Utilization of the human cell line HL-60 for chemiluminescence based detection of microorganisms and related substances.

In this paper we describe a new pyrogen assay using the human leukemia cell line HL-60. The cell line is differentiated using all-trans retinoic acid (ATRA) to generate a cell population that resembles mature granulocytes. The differentiated HL-60 cell is capable of generating reactive oxygen species (ROS) when challenged with pyrogenic substances. In a luminol enhanced chemilumimetric assay the responsiveness of differentiated HL-60 cells is tested towards Salmonella typhimurium, Bacillus subtilis, Saccharomyces cerevisiae, Candida albicans, lipopolysaccharide (LPS) and lipoteichoic acid (LTA). The results show a poor sensitivity to S. typhimurium but displays good sensitivity towards B. subtilis, LTA and LPS. Furthermore, the sensitivity towards the yeasts C. albicans and S. cerevisiae is considerably better than obtained in other in vitro cell systems. Overall these results indicate that the HL-60 cell assay possibly could be evolved to a supplementary assay for the known pyrogenic detection assays. Furthermore, the utilization of the assay for pyrogenic examination of recombinant drugs derived from yeast expression systems would be relevant to examine.

Endotoxin contamination in wound dressings made of natural biomaterials.

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.

Chemical and biological evaluation of endotoxin contamination on natural rubber latex products.

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.

The use of human monocytoid lines as indicators of endotoxin.

We wish to develop an in vitro test system for pyrogenic substances. A major source of pyrogen activity is endotoxin. Here we describe a highly sensitive endotoxin-monitoring system based on cytokine measurement in human cell lines of myelomonocytoid origin. The following measures were taken to develop an endotoxin monitoring system of high sensitivity. (i) Mono Mac 6 (MM6) and THP-1 cells, which both represent advanced stages of myelomonocytic development, were better suited as endotoxin indicators than the more immature U-937 line. (ii) In order to enhance cell surface expression of CD14, a major lipopolysaccharide (LPS) receptor, cells were pretreated for 2 days with calcitriol. (iii) The use of fetal calf serum (FCS) without detectable endotoxin traces was essential for maintaining a high LPS sensitivity. (iv) Selected subclones of either THP-1 or MM6 were significantly more sensitive LPS indicators than bulk cultures from which the clones originated. (v) Based on stimulation indices, a commercial tumor necrosis factor-alpha (TNF-alpha) immunoassay proved to be a more sensitive LPS indicator than other cytokine assays or the expression of procoagulant activity/tissue factor. Thus we were able to eliminate the disadvantage of previous cell line-based systems (i.e., low sensitivity) without loss of reproducibility which is seen when using fresh blood, monocytes or monocyte-derived macrophages. High endotoxin sensitivity is a prerequisite for a test system specifically indicating pyrogen activity, because it permits the testing of substances at higher dilutions, thereby minimizing nonspecific interference.

[Comparison of the Limulus test with the pyrogen test in rabbits]. / Ein Beitrag zum Vergleich des Limulustests mit dem Pyrogentest am Kaninchen.

The Limulus test was compared with the official pyrogen test in rabbits for detecting pyrogens in medical devices for single use. 46 samples were examined. Two of them were positive in the rabbit test and also in the Limulus test; 5 samples were only positive in the Limulus test. This is in agreement with published papers. Against endotoxins of gram-negative bacteria the Limulus test is more sensitive than the common test in rabbits, but not sensitive for other pyrogens. Therefore, the Limulus test cannot replace the official rabbit test in the quality control of medical devices. We recommend the Limulus test as a quick, simple and highly sensitive screening method for gram-negative bacterial endotoxins, the most common pyrogen contaminants in drugs.

The Jeremiah Metzger Lecture. The pathogenesis of fever in human subjects.

The pathogenesis of fever in man begins with the production of endogenous pyrogen by phagocytic leukocytes in response to exogenous pyrogens (toxic, immunologic or infectious agents). Endogenous pyrogen, a protein, is released from a variety of phagocytic leukocytes and enters the circulation after new messenger RNA and protein are synthesized. Fever is caused by an interaction of endogenous pyrogen with specialized receptors on or near thermosensitive neurons in the thermoregulatory center of the anterior hypothalamus. This interaction may cause local hypothalamic production of prostaglandins, monoamines and, possibly, cyclic AMP. From the anterior hypothalamus, information is transmitted through the posterior hypothalamus to the vasomotor center, which directs sympathetic-nerve fibers to constrict peripheral vessels and decrease heat dissipation.

Chemical and biological properties of a purified lymphoreticular-stimulating fraction of Corynebacterium parvum (Propionibacterium acnes type I).

A method is described in which washed whole cells of Corynebacterium parvum were chemically and enzymatically extracted to remove cytoplasm and cell-wall lipids. The resultant insoluble cell-wall residue possessed lympho-reticular stimulating properties as measured by their ability to increase spleen weight and protect against tumour-cell challenge. Analysis of the final product by chromatography and infrared spectroscopy has shown it to consist of carbohydrate and peptidoglycan, both of which appear to be necessary for the activities measured.

Fever: pathogenesis, pathophysiology, and purpose.

Fever appears to have evolved in vertebrate hosts as an adaptive mechanism for controlling infection. This phenomenon is produced by certain exogenous (largely microbial) stimuli that activated bone-marrow-derived phagocytes to release a fever-inducing hormone (endogenous pyrogen). Endogenous pyrogen, in turn, circulates to the thermoregulatory center of the brain (preoptic area of the anterior hypothalamus) where it causes an elevation in the "set-point" for normal body temperature. Warm blooded animals produced fever by increasing heat production (through shivering) or reducing heat loss (by peripheral vasoconstriction), whereas cold blooded animals do so only by behavioral mechanisms (seeking a warmer environment). This paper discusses current concepts that involve the mechanism of endogenous pyrogen production, the role of central transmittors, and the probable function of fever in combating disease.

Purification of influenza virus with Arcton 113.

Arcton 113 has proved effective in the removal of host membrane fragments which cosediment with influenza virus during differential centrifugation. The virus appeared morphologically undamaged by the procedure and retained its infectivity, neuraminidase, and hemagglutinating activity. Pyrogenicity, however, was considerably reduced.

Chemical and biological properties of an extracellular lipopolysaccharide from Escherichia coli grown under lysine-limiting conditions.

Lipopolysaccharide was prepared from the extracellular lipoglycopeptide produced by the lysine-requiring mutant Escherichia coli A.T.C.C. 12408 grown under lysine-limiting conditions. The lipid moiety, containing glucosamine phosphate and four fatty acids (lauric acid, myristic acid, beta-hydroxymyristic acid and palmitic acid) corresponded in composition to lipid A of known bacterial lipopolysaccharides. The components of the polysaccharide moiety were d-glucose, d-galactose, l-glycero-d-manno-heptose, 3-deoxy-2-oxo-octonic acid, ethanolamine and phosphate. These are the constituents of the polysaccharide of the cell-wall antigens from rough strains of E. coli. Lipopolysaccharides were also prepared from whole cells of E. coli 12408 grown with excess or limited amounts of lysine; they were identical in carbohydrate composition with the extracellular lipopolysaccharide. The biological properties of this material also resembled those of known lipopolysaccharides; it was antigenic, pyrogenic, toxic and had adjuvant activity.