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 cytokineproducing 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.

Evidence for the detection of non-endotoxin pyrogens by the whole blood monocyte activation test

“…the surface glycans, e.g., β(1-3) and β(1-6) glucans such as zymosan, laminarin, lichenan, and curdlan interfere with the lAl-test (Vassallo and limper, 1999). the rabbit test does not mirror the human situation regarding pro-inflammatory cytokine release upon stimulation with glucans, as the concentration of glucan needed to induce fever in rabbits (about 100 µg/ml -300 µg/ml) is far higher than for humans (0.01 µg/ml -0.1 µg/ml) (Nakagawa et al, 2002;ellis et al, 2008;Obayashi et al, 1995). this high amount of Human whole blood was stimulated with the LPS, LTA, and zymosan or fungal spores, and IL-1β release was determined by ELISA at the given time points.…”

“…(eU, 2010) As a true pyrogen test, the RPt is considered to be able to detect endotoxin and non-endotoxin pyrogens (NePs) alike (Nakagawa et al, 2002); in some cases, however, the RPt remains negative in response to NeP that induce pyrogenic adverse reactions in humans (Martis et al, 2005).…”

“…they used the rabbit test to determine fever-inducing factors secreted from human blood monocytes. the understanding of the human fever reaction and development of elISA technologies led to the development of test systems based on the in vitro activation of human monocytes (Hansen and Christensen, 1990;WernerFelmayer et al, 1995;eperon et al, 1997;Moesby et al, 1999;Hartung et al, 2001;Dinarello, 2004;Gaines Das et al, 2004;Schindler et al, 2009) detecting lPS, NePs, and mixtures (Hermann et al, 2002;Nakagawa et al, 2002;Kikkert et al, 2007). Peripheral blood mononuclear cells (PBMCs) were first used to detect pyrogens by monitoring the release of pyrogenic cytokines (Duff and Atkins, 1982), such as IL-1β, TNF-α, and Il-6.…”

The human whole blood pyrogen test – lessons learned in twenty years

“…4) (Schindler et al, 2009;Hasiwa et al, 2013a,b). Some independent evaluations were very favorable (Pool et al, 1998(Pool et al, , 1999Nakagawa et al, 2002;Andrade et al, 2003;Francois et al, 2006;Kikkert et al, 2008;Megha et al, 2011;Banerjee and Mohanan, 2011). On the way, the work around the whole blood assay received several awards (Tab.…”

Monocyte Activation Test (MAT) reliably detects pyrogens in parenteral formulations of human serum albumin

“…However, they might be similar to those extracted from cellulose membrane components of hollow-fiber hemodialyzers, which have a molecular weight of around 24,000 (Pearson et al, 1984(Pearson et al, , 1987. this issue is also contradictory in the literature, because other reports indicate that glucans from cellulose filters (Kikkert et al, 2007) or from yeast (Nakagawa et al, 2002) do not induce IL-6 release in monocytes or monocytoid cell lines. This issue would require further studies.…”