Transcriptome Of Peripheral Blood Mononuclear Cells Research Articles

Transcriptomes of whole blood and PBMC in chickens

Global transcriptome analysis of chicken whole blood to discover biomarkers of different phenotypes or physiological disorders has never been investigated so far. Whole blood provides significant advantages, allowing large scale and non-invasive sampling. However, generation of gene expression data from the blood of non-mammalian species remains a challenge, notably due to the nucleated red blood cells, hindering the use of well-established protocols. The aim of this study was to analyze the relevance of using whole blood cells (WB) to find biomarkers, instead of Peripheral Blood Mononuclear Cells (PBMC), usually chosen for immune challenges. RNA sources from WB and PBMC was characterized by microarray analysis. Our results show that the quality and quantity of RNA obtained from WB was suitable for further analyses, although the quality was lower than that from PBMC. The transcriptome profiling comparison revealed that the majority of genes were expressed in both WB and PBMC. Hemoglobin subunits were the major transcripts in WB, whereas the most enriched biological process was related to protein catabolic process. Most of the over-represented transcripts in PBMC were implicated in functions specific to thrombocytes, like coagulation and platelet activation, probably due to the large proportion of this nucleated cell type in chicken PBMC. Functions related to B and T cells and to other immune functions were also enriched in the PBMC subset. We conclude that WB is more suitable for large scale immunity oriented studies and other biological processes that have been poorly investigated so far.

Abstract P1-15-02: Gene expression profiling of doxorubicin cardiotoxicity in peripheral blood cells of breast cancer patients

Abstract Background: Doxorubicin (DOX), a widely used anti-cancer drug for treatment of breast cancer is known for its cardiotoxicity. DOX cardiotoxicity is cumulative-dose-dependent and begins with the first dose of chemotherapy. To date, no biomarker for early presymptomatic detection of DOX cardiotoxicity has been validated. Our previous data indicated that peripheral blood mononuclear cells (PBMCs) can be used as a surrogate tissue for identification of biomarkers for DOX cardiotoxicity. The aim of this study was to analyze PBMC gene expression induced by a single dose of DOX-based chemotherapy in breast cancer patients and correlated the data with DOX-induced cardiotoxicity. Materials and Methods. Blood samples of 33 women treated for breast cancer with DOX-based chemotherapy were collected before the start and after the first cycle of chemotherapy. Total RNA was isolated from PBMC and whole-genome gene expression was performed using Illumina HumanHT-12 v4 Expression BeadChip array. Gene expression data were log2 – transformed and gene transcripts with average log2-intensities > 7 were considered to be expressed. The group-specific means were analyzed via repeated-measures with ANOVA for expression changes after DOX. Genes with p-value<0.05 were considered differentially expressed. Cardiac function was assessed before and after the completion of chemotherapy by echocardiogram and/or multigated acquisition scan. An absolute decrease of left ventricle ejection fraction >10% or <55% was considered abnormal. Differentially expressed genes (DEG) of patients who developed abnormal LVEF decrease were compared with DEG of patients who did not. Results. A single dose of DOX-based chemotherapy resulted in 235 DEG in PBMC (P<0.05, FDR<0.05), mapped to cell death, oxygen transport and iron ion binding. Further analysis identified 87 DEG in the PBMC of eight (n=8) women who developed abnormal decline in LVEF from the baseline in comparison with women who did not (n=25). Most of the 87 DEG encode proteins secreted by activated neutrophils, such as alpha-defensins, arginase, cathepsin G, elastase, haptoglobin. The functional analysis of the 87 DEG showed enrichment for inflammatory response, immune response, cell death and peptidase activity. Discussion. The results from this study indicated that elevated neutrophil-associated transcripts in the early stages of DOX-based chemotherapy were independent of the neutrophil count. These data suggest an association between the neutrophils activation after a single dose of DOX-based chemotherapy and later impairment of cardiac function. The early PBMC transcriptome signature can be used in the future development of biomarkers for DOX-associated cardiotoxicity. Citation Format: Todorova VK, Siegel ER, Makhoul I, Marquette M, Wei JY, Klimberg VS. Gene expression profiling of doxorubicin cardiotoxicity in peripheral blood cells of breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-15-02.

Iron, Expression of the Pattern Recognition Receptor-Inflammasome System, and Early Death in Adults with Sickle Cell Disease

RationalePatients with sickle cell disease (SCD) are marked by chronic inflammation but the cause of this inflammation and the relevance to patient survival are unknown.ObjectiveTo assess the relationship between iron, inflammation and early death in SCD.Methods and ResultsUsing peripheral blood mononuclear cell transcriptome profile hierarchical clustering, we classified 24 patients and 10 controls in clusters with significantly different expression of genes known to be regulated by iron. Subsequent gene set enrichment analysis showed that many genes associated with the high iron cluster were involved in innate immunity pattern recognition receptor (PRR) pathways. These included Toll-like receptors TLR4, TLR7 and TLR8; NOD-like receptors NLRP3 and NLRC4; C-type lectin receptor CLEC7A/Dectin-1; humoral pattern recognition receptor pentraxin-3, the common inflammasome effector caspase-1 and one of its substrates, IL-18. Quantitative PCR confirmed quantitative expression of several representative genes and showed that ferritin light chain, TLR4 and interleukin-6 mRNAs were expressed over 100-fold more highly in SCD patients than in controls (P<0.001). In a Mendelian randomization experiment, 14 patients with ferroportin Q248H variant that causes intracellular iron accumulation had significantly higher levels of interleukin-6 and C-reactive protein (CRP) compared to 14 patients with the wild type allele, implying a causal effect (P<0.05). Finally, level of CRP, also a humoral pattern recognition receptor, predicted mortality in the NIH sickle cell cohort (n=412, ClinicalTrials.gov identifier: NCT00011648). Patients in the highest quartile (CRP>0.8 mg/dL) had the highest mortality, and those with the lowest level (CRP<0.2mg/dL, lower limit of detection) had the lowest mortality (P=0.0017, median follow-up 47 months, IQR 24-82, range 2-132)(figure). CRP was independently associated with mortality in a Cox proportional hazards regression model after adjustment for other covariates previously published as associated with mortality (HR 2.8, 95%CI 1.03-4.2, P<0.001, adjusted for white blood cell count, age, transferrin, NT-proBNP and elevated tricuspid regurgitant flow velocity).ConclusionsThe SCD PBMC transcriptome reflects high intracellular iron exposure, which is associated with inflammation and mortality. The associations found in this study support a hypothetical model in which high intracellular iron in macrophages promotes clinically significant inflammatory pathways involving pattern recognition receptors and innate immune function, identifying potential targets for pharmacological intervention with drugs already approved for inflammasome-mediated disorders. [Display omitted] Figure. Kaplan Meier curve showing a significant difference in survival between patients with low and high CRP. SCD Patients (n=412) of the NIH pulmonary hypertension screening cohort (ClinicalTrials.gov identifier: NCT00011648) Patients were divided in high, intermediate and low CRP groups. Kaplan-Meier survival curves show that mortality rate significantly increased according to CRP group.(P=0.0017).Five year mortality percentages for the low, intermediate and high CRP groups were respectively 12.4%, 20.5 and 25.8%. In a multivariate analysis, CRP was an independent predictor of mortality. Disclosuresvan Beers:Novartis: Research Funding.

118 COMPARISON OF TRANSCRIPTOME PROFILES FROM ENDOMETRIAL CARUNCLES AND PERIPHERAL BLOOD MONONUCLEAR CELLS REVEAL COMMON AND TISSUE-SPECIFIC BIOLOGICAL PROCESSES REGULATED AT IMPLANTATION IN SHEEP

In mammals, implantation is associated with major changes in gene profiles in the female reproductive tract. Molecular signatures of the endometrium have also been shown to vary according to the ability of the embryo to develop to term. Nevertheless, analysing endometrial gene patterns during implantation is incompatible with the maintenance of pregnancy. Therefore early determination of pregnancy issue requires a noninvasive method. Peripheral blood mononuclear cells (PBMC) could represent such an alternative but their reaction to the presence of an implanting embryo has to be investigated. The aim of this study was to investigate gene expression profiles of endometrial caruncular tissue (CAR) and PBMC collected from pregnant ewes (n = 4) and nonpregnant ewes inseminated with inactivated sperm (n = 4) at Day 15 after oestrus. Differentially expressed genes (DEG) were identified using an ovine custom-designed array derived from the ovine 15K Agilent array (Ruscanu et al. 2013 J. Virol. 87, 9333–9343). Data were normalized by Loess and analysed by a linear model in the Limma R package. P-values were corrected using the Benjamini and Hochberg procedure. Comparing pregnancy versus nonpregnancy led to the identification of 2826 DEG in CAR (P &lt; 0.05) and 396 DEG in PBMC (P &lt; 0.10; 265 DEG common with CAR). Ingenuity Pathway Analysis (IPA; Ingenuity Inc., Mountain View, CA, USA) analysis of the 396 PBMC-related DEG revealed 72 overrepresented biological functions (P &lt; 0.001). Among the 15 most overrepresented functions, 13 were common between CAR and PBMC and were mostly related to the immune system, as “infectious disease”, “cell-to-cell signalling and interaction”, “immunological disease”, “immune cell trafficking and inflammatory response”. Using the downstream effect analysis (DEA) of IPA, we identified 163 functions predicted to be increased and 8 functions predicted to be decreased for the CAR DEG dataset, whereas 12 functions were predicted to be increased and 40 functions predicted to be decreased for the PBMC DEG dataset. Interferon (IFN) signalling was strongly present in both datasets, with 44% of PBMC DEG and 29% of CAR DEG found to be related to IFN type I response according to the Interferome database (www.interferome.org). A selection of 12 DEG was validated by qRT-PCR in CAR, intercaruncular areas, and PBMC using 8 pregnant and various groups of nonpregnant ewes (n = 7–9/group). Our data show that PBMC transcriptome is influenced by early pregnancy in sheep, including a major impact of IFN type I such as IFN tau, the signal of maternal recognition of pregnancy. Identifying relevant circulating biomarkers reflecting the quality of the embryo will require further investigation. The authors thank UCEA team (INRA), B. Jost (IGBMC) and F. Moreews (Sigenae).

Osteopontin supplementation of formula shifts the peripheral blood mononuclear cell transcriptome to be more similar to breastfed infants (38.3)

OPN is a key modulator in cell‐mediated immune and anti‐inflammatory responses that is present in breast milk, but not infant formula. A randomized clinical trial was conducted to study the impact of bovine OPN in formula on infant PBMC gene expression. Mothers chose to either breast‐(BF) or formula‐feed their infant. Formula‐fed infants received: formula (FF), formula with 65 mg/L (F65) or 130 mg/L (F130) OPN. Blood samples were collected at 1, 4 and 6 mos of age, PBMC were isolated, RNA extracted and gene expression assessed using the Affymetrix HuGene 2.0ST gene chip. To evaluate patterns of gene expression, Weighted Gene Correlation Network Analysis (WGCNA) was performed on 6,441 probe sets with an overall 4x3 ANOVA FDR p‐value&lt;0.3. Three modules (4066 probe sets) identified patterns where FF65 infants were similar to BF and different from FF and F130 across time. Functional analysis was performed with MetaCore. Pathways with higher expression in BF and F65 focused on cell proliferation and cell‐cell adhesion, supporting increased production and maturation of immune cells. In contrast, PBMC from FF and F130 infants expressed more genes related to immunoglobulin synthesis and receptors, including a gene associated with risk of childhood allergy. Thus, supplementing infant formula with OPN shifted PBMC gene expression to be more similar to BF and may support improved immune development.Grant Funding Source: Supported by: Arla Foods Ingredients and Biostime

Global Analyses of Human Immune Variation Reveal Baseline Predictors of Postvaccination Responses

A major goal of systems biology is the development of models that accurately predict responses to perturbation. Constructing such models requires the collection of dense measurements of system states, yet transformation of data into predictive constructs remains a challenge. To begin to model human immunity, we analyzed immune parameters in depth both at baseline and in response to influenza vaccination. Peripheral blood mononuclear cell transcriptomes, serum titers, cell subpopulation frequencies, and B cell responses were assessed in 63 individuals before and after vaccination and were used to develop a systematic framework to dissect inter- and intra-individual variation and build predictive models of postvaccination antibody responses. Strikingly, independent of age and pre-existing antibody titers, accurate models could be constructed using pre-perturbation cell populations alone, which were validated using independent baseline time points. Most of the parameters contributing to prediction delineated temporally stable baseline differences across individuals, raising the prospect of immune monitoring before intervention.

Peripheral blood mononuclear cell (PBMC) gene expression in human infants shifts from innate to adaptive immunity in the first six months of life (247.5)

The newborn immune system is developmentally naïve, but undergoes rapid maturation upon exposure to environmental antigens. As part of a clinical trial in infants aimed at assessing how diet impacts PBMC transcriptome, genes that were developmentally regulated independent of diet‐ were discovered. Blood samples were obtained from breast‐fed (n=60) and three sets of formula‐fed (n=186 total) infants at 1, 4 and 6 mos of age. PBMC were isolated, RNA extracted and gene expression assessed using the Affymetrix HuGene 2.0ST gene chip. To evaluate patterns of gene expression, Weighted Gene Correlation Network Analysis (WGCNA) was performed on 6,441 probe sets with an overall 4x3 ANOVA FDR p‐value&lt;0.3. Three gene clusters with similar patterns of expression in each diet (1 vs. 4 &amp; 6 mos) encompassing 1085 genes were identified. Functional analysis was performed with MetaCore. Pathways with higher expression at 1‐mo reflected cellular proliferation, innate defense processes (defensins, hydrogen peroxide‐induced killing) and Th17‐derived cytokines. By 4‐ to 6‐mos of age, genes associated with humoral immunoglobulin‐mediated immunity and interferon and IL‐4 signaling predominated. These data illustrate for the first time the developmental ontogeny of gene expression in circulating immune cells of human infants.Grant Funding Source: Arla Foods and Biostime

Transcriptional profiling of left ventricle and peripheral blood mononuclear cells in a rat model of postinfarction heart failure

BackgroundMyocardial infarction (MI) often results in left ventricular (LV) remodeling followed by heart failure (HF). It is of great clinical importance to understand the molecular mechanisms that trigger transition from compensated LV injury to HF and to identify relevant diagnostic biomarkers. The aim of this study was to investigate gene expression in the LV and to evaluate their reflection in peripheral blood mononuclear cells (PBMCs).MethodsMI was induced in rats by ligation of the proximal left coronary artery. Rats with small, moderate, and large MI size were included into the experiment two months after the operation. The development of heart failure was estimated by echocardiography and catheterization. Microarrays were used to compare the LV and PBMCs transcriptomes of control and experimental animals.ResultsOnly rats with a large MI developed extensive LV remodeling and heart failure. 840 transcripts were altered in LV of failing hearts, and especially numerous were those associated with the extracellular matrix. In contrast, no significant gene expression changes were seen in LVs of rats with moderate or small MI that had compensated LV injury. We showed that ceruloplasmin was similarly overexpressed in the heart and blood in response to HF, whereas downregulation of tetraspanin 12 was significant only in the PBMCs.ConclusionA large size of infarcted area is critical for progression of LV remodeling and HF development, associated with altered gene expression in the heart. Ceruloplasmin and tetraspanin 12 are potential convenient markers in readily obtainable PBMCs.

PBMCs reflect the immune component of the WAT transcriptome—Implications as biomarkers of metabolic health in the postprandial state

Food and nutrition studies often require accessing metabolically active tissues, including adipose tissue. This can involve invasive biopsy procedures that can be a limiting factor in study design. In contrast, peripheral blood mononuclear cells (PBMCs) are a population of circulating immune cells that are easily accessible through venipuncture. As transcriptomics is of growing importance in food and metabolism research, understanding the transcriptomic relationship between these tissue types can provide insight into the utility of PBMCs in this field. We examine this relationship within eight subjects, in two postprandial states (following oral lipid tolerance test and oral glucose tolerance test). Multivariate analysis techniques were used to examine variation between tissues, samples, and subjects in order to define which genes havecommon/disparate expression profiles associated with highly defined metabolic phenotypes. We demonstrate global similarities in gene expression between PBMCs and white adipose tissue, irrespective of the metabolic challenge type. Closer examination of individual genes revealed this similarity to be strongest in pathways related to immune response/inflammation. Notably, the expression of metabolism-related nuclear receptors, including PPARs, LXR, etc. was discordant between tissues The PBMC transcriptome may therefore provide a unique insight into the inflammatory component of metabolic health, as opposed to directly reflecting the metabolic component of the adipose tissue transcriptome.

High-Throughput Sequencing of microRNAs in Peripheral Blood Mononuclear Cells: Identification of Potential Weight Loss Biomarkers

IntroductionMicroRNAs (miRNAs) are being increasingly studied in relation to energy metabolism and body composition homeostasis. Indeed, the quantitative analysis of miRNAs expression in different adiposity conditions may contribute to understand the intimate mechanisms participating in body weight control and to find new biomarkers with diagnostic or prognostic value in obesity management.ObjectiveThe aim of this study was the search for miRNAs in blood cells whose expression could be used as prognostic biomarkers of weight loss.MethodsTen Caucasian obese women were selected among the participants in a weight-loss trial that consisted in following an energy-restricted treatment. Weight loss was considered unsuccessful when <5% of initial body weight (non-responders) and successful when >5% (responders). At baseline, total miRNA isolated from peripheral blood mononuclear cells (PBMC) was sequenced with SOLiD v4. The miRNA sequencing data were validated by RT-PCR.ResultsDifferential baseline expression of several miRNAs was found between responders and non-responders. Two miRNAs were up-regulated in the non-responder group (mir-935 and mir-4772) and three others were down-regulated (mir-223, mir-224 and mir-376b). Both mir-935 and mir-4772 showed relevant associations with the magnitude of weight loss, although the expression of other transcripts (mir-874, mir-199b, mir-766, mir-589 and mir-148b) also correlated with weight loss.ConclusionsThis research addresses the use of high-throughput sequencing technologies in the search for miRNA expression biomarkers in obesity, by determining the miRNA transcriptome of PBMC. Basal expression of different miRNAs, particularly mir-935 and mir-4772, could be prognostic biomarkers and may forecast the response to a hypocaloric diet.

Transcriptome Profiling of Peripheral Blood Cells Identifies Potential Biomarkers for Doxorubicin Cardiotoxicity in a Rat Model

AimsDoxorubicin (DOX), a widely used anticancer agent, can cause an unpredictable cardiac toxicity which remains a major limitation in cancer chemotherapy. There is a need for noninvasive, sensitive and specific biomarkers which will allow identifying patients at risk for DOX-induced cardiotoxicity to prevent permanent cardiac damage. The aim of this study was to investigate whether the expression of specific genes in the peripheral blood can be used as surrogate marker(s) for DOX-induced cardiotoxicity.Methods/ResultsRats were treated with a single dose of DOX similar to one single dose that is often administered in humans. The cardiac and peripheral blood mononuclear cells (PBMCs) genome-wide expression profiling were examined using Illumina microarrays. The results showed 4,409 differentially regulated genes (DRG) in the hearts and 4,120 DRG in PBMC. Of these 2411 genes were similarly DRG (SDRG) in both the heart and PBMC. Pathway analysis of the three datasets of DRG using Gene Ontology (GO) enrichment analysis and Ingenuity Pathways Analysis (IPA) showed that most of the genes in these datasets fell into pathways related to oxidative stress response and protein ubiquination. IPA search for potential eligible biomarkers for cardiovascular disease within the SDRG list revealed 188 molecules.ConclusionsWe report the first in-depth comparison of DOX-induced global gene expression profiles of hearts and PBMCs. The high similarity between the gene expression profiles of the heart and PBMC induced by DOX indicates that the PBMC transcriptome may serve as a surrogate marker of DOX-induced cardiotoxicity. Future directions of this research will include analysis of PBMC expression profiles of cancer patients treated with DOX-based chemotherapy to identify the cardiotoxicity risk, predict DOX-treatment response and ultimately to allow individualized anti-cancer therapy.

Turnover of Heme-Bound Iron Is Associated with Activation of TLR4 and Chemokine Receptor Pathways in the Peripheral Blood Mononuclear Cell Transcriptome in Sickle Cell Anemia

AbstractAbstract 819 IntroductionIt is widely accepted that inflammation plays an important role in the pathophysiology of Sickle Cell Anemia (SCA). Recently a number of studies indicated that peripheral blood mononuclear cell (PBMC) iron may contribute to inflammation in some diseases In SCA, PBMCs are continuously exposed to high turnover of iron in haptoglobin/hemoglobin complexes and free heme due to severe intravascular hemolysis. Therefore, we hypothesized that PBMC iron flux is marked by altered expression of iron-regulated genes, and in turn associated with the up regulation of genes in inflammatory pathways. To explore this hypothesis we correlated the expression of a predefined set of iron regulated genes to the SCA PBMC transcriptome as a whole. MethodsSteady state patients with SCA were selected as published earlier. (Bereal-Williams et al. Haematologica 2012) Most noteworthy exclusion criteria were recent blood transfusion, liver or kidney dysfunction or a recent acute complication. Affymetrix Human Genome U133 Plus 2.0 arrays were used to measure PBMC mRNA profile. The primary source of free iron in PBMC in SCA is iron released from heme by heme oxygenase-1 (HMOX1), the first committed biochemical step in production of bilirubin. Therefore, we planned to validate the clustering of the study participants by correlating the cluster rank with bilirubin plasma levels. Genes which are published to be differentially expressed upon iron loading or chelation were used to prospectively create an ‘iron-regulated' gene set (figure). Differentially expressed genes with the filter of a change greater than 40% between the clustering groups and 10% false discovery rate (FDR) were further analyzed with Ingenuity Pathway Analysis (IPA) System. ResultsTwenty-five subjects with SCA and 9 healthy subjects were analyzed. Hierarchical clustering using the predefined iron regulated gene list identified 3 groups of subjects with high, low and intermediate expression of iron activated genes (figure 1). As expected, none of the healthy controls was found in the high iron cluster. The cluster grouping was validated by correlation of serum bilirubin levels to the cluster rank (Spearman rho=0.358 p=0.044) and the expression of HMOX1 (Spearmen rho=0.387 p=0.034). In analysis of these three iron regulated gene clusters, we found 98 genes which were consistently and significantly differentially expressed, notably many genes important to crucial inflammatory pathways, especially Toll-like receptor 4 and 7 (TLR-4,7), chemokine receptor CCR1, and interleukin 15. The 98 markers identified ten canonical pathways in Ingenuity Pathway Analysis, most involving inflammation (each p<0.004, Table ).Expression profiling using a defined set of iron regulated genes identifies co-regulation of genes and pathways related to inflammatory cytokines, signaling cascades and innate immunity pathways. Our human PBMC findings confirm and extend recently presented data from several other groups linking TLR4-mediated heme-iron toxicity to pathological responses in sickle cell mice and in cultured cells. The role of heme-associated iron in sickle cell pathophysiology merits additional investigation.Tabletop ten canonical pathways associated with increased expression of the pre-defined iron regulated gene set. (p<0.004)NameDifferential regulated genesRole of Pattern Recognition Receptors in Recognition of Bacteria and VirusesTLR4, NLRP3, TLR7, PRKCH, PRKCAIL-10 SignalingCCR1, HMOX1, BLVRA, CD14LPS-stimulated MAPK SignalingTLR4, CD14, PRKCH, PRKCAAltered T Cell and B Cell Signaling in Rheumatoid ArthritisTLR4, TNFSF13, IL15(includes EG:16168), TLR7Communication between Innate and Adaptive Immune CellsTLR4, TNFSF13, IL15(includes EG:16168), TLR7Beta-Adrenergic SignalingGNB4, PRKCH, SLC8A1, PRKCAPhospholipase C SignalingHMOX1, GNB4, ZAP70, PRKCH, MARCKS, PRKCAToll-like Receptor SignalingTLR4, TLR7, CD14fMLP Signaling in NeutrophilsGNB4, CYBB, PRKCH, PRKCAIL-8 Signaling.HMOX1, GNB4, CYBB, PRKCH, PRKCA [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Gene expression of peripheral blood mononuclear cells as a tool in dietary intervention studies: What do we know so far?

Peripheral blood mononuclear cells (PBMCs) generally refer to monocytes and lymphocytes, representing cells of the innate and adaptive immune systems. PBMCs are a promising target tissue in the field of nutrigenomics because they seem to reflect the effects of dietary modifications at the level of gene expression. In this review, we describe and discuss the scientific literature concerning the use of gene expression at the mRNA level measured from PBMCs in dietary interventions studies conducted in humans. A search of literature was undertaken using PubMed (last assessed November 24, 2011) and 20 articles were selected for discussion. Currently, results from these studies showed that PBMCs seem to reflect liver environment and complement adipose tissue findings in transcriptomics. PBMC gene expression after dietary intervention studies can be used for studying the response of certain genes related to fatty acid and cholesterol metabolism, and to explore the response of dietary interventions in relation to inflammation. However, PBMC transcriptomics from dietary intervention studies have not resulted yet in clear confirmation of candidate genes related to disease risk. Use of microarray technology in larger well-designed dietary intervention studies is still needed for exploring PBMC potential in the field of nutrigenomics.

Peripheral blood mononuclear cell transcriptome profiles suggest T-cell immunosuppression after uncomplicated mechanical circulatory support device surgery

Mechanical circulatory support device (MCSD) surgery in patients with advanced heart failure patients is often complicated by infections that are linked to altered cell-mediated immunity. Using a transcriptome-wide peripheral blood mononuclear cell (PBMC) gene expression profiling approach, we analyzed expression patterns directly before and after MCSD implantation in 11 patients who had an uncomplicated course after MCSD implantation (Day 0-24 hours before, Day 1-24 hours after, and Day 7-1 week after implantation). Data were analyzed using Significance Analysis of Microarrays (SAM) and High-Throughput GoMiner on post-implantation profiles (Day 1, Day 7) in comparison with baseline (Day 0). Day1 profiles included differential expression of 821 genes (SAM, FDR <0.1, fold change >1.5), enriching >60 Gene Ontology (GO) categories. Grouping by component genes revealed GO clusters, which we term “interleukin related” (primarily upregulated), “T-cell related” (primarily downregulated), and “apoptosis related” (both up- and down-regulated genes). Day 7 profiles included GO categories related to repair processes. In conclusion, transcriptome-wide expression profiling of PBMCs suggests a response pattern to MCSD implantation of inflammatory activation and simultaneous T-cell suppression.

Changes in the peripheral blood transcriptome associated with occupational benzene exposure identified by cross-comparison on two microarray platforms

Benzene is an established cause of leukemia, and possibly lymphoma, in humans, but the underlying molecular pathways remain largely undetermined. We used two microarray platforms to identify global gene expression changes associated with well-characterized occupational benzene exposure in the peripheral blood mononuclear cells (PBMC) of a population of shoe-factory workers. Differential expression of 2692 genes (Affymetrix) and 1828 genes (Illumina) was found and the concordance was 50% (based on an average fold-change ≥ 1.3 from the two platforms), with similar expression ratios among the concordant genes. Four genes ( CXCL16, ZNF331, JUN and PF4), which we previously identified by microarray and confirmed by real-time PCR, were among the top 100 genes identified by both platforms in the current study. Gene ontology analysis showed overrepresentation of genes involved in apoptosis among the concordant genes while pathway analysis identified pathways related to lipid metabolism. The two-platform approach allows for robust changes in the PBMC transcriptome of benzene-exposed individuals to be identified.

Proteomic analysis of mononuclear cells of patients with minimal-change nephrotic syndrome of childhood

Background/Aims. Recently, peripheral blood mononuclear cell transcriptome analysis has identified genes that are upregulated in relapsing minimal-change nephrotic syndrome (MCNS). In order to investigate protein expression in peripheral blood mononuclear cells (PBMC) from relapsing MCNS patients, we performed proteomic comparisons of PBMC from patients with MCNS in relapse and controls. PBMC from a total of 20 patients were analysed. PBMC were taken from five patients with relapsing MCNS, four in remission, five patients with other glomerular diseases and six controls. Two dimensional electrophoresis was performed and proteome patterns were compared. Automatic heuristic clustering analysis allowed us to pool correctly the gels from the MCNS patients in the relapse and in the control groups. Using hierarchical population matching, nine spots were found to be increased in PBMC from MCNS patients in relapse. Four spots were identified by mass spectrometry. Three of the four proteins identified (L-plastin, alpha-tropomyosin and annexin III) were cytoskeletal-associated proteins. Using western blot and immunochemistry, L-plastin and alpha-tropomyosin 3 concentrations were found to be enhanced in PBMC from MCNS patients in relapse. Conclusions. These data indicate that a specific proteomic profile characterizes PBMC from MCNS patients in relapse. Proteins involved in PBMC cytoskeletal rearrangement are increased in relapsing MCNS. We hypothesize that T-cell cytoskeletal rearrangement may play a role in the pathogenesis of MCNS by altering the expression of cell surface receptors and by modifying the interaction of these cells with glomerular cells.

Gene expression profiling of peripheral blood mononuclear cells (PBMC) from Mycobacterium bovis infected cattle after in vitro antigenic stimulation with purified protein derivative of tuberculin (PPD)

Microarray analysis of messenger RNA (mRNA) abundance was used to investigate the gene expression program of peripheral blood mononuclear cells (PBMC) from cattle infected with Mycobacterium bovis, the causative agent of bovine tuberculosis. An immunospecific bovine microarray platform (BOTL-4) with spot features representing 1336 genes was used for transcriptional profiling of PBMC from six M. bovis-infected cattle stimulated in vitro with bovine purified protein derivative of tuberculin (PPD-bovine). Cells were harvested at four time points (3 h, 6 h, 12 h and 24 h post-stimulation) and a split-plot design with pooled samples was used for the microarray experiment to compare gene expression between PPD-bovine stimulated PBMC and unstimulated controls for each time point. Statistical analyses of these data revealed 224 genes (∼17% of transcripts on the array) differentially expressed between stimulated and unstimulated PBMC across the 24 h time course ( P < 0.05). Of the 224 genes, 87 genes were significantly upregulated and 137 genes were significantly downregulated in M. bovis-infected PBMC stimulated with PPD-bovine across the 24 h time course. However, perturbation of the PBMC transcriptome was most apparent at time points 3 h and 12 h post-stimulation, with 81 and 84 genes differentially expressed, respectively. In addition, a more stringent statistical threshold ( P < 0.01) revealed 35 genes (∼3%) that were differentially expressed across the time course. Real-time quantitative reverse transcription PCR (qRT-PCR) of selected genes validated the microarray results and demonstrated a wide range of differentially expressed genes in PPD-bovine-, PPD-avian- and Concanavalin A (ConA) stimulated PBMC, including the interferon-γ gene ( IFNG), which was upregulated in PBMC stimulated with PPD-bovine (40-fold), PPD-avian (10-fold) and ConA (8-fold) after in vitro culture for 12 h. The pattern of expression of these genes in PPD-bovine stimulated PBMC provides the first description of an M. bovis-specific signature of infection that may provide insights into the molecular basis of the host response to infection. Although the present study was carried out with mixed PBMC cell populations, it will guide future studies to dissect immune cell-specific gene expression patterns in response to M. bovis infection.

Collection and Storage of Human Blood Cells for mRNA Expression Profiling: A 15-Month Stability Study

Gene expression profiling is increasingly important in human health research and applications (1). Unfortunately, the human tissue samples required for this process, particularly those from healthy individuals, are not safely and easily available. Therefore, the use of peripheral blood mononuclear cells (PBMCs) as surrogate material for high-throughput analysis of gene expression is currently being explored. These cells are involved in a large variety of immune-related diseases, including infection and cancer. Moreover, in recent studies, characteristic sets of transcriptional changes in PBMCs were associated with physiologic or pathologic states (2)(3)(4). Thus, a PBMC transcriptome may be used as an individual’s health sensor, a concept referred to as the sentinel principle (5). In large multicenter studies, the reliable and reproducible detection of transcript concentrations from PBMCs requires standardization of blood sampling and an efficient method of conservation. Indeed, many preanalytical factors during collection, processing, and storage of blood specimens may affect RNA and its subsequent use as a biomarker (6). Although numerous technical and clinical aspects of blood sampling have been addressed (7)(8)(9), comprehensive data on the long-term storage and stability of RNA from PBMCs are needed. Here we report experiments performed over 15 months to test the preanalytical conditions involved in blood collection procedures and PBMC storage. Blood samples were collected from healthy donors into EDTA and sodium heparin tubes. RNA extractions were performed on isolated PBMCs stored at −80 °C up to 15 months in 4 different lysis buffers. Using spectrometry and real-time PCR, we compared the concentration, purity, integrity, and stability of the total RNA. We propose a quality-assured and controlled protocol of PBMC banking for further mRNA expression analysis. After informed consent was obtained from 12 unrelated adult volunteers, whole blood (10 mL) was collected by standardized venipuncture in …

Aldosteronism and peripheral blood mononuclear cell activation: a neuroendocrine-immune interface.

Aldosteronism eventuates in a proinflammatory/fibrogenic vascular phenotype of the heart and systemic organs. It remains uncertain whether peripheral blood mononuclear cells (PBMCs) are activated before tissue invasion by monocytes/macrophages and lymphocytes, as is the case for responsible pathogenic mechanisms. Uninephrectomized rats treated for 4 weeks with dietary 1% NaCl and aldosterone (ALDOST, 0.75 microg/h) with or without spironolactone (Spi, 100 mg/kg per daily gavage) were compared with unoperated/untreated and uninephrectomized/salt-treated controls. Before intramural coronary vascular lesions appeared at week 4 of ALDOST, we found (1) a reduction of PBMC cytosolic free [Mg2+]i, together with intracellular Mg2+ and Ca2+ loading, whereas plasma and cardiac tissue Mg2+ were no different from controls; (2) increased H2O2 production by monocytes and lymphocytes together with upregulated PBMC gene expression of oxidative stress-inducible tyrosine phosphatase and Mn2+-superoxide dismutase and the presence of 3-nitrotyrosine in CD4+ and ED-1-positive inflammatory cells that had invaded intramural coronary arteries; (3) B-cell activation, including transcription of immunoglobulins, intracellular adhesion molecule-1, and CC and CXC chemokines and their receptors; (4) expansion of B lymphocyte subset and myosin heavy chain class II-expressing lymphocytes; and (5) autoreactivity with gene expression for antibodies to acetylcholine receptors and a downregulation of RT-6.2, which is in keeping with cell activation and associated with autoimmunity. Spi cotreatment attenuated the rise in intracellular Ca2+, the appearance of oxidative/nitrosative stress in PBMCs and invading inflammatory cells, and alterations in PBMC transcriptome. Thus, aldosteronism is associated with an activation of circulating immune cells induced by iterations in PBMC divalent cations and transduced by oxidative/nitrosative stress. ALDO receptor antagonism modulates this neuroendocrine-immune interface. The full text of this article is available online at http://www.circresaha.org.

Aldosteronism: an immunostimulatory state precedes proinflammatory/fibrogenic cardiac phenotype.

Chronic inappropriate (relative to dietary Na+ intake) elevations in circulating aldosterone (ALDO), termed aldosteronism, are associated with remodeling of intramural arteries of the right and left heart. Lesions appear at week 4 of treatment with ALDO and 1% dietary NaCl in uninephrectomized rats (ALDOST) and include invading monocytes, macrophages and lymphocytes with intracellular evidence of oxidative and nitrosative stress, myofibroblasts, and perivascular fibrosis. In this study, we tested the hypothesis that an immunostimulatory state with activated circulating peripheral blood mononuclear cells (PBMCs) precedes this proinflammatory and profibrogenic cardiac phenotype and is initiated by reduction in the cytosolic free Mg2+ concentration ([Mg2+]i). At 1 and 4 wk of ALDOST (preclinical and clinical stages, respectively), we monitored serum Mg2+, PBMC [Mg2+]i and cytosolic free [Ca2+] (via fluorimetry), and expressed genes (via microchip array) as well as markers of oxidative and nitrosative stress in plasma [alpha1-antiproteinase activity (alpha1-AP)] and cardiac tissue (immunohistochemical detection of gp91phox subunit of NADPH oxidase and 3-nitrotyrosine). Age- and gender-matched unoperated and untreated (UO) rats and uninephrectomized salt-treated (UN) rats served as controls. Serum [Mg2+] was unchanged by ALDOST. In contrast with UO and UN, [Mg2+]i and plasma alpha1-AP were each reduced (P < 0.05) at weeks 1 and 4. The decline in PBMC [Mg2+]i was accompanied by Ca2+ loading. Differential (twofold and higher) expression (up- and downregulation) in PBMC transcriptomes was present at week 1 and progressed at week 4. Involved were genes for the alpha1-isoform of Na+-K+-ATPase, the ATP-dependent Ca2+ pump, antioxidant reserves, inducible nitric oxide synthase, and PBMC activation with autoimmune responses. Expression of 3-nitrotyrosine and activation of gp91phox were seen in inflammatory cells that invaded intramural arteries. Thus early in aldosteronism (preclinical stage), an immunostimulatory state featuring activated circulating PBMCs with reduced ionized [Mg2+]i and oxidative and nitrosative stress precedes and may even predispose to coronary vascular lesions that first appear at week 4.