Direct stimulation of cytokines (IL-1β, TNF-α, IL-6, IL-2, IFN-γ and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation

Groote, D. De; Zangerle, P. F.; Gevaert, Y.; Fassotte, Marie-France; Béguin, Yves; Noizat‐Pirenne, F.; Pirenne, Jacques; Gathy, R.; López, Miguel Fraile; Dehart, I.; Igot, D.; Baudrihaye, M.; Delacroix, Dominique L.; Franchimont, P

doi:10.1016/1043-4666(92)90062-v

Cited by 451 publications

(232 citation statements)

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“…PBMC may be depleted of certain populations or activated during the separation procedure, whereas cloned T cells are likely to have been grown in medium containing cytokines and other reagents that may have influenced their cytokine expression phenotype [22]. Recently, it has been reported that higher levels of TNF-a and IFN-g are produced in whole blood cultures then in purified PBMC preparations [23]. Whole blood should reflect more accurately the circulating cells in vivo [24].…”

Section: Introductionmentioning

confidence: 99%

Intracellular cytokine expression in whole blood preparations from normals and patients with atopic dermatitis

Ferry

Antrobus

Hužička

et al. 1997

Clinical and Experimental Immunology

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SUMMARYIn recent years, the importance of characterizing the role of cytokines in a wide range of clinical conditions has resulted in development of new methods to assess cytokine expression in clinical samples. The use of anti-cytokine MoAbs and flow cytometry to detect cytokines intracellularly at the single-cell level has the potential to quantify cytokine production in different diseases. For this technique to be useful in a clinical setting, rapid throughput of clinical samples and a cheap, reliable assay would be required, therefore the development of the above technique using unseparated whole blood samples would be advantageous. Using this technique, only one study to date (Maino et al., 1996) has used unseparated whole blood as the source of cells for detecting intracellular cytokines. In clinical practice, whole blood may be optimal, since this most closely approximates conditions in vivo: as no purification of blood mononuclear cells is required, very little blood is needed to detect a number of cytokines simultaneously in various lymphocyte subpopulations, and the assay can be applied to samples from infants and children. In this study we describe an intracellular cytokine assay using unseparated whole blood from normals. In activated CD8 ¹ T cells, IL-2 and interferon-gamma (IFN-g) were optimally induced after 10 h stimulation with phorbol 12-myristate acetate (PMA)/ionomycin, and in CD8 þ T cells IL-2 was optimally induced after 10 h and IFN-g after 6 h. The levels of IL-2 and IFN-g in CD8 þ and CD8 ¹ T cells in four healthy individuals were consistent on four occasions over a 3-month period. In a large group of 34 normal subjects, there was considerable heterogeneity in CD3/IL-2 þ (range 9 . 7-41 . 3) and CD3/IFN-g þ cells (10 . 1-44), expressed as a percentage of total lymphocytes. In patients with atopic dermatitis (n ¼ 5) there was a significantly decreased percentage of CD3 þ /CD8 þ peripheral blood T cells expressing IFN-g and an increased percentage of CD3 þ /CD8 ¹ T cells expressing IL-4 compared with non-atopic dermatitis controls (n ¼ 5). Possible applications of this technique are discussed.

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Section: Introductionmentioning

confidence: 99%

Intracellular cytokine expression in whole blood preparations from normals and patients with atopic dermatitis

Ferry

Antrobus

Hužička

et al. 1997

Clinical and Experimental Immunology

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“…It is conceivable that comparable results will be obtained using other polyclonal activators. Nevertheless, diluted whole blood stimulated with LPS ϩ PHA offers the most appropriate and reproducible culture condition in order to measure cytokine production and to study the effects of any drugs on the production of cytokines De Groote et al 1992.…”

Section: Discussionmentioning

confidence: 99%

“…The effects of the serotonergic agents on these cytokines were examined by stimulating 1/10 diluted whole blood with PHA (1 g/ml; Murex Diagnostics Ltd, Dartford, England) and LPS (5 g/ml (E.Coli 026 lyophylized and sterilized by gamma-irradiation); Sigma, Belgium). Diluted whole blood stimulated with PHA ϩ LPS offers the most appropriate and reproducible culture condition in order to measure cytokine production De Groote et al 1992. Diluted whole blood cultures reflect the in-vivo immune cellular and humoral interactions and may be employed to examine the effects of drugs on cytokine secretion De Groote et al 1992Maes et al 1998).…”

Section: Methodsmentioning

confidence: 99%

“…Diluted whole blood stimulated with PHA ϩ LPS offers the most appropriate and reproducible culture condition in order to measure cytokine production De Groote et al 1992. Diluted whole blood cultures reflect the in-vivo immune cellular and humoral interactions and may be employed to examine the effects of drugs on cytokine secretion De Groote et al 1992Maes et al 1998). A total of 1.8 ml of RPMI-1640 medium with L-glutamine (Gibco BRL) supplemented with 100 IU/mL penicillin (Sigma), 100 g/mL streptomycin (Sigma), and PHA ϩ LPS were placed into 24 well cell culture plates (Falcone 3047; Becton Dickonsen).…”

Section: Methodsmentioning

confidence: 99%

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Effects of Serotonin and Serotonergic Agonists and Antagonists on the Production of Interferon-γ and Interleukin-10

Kubera¹,

Kenis²,

Bosmans³

et al. 2000

Neuropsychopharmacology

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(5-HT) is a neurotransmitter and an immune modulator. In vitro , antidepressants with a serotonergic mode of action have, at concentrations within the therapeutical range, negative immunoregulatory effects, i.e., they increase the production rate of interleukin-10 (IL-10), a negative immunoregulatory cytokine. We have hypothesized that part of these effects may be explained by the serotonergic activities of antidepressants on immunocytes. This study was carried out to examine the effects of 5-HT, p-chlorophenylalanine (PCPA), a 5-HT depleting agent, flesinoxan , and ritanserin (a 5-HT2A/ 2C antagonist) on the production rate of interferon-␥ (IFN ␥ ), a proinflammatory cytokine, and IL-10 by whole blood stimulated with polyclonal activators. The IFN ␥ /IL-10 production ratio was computed, since this ratio reflects the pro-versus anti-inflammatory capacity of cultured whole blood. We found that: 1) 5-HT, 150 ng/mL, 1.5 g/ mL, and 15 g/mL significantly decreased the IFN ␥ /IL-10 ratio; 2) PCPA (5 M) significantly suppressed the production of 3) Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter, a vasoactive amine released by platelets at sites of inflammation and an immune modulator (Roszman et al. 1985;Mossner and Lesch 1998;Kubera and Maes 1999). In humans, 5-HT is produced, outside the central nervous system, by enterochromaffin cells and it is stored in circulating platelets and to a lesser extent in monocytes and lymphocytes (Essmann 1978). 5-HT is released from platelets and lymphocytes/monocytes following stimulation by platelet activating factors or lectins/interferon-␥ (IFN ␥ ), respectively (Finocchiaro et al. 1988 (Cohen et al. 1985;Felten et al. 1991). Pharmacological and molecular analyses have confirmed the presence of 5HT1A and 5HT2A/C receptors on activated human immunocytes and specific 5-HT transporter sites on macrophages/ lymphocytes (Aune et al. 1994).The effect of 5-HT on cell-mediated immunity and on the inflammatory response system (IRS) is still a matter of controversy. Some studies show that 5-HT has immunosuppressive effects. Devoino et al. (1968) reported that substances, which have the property to increase 5-HT concentrations, inhibit immunogenesis and antibody production in rodents. 5-HT may suppress delayed hypersensitivity and transplantation immunity (for review see Devoino and Morozova 1988). Bonnet et al. (1984) found that 5-HT suppresses murine lymphocytic response to phytohemagglutinin A (PHA) and/or allogeneic cells. p-Chlorophenylalanine (PCPA), an inhibitor of 5-HT synthesis, enhances the production of antibodies . Other immune functions, however, may be stimulated by 5-HT. For example, cutaneous injection of 5-HT can initiate a delayed-type hypersensitivity reaction through local recruitment and activation of CD4 ϩ T-helper cells (Ptak et al. 1991). Low doses (100 ng/ml) of exogenously added 5-HT stimulate T cell proliferation in response to interleukin-2 (IL-2) (Young et al. 1993; Young and Mathews 1995). Enhancement of murine T-cell blastogenesis by 5-HT has b...

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“…For example, it has previously been shown that proteins stimulating the immune-system sometimes can be assessed by measuring the release of inflammatory cytokines with whole blood assays from animals and humans (Groote et al, 1992;House, 2001;Meager, 2006). Figure 2 is an example of the authors' experience with a program where cytokine release assays are complimentary to HCP analyses using process-specific Western blotting.…”

Section: The Biological and Immunological Consequences Of Hcpsmentioning

confidence: 99%

Host cell proteins in biologics development: Identification, quantitation and risk assessment

Wang

Hunter

Mozier

2009

Biotech & Bioengineering

201

184

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Host cell proteins (HCPs) are those produced or encoded by the organisms and unrelated to the intended recombinant product. Some are necessary for growth, survival, and normal cellular processing whereas others may be non-essential, simply carried along as baggage. Like the recombinant product, HCPs may also be modified by the host with a number of post-translational modifications. Regardless of the utility, or lack thereof, HCPs are undesirable in the final drug substance. Though commonly present in small quantities (parts per million expressed as nanograms per milligrams of the intended recombinant protein) much effort and cost is expended by industry to remove them. The purpose of this review is to summarize what is of relevance in regards to the biology, the impact of genomics and proteomics on HCP evaluation, the regulatory expectations, analytical approaches, and various methodologies to remove HCPs with bioprocessing. Historical data, bioinformatics approaches and industrial case study examples are provided. Finally, a proposal for a risk assessment tool is provided which brings these facets together and proposes a means for manufacturers to classify and organize a control strategy leading to meaningful product specifications.

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Direct stimulation of cytokines (IL-1β, TNF-α, IL-6, IL-2, IFN-γ and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation

Cited by 451 publications

References 25 publications

Intracellular cytokine expression in whole blood preparations from normals and patients with atopic dermatitis

Intracellular cytokine expression in whole blood preparations from normals and patients with atopic dermatitis

Effects of Serotonin and Serotonergic Agonists and Antagonists on the Production of Interferon-γ and Interleukin-10

Host cell proteins in biologics development: Identification, quantitation and risk assessment

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