-
1.
Associations between dietary patterns and gene expression pattern in peripheral blood mononuclear cells: A cross-sectional study.
Christensen, JJ, Ulven, SM, Thoresen, M, Westerman, K, Holven, KB, Andersen, LF
Nutrition, metabolism, and cardiovascular diseases : NMCD. 2020;(11):2111-2122
Abstract
BACKGROUND AND AIMS Diet may alter gene expression in immune cells involved in atherosclerotic cardiovascular disease susceptibility. However, we still lack a robust understanding of the association between diet and immune cell-related gene expression in humans. Therefore, we examined associations between dietary patterns (DPs) and gene expression profiles in peripheral blood mononuclear cells (PBMCs) in a population of healthy, Norwegian adults (n = 130 women and 105 men). METHODS AND RESULTS We used factor analysis to define a posteriori DPs from food frequency questionnaire-based dietary assessment data. In addition, we derived interpretable features from microarray-based gene expression data (13 967 transcripts) using two algorithms: CIBERSORT for estimation of cell subtype proportions, and weighted gene co-expression network analysis (WGCNA) for cluster discovery. Finally, we associated DPs with either CIBERSORT-predicted PBMC leukocyte distribution or WGCNA gene clusters using linear regression models. We detected three DPs that broadly reflected Western, Vegetarian, and Low carbohydrate diets. CIBERSORT-predicted percentage of monocytes associated negatively with the Vegetarian DP. For women, the Vegetarian DP associated with a large gene cluster consisting of 600 genes mainly involved in regulation of DNA transcription, whereas for men, the Western DP inversely associated with a smaller cluster of 36 genes mainly involved in regulation of metabolic and inflammatory processes. A subsequent protein-protein interaction network analysis suggested that genes within these clusters might physically interact in biological networks. CONCLUSIONS Although the present findings are exploratory, our analysis pipeline serves as a useful framework for studying the association between diet and gene expression.
-
2.
Expression and Functionality Study of 9 Toll-Like Receptors in 33 Drug-Naïve Non-Affective First Episode Psychosis Individuals: A 3-Month Study.
Juncal-Ruiz, M, Riesco-Davila, L, Vazquez-Bourgon, J, Ortiz-Garcia de la Foz, V, Mayoral-Van Son, J, Ayesa-Arriola, R, Setien-Suero, E, Leza, JC, Lopez-Hoyos, M, Crespo-Facorro, B
International journal of molecular sciences. 2020;(17)
Abstract
Toll-like receptors (TLRs) are a pivotal component of the innate immune system that seem to have a role in the pathogenesis of psychosis. The purpose of this work was to compare the expression and functionality of 9 TLRs in three peripheral blood mononuclear cells (PBMCs) (monocytes, B cells, and T cells) between 33 drug-naïve first-episode psychosis (FEP) individuals and 26 healthy volunteers, at baseline and after 3-month of antipsychotic treatment. The expression of TLRs 1-9 were assessed by flow cytometry. For the assessment of the TLR functionality, cells collected in sodium heparin tubes were polyclonally stimulated for 18 h, with different agonists for human TLR1-9. The results of our study highlight the role that TLR5 and TLR8 might play in the pathophysiology of psychosis. We found a lower expression of these receptors in FEP individuals, regarding healthy volunteers at baseline and after 3-month of treatment on the three PBMCs subsets. Most TLRs showed a lower functionality (especially reduced intracellular levels of TNF-α) in patients than in healthy volunteers. These results, together with previous evidence, suggest that individuals with psychosis might show a pattern of TLR expression that differs from that of healthy volunteers, which could vary according to the intensity of immune/inflammatory response.
-
3.
Decrease of Perforin Expressing Lymphocytes after On-Pump Coronary Artery Bypass Grafting Surgery Irrespective of Carbohydrate Preoperative Oral Feeding.
Sokolic, J, Knezevic, D, Kuharic, J, Medved, I, Sustic, A, Zupan, Z, Laskarin, G, Tadin, T, Sotošek Tokmadžić, V
The heart surgery forum. 2019;(3):E218-E224
Abstract
BACKGROUND Coronary artery bypass grafting (CABG) surgery continues to be the gold standard for treating the patients with coronary artery disease. CABG surgery can be performed on or off cardiopulmonary bypass, termed as on-pump or off-pump CABG, respectively. It has been shown that CABG surgery, preferably on-pump CABG surgery, leads to the changes of cell immunity during perioperative and early postoperative period. The mechanisms of regulation of the immune response in patients during and early after surgical revascularization are not fully understood. The aim of this study was to investigate the influence of carbohydrate preoperative oral feeding on frequency and perforin expression in peripheral blood lymphocytes in patients after on- or off-pump CABG surgery in early postoperative period. PATIENTS AND METHODS In this prospective clinical study, 80 patients scheduled for CABG surgery were included in the study. The patients were randomly allocated into four groups (20 in each group): patients in Group 1 underwent on-pump CABG and did not receive carbohydrate preoperative oral feeding; patients in Group 2 underwent on-pump CABG and were preoperatively fed; patients in Group 3 underwent off-pump CABG and did not receive carbohydrate preoperative oral feeding; while patients in Group 4 underwent off-pump CABG and received carbohydrate preoperative oral feeding. Blood samples were collected immediately before (T1), 24 (T2) and 72 (T3) hours after the surgery. Peripheral blood mononuclear cells were isolated by gradient centrifugation and simultaneously labelled by antigens using fluorochrome-conjugated monoclonal antibodies. Frequency of T lymphocytes, NK and NKT cells, their subsets as well as their perforin expression were detected, and analyzed by flow cytometry. RESULTS There was significant decrease in frequency of CD3+ and CD3+CD4+ cells, as well as perforin expressing CD3+CD8+ cells in patients who underwent on-pump CABG in comparison to patients who underwent off-pump CABG 24 hours after the surgery. Carbohydrate preoperative oral feeding did not effect changes in lymphocytes subpopulations and perforin expression at any time point. CONCLUSION Decreases of CD3+ cells on account of CD3+CD4+ subsets, and perforin expressing cells on account of CD3+CD8+ perforin+ cells were found in patients who had undergone on-pump CABG, but not in patients who had undergone off-pump CABG surgery, irrespectively of carbohydrate preoperative oral feeding.
-
4.
Immunomonitoring of Tacrolimus in Healthy Volunteers: The First Step from PK- to PD-Based Therapeutic Drug Monitoring?
In 't Veld, AE, Grievink, HW, Saghari, M, Stuurman, FE, de Kam, ML, de Vries, APJ, de Winter, BCM, Burggraaf, J, Cohen, AF, Moerland, M
International journal of molecular sciences. 2019;(19)
Abstract
Therapeutic drug monitoring is routinely performed to maintain optimal tacrolimus concentrations in kidney transplant recipients. Nonetheless, toxicity and rejection still occur within an acceptable concentration-range. To have a better understanding of the relationship between tacrolimus dose, tacrolimus concentration, and its effect on the target cell, we developed functional immune tests for the quantification of the tacrolimus effect. Twelve healthy volunteers received a single dose of tacrolimus, after which intracellular and whole blood tacrolimus concentrations were measured and were related to T cell functionality. A significant correlation was found between tacrolimus concentrations in T cells and whole blood concentrations (r = 0.71, p = 0.009), while no correlation was found between tacrolimus concentrations in peripheral blood mononuclear cells (PBMCs) and whole blood (r = 0.35, p = 0.27). Phytohemagglutinin (PHA) induced the production of IL-2 and IFNγ, as well as the inhibition of CD71 and CD154 expression on T cells at 1.5 h post-dose, when maximum tacrolimus levels were observed. Moreover, the in vitro tacrolimus effect of the mentioned markers corresponded with the ex vivo effect after dosing. In conclusion, our results showed that intracellular tacrolimus concentrations mimic whole blood concentrations, and that PHA-induced cytokine production (IL-2 and IFNγ) and activation marker expression (CD71 and CD154) are suitable readout measures to measure the immunosuppressive effect of tacrolimus on the T cell.
-
5.
The effect of retinyl-palmitate on the level of pro and anti-inflammatory cytokines in multiple sclerosis patients: A randomized double blind clinical trial.
Bitarafan, S, Mohammadpour, Z, Jafarirad, S, Harirchian, MH, Yekaninejad, MS, Saboor-Yaraghi, AA
Clinical neurology and neurosurgery. 2019;:101-105
Abstract
OBJECTIVE Multiple sclerosis (MS) is an inflammatory and autoimmune disease associated with the imbalance of cytokines secreted from CD4+ T cells. Studies have shown that vitamin A and its active derivatives are able to modulate the immune system in MS patients. The aim of the present study was to investigate the effect of supplementation of retinyl palmitate (RP), the dietary form of vitamin A, on pro- and anti-inflammatory cytokines in the plasma and supernatants of cultured peripheral blood mononuclear cells (PBMCs) of MS patients. PATIENTS AND METHODS Thirty-six relapsing-remitting MS patients were enrolled in this double-blind randomized clinical trial. Participants received one capsule of 25,000 IU RP or a placebo per day for six months. Blood samples were taken before and after intervention. After intervention, the PBMCs were isolated and cultured. The levels of pro- and anti-inflammatory cytokines in the plasma and supernatant of cells stimulated with myelin oligodendrocyte glycoprotein, phytohemagglutinin or vehicle (media) were determined. The sample t-test and Mann Whitney U test were used to compare data between groups. RESULTS The changes in pro-inflammatory cytokine levels (IL-1β, TNF-α, IFN- γ, IL-2, IL-6, and IL-17) in the serum and supernatant of MS patients were not significant (p > 0.05). There were also no significant changes in the levels of anti-inflammatory cytokines (IL-10, IL-13, IL-4, and TGF-β) (p > 0.05). CONCLUSION Unexpectedly, this study found no significant changes in cytokine levels after six months of RP supplementation in MS patients. The results of other studies by our team have shown significant changes in the gene expression of the cytokines in response to RP supplements. Therefore, we recommend that periodic follow-up of RP supplementation may be needed to reveal changes in the level of the cytokines in the plasma and PBMCs and to clarify the real effect of RP on the immune factor levels in the serum of MS patients.
-
6.
Quantitation of intracellular triphosphate metabolites of antiretroviral agents in peripheral blood mononuclear cells (PBMCs) and corresponding cell count determinations: review of current methods and challenges.
Xiao, D, Ling, KHJ, Custodio, J, Majeed, SR, Tarnowski, T
Expert opinion on drug metabolism & toxicology. 2018;(8):781-802
Abstract
Peripheral blood mononuclear cells (PBMCs) are a critical component of the immune system and the target cells for human immunodeficiency virus, type 1 (HIV-1) infection. Nucleoside/nucleotide analogs for the treatment of HIV infection are prodrugs that require cellular activation to triphosphate (TP) metabolites for antiviral activity. A reliable method of PBMC isolation and subsequent cell counting, as well as an accurate bioanalytical determination of the TPs in PBMCs are important for understanding the intracellular pharmacokinetic (PK) of the TPs and its correlation with plasma PK, the drug effect, and dose determination. Areas covered: The authors review the challenges and solutions in PBMC sample collection, sample processing, cell lysis, cell counting methods, analyte extraction, and liquid chromatography/tandem mass spectrometry (LC-MS/MS) quantitative analysis of the nucleoside reverse transcriptase inhibitor-triphosphate (NRTI-TP) metabolites, and analogs. Expert opinion: Analyzing large numbers of clinical PBMC samples for determination of NRTI-TPs and analogs in PBMCs requires not only a validated LC-MS/MS bioanalytical method but also reliable methods for PBMC isolation, counting, cell lysis, and analyte recovery, and an approach for assessing analyte stability. Furthermore, a simple, consistent, and validated cell counting method often involves DNA quantitation of the PBMCs samples collected from clinical studies.
-
7.
PBMC fixation and processing for Chromium single-cell RNA sequencing.
Chen, J, Cheung, F, Shi, R, Zhou, H, Lu, W, ,
Journal of translational medicine. 2018;(1):198
Abstract
BACKGROUND Interest in single-cell transcriptomic analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. In almost all reported works investigators have used live cells, which introduces cell stress during preparation and hinders complex study designs. Recent studies have indicated that cells fixed by denaturing fixative can be used in single-cell sequencing, however they did not usually work with most types of primary cells including immune cells. METHODS The methanol-fixation and new processing method was introduced to preserve human peripheral blood mononuclear cells (PBMCs) for single-cell RNA sequencing (scRNA-Seq) analysis on 10× Chromium platform. RESULTS When methanol fixation protocol was broken up into three steps: fixation, storage and rehydration, we found that PBMC RNA was degraded during rehydration with PBS, not at cell fixation and up to 3-month storage steps. Resuspension but not rehydration in 3× saline sodium citrate (SSC) buffer instead of PBS preserved PBMC RNA integrity and prevented RNA leakage. Diluted SSC buffer did not interfere with full-length cDNA synthesis. The methanol-fixed PBMCs resuspended in 3× SSC were successfully implemented into 10× Chromium standard scRNA-seq workflows with no elevated low quality cells and cell doublets. The fixation process did not alter the single-cell transcriptional profiles and gene expression levels. Major subpopulations classified by marker genes could be identified in fixed PBMCs at a similar proportion as in live PBMCs. This new fixation processing protocol also worked in several other fixed primary cell types and cell lines as in live ones. CONCLUSIONS We expect that the methanol-based cell fixation procedure presented here will allow better and more effective batching schemes for a complex single cell experimental design with primary cells or tissues.
-
8.
DNA Hydroxymethylation Levels Are Altered in Blood Cells From Down Syndrome Persons Enrolled in the MARK-AGE Project.
Ciccarone, F, Valentini, E, Malavolta, M, Zampieri, M, Bacalini, MG, Calabrese, R, Guastafierro, T, Reale, A, Franceschi, C, Capri, M, et al
The journals of gerontology. Series A, Biological sciences and medical sciences. 2018;(6):737-744
-
-
Free full text
-
Abstract
Down syndrome (DS) is caused by the presence of part or an entire extra copy of chromosome 21, a phenomenon that can cause a wide spectrum of clinically defined phenotypes of the disease. Most of the clinical signs of DS are typical of the aging process including dysregulation of immune system. Beyond the causative genetic defect, DS persons display epigenetic alterations, particularly aberrant DNA methylation patterns that can contribute to the heterogeneity of the disease. In the present work, we investigated the levels of 5-hydroxymethylcytosine and of the Ten-eleven translocation dioxygenase enzymes, which are involved in DNA demethylation processes and are often deregulated in pathological conditions as well as in aging. Analyses were carried out on peripheral blood mononuclear cells of DS volunteers enrolled in the context of the MARK-AGE study, a large-scale cross-sectional population study with subjects representing the general population in eight European countries. We observed a decrease in 5-hydroxymethylcytosine, TET1, and other components of the DNA methylation/demethylation machinery in DS subjects, indicating that aberrant DNA methylation patterns in DS, which may have consequences on the transcriptional status of immune cells, may be due to a global disturbance of methylation control in DS.
-
9.
Proteomic Analysis of Peripheral Blood Mononuclear Cells after a High-Fat, High-Carbohydrate Meal with Orange Juice.
Chaves, DFS, Carvalho, PC, Brasili, E, Rogero, MM, Hassimotto, NA, Diedrich, JK, Moresco, JJ, Yates, JR, Lajolo, FM
Journal of proteome research. 2017;(11):4086-4092
Abstract
Oxidative stress and inflammation play a role in the physiopathology of insulin resistance, diabetes and cardiovascular disease. A single high-fat, high-carbohydrate (HFHC) meal induces an increase in inflammatory and oxidative stress markers in peripheral blood mononuclear cells (PBMC). Previous studies have shown that orange juice is able to prevent this response by inhibiting toll like receptors (TLR) expression and endotoxemia. Our goal was to study the proteome response in PBMC after the consumption of a HFHC meal consumed with water, orange juice or an isocaloric beverage (water with glucose). Twelve healthy individuals completed the protocol in a crossover design, and blood samples were obtained before and 1, 3, and 5 h after consumption. Proteomic profile, glucose, insulin, lipid and cytokines levels were investigated. The glycemic and insulinemic response was higher when the meal was consumed with glucose, while there was no difference in the response between water and orange juice. Proteome analysis in PBMC was carried out using TMT ten-plex. A total of 3813 proteins, originating from 15 662 peptides were identified. Three proteins showed significantly altered expression in the three treatments: apolipoprotein A-II, ceruloplasmin and hemopexin. When the HFHC meal was consumed with water there was an increase in some inflammatory pathways such as the Fc-gamma receptor dependent phagocytosis and the complement cascade, but the immune system as a whole was not significantly altered. However, when the meal was consumed with glucose, the immune system was up regulated. Among the pathways induced after 3 h were those of the adaptive immune system and cytokine signaling. Five hours after the meal, pathways of the complement cascade and classical antibody mediated complement activation were up regulated. When the meal was consumed with orange juice there was an up regulation of proteins involved in signal transduction, DNA replication and cell cycle. The promyelocytic leukemia protein (PML) showed a 28.2-fold increase. This protein was down regulated when the meal was consumed with water. Regarding the immune system, several of the pathways induced by glucose were down regulated when the meal was consumed with orange juice: proteins involved with the adaptive immune system and cytokine signaling. Therefore, we have shown that orange juice can not only suppress diet induced inflammation, but also regulate the expression of proteins such as PML, which may play a key role in the regulation of metabolism.