Assessment of glutathione/glutathione disulphide ratio and S-glutathionylated proteins in human blood, solid tissues, and cultured cells

Abstract We previously demonstrated that Notch ligand Delta-1 in concert with GM-CSF and TGF-β1 promotes the differentiation of human blood monocytes into Langerhans cells that are characterized by the expression of CD1a, Langerhans-associated granules Langerin, cutaneous lymphocyte-associated antigen (CLA), CC chemokine receptor 6 (CCR6), and E-cadherin. These data extended the functional scope of Notch ligand Delta-1 in human adult hematopoiesis. HES-1 is known to be the target gene by Notch signaling. We examined the effect of Delta-1 on the expression of HES-1 mRNA in CD14+ blood monocytes in the presence of GM-CSF and TGF-β1, using real-time RT-PCR. When CD14+ blood monocytes were cultured with Delta-1, GM-CSF, and TGF-β1, the expression level of HES-1 mRNA increased approximately 10-fold at 24 hours of incubation, compared with the expression level in freshly isolated CD14+ monocytes. However, the expression level of HES-1 mRNA declined at 48 hours of incubation. This finding suggests that Delta-1 may operate at the early stage of the differentiation pathway from CD14+ monocytes to Langerhans cells. To explore this issue more precisely, we cultured CD14+ monocytes in the presence of Delta-1, GM-CSF, and TGF-β1 for 2 days, and subsequently replated the cells into the cultures without Delta-1. At day 7 of culture, cultured cells were harvested and characterized by phenotypic analysis. This initial 2-day exposure of CD14+ monocytes to Delta-1 gave rise to Langerhans cells, similar to the observation obtained with the supplementation of Delta-1 throughout 7-day culture. In turn, when Delta-1 was added at day 2 of culture, Langerhans cells were not induced, but instead the resulting cells exhibited the features of macrophages. Our results indicate that in response to Delta-1, human blood monocytes appear to initiate the differentiation program toward Langerhans cells, while they are incapable of differentiating into Langerhans cells after their commitment to macrophages.

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An Indispensable Role of GM-CSF in the Differentiation of Human Blood Monocytes toward Langerhans Cells.

Blood ◽

10.1182/blood.v106.11.3868.3868 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3868-3868

Author(s):

Tetsunori Shibasaki ◽

Naoyuki Katayama ◽

Kohshi Ohishi ◽

Fumihiko Monma ◽

Zhaocai Yu ◽

...

Keyword(s):

Human Blood ◽

Langerhans Cells ◽

Cultured Cells ◽

Expression Profiles ◽

Blood Monocytes ◽

Similar Action ◽

Gm Csf ◽

Tgf Β1 ◽

Cells Cultured ◽

E Cadherin

Abstract We recently demonstrated that GM-CSF, TGFβ1, and Notch ligand Delta-1, all of which are synthesized in the milieu of the skin, instruct human blood monocytes to differentiate into Langerhans cells (LCs) that are characterized by the expression of CD1a, Langerin, CLA, CCR6, and E-cadherin and the presence of Birbeck granules. Previous studies have shown similar biologic activities of GM-CSF and IL-3 on human blood monocytes. In the present study, we investigated whether GM-CSF and IL-3 have a similar action on blood monocytes with respect to their differentiation into LCs. In contrast to CD14+ monocytes cultured for 7 days in the presence of GM-CSF, TGF-β1, and Delta-1, the cells obtained from culture with IL-3, TGF-β1, and Delta-1 were negative for CLA, CCR6, Langerin, and E-cadherin. These cells expressed HLA-ABC, HLA-DR, CD80, CD86, CD40, CD54, and CD11c but not DC-SIGN. The expression level of CD1a declined, while CD14 was upregulated. The difference between cells cultured with GM-CSF, TGF-β1, and Delta-1 and those with IL-3, TGF-β1, and Delta-1 was corroborated by microarray analysis of the gene expression profiles. Thus, GM-CSF and IL-3 exert distinct effects on human blood monocytes in the presence of TGF-β1 and Delta-1. When GM-CSF was added to cultures containing IL-3, TGF-β1, and Delta-1, the phenotype of cultured cells closely resembled that observed with GM-CSF, TGF-β1, and Delta-1. As GM-CSF and IL-3 share common receptor β subunits, the signals mediated through GM-CSF receptor α subunits appear to be required for the development of LCs from monocytes. We also compared the action of GM-CSF with that of IL-3 in terms of the differentiation of monocytes into macrophages and dendritic cells (DCs). When CD14+ monocytes were cultured in the presence of GM-CSF or IL-3 for 7 days, the resulting macrophages obtained from both cultures were phenotypically indistinguishable. Next, we examined whether IL-3 as well as GM-CSF induces CD14+ monocytes to differentiate into DCs in the presence of IL-4. The phenotypic profiles in cells cultured with IL-3 plus IL-4 were parallel to those seen with DCs cultured in the presence of GM-CSF plus IL-4. These data suggest that although IL-3 can substitute for GM-CSF in the differentiation pathway of CD14+ monocytes toward macrophages or DCs, GM-CSF is indispensable for their LC development.

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Activation-dependent surface expression of gC1qR/p33 on human blood platelets

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613450 ◽

2003 ◽

Vol 89 (02) ◽

pp. 331-339 ◽

Cited By ~ 37

Author(s):

Tara Murphy ◽

Berhane Ghebrehiwet ◽

Ellinor Peerschke

Keyword(s):

Amino Acids ◽

Platelet Activation ◽

Human Blood ◽

Cultured Cells ◽

Blood Platelets ◽

Surface Membrane ◽

Protein A ◽

Activated Platelets ◽

Terminal Amino ◽

Human Blood Platelets

SummaryGC1qR/p33 (gC1qR) is expressed by a variety of somatic and cultured cells, including blood platelets. It interacts with several cellular, viral, bacterial, and plasma proteins, suggesting a potential role in thrombosis, inflammation, and infection. Considerable controversy has surrounded the surface membrane localization of gC1qR, however, since its cDNA sequence does not predict a traditional membrane-anchoring domain, and bears a typical mitochondrial targeting sequence. The present study examined gC1qR expression on resting and activated human blood platelets using flow cytometry and confocal microscopy with two monoclonal antibodies, 74.5.2 and 60.11, directed against gC1qR C-terminal amino acids 204-218, and N-terminal amino acids 76-93, respectively. Unstimulated platelets reacted minimally with either antibody. In contrast, platelet activation with TRAP, epinephrine, or ADP produced markedly increased gC1qR expression as reflected by 74.5.2 binding but not 60.11 binding. Platelet activation was verified using PAC-1 and anti CD 62 antibodies. Whereas PAC-1 binding to activated platelets could be reversed following platelet incubation with PGE1, 74.5.2 binding remained unchanged, suggesting the sustained expression of gC1qR following platelet stimulation. The data further demonstrate that detection of cell surface gC1qR may be dependent on antibody specificity. The ability of gC1qR to bind proteins involved in complement, coagulation, and kinin systems, as well as viral and bacterial pathogens including S. aureus protein A, supports the hypothesis that gC1qR expressed on activated platelets may contribute directly to thrombosis, inflammation, and endovascular infections.

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Atherogenic modification of low-density lipoproteins

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20166204391 ◽

2016 ◽

Vol 62 (4) ◽

pp. 391-402 ◽

Cited By ~ 9

Author(s):

V.N. Sukhorukov ◽

V.P. Karagodin ◽

A.N. Orekhov

Keyword(s):

Blood Plasma ◽

Human Blood ◽

Lipid Accumulation ◽

Cultured Cells ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Human Blood Plasma ◽

Low Density ◽

Modified Ldl ◽

Intracellular Lipid Accumulation

One of the first manifestations of atherosclerosis is accumulation of extra- and intracellular cholesterol esters in the arterial intima. Formation of foam cells is considered as a trigger in the pathogenesis of atherosclerosis. Low density lipoprotein (LDL) circulating in human blood is the source of lipids accumulated in the arterial walls. This review considered features and role in atherogenesis different modified forms of LDL: oxidized, small dense, electronegative and especially desialylated LDL. Desialylated LDL of human blood plasma is capable to induce lipid accumulation in cultured cells and it is atherogenic. LDL possesses numerous alterations of protein, carbohydrate and lipid moieties and therefore can be termed multiple-modified LDL. Multiple modification of LDL occurs in human blood plasma and represents a cascade of successive changes in the lipoprotein particle: desialylation, loss of lipids, reduction in the particle size, increase of surface electronegative charge, etc. In addition to intracellular lipid accumulation, stimulatory effects of naturally occurring multiple-modified LDL on other processes involved in the development of atherosclerotic lesions, namely cell proliferation and fibrosis, were shown.

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Tuning the properties of pH responsive nanoparticles to control cellular interactions in vitro and ex vivo

Polymer Chemistry ◽

10.1039/c6py01332e ◽

2016 ◽

Vol 7 (38) ◽

pp. 6015-6024 ◽

Cited By ~ 13

Author(s):

S. K. Mann ◽

A. Dufour ◽

J. J. Glass ◽

R. De Rose ◽

S. J. Kent ◽

...

Keyword(s):

Drug Delivery ◽

Human Blood ◽

Drug Delivery Systems ◽

Cultured Cells ◽

Ex Vivo ◽

Delivery Systems ◽

Effective Drug ◽

Cellular Interactions ◽

Ph Responsive

Engineering the properties of nanoparticles to limit non-specific cellular interactions is critical for developing effective drug delivery systems. Differences between interactions with cultured cells and human blood highlights the need for appropriate assays.

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In Vitro Interaction between Cultured Cells and Human Blood Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648638 ◽

1978 ◽

Vol 40 (01) ◽

pp. 089-102 ◽

Cited By ~ 13

Author(s):

Randi Holme ◽

Reidar Oftebro ◽

Torstein Hovig

Keyword(s):

Platelet Aggregation ◽

Human Blood ◽

Cultured Cells ◽

Blood Platelets ◽

Tumour Cells ◽

Coagulation Activity ◽

Induce Platelet Aggregation ◽

Human Blood Platelets ◽

Chang Cells

SummaryThe effects of washed human cultured cells (tumour cells and Chang liver cells) on human blood platelets in heparinized plasma were studied. Platelet aggregation was induced by suspensions of the tumour cells. Ultrastructural examination showed that the platelets, especially in the central regions of the aggregates, were tightly packed and the α-granules were mostly present. In the periphery of the aggregates the platelets appeared swollen and devoid of organelles, and fibrin strands were seen. The platelet aggregation was not completely abolished by incubation with apyrase. The washing fluids from the tumour cells also induced platelet aggregation, but the aggregation could be abolished by incubation with apyrase.When three different lines of the Chang cells were used, suspensions of two lines of these cells induced platelet aggregation, but the third line did not. Presence of ADP could be demonstrated in the washing fluids from the cultured cells, except for the one line of Chang cells which did not induce platelet aggregation. The experiments indicated that the platelet aggregation induced by the various types of cells was mediated via ADP, but with a possible additional effect of coagulation activity.

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Microglia-like Cells Derived from Hematopoietic Stem and Progenitor Cells Are a Model System to Investigate Chronic Pain in Sickle Cell Disease

Blood ◽

10.1182/blood-2018-99-116936 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1071-1071

Author(s):

Yankai Zhang ◽

Samir K. Ballas ◽

Vivien A Sheehan

Keyword(s):

Chronic Pain ◽

Human Blood ◽

Peripheral Blood ◽

Blood Donors ◽

Cultured Cells ◽

Normal Blood ◽

Hematopoietic Stem ◽

Gm Csf ◽

Normal Human ◽

Normal Human Blood

Abstract Background: Patients with sickle cell disease (SCD) experience acute pain episodes in early childhood; over 50% of adult SCD patients go on to develop chronic pain. In animal models, microglia, derived from hematopoietic stem cells, play an important role in the transition from acute to chronic pain; in patients with chronic pain microglia are often activated with minimal stimuli, releasing TNF-alpha and stimulating neurons. Research involving microglia has been stymied by difficulty in obtaining and culturing microglia, particularly from humans. We therefore propose to culture microglia-like cells from peripheral blood mononuclear cells (PBMC) from pediatric patients with SCD with and without chronic pain (defined as pain for more than 50% of days for at least 3 months) and from normal blood donors. Objectives: Our goal was to determine if our cultured cells exhibited microglia morphology and surface markers, could be activated with inflammatory stimuli, and if donor characteristics were retained in the culture derived microglia-like cells. Methods: PBMC were obtained from peripheral blood by Ficoll separation from three unique patients with SCD and no history of chronic pain (SCD CP-), peripheral blood from three unique patients with SCD with history of chronic pain (SCD CP+), or buffy coat from three human blood donors. PBMC were cultured with recombinant human GM-CSF (10 ng/ml; R&D Systems, MN), recombinant human IL-34 (100 ng/ml; R&D Systems), as previously described (Ohgidani et al, 2014) to induce ramified microglia. On day 7 of culture, cells were collected and morphology analyzed by phase contrast microscopy, phenotyped by flow cytometry by staining with CD11b and CD45 antibodies, indirect immunofluorescence performed with anti-CX3CR1antibodies and imaging by confocal laser scanning microscope. Microglia-like cells from 3 normal blood donors, 3 SCD CP- and 3 SCD CP+, were stimulated by 100 ng/mL LPS for 24 hours, beginning on day 6 of culture. Deramification (activation) was assessed by immunofluorescence using anti-Iba1, and by flow using anti-CD68. Results: PBMCs from patients with SCD ± CP and normal blood donors developed microglial morphology, namely, soma bodies with branched collaterals, after incubation with GM-CSF and IL-34. When subjected to flow cytometry, treated cells were CD11bhigh and CD45low; fluorescence microscopy revealed CX3CR1 positivity in the cultured cells, all consistent with accepted microglia phenotype. PBMCs untreated with GM-CSF and IL-34 maintained their round morphology, and were CD11blow, CD45high by FACS. Microglia-like morphology differed significantly between PBMC sources. Microglia-like cells from SCD CP+ had shorter and fewer branches than normal individuals; branching of microglia-like cells from SCD CP- were intermediate in number and length, greater than to SCD CP+ but less than normal individuals. When treated with LPS, nearly 100% of microglia-like cells derived from patients with SCD CP+ became amoeboid in shape, indicating microglial deramification, or activation, compared to approximately 25% of microglial-like cells derived from normal human blood donors or SCD CP-. We also assessed deramification through flow measurement of CD68, a marker found on activated microglia and found that the microglia-like cells derived from HSPCs from SCD CP+ patients had significantly higher CD68 positivity compared to microglia-like cells derived from HSPCs from normal human blood donors or SCD CP-, and significantly higher Iba1 positivity as measured by immunofluorescence. Conclusions: We established the microglia-like nature of the cultured PBMCs derived from patients with SCD±CP and normal blood donors through morphologic examination and confirmation of characteristic surface markers via flow and fluorescence microscopy, and through demonstration of activation with LPS. Our microglial-like cells derived from patients with SCD CP+ were activated at higher percentages by LPs treatment than microglial-like cells derived from normal human blood donors and SCD CP-, suggesting that donor characteristics are retained by the microglial-like cells developed in culture. We propose to use this model system to derive mechanistic insights into the development of chronic pain in SCD, and to screen pharmacologic agents to treat and prevent chronic pain. Figure Figure. Disclosures No relevant conflicts of interest to declare.

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Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100053711 ◽

1982 ◽

Vol 40 ◽

pp. 210-211

Author(s):

M.J. Murphy ◽

R.R. Price ◽

J.C. Sloman

Keyword(s):

Cultured Cells ◽

Human Tumor ◽

Cell Type ◽

Tumor Mass ◽

Initial Cell ◽

Cloning Assay ◽

Human Tumor Cloning ◽

The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

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Electron Probe Microanalysis of Frozen Hydrated Kidneys, Isolated Cells, and Cultured Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100054595 ◽

1982 ◽

Vol 40 ◽

pp. 402-403 ◽

Cited By ~ 1

Author(s):

Claude Lechene

Keyword(s):

Liquid Nitrogen ◽

Electron Probe Microanalysis ◽

Electron Probe ◽

Cultured Cells ◽

Liquid Nitrogen Temperature ◽

Elemental Content ◽

Isolated Cells ◽

Renal Tubular Cells ◽

Diamond Saw ◽

Saw Blade

Electron probe microanalysis of frozen hydrated kidneysThe goal of the method is to measure on the same preparation the chemical elemental content of the renal luminal tubular fluid and of the surrounding renal tubular cells. The following method has been developed. Rat kidneys are quenched in solid nitrogen. They are trimmed under liquid nitrogen and mounted in a copper holder using a conductive medium. Under liquid nitrogen, a flat surface is exposed by sawing with a diamond saw blade at constant speed and constant pressure using a custom-built cryosaw. Transfer into the electron probe column (Cameca, MBX) is made using a simple transfer device maintaining the sample under liquid nitrogen in an interlock chamber mounted on the electron probe column. After the liquid nitrogen is evaporated by creating a vacuum, the sample is pushed into the special stage of the instrument. The sample is maintained at close to liquid nitrogen temperature by circulation of liquid nitrogen in the special stage.

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The Infection of Cells by Bullet-Shaped Viruses

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100063779 ◽

1969 ◽

Vol 27 ◽

pp. 372-373

Author(s):

Frederick A. Murphy ◽

Alyne K. Harrison ◽

Sylvia G. Whitfield

Keyword(s):

Electron Microscopy ◽

Physical Properties ◽

Host Cell ◽

Thin Section ◽

Cultured Cells ◽

Cell Infection ◽

Thin Section Electron Microscopy ◽

Section Electron ◽

Section Electron Microscopy

The bullet-shaped viruses are currently classified together on the basis of similarities in virion morphology and physical properties. Biologically and ecologically the member viruses are extremely diverse. In searching for further bases for making comparisons of these agents, the nature of host cell infection, both in vivo and in cultured cells, has been explored by thin-section electron microscopy.

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