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Cytometry. Part a : the Journal of the... Nov 2019We developed this comprehensive 28-color flow cytometry panel with the aim to measure a variety of T cell effector functions in combination with T cell differentiation...
We developed this comprehensive 28-color flow cytometry panel with the aim to measure a variety of T cell effector functions in combination with T cell differentiation markers (CCR7, CD27, CD28, CD45RO, CD95) in γδ T cells and CD4 and CD8 αβ T cells (Table 1). The effector functions measured in this panel include activation and co-stimulatory molecules (CD69, CD137, and CD154), cytokines (IL-2, IL-13, IL-17A, IL-21, IL-22, TNF, and IFNγ), the chemokine IL-8, cytotoxic molecules (perforin and granzyme B), and the degranulation marker CD107a. In addition, Ki67 enables the identification and analysis of recently activated T cells. To characterize regulatory T cells (T ), we included CD25, CD39, and the canonical T transcription factor FoxP3. We developed and optimized this panel for cryopreserved human peripheral blood mononuclear cells (PBMC) and stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. However, we successfully tested other types of stimulation such as staphylococcus enterotoxin B (SEB) or a mix of immunodominant peptides (CEF peptide pool) from cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.
Topics: Antigens, CD; Antigens, Differentiation, T-Lymphocyte; CD28 Antigens; CD40 Ligand; Cytokines; Flow Cytometry; Forkhead Transcription Factors; Granzymes; Humans; Immunophenotyping; Interleukin-8; Ki-67 Antigen; Lectins, C-Type; Leukocyte Common Antigens; Leukocytes, Mononuclear; Lymphocyte Activation; Lysosomal-Associated Membrane Protein 1; Perforin; Receptors, CCR7; T-Lymphocytes; T-Lymphocytes, Regulatory; Tumor Necrosis Factor Receptor Superfamily, Member 7; Tumor Necrosis Factor Receptor Superfamily, Member 9; fas Receptor
PubMed: 31334913
DOI: 10.1002/cyto.a.23853 -
Journal of Hepatology Jan 2022Patients with acute-on-chronic liver failure (ACLF) present a systemic hyperinflammatory response associated with increased circulating levels of small-molecule...
BACKGROUND & AIMS
Patients with acute-on-chronic liver failure (ACLF) present a systemic hyperinflammatory response associated with increased circulating levels of small-molecule metabolites. To investigate whether these alterations reflect inadequate cell energy output, we assessed mitochondrial morphology and central metabolic pathways with emphasis on the tricarboxylic acid (TCA) cycle in peripheral leukocytes from patients with acutely decompensated (AD) cirrhosis, with and without ACLF.
METHODS
The study included samples from patients with AD cirrhosis (108 without and 128 with ACLF) and 41 healthy individuals. Leukocyte mitochondrial ultrastructure was visualized by transmission electron microscopy and cytosolic and mitochondrial metabolic fluxes were determined by assessing NADH/FADH production from various substrates. Plasma GDF15 and FGF21 were determined by Luminex and acylcarnitines by LC-MS/MS. Gene expression was analyzed by RNA-sequencing and PCR-based glucose metabolism profiler array.
RESULTS
Mitochondrial ultrastructure in patients with advanced cirrhosis was distinguished by cristae rarefication and swelling. The number of mitochondria per leukocyte was higher in patients, accompanied by a reduction in their size. Increased FGF21 and C6:0- and C8:0-carnitine predicted mortality whereas GDF15 strongly correlated with a gene set signature related to leukocyte activation. Metabolic flux analyses revealed increased energy production in mononuclear leukocytes from patients with preferential involvement of extra-mitochondrial pathways, supported by upregulated expression of genes encoding enzymes of the glycolytic and pentose phosphate pathways. In patients with ACLF, mitochondrial function analysis uncovered break-points in the TCA cycle at the isocitrate dehydrogenase and succinate dehydrogenase level, which were bridged by anaplerotic reactions involving glutaminolysis and nucleoside metabolism.
CONCLUSIONS
Our findings provide evidence at the cellular, organelle and biochemical levels that severe mitochondrial dysfunction governs immunometabolism in leukocytes from patients with AD cirrhosis and ACLF.
LAY SUMMARY
Patients at advanced stages of liver disease have dismal prognosis due to vital organ failures and the lack of treatment options. In this study, we report that the functioning of mitochondria, which are known as the cell powerhouse, is severely impaired in leukocytes of these patients, probably as a consequence of intense inflammation. Mitochondrial dysfunction is therefore a hallmark of advanced liver disease.
Topics: Acute-On-Chronic Liver Failure; Humans; Immunologic Factors; Leukocytes; Leukocytes, Mononuclear; Mitochondrial Diseases; Tandem Mass Spectrometry
PubMed: 34450236
DOI: 10.1016/j.jhep.2021.08.009 -
Nature Genetics Apr 2018Genome-wide association studies have identified thousands of genetic variants that are associated with disease . Most of these variants have small effect sizes, but...
Genome-wide association studies have identified thousands of genetic variants that are associated with disease . Most of these variants have small effect sizes, but their downstream expression effects, so-called expression quantitative trait loci (eQTLs), are often large and celltype-specific. To identify these celltype-specific eQTLs using an unbiased approach, we used single-cell RNA sequencing to generate expression profiles of ~25,000 peripheral blood mononuclear cells from 45 donors. We identified previously reported cis-eQTLs, but also identified new celltype-specific cis-eQTLs. Finally, we generated personalized co-expression networks and identified genetic variants that significantly alter co-expression relationships (which we termed 'co-expression QTLs'). Single-cell eQTL analysis thus allows for the identification of genetic variants that impact regulatory networks.
Topics: Epistasis, Genetic; Gene Regulatory Networks; Genetic Variation; Genome-Wide Association Study; Humans; Leukocytes, Mononuclear; Quantitative Trait Loci; Sequence Analysis, RNA; Single-Cell Analysis; Transcriptome
PubMed: 29610479
DOI: 10.1038/s41588-018-0089-9 -
Cytometry. Part a : the Journal of the... Sep 2023This newly established 24-color (30-marker) panel focuses on the characterization of the main human immune cell subtypes and was optimized for the analysis of human...
This newly established 24-color (30-marker) panel focuses on the characterization of the main human immune cell subtypes and was optimized for the analysis of human whole blood using a full spectrum flow cytometer. The panel covers all main leukocyte populations: neutrophils, eosinophils and basophils, monocytes (with additional subsets), dendritic cells, innate lymphoid cells and lymphocytes. As for lymphocytes, this panel includes CD4+ T helper, Treg cells, and CD8+ cytotoxic T cells. Further T cells subsets are included with special focus on invariant T cells: γδ T cells (including δ2TCR variant), invariant NKT cells and MAIT (mucosal-associated invariant T cells) cells. Additionally, total B cells (including Bregs and plasmocytes), NK cells, and NKT cells are included. For the overall check of activation status of the analyzed immune cells we used HLA-DR, CD38, CD57, CD69, PD-1, and CD94. In addition, we used CD62L, CD45RA, CD27, and CD39 to describe the differentiation status of these cells. The panel was designed to maximize the information that can be obtained from surface markers in order to avoid the need for fixation and permeabilization steps. The presented multimarker panel offers the possibility to discover new immune cell subtypes which in patients and in cohort studies may lead to the identification of altered immune phenotypes and might give a link to immune system based or to certain other diseases. This panel was developed for a full spectrum flow cytometer equipped with a minimum of three lasers. We developed this panel using healthy human fresh blood, however it was also successfully used for staining of isolated human peripheral blood mononuclear cells (PBMC).
Topics: Humans; Immunophenotyping; Leukocytes, Mononuclear; Immunity, Innate; Leukocytes; Killer Cells, Natural; Flow Cytometry
PubMed: 37254600
DOI: 10.1002/cyto.a.24766 -
Arthritis Research & Therapy Aug 2019The in vitro pharmacology of baricitinib, upadacitinib, and tofacitinib was evaluated to understand differences among these JAK inhibitors (JAKis) at the cellular level. (Comparative Study)
Comparative Study
BACKGROUND
The in vitro pharmacology of baricitinib, upadacitinib, and tofacitinib was evaluated to understand differences among these JAK inhibitors (JAKis) at the cellular level.
METHODS
Peripheral blood mononuclear cells from healthy donors were incubated with different JAKis, levels of phosphorylated signal transducer and activator of transcription (pSTAT) were measured following cytokine stimulation, and half maximum inhibitory concentration (IC) values were calculated in phenotypically gated leukocyte subpopulations. Therapeutic dose relevance of the in vitro analysis was assessed using calculated mean concentration-time profiles over 24 h obtained from JAKi-treated subjects. Time above IC and average daily percent inhibition of pSTAT formation were calculated for each JAKi, cytokine, and cell type.
RESULTS
Distinct JAKis displayed different in vitro pharmacologic profiles. For example, tofacitinib and upadacitinib were the most potent inhibitors of the JAK1/3-dependent cytokines tested (interleukin [IL]-2, IL-4, IL-15, and IL-21) with lower IC values and increased time above IC translating to a greater overall inhibition of STAT signaling during the dosing interval. All JAKis tested inhibited JAK1/2-dependent cytokines (e.g., IL-6 and interferon [IFN]-γ), the JAK1/tyrosine kinase 2 (TYK2)-dependent cytokines IL-10 and IFN-α, the JAK2/2-dependent cytokines IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF), and the JAK2/TYK2-dependent cytokine granulocyte colony-stimulating factor (G-CSF), but often to significantly differing degrees.
CONCLUSIONS
Different JAKis modulated distinct cytokine pathways to varying degrees, and no agent potently or continuously inhibited an individual cytokine signaling pathway throughout the dosing interval. Notably, baricitinib inhibited JAK1/3 signaling to a lesser extent than upadacitinib and tofacitinib, while upadacitinib, baricitinib, and tofacitinib inhibited the signaling of JAK2/2-dependent cytokines, including GM-CSF and IL-3, as well as the signaling of the JAK2/TYK2-dependent cytokine G-CSF.
Topics: Arthritis, Rheumatoid; Azetidines; Biomarkers; Cytokines; Flow Cytometry; Heterocyclic Compounds, 3-Ring; Humans; Janus Kinase Inhibitors; Leukocytes, Mononuclear; Piperidines; Protein Kinase Inhibitors; Purines; Pyrazoles; Pyrimidines; Pyrroles; Signal Transduction; Sulfonamides
PubMed: 31375130
DOI: 10.1186/s13075-019-1964-1 -
Cytometry. Part a : the Journal of the... Jan 2024We have developed a 31-color panel to define the steady-state phenotype of T cells in human peripheral blood (Table 1). The panel presented here was optimized using...
We have developed a 31-color panel to define the steady-state phenotype of T cells in human peripheral blood (Table 1). The panel presented here was optimized using cryopreserved peripheral blood mononuclear cells (PBMC). The markers included in this panel were chosen in order to characterize the steady-state phenotype of T cells and includes markers (CD45RA, CD45RO, CCR7, CD95) to distinguish the main subsets (e.g., naïve, T , T , T , T etc.) of CD4, CD8, and γδ T cells. This panel also includes markers for the identification of differentiation status (CD27, CD28), activation/antigen experience status (CD11a, CD49d, CD38, HLA-DR, CD56, and CD39), co-inhibitory marker expression (PD-1, TIM-3), and CD4 T helper subsets (CXCR3, CXCR5, CCR4, CCR6, Foxp3, CD25, and CD127). This optimized panel provides a broad assessment of the steady-state phenotype of human T cells.
Topics: Humans; Leukocytes, Mononuclear; Flow Cytometry; T-Lymphocytes; Leukocyte Common Antigens; Phenotype; T-Lymphocyte Subsets
PubMed: 37814476
DOI: 10.1002/cyto.a.24799 -
Psychiatria Danubina Oct 2023Schizophrenia is a severe mental illness causing significant impairment in personal, family, social, educational, occupational, and other important areas of life. While... (Review)
Review
INTRODUCTION
Schizophrenia is a severe mental illness causing significant impairment in personal, family, social, educational, occupational, and other important areas of life. While there is no widely accepted endophenotype, peripheral blood cells may serve as an accessible model of intracellular changes in schizophrenia.
METHODS
We reviewed the literature on the query "peripheral blood mononuclear cells AND schizophrenia" in Medline (Pubmed), selecting studies that searched for specific biomarkers of schizophrenia. We considered both diagnostic biomarkers and biomarkers of therapeutic response, specific schizophrenia disorders or differential diagnostic biomarkers.
RESULTS
We retrieved 41 articles matching the search criteria, among which were studies that considered changes in the production of pro-inflammatory and anti-inflammatory markers, proteins, receptors, enzyme activity, and gene expression as potential biomarkers.
CONCLUSION
Approaches analysing a biological axis or a group of related biomarkers may hold the greatest promise for identifying schizophrenia. In addition, pharmacological status, smoking status, inflammatory markers and glucose metabolites, the presence of comorbidities should be considered. Certain biomarkers, while not specific for the diagnosis of schizophrenia, may indicate the prognosis and effectiveness of treatment in the established diagnosis.
Topics: Humans; Schizophrenia; Leukocytes, Mononuclear; Biomarkers; Endophenotypes; Prognosis
PubMed: 37800212
DOI: No ID Found -
International Journal of Molecular... Mar 2023Huntington's disease (HD) is characterized by clinical motor impairment (e.g., involuntary movements, poor coordination, parkinsonism), cognitive deficits, and... (Review)
Review
Huntington's disease (HD) is characterized by clinical motor impairment (e.g., involuntary movements, poor coordination, parkinsonism), cognitive deficits, and psychiatric symptoms. An inhered expansion of the CAG triplet in the huntingtin gene causing a pathogenic gain-of-function of the mutant huntingtin () protein has been identified. In this review, we focus on known biomarkers (e.g., mHTT, neurofilament light chains) and on new biofluid biomarkers that can be quantified in plasma or peripheral blood mononuclear cells from mHTT carriers. Circulating biomarkers may fill current unmet needs in HD management: better stratification of patients amenable to etiologic treatment; the initiation of preventive treatment in premanifest HD; and the identification of peripheral pathogenic central nervous system cascades.
Topics: Humans; Huntington Disease; Leukocytes, Mononuclear; Cognition Disorders; Biomarkers; Cognitive Dysfunction; Huntingtin Protein
PubMed: 37047023
DOI: 10.3390/ijms24076051 -
Frontiers in Immunology 2018Antibody-mediated rejection (AMR) of solid organ transplants is characterized by the activation and injury of the allograft endothelium. Histological and transcriptomic... (Review)
Review
Antibody-mediated rejection (AMR) of solid organ transplants is characterized by the activation and injury of the allograft endothelium. Histological and transcriptomic studies have associated microvascular inflammation and endothelial lesions with the severity of rejection and poor graft outcomes. The allograft endothelium forms the physical barrier between the donor organ and the recipient; this position directly exposes the endothelium to alloimmune responses. However, endothelial cells are not just victims and can actively participate in the pathogenesis of rejection. In healthy tissues, the endothelium plays a major role in vascular and immune homeostasis. Organ transplantation, however, subjects the endothelium to an environment of inflammation, alloreactive lymphocytes, donor-specific antibodies, and potentially complement activation. As a result, endothelial cells become activated and have modified interactions with the cellular effectors of allograft damage: lymphocytes, natural killer, and myeloid cells. Activated endothelial cells participate in leukocyte adhesion and recruitment, lymphocyte activation and differentiation, as well as the secretion of cytokines and chemokines. Ultimately, highly activated endothelial cells promote pro-inflammatory alloresponses and become accomplices to AMR.
Topics: Animals; Antibodies; Endothelial Cells; Endothelium, Vascular; Graft Rejection; Humans; Leukocytes, Mononuclear
PubMed: 29434607
DOI: 10.3389/fimmu.2018.00106 -
Hypertension (Dallas, Tex. : 1979) Jul 2020Endothelial inflammation and mitochondrial dysfunction have been implicated in cardiovascular diseases, yet, a unifying mechanism tying them together remains limited....
Endothelial inflammation and mitochondrial dysfunction have been implicated in cardiovascular diseases, yet, a unifying mechanism tying them together remains limited. Mitochondrial dysfunction is frequently associated with mitochondrial fission/fragmentation mediated by the GTPase Drp1 (dynamin-related protein 1). Nuclear factor (NF)-κB, a master regulator of inflammation, is implicated in endothelial dysfunction and resultant complications. Here, we explore a causal relationship between mitochondrial fission and NF-κB activation in endothelial inflammatory responses. In cultured endothelial cells, TNF-α (tumor necrosis factor-α) or lipopolysaccharide induces mitochondrial fragmentation. Inhibition of Drp1 activity or expression suppresses mitochondrial fission, NF-κB activation, vascular cell adhesion molecule-1 induction, and leukocyte adhesion induced by these proinflammatory factors. Moreover, attenuations of inflammatory leukocyte adhesion were observed in Drp1 heterodeficient mice as well as endothelial Drp1 silenced mice. Intriguingly, inhibition of the canonical NF-κB signaling suppresses endothelial mitochondrial fission. Mechanistically, NF-κB p65/RelA seems to mediate inflammatory mitochondrial fission in endothelial cells. In addition, the classical anti-inflammatory drug, salicylate, seems to maintain mitochondrial fission/fusion balance against TNF-α via inhibition of NF-κB. In conclusion, our results suggest a previously unknown mechanism whereby the canonical NF-κB cascade and a mitochondrial fission pathway interdependently regulate endothelial inflammation.
Topics: 3T3 Cells; Animals; Aorta; Cell Adhesion; Cells, Cultured; Dynamins; Endothelial Cells; Endothelium, Vascular; Leukocytes, Mononuclear; Membrane Proteins; Mice; Mitochondrial Dynamics; Mitochondrial Proteins; Mutation, Missense; NF-kappa B; Phosphorylation; Phosphoserine; Protein Processing, Post-Translational; Proteome; RNA Interference; RNA, Small Interfering; Rats; Sodium Salicylate; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1; Vasculitis
PubMed: 32389075
DOI: 10.1161/HYPERTENSIONAHA.120.14686