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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.
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Association of Folate Metabolites and Mitochondrial Function in Peripheral Blood Cells in Alzheimer's Disease: A Matched Case-Control Study.
Lv, X, Zhou, D, Ge, B, Chen, H, Du, Y, Liu, S, Ji, Y, Sun, C, Wang, G, Gao, Y, et al
Journal of Alzheimer's disease : JAD. 2019;(4):1133-1142
Abstract
BACKGROUND The nutrition state plays an important role in the progress of aging. Folate may play a role in protecting mitochondrial (mt) DNA by reducing oxidative stress. OBJECTIVE The primary aim of this study was to examine the association of mitochondrial oxidative damage with risk of Alzheimer's disease (AD), and to explore the possible role of folate metabolites in this association in a matched case-control study. METHODS Serum folate metabolites and mitochondrial function in peripheral blood cells were determined in 82 AD cases and 82 healthy controls, individually matched by age, gender, and education. RESULTS AD patients had lower serum levels of folate and higher homocysteine (Hcy) concentration. AD patients had a reduced mtDNA copy number, higher mtDNA deletions, and increased 8-OHdG content in mtDNA indicative of reduced mitochondrial function. The highest level of mtDNA copy number would decrease the risk of AD (OR = 0.157, 95% CI: 0.058-0.422) compared to the lowest level, independently of serum folate, and Hcy levels. Serum folate levels correlated with low 8-OHdG content in mtDNA both in AD patients and controls, independently of serum Hcy level. Moreover, serum Hcy levels correlated with low copy number in mtDNA both in AD patients and controls, independently of serum folate levels. CONCLUSION In conclusion, mitochondrial function in peripheral blood cells could be associated with risk of AD independent of multiple covariates. AD patients with a folate deficiency or hyperhomocysteinemia had low mitochondrial function in peripheral blood cells. However, further randomized controlled trials are need to determine a causal effect.
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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.
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The influence of biotinylation on the ability of a computer designed protein to detect B-cells producing anti-HIV-1 2F5 antibodies.
Coêlho, DF, Ferraz, MVF, Marques, ETA, Lins, RD, Viana, IFT
Journal of molecular graphics & modelling. 2019;:107442
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Abstract
Antibodies against the HIV-1 2F5 epitope are known as one of the most powerful and broadly protective anti-HIV antibodies. Therefore, vaccine strategies that include the 2F5 epitope in their formulation require a robust method to detect specific anti-2F5 antibody production by B cells. Towards this goal, we have biotinylated a previously reported computer-designed protein carrying the HIV-1 2F5 epitope aiming the further development of a platform to detect human B-cells expressing anti-2F5 antibodies through flow cytometry. Biophysical and immunological properties of our devised protein were characterized by computer simulation and experimental methods. Biotinylation did not affect folding and improved protein stability and solubility. The biotinylated protein exhibited similar binding affinity trends compared to its unbiotinylated counterpart and was recognized by anti-HIV-1 2F5 antibodies expressed on the surface of patient-derived peripheral blood mononuclear cells. Moreover, we present a high affinity marker for the identification of epitope-specific B cells that can be used to measure the efficacy of vaccine strategies based on the HIV-1 envelope protein.
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Investigating gene expression profiles of whole blood and peripheral blood mononuclear cells using multiple collection and processing methods.
Gautam, A, Donohue, D, Hoke, A, Miller, SA, Srinivasan, S, Sowe, B, Detwiler, L, Lynch, J, Levangie, M, Hammamieh, R, et al
PloS one. 2019;(12):e0225137
Abstract
Gene expression profiling using blood samples is a valuable tool for biomarker discovery in clinical studies. Different whole blood RNA collection and processing methods are highly variable and might confound comparisons of results across studies. The main aim of the current study is to compare how blood storage, extraction methodologies, and the blood components themselves may influence gene expression profiling. Whole blood and peripheral blood mononuclear cell (PBMC) samples were collected in triplicate from five healthy donors. Whole blood was collected in RNAgard® and PAXgene® Blood RNA Tubes, as well as in collection tubes with anticoagulants such as dipotassium ethylenediaminetetraacetic acid (K2EDTA) and Acid Citrate Dextrose Solution A (ACD-A). PBMCs were separated using sodium citrate Cell Preparation Tubes (CPT™), FICOLL™, magnetic separation, and the LeukoLOCK™ methods. After blood collection, the LeukoLOCK™, K2EDTA and ACD-A blood tubes were shipped overnight using cold conditions and samples from the rest of the collection were immediately frozen with or without pre-processing. The RNA was isolated from whole blood and PBMCs using a total of 10 different experimental conditions employing several widely utilized RNA isolation methods. The RNA quality was assessed by RNA Integrity Number (RIN), which showed that all PBMC procedures had the highest RIN values when blood was stabilized in TRIzol® Reagent before RNA extraction. Initial data analysis showed that human blood stored and shipped at 4°C overnight performed equally well when checked for quality using RNA integrity number when compared to frozen stabilized blood. Comparisons within and across donor/method replicates showed signal-to-noise patterns which were not captured by RIN value alone. Pathway analysis using the top 1000 false discovery rate (FDR) corrected differentially expressed genes (DEGs) showed frozen vs. cold shipping conditions greatly impacted gene expression patterns in whole blood. However, the top 1000 FDR corrected DEGs from PBMCs preserved after frozen vs. cold shipping conditions (LeukoLOCK™ preserved in RNAlater®) revealed no significantly affected pathways. Our results provide novel insight into how RNA isolation, various storage, handling, and processing methodologies can influence RNA quality and apparent gene expression using blood samples. Careful consideration is necessary to avoid bias resulting from downstream processing. Better characterization of the effects of collection method idiosyncrasies will facilitate further research in understanding the effect of gene expression variability in human sample types.
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Mitochondrial implications in human pregnancies with intrauterine growth restriction and associated cardiac remodelling.
Guitart-Mampel, M, Juarez-Flores, DL, Youssef, L, Moren, C, Garcia-Otero, L, Roca-Agujetas, V, Catalan-Garcia, M, Gonzalez-Casacuberta, I, Tobias, E, Milisenda, JC, et al
Journal of cellular and molecular medicine. 2019;(6):3962-3973
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Abstract
Intrauterine growth restriction (IUGR) is an obstetric complication characterised by placental insufficiency and secondary cardiovascular remodelling that can lead to cardiomyopathy in adulthood. Despite its aetiology and potential therapeutics are poorly understood, bioenergetic deficits have been demonstrated in adverse foetal and cardiac development. We aimed to evaluate the role of mitochondria in human pregnancies with IUGR. In a single-site, cross-sectional and observational study, we included placenta and maternal peripheral and neonatal cord blood mononuclear cells (PBMC and CBMC) from 14 IUGR and 22 control pregnancies. The following mitochondrial measurements were assessed: enzymatic activities of mitochondrial respiratory chain (MRC) complexes I, II, IV, I + III and II + III, oxygen consumption (cell and complex I-stimulated respiration), mitochondrial content (citrate synthase [CS] activity and mitochondrial DNA copy number), total ATP levels and lipid peroxidation. Sirtuin3 expression was evaluated as a potential regulator of bioenergetic imbalance. Intrauterine growth restriction placental tissue showed a significant decrease of MRC CI enzymatic activity (P < 0.05) and CI-stimulated oxygen consumption (P < 0.05) accompanied by a significant increase of Sirtuin3/β-actin protein levels (P < 0.05). Maternal PBMC and neonatal CBMC from IUGR patients presented a not significant decrease in oxygen consumption (cell and CI-stimulated respiration) and MRC enzymatic activities (CII and CIV). Moreover, CS activity was significantly reduced in IUGR new-borns (P < 0.05). Total ATP levels and lipid peroxidation were preserved in all the studied tissues. Altered mitochondrial function of IUGR is especially present at placental and neonatal level, conveying potential targets to modulate obstetric outcome through dietary interventions aimed to regulate Sirtuin3 function.
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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.
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Stimulatory Response of Celiac Disease Peripheral Blood Mononuclear Cells Induced by RNAi Wheat Lines Differing in Grain Protein Composition.
Sánchez-León, S, Giménez, MJ, Comino, I, Sousa, C, López Casado, MÁ, Torres, MI, Barro, F
Nutrients. 2019;(12)
Abstract
Wheat gluten proteins are responsible for the bread-making properties of the dough but also for triggering important gastrointestinal disorders. Celiac disease (CD) affects approximately 1% of the population in Western countries. The only treatment available is the strict avoidance of gluten in the diet. Interference RNA (RNAi) is an excellent approach for the down-regulation of genes coding for immunogenic proteins related to celiac disease, providing an alternative for the development of cereals suitable for CD patients. In the present work, we report a comparative study of the stimulatory capacity of seven low-gluten RNAi lines differing in grain gluten and non-gluten protein composition, relevant for CD and other gluten pathologies. Peripheral blood mononuclear cells (PBMCs) of 35 patients with active CD were included in this study to assess the stimulatory response induced by protein extracts from the RNAi lines. Analysis of the proliferative response and interferon-gamma (INF-γ) release of PBMCs demonstrated impaired stimulation in response to all RNAi lines. The lower response was provided by lines with a very low content of α- and γ-gliadins, and low or almost devoid of DQ2.5 and p31-43 α-gliadin epitopes. The non-gluten protein seems not to play a key role in PBMC stimulation.
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Impact of Mon2 monocyte-platelet aggregates on human coronary artery disease.
Brown, RA, Lip, GYH, Varma, C, Shantsila, E
European journal of clinical investigation. 2018;(5):e12911
Abstract
BACKGROUND Monocyte-platelet aggregates (MPAs) form when Mon1, Mon2 or Mon3 monocyte subsets adhere to platelets. They are pathophysiologically linked to coronary artery disease (CAD). However, their individual roles in the occurrence of diffuse CAD remain unknown. MATERIALS AND METHODS Peripheral blood from 50 patients with diffuse CAD, 40 patients with focal CAD and 50 age-matched patients with normal coronary arteries was analysed by flow cytometry to quantify MPAs associated with individual monocyte subsets. Cutaneous forearm microcirculation was assessed using laser Doppler flowmetry at rest and after iontophoresis of acetylcholine (endothelium-dependent vasodilation) and sodium nitroprusside (endothelium-independent vasodilation) at 100 μA for 60 seconds. Patients with CAD had repeat assessment at 6 and 12 months. RESULTS Baseline counts of MPAs with Mon2 subset (CD14++CD16+CC2+ monocytes) were significantly higher in patients with diffuse CAD compared to focal CAD (P = .001) and patients without CAD (P = .006). On multivariate regression, MPAs with Mon2 independently predicted diffuse CAD (odds ratio 1.10, 95% confidence interval 1.02-1.19, P = .01) and correlated negatively with endothelium-dependent microvascular vasodilation (r = -.37, P = .008), an association which persisted after adjustment for covariates. Longitudinal observation confirmed the persistence of an inverse relationship between MPAs with Mon2 and endothelium-dependent microvascular function. CONCLUSION Monocyte-platelet aggregates with Mon2 are increased in patients with diffuse CAD and therefore could represent an important contributor to accelerated coronary atherosclerotic progression by a mechanism involving microvascular endothelial dysfunction.
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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
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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.