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1.  Gene Expression Profiles from Peripheral Blood Mononuclear Cells Are Sensitive to Short Processing Delays 
Biopreservation and Biobanking  2010;8(3):153-162.
In the analysis of peripheral blood gene expression, timely processing of samples is essential to ensure that measurements reflect in vivo biology, rather than ex vivo sample processing variables. The effect of processing delays on global gene expression patterns in peripheral blood mononuclear cells (PBMCs) was assessed by isolating and stabilizing PBMC-derived RNA from 3 individuals either immediately after phlebotomy or after a 4 h delay. RNA was labeled using NuGEN Ovation labeling and probed using the Affymetrix HG U133 Plus 2.0 GeneChip®. Comparison of gene expression levels (≥2-fold expression change and P < 0.05) identified 307 probe sets representing genes with increased expression and 46 indicating decreased expression after 4 h. These differentially expressed genes include many that are important to inflammatory, immunologic, and cancer pathways. Among others, CCR2, CCR5, TLR10, CD180, and IL-16 have decreased expression, whereas VEGF, IL8, SOCS2, SOCS3, CD69, and CD83 have increased expression after a 4 h processing delay. The trends in expression patterns associated with delayed processing were also apparent in an independent set of 276 arrays of RNA from human PBMC samples with varying processing times. These data indicate that the time between sample acquisition, initiation of processing, and when the RNA is stabilized should be a prime consideration when designing protocols for translational studies involving PBMC gene expression analysis.
doi:10.1089/bio.2010.0009
PMCID: PMC3129811  PMID: 21743826
2.  Biologic similarities based on age at onset in oligoarticular and polyarticular subtypes of juvenile idiopathic arthritis 
Arthritis and rheumatism  2010;62(11):3249-3258.
Objective
To explore biologic correlates to age at onset in patients with juvenile idiopathic arthritis (JIA) using peripheral blood mononuclear cell (PBMC) gene expression analysis.
Methods
PBMCs were isolated from 56 healthy controls and 104 patients with recent-onset JIA (39 with persistent oligoarticular JIA, 45 with rheumatoid factor–negative polyarticular JIA, and 20 with systemic JIA). RNA was amplified and labeled using NuGEN Ovation, and gene expression was assessed with Affymetrix HG-U133 Plus 2.0 GeneChips.
Results
A total of 832 probe sets revealed gene expression differences (false discovery rate 5%) in PBMCs from children with oligoarticular JIA whose disease began before age 6 years (early-onset disease) compared with those whose disease began at or after age 6 years (late-onset disease). In patients with early-onset disease, there was greater expression of genes related to B cells and less expression of genes related to cells of the myeloid lineage. Support vector machine analyses identified samples from patients with early- or late-onset oligoarticular JIA (with 97% accuracy) or from patients with early- or late-onset polyarticular JIA (with 89% accuracy), but not from patients with systemic JIA or healthy controls. Principal components analysis showed that age at onset was the major classifier of samples from patients with oligoarticular JIA and patients with polyarticular JIA.
Conclusion
PBMC gene expression analysis reveals biologic differences between patients with early-and late-onset JIA, independent of classification based on the number of joints involved. These data suggest that age at onset may be an important parameter to consider in JIA classification. Furthermore, pathologic mechanisms may vary with age at onset, and understanding these processes may lead to improved treatment of JIA.
doi:10.1002/art.27657
PMCID: PMC3018072  PMID: 20662067
3.  Identification of blood biomarkers of rheumatoid arthritis by transcript profiling of peripheral blood mononuclear cells from the rat collagen-induced arthritis model 
Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disease that results in joint destruction and subsequent loss of function. To better understand its pathogenesis and to facilitate the search for novel RA therapeutics, we profiled the rat model of collagen-induced arthritis (CIA) to discover and characterize blood biomarkers for RA. Peripheral blood mononuclear cells (PBMCs) were purified using a Ficoll gradient at various time points after type II collagen immunization for RNA preparation. Total RNA was processed for a microarray analysis using Affymetrix GeneChip technology. Statistical comparison analyses identified differentially expressed genes that distinguished CIA from control rats. Clustering analyses indicated that gene expression patterns correlated with laboratory indices of disease progression. A set of 28 probe sets showed significant differences in expression between blood from arthritic rats and that from controls at the earliest time after induction, and the difference persisted for the entire time course. Gene Ontology comparison of the present study with previous published murine microarray studies showed conserved Biological Processes during disease induction between the local joint and PBMC responses. Genes known to be involved in autoimmune response and arthritis, such as those encoding Galectin-3, Versican, and Socs3, were identified and validated by quantitative TaqMan RT-PCR analysis using independent blood samples. Finally, immunoblot analysis confirmed that Galectin-3 was secreted over time in plasma as well as in supernatant of cultured tissue synoviocytes of the arthritic rats, which is consistent with disease progression. Our data indicate that gene expression in PBMCs from the CIA model can be utilized to identify candidate blood biomarkers for RA.
doi:10.1186/ar1883
PMCID: PMC1526586  PMID: 16507131
4.  Gene Expression Signatures in Polyarticular Juvenile Idiopathic Arthritis Demonstrate Disease Heterogeneity and Offer a Molecular Classification of Disease Subsets 
Arthritis and rheumatism  2009;60(7):2113-2123.
Objective
Microarray analysis was used to determine whether children with recent onset polyarticular juvenile idiopathic arthritis (JIA) exhibit biologically or clinically informative gene expression signatures in peripheral blood mononuclear cells (PBMC).
Methods
Peripheral blood samples were obtained from 59 healthy children and 61 children with polyarticular JIA prior to treatment with second-line medications, such as methotrexate or biological agents. RNA was extracted from Ficoll-isolated mononuclear cells, fluorescently labeled and hybridized to Affymetrix U133 Plus 2.0 GeneChips. Data were analyzed using ANOVA at a 5% false discovery rate threshold after Robust Multi-Array Average pre-processing and Distance Weighted Discrimination normalization.
Results
Initial analysis revealed 873 probe sets for genes that were differentially expressed between polyarticular JIA and controls. Hierarchical clustering of these probe sets distinguished three subgroups within polyarticular JIA. Prototypical subjects within each subgroup were identified and used to define subgroup-specific gene expression signatures. One of these signatures was associated with monocyte markers, another with transforming growth factor β-inducible genes, and a third with immediate-early genes. Correlation of gene expression signatures with clinical and biological features of JIA subgroups suggests relevance to aspects of disease activity and supports the division of polyarticular JIA into distinct subsets.
Conclusions
PBMC gene expression signatures in recent onset polyarticular JIA reflect discrete disease processes and offer a molecular classification of disease.
doi:10.1002/art.24534
PMCID: PMC2741130  PMID: 19565504
5.  Subtype-specific peripheral blood gene expression profiles in recent onset juvenile idiopathic arthritis 
Arthritis and rheumatism  2009;60(7):2102-2112.
Objective
A multi-center study of recent onset juvenile idiopathic arthritis (JIA) subjects prior to treatment with DMARDS or biologics was undertaken to identify peripheral blood gene expression differences between JIA subclasses and controls.
Methods
PBMC from 59 healthy children and 136 JIA subjects (28 enthesitis-related arthritis [ERA], 42 persistent oligoarthritis, 45 RF- polyarthritis, and 21 systemic) were isolated over Ficoll. Poly-A RNA was labeled using NuGEN Ovation and gene expression profiles were obtained using Affymetrix HG-U133 plus 2.0 Arrays.
Results
9,501 differentially expressed probe sets were identified among JIA subtypes and controls (ANOVA, FDR 5%). Specifically, 193, 1036, 873 and 7595 probe sets were different between controls and ERA, persistent oligoarthritis, RF- polyarthritis and systemic JIA samples respectively. In persistent oligoarthritis, RF- polyarthritis and systemic JIA subtypes, up-regulation of genes associated with IL-10 signaling was prominent. A hemoglobin cluster was identified that was under-expressed in ERA patients but over-expressed in systemic JIA. The influence of JAK/STAT, ERK/MAPK, IL-2 and B cell receptor signaling pathways was evident in persistent oligoarthritis. In systemic JIA, up-regulation of innate immune pathways, including IL-6, TLR/IL1R, and PPAR signaling were noted, along with down-regulation of gene networks related to NK and T cells. Complement and coagulation pathways were up-regulated in systemic JIA with a subset of these genes differentially-expressed in other subtypes as well.
Conclusions
Expression analysis identified differentially expressed genes in PBMCs between subtypes of JIA early in disease and controls, thus providing evidence for immunobiologic differences between these forms of childhood arthritis.
doi:10.1002/art.24601
PMCID: PMC2782469  PMID: 19565513
6.  57 Increased Frequency of CD4+ CCR4+ CCR9+ Cells in Patients With Allergic Conjunctivitis 
Background
Allergic conjunctivitis is one of the most common diseases affecting the ocular surface, it has been suggested that T CD4+ cells regulate immune response in allergic diseases such as asthma and rhinitis, in a predominant Th2 response. In animal models, it has been observed a selective migration of CD4+ T cells to conjunctiva directed by chemokines; however molecules involved in CD4+ T cell migration in humans is unknown, thus it was the aim of this study.
Methods
Peripheral blood mononuclear cells (PBMC) were isolated from blood samples of healthy donors (HD) and AC-patients. PBMC were labeled with mAbs against CD4, CCR4, CCR5, and CRR9, and then labeled cells were analyzed by flow cytometry. T test was used to perform statistical analysis, P < 0.05 were considered statistically significant.
Results
We observed increased frequency of CCR4+ and CCR9+ on PBMC cells; interestingly, expression of CCR4+ was 1.46 times increased on CD4+ T cells of AC-patients compared to CD4+ T cells of HD (P = 0.01). Similarly, we observed higher frequency of CCR9 expression on CD4+ cells of AC-patients than on CD4+ T cells of HD (P = 0.01). On the other hand, CCR5 expression was diminished on PBMC from AC-patients than in HD (P = 0.0002).
Conclusions
Increased frequency of CD4+ CCR4+ CCR9+ was observed in AC patients with diminished frequency of CCR5 expression on PBMC. CCR4 and CCR9 have been involved in inflammatory process such arthritis and asthma, both could be related to inflammatory reaction at conjunctiva. CCR5 expression is mainly on Th1 cells, diminished frequency on PBMC in allergic conjunctivitis patients could be related with imbalance of immune response favoring a Th2 chronic inflammation.
doi:10.1097/01.WOX.0000411802.41044.6d
PMCID: PMC3512702
7.  Impact of blood processing variations on Natural Killer cell frequency, activation, chemokine receptor expression and function 
Journal of immunological methods  2011;366(1-2):28-35.
Understanding the role of natural killer (NK) cells in human disease pathogenesis is crucial and necessitates study of patient samples directly ex vivo. Manipulation of whole blood by density gradient centrifugation or delays in sample processing due to shipping, however, may lead to artifactual changes in immune response measures. Here, we assessed the impact of density gradient centrifugation and delayed processing of both whole blood and peripheral blood mononuclear cells (PBMC) at multiple timepoints (2–24 hrs) on flow cytometric measures of NK cell frequency, activation status, chemokine receptor expression, and effector functions. We found that density gradient centrifugation activated NK cells and modified chemokine receptor expression. Delays in processing beyond 8 hours activated NK cells in PBMC but not in whole blood. Likewise, processing delays decreased chemokine receptor (CCR4 and CCR7) expression in both PBMC and whole blood. Finally, delays in processing PBMC were associated with a decreased ability of NK cells to degranulate (as measured by CD107a expression) or secrete cytokines (IFN-γ and TNF-α). In summary, our findings suggest that density gradient centrifugation and delayed processing of PBMC can alter measures of clinically relevant NK cell characteristics including effector functions; and therefore should be taken into account in designing clinical research studies.
doi:10.1016/j.jim.2011.01.001
PMCID: PMC3051017  PMID: 21255578
Natural Killer cells; flow cytometry; activation; delayed processing; PBMC; whole blood; ficoll; chemokine receptor
8.  Sample Labeling and Analysis Approaches for the Affymetrix Whole-Transcript Gene ST Array 
RP-31
The Affymetrix Gene 1.0 ST arrays provide an alternative platform for performing expression analysis than traditional 3' gene expression analysis arrays. These arrays offers several advantages, including updated content, lower cost, and probe sets designed to span the entire length of a given transcript. However, since its release in 2007, adoption of this platform has been limited and questions remain as to optimal approaches for sample labeling and subsequent data analysis. To address these issues we compared two labeling chemistries to generate the biotinylated sense DNA using the Microarray Quality Control Consortium (MAQC) total RNA titration samples: Stratagene Universal Reference RNA (A), Ambion Human Brain RNA (B) and the two titration samples (C and D, consisting of 3:1 and 1:3 ratios of A to B, respectively). Samples were labeled in triplicate, using either the Affymetrix GeneChip® WT Sense Target Labeling kit or the NuGEN WT-Ovation™ Pico RNA Amplification System, and hybridized to the Human Gene 1.0 ST Array. We next evaluated different data preprocessing approaches for background correction, normalization and probe set summarization, including the RMA, PLIER and dCHIP methods. Statistical analysis was performed using the LIMMA package in R, to generate lists of significantly differentially expressed genes (P < 0.001) and a fold change > 2. Dependent upon the preprocessing method used, the Affymetrix labeling chemistry consistently identified 30-50% more significantly differentially expressed genes than were detected with the NuGEN chemistry. Analysis of titration data showed that both chemistries had similar performance, and enabled us to determine optimal data preprocessing approaches. Our data demonstrate that even though the NuGEN chemistry provides a solution for sample labeling limited amounts of starting material, it results in significant data compression. However, higher quality data is obtained with the Affymetrix WT Sense Target Labeling kit if sufficient starting material (100 ng) is available.
PMCID: PMC2918114
9.  CCR5Δ32 Protein Expression and Stability Are Critical for Resistance to Human Immunodeficiency Virus Type 1 In Vivo▿  
Journal of Virology  2007;81(15):8041-8049.
Human immunodeficiency virus type 1 (HIV-1) infection of individuals carrying the two alleles of the CCR5Δ32 mutation (CCR5−/−) has rarely been reported, but how the virus overcomes the CCR5Δ32 protective effect in these cases has not been delineated. We have investigated this in 6 infected (HIV+) and 25 HIV− CCR5−/− individuals. CD4+ T lymphocytes isolated from HIV− CCR5−/− peripheral blood mononuclear cells (PBMCs) showed lower levels of CXCR4 expression that correlated with lower X4 Env-mediated fusion. Endogenous CCR5Δ32 protein was detected in all HIV− CCR5−/− PBMC samples (n = 25) but not in four of six unrelated HIV+ CCR5−/− PBMC samples. Low levels were detected in another two HIV+ CCR5−/− PBMC samples. The expression of adenovirus 5 (Ad5)-encoded CCR5Δ32 protein restored the protective effect in PBMCs from three HIV+ CCR5−/− individuals but failed to restore the protective effect in PBMCs isolated from another three HIV+ CCR5−/− individuals. In the latter samples, pulse-chase analyses demonstrated the disappearance of endogenous Ad5-encoded CCR5Δ32 protein and the accumulation of Ad5-encoded CCR5 during the chase periods. PBMCs isolated from CCR5−/− individuals showed resistance to primary X4 but were readily infected by a lab-adapted X4 strain. Low levels of Ad5-encoded CCR5Δ32 protein conferred resistance to primary X4 but not to lab-adapted X4 virus. These data provide strong support for the hypothesis that the CCR5Δ32 protein actively confers resistance to HIV-1 in vivo and suggest that the loss or reduction of CCR5Δ32 protein expression may account for HIV-1 infection of CCR5−/− individuals. The results also suggest that other cellular or virally induced factors may be involved in the stability of CCR5Δ32 protein.
doi:10.1128/JVI.00068-07
PMCID: PMC1951285  PMID: 17522201
10.  Expression Profiling Smackdown: Human Transcriptome Array HTA 2.0 vs. RNA-Seq 
The advent of both microarray and massively parallel sequencing have revolutionized high-throughput analysis of the human transcriptome. Due to limitations in microarray technology, detecting and quantifying coding transcript isoforms, in addition to non-coding transcripts, has been challenging. As a result, RNA-Seq has been the preferred method for characterizing the full human transcriptome, until now. A new high-resolution array from Affymetrix, GeneChip Human Transcriptome Array 2.0 (HTA 2.0), has been designed to interrogate all transcript isoforms in the human transcriptome with >6 million probes targeting coding transcripts, exon-exon splice junctions, and non-coding transcripts. Here we compare expression results from GeneChip HTA 2.0 and RNA-Seq data using identical RNA extractions from three samples each of healthy human mesothelial cells in culture, LP9-C1, and healthy mesothelial cells treated with asbestos, LP9-A1. For GeneChip HTA 2.0 sample preparation, we chose to compare two target preparation methods, NuGEN Ovation Pico WTA V2 with the Encore Biotin Module versus Affymetrix's GeneChip WT PLUS with the WT Terminal Labeling Kit, on identical RNA extractions from both untreated and treated samples. These same RNA extractions were used for the RNA-Seq library preparation. All analyses were performed in Partek Genomics Suite 6.6. Expression profiles for control and asbestos-treated mesothelial cells prepared with NuGEN versus Affymetrix target preparation methods (GeneChip HTA 2.0) are compared to each other as well as to RNA-Seq results.
PMCID: PMC4162206
11.  Discovery of Novel Biomarkers by Microarray Analysis of Peripheral Blood Mononuclear Cell Gene Expression in Benzene-Exposed Workers 
Environmental Health Perspectives  2005;113(6):801-807.
Benzene is an industrial chemical and component of gasoline that is an established cause of leukemia. To better understand the risk benzene poses, we examined the effect of benzene exposure on peripheral blood mononuclear cell (PBMC) gene expression in a population of shoe-factory workers with well-characterized occupational exposures using microarrays and real-time polymerase chain reaction (PCR). PBMC RNA was stabilized in the field and analyzed using a comprehensive human array, the U133A/B Affymetrix GeneChip set. A matched analysis of six exposed–control pairs was performed. A combination of robust multiarray analysis and ordering of genes using paired t-statistics, along with bootstrapping to control for a 5% familywise error rate, was used to identify differentially expressed genes in a global analysis. This resulted in a set of 29 known genes being identified that were highly likely to be differentially expressed. We also repeated these analyses on a smaller subset of 508 cytokine probe sets and found that the expression of 19 known cytokine genes was significantly different between the exposed and the control subjects. Six genes were selected for confirmation by real-time PCR, and of these, CXCL16, ZNF331, JUN, and PF4 were the most significantly affected by benzene exposure, a finding that was confirmed in a larger data set from 28 subjects. The altered expression was not caused by changes in the makeup of the PBMC fraction. Thus, microarray analysis along with real-time PCR confirmation reveals that altered expressions of CXCL16, ZNF331, JUN, and PF4 are potential biomarkers of benzene exposure.
doi:10.1289/ehp.7635
PMCID: PMC1257610  PMID: 15929907
Affymetrix; benzene; biomarkers; blood; expression profiling; leukemia; lymphocyte; microarray; molecular epidemiology; occupational exposure; real-time PCR
12.  Identification and utilization of inter-species conserved (ISC) probesets on Affymetrix human GeneChip® platforms for the optimization of the assessment of expression patterns in non human primate (NHP) samples 
BMC Bioinformatics  2004;5:165.
Background
While researchers have utilized versions of the Affymetrix human GeneChip® for the assessment of expression patterns in non human primate (NHP) samples, there has been no comprehensive sequence analysis study undertaken to demonstrate that the probe sequences designed to detect human transcripts are reliably hybridizing with their orthologs in NHP. By aligning probe sequences with expressed sequence tags (ESTs) in NHP, inter-species conserved (ISC) probesets, which have two or more probes complementary to ESTs in NHP, were identified on human GeneChip® platforms. The utility of human GeneChips® for the assessment of NHP expression patterns can be effectively evaluated by analyzing the hybridization behaviour of ISC probesets. Appropriate normalization methods were identified that further improve the reliability of human GeneChips® for interspecies (human vs NHP) comparisons.
Results
ISC probesets in each of the seven Affymetrix GeneChip® platforms (U133Plus2.0, U133A, U133B, U95Av2, U95B, Focus and HuGeneFL) were identified for both monkey and chimpanzee. Expression data was generated from peripheral blood mononuclear cells (PBMCs) of 12 human and 8 monkey (Indian origin Rhesus macaque) samples using the Focus GeneChip®. Analysis of both qualitative detection calls and quantitative signal intensities showed that intra-species reproducibility (human vs. human or monkey vs. monkey) was much higher than interspecies reproducibility (human vs. monkey). ISC probesets exhibited higher interspecies reproducibility than the overall expressed probesets. Importantly, appropriate normalization methods could be leveraged to greatly improve interspecies correlations. The correlation coefficients between human (average of 12 samples) and monkey (average of 8 Rhesus macaque samples) are 0.725, 0.821 and 0.893 for MAS5.0 (Microarray Suite version 5.0), dChip and RMA (Robust Multi-chip Average) normalization method, respectively.
Conclusion
It is feasible to use Affymetrix human GeneChip® platforms to assess the expression profiles of NHP for intra-species studies. Caution must be taken for interspecies studies since unsuitable probesets will result in spurious differentially regulated genes between human and NHP. RMA normalization method and ISC probesets are recommended for interspecies studies.
doi:10.1186/1471-2105-5-165
PMCID: PMC526766  PMID: 15507140
13.  The DNA Methylome of Human Peripheral Blood Mononuclear Cells 
PLoS Biology  2010;8(11):e1000533.
Analysis across the genome of patterns of DNA methylation reveals a rich landscape of allele-specific epigenetic modification and consequent effects on allele-specific gene expression.
DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome) analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold per strand), we report a comprehensive (92.62%) methylome and analysis of the unique sequences in human peripheral blood mononuclear cells (PBMC) from the same Asian individual whose genome was deciphered in the YH project. PBMC constitute an important source for clinical blood tests world-wide. We found that 68.4% of CpG sites and <0.2% of non-CpG sites were methylated, demonstrating that non-CpG cytosine methylation is minor in human PBMC. Analysis of the PBMC methylome revealed a rich epigenomic landscape for 20 distinct genomic features, including regulatory, protein-coding, non-coding, RNA-coding, and repeat sequences. Integration of our methylome data with the YH genome sequence enabled a first comprehensive assessment of allele-specific methylation (ASM) between the two haploid methylomes of any individual and allowed the identification of 599 haploid differentially methylated regions (hDMRs) covering 287 genes. Of these, 76 genes had hDMRs within 2 kb of their transcriptional start sites of which >80% displayed allele-specific expression (ASE). These data demonstrate that ASM is a recurrent phenomenon and is highly correlated with ASE in human PBMCs. Together with recently reported similar studies, our study provides a comprehensive resource for future epigenomic research and confirms new sequencing technology as a paradigm for large-scale epigenomics studies.
Author Summary
Epigenetic modifications such as addition of methyl groups to cytosine in DNA play a role in regulating gene expression. To better understand these processes, knowledge of the methylation status of all cytosine bases in the genome (the methylome) is required. DNA methylation can differ between the two gene copies (alleles) in each cell. Such allele-specific methylation (ASM) can be due to parental origin of the alleles (imprinting), X chromosome inactivation in females, and other as yet unknown mechanisms. This may significantly alter the expression profile arising from different allele combinations in different individuals. Using advanced sequencing technology, we have determined the methylome of human peripheral blood mononuclear cells (PBMC). Importantly, the PBMC were obtained from the same male Han Chinese individual whose complete genome had previously been determined. This allowed us, for the first time, to study genome-wide differences in ASM. Our analysis shows that ASM in PBMC is higher than can be accounted for by regions known to undergo parent-of-origin imprinting and frequently (>80%) correlates with allele-specific expression (ASE) of the corresponding gene. In addition, our data reveal a rich landscape of epigenomic variation for 20 genomic features, including regulatory, coding, and non-coding sequences, and provide a valuable resource for future studies. Our work further establishes whole-genome sequencing as an efficient method for methylome analysis.
doi:10.1371/journal.pbio.1000533
PMCID: PMC2976721  PMID: 21085693
14.  Selection of Reference Genes for Gene Expression Studies in Porcine Whole Blood and Peripheral Blood Mononuclear Cells under Polyinosinic:Polycytidylic Acid Stimulation 
Investigating gene expression of immune cells of whole blood or peripheral blood mononuclear cells (PBMC) under polyinosinic:polycytidylic acid (poly I:C) stimulation is valuable for understanding the immune response of organism to RNA viruses. Quantitative real-time PCR (qRT-PCR) is a standard method for quantification of gene expression studies. However, the reliability of qRT-PCR data critically depends on proper selection of reference genes. In the study, using two different analysis programs, geNorm and NormFinder, we systematically evaluated the gene expression stability of six candidate reference genes (GAPDH, ACTB, B2M, RPL4, TBP, and PPIA) in samples of whole blood and PBMC with or without poly I:C stimulation. Generally, the six candidate genes performed a similar trend of expression stability in the samples of whole blood and PBMC, but more stably expressed in whole blood than in PBMC. geNorm ranked B2M and PPIA as the best combination for gene expression normalization, while according to NormFinder, TBP was ranked as the most stable reference gene, followed by B2M and PPIA. Comprehensively considering the results from the two programs, we recommended using the geometric mean of the three genes, TBP, PPIA and B2M, to normalize the gene expression of whole blood and PBMC with poly I:C stimulation. Our study is the first detailed survey of the gene expression stability in whole blood and PBMC with or without poly I:C stimulation and should be helpful for investigating the molecular mechanism involved in porcine whole blood and PBMC in response to poly I:C stimulation.
doi:10.5713/ajas.2013.13471
PMCID: PMC4093518  PMID: 25049976
Pigs; Reference Genes; Whole Blood; Peripheral Blood Mononuclear Cells (PBMC); Polyinosinic:Polycytidylic Acid (Poly I:C)
15.  CCR2-64I Polymorphism Is Not Associated with Altered CCR5 Expression or Coreceptor Function 
Journal of Virology  1999;73(3):2450-2459.
A polymorphism in the gene encoding CCR2 is associated with a delay in progression to AIDS in human immunodeficiency virus (HIV)-infected individuals. The polymorphism, CCR2-64I, changes valine 64 of CCR2 to isoleucine. However, it is not clear whether the effect on AIDS progression results from the amino acid change or whether the polymorphism marks a genetically linked, yet unidentified mutation that mediates the effect. Because the gene encoding CCR5, the major coreceptor for HIV type 1 primary isolates, lies 15 kb 3′ to CCR2, linked mutations in the CCR5 promoter or other regulatory sequences could explain the association of CCR2-64I with slowed AIDS pathogenesis. Here, we show that CCR2-64I is efficiently expressed on the cell surface but does not have dominant negative activity on CCR5 coreceptor function. A panel of peripheral blood mononuclear cells (PBMC) from uninfected donors representing the various CCR5/CCR2 genotypes was assembled. Activated primary CD4+ T cells of CCR2 64I/64I donors expressed cell surface CCR5 at levels comparable to those of CCR2 +/+ donors. A slight reduction in CCR5 expression was noted, although this was not statistically significant. CCR5 and CCR2 mRNA levels were nearly identical for each of the donor PBMC, regardless of genotype. Cell surface CCR5 and CCR2 levels were more variable than mRNA transcript levels, suggesting that an alternative mechanism may influence CCR5 cell surface levels. CCR2-64I is linked to the CCR5 promoter polymorphisms 208G, 303A, 627C, and 676A; however, in transfected promoter reporter constructs, these did not affect transcriptional activity. Taken together, these findings suggest that CCR2-64I does not act by influencing CCR5 transcription or mRNA levels.
PMCID: PMC104492  PMID: 9971830
16.  Possible Association between Expression of Chemokine Receptor-2 (CCR2) and Amyotrophic Lateral Sclerosis (ALS) Patients of North India 
PLoS ONE  2012;7(6):e38382.
Background and Objectives
We earlier reported elevated chemokine ligand-2 (CCL2) in Indian amyotrophic lateral sclerosis (ALS) patients. We now analysed chemokine receptor-2 (CCR2), the receptor of CCL2, in these ALS patients.
Methods
Indian sporadic ALS patients (n = 50) were included on the basis of El Escorial criteria. Percentage (%) of CCR2 expressing peripheral blood mononuclear cells (PBMCs) was evaluated using Flow Cytometry. Real Time Polymerase Chain Reaction (PCR) was used to quantitate CCR2 mRNA expression in PBMCs. Normal controls (n = 40) were also included for comparison.
Results
Flow Cytometry revealed significantly reduced CCR2 expressing PBMCs in the ALS patients. We also found a significant decline in number of CCR2 expressing PBMCs in limb onset ALS when compared to bulbar onset ALS. PBMCs from ALS patients showed substantial down-regulation of CCR2 mRNA. CCR2 mRNA expression was found to be decreased among limb ALS patients as compared to bulbar onset ALS. Further, the count of CCR2+ PBMCs and CCR2 mRNA transcript in PBMCs was significantly lower in severe and moderate ALS as compared to ALS patients with mild impairments.
Conclusions
Downregulation of PBMCs CCR2 may indicate its etio-pathological relevance in ALS pathogenesis. Reduced PBMCs CCR2 may result in decreased infiltration of leukocytes at the site of degeneration as a compensatory response to ALS. CCR2 levels measurements in hematopoietic stem cells and estimation of comparative PBMCs count among ALS, disease controls and normal controls can unveil its direct neuroprotective role. However, the conclusions are restricted by the absence of neurological/non-neurological disease controls in the study.
doi:10.1371/journal.pone.0038382
PMCID: PMC3369904  PMID: 22685564
17.  Influence of the CCR2-V64I Polymorphism on Human Immunodeficiency Virus Type 1 Coreceptor Activity and on Chemokine Receptor Function of CCR2b, CCR3, CCR5, and CXCR4 
Journal of Virology  1998;72(9):7450-7458.
The chemokine receptors CCR5 and CXCR4 are used by human immunodeficiency virus type 1 (HIV-1) in conjunction with CD4 to infect cells. In addition, some virus strains can use alternative chemokine receptors, including CCR2b and CCR3, for infection. A polymorphism in CCR2 (CCR2-V64I) is associated with a 2- to 4-year delay in the progression to AIDS. To investigate the mechanism of this protective effect, we studied the expression of CCR2b and CCR2b-V64I, their chemokine and HIV-1 coreceptor activities, and their effects on the expression and receptor activities of the major HIV-1 coreceptors. CCR2b and CCR2b-V64I were expressed at similar levels, and neither molecule affected the expression or coreceptor activity of CCR3, CCR5, or CXCR4 in cotransfected cell lines. Peripheral blood mononuclear cells (PBMCs) from CCR2-V64I heterozygotes had normal levels of CCR2b and CCR5 but slightly reduced levels of CXCR4. CCR2b and CCR2b-V64I functioned equally well as HIV-1 coreceptors, and CCR2-V64I PBMCs were permissive for HIV-1 infection regardless of viral tropism. The MCP-1-induced calcium mobilization mediated by CCR2b signaling was unaffected by the polymorphism, but MCP-1 signaling mediated by either CCR2b- or CCR2-V64I-encoded receptors resulted in heterologous desensitization (i.e., limiting the signal response of other receptors) of both CCR5 and CXCR4. The heterologous desensitization of CCR5 and CXCR4 signaling by both CCR2 allele receptor types provides a mechanistic link that might help explain the in vivo effects of CCR2 gene variants on progression to AIDS as well as the reported antiviral activity of natural CCR2 ligands.
PMCID: PMC109977  PMID: 9696841
18.  Kinetic Analysis of Human T-Cell Leukemia Virus Type 1 Gene Expression in Cell Culture and Infected Animals▿  
Journal of Virology  2009;83(8):3788-3797.
Human T-cell leukemia virus type 1 (HTLV-1) infection causes adult T-cell leukemia and is associated with a variety of lymphocyte-mediated disorders. It has been hypothesized that a highly regulated pattern of HTLV-1 gene expression is critical for virus survival and disease pathogenesis. In this study, real-time reverse transcriptase PCR was used to determine the kinetics of viral gene expression in cells transiently transfected with an HTLV-1 proviral plasmid, in newly infected human peripheral blood mononuclear cells (PBMCs), and in PBMCs from newly infected rabbits. The HTLV-1 gene expression profiles in transiently transfected and infected cells were similar; over time, all transcripts increased and then maintained stable levels. gag/pol, tax/rex, and env mRNA were detected first and at the highest levels, whereas the expression levels of the accessory genes, including the antisense Hbz, were significantly lower than the tax/rex levels (ranging from 1 to 4 logs depending on the specific mRNA). In infected rabbits, tax/rex and gag/pol mRNA levels peaked early after inoculation and progressively decreased, which correlated inversely with the proviral load and host antibody response against viral proteins. Interestingly, Hbz mRNA was detectable at 1 week postinfection and increased and stabilized. The expression levels of all other HTLV-1 genes in infected rabbit PBMCs were at or below our limit of detection. This analysis provides insight into viral gene expression under various in vitro and in vivo experimental conditions. Our in vivo data indicate that in infected rabbits, Hbz mRNA expression over time directly correlates with the proviral load, which provides the first evidence linking Hbz expression to proviral load and the survival of the virus-infected cell in the host.
doi:10.1128/JVI.02315-08
PMCID: PMC2663277  PMID: 19193802
19.  Analysis Of Whole Genome Biomarker Expression In Blood And Brain 
The consistency of peripheral gene expression data and the overlap with brain expression has not been evaluated in biomarker discovery, nor has it been reported in multiple tissues from the same subjects on a genome wide transcript level. The effects of processing whole blood, transformation, and passaged cell lines on gene expression profiling was studied in healthy subjects using Affymetrix arrays. Ficoll extracted peripheral blood mononuclear cells (PBMCs), Epstein-Barr virus (EBV) transformed lymphocytes, passaged lymphoblastic cell lines (LCLs), and whole blood from Tempus tubes were compared. There were 6,813 transcripts differentially expressed between different methods of blood preparation. Principal component analysis resolved two partitions involving pre- and post-transformation EBV effects.
Combining results from Affymetrix arrays, postmortem subjects' brain and PBMC profiles showed co-expression levels of summarized transcripts for 4,103 of 17,859 (22.9%) RefSeq transcripts. In a control experiment, rat hemi-brain and blood showed similar expression levels for 19% of RefSeq transcripts. After filtering transcripts that were not significantly different in abundance between human cerebellum and PBMCs from the Affymetrix exon array the correlation in mean transcript abundance was high as expected (r = 0.98). Differences in the alternative splicing index in brain and blood were found for about 90% of all transcripts examined. This study demonstrates over 4,100 brain transcripts co-expressed in blood samples can be further examined by in vitro and in vivo experimental studies of blood and cell lines from patients with psychiatric disorders.
doi:10.1002/ajmg.b.31062
PMCID: PMC3098564  PMID: 20127885
biomarker; gene expression; whole genome
20.  Genotyping of the CCR5 Chemokine Receptor by Isothermal NASBA Amplification and Differential Probe Hybridization 
The human CCR5 chemokine receptor functions as a coreceptor with CD4 for infection by macrophage-tropic isolates of human immunodeficiency virus type 1 (HIV-1). A mutated CCR5 allele which encodes a protein that does not function as a coreceptor for HIV-1 has been identified. Thus, expression of the wild-type and/or mutation allele is relevant to determining the infectibility of patient peripheral blood mononuclear cells (PBMC) and affects disease progression in vivo. We developed a qualitative CCR5 genotyping assay using NASBA, an isothermal nucleic acid amplification technology. The method involves three enzymes and two oligonucleotides and targets the CCR5 mRNA, which is expressed in PBMC at a copy number higher than 2, the number of copies of DNA present encoding the gene. The single oligonucleotide set amplifies both alleles, and genotyping is achieved by separate hybridizations of wild-type- and mutation-specific probes directly to the single-stranded RNA amplification product. Assay sensitivity and specificity were demonstrated with RNAs produced in vitro from plasmid clones bearing the DNA encoding each allele. No detectable cross-reactivity between wild-type and mutation probes was found, and 50 copies of each allele were readily detectable. Analysis of patient samples found that 20% were heterozygous and 1% were homozygous for the CCR5 mutation. Thus, NASBA is a sensitive and specific means of rapidly determining CCR5 genotype and provides several technical advantages over alternative assay systems.
PMCID: PMC95805  PMID: 10548593
21.  Transcriptomic Response of Porcine PBMCs to Vaccination with Tetanus Toxoid as a Model Antigen 
PLoS ONE  2013;8(3):e58306.
The aim of the present study was to characterize in vivo genome-wide transcriptional responses to immune stimulation in order to get insight into the resulting changes of allocation of resources. Vaccination with tetanus toxoid was used as a model for a mixed Th1 and Th2 immune response in pig. Expression profiles of PBMCs (peripheral blood mononuclear cells) before and at 12 time points over a period of four weeks after initial and booster vaccination at day 14 were studied by use of Affymetrix GeneChip microarrays and Ingenuity Pathway Analysis (IPA). The transcriptome data in total comprised more than 5000 genes with different transcript abundances (DE-genes). Within the single time stages the numbers of DE-genes were between several hundred and more than 1000. Ingenuity Pathway Analysis mainly revealed canonical pathways of cellular immune response and cytokine signaling as well as a broad range of processes in cellular and organismal growth, proliferation and development, cell signaling, biosynthesis and metabolism. Significant changes in the expression profiles of PBMCs already occurred very early after immune stimulation. At two hours after the first vaccination 679 DE-genes corresponding to 110 canonical pathways of cytokine signaling, cellular immune response and other multiple cellular functions were found. Immune competence and global disease resistance are heritable but difficult to measure and to address by breeding. Besides QTL mapping of immune traits gene expression profiling facilitates the detection of functional gene networks and thus functional candidate genes.
doi:10.1371/journal.pone.0058306
PMCID: PMC3607572  PMID: 23536793
22.  Identification of Candidate Genes in Scleroderma-Related Pulmonary Arterial Hypertension 
We hypothesize that pulmonary arterial hypertension (PAH)-associated genes identified by expression profiling of peripheral blood mononuclear cells (PBMCs) from patients with idiopathic pulmonary arterial hypertension (IPAH) can also be identified in PBMCs from scleroderma patients with PAH (PAH-SSc). Gene expression profiles of PBMCs collected from IPAH (n=9), PAH-SSc (n=10) patients and healthy controls (n=5) were generated using HG_U133A_2.0 GeneChips and processed by RMA/GCOS_1.4/SAM_1.21 data analysis pipeline. Disease severity in consecutive patients was assessed by functional status and hemodynamic measurements. The expression profiles were analyzed using PAH severity-stratification, and identified candidate genes were validated with real time PCR (rtPCR). Transcriptomics of PBMCs from IPAH patients was highly comparable with that of PMBCs from PAH-SSc patients. The PBMC gene expression patterns significantly correlate with right atrium pressure (RA) and cardiac index (CI), known predictors of survival in PAH. Array stratification by RA and CI identified 364 PAH-associated candidate genes. Gene ontology analysis revealed significant (Zscore > 1.96) alterations in angiogenesis genes according to PAH severity: MMP9 and VEGF were significantly upregulated in mild as compared to severe PAH and healthy controls, as confirmed by rtPCR. These data demonstrate that PBMCs from patients with PAH-SSc carry distinct transcriptional expression. Furthermore, our findings suggest an association between angiogenesis-related gene expression and severity of PAH in PAH-SSc patients. Deciphering the role of genes involved in vascular remodeling and PAH development may reveal new treatment targets for this devastating disorder.
doi:10.1016/j.trsl.2007.12.010
PMCID: PMC2359723  PMID: 18355767
23.  Evaluation of suitable reference genes for gene expression studies in porcine PBMCs in response to LPS and LTA 
BMC Research Notes  2013;6:56.
Background
As an in vitro model porcine peripheral blood mononuclear cells (PBMCs) is frequently used as for immunogenetic research with the stimulation of bacterial antigens. To investigate the immunocompetence of PBMCs for recognition of Gram-positive and Gram-negative bacteria and in order to dissect the pathogenesis of diseases, gene expression assay is most commonly used. The gene expressions are required to normalize for reference genes which have tremendous effect on the results of expression study. The reference genes should be stably expressed between different cells under a variety of experimental conditions, but recent influx of data showed that expression stability of reference genes are varied under different experimental conditions. But data regarding the expression stability of reference genes in porcine PBMCs are limited. Therefore, this study was aimed to know whether the expression stability of commonly used reference genes in PBMCs is affected by various bacterial antigens under different experimental conditions in pigs.
Results
The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined by RT-qPCR in PBMCs that were stimulated by LPS and LTA in vitro as well as cells un-stimulated control and non-cultured were also consider for this experiment. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P < 0.05). geNorm software revealed that in case of irrespective of stimulation (without considering the type of stimulation), RPL4, PPIA and B2M were the most stable reference genes in PBMCs; in case of the control group, PPIA, BLM and GAPDH were the most stable reference genes. PPIA, B2M and RPL4 were the most stable reference genes in LPS stimulated PBMCs; and YWHAZ, RPL4 and PPIA were the most stably expressed reference genes in the case of LTA stimulated PBMCs. When LPS was used combined with LTA for the stimulation, YWHAZ, B2M and SDHA remained the most stable genes. PPIA, BLM and GAPDH were found to be most stably expressed reference genes when PBMCs were not cultured. NormFinder revealed different sets of stably expressed reference genes in PBMCs under different experimental conditions. Moreover, geNorm software suggested that the geometric mean of the three most stable genes would be the suitable combination for accurate normalization of gene expression study.
Conclusion
There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms (geNorm and NormFinder). In conclusion, the geometric mean of the RPL4, B2M and PPIA seemed to be the most appropriate combination of reference genes for accurate normalization of gene expression data in porcine PBMCs without knowing the type of bacterial pathogenic status of the animals and in the case of mixed infection with Gram-negative and Gram-positive bacteria. In case of PBMCs without any stimulation, PPIA, BLM and GAPDH could be suggested as suitable reference genes.
doi:10.1186/1756-0500-6-56
PMCID: PMC3584940  PMID: 23394600
Reference genes; PBMC; LPS; LTA; Pigs
24.  Relationship between quantity of IFNT estimated by IFN-stimulated gene expression in peripheral blood mononuclear cells and bovine embryonic mortality after AI or ET 
Background
Interferon tau (IFNT), which is secreted into the uterine cavity during the maternal recognition period (MRP), is a key factor for establishment of pregnancy. The present study aims to clarify the relationship between the ability of a bovine conceptus to produce IFNT during the MRP and the conceptus's ability to establish pregnancy.
Methods
In the first experiment, IFNT (0, 500, or 1000 micrograms) was administered into the uterine horn ipsilateral to the CL 16 or 17 d after standing estrus, and mRNA levels of IFN-stimulated gene 15-kDa protein (ISG15) and Mx2 in peripheral blood mononuclear cells (PBMCs) were determined. In the second experiment, we investigated ISG15 mRNA expression in PBMCs during the MRP in cattle after either artificial insemination (AI) or embryo transfer (ET).
Results
Intrauterine administration of IFNT stimulated ISG15 and Mx2 gene expressions in PBMCs in cattle, and there was a positive correlation between the expressions of peripheral markers and the quantity of IFNT administered. In pregnant and normal interestrous interval (< 25 d) cattle (nIEI cattle), expression levels of the ISG15 gene showed similar patterns after AI and ET, and ISG15 mRNA expression was increased in pregnant cattle but unchanged in nIEI cattle. In contrast, ISG15 gene expression in extended interestrous interval (greater than or equal to 25 d) cattle (eIEI cattle) differed after ET compared with AI. In eIEI cattle after ET, ISG15 gene expression increased, such that the value on day 18 was intermediate between those of pregnant and nIEI cattle. In eIEI cattle after AI, ISG15 gene expression did not increase throughout the observation period.
Conclusions
The results of the current study indicate that the quantity of conceptus-derived IFNT can be estimated by measuring ISG15 mRNA levels in PBMCs from cattle. Using this approach, we demonstrate that ISG15 gene expression during the MRP in eIEI cattle differed after ET compared with AI. In addition, the modest increase in ISG15 gene expression in eIEI cattle after ET suggests that late embryo losses were due to delayed or insufficient growth of the conceptus during the MRP in cattle.
doi:10.1186/1477-7827-10-21
PMCID: PMC3364858  PMID: 22439976
Interferon tau; Interferon-stimulated gene 15-kDa protein; Embryonic mortality; Maternal recognition period; Cattle; Peripheral blood mononuclear cells; Corpus luteum; Estrous cycle
25.  RRM1 single nucleotide polymorphism -37C→A correlates with progression-free survival in NSCLC patients after gemcitabine-based chemotherapy 
Background
The ribonucleotide reductase M1 (RRM1) gene encodes the regulatory subunit of ribonucleotide reductase, the molecular target of gemcitabine. The overexpression of RRM1 mRNA in tumor tissues is reported to be associated with gemcitabine resistance. Thus, single nucleotide polymorphisms (SNPs) of the RRM1 gene are potential biomarkers of the response to gemcitabine chemotherapy. We investigated whether RRM1 expression in peripheral blood mononuclear cells (PBMCs) or SNPs were associated with clinical outcome after gemcitabine-based chemotherapy in advanced non-small cell lung cancer (NSCLC) patients.
Methods
PBMC samples were obtained from 62 stage IIIB and IV patients treated with gemcitabine-based chemotherapy. RRM1 mRNA expression levels were assessed by real-time PCR. Three RRM1 SNPs, -37C→A, 2455A→G and 2464G→A, were assessed by direct sequencing.
Results
RRM1 expression was detectable in 57 PBMC samples, and SNPs were sequenced in 56 samples. The overall response rate to gemcitabine was 18%; there was no significant association between RRM1 mRNA expression and response rate (P = 0.560). The median progression-free survival (PFS) was 23.3 weeks in the lower expression group and 26.9 weeks in the higher expression group (P = 0.659). For the -37C→A polymorphism, the median PFS was 30.7 weeks in the C(-)37A group, 24.7 weeks in the A(-)37A group, and 23.3 weeks in the C(-)37C group (P = 0.043). No significant difference in PFS was observed for the SNP 2455A→G or 2464G→A.
Conclusions
The RRM1 polymorphism -37C→A correlated with PFS in NSCLC patients treated with gemcitabine-based chemotherapy. No significant correlation was found between PBMC RRM1 mRNA expression and the efficacy of gemcitabine.
doi:10.1186/1756-8722-3-10
PMCID: PMC2855513  PMID: 20226083

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