Peripheral Blood Mononuclear Cells Gene Expression Research Articles

Soluble CD40L is associated with increased oxidative burst and neutrophil extracellular trap release in Beh\xe7et\u2019s disease

BackgroundStudies have suggested that soluble factors in plasma from patients with active (aBD) and inactive (iBD) Behçet’s disease (BD) stimulate neutrophil function. Soluble CD40 ligand (sCD40L) is an important mediator of inflammation in BD. Its expression and effect on neutrophil oxidative burst and neutrophil extracellular trap (NET) release have not been characterized. In this study, we sought to investigate the role of plasma and the CD40L pathway on NET release and the oxidative burst profile in patients with aBD and iBD.MethodsNeutrophils and peripheral blood mononuclear cells (PBMCs) were obtained from patients with aBD (n = 30), patients with iBD (n = 31), and healthy control subjects (HCs; n = 30). sCD40L plasma concentration was determined in individual samples. A pool of plasma for each group was created. In some experiments, plasma pools were treated with recombinant CD40 (rhCD40-muIg) for sCD40L blockade. NET release and H2O2/O2− production were determined after stimulation with phorbol 12-myristate 13-acetate, sCD40L, or plasma pool. Flow cytometric analysis was performed to evaluate the expression of (1) CD40, Mac-1, and phosphorylated NF-κB p65 on neutrophils and monocytes and (2) CD40L on activated T cells and platelets. CD40L gene expression in PBMCs was determined by qRT-PCR.ResultssCD40L plasma levels were significantly higher in patients with iBD (median 17,234, range 2346–19,279 pg/ml) and patients with aBD (median 18,289, range 413–19,883 pg/ml) than in HCs (median 47.5, range 33.7–26.7 pg/ml; p < 0.001). NET release was constitutively increased in BD compared with HC. NET release and H2O2/O2− were higher after stimulation with sCD40L or BD plasma and decreased after sCD40L blockade. Mac-1 expression was constitutively increased in neutrophils of patients with aBD (88.7 ± 13.2% of cells) and patients with iBD (89.2 ± 20.1% of cells) compared with HC (27.1 ± 18.8% of cells; p < 0.01). CD40 expression on phagocytes and CD40L expression on platelets were similar in the three groups. PBMCs as well as nonactivated and activated CD4+ T cells from patients with BD showed higher CD40L expression.ConclusionsPlasma from patients with aBD exerts a stimulus on NET release and oxidative burst, probably induced by sCD40L.

The role of the ovarian cycle and the effects of mitogen-induced cytokines on human prolactin gene expression in peripheral blood mononuclear cells

ABSTRACT Background: little is known on the influences of normal menstrual cycle on prolactin gene expression in immune cells. Aim of the study: to determine the effects of the ovarian cycle on prolactin and its receptor expression. Methods: peripheral blood mononuclear cells (PBMC) were obtained from twenty-six normal menstruating women at different intervals of their menstrual cycle. The PBMC were incubated during 24 h in the presence or absence of Concanavalin-A (Con-A) and the gene expression of PRL, PRLR and cytokines was evaluated by qPCR. Prolactin, IL-2 and cAMP were determined in each culture by specific immunoassays. Results: neither PRL nor its receptor expression in PBMC changed significantly among groups, including the cytokines (IL-2, IL-10, and IFNG) studied. Similar results, among groups, were obtained, when PRL expression was stimulated by PGE2 or 8-Br-cAMP. Concanavalin A-stimulated PBMC expressed significantly less prolactin and a significant negative correlation between secreted IL-2 and PRL expression was found. The presence of anti-IL-2 antibodies in Con-A stimulated-cultures significantly increased PRL expression when compared to control cells regardless the hormonal status. Conclusions: these data suggest that the menstrual cycle does not significantly modulate or influence prolactin and cytokines gene expression in PBMC, and indicate that IL-2 may be involved in the Con-A regulation of PRL expression in immune cells.

Changes in Global Transcriptional Profiling of Women Following Obesity Surgery Bypass.

Differential gene expression in peripheral blood mononuclear cells (PBMCs) after Roux-en-Y gastric bypass (RYGB) is poorly characterized. Markers of these processes may provide a deeper understanding of the mechanisms that underlie these events. The main goal of this study was to identify changes in PBMC gene expression in women with obesity before and 6months after RYGB-induced weight loss. The ribonucleic acid (RNA) of PBMCs from 13 obese women was analyzed before and 6months after RYGB; the RNA of PBMCs from nine healthy women served as control. The gene expression levels were determined by microarray analysis. Significant differences in gene expression were validated by real-time quantitative polymerase chain reaction (RT-qPCR). Microarray analysis for comparison of the pre- and postoperative periods showed that 1366 genes were differentially expressed genes (DEGs). The main pathways were related to gene transcription; lipid, energy, and glycide metabolism; inflammatory and immunological response; cell differentiation; oxidative stress regulation; response to endogenous and exogenous stimuli; substrate oxidation; mTOR signaling pathway; interferon signaling; mitogen-activated protein kinases (MAPK), cAMP response element binding protein (CREB1), heat shock factor 1 (HSF1), and sterol regulatory element binding protein 1c (SREBP-1c) gene expression; adipocyte differentiation; and methylation. Six months after bariatric surgery and significant weight loss, many molecular pathways involved in obesity and metabolic diseases change. These findings are an important tool to identify potential targets for therapeutic intervention and clinical practice of nutritional genomics in obesity.

Immunomodulation of TLR2 and TLR4 by G2013 (alfa-L-Guluronic acid) in CVID Patients

Background: Common variable immunodeficiency (CVID) is a primary immune disorder associated with hypogammaglobulinemia, recurrent infections and autoimmune diseases. CVID patients are frequently in contact with infectious pathogens leading to the activation of innate immunity through Toll-like receptors (TLR) affecting adaptive immunity. The aim of the present study was to test the immunomedulatory effect of small molecule G2013, a novel designed non-steroidal anti-inflammatory agent in CVID. Materials and Methods: After blood sampling from 16 CVID patients and 16 age- and sex-matched healthy controls, peripheral blood mononuclear cells (PBMCs) were isolated and treated with/without lipopolysaccharide (LPS), lipopolyteichoic acid (LTA), and G2013. Assessing the immunomodulatory effect of G2013, flowcytometry was done for quantify the protein expression of TLR2 and TLR4. Gene expressions of signaling molecules involved in the TLR2 and TLR4 pathways were assessed by real-time PCR. ELISA performed assessing the production of IL-1b and IL-6. Results: G2013 significantly decreased the intensity of TLR2 expression in CVID PBMCs (p=0.001) also G2013 decreased significantly the NF-kB gene expression in PBMCs of CVID patients (p=0.006). Conclusion: These results indicated that G2013 had immunomodulatory effect at least in part via TLR2 and NF-kB expression. G2013 by decreasing MFI of TLR2 expression and NFkB gene expression provide the possibility of designing new drugs for preventing or controlling autoimmunity in CVID patients.

Identification of Jak-STAT signaling involvement in sarcoidosis severity via a novel microRNA-regulated peripheral blood mononuclear cell gene signature

Sarcoidosis is a granulomatous lung disorder of unknown cause. The majority of individuals with sarcoidosis spontaneously achieve full remission (uncomplicated sarcoidosis), however, ~20% of sarcoidosis-affected individuals experience progressive lung disease or cardiac and nervous system involvement (complicated sarcoidosis). We investigated peripheral blood mononuclear cell (PBMC) microRNA and protein-coding gene expression data from healthy controls and patients with uncomplicated or complicated sarcoidosis. We identified 46 microRNAs and 1,559 genes that were differentially expressed across a continuum of sarcoidosis severity (healthy control → uncomplicated sarcoidosis → complicated sarcoidosis). A total of 19 microRNA-mRNA regulatory pairs were identified within these deregulated microRNAs and mRNAs, which consisted of 17 unique protein-coding genes yielding a 17-gene signature. Pathway analysis of the 17-gene signature revealed Jak-STAT signaling pathway as the most significantly represented pathway. A severity score was assigned to each patient based on the expression of the 17-gene signature and a significant increasing trend in the severity score was observed from healthy control, to uncomplicated sarcoidosis, and finally to complicated sarcoidosis. In addition, this microRNA-regulated gene signature differentiates sarcoidosis patients from healthy controls in independent validation cohorts. Our study suggests that PBMC gene expression is useful in diagnosis of sarcoidosis.

Effect of Lang-Chuang-Ding Decoction () on DNA Methylation of CD70 Gene Promoter in Peripheral Blood Mononuclear Cells of Female Patients with Systemic Lupus Erythematosus.

To investigate the effect of Lang-chuang-ding Decoction (, LCD) on the expression of DNA methylation of CD70 gene promoter in peripheral blood mononuclear cells (PBMCs) of females with systemic lupus erythematosus (SLE). PBMCs isolated from female patients with SLE or healthy donors were cultured and treated with LCD medicated serum or normal serum for 24 or 48 h. The mRNA expressions of CD70 gene in PBMCs were detected by reverse transcription polymerase chain reaction (PCR); the DNA methylation of the CD70 gene promoter region was detected by methylation-specific PCR. After treated with medicated serum for 48 h, the mRNA expression levels of CD70 in PBMCs of SLE patients were signifificantly higher than those of healthy donors (P<0.05); the DNA methylation levels of CD70 promoter region in PBMCs of SLE patients treated with medicated serum for 48 h were signifificantly higher than those treated with fetal bovine serum (P<0.01). LCD could inhibit CD70 gene expression in PBMCs of SLE patients by promoting the DNA methylation of CD70 gene promoter.

Relationship between soluble CD25 and gene expression in healthy individuals and patients with multiple sclerosis

Genome wide association studies and fine mapping has established a firm link between the IL2RA gene, encoding the interleukin-2 receptor α-chain CD25, and susceptibility to multiple sclerosis (MS).We hypothesized that gene expression in peripheral blood mononuclear cells (PBMCs) from healthy controls (HCs) and MS patients are associated with IL2RA SNP rs2104286 and that gene expression levels correlate with soluble CD25 (sCD25) concentrations – that are affected by rs2104286.We used the Affymetrix Human Gene ST 1.0 microarray to analyze gene expression levels in PBMCs from 18 HCs and 51MS patients. Plasma concentrations of sCD25 were measured by ELISA in all individuals.In HCs 266 genes correlated with sCD25 with Spearman’s rho≥0.707; 70 of these genes had a false discovery rate (FDR) value of q<0.05. These genes were highest expressed in cells belonging to the innate immune system. Gene-networks were focused around NFKB1, TNF, BCL6 and STAT1. Eighteen genes correlated with sCD25 with rho≥0.707 in relapsing remitting MS versus 33 in secondary progressive and 34 in primary progressive MS. None had a FDR<0.05. Thirty-eight and 23 genes were differentially expressed between rs2104286 genotype-groups in MS patients and HCs respectively, however they were not significant after FDR correction.Our study indicates that rs2104286 influences gene expression in PBMCs in HCs as shown by the high correlations with the rs2104286-affected sCD25 protein. Correlations were strongest in HCs suggesting that immunological alterations may obscure the role of the IL2RA SNP rs2104286 in established MS.

Integration of Immune Cell Populations, mRNA-Seq, and CpG Methylation to Better Predict Humoral Immunity to Influenza Vaccination: Dependence of mRNA-Seq/CpG Methylation on Immune Cell Populations.

The development of a humoral immune response to influenza vaccines occurs on a multisystems level. Due to the orchestration required for robust immune responses when multiple genes and their regulatory components across multiple cell types are involved, we examined an influenza vaccination cohort using multiple high-throughput technologies. In this study, we sought a more thorough understanding of how immune cell composition and gene expression relate to each other and contribute to interindividual variation in response to influenza vaccination. We first hypothesized that many of the differentially expressed (DE) genes observed after influenza vaccination result from changes in the composition of participants’ peripheral blood mononuclear cells (PBMCs), which were assessed using flow cytometry. We demonstrated that DE genes in our study are correlated with changes in PBMC composition. We gathered DE genes from 128 other publically available PBMC-based vaccine studies and identified that an average of 57% correlated with specific cell subset levels in our study (permutation used to control false discovery), suggesting that the associations we have identified are likely general features of PBMC-based transcriptomics. Second, we hypothesized that more robust models of vaccine response could be generated by accounting for the interplay between PBMC composition, gene expression, and gene regulation. We employed machine learning to generate predictive models of B-cell ELISPOT response outcomes and hemagglutination inhibition (HAI) antibody titers. The top HAI and B-cell ELISPOT model achieved an area under the receiver operating curve (AUC) of 0.64 and 0.79, respectively, with linear model coefficients of determination of 0.08 and 0.28. For the B-cell ELISPOT outcomes, CpG methylation had the greatest predictive ability, highlighting potentially novel regulatory features important for immune response. B-cell ELISOT models using only PBMC composition had lower performance (AUC = 0.67), but highlighted well-known mechanisms. Our analysis demonstrated that each of the three data sets (cell composition, mRNA-Seq, and DNA methylation) may provide distinct information for the prediction of humoral immune response outcomes. We believe that these findings are important for the interpretation of current omics-based studies and set the stage for a more thorough understanding of interindividual immune responses to influenza vaccination.

Molecular effect of fenofibrate on PBMC gene transcription related to lipid metabolism in patients with metabolic syndrome.

Both fasting and postprandial hypertriglyceridaemia are considered independent risk factors for atherosclerosis. Treatment of hypertriglyceridaemia is based on fibrates, which activate the peroxisome proliferator-activated receptor alpha (PPARα). However, the metabolic pathways that activate or inhibit fibrates, and how the postprandial triglyceride levels are modified, have not yet been fully described. Accordingly, the aim of this study was to determine the feasibility of peripheral blood mononuclear cells (PBMC) to study the effects of fenofibrate in patients with the metabolic syndrome. A fat overload was given to 50 patients before and after treatment with fenofibrate for 3 months. Anthropometric and biochemical variables as well as gene expression in PBMC were analysed. After treatment with fenofibrate, we observed a decrease in both baseline and postprandial (3 h after the fat overload) levels of serum triglycerides, cholesterol and uric acid and an increase in HDL cholesterol and apolipoprotein AI levels. After treatment, there was also a rise in PPARα and RXRα expression and changes in genes regulated by PPARα, both baseline and postprandial. Furthermore, in vitro experiments showed that a PPARα agonist changed the expression of genes related with lipid metabolism. Treatment with fenofibrate reduced fasting and postprandial serum triglyceride levels, possibly through a mechanism related with an increase in the expression of RXRα and PPARα, by activating the pathways involved in the uptake and degradation of triglycerides and increasing the synthesis of apolipoprotein. These results suggest that PBMC may be useful for the easy study of fenofibrate actions.

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.

Effects of Lavandula angustifolia essential oil on Interleukin 23 and Brain-Derived neurotrophic factor gene expression in peripheral blood mononuclear cells of multiple sclerosis patients

Introduction: We aimed to determine the effect of lavandula angustifolia essential oil (LEO) on IL-23 and brain-derived neurotrophic factor (BDNF) gene expressions in peripheral blood mononuclear cells (PBMCs) of relapse-remitting MS (RRMS) patients. Methods : LEO was prepared using the hydrodistilation method on the plants aerial parts. 8 female RRMS patients and 8 healthy sex and age matched controls were entered into this study. PBMC cells were separated using Ficoll method and were treated with a concentration of 225 µg/ml LEO which and then the mRNAs were used for determining the effects of LEO on IL-23 and BDNF gene expressions using Quantitative Real Time PCR technique. Moreover in order to determine the anti-inflammatory effects of LEO, we measured the gene expression of IL-6 and IL-23 in stimulated healthy PBMC cells treated with LEO. Results: Results showed that there is no significant difference between PBMC of patients compared to healthy controls in case of IL-23 gene expression. Moreover, LEO has no significant effect on gene expression of IL-23 in PBMC of neither patients nor control. Also the results showed that BDNF gene expression is reduced to 41% compared to healthy controls and LEO can increase the BDNF gene expression by 81% in patients PBMCs. Moreover we observed that LEO can significantly reduce the LPS stimulated IL-6 gene expression in healthy PBMCs but had no significant effect on IL-23 gene expression. Conclusion : The present study demonstrated that L.angustifolia essential oil may have a protective effect against neuron damage via increasing the gene expression of BDNF in PBMCs from RRMS patients. However, further studies are necessary to confirm our results.

Beneficial Effects of Pterocarpan-High Soybean Leaf Extract on Metabolic Syndrome in Overweight and Obese Korean Subjects: Randomized Controlled Trial.

Pterocarpans are known to have antifungal and anti-inflammatory properties. However, little is known about the changes in transcriptional profiles in response to a pterocarpan-high soybean leaf extract (PT). Therefore, this study investigated the effects of PT on blood glucose and lipid levels, as well as on the inflammation-related gene expression based on a peripheral blood mononuclear cells (PBMCs) mRNA sequencing analysis in Korean overweight and obese subjects with mild metabolic syndrome. The participants were randomly assigned to two groups and were administered either placebo (starch, 3 g/day) or PT (2 g/day) for 12 weeks. The PT intervention did not change body weight, body fat percentage and body mass index (BMI). However, PT significantly decreased the glycosylated hemoglobin (HbA1c), plasma glucose, free fatty acid, total cholesterol, and non-HDL cholesterol levels after 12 weeks. Furthermore, PT supplementation significantly lowered the homeostatic index of insulin resistance, as well as the plasma levels of inflammatory markers. Finally, the mRNA sequencing analysis revealed that PT downregulated genes related to immune responses. PT supplementation is beneficial for the improvement of metabolic syndrome by altering the fasting blood and plasma glucose, HbA1c, plasma lipid levels and inflammation-related gene expression in PBMCs.

Abstract 083: γ/δ T Cells Play a Role in Development of Hypertension

Objective: Both innate antigen-presenting cells and the adaptive immune system have been shown to play a role in the development of hypertension. Nevertheless, the T cell subsets involved in the pathophysiology of hypertension remains unclear. There is a small subset of “innate-like” T cells expressing the γ/δ T cell receptor (TCR) rather than the α/β TCR that could play a role bridging the innate and adaptive immune systems. We previously observed that angiotensin (Ang) II caused an increase in number and activation of γ/δ T cells and Ang II-induced systolic blood pressure (SBP) rise and vascular injury were blunted in Tcr δ -/- mice, which are devoid of γ/δ T cells. In order to further characterize the role of γ/δ T cells in hypertension, we determined whether Ang II-effects would be blunted by antibody-induced depletion of γ/δ T cells. In addition, we tested whether SBP in human could be predicted by combining the expression of genes encoding TCRGC (TCR gamma constant region) and pro-inflammatory markers of γ/δ T cells in peripheral blood mononuclear cells (PBMC). Method and Results: Thirteen to 15-week old male C57BL/6 wild-type (WT) mice were infused with Ang II (490 ng/kg/min, SC) for 14 days and injected IP with anti-γ/δ TCR or control isotype antibodies 1 day before and at day 6 of Ang II infusion. Depletion of γ/δ T cells decreased SBP (147±2 vs 167 ± 3 mm Hg, P &lt;0.05) and restored mesenteric artery relaxation responses to acetylcholine (E max : 90±4 vs 62 ± 8%, P &lt;0.05) compared to isotype antibody-treated mice. Using the SBP data and the PBMC gene expression profile (GSE12288) of 222 human subjects, we predicted with a supervised machine learning approach SBP by combining the gene expression of TCRGC and pro-inflammatory makers including interleukin-17A, interferon-γ and their receptors (R=0.23, P &lt;0.001). Conclusion: Antibody-induced depletion further demonstrates the role of γ/δ T cells in Ang II-induced SBP elevation and vascular injury. Prediction of SBP using PBMC gene expression of γ/δ T cells and pro-inflammatory markers suggests that γ/δ T cells contribute to the development of human hypertension.

Methylation of SOCS3 is inversely associated with metabolic syndrome in an epigenome-wide association study of obesity

ABSTRACTEpigenetic mechanisms, including DNA methylation, mediate the interaction between gene and environment and may play an important role in the obesity epidemic. We assessed the relationship between DNA methylation and obesity in peripheral blood mononuclear cells (PBMCs) at 485,000 CpG sites across the genome in family members (8-90 y of age) using a discovery cohort (192 individuals) and a validation cohort (1,052 individuals) of Northern European ancestry. After Bonferroni-correction (Pα=0.05 = 1.31 × 10−7) for genome-wide significance, we identified 3 loci, cg18181703 (SOCS3), cg04502490 (ZNF771), and cg02988947 (LIMD2), where methylation status was associated with body mass index percentile (BMI%), a clinical index for obesity in children, adolescents, and adults. These sites were also associated with multiple metabolic syndrome (MetS) traits, including central obesity, fat depots, insulin responsiveness, and plasma lipids. The SOCS3 methylation locus was also associated with the clinical definition of MetS. In the validation cohort, SOCS3 methylation status was found to be inversely associated with BMI% (P = 1.75 × 10−6), waist to height ratio (P = 4.18 × 10−7), triglycerides (P = 4.01 × 10−4), and MetS (P = 4.01 × 10−7), and positively correlated with HDL-c (P = 4.57 × 10−8). Functional analysis in a sub cohort (333 individuals) demonstrated SOCS3 methylation and gene expression in PBMCs were inversely correlated (P = 2.93 × 10−4) and expression of SOCS3 was positively correlated with status of MetS (P = 0.012). We conclude that epigenetic modulation of SOCS3, a gene involved in leptin and insulin signaling, may play an important role in obesity and MetS.

Dynamics of PBMC gene expression in hepatitis C virus genotype 1-infected patients during combined peginterferon/ribavirin therapy.

Hepatitis C virus (HCV) can replicate in peripheral blood mononuclear cells (PBMCs), which can produce interferon to defend against virus infection. We hypothesized that dynamic gene expression in PBMCs might impact the treatment efficacy of peginterferon/ribavirin in HCV patients. PBMCs were collected at baseline, 1st week and 4th week of treatment from 27 chronic HCV-1 patients with 48-week peginterferon/ribavirin therapy (screening dataset n = 7; validation dataset n = 20). A sustained virologic response (SVR) was defined as undetectable HCV RNA throughout the 24 weeks after end-of-treatment. A complete early virologic response (cEVR) was defined as negative HCV RNA at treatment week 12. Forty-three differentially expressed genes identified by Affymetrix microarray were validated by quantitative polymerase chain reaction. Thirteen genes at week 1 and 24 genes at week 4 were upregulated in the SVR group compared with the non-SVR group. We selected 8 target genes (RSAD2, LOC26010, HERC5, HERC6, IFI44, SERPING1, IFITM3, and DDX60) at week 1 as the major components of the predictive model. This predictive model reliably stratified the responders and non-responders at week 1 (AUC = 0.89, p = 0.007 for SVR; AUC = 0.95, p = 0.003 for cEVR), especially among patients carrying the IL28B rs8099917 TT genotype (AUC = 0.89, p = 0.02 for SVR; AUC = 1.0, p = 0.008 for cEVR). The performance of this predictive model was superior to traditional predictors, including the rapid virologic response, viral load and IL28B genotype.

Differences in metabolic biomarkers in the blood and gene expression profiles of peripheral blood mononuclear cells among normal weight, mildly obese and moderately obese subjects.

We compared metabolic biomarkers in the blood and peripheral blood mononuclear cell (PBMC) gene expression profiles among normal weight (BMI, 18·5-23 kg/m2), mildly obese (BMI, 25-27·5 kg/m2) and moderately obese Korean adult men (BMI, 27·5-30 kg/m2). High leptin, lipids (except LDL- and HDL-cholesterol) and apoB levels and low adiponectin and HDL-cholesterol levels were present in the plasma of both mildly and moderately obese subjects. Circulating levels of inflammatory cytokines and markers of insulin resistance, oxidative stress and liver damage were altered in moderately obese subjects but not in mildly obese subjects. PBMC transcriptome data showed enrichment of pathways involved in energy metabolism, insulin resistance, bone metabolism, cancer, inflammation and fibrosis in both mildly and moderately obese subjects. Signalling pathways involved in oxidative phosphorylation, TAG synthesis, carbohydrate metabolism and insulin production; mammalian target of rapamycin, forkhead box O, ras-proximate-1, RAS and transforming growth factor-β signalling; as well as extracellular matrix-receptor interaction were enriched only in moderately obese subjects, indicating that changes in PBMC gene expression profiles, according to metabolic disturbances, were associated with the development and/or aggravation of obesity. In particular, fourteen and fifteen genes differentially expressed only in mildly obese subjects and in both mildly and moderately obese subjects, respectively, could be used as early or stable biomarkers for diagnosing and treating obesity-associated metabolic disturbance. We characterised BMI-associated metabolic and molecular biomarkers in the blood and provided clues about potential blood-based targets for preventing or treating obesity-related complications.

In vitro free radicals scavenging activity and antioxidant capacity of solid-state fermented wheat bran and its potential modulation of antioxidative molecular targets in chicken PBMC

The purpose of this study was to demonstrate the in vitro free radical scavenging activity and antioxidant capacity in solid-state fermented wheat bran and its potential modulation of antioxidative molecular targets in chicken peripheral blood mononuclear cells (PBMC). After solid-state fermentation of wheat bran by white rot fungi for 12 d, the scavenging action of the fermented wheat bran extracts was 1, 1-diphenyl-2-picrylhydrazyl (DPPH), and the free radicals increased significantly, approximately 1.5-fold. Trolox equivalent antioxidant capacity of 1 mg/mL fermented wheat bran extracts was increased from 100 to 150 mM trolox antioxidant capacity after 12 d of fermentation. Moreover, the extracts exhibited 50% of the chelating capacity observed for ferrous iron (Fe2+) after fermenting for 12 d. In vitro, and under the stimulus of fermented wheat bran, the antioxidant gene expression (GST, HO-1, Nrf2, and GCLC genes) of PBMC was more than double that of the PBS, ascorbic acid, and unfermented wheat bran. The expression of fermented wheat bran was the lowest for the NOX1 and ROMO1 genes. Solid-state wheat bran fermented by white rot fungi can increase the scavenging action of DPPH, the trolox equivalent antioxidant capacity, and the chelating capacity of ferrous iron; in addition, in vitro, it can regulate the expression of antioxidant molecular targets in chicken PBMC.

Ex Vivo Model of Human Penile Transplantation and Rejection: Implications for Erectile Tissue Physiology

BackgroundPenile transplantation is a potential treatment option for severe penile tissue loss. Models of human penile rejection are lacking. ObjectiveEvaluate effects of rejection and immunosuppression on cavernous tissue using a novel ex vivo mixed lymphocyte reaction (MLR) model. Design, setting, and participantsCavernous tissue and peripheral blood mononuclear cells (PBMCs) from 10 patients undergoing penile prosthesis operations and PBMCs from a healthy volunteer were obtained. Ex vivo MLRs were prepared by culturing cavernous tissue for 48h in media alone, in media with autologous PBMCs, or in media with allogenic PBMCs to simulate control, autotransplant, and allogenic transplant conditions with or without 1μM cyclosporine A (CsA) or 20nM tacrolimus (FK506) treatment. Outcome measurements and statistical analysisRejection was characterized by PBMC flow cytometry and gene expression transplant array. Cavernous tissues were evaluated by histomorphology and myography to assess contraction and relaxation. Data were analyzed using two-way analysis of variance and unpaired Student t test. Results and limitationsFlow cytometry and tissue array demonstrated allogenic PBMC activation consistent with rejection. Rejection impaired cavernous tissue physiology and was associated with cellular infiltration and apoptosis. CsA prevented rejection but did not improve tissue relaxation. CsA treatment impaired relaxation in tissues cultured without PBMCs compared with media and FK506. Study limitations included the use of penile tissue with erectile dysfunction and lack of cross-matching data. ConclusionsThis model could be used to investigate the effects of penile rejection and immunosuppression. Additional studies are needed to optimize immunosuppression to prevent rejection and maximize corporal tissue physiology. Patient summaryThis report describes a novel ex vivo model of human penile transplantation rejection. Tissue rejection impaired erectile tissue physiology. This report suggests that cyclosporin A might hinder corporal physiology and that other immunosuppressant agents, such as FK506, might be better suited to penile transplantation.

The immunomodulatory activity of secondary metabolites isolated from Streptomyces calvus on human peripheral blood mononuclear cells

Background: The natural products derived from micro-organisms are potential candidates for the discovery of novel drugs. Streptomyces bacteria are prolific sources of secondary metabolites with a wide variety of biological activities. Streptomyces calvus (S. calvus) is one strain of this genus and may be an appropriate candidate for isolating new compounds. In this study, the immunomodulatory effects of S. calvus secondary metabolites on the expression of various cytokine genes by human peripheral blood mononuclear cells (PBMCs) were evaluated.Methods: A bacterial sample was inoculated in Mueller Hinton Broth and secondary metabolites were extracted. PBMCs were isolated from venous blood and were treated with S. calvus secondary metabolites for 48 h. The cell proliferation was assessed by Methyl tetrazolium bromide (MTT) assay and quantitative real-time polymerase chain reaction (qRT-PCR) assays to survey mRNA expressions of selected pro-inflammatory and inhibitory cytokine genes.Results: Secondary metabolites augmented interleukin-2 and interferon-γ gene expression in PBMCs at low doses and also reduced the levels of immunosuppressive cytokine interleukin-10. In addition, the proliferation of PBMCs substantially increased in response to metabolite treatment in a concentration-dependent manner (p < 0.001).Conclusion: This in vitro study revealed that the secondary metabolites from S. calvus can successfully stimulate human PBMCs. Therefore, these metabolites have the potential to serve as robust immunomodulators.

Dynamics of salivary immunoglobulin A and serum interleukin 6 levels in newborn calves

Mucosal immunoglobulin A (IgA) is an important component of the adaptive first line immune defense. Especially in newborn calves, where diarrheal pathogens can cause severe diseases, the adaptive mucosal immunity is crucial. Regulating local IgA production is complex. Cytokines and in particular, interleukin 6 (IL-6), are described to be main actors in this process. In vaccination trails, IL-6 is used to improve the immune response with ambiguous results. Therefore, this study investigated the influence of IL-6 on the physiological development of salivary IgA production in newborn calves. Ten clinically healthy female Holstein calves were sampled over a period of four weeks. Immunoglobulin A and IL-6 were measured in serum, colostrum, milk and saliva. In addition, to compare the mucosal immune response with the systemic immune response, immunoglobulin G was measured in serum, colostrum and milk, too, using a commercial enzyme-linked immunosorbent assay (ELISA). In order to determine the ability of newborn calves to produce IL-6 actively by themselves, the relative gene expression was analyzed in peripheral blood mononuclear cells (PBMC) using a quantitative reverse transcriptase polymerase chain reaction (qPCR). Interleukin 6 gene expression in PBMCs was detected directly after birth. A significant positive correlation between serum IL-6 and salivary IgA was found on day seven (p=0.043). Furthermore, the IL-6 serum concentration peaked on day 14 and coincided with a remarkable IgA increase in saliva (p<0.01). Therefore, IL-6 seemed to play a role during the first initialization phase of a local IgA response.