant interactions between resident cells of the tissue and invading immunocytes. Here, we unraveled basic functions of epithelial cells and immune cells and the sequence of their interactions in an inflammatory skin disease. Ubiquitous deficiency of the I?B? protein (Ikba?/?) as well as concomitant deletion of Ikba specifically in keratinocytes and T cells (IkbaK5?/K5? lck?/lck?) resulted in an inflammatory skin phenotype that involved the epithelial compartment and depended on the presence of lymphocytes as well as tumor necrosis factor and lymphotoxin signaling. In contrast, mice with selective ablation of Ikba in keratinocytes or lymphocytes showed inflammation limited to the dermal compartment or a normal skin phenotype, respectively. Targeted deletion of RelA from epidermal keratinocytes completely rescued the inflammatory skin phenotype of Ikba?/? mice. This finding emphasizes the important role of aberrant NF-?B activation in both keratinocytes and lymphocytes in the development of the observed inflammatory skin changes

Comparison of Blood RNA Extraction Methods Used for Gene Expression Profiling in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that causes death within a mean of 2-3 years from symptom onset. There is no diagnostic test and the delay from symptom onset to diagnosis averages 12 months. The identification of prognostic and diagnostic biomarkers in ALS would facilitate earlier diagnosis and faster monitoring of treatments. Gene expression profiling (GEP) can help to identify these markers as well as therapeutic targets in neurological diseases. One source of genetic material for GEP in ALS is peripheral blood, which is routinely accessed from patients. However, a high proportion of globin mRNA in blood can mask important genetic information. A number of methods allow safe collection, storage and transport of blood as well as RNA stabilisation, including the PAXGENE and TEMPUS systems for the collection of whole blood and LEUKOLOCK which enriches for the leukocyte population. Here we compared these three systems and assess their suitability for GEP in ALS. We collected blood from 8 sporadic ALS patients and 7 controls. PAXGENE and TEMPUS RNA extracted samples additionally underwent globin depletion using GlobinClear. RNA was amplified and hybridised onto Affymetrix U133 Plus 2.0 arrays. Lists of genes differentially regulated in ALS patients and controls were created for each method using the R package PUMA, and RT-PCR validation was carried out on selected genes. TEMPUS/GlobinClear, and LEUKOLOCK produced high quality RNA with sufficient yield, and consistent array expression profiles. PAXGENE/GlobinClear yield and quality were lower. Globin depletion for PAXGENE and TEMPUS uncovered the presence of over 60% more transcripts than when samples were not depleted. TEMPUS/GlobinClear and LEUKOLOCK gene lists respectively contained 3619 and 3047 genes differentially expressed between patients and controls. Real-time PCR validation revealed similar reliability between these two methods and gene ontology analyses revealed similar pathways differentially regulated in disease compared to controls