Comparison of pyrogen assays by testing products exhibiting low endotoxin recovery.

The use of pyrogen tests to assess the risk of endotoxin in biological products has increased recently due to concerns of some regulatory authorities about products exhibiting low endotoxin recovery (LER). Manufacturers increasingly seek to reduce the use of animals unless essential to assure patient safety. The current study compares the ability of the monocyte activation test (MAT) and the bacterial endotoxin test (BET) to the rabbit pyrogen test (RPT) to detect endotoxin spikes in samples of products shown to exhibit LER. Product samples or water were spiked with endotoxin and held for three days or tested immediately in the BET, the RPT, and two variations of the MAT at the same time. Results show high sensitivity to endotoxin of both the BET and MAT, and much lower sensitivity of the RPT, indicating that much higher levels of reference standard endotoxin are required to induce pyrogenicity in the RPT than the 5 endotoxin units (EU) per kg common threshold. The results of the BET and MAT correlated well for the detection of endotoxin spike in water. We also show that LER (masking of endotoxin) found in the BET is also seen in the MAT and RPT, suggesting that the products themselves elicit a biological inactivation of spiked endotoxin over time, thereby rendering it less or non-pyrogenic. We conclude that the non-animal MAT option is a suitable replacement for the RPT to measure spiked endotoxin in biopharmaceuticals.

Pyrogen testing revisited on occasion of the 25th anniversary of the whole blood monocyte activation test.

The whole blood pyrogen test invented 25 years ago, and its variant based on cryo-preserved blood one year later, brought momentum into the field of pyrogen testing, which, despite the broad application of the Limulus amebocyte lysate (LAL) assay, aka bacterial endotoxin test (BET), consumed several hundred thousand rabbits per year world-wide. The resulting international validation and lengthy acceptance and implementation process of what are called now monocyte activation tests (MATs) finally is impacting on animal numbers - at least in Europe - reducing them by more than 70% and counting. The author sees no reason for continuing any regulatory rabbit testing for pyrogens except the lack of acceptance of MATs in some regions of the world. The availability of MATs has opened also the discussion about the shortcomings of LAL/BET, namely its restriction to Gram-negative pyrogens, non-reflection of the potency of these in humans, interference and masking by many products, and animal welfare concerns for horseshoe crabs. The obvious advantages of MATs in all these respects should lead to a shift from LAL/BET to MATs. We are starting to see this for vac-cines and medical devices, but other areas like safety testing of blood transfusions, cell therapies and nanomaterials, and the assessment of air-borne pyrogens still need to grasp the opportunity provided by MATs. While the different MATs can jointly serve these needs, the whole blood MAT has some advantages as discussed here.

Optimization of the monocyte activation test for evaluating pyrogenicity of tick-borne encephalitis virus vaccine

Pyrogen content is a key quality feature that must be checked in all injectable products, including vaccines. Four tests are currently available in the European Pharmacopoeia to monitor pyrogen/endotoxin presence: the rabbit pyrogen test (RPT), the bacterial endotoxin test, the recombinant factor C test, and the monocyte activation test (MAT). Here, we explored the possibility to replace the RPT with the MAT in the quality control of a vaccine against tick-borne encephalitis virus (TBEV). The testing was carried out using cryopreserved peripheral blood mononuclear cells as cell source. IL-6 release was selected as readout for the detection of both endotoxin and non-endotoxin contaminants. MAT applicability for pyrogen testing of the TBEV vaccine was assessed through preparatory tests and resulted in the establishment of a very sensitive assay (limit of detection (LOD) = 0.04 EU/mL; sensitivity = 0.1 EU/mL). Both quantitative Method A and semiquantitative Method B were used for data analysis. Our studies revealed that for a vaccine without intrinsic pyrogenicity, such as that against TBEV, sensitivity (the lowest endotoxin value of the standard curve) should be used instead of LOD to define a stable maximum valid dilution of the product. In conclusion, we describe the challenges of MAT implementation for anti-TBEV vaccine following the current Ph. Eur. chapter 2.6.30 and propose a re-evaluation of the validity criteria of Methods A and B in order to set a semi-quantitative or limit test suitable for those products for which a reference lot comparison analysis is not applicable or favorable.

Lipopolysaccharides from Microcystis Cyanobacteria-Dominated Water Bloom and from Laboratory Cultures Trigger Human Immune Innate Response.

Massive toxic blooms of cyanobacteria represent a major threat to water supplies worldwide. Here, the biological activities of lipopolysaccharide (LPS) isolated from Microcystis aeruginosa, the most prominent cyanobacteria in water bloom, were studied. LPS was isolated from complex environmental water bloom samples dominated by M. aeruginosa, and from laboratory cultures of non-axenic as well as axenic M. aeruginosa strains PCC7806 and HAMBI/UHCC130. Employing human blood-based in vitro tests, the LPS isolated from complex water bloom revealed the priming of both major blood phagocyte population monocytes and polymorphonuclear leukocytes documented by the increased surface expression of CD11b and CD66b. This was accompanied by a water bloom LPS-mediated dose-dependent induction of tumor necrosis factor α, interleukin-1ß, and interleukin-6 production. In accordance with its priming effects, water bloom LPS induced significant activation of p38 and ERK1/2 kinases, as well as NF-κB phosphorylation, in isolated polymorphonuclear leukocytes. Interestingly, the pro-inflammatory potential of LPS from the axenic strain of M. aeruginosa was not lower compared to that of LPS isolated from non-axenic strains. In contrast to the biological activity, water bloom LPS revealed almost twice higher pyrogenicity levels compared to Escherichia coli LPS, as analyzed by the PyroGene test. Moreover, LPS from the non-axenic culture exhibited higher endotoxin activity in comparison to LPS from axenic strains. Taking the above findings together, M. aeruginosa LPS can contribute to the health risks associated with contamination by complex water bloom mass.

Staphylococcal Superantigens: Pyrogenic Toxins Induce Toxic Shock.

Staphylococcal enterotoxin B (SEB) and related superantigenic toxins produced by Staphylococcus aureus are potent activators of the immune system. These protein toxins bind to major histocompatibility complex (MHC) class II molecules and specific Vß regions of T-cell receptors (TCRs), resulting in the activation of both monocytes/macrophages and T lymphocytes. The bridging of TCRs with MHC class II molecules by superantigens triggers an early "cytokine storm" and massive polyclonal T-cell proliferation. Proinflammatory cytokines, tumor necrosis factor α, interleukin 1 (IL-1), IL-2, interferon γ (IFNγ), and macrophage chemoattractant protein 1 elicit fever, inflammation, multiple organ injury, hypotension, and lethal shock. Upon MHC/TCR ligation, superantigens induce signaling pathways, including mitogen-activated protein kinase cascades and cytokine receptor signaling, which results in NFκB activation and the phosphoinositide 3-kinase/mammalian target of rapamycin pathways. In addition, gene profiling studies have revealed the essential roles of innate antimicrobial defense genes in the pathogenesis of SEB. The genes expressed in a murine model of SEB-induced shock include intracellular DNA/RNA sensors, apoptosis/DNA damage-related molecules, endoplasmic reticulum/mitochondrial stress responses, immunoproteasome components, and IFN-stimulated genes. This review focuses on the signaling pathways induced by superantigens that lead to the activation of inflammation and damage response genes. The induction of these damage response genes provides evidence that SEB induces danger signals in host cells, resulting in multiorgan injury and toxic shock. Therapeutics targeting both host inflammatory and cell death pathways can potentially mitigate the toxic effects of staphylococcal superantigens.

Preclinical safety and efficacy evaluation of 'BioCaS' bioactive calcium sulfate bone cement.

A new bioactive calcium sulfate-based formulation (named 'BioCaS') has been developed for bone filler applications. This is a self-setting injectable cement where the preset form comprises bassanite obtained from the uniform submicron-sized precursor crystals of gypsum, modified with hydrogen orthophosphate ions. The results of the safety and efficacy evaluation of BioCaS cement, done as per the International Standards and guidelines, are presented in this paper. The study plan consisted of in vitro screening tests of cytotoxicity and haemolysis and in vivo biocompatibility evaluation, including an acute systemic toxicity test (in mice), an intracutaneous reactivity test (in rabbits), a pyrogen test (in rabbits) and a maximization sensitization test (in guinea pigs). The efficacy of the material in healing bone defects was investigated by implanting it in artificially created defects in rabbit femora, with clinically established hydroxyapatite porous ceramic as the control, followed by histological analysis at 12, 26 and 52 weeks. Set BioCaS cement consisted of hydrogen orthophosphate incorporating low-dimensional gypsum crystal lattices, the bioactivity of which has been identified by immersion in simulated body fluid. BioCaS was proved to be non-cytotoxic and non-haemolytic in the screening tests. In the live/dead assay, human osteoblast-like human osteosarcoma cells adhered well and spread on the surface of the material, attaining typical morphology and affirming the bone cell compatibility of the material. In the biocompatibility evaluation there were no acute systemic effects and the material proved non-pyrogenic. There was no intracutaneous erythemic or oedematous reactivity and no hypersensitivity observed in the Magnusson-Kligman method. The material satisfied the biocompatibility requirements. Bone implantation study revealed BioCaS to be osteoconductive and its efficacy of healing the experimental bone defects in rabbit femora is on a par with that of hydroxyapatite ceramic. The material resorbed at a pace matching that of new bone formation. This property of osteotransductivity will help the defect to heal and gain strength faster.

More than 70 years of pyrogen detection: Current state and future perspectives.

In the quality assurance of medical products, tests for sterility are essential. For parenteral pharmaceuticals, avoiding the presence of pyrogens is crucial. These fever-inducing substances (endotoxins and non-endotoxins) are not eliminated by standard sterilisation processes, and are biologically active once in the bloodstream, causing risks to human health, ranging from mild reactions (e.g. fever) to septic shock and death. Therefore, for injectable formulations, pyrogen testing is mandatory. Over the years, various pyrogen testing methods have been introduced, namely: in the 1940s, the rabbit pyrogen test, which is an in vivo test that measures the fever reaction as an endpoint; in the 1970s, the Limulus Amoebocyte Lysate (LAL) test, which is an in vitro test (with the haemolymph of the horseshoe crab) that specifically detects endotoxin; and in 2010, the Monocyte-Activation Test (MAT), which is a non-animal based in vitro pyrogen test that represents a full replacement of the rabbit test. Due to the ubiquity and biological significance of pyrogens, we are currently further developing the MAT so that it can be used for other applications. More specifically, our focus is on the detection of pyrogenic contamination on medical devices, as well as on the measurement of air quality. In addition, further improvements to permit the use of cryopreserved blood in the MAT, to overcome the limitations in the availability of freshly-drawn blood from human donors, are ongoing.

Simulated acute central Mycoplasma infections in rats induce fever, anorexia, body mass stunting and lethargy but spare memory.

Despite the documented post-infectious neurological complications of a central nervous system (CNS) Mycoplasma infection in humans, very few studies have investigated the acute inflammatory responses and sickness behaviours induced by CNS Mycoplasma infections. We therefore determined the effect of acute central administration of fibroblast-stimulating lipopeptide-1 (FSL-1), derived from Mycoplasma salivarium, and FAM-20 from a more pathogenic species, namely Mycoplasma pneumoniae, on behavioural and inflammatory responses in rats. Male Sprague-Dawley rats had radiotransmitters implanted, intra-abdominally, to measure body temperature and cage activity continuously. After recovery from surgery, rats were conditioned in a fear conditioning task and then immediately received an intra-cisterna magna (i.c.m.) injection of either: (1) FSL-1 (10 or 100µg/5µl) or its vehicle (phosphate-buffered saline, 5µl), or (2) FAM-20 (10 or 100µg/5µl) or its vehicle (dimethyl sulfoxide, 5µl). Body mass and food intake were measured daily. Memory was assessed seven days after injection using fear conditioning tests. A single, i.c.m. injection of either FSL-1 or FAM-20 induced profound, dose-dependent fever, anorexia, lethargy and body mass stunting in rats. Moreover, rats that received an i.c.m. injection of 100µg/5µl FAM-20 had a significant increase in the concentration of IL-1ß in both the hypothalamus and the hippocampus for ~27h after injection. Seven days after FSL-1 or FAM-20 injection, when body mass of rats still was stunted, they maintained their memory for fear of the context and for fear of the tone, despite the increase in hippocampal IL-1ß concentration after FAM-20 administration. Thus, acute simulated CNS Mycoplasma infections caused pronounced sickness responses and brain inflammation in rats, but spared fear memory.

Assessing endotoxins in equine-derived snake antivenoms: Comparison of the USP pyrogen test and the Limulus Amoebocyte Lysate assay (LAL).

Snake antivenoms are parenterally administered; therefore, endotoxin content must be strictly controlled. Following international indications to calculate endotoxin limits, it was determined that antivenom doses between 20 mL and 120 mL should not exceed 17.5 Endotoxin Units per milliliter (EU/mL) and 2.9 EU/mL, respectively. The rabbit pyrogen test (RPT) has been used to evaluate endotoxin contamination in antivenoms, but some laboratories have recently implemented the LAL assay. We compared the capability of both tests to evaluate endotoxin contamination in antivenoms, and we found that both methods can detect all endotoxin concentrations in the range of the antivenom specifications. The acceptance criteria of RPT and LAL must be harmonized by calculating the endotoxin limit as the quotient of the threshold pyrogenic dose and the therapeutic dose and the dose administered to rabbits as the quotient of the threshold pyrogenic dose and the endotoxin limit. Since endotoxins from Gram-negative bacteria exert different pyrogenicity, if contamination occurred, antivenom batches that induce pyrogenic reactions may be found in spite of passing LAL specifications. Although LAL assay can be used to assess endotoxin content throughout the antivenom manufacturing process, we recommend that the release of final products be based on the results of both methods.

Heme oxygenase-1 induction by cobalt protoporphyrin enhances fever and inhibits pyrogenic tolerance to lipopolysaccharide.

Heme oxygenase-1 (HO-1) is an enzyme that catalyzes degradation of the heme and regulates its availability for newly synthetized hemeproteins such as cyclooxygenases, NO synthases and cytochrome P450. Moreover, HO-1 activity modulates synthesis of cytokines and prostaglandins. All of these factors are well-defined components of fever and pyrogenic tolerance mechanisms. We examine the effect of HO-1 induction and activation using cobalt protoporphyrin (CoPP) on changes in body temperature (Tb), plasma levels of interleukin-6 (IL-6), prostaglandin E2 (PGE2) and HO-1 protein in the course of these processes. Intraperitoneally (i.p.) pre-treatment of rats with CoPP (5 mg kg(-1)) significantly accelerated and enhanced the early stage of lipopolysaccharide (LPS)-induced fever and shortened a post-fever recovery to normal temperature. Pre-treatment with CoPP significantly potentiated the increase in plasma IL-6, PGE2 and HO-1 levels measured 4h after the LPS administration. Furthermore, induction of HO-1 attenuated the development of pyrogenic tolerance to repeated injections of LPS. Based on these data we conclude that heme oxygenase-1 may act as a physiological regulator of the febrile response intensity to bacterial infections.

Mechanisms of fever production and lysis: lessons from experimental LPS fever.

Fever is a cardinal symptom of infectious or inflammatory insults, but it can also arise from noninfectious causes. The fever-inducing agent that has been used most frequently in experimental studies designed to characterize the physiological, immunological and neuroendocrine processes and to identify the neuronal circuits that underlie the manifestation of the febrile response is lipopolysaccharide (LPS). Our knowledge of the mechanisms of fever production and lysis is largely based on this model. Fever is usually initiated in the periphery of the challenged host by the immediate activation of the innate immune system by LPS, specifically of the complement (C) cascade and Toll-like receptors. The first results in the immediate generation of the C component C5a and the subsequent rapid production of prostaglandin E2 (PGE2). The second, occurring after some delay, induces the further production of PGE2 by induction of its synthesizing enzymes and transcription and translation of proinflammatory cytokines. The Kupffer cells (Kc) of the liver seem to be essential for these initial processes. The subsequent transfer of the pyrogenic message from the periphery to the brain is achieved by neuronal and humoral mechanisms. These pathways subserve the genesis of early (neuronal signals) and late (humoral signals) phases of the characteristically biphasic febrile response to LPS. During the course of fever, counterinflammatory factors, "endogenous antipyretics," are elaborated peripherally and centrally to limit fever in strength and duration. The multiple interacting pro- and antipyretic signals and their mechanistic effects that underlie endotoxic fever are the subjects of this review.

Hemocompatibility testing according to ISO 10993-4: discrimination between pyrogen- and device-induced hemostatic activation.

Next to good hemocompatibility performance of new medical devices, which has to be tested according to the ISO 10993-4, the detection of pyrogen-contaminated devices plays a pivotal role for safe device application. During blood contact with pyrogen-contaminated devices, intense inflammatory and hemostatic reactions are feared. The aim of our study was to investigate the influence of pyrogenic contaminations on stents according to the ISO 10993-4. The pyrogens of different origins like lipopolysaccharides (LPS), purified lipoteichoic acid (LTA) or zymosan were used. These pyrogens were dried on stents or dissolved and circulated in a Chandler-loop model for 90 min at 37°C with human blood. Before and after circulation, parameters of the hemostatic system including coagulation, platelets, complement and leukocyte activation were investigated. The complement system was activated by LPS isolated from Klebsiella pneumoniae and Pseudomonas aeruginosa and by LTA. Leukocyte activation was triggered by LPS isolated from K. pneumoniae, LTA and zymosan, whereas coagulation and platelet activation were only slightly influenced. Our data indicate that pyrogen-contaminated devices lead to an alteration in the hemostatic response when compared to depyrogenized devices. Therefore, pyrogenicity testing should be performed prior to hemocompatibility tests according to ISO 10993-4 in order to exclude hemostatic activation induced by pyrogen contaminations.

Highly sensitive pyrogen detection on medical devices by the monocyte activation test.

Pyrogens are components of microorganisms, like bacteria, viruses or fungi, which can induce a complex inflammatory response in the human body. Pyrogen contamination on medical devices prior operation is still critical and associated with severe complications for the patients. The aim of our study was to develop a reliable test, which allows detection of pyrogen contamination on the surface of medical devices. After in vitro pyrogen contamination of different medical devices and incubation in a rotation model, the human whole blood monocyte activation test (MAT), which is based on an IL-1ß-specific ELISA, was employed. Our results show that when combining a modified MAT protocol and a dynamic incubation system, even smallest amounts of pyrogens can be directly detected on the surface of medical devices. Therefore, screening of medical devices prior clinical application using our novel assay, has the potential to significantly reduce complications associated with pyrogen-contaminated medical devices.

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

Threats of pyrogenicity were discovered more than a century ago. Measures to determine the safety of parenterals and, more recently, medical devices and cell therapies for human use have been in place for 70 years. Currently, there are three testing possibilities available: the Rabbit Pyrogen Test, the Limulus Amebocyte Lysate test (Bacterial Endotoxin Test), and test systems using human whole blood or human monocytes, called Monocyte Activation Test (MAT). The MAT is based on the human fever reaction and thus most closely reflects the human situation. Unfortunately, regulations and testing guidelines are not fully harmonized, despite formal international validation. Furthermore, data showing that the MAT is capable of covering the totality of possible pyrogens relevant to humans were not included in the MAT validations of the last decade. For this review we collate evidence from published literature, unpublished data of our own, and results from the international validation study to show that there is overwhelming scientific evidence to conclude that the whole blood MAT reliably detects non-endotoxin pyrogens. Therefore, further validation exercises do not seem warranted.

Monocyte activation test (MAT) reliably detects pyrogens in parenteral formulations of human serum albumin.

Disadvantages of the regulatory pyrogen test to assure safety of the end-product Human Serum Albumin (HSA) for parenteral use call for the implementation of an alternative test. In the current study, 16 HSA batches were assayed for pyrogens in parallel with the Rabbit Pyrogen Test, conventional and endotoxin-specific LAL assay and monocyte activation test (MAT). It was found that all HSA batches were contaminated with (1,3)-beta-glucans, which interfere with the conventional LAL. Endotoxin-specific LAL was not suitable to test HSA due to unacceptable endotoxin recovery. Experiments combining polymyxin B and MAT demonstrated that pyrogenic batches were mainly contaminated with endotoxins. However, endotoxin-specific LAL failed to detect one of them. The contaminating (1,3)-beta-glucans enhanced the MAT/IL-6 response to endotoxin, but not that of MAT/IL-1beta. The endotoxin equivalent concentrations obtained using the IL-6 readout were usually higher than those using IL-1beta, probably owing to the direct induction of IL-6 release from monocytes by (1,3)-beta-glucans. The MAT correlates with the rabbit pyrogen test, providing a higher safety level for pyrogenicity testing of HSA and probably other therapeutic proteins.

Identification of a possible role for atrial natriuretic peptide in MDMA-induced hyperthermia.

MDMA (3,4-methylenedioxymethamphetamine) induces thermogenesis in a mitochondrial uncoupling protein 3-dependent manner. There is evidence that this hyperthermia is mediated in part by the lipolytic release of free fatty acids, that subsequently activate uncoupling protein 3 in skeletal muscle mitochondria. We hypothesize that atrial natriuretic peptide (ANP), a strong lipolytic mediator, may contribute to the induction and maintenance of MDMA-induced thermogenesis. The specific aims of this study were to (1) determine if ANP is released following MDMA administration, and (2) use the ANP receptor antagonist, Anantin, to ascertain the role of ANP in MDMA-induced hyperthermia. ANP levels were measured in plasma at baseline, 10, 20, 30 and 60 min following MDMA (40 mg/kg, sc) administration in 16 male Sprague-Dawley rats. A robust increase in ANP was seen within 10 min of MDMA administration. ANP levels returned to baseline at 20 min and then gradually rose over the 60 min monitoring period. The administration of Anantin (40 mg, ip), 15 min before and after MDMA, significantly attenuated the MDMA-induced hyperthermia. We conclude that ANP signaling contributes to the hyperthermia induced by MDMA.

Anti-inflammatory, analgesic, and antipyretic activities of virgin coconut oil.

This study investigated some pharmacological properties of virgin coconut oil (VCO), the natural pure oil from coconut [Cocos nucifera Linn (Palmae)] milk, which was prepared without using chemical or high-heat treatment. The anti-inflammatory, analgesic, and antipyretic effects of VCO were assessed. In acute inflammatory models, VCO showed moderate anti-inflammatory effects on ethyl phenylpropiolate-induced ear edema in rats, and carrageenin- and arachidonic acid-induced paw edema. VCO exhibited an inhibitory effect on chronic inflammation by reducing the transudative weight, granuloma formation, and serum alkaline phosphatase activity. VCO also showed a moderate analgesic effect on the acetic acid-induced writhing response as well as an antipyretic effect in yeast-induced hyperthermia. The results obtained suggest anti-inflammatory, analgesic, and antipyretic properties of VCO.

Development, validation and applications of the monocyte activation test for pyrogens based on human whole blood.

Microorganisms such as Gram-negative or Gram-positive bacteria, viruses and fungi contain components that activate the innate immune system. These components, called pyrogens (Greek: pyros=fire), can occur independently of viable microorganisms and are a major safety concern in parenterally administered drugs, since they can cause severe reactions such as fever, organ failure, and shock in the recipient. So far these drugs have been tested by injecting them intravenously into rabbits and measuring their fever reaction or, alternatively, by the Limulus Amoebocyte Lysate (LAL) test, employing the coagulation of the hemolymph lysate of Limulus polyphemus. Both tests have inherent limitations. A new in vitro monocyte activation test (MAT) based on human whole blood, capable of measuring all pyrogens relevant to the human patient, introduced in this journal in 1995, was validated and recently accepted by European Pharmacopoeia and US FDA. This review describes its principle, development, validation and the wide spectrum of applications, such as for testing of medical devices, blood products, toxic or immunomodulatory drugs, dialysis liquids, lipidic parenterals, and air quality. This alternative method promises to replace the rabbit pyrogen test fully and to overcome several limitations of the LAL assay.

[Chemical characteristics and endotoxic activity of the lipopolysaccharide of Rahnella aquatilis 2-95].

The lipopolysaccharide (LPS) from a new Enterobacteriaceae species, Rahnella aquatilis 2-95, was isolated and investigated. The structural components of the LPS molecule, namely, lipid A, core oligosaccharide, and O-specific polysaccharide, were obtained by mild acid hydrolysis. In lipid A, 3-oxytetradecanoic and tetradecanoic acids were found to be the predominant fatty acids. The major monosaccharides of the core oligosaccharide were galactose, arabinose, fucose, rhamnose, and an unidentified component. The O-specific polysaccharide was found to be assembled of a repeated trisaccharide unit of the following structure: [structure: see text]. The R. aquatilis 2-95 LPS is less toxic and more pyrogenic as compared to the one from the R. aquatilis 1-95 strain studied earlier. Both acyl and phosphate groups are essential for toxic and pyrogenic activity of R. aquatilis 2-95 LPS.

An in vitro pyrogen safety test for immune-stimulating components on surfaces.

Due to the discovery of novel surgical techniques and new materials medical devices are increasingly used. Contact of these biomaterials with human tissue or blood commonly leads to inflammation of varying degrees, sometimes resulting in severe health problems. Possible causes are limited biocompatibility or pyrogenic contamination of the material. We adapted the recently validated in vitro pyrogen test (IPT), based on human whole blood cytokine release, to test the safety of biomaterials. Human whole blood is brought into direct contact with the surface of the test material and the release of the pro-inflammatory cytokine IL-1beta is measured. This procedure represents a human-relevant assay allowing the detection of pyrogens of different origins, e.g. Gram-negative (lipopolysaccharide, LPS) or Gram-positive (lipoteichoic acid, LTA), peptidoglycan (cell wall components of most bacteria) and fungal zymosan by direct material contact. The sensitivity of the test system allows a starting concentration of 10 pg/ml for LPS, 10 ng/ml for zymosan and 1 microg/ml for LTA and peptidoglycan from different strains. Furthermore, we have shown that the test for solid materials can be carried out with cryo-preserved blood, which results in an even lower detection limit.