Analysis of IL-1βRelease from Cryopreserved Pooled Lymphocytes in Response to Lipopolysaccharide and Lipoteichoic Acid

Abstract

Pyrogens are heterogeneous group of fever-inducing substances derived from Gram-positive and Gram-negative bacteria, fungi, and viruses. They incite immune response by producing endogenous pyrogens such as prostaglandins and other proinflammatory cytokines like IL-1β, IL-6, and TNF-α. The present study was to analyze the influence of cryopreservation in IL-1β release, a marker for inflammatory response from human lymphocytes, in response to exogenous pyrogenic stimulants. Lymphocytes isolated from pooled blood of multiple healthy individuals were cryopreserved in DMSO and glycerol for periods of 7, 14, 30, and 60 days and were challenged with LPS and LTA in vitro. The inflammatory cytokine, IL-1β release, was measured by ELISA method. It was observed that the release of IL-1β increases instantaneously after the initiation of incubation and reaches a maximum at 3 to 5 hours and then gradually decreases and gets stabilized for both pyrogens. Moreover it was also observed that the effect of cryoprotectants, DMSO (10%) and glycerol (10%), showed almost similar results for short-term storage, but DMSO-preserved lymphocytes yielded a better viability for long-term storage. Thus, the isolated cryopreserved lymphocytes system can be a promising approach for the total replacement/alteration to animal experimentation for pyrogenicity evaluation.

Escherichia coli Braun Lipoprotein (BLP) exhibits endotoxemia – like pathology in Swiss albino mice

“…It is estimated that single Gram negative bacterial cell contains two million molecules of LPS 12 , covering approximately 75% of the outer membrane surface 13 . LPS exerts its action through Toll-like receptor 4 (TLR4) 14 , a pattern recognizing receptor that ligates structurally conserved pathogen associated molecular patterns (PAMPs) of microbial origin 15 .…”

A Novel Reporter Gene Assay for Pyrogen Detection

“…Compared with other sources of monocytes and macrophages, such as whole blood (15)(16)(17) and peripheral blood monocytes (18)(19)(20), monocytic and macrophage cell lines are more stable and easier to use (21)(22)(23)(24). RAW264.7 cells are mouse-derived macrophages and express the main receptors that can bind pyrogens, including TLR2, TLR4, and TLR6.…”

One-step immunoassay for the detection of food-poisoning related bacteria using a switching peptide

“…For the detection of such antigens, the lysis of bacteria cell has been required. As described in previous works, [22][23][24] the outer membranes of Gram-negative bacteria and Gram-positive bacteria have endotoxins, such as lipopolysaccharide (LPS) for Gram-negative bacteria and lipoteichoic acid (LTA) for Grampositive bacteria. These endotoxins are known to be strong pyrogens which stimulate monocytes or macrophages and T lymphocytes to produce inflammatory cytokines.…”