Profiling Of Peripheral Blood Mononuclear Cells Research Articles

RNA expression profiling in sulfamethoxazole-treated patients with a range of invitro lymphocyte cytotoxicity phenotypes.

The lymphocyte toxicity assay (LTA) is a proposed surrogate marker of sulfonamide antibiotic hypersensitivity. In the LTA, peripheral blood mononuclear cells (PBMCs) undergo apoptosis more readily in hypersensitive versus tolerant patients when exposed to drug‐hydroxylamine metabolites in vitro. The purpose of this study was to identify key gene transcripts associated with increased cytotoxicity from sulfamethoxazole‐hydroxylamine in human PBMCs in the LTA. The LTA was performed on PBMCs of 10 patients hypersensitive to trimethoprim‐sulfamethoxazole (HS) and 10 drug‐tolerant controls (TOL), using two cytotoxicity assays: YO‐PRO (n = 20) and MTT (n = 12). mRNA expression profiles of PBMCs, enriched for CD8+ T cells, were compared between HS and TOL patients. Transcript expression was interrogated for correlation with % cytotoxicity from YO‐PRO and MTT assays. Correlated transcripts of interest were validated by qPCR. LTA results were not significantly different between HS and TOL patients, and no transcripts were found to be differentially expressed between the two groups. 96 transcripts were correlated with cytotoxicity by YO‐PRO (r = ±.63‐.75, FDR 0.188). Transcripts were selected for validation based on mechanistic plausibility and three were significantly over‐expressed by qPCR in high cytotoxicity patients: multi‐specific organic anion transporter C (ABCC5), mitoferrin‐1 (SLC25A37), and Porimin (TMEM123). These data identify novel transcripts that could contribute to sulfonamide‐hydroxylamine induced cytotoxicity. These include SLC25A37, encoding a mitochondrial iron transporter, ABCC5, encoding an arylamine drug transporter, and TMEM123, encoding a transmembrane protein that mediates cell death.

Long Noncoding RNAs and mRNA Regulation in Peripheral Blood Mononuclear Cells of Patients with Chronic Obstructive Pulmonary Disease.

Background Inflammation plays a pivotal role in the pathogenesis of chronic obstructive pulmonary disease (COPD). We evaluated the lncRNA and mRNA expression profile of peripheral blood mononuclear cells (PBMCs) from healthy nonsmokers, smokers without airflow limitation, and COPD patients. Methods lncRNA and mRNA profiling of PBMCs from 17 smokers and 14 COPD subjects was detected by high-throughput microarray. The expression of dysregulated lncRNAs was validated by qPCR. The lncRNA targets in dysregulated mRNAs were predicted and the GO enrichment was analyzed. The regulatory role of lncRNA ENST00000502883.1 on CXCL16 expression and consequently the effect on PBMC recruitment were investigated by siRNA knockdown and chemotaxis analysis. Results We identified 158 differentially expressed lncRNAs in PBMCs from COPD subjects compared with smokers. The dysregulated expression of 5 selected lncRNAs NR_026891.1 (FLJ10038), ENST00000502883.1 (RP11-499E18.1), HIT000648516, XR_429541.1, and ENST00000597550.1 (CTD-2245F17.3), was validated. The GO enrichment showed that leukocyte migration, immune response, and apoptosis are the main enriched processes that previously reported to be involved in the pathogenesis of COPD. The regulatory role of ENST00000502883.1 on CXCL16 expression and consequently the effect on PBMC recruitment was confirmed. Conclusion This study may provide clues for further studies targeting lncRNAs to control inflammation in COPD.

MIF promotes a differential Th1/Th2/Th17 inflammatory response in human primary cell cultures: Predominance of Th17 cytokine profile in PBMC from healthy subjects and increase of IL-6 and TNF-α in PBMC from active SLE patients

Macrophage migration Inhibitory Factor (MIF) is a cytokine associated with the pathogenesis of autoimmune inflammatory diseases. There is evidence that MIF functions in a positive-feedback-loop with proinflammatory cytokines and could perpetuate the inflammatory process in Systemic Lupus Erythematosus (SLE).The aim of this study was to assess the effect of recombinant-human-MIF (rhMIF) on the expression of Th1, Th2 and Th17 cytokines in Peripheral Blood Mononuclear Cells (PBMC) from Healthy Subjects (HS) and SLE patients. The PBMC were isolated from SLE patients classified according to the 1997 SLE ACR criteria and HS donors; all subjects included were women from an unrelated Mexican-Mestizo population. The PBMC isolated were stimulated with rhMIF, LPS and ISO-1 in different combinations; Th1, Th2 and Th17cytokine profiles levels were determined by MAGPIX Bio-plex assay in supernatants from cell cultures. We observed in supernatants of PBMCs from HS treated with rhMIF a predominance of Th17 cytokine profile with an increase of IL-17A, IL-17F and IL-21 versus PBMCs from SLE patients, which showed an inflammatory profile represented by increase of IL-6 cytokine. According to SLE remission/activity presented at enrollment in the study (Mex-SLEDAI index), the PBMC from active SLE patients showed higher levels of TNF-α and IL-6 versus PBMC from remission SLE patients. In conclusion, our results suggest that MIF can induce a differential inflammatory response in physiological and pathological conditions with a predominance of a Th17 cytokine profile in PBMC from HS and an increase in TNF-α and IL-6 expression in PBMC from active SLE patients.

Abstract 36: Changes in cellular and molecular immune markers in the peripheral blood of patients undergoing chemotherapy and radiation for squamous cell carcinoma of head and neck: A prospective pilot study

Abstract Objective: In order to help further development of novel immunotherapeutic targets related to signal-transducer and activator of transcription-3 (STAT3) in squamous cell carcinoma of head and neck (SCCHN), data are needed to help understand changes in immune-mediated signaling during treatment. The purpose of this study was to analyze changes in the mRNA or protein expression of inflammatory or immunosuppressive mediators in peripheral blood mononuclear cells (PBMC) or plasma, in SCCHN patients receiving concurrent chemotherapy and radiation therapy (CRT). Methods: This was a prospective biospecimen collection study of 15 patients with stage III-IV SCCHN who received CRT with curative intent. Blood specimens were obtained before, during weeks 1, 2, 4, 6, and 7 of CRT, and 6-12 weeks after treatment completion. Plasma and PBMC were separated from patients' blood using Vacutainer CPT tubes (BD Bioscience) within 2 hours after collection by centrifugation at 1800×g for 20 min at room temperature and stored in -80°C. Plasma samples were analyzed for 30 human cytokines and chemokines using Luminex FLEXMAP 3D system at the Clinical Immunobiology Correlative Studies (CICSL) core at City of Hope. mRNA was isolated from PBMCs acquired at the baseline and at the second week draw during RT from 6 patients and expression of 730 genes selected for immune profiling was evaluated using nCounter Immune Cell Profiling Assay, and data were analyzed using nSolver software (Nanostring). Results: Among the plasma level of 30 cytokines tested by Luminex Assay, IL-6 showed cumulative increase during RT (one-way ANOVA, p=0.046). At 6 week post RT, IL-6 level decreased comparable to baseline level. Plasma level of IFN-gamma was significantly increased at 2 week of RT and the level remained unchanged until 12 week post CRT (p=0.0004). In contrast, we observed pro-inflammatory cytokine IL-12 was significantly decreased during CRT (p=0.0004). Plasma level of a tumor promoting cytokine HGF was significantly increased during and 12 week post CRT (p=0.0035). Comparison of gene expression profiles in PBMC from 6 patients between baseline and the second week of CRT indicates that mRNA expression of 335 immune-regulators are differentially expressed (p<0.05) among 740 tested. Consistent with the plasma cytokine analysis, mRNA level of IL-6 in PBMC was 13.9 fold increased (p=0.003). Subsequently, increase in STAT3 expression was observed (1.7 fold, p=0.002) since IL-6 stimulates Janus kinase (JAK)-STAT pathway. In addition, mRNA expression of Th2 cytokines including IL-10, IL-5, and TGFb1 were significantly increased by greater than 2 folds (p<0.05). Conclusion: These results suggest CRT affects immune activation in SCCHN patients systemically, which may contribute to immune suppression and disease relapse after treatment. Our data provide an impetus for investigating STAT3-targeted therapies in SCCHN. Citation Format: Sagus Sampath, Hae Jung Won, Ellie Maghami, Erminia Massarelli, Nayana Vora, Lalit Vora, Marcin Kortylewski. Changes in cellular and molecular immune markers in the peripheral blood of patients undergoing chemotherapy and radiation for squamous cell carcinoma of head and neck: A prospective pilot study [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 36.

Abstract 5706: Gene expression profile of peripheral blood mononuclear cells in breast cancer patients may be contribute to the identification and the immunological classification of breast cancer patients by blood test

Abstract It is reported that gene expression profile of peripheral blood mononuclear cells (PBMCs) exhibits unique gene expression signature in cancer patients including renal cell carcinoma, pancreatic cancer and lung cancer. Since pancreatic cancer diagnosis is difficult in not only early detection of the disease but also in the diagnosis itself, development of novel diagnostic tools in addition to conventional diagnostic strategy has been awaited. On the other hand, in breast cancer, because early diagnosis by mammography and ultra sound examination on breast is established successfully, exploration of gene expression profile of PBMCs may be important in terms of insight to host antitumor immune response aspects. In the current study, we performed RNA sequencing (RNA-seq) analysis on RNA of PBMCs from 3 healthy volunteers, 6 early and 7 metastatic breast cancer patients including all phenotypes defined by ER, PgR and HER2. Genes that showed FDR<0.05 and fold change of <0.5 and 2.0< in comparison of healthy volunteers and breast cancer patients were applied to clustering analysis by Ward method. We found significant unique gene expression signature between healthy volunteers and breast cancer patients. Furthermore, it was shown that HER2 overexpressing breast cancer patients, which breast cancer patients were divided into 3 clusters including HER2 type and other 2 different groups, seemed to be classified into healthy group different from other phenotypes of breast cancer patients. These findings suggested that evaluation of gene expression patterns of PBMCs of breast cancer patients might distinguish breast cancer patients from healthy subjects and the gene expression signature of PBMCs which divided breast cancer patients into 3 groups might reveal immunologically important biologic properties such as response prediction of cancer immunotherapy including immune checkpoint inhibition treatment. Citation Format: Eiji Suzuki, Kosuke Kawaguchi, Masahiro Sugimoto, Fengling Pu, Ryuji Uozumi, Ayane Yamaguchi, Mariko Nishie, Moe Tsuda, Takeshi Kotake, Satoshi Morita, Masakazu Toi. Gene expression profile of peripheral blood mononuclear cells in breast cancer patients may be contribute to the identification and the immunological classification of breast cancer patients by blood test [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5706. doi:10.1158/1538-7445.AM2017-5706

Gene Expression Profile of Peripheral Blood Mononuclear Cells in Response to Intracerebral Hemorrhage.

RNA-sequencing, a powerful tool, yields a comprehensive view of whole transcriptome. Intracerebral hemorrhage (ICH) is a devastating form of stroke. To date, RNA-sequencing analysis of ICH has not been reported. Peripheral blood mononuclear cells (PBMCs) were used as a source of mRNA for gene expression profile analysis in stroke. In this study, we performed transcriptome analyses for PBMCs from four ICH patients and four healthy volunteers on Illumina platform. We identified 4040 significantly differentially expressed genes (DEGs). Functional annotation of DEGs with DAVID Bioinformatics Resources indicated that genes associated with cell apoptosis, autophagy, cell-cell adhesion, inflammatory response, protein binding, positive regulation of gene expression, and signal transduction were most significantly enriched by DEGs. Gene set enrichment analysis identified 40 significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including chemokine signaling, cytokine-cytokine receptor interaction, oxidative phosphorylation, and glutathione metabolism processes. These data point to a complex mechanism for ICH pathogenesis. Overall, the present study demonstrated an altered gene expression profile of PBMCs in response to acute ICH. Our study provided important information for understanding the molecular mechanisms of ICH pathogenesis at system-wide levels.

Curcumin Improves Systemic Responses to Exertional Hyperthermia but Doesn’t Alter Protein Expression in Circulating Leukocytes

Exertional heat stress increases gastrointestinal barrier permeability and risk of exertional heatstroke (EHS) via a TLR4-mediated inflammatory pathway. Curcumin has been shown to inhibit TLR4 signaling in vitro but has not been examined in a human exertional heat stress model. PURPOSE: This work investigated the effect of 3d of 500mg/d dietary curcumin supplementation on the cellular and systemic responses to exertional heat stress in non-heat acclimated humans. METHODS: Subjects (N=6) ran (65%VO2max) for 60min inside an environmental chamber (37°C/26%RH) two times (CURCUMIN/PLACEBO). Core temperature (Tc), heart rate (HR), and physiological strain index (PSI) were measured throughout exercise. Peripheral blood mononuclear cells (PBMC) were isolated from blood samples that were taken before (PRE), after (POST), 1hr (1-POST), and 4hrs after (4-POST) exercise. The protein content of markers along the TLR4 signaling pathway (TLR4, MyD88, pNFKB, NFKB) and indicators of cellular energy status (SIRT1 & p-AMPK) were determined with Western Blot. Group differences were determined with 2-Way (Condition x Time) RM ANOVAs. RESULTS: Under CURCUMIN, Tc rose less (0.23±0.15°C; p<0.01) and both HR and PSI were lower from 45-60min of exercise (HR: 9±2 bpm, PSI: 12±1%; p< 0.05). In PBMC, the ratio of pNFKB to NFKB at 1-POST was increased by 64% in PLACEBO and 51% in CURCUMIN (p=0.05). Intriguingly, TLR4 was reduced at 1POST in both conditions (PLACEBO: -28%, CURCUMIN: -17%; p=0.05), as were pAMPK (PLACEBO: -62%, CURCUMIN: -57%; p<0.01) and SIRT1 (PLACEBO: -61%, CURCUMIN: -48%; p=0.02). CONCLUSIONS: Despite robust improvements in systemic responses to exertional heat stress under CURCUMIN, there was no difference in the protein expression profile of PBMC that were collected under CURCUMIN and PLACEBO conditions. However, in both conditions we did note a significant elevation in pNFKB:NFKB at 1-POST, which (ironically) coincided with a significant reduction in TLR4 (but not MyD88). At present, our working hypothesis is that pAMPK and SIRT1 were downregulated in an effort to maintain the pro-inflammatory capacity of PBMC during this “open window”, an effect that has not previously been described in PBMC collected under exertional heat stress conditions.

Identification of the miRNA-mRNA regulatory network of small cell osteosarcoma based on RNA-seq

Small cell osteosarcoma (SCO) is a rare subtype of osteosarcoma characterized by highly aggressive progression and a poor prognosis. The miRNA and mRNA expression profiles of peripheral blood mononuclear cells (PBMCs) were obtained in 3 patients with SCO and 10 healthy individuals using high-throughput RNA-sequencing. We identified 37 dysregulated miRNAs and 1636 dysregulated mRNAs in patients with SCO compared to the healthy controls. Specifically, the 37 dysregulated miRNAs consisted of 27 up-regulated miRNAs and 10 down-regulated miRNAs; the 1636 dysregulated mRNAs consisted of 555 up-regulated mRNAs and 1081 down-regulated mRNAs. The target-genes of miRNAs were predicted, and 1334 negative correlations between miRNAs and mRNAs were used to construct an miRNA-mRNA regulatory network. Dysregulated genes were significantly enriched in pathways related to cancer, mTOR signaling and cell cycle signaling. Specifically, hsa-miR-26b-5p, hsa-miR-221-3p and hsa-miR-125b-2-3p were significantly dysregulated miRNAs and exhibited a high degree of connectivity with target genes. Overall, the expression of dysregulated genes in tumor tissues and peripheral blood samples of patients with SCO measured by quantitative real-time polymerase chain reaction corroborated with our bioinformatics analyses based on the expression profiles of PBMCs from patients with SCO. Thus, hsa-miR-26b-5p, hsa-miR-221-3p and hsa-miR-125b-2-3p may be involved in SCO tumorigenesis.

Increased miR-424-5p expression in peripheral blood mononuclear cells from patients with pemphigus.

Pemphigus is an autoimmune disease that causes blisters and erosions in the skin and mucous membranes. The development of pemphigus is associated with the imbalance of T-cell and humoral responses. MicroRNAs (miRNAs) can regulate many cell functions. However, whether miRNA expression is altered in peripheral blood mononuclear cells (PBMCs) during the pathogenesis of pemphigus has not been clarified. The aim of the present study was to examine the miRNA expression profiles of PBMCs from patients with pemphigus. The expression profiles of miRNAs in PBMCs from patients with active pemphigus (n=3) and healthy subjects (n=3) were analyzed by microarray. The relative levels of miR-424-5p expression in PBMCs from 9 patients and controls were validated by RT-qPCR. The functional and biological processes of the differentially expressed miRNAs were analyzed by bioinformatics. There were 124 differentially expressed miRNAs in PBMCs from the patients with pemphigus, compared with healthy controls, including 71 that were upregulated (P<0.05, fold change >2), and 53 that were downregulated (P<0.05, fold change <0.5). miR-424-5p was highly expressed in patients with pemphigus. Bioinformatics analysis indicated that the genes targeted by miR-424-5p were involved in intracellular signaling cascades, phosphate metabolism and regulation of kinase activity. The predicted target genes were associated with the T-cell receptor and mitogen-activated protein kinase signaling pathways as well as others. In conclusion, the results have demonstrated the miRNA expression profile, and verified that miR-424-5p was upregulated in PBMCs from patients with pemphigus. The biological function and potential pathways of miR-424-5p in pemphigus were predicted. Thus, miR-424-5p may contribute to the pathogenesis of pemphigus.

Transcriptional profiling of leukocytes from rheumatoid arthritis patients before and after anti-tumor necrosis factor therapy: A comparison of anti-nuclear antibody positive and negative subsets.

Anti-nuclear antibodies (ANAs) may be induced in patients with rheumatoid arthritis (RA) receiving anti-tumor necrosis factor (TNF) therapy with TNF inhibitors (TNFi), etanercept, infliximab or adalimumab. In the present study, 11 patients who were TNFi drug naive were started on TNFi at a time of high disease activity. Of these, all cases were positive for rheumatoid factor and 9 cases tested were positive for anti-citrullinated peptide (anti-CCP) antibodies prior to TNFi treatment. Peripheral blood mononuclear cells (PBMCs) and serum were collected from all patients before and after TNFi therapy. Serum was assayed for ANAs over time. Total cellular RNA was extracted from PBMCs and assessed using Illumina arrays. Gene expression profiles were examined for alterations in key effector pathways. After 3 or more months on TNFi, 6 patients converted to ANA-positivity. Analysis of transcripts from patients with RA who converted to ANA-positivity after 3 months on TNFi identified complex gene expression profiles that reflected a reduction in cell adhesion, cell stress and lipid metabolism transcripts. In summary, unique transcriptional profiles in PBMCs from patients with RA were observed after TNFi therapy. This pilot study suggests that transcriptional profiling is a precise method of measuring the impact of TNFi therapies and reveals novel pathways that likely influence the immune response.

PBMC transcriptome profiles identifies potential candidate genes and functional networks controlling the innate and the adaptive immune response to PRRSV vaccine in Pietrain pig.

The porcine reproductive and respiratory syndrome (PRRS) is a devastating viral disease affecting swine production, health and welfare throughout the world. A synergistic action of the innate and the adaptive immune system of the host is essential for mounting a durable protective immunity through vaccination. Therefore, the current study aimed to investigate the transcriptome profiles of peripheral blood mononuclear cells (PBMCs) to characterize the innate and the adaptive immune response to PRRS Virus (PRRSV) vaccination in Pietrain pigs. The Affymetrix gene chip porcine gene 1.0 ST array was used for the transcriptome profiling of PBMCs collected at immediately before (D0), at one (D1) and 28 days (D28) post PRRSV vaccination with three biological replications. With FDR <0.05 and log2 fold change ±1.5 as cutoff criteria, 295 and 115 transcripts were found to be differentially expressed in PBMCs during the stage of innate and adaptive response, respectively. The microarray expression results were technically validated by qRT-PCR. The gene ontology terms such as viral life cycle, regulation of lymphocyte activation, cytokine activity and inflammatory response were enriched during the innate immunity; cytolysis, T cell mediated cytotoxicity, immunoglobulin production were enriched during adaptive immunity to PRRSV vaccination. Significant enrichment of cytokine-cytokine receptor interaction, signaling by interleukins, signaling by the B cell receptor (BCR), viral mRNA translation, IFN-gamma pathway and AP-1 transcription factor network pathways were indicating the involvement of altered genes in the antiviral defense. Network analysis revealed that four network modules were functionally involved with the transcriptional network of innate immunity, and five modules were linked to adaptive immunity in PBMCs. The innate immune transcriptional network was found to be regulated by LCK, STAT3, ATP5B, UBB and RSP17. While TGFß1, IL7R, RAD21, SP1 and GZMB are likely to be predictive for the adaptive immune transcriptional response to PRRSV vaccine in PBMCs. Results of the current immunogenomics study advances our understanding of PRRS in term of host-vaccine interaction, and thereby contribute to design a rationale for disease control strategy.

Gene expression profiles in peripheral blood mononuclear cells correlate with salience network activity in chronic visceral pain: A pilot study.

Distinct gene expression profiles in peripheral blood mononuclear cells (PBMCs) consistent with increased sympathetic nervous system activity have been described in different populations under chronic stress. Neuroinflammatory brain changes, possibly related to the migration of primed monocytes to the brain, have been implicated in the pathophysiology of chronic pain. Irritable bowel syndrome (IBS) is a stress-sensitive gastrointestinal disorder associated with altered brain-gut interactions and increased sympathetic/vagal tone and anxiety. Reports about immune alterations in IBS are conflicting. This pilot study aimed to test how PBMC gene expression inflammatory profiles are correlated with altered brain signatures in the salience system. Sixteen IBS and 16 healthy controls (HCs) completed resting state MRI scans. Gene expression profiles in PBMCs were assessed using human transcriptome array-2. Bioinformatic analyses determined differential expression of PBMCs between IBS and HCs. Partial least squares, a multivariate analysis technique, was used to identify disease correlations between PBMC gene expression profiles and functional activity in the brain's salience network. Regions of the salience network, including the mid cingulate cortex, and mid and superior temporal gyrus were positively correlated with several pro-inflammatory genes (interleukin 6, APOL2) in IBS, but negatively correlated with several anti-inflammatory genes (KRT8, APOA4) in HCs. Based on rodent studies, one may speculate that chronically activated stress signaling pathways in IBS maintain a pro-inflammatory state in the periphery. Alternatively, primed monocytes may migrate to the brain during stress, inducing regional neuroinflammatory changes in salience regions involved in the modulation of visceral sensitivity.

Prevailing vitamin D status influences mitochondrial and glycolytic bioenergetics in peripheral blood mononuclear cells obtained from adults

BackgroundCirculating peripheral blood mononuclear cells (PBMCs) are exposed to metabolic and immunological stimuli that influence their functionality. We hypothesized that prevailing vitamin D status [25(OH)D] would modulate the bioenergetic profile of PBMCs derived from humans. Materials and methods38 participants (16 males, 22 females) ranging in body fat from 14–51% were studied. PBMCs were isolated from whole blood, counted and freshly seeded for bioenergetic analysis using the Seahorse XFe96 flux analyser. Whole body energy metabolism via indirect calorimetry, body composition by dual-energy X-ray absorptiometry, and relevant clinical biochemistry were measured. Data was analysed based on 25(OH)D cut-offs of <50nmol/L (Group 1, n=12), 50–75nmol/L (Group 2, n=15) and ≥75nmol/L (Group 3, n=11). A multivariate general linear model adjusting for age, fat mass, fat-free mass, parathyroid hormone and insulin sensitivity was used. ResultsThere were significant differences in cellular mitochondrial function between groups. Group 1 had significantly higher basal respiration (p=0.001), non-mitochondrial respiration (p=0.009), ATP production (p=0.001), proton leak (p=0.018), background glycolysis (p=0.023) and glycolytic reserve (p=0.039) relative to either Group 2 or Group 3; the latter two did not differ on any measures. There were no differences in bioenergetic health index (BHI), resting metabolic rates and systemic inflammatory markers between groups. ConclusionsInadequate vitamin D status adversely influenced bioenergetic parameters of PBMCs obtained from adults, in a pattern consistent with increased oxidative metabolism and activation of these cells.

Different microRNA alterations contribute to diverse outcomes following EV71 and CA16 infections: Insights from high-throughput sequencing in rhesus monkey peripheral blood mononuclear cells

Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) are the predominant pathogens of hand, foot, and mouth disease (HFMD). Although these viruses exhibit genetic homology, the clinical manifestations caused by the two viruses have some discrepancies. In addition, the underlying mechanisms leading to these differences remain unclear. microRNAs (miRNAs) participate in numerous biological or pathological processes, including host responses to viral infections. Here, we focused on differences in miRNA expression patterns in rhesus monkey peripheral blood mononuclear cells (PBMCs) infected with EV71 and CA16 at various time points using high-throughput sequencing. The results demonstrated that 106 known and 13 novel miRNAs exhibited significant differences, and 32 key miRNAs among them for target prediction presented opposite trends in the EV71- and CA16-infected samples. GO and pathway analysis of the predicted targets showed enrichment in 14 biological processes, 10 molecular functions, 8 cellular components and 104 pathways. Subsequently, regulatory networks of miRNA-transcription factors, miRNA-predicted targets, miRNA-GOs and miRNA-pathways were constructed to reveal the complex regulatory mechanisms of miRNAs during the infection phase. Ultimately, we analysed hierarchical GO categories of the predicted targets involved in immune system processes, which indicated that the innate and adaptive immunity following EV71 and CA16 infections may be remarkably distinct. In conclusion, this report is the first describing miRNA expression profiles in PBMCs with EV71 and CA16 infections using high-throughput sequencing. Our findings could provide a valuable basis for further studies on the regulatory roles of miRNAs related to the different immune responses caused by EV71 and CA16 infections.

P6011 Transcriptome profiling of the peripheral blood mononuclear cells following PRRSV vaccination in Pietrain pig

Porcine reproductive and respiratory syndrome (PRRS) is a devastating viral disease affecting swine production, health and welfare throughout the world. Vaccination has been considered as one of the most economic tools for PRRS control. A synergistic action of the innate and the adaptive immune system of host is essential for developing a durable protective immunity to vaccine antigen. The peripheral blood mononuclear cells (PBMCs) play central role in immune system and are able to display gene expression patterns characteristics for certain infection. Therefore, the current study aimed to investigate the global transcriptome profiles of PBMCs to characterize the innate and the adaptive immune response to PRRS Virus (PRRSV) vaccine in Pietrain pigs. We employed nine Affymetrix gene chip porcine gene 1.0 ST array for the transcriptome profiling of PBMCs collected from three female piglets at immediately before (D0), at one (D1) and 28 d (D28) post PRRSV vaccination given at 4 wk (D0) of their age. Two pairwise contrasts were tested to characterize transcriptome alterations associated with the innate immune response (D1 vs. D0) and the adaptive immune response (D28 vs. D0). Normalization and statistical analysis of microarray data was performed with the ‘oligo’ and ‘limma’ R/Bioconductor package. With FDR < 0.05 and log2 fold change ± 1.5 as cutoff criteria, 83 and 53 transcripts were found to be differentially expressed in PBMCs during innate and adaptive response, respectively. The microarray expression results were technically validated by qRT-PCR. The gene ontology (GO) terms such as viral life cycle, regulation of lymphocyte activation, cytokine activity and inflammatory response were enriched during the innate immune response. The GO terms enriched during adaptive response includes cytolysis, T cell mediated cytotoxicity, immunoglobulin production. Significant enrichment of cytokine-cytokine receptor interaction, signaling by interleukins, viral mRNA translation, IFN-γ pathway and AP-1 transcription factor network pathways was indicating the involvement of altered genes in the antiviral defense. Network analysis has detected four module were functionally involved with functional network of innate immune transcriptional response and five modules were detected for adaptive immune responses. The innate immune transcriptional network found to be regulated by LCK, STAT3, ATP5B, UBB and RSP17. While TGFβ, IL7R, RAD21, SP1 and GZMB are responsible for coordinating the adaptive immune transcriptional response to PRRSV vaccine in PBMCs. Further work is required to determine whether polymorphisms linked to these genes affect the immune response to PRRSV vaccine in pigs.

Clinical and kinomic analysis identifies peripheral blood mononuclear cells as a potential pharmacodynamic biomarker in metastatic renal cell carcinoma patients treated with sunitinib.

BackgroundSunitinib is a protein tyrosine kinase (PTK) inhibitor that has immune-modulating properties. In this context, peripheral blood mononuclear cells (PBMC), mainly constituted by lymphocytes, could be a perfect surrogate tissue for identifying and assaying pharmacodynamic biomarkers of sunitinib. In this study, we investigated the changes in lymphocytes count as pharmacodynamic biomarker in metastatic renal cell carcinoma (mRCC) patients under sunitinib therapy. Thereafter, we studied the ex vivo effect of sunitinib and SU12262 (active metabolite) on PBMC from naïve mRCC patients using a high throughput kinomic profiling method.MethodsThe prognostic value of total lymphocytes count between Day 0 and Day 21 (expressed as a ratio D21/D0) was retrospectively investigated in 88 mRCC patients under sunitinib therapy. PTK PamChip® microarrays were used to explore prospectively the ex vivo effect of sunitinib and SU12662 on PTK activity in PBMC from 21 naïve mRCC patients.ResultsIn this retrospective study, D21/D0 lymphocytes ratio (Hazard Ratio, 1.83; CI95%, 1.24-2.71; p=0.0023) was independently associated with PFS. Interestingly, kinomic analysis showed that D21/D0 lymphocytes ratio and Heng prognostic model was statistically associated with the ex vivo sunitinib and SU12662 effect in PBMC.ConclusionThe present study highlights that D21/D0 total lymphocytes ratio could be a promising pharmacodynamic biomarker in mRCC patients treated with sunitinib. Additionally, it paves the way to investigate the kinomic profile in PBMC as a prognostic factor in a larger cohort of mRCC patients under sunitinib therapy.

Deciphering transcriptome profiles of peripheral blood mononuclear cells in response to PRRSV vaccination in pigs.

BackgroundPorcine reproductive and respiratory syndrome (PRRS) is one of the most economically important viral diseases affecting swine industry worldwide. Despite routine farm vaccination, effective control strategies for PRRS remained elusive which underscores the need for in-depth studies to gain insight into the host immune response to vaccines. The current study aimed to investigate transcriptional responses to PRRS Virus (PRRSV) vaccine in the peripheral blood mononuclear cells (PBMCs) within 3 days following vaccination in German Landrace pigs.ResultsTranscriptome profiling of PBMCs from PRRSV vaccinated and age-matched unvaccinated pigs at right before (0 h), and at 6, 24 and 72 h after PRRSV vaccination was performed using the Affymetrix gene chip porcine gene 1.0 st array. Comparison of PBMCs transcriptome profiles between vaccinated and unvaccinated pigs revealed a distinct host innate immune transcriptional response to PRRSV vaccine. There was a significant temporal variation in transcriptional responses of PRRSV vaccine in PBMCs accounting 542, 2,263 and 357 differentially expressed genes (DEGs) at 6, 24 and 72 h post vaccination, respectively compared to the time point before vaccination (controls). Gene ontology analysis revealed the involvement of these DEGs in various biological process including innate immune response, signal transduction, positive regulation of MAP kinase activity, TRIF-dependent toll-like receptor signaling pathway, T cell differentiation and apoptosis. Immune response specific pathways such as cytokine-cytokine receptor interaction, chemokine signaling pathway, signal transduction, JAK-STAT pathway and regulation, TRAF6 mediated induction of NF-kB and MAPK, the NLRP3 inflammasome, endocytosis and interferon signaling were under regulation during the early stage of PRRSV vaccination. Network enrichment analysis revealed APP, TRAF6, PIN1, FOS, CTNNB1, TNFAIP3, TIP1, CDKN1, SIRT1, ESR1 and HDAC5 as the highly interconnected hubs of the functional network of PRRSV vaccine induced transcriptome changes in PBMCs.ConclusionsThis study showed that a massive gene expression change occurred in PBMCs following PRRSV vaccination in German Landrace pigs. Within first 3 days of vaccine exposure, the highest transcript abundance was observed at 24 h after vaccination compared to that of control. Results of this study suggest that APP, TRAF6, PIN1, FOS, CDKN1A and TNFAIP3 could be considered as potential candidate genes for PRRSV vaccine responsiveness.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2849-1) contains supplementary material, which is available to authorized users.

Abstract 2038: Sunitinib impact on kinome profiles of peripheral blood mononuclear cells from renal cell carcinoma patients: Do molecular effects correlate with clinical data

Abstract Introduction: Sunitinib, a potent multi-tyrosine kinase (TK) inhibitor, is a standard first-line treatment for metastatic renal cell carcinoma (mRCC). In addition to its antiangiogenic activity, sunitinib is known to have immune-modulating properties especially on regulatory T-cells and tumor-infiltrating lymphocytes. However, data is sparse about sunitinib impact on peripheral lymphocytes and new data is needed to gain insights into the angiogenesis and immunity bidirectional link. This study aims to fill such a gap by investigating the clinical and intracellular modifications of sunitinib on peripheral blood mononuclear cells (PBMC) from naïve mRCC patients. Methods: Initially, a retrospective study was conducted in 88 mRCC patients treated with first-line sunitinib therapy to assess the evolution of lymphocyte count (expressed as a ratio between Day 21 and Day 0 i.e. D21/D0) during the first cycle of treatment. A new prospective study was carried out to determine kinomic profiles in PBMC from 21 naïve mRCC patients and 12 healthy volunteers. TK activity profiles of PBMC lysates were generated on TK PamChip® microarrays. The ex vivo effect of sunitinib and its active metabolite SU12662 were also determined in PBMCs. All data were analyzed using BioNavigator software. Results: The retrospective preliminary study showed that an increased D21/D0 lymphocytes ratio was significantly associated with a shorter Progression Free Survival (PFS) in multivariate analysis (p = 0.0023). In the prospective study, the phosphorylation level in PBMCs from mRCC patients was significantly lower than in healthy volunteers for 74 peptides (p&amp;lt;0.05). Ex vivo exposure to sunitinib or SU12662 led to a decreased phosphorylation level for majority of peptides in PBMCs from mRCC patients. Moreover, sunitinib had a stronger inhibitory profile than SU12662 for 80 peptides (p&amp;lt;0.05). The ex vivo sunitinib effect was statistically correlated with the IMDC (“Heng”) prognostic model and D21/D0 lymphocytes ratio and 53 and 16 peptides, respectively were found to be significant. Less ex vivo inhibition was associated with both a poor prognosis according to Heng and an increased D21/D0 lymphocytes ratio. Conclusions: Our retrospective study shows a decreased lymphocyte count on D21 after sunitinib initiation is a favorable prognostic factor in mRCC patients. The kinomic analysis of TKs in PBMCs after ex vivo exposure to sunitinib correlates with both Heng prognostic score and lymphocyte D21/D0 ratio, suggesting that PBMCs could be an interesting biological matrix to seek future biomarkers regarding clinical efficacy of sunitinib. Further investigations are underway to determine the involvement of signaling pathways contributing to the inter-individual variability in kinomic profiles of PBMCs from mRCC patients treated with sunitinib. Citation Format: Audrey Bellesoeur, Gaelle Noe, Audrey Thomas-Schoemann, Olivier Huillard, Faris Naji, Savithri Rangarajan, Alicja Puszkiel, Jerome Alexandre, François Goldwasser, Benoit Blanchet, Michel Vidal. Sunitinib impact on kinome profiles of peripheral blood mononuclear cells from renal cell carcinoma patients: Do molecular effects correlate with clinical data. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2038.

A risk assessment-driven quantitative comparison of gene expression profiles in PBMCs and white adipose tissue of humans and rats after isoflavone supplementation

Quantitative insight into species differences in risk assessment is expected to reduce uncertainty and variability related to extrapolation from animals to humans. This paper explores quantification and comparison of gene expression data between tissues and species from intervention studies with isoflavones.Gene expression data from peripheral blood mononuclear cells (PBMCs) and white adipose tissue (WAT) after 8wk isoflavone interventions in postmenopausal women and ovariectomized F344 rats were used. A multivariate model was applied to quantify gene expression effects, which showed 3–5-fold larger effect sizes in rats compared to humans. For estrogen responsive genes, a 5-fold greater effect size was found in rats than in humans. For these genes, intertissue correlations (r = 0.23 in humans, r = 0.22 in rats) and interspecies correlation in WAT (r = 0.31) were statistically significant. Effect sizes, intertissue and interspecies correlations for some groups of genes within energy metabolism, inflammation and cell cycle processes were significant, but weak.Quantification of gene expression data reveals differences between rats and women in effect magnitude after isoflavone supplementation. For risk assessment, quantification of gene expression data and subsequent calculation of intertissue and interspecies correlations within biological pathways will further strengthen knowledge on comparability between tissues and species.

Replacing carbohydrate with protein and fat in prediabetes or type-2 diabetes: greater effect on metabolites in PBMC than plasma

BackgroundActive metabolism of peripheral blood mononuclear cells (PBMC) could suggest their suitability for metabolomics studies. This study examined whether reductions in PBMCs and plasma lipoprotein-associated phospholipase A2 (Lp-PLA2) activities induced by dietary intervention affected the overall metabolic profiles of PBMC and plasma.MethodsEighty nonobese subjects aged 40–70 years (18.5 ≤ BMI < 30 kg/m2) with prediabetes or newly-diagnosed type-2 diabetes were assigned to consume either the usual refined-rice diet (control group, n = 40) or to replace refined rice with whole grains and legumes as carbohydrates (whole-grain group, n = 40) for three meals per day during the 12-week intervention. Fasting PBMC and plasma metabolomes were profiled using UPLC-LTQ-Orbitrap mass spectrometry.ResultsAfter 12 weeks, changes in fasting glucose, HbA1c, HOMA-IR, MDA, ox-LDL, LDL particle size, plasma Lp-PLA2 activity, and PBMC enzyme activity in the whole-grain group were significantly different from those in the control group before and after adjusting for baseline levels. The PBMC levels of L-leucine, oleamide, lysoPC (16:0), and lysoPC (18:0) in the whole-grain group showed greater reductions compared with those of the control group. Changes in plasma metabolites were not significantly different between the two groups. Changes in PBMC Lp-PLA2 activity positively correlated with changes in L-leucine, oleamide, lysoPC (16:0), lysoPC (18:0), glucose, and ox-LDL, and negatively correlated with changes in LDL particle size.ConclusionsThis study showed that dietary intervention in prediabetic or type-2 diabetic patients had a greater effect on PBMC Lp-PLA2 activity and metabolites compared with those of plasma metabolites.Trial registrationNCT02191644