scholarly journals FACS-Based Proteomics Enables Profiling of Proteins in Rare Cell Populations

2020 ◽  
Vol 21 (18) ◽  
pp. 6557
Author(s):  
Evelyne Maes ◽  
Nathalie Cools ◽  
Hanny Willems ◽  
Geert Baggerman
Keyword(s):  
Proteomic Analysis ◽  
Mononuclear Cells ◽  
Protein Extraction ◽  
Cell Types ◽  
Clinical Samples ◽  
Cell Populations ◽  
Peripheral Blood Mononuclear ◽  
Limited Protein ◽  
Instrument Sensitivity ◽  
Acquisition Method

Understanding disease pathology often does not require an overall proteomic analysis of clinical samples but rather the analysis of different, often rare, subpopulations of cells in a heterogeneous mixture of cell types. For the isolation of pre-specified cellular subtypes, fluorescence activated cell sorting (FACS) is commonly used for its ability to isolate the required cell populations with high purity, even of scarce cell types. The proteomic analysis of a limited number of FACS-sorted cells, however, is very challenging as both sample preparation inefficiencies and limits in terms of instrument sensitivity are present. In this study, we used CD14+CD15+ immune cells sorted out of peripheral blood mononuclear cells isolated from whole blood to improve and evaluate FACS-based proteomics. To optimize both the protein extraction protocol and the mass spectrometry (MS) data acquisition method, PBMCs as well as commercialized HeLa digest were used. To reflect the limited number of sorted cells in some clinical samples, different numbers of sorted cells (1000, 5000, 10,000, or 50,000) were used. This allowed comparing protein profiles across samples with limited protein material and provided further insights in the benefits and limitations of using a very limited numbers of cells.

scholarly journals Made to Measure: Patient-Tailored Treatment of Multiple Sclerosis Using Cell-Based Therapies

2021 ◽  
Vol 22 (14) ◽  
pp. 7536
Author(s):  
Inez Wens ◽  
Ibo Janssens ◽  
Judith Derdelinckx ◽  
Megha Meena ◽  
Barbara Willekens ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Autoimmune Diseases ◽  
Stromal Cells ◽  
Mononuclear Cells ◽  
Treatment Options ◽  
Cell Types ◽  
Peripheral Blood Mononuclear ◽  
Tailored Treatment ◽  
Key Issues ◽  
The Central Nervous System

Currently, there is still no cure for multiple sclerosis (MS), which is an autoimmune and neurodegenerative disease of the central nervous system. Treatment options predominantly consist of drugs that affect adaptive immunity and lead to a reduction of the inflammatory disease activity. A broad range of possible cell-based therapeutic options are being explored in the treatment of autoimmune diseases, including MS. This review aims to provide an overview of recent and future advances in the development of cell-based treatment options for the induction of tolerance in MS. Here, we will focus on haematopoietic stem cells, mesenchymal stromal cells, regulatory T cells and dendritic cells. We will also focus on less familiar cell types that are used in cell therapy, including B cells, natural killer cells and peripheral blood mononuclear cells. We will address key issues regarding the depicted therapies and highlight the major challenges that lie ahead to successfully reverse autoimmune diseases, such as MS, while minimising the side effects. Although cell-based therapies are well known and used in the treatment of several cancers, cell-based treatment options hold promise for the future treatment of autoimmune diseases in general, and MS in particular.

scholarly journals Selection following isolation of human immunodeficiency virus type 1 in peripheral blood mononuclear cells and herpesvirus saimiri-transformed T cells is comparable

2002 ◽  
Vol 83 (6) ◽  
pp. 1343-1352 ◽  
Author(s):  
Natalie N. Zheng ◽  
Cherelyn Vella ◽  
Philippa J. Easterbrook ◽  
Rod S. Daniels
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Mononuclear Cells ◽  
Cell Types ◽  
Herpesvirus Saimiri ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Blood Mononuclear Cells ◽  
Hiv 1

In attempts to improve isolation rates and virus yields for human immunodeficiency virus (HIV), the use of herpesvirus saimiri-immortalized T cells (HVS T cells) has been investigated as an alternative to/improvement over peripheral blood mononuclear cells (PBMCs). Here we characterize isolates rescued, in the two cell types, from two asymptomatic, long-term non-progressing HIV-1-infected individuals. All rescued viruses replicated in PBMCs and HVS T cells only, displaying a non-syncytium inducing (NSI) phenotype, and using CCR5 as co-receptor. Furthemore, PBMC/HVS T cell virus pairs displayed similar neutralization profiles. Full-length, expression-competent env genes were rescued from all virus isolates and directly from the patient samples using proviral DNA and viral RNA as templates. Compared with the sequences retrieved directly from the patient samples, both cell types showed similar selection characteristics. Whilst the selections were distinct for individual patient samples, they shared a common characteristic in selecting for viruses with increased negative charge across the V2 domain of the viral glycoproteins. The latter was observed at the env gene sequencing level for three other patients whose HIV strains were isolated in PBMCs only. This further supports a common selection for viral sequences that display a macrophage-tropic/NSI phenotype and shows that HVS T cells are a viable alternative to PBMCs for HIV-1 isolation.

scholarly journals Molecular mechanisms governing circulating immune cell heterogeneity across different species revealed by single cell sequencing

2021 ◽  
Author(s):  
Zhibin Li ◽  
chengcheng Sun ◽  
Fei Wang ◽  
Xiran Wang ◽  
Jiacheng Zhu ◽  
...  
Keyword(s):  
Single Cell ◽  
Immune Cells ◽  
Evolutionary Biology ◽  
Regulatory Networks ◽  
Molecular Mechanisms ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Cell Types ◽  
Genetic Regulatory Networks ◽  
Peripheral Blood Mononuclear

Background: Immune cells play important roles in mediating immune response and host defense against invading pathogens. However, insights into the molecular mechanisms governing circulating immune cell diversity among multiple species are limited. Methods: In this study, we compared the single-cell transcriptomes of 77 957 immune cells from 12 species using single-cell RNA-sequencing (scRNA-seq). Distinct molecular profiles were characterized for different immune cell types, including T cells, B cells, natural killer cells, monocytes, and dendritic cells. Results: The results revealed the heterogeneity and compositions of circulating immune cells among 12 different species. Additionally, we explored the conserved and divergent cellular cross-talks and genetic regulatory networks among vertebrate immune cells. Notably, the ligand and receptor pair VIM-CD44 was highly conserved among the immune cells. Conclusions: This study is the first to provide a comprehensive analysis of the cross-species single-cell atlas for peripheral blood mononuclear cells (PBMCs). This research should advance our understanding of the cellular taxonomy and fundamental functions of PBMCs, with important implications in evolutionary biology, developmental biology, and immune system disorders

scholarly journals CD4-Negative Cells Bind Human Immunodeficiency Virus Type 1 and Efficiently Transfer Virus to T Cells

2000 ◽  
Vol 74 (18) ◽  
pp. 8550-8557 ◽  
Author(s):  
Gene G. Olinger ◽  
Mohammed Saifuddin ◽  
Gregory T. Spear
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Mononuclear Cells ◽  
Cell Types ◽  
Virus Binding ◽  
Peripheral Blood Mononuclear ◽  
Human Immunodeficiency Virus Strain ◽  
Immunodeficiency Virus

ABSTRACT The ability of human immunodeficiency virus strain MN (HIVMN), a T-cell line-adapted strain of HIV, and X4 and R5 primary isolates to bind to various cell types was investigated. In general, HIVMN bound to cells at higher levels than did the primary isolates. Virus bound to both CD4-positive (CD4+) and CD4-negative (CD4−) cells, including neutrophils, Raji cells, tonsil mononuclear cells, erythrocytes, platelets, and peripheral blood mononuclear cells (PBMC), although virus bound at significantly higher levels to PBMC. However, there was no difference in the amount of HIV that bound to CD4-enriched or CD4-depleted PBMC. Virus bound to CD4− cells was up to 17 times more infectious for T cells in cocultures than was the same amount of cell-free virus. Virus bound to nucleated cells was significantly more infectious than virus bound to erythrocytes or platelets. The enhanced infection of T cells by virus bound to CD4− cells was not due to stimulatory signals provided by CD4− cells or infection of CD4− cells. However, anti-CD18 antibody substantially reduced the enhanced virus replication in T cells, suggesting that virus that bound to the surface of CD4−cells is efficiently passed to CD4+ T cells during cell-cell adhesion. These studies show that HIV binds at relatively high levels to CD4− cells and, once bound, is highly infectious for T cells. This suggests that virus binding to the surface of CD4− cells is an important route for infection of T cells in vivo.

scholarly journals Cells Derived from Human Long Bone Appear More Differentiated and More Actively Stimulate Osteoclastogenesis Compared to Alveolar Bone-Derived Cells

2020 ◽  
Vol 21 (14) ◽  
pp. 5072
Author(s):  
Cindy Kelder ◽  
Cornelis J. Kleverlaan ◽  
Marjolijn Gilijamse ◽  
Astrid D. Bakker ◽  
Teun J. de Vries
Keyword(s):  
Tissue Engineering ◽  
Mononuclear Cells ◽  
Alveolar Bone ◽  
Bone Cells ◽  
Transmembrane Protein ◽  
Cell Types ◽  
Long Bone ◽  
Long Bones ◽  
Peripheral Blood Mononuclear ◽  
Alveolar Bone Cells

Osteoblasts derived from mouse skulls have increased osteoclastogenic potential compared to long bone osteoblasts when stimulated with 1,25(OH)2 vitamin D3 (vitD3). This indicates that bone cells from specific sites can react differently to biochemical signals, e.g., during inflammation or as emitted by bioactive bone tissue-engineering constructs. Given the high turn-over of alveolar bone, we hypothesized that human alveolar bone-derived osteoblasts have an increased osteogenic and osteoclastogenic potential compared to the osteoblasts derived from long bone. The osteogenic and osteoclastogenic capacity of alveolar bone cells and long bone cells were assessed in the presence and absence of osteotropic agent vitD3. Both cell types were studied in osteogenesis experiments, using an osteogenic medium, and in osteoclastogenesis experiments by co-culturing osteoblasts with peripheral blood mononuclear cells (PBMCs). Both osteogenic and osteoclastic markers were measured. At day 0, long bones seem to have a more late-osteoblastic/preosteocyte-like phenotype compared to the alveolar bone cells as shown by slower proliferation, the higher expression of the matrix molecule Osteopontin (OPN) and the osteocyte-enriched cytoskeletal component Actin alpha 1 (ACTA1). This phenotype was maintained during the osteogenesis assays, where long bone-derived cells still expressed more OPN and ACTA1. Under co-culture conditions with PBMCs, long bone cells also had a higher Tumor necrose factor-alfa (TNF-α) expression and induced the formation of osteoclasts more than alveolar bone cells. Correspondingly, the expression of osteoclast genes dendritic cell specific transmembrane protein (DC-STAMP) and Receptor activator of nuclear factor kappa-Β ligand (RankL) was higher in long bone co-cultures. Together, our results indicate that long bone-derived osteoblasts are more active in bone-remodeling processes, especially in osteoclastogenesis, than alveolar bone-derived cells. This indicates that tissue-engineering solutions need to be specifically designed for the site of application, such as defects in long bones vs. the regeneration of alveolar bone after severe periodontitis.

scholarly journals Decreased PERP Expression on Peripheral Blood Mononuclear Cells from Patient with Rheumatoid Arthritis Negatively Correlates with Disease Activity

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Yanchun Du ◽  
Lin Deng ◽  
Yan Li ◽  
Lu Gan ◽  
Yantang Wang ◽  
...  
Keyword(s):  
Disease Activity ◽  
Mrna Expression ◽  
Mononuclear Cells ◽  
Cell Types ◽  
Healthy Controls ◽  
Peripheral Blood Mononuclear ◽  
Expression Levels ◽  
Cell Type Specific ◽  
Mrna Expression Levels

Background. PERP, p53 apoptosis effector related to PMP-22, is a p53-dependent apoptosis in diverse cell types and has cell type-specific roles in p53-mediated apoptosis. However, its role in PBMCs of RA patients has remained largely unclear.Objectives. The aim of this study was to detect the expression levels of PERP on PBMCs of RA patients and healthy controls and analyze the role of PERP in the pathogenesis of RA.Methods. The mRNA expression levels of PERP and IL-17 were detected by real-time PCR in PBMCs from patients with RA (n=40) and healthy controls (n=40). The correlations of PERP expression levels to IL-17 transcripts and disease activity parameters were analyzed.Results. The PERP and IL-17 expression levels in the PBMCs were significantly decreased and increased in comparison of which in healthy controls. The mRNA expression levels of PERP in PBMCs from patients with RA were negatively correlated with IL-17 and disease activity parameters DAS28, RF, CRP, and ESR rather than Anti-CCP and ANA.Conclusions. These results demonstrated that PERP might be involved in the pathogenesis and a potential therapeutic target of RA by regulating the expression of IL-17.

Hydrogen Peroxide and Lipopolysaccharide Differentially Affect the Expression of MicroRNAs 10a, 33a, 21, 221 in Endothelial Cells Before and After Coculture With Monocytes

2017 ◽  
Vol 36 (2) ◽  
pp. 133-141 ◽  
Author(s):  
Atefe Ghamar Talepoor ◽  
Mehdi Kalani ◽  
Alamtaj Samsami Dahaghani ◽  
Mehrnoosh Doroudchi
Keyword(s):  
Endothelial Cells ◽  
Mononuclear Cells ◽  
Cell Types ◽  
Magnetic Bead ◽  
Complete Medium ◽  
Peripheral Blood Mononuclear ◽  
Factors Affecting ◽  
Before And After ◽  
Polymerase Chain ◽  
Cellular Stressors

Inflammation and oxidative stress are important risk factors affecting various cells in the formation of atherosclerosis. MicroRNAs (miRs) are regulators of inflammation and atherogenesis. The expressions of endothelial cell (EC)-specific miR-10a and miR-21 and monocyte-specific miR-33a and miR-221 were investigated using coculture of the ECs and monocytes upon exposure to H2O2 as an oxidative stressor, and endotoxin/lipopolysaccharide (LPS) as a microbial stressor. Human umbilical endothelial cells (HUVECs) and peripheral blood mononuclear cells (or monocytes) were cocultured in M199 complete medium and were incubated with LPS (20 ng/mL) or H2O2 (1%) for 8 hours at 37°C. The HUVECs and monocytes were then separated from the cellular mix using a magnetic bead negative selection technique. The relative expression of miRs was determined by real-time polymerase chain reaction. In both cell types, H2O2 induced miR10a ( P = 0.05) and LPS induced miR21 ( P = 0.0003) compared to the untreated controls. Coculture increased miR-10a and miR-21 expression in monocytes ( P = 0.0008 and <0.0001); however when cultured alone, HUVECs expressed higher levels of miR-10a and miR-21 ( P < 0.0001 and <0.0001). Coculture decreased the expression of miR-33a in monocytes ( P < 0.0001) while increasing miR221 in HUVECs and monocytes ( P < 0.0001 and <0.0001). The expression pattern of miRs in HUVECs and monocytes changes in the coculture compared to culturing alone in response to oxidative and microbial toxic compounds. Moreover, different cellular stressors induce different athero-miRs, which may affect the course of inflammation.

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