Evaluation of the In Vitro Pyrogen Test System Based on Proinflammatory Cytokine Release from Human Monocytes: Comparison with a Human Whole Blood Culture Test System and with the Rabbit Pyrogen Test

ABSTRACT The reliability of an in vitro pyrogen test system based on proinflammatory cytokine release from human monocytic cells was assessed by comparison with a test system based on a human whole blood culture as well as with the conventional rabbit pyrogen test. The human cells used as the pyrogen indicator cells were newly selected by subcloning of a human monocytic cell line, Mono-Mac-6. The selected cells, named MM6-CA8, responded to various pyrogens, including endotoxin, peptidoglycan (PG), Staphylococcus aureus Cowan 1 (SAC), and poly(I · C), with a high sensitivity and produced proinflammatory cytokines, such as interleukin 1 (IL-1), IL-6, and tumor necrosis factor alpha. Among these cytokines, IL-6 was produced most sensitively in response to traces of the pyrogens and detected in the largest quantities in the culture medium. The cytokine-producing responses of MM6-CA8 cells correlated significantly with the responses of cultured human whole blood, which represents an ex vivo culture test system reproducing pyrogen-induced cytokine production in the human body. In terms of cytokine inducibility, the pyrogens were ranked in the order endotoxin > PG > poly (I · C) > SAC in both culture systems, a ranking which almost agreed with the ranking of their pyrogenicity as assessed by the rabbit pyrogen test. These results suggest that the in vitro responsiveness of MM6-CA8 cells to various pyrogens is highly relevant for human pyrogenic reactions. Therefore, the in vitro test system is useful and reliable for detecting the presence of materials that are pyrogenic for humans.

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Tigecycline has no effect on cytokine release in an ex vivo endotoxin model of human whole blood

International Journal of Antimicrobial Agents ◽

10.1016/j.ijantimicag.2008.11.008 ◽

2009 ◽

Vol 33 (6) ◽

pp. 583-586 ◽

Cited By ~ 1

Author(s):

Friederike Traunmüller ◽

Christiane Thallinger ◽

Johann Hausdorfer ◽

Christopher Lambers ◽

Stanislava Tzaneva ◽

...

Keyword(s):

Whole Blood ◽

Ex Vivo ◽

Cytokine Release ◽

Human Whole Blood

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Human whole-blood culture system for ex vivo characterization of designer-cell function

Biotechnology and Bioengineering ◽

10.1002/bit.25828 ◽

2015 ◽

Vol 113 (3) ◽

pp. 588-597 ◽

Cited By ~ 9

Author(s):

Lina Schukur ◽

Barbara Geering ◽

Martin Fussenegger

Keyword(s):

Blood Culture ◽

Whole Blood ◽

Culture System ◽

Cell Function ◽

Ex Vivo ◽

Blood Culture System ◽

Human Whole Blood

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Evaluation and Prevalidation of an Immunotoxicity Test Based on Human Whole-blood Cytokine Release

Alternatives to Laboratory Animals ◽

10.1177/026119290203000605 ◽

2002 ◽

Vol 30 (6) ◽

pp. 581-595 ◽

Cited By ~ 33

Author(s):

Ingrid Langezaal ◽

Sebastian Hoffmann ◽

Thomas Hartung ◽

Sandra Coecke

Keyword(s):

Immune System ◽

Whole Blood ◽

Animal Studies ◽

Ex Vivo ◽

Interleukin 4 ◽

Cytokine Release ◽

Fourfold Increase ◽

Ic50 Values

Immunotoxicology is a relatively new field in toxicology, and is one of emerging importance, because immunotoxicity appears to contribute to the development of cancer, autoimmune disorders, allergies and other diseases. At present, there is a lack of human cell-based immunotoxicity assays for predicting the toxicity of xenobiotics toward the immune system in a simple, fast, economical and reliable way. Existing immunotoxicity tests are mainly performed in animals, although species differences favour human-based testing. Whole-blood cytokine release models have attracted increasing interest, and are broadly used for pharmacological in vitro and ex vivo studies, as well as for pyrogenicity testing. We have adapted those methods for immunotoxicity testing, to permit the potency testing of immunostimulants and immunosuppressants. Following stimulation with a lipopolysaccharide or staphylococcal enterotoxin B, monocytes and lymphocytes release interleukin-1β and interleukin-4, respectively. Thirty-one pharmaceutical compounds, with known effects on the immune system, were used to optimise and standardise the method, by analysing their effects on cytokine release. The in vitro results were expressed as IC50 values for immunosuppression, and SC4 (fourfold increase) values for immunostimulation, and compared with therapeutic serum concentrations of the compounds in patients, and in vivo LD50 values from animal studies. The in vitro results correlated well with the in vivo data, so the test appears to reflect immunomodulation. Results were reproducible (CV = 20 ± 5%), and the method could be transferred to another laboratory (r2 = 0.99). We therefore propose this method for further validation and for use in immunotoxicity testing strategies.

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A model of human whole blood lymphokine release for in vitro and ex vivo use

Journal of Immunological Methods ◽

10.1016/s0022-1759(03)00003-6 ◽

2003 ◽

Vol 275 (1-2) ◽

pp. 69-79 ◽

Cited By ~ 28

Author(s):

Corinna Hermann ◽

Sonja von Aulock ◽

Kathrin Graf ◽

Thomas Hartung

Keyword(s):

Whole Blood ◽

Ex Vivo ◽

Human Whole Blood

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Ketamine Suppresses Proinflammatory Cytokine Production in Human Whole Blood In Vitro

Anesthesia & Analgesia ◽

10.1097/00000539-199909000-00024 ◽

1999 ◽

Vol 89 (3) ◽

pp. 665 ◽

Cited By ~ 16

Author(s):

Takashi Kawasaki ◽

Masanori Ogata ◽

Chika Kawasaki ◽

Jun-ichi Ogata ◽

Yoshitaka Inoue ◽

...

Keyword(s):

Whole Blood ◽

Proinflammatory Cytokine ◽

Cytokine Production ◽

Proinflammatory Cytokine Production ◽

Human Whole Blood

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Modulation of Blood Cell Activation by Four Commonly Used Anticoagulants

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656035 ◽

1997 ◽

Vol 77 (04) ◽

pp. 690-696 ◽

Cited By ~ 63

Author(s):

Charlotte Sissener Engstad ◽

Tore Jarl Gutteberg ◽

Bjarne Østerud

Keyword(s):

Whole Blood ◽

Cell Activation ◽

Ex Vivo ◽

Ex Vivo Model ◽

Platelet Factor ◽

Human Whole Blood ◽

Platelet Parameter ◽

Factor Α ◽

Edta Blood

SummaryIn the past years, our group has made several observations suggesting that blood cells behave differently and when stimulated, release different levels of cytokines, depending on which anticoagulant the blood has been drawn into. The aim of this study was therefore to compare the effect of the four anticoagulants EDTA, citrate, heparin and hirudin on monocyte, neutrophil (PMN), and platelet function in human whole blood. Human whole blood was employed as an ex vivo model of cytokine production and protein secretion, and lipopolysaccharide (LPS) induced tissue factor (TF) activity in monocytes and LPS induced tumor necrosis factor α (TNFα) release were chosen as parameters of monocyte activation. Platelet factor 4 (PF4) secretion and LPS induced lacto ferrin release were chosen as parameters of platelet and PMN activation, respectively. When human whole blood was stimulated with 5 ng/ml LPS for 2 h, TF activity in monocytes isolated from EDTA blood was found to be 2.9 mU/106 cells, whereas TF activity in monocytes isolated from citrated, heparinized and hirudinized blood was 14.7, 24.7 and 28.5 mU/106 cells, respectively. TNFα concentrations in platelet poor plasma (PPP) isolated from whole blood stimulated with 5 ng/ml LPS for 2 h was increased with 200,400 and 350% in citrated, heparinized and hirudinized blood respectively, as compared to EDTA blood. Next, the effect of the anticoagulant on PMN secretion was measured. PPP isolated from whole blood incubated with 5 ng/ml LPS for 90 min contained 1170 ng/ml (EDTA blood), 2880 ng/ml (citrated blood), 4220 ng/ml (heparinized blood), and 5520 ng/ml lactoferrin (hirudinized blood). When studying the platelet parameter PF4, whole blood was incubated without any stimuli for 60 min, and we found that heparin PPP contained 1180 ng/ml PF4, while hirudin PPP contained 469 ng/ml, citrate PPP 440 ng/ml, and EDTA PPP 217 ng/ml PF4, respectively. Finally, the low molecular weight heparin compound Fragmin had no enhancing effect on PF4 levels in whole blood. It is concluded that the anticoagulant used in in vitro experiments has a large influence on the parameters measured.

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An Investigation of Aluminium Neurotoxicity using some In Vitro Systems

Alternatives to Laboratory Animals ◽

10.1177/026119299001800119.1 ◽

1990 ◽

Vol 18 (1_part_1) ◽

pp. 181-190

Author(s):

Christopher K. Atterwill ◽

Wendy J. Davies ◽

Michael A. Kyriakides

Keyword(s):

Ex Vivo ◽

Test System ◽

Aluminium Chloride ◽

Acute Exposure ◽

In Vitro Test ◽

Mixed Cell ◽

Lower Vertebrate ◽

Short Term Exposure ◽

Culture Model

It has been shown that acute exposure in vitro to high concentrations of aluminium chloride does not appear to perturb neural function in terms of the electrophysiological properties of lower vertebrate leech neurones. Longer term exposure in vitro, however, both non-specifically inhibits cellular differentiation and also produces neural cytotoxicity in the rat midbrain micromass, mixed cell culture model. Furthermore, previous studies from this laboratory have demonstrated a reduction of cholinergic neuronal function in brain organotypic reaggregate cultures following long-term, but not short-term, exposure. More-immature neural cells appear to be most sensitive to the effects of aluminium. Relating these data to the tiered in vitro test system for neurotoxicants previously proposed by Atterwill (13), it is apparent that the neurotoxic effects of aluminium are detectable in a first-stage procedure using the micromass culture model, but not following acute exposure in freshly isolated, ex vivo leech neurones. Functional cholinergic toxicity was also detected in the organotypic reaggregate cultures proposed as a second level screen.

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