Profiling Of Peripheral Blood Mononuclear Cells Research Articles

Impaired Cell Cycle Regulation in a Natural Equine Model of Asthma.

Recurrent airway obstruction (RAO) is a common and potentially debilitating lower airway disease in horses, which shares many similarities with human asthma. In susceptible horses RAO exacerbation is caused by environmental allergens and irritants present in hay dust. The objective of this study was the identification of genes and pathways involved in the pathology of RAO by global transcriptome analyses in stimulated peripheral blood mononuclear cells (PBMCs). We performed RNA-seq on PBMCs derived from 40 RAO affected and 45 control horses belonging to three cohorts of Warmblood horses: two half-sib families and one group of unrelated horses. PBMCs were stimulated with hay dust extract, lipopolysaccharides, a recombinant parasite antigen, or left unstimulated. The total dataset consisted of 561 individual samples. We detected significant differences in the expression profiles between RAO and control horses. Differential expression (DE) was most marked upon stimulation with hay dust extract. An important novel finding was a strong upregulation of CXCL13 together with many genes involved in cell cycle regulation in stimulated samples from RAO affected horses, in addition to changes in the expression of several HIF-1 transcription factor target genes. The RAO condition alters systemic changes observed as differential expression profiles of PBMCs. Those changes also depended on the cohort and stimulation of the samples and were dominated by genes involved in immune cell trafficking, development, and cell cycle regulation. Our findings indicate an important role of CXCL13, likely macrophage or Th17 derived, and the cell cycle regulator CDC20 in the immune response in RAO.

Characterisation of peripheral blood mononuclear cell microRNA in hepatitis B-related acute-on-chronic liver failure.

Hepatitis B-related acute-on-chronic liver failure (HBV-ACLF) is a life-threatening condition and the mechanisms of its development and progression remain unclear. The aim of this study was to define the characteristics of peripheral blood mononuclear cell microRNAs in patients with HBV-ACLF. In this study, novel microRNA (miRNA) biomarkers of peripheral blood mononuclear cells (PBMCs) in patients with HBV-ACLF were characterised by high-throughput sequencing and validated by quantitative real-time polymerase chain reaction (qRT-PCR). The results showed 78 miRNAs were significantly differentially expressed in patients with HBV-ACLF compared to patients with chronic hepatitis B (CHB) and healthy controls. Among patients with HBV-ACLF, 17 dysregulated miRNAs increased or decreased more than 4-fold, of which eight miRNAs had higher expression levels than median level. qRT-PCR validation demonstrated that six miRNAs (hsa-miR-21-5p, hsa-miR-34c-5p, hsa-miR-143-3p, hsa-miR-143-5p, hsa-miR-374a-3p and hsa-miR-542-3p) may be useful as novel biomarkers for the diagnosis of HBV-ACLF. Five novel miRNAs (L-miR-1~5) were detected and two (L-miR-1 and L-miR-3) were significantly differentially expressed in patients with HBV-ACLF. Conclusions: The miRNA expression profile of PBMCs is altered in patients with HBV-ACLF, and a signature of six miRNAs may be a promising biomarker for HBV-ACLF progression.

A type I interferon signature characterizes chronic antibody‐mediated rejection in kidney transplantation

Chronic antibody-mediated rejection (CAMR) represents the main cause of kidney graft loss. To uncover the molecular mechanisms underlying this condition, we characterized the molecular signature of peripheral blood mononuclear cells (PBMCs) and, separately, of CD4(+) T lymphocytes isolated from CAMR patients, compared to kidney transplant recipients with normal graft function and histology. We enrolled 29 patients with biopsy-proven CAMR, 29 stable transplant recipients (controls), and 8 transplant recipients with clinical and histological evidence of interstitial fibrosis/tubular atrophy. Messenger RNA and microRNA profiling of PBMCs and CD4(+) T lymphocytes was performed using Agilent microarrays in eight randomly selected patients per group from CAMR and control subjects. Results were evaluated statistically and by functional pathway analysis (Ingenuity Pathway Analysis) and validated in the remaining subjects. In PBMCs, 45 genes were differentially expressed between the two groups, most of which were up-regulated in CAMR and were involved in type I interferon signalling. In the same patients, 16 microRNAs were down-regulated in CAMR subjects compared to controls: four were predicted modulators of six mRNAs identified in the transcriptional analysis. In silico functional analysis supported the involvement of type I interferon signalling. To further confirm this result, we investigated the transcriptomic profiles of CD4(+) T lymphocytes in an independent group of patients, observing that the activation of type I interferon signalling was a specific hallmark of CAMR. In addition, in CAMR patients, we detected a reduction of circulating BDCA2(+) dendritic cells, the natural type I interferon-producing cells, and their recruitment into the graft along with increased expression of MXA, a type I interferon-induced protein, at the tubulointerstitial and vascular level. Finally, interferon alpha mRNA expression was significantly increased in CAMR compared to control biopsies. We conclude that type I interferon signalling may represent the molecular signature of CAMR.

Peripheral blood mononuclear cells from severe asthmatic children release lower amounts of IL-12 and IL-4 after LPS stimulation

IntroductionAsthma is an inflammatory disorder of the airways associated with bronchial hyperresponsiveness, airway obstruction, and increased mucus production, with a predominance of type 2 immune response (Th2). According to the hygiene hypothesis, exposure to environmental bacterial lipopolysaccharide (LPS) may induce a type 1 immune response (Th1), modulating the development of asthma. ObjectiveIn this study we investigated cytokine production by peripheral blood mononuclear cells (PBMC) from children and adolescents with severe asthma, in response to LPS stimulation in vitro. Materials and methods26 children were selected: 13 severe asthmatics and 13 healthy controls, aged between 5 and 18 years. They were evaluated through routine medical history, physical examination and lung function test to diagnose severe asthma. Allergy status was confirmed by skin prick test and specific IgE assay. We collected blood samples to analyse in vitro LPS-induced cytokines release by PBMC. ResultsPBMC from severe asthmatic children produced lower levels of IL-12p70 in basal conditions and after 12 and 24h stimulation with LPS compared to healthy controls. PBMC from severe asthmatic children produced lower levels of IL-4 after 24h LPS stimulation compared to healthy controls. PBMC from severe asthmatic children produced more levels IL-17 and IL-10 after stimulus with LPS compared to healthy controls. The release of IFN-γ, IL-5 and TNF-α by PBMC from severe asthmatic children was similar to healthy controls. ConclusionOur results demonstrate that LPS directly influence the cytokine profile of PBMC in children with severe asthma. These observations may be potentially helpful in developing new treatment strategies.

Synovial fluid and plasma n3 long chain polyunsaturated fatty acids in patients with inflammatory arthritis

Relationships between n-3 long chain polyunsaturated fatty acids (LC-PUFA) in plasma and synovial fluid (SF) were examined in 36 patients with knee effusion within the context of a variety of rheumatic diagnoses and various stated fish oil (FO) intakes (from 0 to 30mL of standard FO daily) of variable duration. In a sub-group of patients, correlations between PUFA in SF mononuclear cells (MNC) and cell-free supernatants of SF and between SF MNC and peripheral blood (PB) MNC were examined. Correlations were also sought between clinical data (stated FO intake, pain score) and n-3 LC-PUFA.Correlations between plasma n-3 LC-PUFA and SF n-3 LC-PUFA were very strong (r2>0.9, p<0.001). The LC-PUFA profiles of SF supernatants differed from those of MNC. PUFA profiles in PB MNC and SF MNC were similar, except for a higher proportion of DHA in the latter. Positive correlations were observed between stated intakes of FO and EPA in plasma and SF (for both r=0.37, p=0.02) and DHA in plasma (r=0.37, p=0.02) and SF (r=0.36, p=0.03). n-3 LC-PUFA in plasma and SF correlated inversely with pain score (plasma r2=0.16, p<0.02; SF r2 0.32, p=0.001).In conclusion, plasma n-3 LC-PUFA is a strong indicator of SF n-3 LC-PUFA status across a broad range of rheumatic diagnoses and FO intakes. Higher n-3 LC-PUFA in plasma and SF were associated with lesser pain experience.

Gene expression profiling reveals potential prognostic biomarkers associated with the progression of heart failure.

BackgroundHeart failure (HF) is the most common cause of morbidity and mortality in developed countries. Here, we identify biologically relevant transcripts that are significantly altered in the early phase of myocardial infarction and are associated with the development of post-myocardial infarction HF.MethodsWe collected peripheral blood samples from patients with ST-segment elevation myocardial infarction (STEMI): n = 111 and n = 41 patients from the study and validation groups, respectively. Control groups comprised patients with a stable coronary artery disease and without a history of myocardial infarction. Based on plasma NT-proBNP level and left ventricular ejection fraction parameters the STEMI patients were divided into HF and non-HF groups. Microarrays were used to analyze mRNA levels in peripheral blood mononuclear cells (PBMCs) isolated from the study group at four time points and control group. Microarray results were validated by RT-qPCR using whole blood RNA from the validation group.ResultsSamples from the first three time points (admission, discharge, and 1 month after AMI) were compared with the samples from the same patients collected 6 months after AMI (stable phase) and with the control group. The greatest differences in transcriptional profiles were observed on admission and they gradually stabilized during the follow-up. We have also identified a set of genes the expression of which on the first day of STEMI differed significantly between patients who developed HF after 6 months of observation and those who did not. RNASE1, FMN1, and JDP2 were selected for further analysis and their early up-regulation was confirmed in HF patients from both the study and validation groups. Significant correlations were found between expression levels of these biomarkers and clinical parameters. The receiver operating characteristic (ROC) curves indicated a good prognostic value of the genes chosen.ConclusionsThis study demonstrates an altered gene expression profile in PBMCs during acute myocardial infarction and through the follow-up. The identified gene expression changes at the early phase of STEMI that differentiated the patients who developed HF from those who did not could serve as a convenient tool contributing to the prognosis of heart failure.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-015-0149-z) contains supplementary material, which is available to authorized users.

Changes in Gene Expression in PBMCs Profiles of PPARα Target Genes in Obese and Non-Obese Individuals During Fasting

Background: The prevalence of obesity has risen dramatically and the World Health Organization estimates that 700 million people will be obese worldwide by 2015. Approximately, 50% of the Brazilian population above 20 years of age is overweight, and 16% is obese. Aim: This study aimed to evaluate the differences in the expression of PPARα target genes in human peripheral blood mononuclear cells (PBMCs) and free fatty acids (FFA) in obese and non-obese individuals after 24 h of fasting. We first presented evidence that Brazilian people exhibit expression changes in PPARα target genes in PBMCs under fasting conditions. Methods: Q-PCR was utilized to assess the mRNA expression levels of target genes. Results: In both groups, the FFA concentrations increased significantly after 24 h of fasting. The basal FFA mean concentration was two-fold higher in the obese group compared with the non-obese group. After fasting, all genes evaluated in this study showed increased expression levels compared with basal expression in both groups. Conclusion: However, our results reveal no differences in gene expression between the obese and non-obese, more studies are necessary to precisely delineate the associated mechanisms, particularly those that include groups with different degrees of obesity and patients with diabetes mellitus type 2 because the expression of the main genes that are involved in β-oxidation and glucose level maintenance are affected by these factors. © 2014 S. Karger AG, Basel

Immune-metabolic profiling of anorexic patients reveals an anti-oxidant and anti-inflammatory phenotype

ContextAnorexia nervosa (AN) is an excessive form of calorie restriction (CR) associated with pathological weight loss and alterations of the immune system. However, AN patients seem to be protected from common viral infections. ObjectivesTo investigate the metabolic and molecular adaptations induced by sustained extreme CR in the peripheral blood mononuclear cells (PBMCs) of patients with restrictive alimentary AN. DesignInflammatory cytokines and adipokines were measured in 15 young (age range, 15–24 years) AN female patients and 20 age-matched healthy controls. Isolated PBMCs were immunophenotyped by flow cytometry, and glycolysis and mitochondrial respiration were determined by measuring the extracellular acidification and oxygen consumption rate. Stress resistance to H2O2 and the antioxidant transcriptional profile of PBMCs and human fibroblasts incubated with sera from AN patients were also determined. ResultsCompared with controls, AN patients (BMI, 15.9±0.4kg/m2) had significantly fewer leucocytes, lymphocytes and NK cells, lower serum concentrations of leptin, IGF-1 and sTNFR1, and higher levels of adiponectin, sCD40L and sICAM-1 (p<0.05). IL-1β, TNFα, and IL-6 produced by PBMC cultured with autologous serum for 48h were significantly lower in AN patients than in controls (p<0.01). Moreover, glycolysis and mitochondrial respiration were lower, and the antioxidant transcriptional profile was higher in the PBMCs of AN patients. Fibroblasts cultured in serum from AN patients showed a 24% increase in resistance to H2O2 damage. ConclusionsExtreme CR in AN patients is associated with a reduction in several immune cell populations, but with higher antioxidant potential, stress resistance and an anti-inflammatory status.

Metabolic profiling of human peripheral blood mononuclear cells: influence of vitamin d status and gender.

Metabolic profiling of peripheral blood mononuclear cells (PBMC) could serve as a less invasive and more direct alternative to tissue biopsies or serum in metabolomic research. We conducted two exploratory independent studies in order to characterise PBMC’s metabolomic profile following short-term vitamin D3 supplementation and to determine gender effects. In the first study, eight healthy males and females aged 40–65 y were randomly selected for profiling of PBMCs after receiving either 15 µg of vitamin D3 or placebo for four weeks. In the second study, twenty younger healthy males and females were studied. Cell metabolites were extracted and deproteinised using methanol/chloroform/water method and analysed by GC-MS. Higher vitamin D status had no effect on the fatty acid profile of PBMCs, but inflammatory biomarkers and adipokines correlated positively with stearic acid levels. In the second study, no gender-specific metabolites were identified. Valine, leucine and aspartic acid were identified as potential BMI-sensitive amino acids. Larger studies are needed to confirm the influence of BMI on these parameters. This work clearly demonstrates the utility of metabolomics profiling of PBMCs and paves the way for future applications of metabolomics in identifying metabolic profiles of blood cells as a measure for dietary intakes or physiological status.

Peripheral blood mononuclear cells microRNA predicts treatment outcome of hepatitis C virus genotype 1 infection

BackgroundsChronic hepatitis C virus (HCV) infection has been associated with induction of microRNAs (miRNAs) in peripheral blood mononuclear cells (PBMC). We aimed to evaluate the role of PBMC–miRNAs in the treatment outcome to antiviral therapy for HCV genotype 1 (HCV-1) patients. MethodsTreatment-naive chronic HCV-1 patients, including 13 in screening phase and 48 in validation phase, were treated with 48weeks of peginterferon/ribavirin. The primary end-point was the achievement of a sustained virological response (SVR, HCV RNA undetectable during 24weeks post-treatment follow-up). Expression profiling of PBMC–miRNAs was performed by quantitative PCR-based array in typical responders and null-responders. Then candidate PBMC–miRNAs were validated by quantitative PCR in an independent validation set. ResultsPBMC–miR-125b was significantly predictive of an SVR, with expression levels of 5.28-fold lower in sustained responders versus null-responders (p=0.0163). In multivariate analysis, PBMC–miR-125b was significantly associated with the achievement of SVR (per 2-fold decrease, odds ratio/95% confidence interval (OR/CI): 2.07/1.14-6.31) independent of sex, age and interleukin-28B genotype. In patients who did not achieve a rapid virological response (RVR, undetectable HCV RNA at treatment week 4), PBMC–miR-125b was the only predictive factor of an SVR (per 2-fold decrease, OR/CI: 2.07/1.14-6.31). However, the circulating and hepatic miR-125b did not show significant difference between responders and non-responders. ConclusionsPBMC–miR-125b expression levels were inversely related to the achievement of an SVR in HCV-1 patients, independent of interleukin-28B genotype, and was the single predictor of SVR in non-RVR patients.

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

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

A Non-invasive Diagnosis of Histiocytic Necrotizing Lymphadenitis by Means of Gene Expression Profile Analysis of Peripheral Blood Mononuclear Cells

Histiocytic necrotizing lymphadenitis (HNL), also called Kikuchi-Fujimoto disease, is a benign, self-limiting inflammatory disease with fever and painful cervical lymphadenopathy of unknown etiology. A lymph node biopsy is required for the definitive diagnosis because of no specific symptoms or laboratory findings for HNL. To establish the rapid non-invasive diagnostic method for this disease, we investigated genes specifically expressed in the patients by analyzing whole transcriptome using microarray analysis of peripheral blood mononuclear cells (PBMC). The top five up-regulated genes (IFI44L, CXCL10, GBP1, EPSTI1 and IFI27) in HNL were interferon-induced genes (ISGs). The expression levels of the up-regulated genes by microarray were verified by quantitative PCR. High levels of serum CXCL10 concentration were confirmed at the symptomatic phase of HNL patients. The expression levels of these 5 genes positively correlated with each other (r(2) = 0.28-0.60). The genes were also highly expressed in HNL lymph nodes. The discriminant analysis using the expression levels of these five genes distinguished HNL with 84% accuracy. The combination of up-regulated ISGs in HNL seemed to be a specific response induced by viral infections or autoantigens. An analysis of the gene expression profile of PBMC may provide a rapid non-invasive diagnosis of HNL.

Transcriptomic Response of Porcine PBMCs to Vaccination with Tetanus Toxoid as a Model Antigen

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.

Altered Cytokine Gene Expression in Peripheral Blood Monocytes across the Menstrual Cycle in Primary Dysmenorrhea: A Case-Control Study

Primary dysmenorrhea is one of the most common gynecological complaints in young women, but potential peripheral immunologic features underlying this condition remain undefined. In this paper, we compared 84 common cytokine gene expression profiles of peripheral blood mononuclear cells (PBMCs) from six primary dysmenorrheic young women and three unaffected controls on the seventh day before (secretory phase), and the first (menstrual phase) and the fifth (regenerative phase) days of menstruation, using a real-time PCR array assay combined with pattern recognition and gene function annotation methods. Comparisons between dysmenorrhea and normal control groups identified 11 (nine increased and two decreased), 14 (five increased and nine decreased), and 15 (seven increased and eight decreased) genes with ≥2-fold difference in expression (P<0.05) in the three phases of menstruation, respectively. In the menstrual phase, genes encoding pro-inflammatory cytokines (IL1B, TNF, IL6, and IL8) were up-regulated, and genes encoding TGF-β superfamily members (BMP4, BMP6, GDF5, GDF11, LEFTY2, NODAL, and MSTN) were down-regulated. Functional annotation revealed an excessive inflammatory response and insufficient TGF-β superfamily member signals with anti-inflammatory consequences, which may directly contribute to menstrual pain. In the secretory and regenerative phases, increased expression of pro-inflammatory cytokines and decreased expression of growth factors were also observed. These factors may be involved in the regulation of decidualization, endometrium breakdown and repair, and indirectly exacerbate primary dysmenorrhea. This first study of cytokine gene expression profiles in PBMCs from young primary dysmenorrheic women demonstrates a shift in the balance between expression patterns of pro-inflammatory cytokines and TGF-β superfamily members across the whole menstrual cycle, underlying the peripheral immunologic features of primary dysmenorrhea.

Alteration of proteomic profiles in PBMC isolated from patients with Fabry disease: preliminary findings

Fabry disease (FD) is an X-linked progressive multisystem disease due to mutations in the gene encoding the lysosomal enzyme α-galactosidase A (α-GalA). The deficiency in α-GalA activity leads to an intra-lysosomal accumulation of neutral glycosphingolipids, mainly globotriaosylceramide (Gb3), in various organs and systems. Enzyme replacement therapy is available and alternative therapeutic approaches are being explored. No diagnostic test, other than sequencing of the α-galactosidase A gene, is available, no biomarker has been proven useful to screen for and predict the disease, and underlying mechanisms are still elusive. The aim of this study is to identify FD specific biomarkers and to better understand the pathophysiological changes that occur over time in FD. We compared peripheral blood mononuclear cells (PBMC) from FD patients (n = 8) with control PBMC from healthy individuals (n = 6), by two-dimensional electrophoresis (2DE) and the detected differentially expressed proteins were then subjected to matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). In FD patients we identified, among the down-regulated proteins, Calnexin, Rho GDP-dissociation inhibitor 2, Rho GDP-dissociation inhibitor 1, Chloride intracellular channel protein 1; on the other hand γ-enolase, 14-3-3 protein theta, 14-3-3 protein zeta/delta, and galectin-1 were identified as up-regulated proteins. Calnexin and Rho GDP-dissociation inhibitor-1,2 are related to protein folding, signal transduction and cell proliferation. This is the first time that γ-enolase and galectin-1 are described to be up-regulated in Fabry patients. Levels of γ-enolase increase dramatically in cardiovascular accidents and cerebral trauma, whereas galectins are regulators of acute and chronic inflammation. These findings may improve our understanding of the molecular mechanisms underlying the pathology and provide new insight and knowledge for future studies in this field.

Dendritic cells fused with different pancreatic carcinoma cells induce different T-cell responses

BackgroundIt is unclear whether there are any differences in the induction of cytotoxic T lymphocytes (CTL) and CD4+CD25high regulatory T-cells (Tregs) among dendritic cells (DCs) fused with different pancreatic carcinomas. The aim of this study was to compare the ability to induce cytotoxicity by human DCs fused with different human pancreatic carcinoma cell lines and to elucidate the causes of variable cytotoxicity among cell lines.MethodsMonocyte-derived DCs, which were generated from peripheral blood mononuclear cells (PBMCs), were fused with carcinoma cells such as Panc-1, KP-1NL, QGP-1, and KP-3L. The induction of CTL and Tregs, and cytokine profile of PBMCs stimulated by fused DCs were evaluated.ResultsThe cytotoxicity against tumor targets induced by PBMCs cocultured with DCs fused with QGP-1 (DC/QGP-1) was very low, even though PBMCs cocultured with DCs fused with other cell lines induced significant cytotoxicity against the respective tumor target. The factors causing this low cytotoxicity were subsequently investigated. DC/QGP-1 induced a significant expansion of Tregs in cocultured PBMCs compared with DC/KP-3L. The level of interleukin-10 secreted in the supernatants of PBMCs cocultured with DC/QGP-1 was increased significantly compared with that in DC/KP-3L. Downregulation of major histocompatibility complex class I expression and increased secretion of vascular endothelial growth factor were observed with QGP-1, as well as in the other cell lines.ConclusionThe present study demonstrated that the cytotoxicity induced by DCs fused with pancreatic cancer cell lines was different between each cell line, and that the reduced cytotoxicity of DC/QGP-1 might be related to the increased secretion of interleukin-10 and the extensive induction of Tregs.

Gene expression profiles displayed by peripheral blood mononuclear cells from patients with type 2 diabetes mellitus focusing on biological processes implicated on the pathogenesis of the disease

Patients with type 2 diabetes mellitus (T2DM) exhibit insulin resistance associated with obesity and inflammatory response, besides an increased level of oxidative DNA damage as a consequence of the hyperglycemic condition and the generation of reactive oxygen species (ROS). In order to provide information on the mechanisms involved in the pathophysiology of T2DM, we analyzed the transcriptional expression patterns exhibited by peripheral blood mononuclear cells (PBMCs) from patients with T2DM compared to non-diabetic subjects, by investigating several biological processes: inflammatory and immune responses, responses to oxidative stress and hypoxia, fatty acid processing, and DNA repair. PBMCs were obtained from 20 T2DM patients and eight non-diabetic subjects. Total RNA was hybridized to Agilent whole human genome 4×44K one-color oligo-microarray. Microarray data were analyzed using the GeneSpring GX 11.0 software (Agilent). We used BRB-ArrayTools software (gene set analysis — GSA) to investigate significant gene sets and the Genomica tool to study a possible influence of clinical features on gene expression profiles. We showed that PBMCs from T2DM patients presented significant changes in gene expression, exhibiting 1320 differentially expressed genes compared to the control group. A great number of genes were involved in biological processes implicated in the pathogenesis of T2DM. Among the genes with high fold-change values, the up-regulated ones were associated with fatty acid metabolism and protection against lipid-induced oxidative stress, while the down-regulated ones were implicated in the suppression of pro-inflammatory cytokines production and DNA repair. Moreover, we identified two significant signaling pathways: adipocytokine, related to insulin resistance; and ceramide, related to oxidative stress and induction of apoptosis. In addition, expression profiles were not influenced by patient features, such as age, gender, obesity, pre/post-menopause age, neuropathy, glycemia, and HbA1c percentage. Hence, by studying expression profiles of PBMCs, we provided quantitative and qualitative differences and similarities between T2DM patients and non-diabetic individuals, contributing with new perspectives for a better understanding of the disease.

Transcriptome analysis of the human T lymphocyte cell line Jurkat and human peripheral blood mononuclear cells exposed to deoxynivalenol (DON): New mechanistic insights

Deoxynivalenol (DON) or vomitoxin is a commonly encountered type-B trichothecene mycotoxin, produced by Fusarium species predominantly found in cereals and grains. DON is known to exert toxic effects on the gastrointestinal, reproductive and neuroendocrine systems, and particularly on the immune system. Depending on dose and exposure time, it can either stimulate or suppress immune function. The main objective of this study was to obtain a deeper insight into DON-induced effects on lymphoid cells. For this, we exposed the human T-lymphocyte cell line Jurkat and human peripheral blood mononuclear cells (PBMCs) to various concentrations of DON for various times and examined gene expression changes by DNA microarray analysis. Jurkat cells were exposed to 0.25 and 0.5μM DON for 3, 6 and 24h. Biological interpretation of the microarray data indicated that DON affects various processes in these cells: It upregulates genes involved in ribosome structure and function, RNA/protein synthesis and processing, endoplasmic reticulum (ER) stress, calcium-mediated signaling, mitochondrial function, oxidative stress, the NFAT and NF-κB/TNF-α pathways, T cell activation and apoptosis. The effects of DON on the expression of genes involved in ER stress, NFAT activation and apoptosis were confirmed by qRT-PCR. Other biochemical experiments confirmed that DON activates calcium-dependent proteins such as calcineurin and M-calpain that are known to be involved in T cell activation and apoptosis. Induction of T cell activation was also confirmed by demonstrating that DON activates NFATC1 and induces its translocation from the cytoplasm to the nucleus. For the gene expression profiling of PBMCs, cells were exposed to 2 and 4μM DON for 6 and 24h. Comparison of the Jurkat microarray data with those obtained with PBMCs showed that most of the processes affected by DON in the Jurkat cell line were also affected in the PBMCs.

Gene expression profile of human peripheral blood mononuclear cells induced by Staphylococcus aureus lipoteichoic acid

Lipoteichoic acid (LTA) is a major virulence factor of Gram-positive bacteria including Staphylococcus aureus. Despite its pivotal role in causing sepsis, the systemic immune responses to LTA in human cells are poorly understood. Here, we produced highly-pure and structurally-intact LTA from S. aureus and examined the gene expression profile of LTA-stimulated human peripheral blood mononuclear cells (PBMCs). The LTA preparation did not contain any detectable biologically-active impurities and stimulated Toll-like receptor 2. Protein expression profiling using a cytokine array kit and ELISA revealed expression of MCP-1/CCL2, IL-6, and IL-1β. We performed transcriptional profiling of PBMCs in response to S. aureus LTA using an Affymetrix genechip microarray. A total of 208 genes were significantly (fold change>1.5 and P<0.05) altered, with 157 up-regulated and 51 down-regulated genes in response to S. aureus LTA treatment. The up-regulated genes were involved in recognition (30 genes), cellular adhesion (6 genes), signal transduction (42 genes), co-stimulation (4 genes), chemokines, cytokines and their receptors (51 genes), apoptosis (9 genes), and negative regulation (15 genes). The down-regulated genes were involved in recognition (12 genes), antigen processing and presentation (9 genes), signal transduction (27 genes), and chemotaxis (3 genes). The microarray results were validated using real-time RT-PCR with 21 up-regulated genes and 9 down-regulated genes. Our results provide a more comprehensive overview of the transcriptional changes in PBMCs in response to S. aureus LTA, and contribute to the understanding of the pathophysiological role of S. aureus LTA during the systemic inflammatory response.

Erythroid-Specific Transcriptional Changes in PBMCs from Pulmonary Hypertension Patients

BackgroundGene expression profiling of peripheral blood mononuclear cells (PBMCs) is a powerful tool for the identification of surrogate markers involved in disease processes. The hypothesis tested in this study was that chronic exposure of PBMCs to a hypertensive environment in remodeled pulmonary vessels would be reflected by specific transcriptional changes in these cells.Methodology/Principal FindingsThe transcript profiles of PBMCs from 30 idiopathic pulmonary arterial hypertension patients (IPAH), 19 patients with systemic sclerosis without pulmonary hypertension (SSc), 42 scleroderma-associated pulmonary arterial hypertensio patients (SSc-PAH), and 8 patients with SSc complicated by interstitial lung disease and pulmonary hypertension (SSc-PH-ILD) were compared to the gene expression profiles of PBMCs from 41 healthy individuals. Multiple gene expression signatures were identified which could distinguish various disease groups from controls. One of these signatures, specific for erythrocyte maturation, is enriched specifically in patients with PH. This association was validated in multiple published datasets. The erythropoiesis signature was strongly correlated with hemodynamic measures of increasing disease severity in IPAH patients. No significant correlation of the same type was noted for SSc-PAH patients, this despite a clear signature enrichment within this group overall. These findings suggest an association of the erythropoiesis signature in PBMCs from patients with PH with a variable presentation among different subtypes of disease.Conclusions/SignificanceIn PH, the expansion of immature red blood cell precursors may constitute a response to the increasingly hypoxic conditions prevalent in this syndrome. A correlation of this erythrocyte signature with more severe hypertension cases may provide an important biomarker of disease progression.