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.

The effect of a soluble beta-glucan from Candida albicans (CSBG) on cytokine production by cultured human peripheral blood mononuclear cells (PBMC) was assessed. CSBG induced a slight increase in the spontaneous release of proinflammatory cytokines, such as interleukin (IL)-6, but significantly suppressed endotoxin-induced IL-6 production in cultures of PBMC and monocytes isolated from PBMC. CSBG also suppressed the release of type 1 cytokines, IL-2, and interferon-gamma. These findings suggest that CSBG suppresses monocyte functions directly and thus suppresses T cell function indirectly. CSBG may play a role in the development of candidiasis.

Contamination by endotoxin of nine kinds of wound dressings made of natural biomaterials (calcium alginate, collagen, chitin, and poly-L-leucine) was examined with the use of water extracts. By applying the Limulus amoebocyte lysate (LAL) test, high concentrations of endotoxin were detected in extracts from three kinds of products made of calcium alginate. These extracts evoked fever in rabbits and induced the release of a proinflammatory (pyrogenic) cytokine, interleukin-6 (IL-6), from human monocytic cells (MM6-CA8). The effects disappeared when the extracts were treated with endotoxin-removing gel column chromatography or with an endotoxin antagonist, B464, confirming that the contaminating pyrogen was endotoxin. A noteworthy finding was that one of the endotoxin-containing extracts showed very weak IL-6-inducibility in human monocytic cells in contrast to its high pyrogenicity to rabbits. The discrepancy could be explained based on differences between humans and rabbits in sensitivity to the endotoxin, because the extract showed higher proinflammatory-cytokine (TNF-alpha)-inducibility in rabbit whole-blood cells (WBCs) than human WBCs. The results suggest that the LAL test is a useful method of detecting endotoxin contamination in wound dressings and the MM6-CA8 assay is a good supplement to the LAL test for evaluating pyrogenicity in humans accurately.

Resident alveolar macrophages (AMs) and peritoneal macrophages (PMs), though they originate from common precursor cells, differ morphologically and functionally. The two types of macrophages residing in different tissues may respond differently to glucocorticoids.In the present study, we compared the effects of a synthetic glucocorticoid, dexamethasone (Dex), on rat AMs and PMs with regard to their phagocytic activity and tumour necrosis factor-α (TNF-α) releasability.In vitro exposure of the macrophages to Dex caused the depression of phagocytic activity of AMs but not of PMs. In contrast, TNF-α releasability was depressed in the both types of macrophages, and no difference was found between AMs and PMs in their susceptibility to TNF-α regulation by Dex. When Dex was administered subcutaneously into rats, phagocytic activity was severely depressed in AMs but not in PMs. On the other hand, TNF-α releasability was depressed both in AMs and PMs by the in vivo Dex administration. The depression in PMs, however, was transitory and less severe than that in AMs.These results suggest that alveolar macrophages and peritoneal macrophages differ intrinsically in responses to glucocorticoid, and that the cell location and the cell's microenvironment can also modulate the effects of glucocorticoid on macrophage functions.

Following heat shock the expression of heat shock genes is regulated by the heat shock transcription factor, HSF, known to bind to arrays of the heat shock element, NGAAN, upstream of the heat shock genes. Phosphorylation of HSF is necessary for its activation. We report that the treatment of Chinese hamster HA-1 cells with 250 nM of okadaic acid (OA), a ser/thr phosphatase inhibitor, leads to an increase in activated HSF after heat shock. This is followed by the activation of the transcription of heat shock genes as assayed by the increase in the synthesis of beta-galactosidase in an HA-1 cell line containing the heat shock promoter ligated to the beta-galactosidase gene. To investigate the specificity of OA, we used other phosphatase inhibitors. We found that treatment of HA-1 cells with 500 microM of sodium vanadate, an inhibitor of tyr/phosphatases, resulted in a three to fivefold reduction in HSF activation and binding to the heat shock element following heat shock. Such reduction in HSF activation virtually abolished beta-galactosidase induction. Reduced HSP synthesis was further confirmed by SDS-PAGE and Western blot analysis using anti-HSP-70 and 28 antibodies. Sodium vanadate treatment of heat shocked cells greatly reduced levels of thermotolerance. These results show that ser/thr and specifically tyr/phosphatase inhibitors modulate the signal transduction pathway of HSF activation.

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The effect of sonication on the particle size of lipopolysaccharide (LPS) in aqueous media was studied, in order to examine the relation of particle size to pyrogenicity in rabbits, by the sucrose density gradient ultracentrifugation technique. LPS extracted from E. coli UKT-B according to the phenol/water method showed a polydispersed profile on the gradients, but after sonication for 3 min it formed a single peak in the lower density regions. From the results of electron micrographic observations, partial specific volume, viscosity and turbidity measurements, and density gradient data, it was revealed that sonication produced a decrease in the particle size of LPS. A more marked pyrogenicity in rabbits was observed in the LPS fractions in the lower density regions than in the higher ones, or in the fractions having smaller-sized particles of LPS than in the fractions having larger particles.

Relationship between pyrogenicity and bacterial endotoxin contamination on latex products was demonstrated by chemical analysis and biological assays. In commercially available latex products' surveillance, water extracts prepared from one surgical glove and two silicone elastomer-coated Foley catheters sterilized by gamma-irradiation were obviously pyrogenic in rabbits. The induced fever was monophasic at low dose of the pyrogenic extracts and biphasic at high dose. These extracts exhibited limulus amebocyte lysate gelation activity, and induced inflammatory cytokine (interleukin-1, interleukin-6, and tumor necrosis factor-alpha) production from MM6-CA8 human monocytoid cells. These biological properties, including pyrogenicity, completely disappeared by treating the pyrogenic extracts with endotoxin-adsorbent affinity column. Limulus amebocyte lysate activity and cytokine production from MM6-CA8 cells induced by the extracts were significantly decreased by endotoxin inhibitors, an active fragment peptide of an 18-kDa cationic antimicrobial protein and a synthetic lipid A B464 analogue. Furthermore, very small amounts of 2-keto-3-deoxyoctonate and 3-hydroxy fatty acid, which are common constituents of bacterial endotoxins, were detected by gas chromatography-mass spectrometry analysis of the pyrogenic extracts. These findings clearly showed that the pyrogenicity found in these latex products originated from endotoxins contaminating the products.