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Scientific Reports Nov 2022Same day processing of biospecimens such as blood is not always feasible, which presents a challenge for research programs seeking to study a broad population or to...
Same day processing of biospecimens such as blood is not always feasible, which presents a challenge for research programs seeking to study a broad population or to characterize patients with rare diseases. Recruiting sites may not be equipped to process blood samples and variability in timing and technique employed to isolate peripheral blood mononuclear cells (PBMCs) at local sites may compromise reproducibility across patients. One solution is to send whole blood collected by routine phlebotomy via overnight courier to the testing site under ambient conditions. Determining the impact of shipping on subsequent leukocyte responses is a necessary prerequisite to any experimental analysis derived from transported samples. To this end, whole blood was collected from healthy control subjects and processed fresh or at 6, 24 and 48 h after collection and handling under modeled shipping conditions. At endpoint, whole blood was assessed via a complete blood count with differential and immunophenotyped using a standardized panel of antibodies [HLADR, CD66b, CD3, CD14, CD16]. PBMCs and neutrophils were isolated from whole blood and subjected to ex vivo stimulation with lipopolysaccharide and heat-killed Staphylococcus aureus. Stimulated release of cytokines and chemokines was assessed by cytometric bead array. RNA was also isolated from PBMCs to analyze transcriptional changes induced by shipping. The complete blood count with differential revealed that most parameters were maintained in shipped blood held for 24 h at ambient temperature. Immunophenotyping indicated preservation of cellular profiles at 24 h, although with broadening of some populations and a decrease in CD16 intensity on classical monocytes. At the transcriptional level, RNAseq analysis identified upregulation of a transcription factor module associated with inflammation in unstimulated PBMCs derived from whole blood shipped overnight. However, these changes were limited in both scale and number of impacted genes. Ex vivo stimulation of PBMCs further revealed preservation of functional responses in cells isolated from shipped blood held for 24 h at ambient temperature. However, neutrophil responses were largely abrogated by this time. By 48 h neither cell population responded within normal parameters. These findings indicate that robust immunophenotyping and PBMC stimulated response profiles are maintained in whole blood shipped overnight and processed within 24 h of collection, yielding results that are representative of those obtained from the sample immediately following venipuncture. This methodology is feasible for many patient recruitment sites to implement and allows for sophisticated immunological analysis of patient populations derived from large geographic areas. With regard to rare disease research, this meets a universal need to enroll patients in sufficient numbers for immunoprofiling and discovery of underlying pathogenic mechanisms.
Topics: Leukocytes, Mononuclear; Reproducibility of Results; Monocytes; Blood Preservation; Phenotype
PubMed: 36402888
DOI: 10.1038/s41598-022-24550-6 -
Methods in Molecular Biology (Clifton,... 2021Cytotoxicity is the primary function of CD8 T-cells, also called cytotoxic CD8 T lymphocytes (or CTLs). Quantification of this capacity is of major importance in...
Cytotoxicity is the primary function of CD8 T-cells, also called cytotoxic CD8 T lymphocytes (or CTLs). Quantification of this capacity is of major importance in diagnostic and research tools. While phenotypic characterization of CTLs is frequent and easily performed, their function is indeed more difficult to assess. CTLs are responsible for the lysis of cells expressing foreign or modified antigen peptides on their MHC class I molecules. Here we describe the detailed protocol used for the in vitro specific lysis of target cells.
Topics: CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Separation; Coculture Techniques; Cytotoxicity, Immunologic; Humans; In Vitro Techniques; Leukocyte Common Antigens; Leukocytes, Mononuclear; Neoplasms; T-Lymphocytes, Cytotoxic
PubMed: 34053049
DOI: 10.1007/978-1-0716-1507-2_3 -
Free Radical Research 2022Vitamin B is involved in biochemical metabolic pathways. B deficiency is common in childhood when the need for the vitamin increases and growth and development occur....
Beneficial effects of vitamin B treatment in pediatric patients diagnosed with vitamin B deficiency regarding total-native thiol, oxidative stress, and mononuclear leukocyte DNA damage.
Vitamin B is involved in biochemical metabolic pathways. B deficiency is common in childhood when the need for the vitamin increases and growth and development occur. Various hematological, neurological, psychiatric, and gastrointestinal disorders are observed in its deficiency. In addition, B deficiency is associated with oxidative stress and DNA damage. Therefore, the aim of our study is to evaluate oxidative stress, thiol/disulfide homeostasis, and DNA damage pre and post-treatment in children diagnosed with B deficiency. A total of 40 children with B deficiency were included in the study after the consent form was approved. Blood was drawn from children pre and posttreatment. Hemoglobin (HGB), hematocrit (HCT), and red blood cells (RBC) were measured by autoanalyzer; total antioxidant status (TAS), total oxidant status (TOS), total thiol (TT), and native thiol (NT) were measured by the photometric method, and DNA damage was analyzed by the comet assay method. Oxidative stress index (OSI) and disulfide (DIS) values were calculated. As a result of the experiments, HGB, HCT, and RBC increased with treatment. While TAS, TT, and NT as antioxidant parameters increased; TOS, OSI, and DIS decreased with treatment compared to pretreatment. DNA damage was also found to decrease with treatment. Additionally, these data were statistically significant ( < 0.001). It was found that oxidative stress and DNA damage decreased with oral B treatment in children with B deficiency, and clinical parameters were also improved.
Topics: Humans; Child; Antioxidants; Vitamin B 12; Sulfhydryl Compounds; Oxidative Stress; Oxidants; DNA Damage; Hemoglobins; Vitamins; Leukocytes, Mononuclear; Disulfides
PubMed: 36571212
DOI: 10.1080/10715762.2022.2162392 -
Shock (Augusta, Ga.) Jan 2020Sepsis is a complex host response triggered by an infection, with the patient's immune system between hyper- and hypo-responsiveness being the main reason for the...
Sepsis is a complex host response triggered by an infection, with the patient's immune system between hyper- and hypo-responsiveness being the main reason for the syndromes' development and propagation. Studies conducted in peripheral blood mononuclear cells uncovered an association between an impaired immunometabolism and the severity and outcome of the disease. With this prospective observational study, we aimed to evaluate the immunometabolic phenotype of monocytes and B cells and its association with the cell function.Monocytes and B cells were isolated from patients with sepsis (n = 10; onset, days 4 and 8) and healthy volunteers (n = 10) and subsequently analyzed for metabolic changes and human leukocyte antigen-DR (HLA-DR) expression. Contemporaneously, immune checkpoints on monocytes and the ex vivo cytokine responses (interleukins 6 and 8) upon lipopolysaccharide or zymosan stimulation were analyzed. The distribution of B cell subsets was assessed, and plasma levels of immunoglobulins and tricarboxylic acid cycle intermediates were quantified.Both monocytes and B cells exhibited decreased HLA-DR expression in patients with sepsis. Monocytes displayed a stable upregulated glycolysis while B cells augmented glycolysis and respiration over time. The monocytes' ability to respond to stimulation was stimuli-dependently reduced but recovered over time. The B cell compartment shifted toward antibody-producing subsets and elevated immunoglobulins within the first days.Our results provide evidence for the induction of a state of trained immunity in monocytes and an early but transient immunosuppressive phenotype accounting for peripheral sepsis-induced vulnerability to infections. B cells exhibit an unsustainable activation contributing to adaptive immunosuppression.
Topics: Antigen-Presenting Cells; B-Lymphocytes; Cytokines; Flow Cytometry; Glycolysis; Humans; Immunoglobulins; Immunosuppression Therapy; Leukocytes, Mononuclear; Sepsis
PubMed: 31738315
DOI: 10.1097/SHK.0000000000001337 -
Innate Immunity Nov 2020(PsV) is an immune-mediated inflammatory disorder with devastating psychosocial consequences. Expression of immunoregulator molecules on leukocytes in PsV remains...
(PsV) is an immune-mediated inflammatory disorder with devastating psychosocial consequences. Expression of immunoregulator molecules on leukocytes in PsV remains unclear. Leukocyte-associated Ig-like receptor-1 (LAIR-1) and complement receptor-1 (CR-1) are immunoregulator receptors reported to bind complement component 1q involved in phagocytosis. We aimed to explore if altered leukocyte expression of LAIR-1 and CR-1 is associated with PsV. This case-control study included 36 PsV patients and 36 healthy controls. Neutrophils, monocytes and B and T cells were examined by flow cytometry for LAIR-1 and CR-1 mean fluorescence intensity (MFI) and positive cell percentage. Comparison between both groups revealed a significant decrease in LAIR-1 MFI on neutrophils and T cells ( < 0.001 and = 0.003, respectively). CR-1 MFI on neutrophils, monocytes and T cells also showed a significant decrease in patients ( = 0.033, = 0.001 and = 0.040, respectively). There was a significant positive correlation of LAIR-1 MFI on neutrophils with CR-1 MFI on neutrophils ( = 0.503; = 0.002) and LAIR-1 MFI on monocytes with CR-1 MFI on monocytes ( = 0.371; = 0.026). Receiver operating characteristic curves revealed that CR-1 MFI on monocytes had the highest discrimination power to differentiate patients from controls, with 86.1% specificity and 75% sensitivity ( = 0.001). In conclusion, altered leukocytes expression of LAIR-1 and CR-1 is associated with PsV. Down-regulated CR-1 MFI on monocytes is a promising diagnostic biomarker for PsV.
Topics: Adult; Biomarkers; Case-Control Studies; Complement C1q; Female; Gene Expression Regulation; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Neutrophils; Phagocytosis; Prospective Studies; Psoriasis; Receptors, Complement; Receptors, Immunologic; T-Lymphocytes
PubMed: 32731787
DOI: 10.1177/1753425920942570 -
Cellular Physiology and Biochemistry :... 2018There is a lack of reliable biological markers for the early diagnosis of diabetic nephropathy (DN) during type 2 diabetes. In this pilot study we aim to assess whether...
BACKGROUND/AIMS
There is a lack of reliable biological markers for the early diagnosis of diabetic nephropathy (DN) during type 2 diabetes. In this pilot study we aim to assess whether miR-31 levels are modulated by the presence of DN and whether the expression of this miRNA is related to leukocyte-endothelial interactions and inflammation.
METHODS
Thirty-one T2D patients were enrolled in this pilot study; 18 with no diabetic complications and 13 with diabetic nephropathy. 24 non-diabetic subjects and 13 T2D patients with retinopathy (absent of other complications) were included to test the specificity of miR-31. Following anthropometric and biochemical evaluation, serum miR-31 levels were assessed by Real Time-PCR. Leukocyte-endothelial interactions were evaluated by a parallel flow chamber in vitro model. Serum TNFα, IL-6 and ICAM-1 levels were determined by XMAP-technology in a flow cytometry-based Luminex 200 instrument.
RESULTS
Serum miR-31 levels were similar between control and T2D subjects. However, T2D patients with DN displayed reduced levels of miR-31 with respect to patients without complications. This decrease in miR-31 was more pronounced in patients with macroalbuminuria than in those with microalbuminuria and was specific for DN, since patients with retinopathy displayed unaltered miR-31 levels. The presence of DN involved a lower leukocyte rolling velocity and an increased rolling flux and adhesion. miR-31 levels were positively correlated with leukocyte rolling velocity and negatively associated to leukocyte adhesion, TNFα, IL-6 and ICAM-1 levels.
CONCLUSION
Serum miR-31 may be a biomarker for DN in T2D patients. The regulation of this miRNA seems to be related to the recruitment of leukocytes to vascular walls induced by pro-inflammatory and adhesion molecules.
Topics: Aged; Albuminuria; Biomarkers; Cell Adhesion; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Down-Regulation; Endothelial Cells; Female; Humans; Inflammation; Intercellular Adhesion Molecule-1; Interleukin-6; Leukocytes, Mononuclear; Male; MicroRNAs; Middle Aged; Tumor Necrosis Factor-alpha
PubMed: 30355913
DOI: 10.1159/000494485 -
International Immunopharmacology Nov 2022Studies have described the role of microRNAs (miRNAs) in thymic function, along with directly observing the altered expression of miRNAs in thymuses of myasthenia gravis...
BACKGROUND
Studies have described the role of microRNAs (miRNAs) in thymic function, along with directly observing the altered expression of miRNAs in thymuses of myasthenia gravis (MG) patients; so, miRNAs became a core component in the pathophysiology of MG. However, because the miRNA analysis results are contradictory, the identification of MG-related miRNAs is daunting.
OBJECTIVE
We did a systematic review of studies analyzing the miRNA expression profile of peripheral blood and mononuclear cells for patients with MG.
METHODS
We ran a database search in PubMed, Scopus, and Web of Science on August 17, 2021. Original articles that analyzed miRNA profiles in peripheral blood (serum, plasma, and whole blood) and peripheral blood mononuclear cells (PBMCs) for patients with MG in comparison with a non-MG or healthy control (HC) group were eligible. The quality of studies was assessed using the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2).
RESULTS
26 studies were included. The quality of studies was fair (median score, 5). Among 226 different miRNAs that were deregulated in at least one study (range, 1-87), ten miRNAs were significantly deregulated in three or more studies. Five miRNAs (50%) showed the same deregulation: miR-106b-3p and miR-21-5p were consistently upregulated, and miR-20b, miR-15b, and miR-16 were consistently downregulated. Also, there were five miRNAs that were mostly upregulated, miR-150-5p, miR-146a, miR-30e-5p, and miR-338-3p, or downregulated, miR-324-3p, across studies.
CONCLUSION
These miRNAs contribute to different pathways, importantly neural apoptosis and autophagy, inflammation, T regulatory cell development, and T helper cell balance. Prior to being used for diagnostic and therapeutic purposes, it is required to pursue molecular mechanisms these consistently and mostly dysregulated miRNAs specifically use in the context of MG.
Topics: Humans; Gene Expression Profiling; Leukocytes, Mononuclear; MicroRNAs; Myasthenia Gravis; Leukocyte Count
PubMed: 36087508
DOI: 10.1016/j.intimp.2022.109205 -
Scientific Reports Mar 2017Leukocyte adhesion to brain endothelial cells, the blood-brain barrier main component, is a critical step in the pathogenesis of neuroinflammatory diseases such as...
Leukocyte adhesion to brain endothelial cells, the blood-brain barrier main component, is a critical step in the pathogenesis of neuroinflammatory diseases such as multiple sclerosis (MS). Leukocyte adhesion is mediated mainly by selectins, cell adhesion molecules and chemokines induced by pro-inflammatory cytokines such as TNFα and IFNγ, but the regulation of this process is not fully clear. This study investigated the regulation of firm leukocyte adhesion to human brain endothelium by two different brain endothelial microRNAs (miRs), miR-126 and miR-126*, that are downregulated by TNFα and IFNγ in a human brain endothelial cell line, hCMEC/D3. Using a leukocyte adhesion in vitro assay under shear forces mimicking blood flow, we observed that reduction of endothelial miR-126 and miR-126* enhanced firm monocyte and T cell adhesion to hCMEC/D3 cells, whereas their increased expression partially prevented THP1, Jurkat and primary MS patient-derived PBMC firm adhesion. Furthermore, we observed that miR-126* and miR-126 downregulation increased E-selectin and VCAM1, respectively, while miR-126 overexpression reduced VCAM1 and CCL2 expression by hCMEC/D3 cells, suggesting that these miRs regulate leukocyte adhesion by modulating the expression of adhesion-associated endothelial mRNA targets. Hence, human brain endothelial miR-126 and miR-126* could be used as a therapeutic tool to reduce leukocyte adhesion and thus reduce neuroinflammation.
Topics: Blood-Brain Barrier; Brain; Cell Adhesion; Cell Adhesion Molecules; Cell Line; E-Selectin; Endothelium; Gene Expression Regulation; Humans; Interferon-gamma; Jurkat Cells; Leukocytes, Mononuclear; MicroRNAs; Multiple Sclerosis; Shear Strength; THP-1 Cells; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1
PubMed: 28358058
DOI: 10.1038/srep45284 -
Immunological Investigations Nov 2023To investigate the expression of layilin (LAYN) in human circulating monocytes and lymphocytes and its clinical significance in systemic lupus erythematosus (SLE).
OBJECTIVE
To investigate the expression of layilin (LAYN) in human circulating monocytes and lymphocytes and its clinical significance in systemic lupus erythematosus (SLE).
METHODS
Blood samples were collected from 51 SLE patients and 50 healthy controls. Flow cytometry was used to analyze LAYN in lymphocytes and monocyte subsets. Functionally characterized molecules including human HLA, CD74 and CD62L were studied in LAYN+ monocytes. A correlation analysis was conducted between LAYN-related subsets and clinical indicators of SLE such as anti-double-stranded DNA and complements levels. ROC curves were used to explore the potential clinical diagnostic value of LAYN in SLE.
RESULTS
LAYN was significantly higher in monocytes than in lymphocytes and higher in CD14+CD16+ monocytes than in CD14-CD16+ and CD14+CD16- monocytes. CD74 was upregulated and CD62L was downregulated in LAYN+ monocytes compared with LAYN- monocytes. The absolute number of LAYN+ monocytes was increased in SLE patients, and the median fluorescence intensity of HLA was decreased. LAYN+ monocytes were positively correlated with complement C4, while decreased CD62L+ percentages in LAYN+ monocytes were negatively correlated with C4. The ROC analysis revealed that the area under the curve (AUCs) for CD62L+ percentages in LAYN+ monocytes, LAYN+ lymphocyte numbers, and LAYN+ monocyte numbers to distinguish SLE from healthy individuals were 0.6245, 0.6196 and 0.6173, respectively.
CONCLUSION
LAYN is differentially expressed in monocytes and their subpopulations and has corresponding functional differences. Changes in LAYN expression on monocytes are associated with complement C4 levels in SLE patients. These suggest that LAYN may be involved in the pathogenesis of SLE.
ABBREVIATION
ANOVA: analysis of variance; anti-dsDNA: anti-double-stranded DNA; anti-ENA: anti-extractable nuclear antigen; anti-SSA: anti-Sjogren syndrome A; anti-SSB: anti-Sjogren syndrome B; anti-U1RNP: anti-U1 ribonucleoprotein; AUC: area under the ROC curve; CBC: complete blood count; CD62L: L-selectin; CD74/Ii: MHC class II invariant chain; CD44/HCAM: homing cell adhesion molecule; cMos: classical monocytes; CRP: C-reactive protein; CXCR2: C-X-C motif chemokine receptor 2; CXCR4: C-X-C motif chemokine receptor 4; ESR: erythrocyte sedimentation rate; HCs: healthy controls; HA: hyaluronan; HLA: human leukocyte antigen; Ig: immunoglobulin; iMos: intermediate monocytes; LAYN: layilin; MFI: median fluorescence intensity; MIF: migration inhibitory factor; ncMos: nonclassical monocytes; PBMCs: peripheral blood mononuclear cells; ROC: receiver operating characteristic curve; SLE: systemic lupus erythematosus; SLEDAI, SLE disease activity index; Treg: regulatory T cells; WBCs: white blood cells.
Topics: Humans; Monocytes; Leukocytes, Mononuclear; Lupus Erythematosus, Systemic; Complement C4; Antibodies, Antinuclear; Receptors, Chemokine; Lectins, C-Type
PubMed: 37642473
DOI: 10.1080/08820139.2023.2249531 -
PloS One 2022Vitamin D deficiency is common among postmenopausal women. Telomere length can be a potential protective mechanism for age-related diseases. The objective of our study... (Randomized Controlled Trial)
Randomized Controlled Trial
Vitamin D deficiency is common among postmenopausal women. Telomere length can be a potential protective mechanism for age-related diseases. The objective of our study is to examine the association of vitamin D supplementation on leukocyte telomere length (LTL) in healthy postmenopausal women with vitamin D deficiency. The study was designed as a placebo-controlled study to investigate the short-term effects of vitamin D supplementation and seasonal changes on vitamin D related parameters, including 25(OH)D, 1,25(OH)2D parathormone (PTH), Vitamin D binding protein (VDBP), vitamin D receptor (VDR), and telomere length in a cohort of postmenopausal women (n = 102). The group was divided as supplementation (n = 52) and placebo groups (n = 50). All parameters were measured before and after treatment. Serum VDBP levels were measured by ELISA method and VDR, GC (VDBP) gene expressions and relative telomere lengths were measured in peripheral blood mononuclear cells (PBMC) using a quantitative real-time PCR method. The results demonstrate that baseline levels were similar between the groups. After vitamin D supplementation 25(OH)D, 1,25(OH)2D, PTH and VDBP levels were changed significantly compared to the placebo group. At the end of the study period, LTL levels were significantly increased in both groups and this change was more prominent in placebo group. The change in GC expression was significant between treatment and placebo groups but VDR expression remained unchanged. Even though the study was designed to solely assess the effects of vitamin D supplementation, LTL was significantly increased in the whole study group in summer months suggesting that LTL levels are affected by sun exposure and seasonal changes rather than supplementation. The study displayed the short-term effect of Vitamin D supplementation on vitamin D, PTH levels, LTL and vitamin D associated gene expressions. The relation between Vitamin D and LTL is not linear and could be confounded by several factors such as the population differences, regional and seasonal changes in sun exposure.
Topics: Aged; Cohort Studies; Female; Humans; Leukocytes, Mononuclear; Middle Aged; Postmenopause; Receptors, Calcitriol; Telomere; Telomere Homeostasis; Transcriptome; Vitamin D; Vitamin D Deficiency
PubMed: 35202418
DOI: 10.1371/journal.pone.0264337