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Pyrogen, often as a contaminant, is a key indicator affecting the safety of almost all parenteral drugs (including biologicals, chemicals, traditional Chinese medicines and medical devices). It has become a goal to completely replace the in vivo rabbit pyrogen test by using the in vitro pyrogen test based on the promoted 'reduction, replacement and refinement' principle, which has been highly considered by regulatory agencies from different countries. We used NF-κB, a central signalling molecule mediating inflammatory responses, as a pyrogenic marker and the monocyte line THP-1 transfected with a luciferase reporter gene regulated by NF-κB as an in vitro model to detect pyrogens by measuring the intensity of a fluorescence signal. Here, we show that this test can quantitatively and sensitively detect endotoxin (lipopolysaccharide from different strains) and nonendotoxin (lipoteichoic acid, zymosan, peptidoglycan, lectin and glucan), has good stability in terms of NF-κB activity and cell phenotypes at 39 cell passages and can be applied to detect pyrogens in biologicals (group A & C meningococcal polysaccharide vaccine; basiliximab; rabies vaccine (Vero cells) for human use, freeze-dried; Japanese encephalitis vaccine (Vero cells), inactivated; insulin aspart injection; human albumin; recombinant human erythropoietin injection (CHO Cell)). The within-laboratory reproducibility of the test in three independent laboratories was 85%, 80% and 80% and the interlaboratory reproducibility among laboratories was 83.3%, 95.6% and 86.7%. The sensitivity (true positive rate) and specificity (true negative rate) of the test were 89.9% and 90.9%, respectively. In summary, the test provides a novel alternative for pyrogen detection., (© 2024. The Author(s).)

Objective: To detect the pyrogen in CAR-T cells product employing the HL60-IL-6 assay., Methods: The HL60 cells were incubated with CAR-T cells injection or endotoxin standard for 48 hours. After then, the secreted cytokine interleukin-6 (IL-6) from HL60 cells was determined by ELISA. According to the four-parameter logistic curve fitted by Optical Density (OD) value corresponding to IL-6 and endotoxin standard concentration, the endotoxin equivalents of pyrogen content in the CAR-T cells products can be measured. Then, the method was validated, including the limit of detection (LOD), limit of quantitation, the recovery rate and the comparison of the determined results by HL60-IL-6 assay with that by the conventional pyrogen test, the Rabbit Pyrogen Test (RPT)., Results: The HL60-IL-6 assay applied to pyrogen test in CAR-T cells products has been established and validated, The LOD was 0.03 EU/mL while the LOQ was 0.07 EU/mL, the recovery rates were 121.4% and 94.5% respectively. The results determined by HL60-IL-6 assay were consistent with that by the RPT., Conclusion: The HL60-IL-6 assay can be employed in CAR-T cell products in vitro pyrogen test., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Cochliomyia hominivorax is the major fly causing primary myiasis in livestock animals in Brazil; its larvae develop in the host's living tissues, causing mutilations, which can even lead to death. In conventional treatments of myiasis, chemo-synthetic insecticides have been employed directly on larvae present in the wounds. Homeopathy may represent a healthy and sustainable alternative both to prevent and to treat myiasis in animals and humans. The current study evaluated how the emergence of adult insects is affected by the use of the homeopathic medicines Sulfur 12cH and Pyrogenium 12cH, and the nosode produced from C. hominivorax larvae at potencies 8cH and 12cH, on third-stage larvae of a C. hominivorax colony., Materials and Methods: The homeopathic medicines and the nosodes were produced according to the Brazilian Homeopathic Pharmacopoeia. Control groups were distilled water, alcohol, no substance, and the organophosphate insecticide trichlorfon. For each group, 10 replicates were performed. Emergence rate of adult insects was evaluated by descriptive statistics followed by analysis of variance. Homogeneity of variances was verified by F -test and group means were compared with Tukey's test., Results: Mortality rates in control groups were 2.7% for 30% (v/v) alcohol, 4.3% for distilled water, 3.2% for no substance ( p  > 0.05). In the trichlorfon group, the mortality rate of larvae was 90.8%. For Sulfur 12cH, the mortality of larvae was 94.6%, and for Pyrogenium 12cH it was 98.6%. The latter three means were not statistically different from each other or from the mean found for the trichlorfon group. The mortality rates of larvae were 61.3% and 66.6% for nosode C. hominivorax 8cH and 12cH, respectively ( p  > 0.05)., Conclusions: Results suggest that homeopathy could be used therapeutically to prevent and treat animals and humans with myiasis caused by C. hominivorax ., Competing Interests: None declared., (The Faculty of Homeopathy.)

Pyrogens are components derived from microorganisms that induce complex inflammatory responses. Current approaches to detect pyrogens are complex and difficult to replicate, thus there is a need for new methods to detect pyrogens. We successfully constructed a pyrogen-sensitive cell model by overexpressing Toll-like receptor (TLR)2, TLR4, MD2, and CD14 in HEK293 cells. Since the cytokine IL-6 is specifically released upon stimulation of the TLR2 and TLR4 signaling pathways in response to pyrogen stimulation, we used it as a read out for our assay. Our results show that IL-6 is released in response to trace amounts of pyrogens in our cell model. Pyrogen incubation times and concentrations were explored to determine the sensitivity of our cell model, and was found to be sensitive to 0.05 EU/ml of LPS and 0.05 ug/ml of LTA after stimulation for 5 hr. Our TLR overexpressing cell model, with IL-6 as readout, could be a new method for in vitro testing of pyrogens and applicable for evaluating the safety of drugs., (© 2019 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.)

Cardiovascular disease is a major concern in patients with end-stage kidney disease (ESKD). Inflammation induced by retention of uremic toxins, of which a substantial fraction has a molecular weight in the middle molecular range, has been associated with increased cardiovascular risk. In an attempt to reduce inflammation and thus cardiovascular toxicity in patients with ESKD, hemodiafiltration (HDF) has been promoted to enhance the clearance of middle molecular weight substances during dialysis. However, HDF increases the technical complexity and costs, and requires ultrapure dialysis fluid. Also, HDF becomes less beneficial when it is not possible to achieve sufficient convective volume in all patients. Over the last years, membranes with larger pore sizes, such as medium cut-off and high cut-off, have been introduced. These membranes, applied in hemodialysis mode, appear to have removal rates of middle molecular weight molecules that are comparable to those achieved with HDF, and could thus obviate the need for HDF. However, ultrapure dialysis fluid might still be required if there is a risk of transmigration of contaminants from the dialysate side into the blood because of the increased pore size. This transmigration of pyrogens might upregulate the expression of cytokines and other pro-inflammatory factors, and thus completely neutralize the beneficial effects of higher clearance of middle molecules. This chapter will explore the existing evidence on permeability of membranes with large pores for bacterial degradation products, and based on this information we will try to define the position of these novel membranes among the spectrum of existing membranes., (© 2017 S. Karger AG, Basel.)

Objective: To detect the toxic reaction degree for sheep acellular dermal matrix (ADM) in vivo or vitro by using hemolytic, pyrogen and cell-cytotoxic reaction experiments, respectively.
 Methods: Leach liquor of cross-linked and non-cross-linked sheep ADMs were set for cross-linked group and non-cross-linked group, respectively, with a positive control group (10 mL sterile water for injection in test tube) and a negative control group (10 mL 0.9% sodium chloride solution in test tube). The supernatants were obtained from each group and were measured for the absorbance. The hemolysis degree was calculated; 16 New-Zealand rabbits were selected and then divided into 4 groups, A, B, C and D group. The leach liquor of cross-linked and non-cross-linked sheep ADMs were injected into bodies of the 6 New-Zealand rabbits in the A and B groups, and then the body temperatures were measured in every half hour after injection, 6 times in total. The value of highest temperature among 6 measurements minus the normal temperature was the fever degree for the body temperature. Based on these fever degree, the criterion of biological pyrogen reaction for sheep ADM pyrogen experiment was evaluated; the mice fibroblasts were collected during logarithmic phase and were cultured in the nutrient medium containing sheep ADM leach liquor with different density. The absorbance was measured to evaluate relative growth rate for fibroblast.
 Results: The hemolysis degree for the group A and B are less than 5%. The summary of fever degree for New-Zealand rabbits were lower than 1.8 ℃. MTT experiment showed that the toxicity of 10%-90% or 100% leach liquor nutrient medium with sheep ADM for the mice fibroblast is at level 1 or level 2. There was no significant difference between leach liquor of cross-linked and non-cross-linked sheep ADMs (P>0.05). The effects on relative growth rate for mice fibroblasts were minor. 
 Conclusion: The hemolytic and pyrogen reactions for the sheep ADMs embedded in New-Zealand rabbit were within the evaluation criterion, and the effects on vitality and growth rate for the fibroblast were not significant.

Pyrogen tests are safety assays performed during the routine quality control of injectable products required by regulatory agencies. Currently, there are three available testing possibilities: 1) the Rabbit Pyrogen Test (RPT); 2) the Bacterial Endotoxin Test (BET); and 3) test systems using human whole-blood or monocytes, termed Monocyte Activation Test (MAT). Although BET is often considered as a replacement for the animal test, it is unable to detect pyrogens other than endotoxin. MAT is based on the human fever reaction and thus, most closely reflects the human response. The aim of this study was to conduct a parallel comparison of the RPT and MAT for hyperimmune sera (HS) batches analyzed during the routine of a quality control laboratory. MAT was performed in the same 43 batches of HS previously tested using RPT. The results showed that MAT presented 100% sensitivity and approximately 85% specificity as compared to RPT, i.e., no false-negative results were obtained. Few suspicious samples, which were negative in the RPT after retesting, provided divergent positive results suggesting a lower limit of detection of MAT. MAT is thus able to detect contaminants in biological products such as HS batches., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In the present study, we aimed to determine the influence of β-(1,3)-d-glucans on the LPS-induced pro-inflammatory cytokine response in the Monocyte Activation Test (MAT) for pyrogens, and on the LPS-induced febrile response in the Rabbit Pyrogen Test (RPT), thus evaluating the resulting effect in the outcome of each test. It was found that β-(1,3)-d-glucans elicited the production of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, also known as endogenous pyrogens, but not enough to classify them as pyrogenic according to MAT. The same β-(1,3)-d-glucans samples were non-pyrogenic by RPT. However, β-(1,3)-d-glucans significantly enhanced the LPS-induced pro-inflammatory cytokines response in MAT, insomuch that samples containing non-pyrogenic concentrations of LPS become pyrogenic. On the other hand, β-(1,3)-d-glucans had no effect on sub-pyrogenic LPS doses in the RPT, but surprisingly, inhibited the LPS-induced febrile response of pyrogenic LPS concentrations. Thus, while β-(1,3)-d-glucans could mask the LPS pyrogenic activity in the RPT, they exerted an overstimulation of pro-inflammatory cytokines in the MAT. Hence, MAT provides higher safety since it evidences an unwanted biological response, which is not completely controlled and is overlooked by the RPT., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The whole blood pyrogen test was first described in this journal exactly twenty years ago. It employs the cytokine response of blood monocytes for the detection of microbiological contaminants with the potential to finally replace the still broadly used rabbit pyrogen test. The article reviews its development process, the current status of the test as well as the challenges and missed opportunities. The article highlights the enormous efforts of many people to get the test to where it is today. But it also shows the incredible missed opportunities for implementation and thus sparing about 400,000 rabbits still used for this purpose per year worldwide; in the EU, since the official acceptance of the test, the number of animals used for pyrogen testing did not fall but increased by about 10,000 to 170,000. The test is the first solution enabling adequate pyrogen testing of cell therapies, including blood transfusions, and medical devices, but has not been implemented for either application by authorities. As the test can quantitatively assess human-relevant airborne pyrogens, the contribution of pyrogens to chronic obstructive lung diseases and childhood asthma can for the first time be defined and home and workplace safety improved in the future.

EU Directive 2010/63/EU regarding the protection of experimental animals came into force in November 2010 with an obligation for EU member states to incorporate its requirements into their respective national legislations by 1st of January 2013. The directive stipulates the application of in vitro methods to replace animal experiments whenever such an in vitro method exits and is recognized by EU legislation. The monocyte activation test (MAT) for the detection and quantification of pyrogenic contamination in medicines is recognized by the European Directorate for the Quality of Medicines & Health Care (EDQM) and was published in the European Pharmacopeia (Pharm. Eur.) in April 2010. The methodology described here facilitates the use of the MAT by making monocytes available, in the form of cryopreserved human peripheral blood mononuclear cells (PBMCs). We have developed and qualified a procedure to prepare functional monocytes in the form of PBMCs from the leukocyte filters that are used for the separation of blood in blood donation centers. Once used, these filters are normally treated as biological waste. Here we describe the procedures that are critical for the successful cryopreservation of PBMCs, demonstrate protection of PBMC functionality using various ligands for the toll-like receptors (TLRs) that mediate pyrogenic responses, report validation of the methodology for linearity, precision and robustness and show examples of the practical application of cryopreserved in MATs with samples of drugs and vaccines. Another application of cryopreserved PBMCs, only mentioned here, is to serve as an alternative to freshly isolated PBMCs in tests for unwanted intrinsic pro-inflammatory activities of new biological therapeutics. Such tests use PBMCs or PBMCs over a layer of endothelial cells to detect (unwanted) cytokine release, PBMCs being more suited to this purpose than tests using whole blood., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

The in vivo pyrogen test is the main toxicological assay used in the quality control of injectable products, especially immunobiologicals. Pharmacopoeias state that, before the main test, a preliminary test must be conducted on all animals, and must follow the same conditions as the main test. The aim of this study was to determine the normal temperature variation in New Zealand white rabbits during restraint and propose a limit value for considering an animal as suitable for testing. Results of the temperature variation in 4,689 rabbits during preliminary tests were obtained from the routine database of the Pharmacology and Toxicology Department of the National Institute of Quality Control in Health (INCQS/FIOCRUZ), Brazil. From these preliminary tests, 3,364 rabbits were considered suitable for testing according to the Brazilian Pharmacopoeia criteria (temperature variation < 0.5 °C). Results showed that about 92 per cent of the rabbits presented a normal individual temperature variation equal to or below 0.30 °C. Animals presenting a temperature variation close to the fever temperature must not be included in the main test, since they can be stressed or sick. Consequently, the temperature variation of 0.30 °C could be adopted by pharmacopoeias as a limit temperature to be considered in the preliminary test to determine which animals can be used in the main rabbit pyrogen test. Animals can be pre-tested until presenting this safe variation, in order to ensure they are healthy and minimise interference in the result.

Fever is regarded as a physiological response to infection both in endothermic and ectothermic animals. In ectotherms, fevers are achieved only behaviorally, and has been described in many vertebrates' and few invertebrates' groups. In snails only symptoms of reverse fever as a response to trematode invasion were found. Present work reports on the effects of two different pyrogens - lipopolysaccharide extracted from Escherichia coli (LPS), and zymosan - from Saccharomyces cerevisiae on the thermal behavior of wintering (studied during a winter season) specimens of the Planorbarius corneus (L.). Using the thermal gradient protocol we demonstrate that the individuals of this snail species responded with behavioral fevers to dosages of pyrogens. LPS injection to the surface of the snail's foot at a dose of 10 μg/g resulted in a significant increase in preferred temperature at 5h after injection. Similarly zymosan at a dose of 0.5 and 1.0 μg/g - caused fever at 8h and 9h respectively. Average temperature chosen by feverish animals after latency period reached 28.7±0.41 °C (LPS), 28.1±0.43 °C (zymosan 1.0 μg/g) or 25.5±0.33 °C (zymosan 0.5 μg/g). We conclude, therefore, that snails are capable of reacting with fever to selected pathogen associated factors, and P. corneus can be used as a model to study a behavioral fever phenomenon in invertebrate animals., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aim: To detect the interleukin -1β levels from single and pooled isolated human lymphocytes in response to lipolysaccharide and lipoteichoic acid., Materials and Methods: Blood collected from healthy individuals (O +ve, A +ve, B +ve, and AB +ve) were subjected to gradient centrifugation to isolate lymphocytes. Different lymphocyte concentrations were used for in vitro pyrogen assay. Lymphocytes isolated were challenged with 5 EU of Gram negative (LPS) and 1 μg/μl of Gram positive (LTA) pyrogens in vitro and the inflammatory cytokine, Interleukin 1β (IL-1β) release was measured by Sandwich ELISA method., Results: The results indicated that the release of IL-1β increases immediately after the initiation of incubation and reaches a maximum at 4 to 6th hour and then stabilizes for both the pyrogens. Furthermore, IL-1β release by 5 EU of LPS and 1 μg/μl of LTA is dependent on lymphocytes concentration. It was also observed that the difference in blood group did not interfere with the IL-1β release., Conclusion: The isolated lymphocyte system can be used as an alternative to the in vivo rabbit pyrogen assay.

The results of the comparative toxicity studies of native lipopolysaccharide (LPS) of Rahnella aquatilis 96U037 and that modified by tin complexes indicates that, due to the modification of LPS by tin complex with benzoylhydrazone of 4-dimethylaminobenzaldehyde, a decrease of its toxicity was observed that led to disappearance of the pyrogenic effect. All obtained derivatives lost completely the antigenic activity both in homologous and heterologous systems which may indicate to the interaction of modifying complexes with certain groups being the components of antigenic determinant. The paper is presented in Ukrainian.

To investigate potential consequences for learning and memory, we have simulated the effects of Mycoplasma infection, in rats, by administering fibroblast-stimulating lipopepide-1 (FSL-1), a pyrogenic moiety of Mycoplasma salivarium. We measured the effects on body temperature, cage activity, food intake, and on spatial learning and memory in a Morris Water Maze. Male Sprague-Dawley rats had radio transponders implanted to measure abdominal temperature and cage activity. After recovery, rats were assigned randomly to receive intraperitoneal (I.P.) injections of FSL-1 (500 or 1000 μg kg(-1) in 1 ml kg(-1) phosphate-buffered saline; PBS) or vehicle (PBS, 1 ml kg(-1)). Body mass and food intake were measured daily. Training in the Maze commenced 18 h after injections and continued daily for four days. Spatial memory was assessed on the fifth day. In other rats, we measured concentrations of brain pro-inflammatory cytokines, interleukin (IL)-1β and IL-6, at 3 and 18 h after injections. FSL-1 administration induced a dose-dependent fever (∼1°C) for two days, lethargy (∼78%) for four days, anorexia (∼65%) for three days and body mass stunting (∼6%) for at least four days. Eighteen hours after FSL-1 administration, when concentrations of IL-1β, but not that of IL-6, were elevated in both the hypothalamus and the hippocampus, and when rats were febrile, lethargic and anorexic, learning in the Maze was unaffected. There also was no memory impairment. Our results support emerging evidence that impaired learning and memory is not inevitable during simulated infection., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The causative/regulatory connections between changes in tissue redox state and fever induction were investigated herein. Wherefore, LPS, the primary element of bacterial cell wall, in addition to inducing pro-inflammatory cytokines, activated macrophages and other leukocytes to secrete hydroxyl radical (OH), nitric oxide metabolites (NO(x)(-)), superoxide (O(2)) and other reactive oxygen/nitrogen species. Furthermore, inflammation response-associated hypoxia stimulated glutamate release, which caused excitotoxicity of cells by increasing extracellular Ca(2+). Cytokines and glutamate in turn also triggered the release of large amounts of NO(x)(-), OH, O(2), and other radicals. Those reactive nitrogen species in turn caused cellular injury via the peroxidation of membrane lipids and oxidative damage of proteins and DNA. Glutamate, NO(x)(-), OH and antioxidants participated in the pathogenesis and regulation of LPS- or cytokines-induced fever. In particular, to highlight the role of glutamate, prostaglandin E(2), NO(x)(-) and OH generated in the hypothalamus during pyrogenic fever was attempted hereby. To find the link among the signaling with the glutamate, NO(x)(-) and OH/prostaglandin E(2) in the hypothalamus during pyrogenic fever will be challenging and could now clinically suppress pyrogenic fever., (Copyright © 2011 Elsevier B.V. All rights reserved.)

CC Chemokine ligand 22 (Ccl22) is a selective, high affinity ligand at the CC chemokine receptor 4 (Ccr4). We have identified cDNAs encoding both ligand and receptor of the Ccl22-Ccr4 pair in cDNA libraries of the anterior hypothalamus/pre-optic area (AH/POA) by PCR. The AH/POA is the key brain region where endogenous pyrogens have been shown to act on warm sensitive neurons to affect thermogenesis in brown adipose tissue (BAT) and other thermogenically responsive tissues. We show that functional Ccr4 receptors are present in the AH/POA neurons as injection of Ccl22 into the POA but not to other hypothalamic nuclei induces an increase in core body temperature as measured by radiotelemetry. Indomethacin (5 mg/kg s.c) pre-treatment markedly reduced the hyperthermia evoked by POA injection of Ccl22 (10 ng/0.5 ul) and thus suggests that this hyperthermia is mediated through cyclooxygenase activation and thus likely through the formation and action of the pyrogen prostaglandin E2. The temperature elevation involves a decrease in the respiratory exchange ratio and increased activation of the brown adipose tissue as demonstrated by ¹⁸F-FDG-PET imaging. We describe a novel role to the ligand Ccl22 and its receptor Ccr4 in the anterior hypothalamus in temperature regulation that depends on the synthesis of the endogenous pyrogen, prostaglandin E2., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background and Purpose: Bacterial lipopolysaccharide (LPS) induces fever through two parallel pathways; one, prostaglandin (PG)-dependent and the other, PG-independent and involving endothelin-1 (ET-1). For a better understanding of the mechanisms by which dipyrone exerts antipyresis, we have investigated its effects on fever and changes in PGE(2) content in plasma, CSF and hypothalamus induced by either LPS or ET-1., Experimental Approach: Rats were given (i.p.) dipyrone (120 mg·kg(-1)) or indomethacin (2 mg·kg(-1)) 30 min before injection of LPS (5 µg·kg(-1), i.v.) or ET-1 (1 pmol, i.c.v.). Rectal temperature was measured by tele-thermometry. PGE(2) levels were determined in the plasma, CSF and hypothalamus by elisa., Key Results: LPS or ET-1 induced fever and increased CSF and hypothalamic PGE(2) levels. Two hours after LPS, indomethacin reduced CSF and hypothalamic PGE(2) but did not inhibit fever, while at 3 h it reduced all three parameters. Three hours after ET-1, indomethacin inhibited the increase in CSF and hypothalamic PGE(2) levels but did not affect fever. Dipyrone abolished both the fever and the increased CSF PGE(2) levels induced by LPS or ET-1 but did not affect the increased hypothalamic PGE(2) levels. Dipyrone also reduced the increase in the venous plasma PGE(2) concentration induced by LPS., Conclusions and Implications: These findings confirm that PGE(2) does not play a relevant role in ET-1-induced fever. They also demonstrate for the first time that the antipyretic effect of dipyrone was not mechanistically linked to the inhibition of hypothalamic PGE(2) synthesis., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

During systemic immune challenge, the organum vasculosum laminae terminalis (OVLT) with its dense vascularization by fenestrated capillaries lacking blood-brain barrier function allows direct access of circulating pyrogens to brain tissue located in close vicinity to the preoptic area. We aimed to analyze direct responses of OVLT cells to exposure to lipopolysaccharide (LPS) and fibroblast-stimulating lipopeptide-1 (FSL-1) or the cytokines tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6. A primary microculture of the OVLT was established from topographically excised rat pup brain tissue, with cellular identification by marker protein-specific immunocytochemistry. Employing the ratio calcium imaging technique, pyrogen-induced calcium signaling in single OVLT cells could be characterized. LPS--as opposed to FSL-1--stimulation caused fast, transient rises in intracellular calcium concentration in 17% of neurons, 9% of astrocytes, and <5% of microglial cells investigated. LPS additionally led to enhanced expression of TNF-α and IL-1β exclusively in microglial cells, as well as a time-dependent release of TNF-α and IL-6 from OVLT microcultures. TNF-α evoked calcium signals in 11% of neurons, 22% of astrocytes, and 5% of microglial cells tested. A considerable population of neurons (11%) but only few astrocytes and microglial cells responded to IL-6, whereas 8% of microglial cells and 3% of astrocytes or neurons were activated by IL-1β. The demonstration of direct cellular responses of OVLT-intrinsic cells to stimulations with LPS or cytokines reinforces the suggested role of this brain structure as a responsive brain site to circulating pyrogens., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The pattern recognition receptors (PRRs) of the innate immune system are the first defence line of the immune system. Toll-like receptors (TLRs) are the most well known and the best examined of the PR receptors. In the last years TLRs had been studied in different ways resulting in a lot of new insights in the function and signalling pathways of these receptors. However, it was not possible to investigate individual combinations of the TLRs and their specific ligands, because of the complex network in immune signalling resulting in interference with each other. This work shows a new cell-based assay, established for the analysis of single PRRs or heterodimers. For this purpose NIH3T3 (mouse fibroblasts) were stably transfected with the NF-kappaB-inducible reporter gene secreted alkaline phosphatase (SEAP) together with the corresponding combinations of human TLRs and their co-receptors (e.g. TLR1/2, TLR2/6 and TLR4/CD14). The specificity of the respective cell lines was shown by induction with variations of specific and unspecific ligands (immune-stimulating components of microorganisms or synthetic ligands). Analysis via the NF-kappaB-dependent reporter gene SEAP allows a direct way to detect the human TLR-activity. Our results showed that this assay is highly sensitive and specific for the respective ligands. For the synthetic ligands Pam(2)CysSK(4) the assay demonstrates a detection limit of 1 pg/ml for TLR2/6. In summary, this test system allows the investigation of individual human PRR-receptors in a highly specific way, without interference with other immune components opening new avenues for novel insights in the innate immune system and its applications., (Copyright 2010 Elsevier B.V. All rights reserved.)

The present study was attempted to determine whether interleukin-1 receptor antagonist (IL-1ra) pretreatment exerts its antipyresis by reducing organum vasculosum laminae terminalis (OVLT) release of glutamate, hydroxyl radicals and prostaglandin E(2) in rabbits. It was found that systemic administration of lipopolysaccharide induced increased levels of both core temperature and OVLT levels of glutamate, hydroxyl radicals, and prostaglandin E(2). The rise in both the core temperature and OVLT glutamate, hydroxyl radicals and prostaglandin E(2) could also be induced by intracerebroventricular injection of interleukin-1beta. Pretreatment with an intracerebroventricular dose of IL-1ra significantly prevented the lipopolysaccharide or IL-1beta-induced overproduction of glutamate, hydroxyl radicals, and prostaglandin E(2) in OVLT of rabbit's brain. The febrile response caused by systemic administration of lipopolysaccharide or central injection of interleukin-1beta could also be IL-1ra pretreatment-ameliorated. These results indicate that IL-1ra pretreatment may exert its antipyresis by inhibiting the glutamate-hydroxyl radicals-prostaglandin E(2) pathways in the OVLT of rabbit's brain during lipopolysaccharide fever., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

We examined the structure of the rat kinin B2 receptor gene (KB2r) and encoding messenger RNA (mRNA) processing. Differently from the closely related mouse and rabbit genes that have three exons and two introns, the rat gene purportedly consists of four exons and three introns. There are two purported gene products; one of them contains an upstream approximately 180-bp open reading frame region ("exon-X") potentially expressed as a result of alternative processing. To examine the processing of rat KB2r mRNA, cDNA amplicons were generated using primer pairs directed towards 5' or 3' exon or intron flanking regions. Analyses of intron/exon primary cDNA amplicons showed that introns 1 to 3 are removed sequentially and that "exon-X" removal follows that of intron-3. No evidence was found for "exon-X" expression in polyadenylated (mature) mRNA of adult Wistar, Wistar Kyoto, spontaneously hypertensive or Sprague-Dawley rat tissues. Nor was "exon-X" detected in tissues subject to inflammatory stimulus expressing B1 kinin receptor mRNA or in 1- to 21-day-old rat embryos or fetuses. The lack of evidence for the expression of "exon-X" in mature mRNA indicates that the structure of the rat gene is similar to that of the mouse, rabbit and human genes, all consisting of three exons and two introns. The "exon-X" fragment may result from interstitial gene duplication, be a fragment of the ancestral gene, or most likely heterologous transposon insertion of an exon-like fragment into intron-2 of the KB2r gene.

Experiments on rats showed that complete Freund's adjuvant-induced low-grade fever and pyrogenal-induced fever of different duration are accompanied by an increase in the content of lipid peroxidation products and components of the antioxidant system not only in blood plasma, but also in the central lymph. These changes reflect activation of drainage, evacuation, and transport function of the lymphatic system. We conclude that the lymphatic system plays an important role in the maintenance of the prooxidant-antioxidant balance under these pathological conditions.

Modified lipopolysaccharides (LPS) of Pragia fontium were obtained with germanium complexes (IV) of 2-aminobenzoylhydrazon of salicylic aldehyde (2-NH2-H2Bs), 2-hydroxybenzoylhydrazon salicylic aldehyde (2-OH-H2Bs) and nicotinoylhydrazon of salicylic aldehyde (H2Ns). The modification of LPS was confirmed by IR spectroscopy. Comparative investigations of pyrogenic activity of native and modified LPS showed, that only P. fontium 20125 LPS, modified by germanium complexes with 2-hydroxybenzoylhydrazon of salicylic aldehyde (2-OH-H2Bs) has lost the pyrogenic activity. In the homological reactions of double immunodiffusion in agar it was shown that all modified LPS unlike the native ones lose completely antigenic activity.

All-trans retinoic-acid (ATRA) differentiated HL-60 cells can be used to detect pyrogens such as bacteria, bacterial components, yeasts and fungi. Differentiated HL-60 cells obtain neutrophil like characteristics and if stimulated the differentiated HL-60 cells produce reactive oxygen species in a dose dependent manner. Culturing and differentiation of cell lines are time consuming activities and require suitable facilities; cryopreservation of pre-differentiated cells could provide the basis for an easily distributable pyrogen testing kit. Cryopreservation of granulocytes has proven to be very complicated and neutrophils are especially difficult to cryopreserve, most likely due to their large degree of granulation. Here we present evidence that HL-60 cells can be differentiated with ATRA and subsequently cryopreserved. Upon thawing the cells retain their ROS producing capabilities and reactivity towards pyrogens. Further, the cells retain their ability to react dose dependently towards lipopolysaccharide (LPS), lipoteichoic acid (LTA) and zymosan. At pathophysiologically relevant concentrations of LPS, LTA and zymosan the cells retain full reactivity for at least two months when stored in liquid nitrogen. In conclusion, ATRA differentiated HL-60 cells are cryopreservable and retain reactivity upon thawing. It is therefore possible to produce an in-vitro in-house pyrogen test kit for medicines and related products.

As opposed to endotoxins very little is known about the heat resistance of Gram positive pyrogens. The aim of this study is to examine the pyrogenic activity of the cell wall components lipoteichoic acid and peptidoglycan from Staphylococcus aureus after moist heat sterilisation. The pyrogenic activity is determined as the ability of the substances to induce interleukin-6 secretion in Mono Mac 6 cells. The standard terminal moist heat sterilisation procedures (121 degrees C for 15min and 134 degrees C for 3min) are not able to inactivate the pyrogenic activity of S. aureus lipoteichoic acid and peptidoglycan. However after longer treatment times the pyrogenic activity of lipoteichoic acid is removed at both 121 degrees C and 134 degrees C. In contrast the activity of peptidoglycan is not removed after 160min at neither 121 degrees C nor 134 degrees C where only a 2-log reduction is obtained. In conclusion the terminal moist heat sterilisation procedures described by the European Pharmacopoeia are not able to inactivate the interleukin-6 inducing activity of S. aureus lipoteichoic acid and peptidoglycan.

Given the shortcomings in the measurement of pyrogenic contamination of pharmaceuticals and/or test substances by means of the rabbit pyrogen test and the Limulus amoebocyte lysate (LAL) test, several in vitro pyrogen tests have been developed based on the measurement of cytokine production by monocytes. In this study we measured cytokine production (IL-6, IL-8, IL-1beta, and TNF) in diluted whole blood (WB), mononuclear cells (MNC), and HEK cells stably transfected with CD14 and Toll-like Receptor-2 (TLR2) or TLR4, after stimulation with both standard pyrogens and contaminated substances. Our study demonstrated that in MNC, IL-6 production was more sensitive to pyrogen stimulation than IL-1beta and TNF production. The sensitivity of WB IL-8 production for pyrogens was comparable with that of MNC IL-6 production, but higher than WB IL-6 production. MNC IL-8 production as readout for pyrogenic stimulation was not useful due to high background IL-8 production. Surprisingly, contaminated culture media potently stimulated WB IL-8 production, but not MNC IL-6 production. Finally, the value of TLR-transfected HEK cells in the detection of pyrogenic contamination as well as the role of IL-10 in interindividual differences in cytokine production, is discussed. To summarize, the results presented herein together with literature data indicate that the measurement of WB IL-8 production may represent an advantageous alternative to the measurement of MNC IL-6 production, for the detection of pyrogenic contamination of pharmaceuticals.

In mammals, procedures such as handling, restraint, or exposure to open spaces induces an increase in body temperature (T(b)). The increase in temperature shares some characteristics with pyrogen-induced fever and so is often called "stress fever." Birds also respond to acute handling with a stress fever, which may confound thermoregulatory studies that involve animal restraint. We have measured the T(b) responses of Pekin ducks on days when they were restrained and compared them to days when the birds remained unrestrained. Restraint induced a 0.5 degrees C increase in T(b) that was sustained for the entire 8 h of restraint. To determine whether the restraint-induced increase in T(b) is mediated by prostaglandins (PGs) we compared the T(b) responses during restraint after intraperitoneal injection with saline to the responses during restraint after injection with diclofenac sodium (15 mg/kg). There was no difference in response, suggesting that restraint affects T(b) by a PG-independent mechanism. We also compared the T(b) response to intramuscular injection of lipopolysaccharide (LPS; 100 microg/kg), a bacterial pyrogen, when the ducks were restrained or unrestrained. Despite T(b) being higher at the time of LPS injection when the ducks were restrained, the maximum temperature reached after LPS injection was higher, and the period that T(b) remained elevated was longer when the ducks were unrestrained. We conclude that restraint should be considered as a potential confounder in thermoregulatory studies in birds and presumably other species too.

The human whole blood IL-1 test exploits the reaction of monocytes/macrophages for the detection of pyrogens: human whole blood taken from healthy volunteers is incubated in the presence of the test sample in any form, be it a solution, a powder or even solid material. Pyrogenic contaminations initiate the release of the "endogenous pyrogen" Interleukin-1beta determined by ELISA after incubation. In order to understand any differences between the pyrogenic activity in this test and the existing live rabbit test (species differences versus aberrant response of the particular blood sample), the rabbit whole blood test was developed. This approach could also help to avoid the use of putatively infectious human blood for pyrogen testing in vitro.

The European Partnership for Alternative Approaches to Animal Testing (EPAA) pointed out the need to involve authorities throughout the process of validation and legal acceptance of alternatives to animal experiments. The Paul-Ehrlich-Institute (PEI), Federal Agency for Sera and Vaccines, is the national competent authority in Germany which is responsible for the quality and safety of biologicals including blood and cell-based products. This paper is intended to contribute to the discussion concerning the use of alternative methods in safety testing of medicinal products and considers the scientific work of the PEI in this field. From a regulator's perspective, adequate demonstration of safety and quality of medicinal products are of major interest. Additionally, the availability of the products to the patient has to be taken into consideration. It has to be carefully explored whether the respective in vitro method for demonstration of non-clinical safety as part of the non-clinical development programme is able to guarantee safety level comparable to the corresponding experiment in animals. The topics cited above shall be discussed in this paper using the example of the Alternative Pyrogen Test or also called Monocyte Activation Test. The Alternative Pyrogen Test could serve as paradigm to exemplify how an alternative test can provide at least a comparable level of safety estimation in comparison with a conventional animal test. Furthermore, this alternative test creates additional information which cannot be obtained from the animal experiment, and might also open further scientific insight into the mechanisms of pyrogenicity and acute pro-inflammatory reactions in patients. This test method allows the definition of pyrogen limits for medicinal products. Due to its use of relevant cell systems this in vitro test might contribute significantly to safety assessments of advanced medicinal products during the pre-clinical phase.

Bacterial DNA containing unmethylated cytosine-phosphate-guanosine motif (CpG-DNA) has been identified as a pathogen-associated molecular pattern, which is recognized by Toll-like receptors and activates immune cells to produce cytokines. The aim of the study was to characterize the ability of CpG-DNA to induce fever in mice. Intravenous administration of unmethylated CpG-DNA 1826 triggered an elevation of body temperature (T(b)) lasting several hours. The magnitude of T(b) elevation increased with an increase of dose of the oligonucleotide (administered in a range from 0.01 mg/kg to 1.0 mg/kg). A fever-like increase of T(b) in mice was partially dependent on IL-6, as IL-6 deficient mice responded with reduced fever to the CpG-DNA 1826. Meloxicam and sulindac sulfide, inhibitors of cyclooxygenases, reduced fever in mice challenged with CpG-DNA 1826, indicating that the process may also depend on prostaglandins. In fact, plasma levels of prostaglandin E(2), as well as IL-6, increased at 4 h postinjection of CpG-DNA 1826 into mice. These data demonstrate that the pathophysiological mechanism of the increase of T(b) induced by CpG-DNA 1826 is similar to fever induced by LPS. Both LPS and CpG-DNA 1826 failed to produce elevation of T(b) in mice deficient for a nuclear factor-kappaB (NF-kappaB) gene, further supporting the hypothesis that the two pyrogens provoke fever, using the same components of the cellular signaling metabolism. However, parthenolide, an inhibitor of I-kappaB kinase reduced fever due to CpG-DNA 1826, and did not affect fever to LPS, suggesting that the two structurally dissimilar pyrogens may affect different intracellular pathways leading to the upregulation of NF-kappaB. In support of this hypothesis, we demonstrate that C3H/HeJ mice, known to exhibit a mutation in the Toll-like receptor-4 gene, do not respond with fever to LPS. They respond, however, with fever after injection of CpG-DNA 1826. We conclude that bacterial DNA shares with components of the bacterial wall the capacity to elicit fever and may, consequently, be part of a novel class of exogenous pyrogens.

Blockade of central endothelin ET(B) receptors inhibits fever induced by LPS in conscious rats. The contribution of ET(B) receptor-mediated mechanisms to fever triggered by intracerebroventricular IL-6, PGE2, PGF(2alpha), corticotropin-releasing factor (CRF), and preformed pyrogenic factor derived from LPS-stimulated macrophages (PFPF) was examined. The influence of natural IL-1 receptor antagonist or soluble TNF receptor I on endothelin (ET)-1-induced fever was also assessed. The selective ET(B) receptor antagonist BQ-788 (3 pmol icv) abolished fever induced by intracerebroventricular ET-1 (1 pmol) or PFPF (200 ng) and reduced that caused by ICV CRF (1 nmol) but not by IL-6 (14.6 pmol), PGE2 (1.4 nmol), or PGF(2alpha) (2 nmol). CRF-induced fever was also attenuated by bosentan (dual ET(A)/ET(B) receptor antagonist; 10 mg/kg iv) but unaffected by BQ-123 (selective ET(A) receptor antagonist; 3 pmol icv). alpha-Helical CRF(9-41) (dual CRF1/CRF2 receptor antagonist; 6.5 nmol icv) attenuated fever induced by CRF but not by ET-1. Human IL-1 receptor antagonist (9.1 pmol) markedly reduced fever to IL-1beta (180 fmol) or ET-1 and attenuated that caused by PFPF or CRF. Murine soluble TNF receptor I (23.8 pmol) reduced fever to TNF-alpha (14.7 pmol) but not to ET-1. The results of the present study suggest that PFPF and CRF recruit the brain ET system to cause ET(B) receptor-mediated IL-1-dependent fever.

Fever is induced by a neuronal mechanism in the brain. Prostaglandin (PG) E2 acts as a pyrogenic mediator in the preoptic area (POA) probably through the EP3 subtype of PGE receptor expressed on GABAergic neurons, and this PGE2 action triggers neuronal pathways for sympathetic thermogenesis in peripheral effector organs including brown adipose tissue (BAT). To explore pyrogenic efferent pathways from the POA, we determined projection targets of EP3 receptor-expressing POA neurons with a special focus on rat hypothalamic regions including the dorsomedial hypothalamic nucleus (DMH), which is known as a center for autonomic responses to stress. Among injections of cholera toxin b-subunit (CTb), a retrograde tracer, into hypothalamic regions at the rostrocaudal level of the DMH, injections into the DMH, lateral hypothalamic area (LH) and dorsal hypothalamic area (DH) resulted in EP3 receptor immunolabelling in substantial populations of CTb-labeled neurons in the POA. Bilateral microinjections of muscimol, a GABA(A) receptor agonist, into the DMH and a ventral region of the DH, but not those into the LH, inhibited thermogenic (BAT sympathetic nerve activity, BAT temperature, core body temperature and expired CO2) and cardiovascular (arterial pressure and heart rate) responses to an intra-POA PGE2 microinjection. Further immunohistochemical observations revealed a close association of POA-derived GABAergic axon swellings with DMH neurons projecting to the medullary raphe regions where sympathetic premotor neurons for febrile and thermoregulatory responses are localized. These results suggest that a direct projection of EP3 receptor-expressing POA neurons to the DMH/DH region mediates febrile responses via a GABAergic mechanism.

Background: Formulations of amphotericin include a deoxycholate suspension (d-Amph), an amphotericin-B lipid complex (Ablc), and a liposomal product (L-Amph). Fever is most frequent with d-Amph, intermediate with Ablc, and lowest with L-Amph., Objective: To determine if the release of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1) from brain endothelium corresponds to the incidence of amphotericin fever., Results: Release of TNF-alpha and IL-1beta after L-Amph treatment was similar to negative controls while after d-Amph treatment release was similar to lipopolysaccharide. Ablc treatment produced intermediate pyrogen release.NF-kappaB expression, a transcriptional regulator for TNF-alpha and IL-1beta genes, corresponded to this secretion pattern. TNF-alpha release was elevated 2 hours (p = 0.0021) after treatment while significant elevations in IL-1beta required 6 hours (p = 0.0009)., Conclusion: Results from this in vitro study suggest that amphotericin fever may be directly mediated by brain endothelium. These experiments also suggest that amphotericin fever is initially mediated by TNF-alpha.

The lipopolysaccharide from the freshwater bacterium Rahnella aquatilis 1-95 has been isolated and investigated for the first time. The structural components of the lipopolysaccharide molecule: lipid A, core oligosaccharide, and O-specific polysaccharide were isolated by mild acidic hydrolysis. In lipid A, 3-hydroxytetradecanoic and tetradecanoic acids were found to be the predominant fatty acids. In the core oligosaccharide, galactose, arabinose, fucose, and an unidentified component were shown to be the major monosaccharides. The O-specific polysaccharide consists of a regularly repeating trisaccharide unit with the acyl and phosphate following structure: [structure: see text] groups have been shown to be responsible for the toxic and pyrogenic properties of the lipopolysaccharide of R. aquatilis.

We have earlier found that a trivalent vaccine, containing antigenic components from both Gram-positive and Gram-negative bacteria, induced secretion of the endogenous pyrogen interleukin 6 (IL-6) when added to fresh human blood in vitro. The results of the present study showed that the IL-6 secretion was induced by toxoids derived from the Gram-positive bacterium Corynebacterium diphtheriae. However, fresh whole blood from different donors reacted differently to the stimulation. The blood from some donors induced secretion of large concentrations of IL-6, while the blood from other donors induced essentially no IL-6 secretion as a response to stimulation with diphtheria toxoid or a mixture of diphtheria and tetanus toxoids. Repeated testing over several years using blood from the same donor confirmed a donor-dependency of the reaction. This donor-dependency was only found for the toxoid, since blood from all donors reacted with approximately similar IL-6 production to stimulation by endotoxin from the Gram-negative bacterium Escherichia coli, known to be mediated via the toll-like receptor (TLR) 4. Also, no donor-dependecy was found to highly purified lipoteichoic acid from the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus, known to be mediated via TLR-2 and TLR-6. The receptors involved in stimulation by diphtheria toxoid are not known, but may differ from those used by endotoxin and lipoteichoic acid.

TRPV1, the capsaicin receptor, is expressed not only in nociceptive neurons, but also in other locations, including the hypothalamus. Studies involving systemic or intrahypothalamic capsaicin administration have suggested a role for TRPV1 in body temperature control. To explore this possibility, we examined thermoregulatory responses in TRPV1-/- mice. These mutant animals exhibited no obvious changes in circadian body temperature fluctuation, tolerance to increased (35 degrees C) or decreased (4 degrees C) ambient temperature or ethanol-induced hypothermia. In contrast, fever production in response to the bacterial pyrogen, lipopolysaccharide (LPS) was significantly attenuated in TRPV1-/- mice. Despite this finding, we detected no significant differences between TRPV1-/- and control mice in the extent of LPS-induced c-Fos expression in numerous fever-related brain subregions. These results suggest that TRPV1 participates in the generation of polyphasic fever, perhaps at sites outside the brain.

Human whole blood assays are increasingly employed to test immune function or detect pyrogenic contamination, since they offer advantages, such as ease of performance, few preparation artifacts and a physiological cell environment. However, the approach is often limited by the availability of freshly drawn blood, putative safety concerns in the case of infected donors and interindividual donor differences. To overcome these limitations, a method was developed and optimized to produce batches of cryopreserved blood that can be used directly after thawing without any washing steps. Mononuclear cells remained intact as shown by FACS analysis. Cytokine release could be induced by a variety of immunological stimuli. The cell preparation released higher amounts of interleukin-1beta (IL-1beta) and IL-6 compared to fresh blood, but no TNF. These differences could be attributed to the presence of the cryoprotectant dimethylsulfoxide (DMSO) alone by addition of DMSO to fresh blood. Large batches of cryopreserved blood could be produced by mixing blood donations of up to 10 donors, independent of differing blood groups. The detection limit for the World Health Organization (WHO) lipopolysaccharides (endotoxin, LPS) reference preparation (EC-6) with regard to the induction of IL-1beta release was at least 0.5 endotoxin equivalent units (EU)/ml. Endotoxin spikes at the limit concentrations prescribed in the European Pharmacopoeia could be detected in a series of drugs, showing that the In vitro Pyrogen Test (IPT) can also be run with cryopreserved blood. Further possible applications include high-throughput screening for immunomodulators or toxins as well as preservation of patient samples for later analysis of cell functions.

Most experimentally induced fevers are acute, usually lasting approximately 6-12 h, and thus do not mimic chronic natural fevers, which can extend over several days or more. To produce a model of chronic natural fever, we infused eight goats (Capra hircus) intravenously with 2 ml of 2 x 10(11) cell walls of Staphylococcus aureus (S. aureus) for 6 days using osmotic infusion pumps (10 microl/h) while measuring changes in body temperature, behavior, and plasma cortisol concentration. Seven control animals were infused with sterile saline. Abdominal temperature-sensitive data loggers and osmotic infusion pumps were implanted under halothane anesthesia. To compare our new model with existing models of experimental fever, we also administered 2-ml bolus intravenous injections of 2 x 10(11) S. aureus cell walls, 0.1 microg/kg lipopolysaccharide (Escherichia coli, serotype 0111:B4), and sterile saline in random order to six other goats. Bolus injection of lipopolysaccharide and S. aureus induced typical acute phase responses, characterized by fevers lasting approximately 6 h, sickness behavior, and increased plasma cortisol concentration. Infusion of S. aureus evoked prolonged fevers, which lasted for approximately 3 days, starting on day 4 of infusion (ANOVA, P < 0.05), and did not disrupt the normal circadian rhythm of body temperature. However, pyrogen infusion did not cause plasma cortisol concentration to rise (ANOVA, P > 0.05) or the expression of sickness behavior. In conclusion, infusion of S. aureus produced a fever response resembling that of sustained natural fevers but did not elicit the cortisol and behavioral responses that often are described clinically and during short-term experimental fevers.

For many years, it was thought that bacterial products caused fever via the intermediate production of a host-derived, fever-producing molecule, called endogenous pyrogen (EP). Bacterial products and other fever-producing substances were termed exogenous pyrogens. It was considered highly unlikely that exogenous pyrogens caused fever by acting directly on the hypothalamic thermoregulatory center since there were countless fever-producing microbial products, mostly large molecules, with no common physical structure. In vivo and in vitro, lipopolysaccharides (LPSs) and other microbial products induced EP, subsequently shown to be interleukin-1 (IL-1). The concept of the 'endogenous pyrogen' cause of fever gained considerable support when pure, recombinant IL-1 produced fever in humans and in animals at subnanomolar concentrations. Subsequently, recombinant tumor necrosis factor-alpha (TNF-alpha), IL-6 and other cytokines were also shown to cause fever and EPs are now termed pyrogenic cytokines. However, the concept was challenged when specific blockade of either IL-1 or TNF activity did not diminish the febrile response to LPS, to other microbial products or to natural infections in animals and in humans. During infection, fever could occur independently of IL-1 or TNF activity. The cytokine-like property of Toll-like receptor (TLR) signal transduction provides an explanation by which any microbial product can cause fever by engaging its specific TLR on the vascular network supplying the thermoregulatory center in the anterior hypothalamus. Since fever induced by IL-1, TNF-alpha, IL-6 or TLR ligands requires cyclooxygenase-2, production of prostaglandin E2 (PGE2) and activation of hypothalamic PGE2 receptors provides a unifying mechanism for fever by endogenous and exogenous pyrogens. Thus, fever is the result of either cytokine receptor or TLR triggering; in autoimmune diseases, fever is mostly cytokine mediated whereas both cytokine and TLR account for fever during infection.

Two different in vitro tests for pyrogens, using human peripheral blood monocytes (PBMNC) and diluted whole blood (WBC), respectively, were applied to different classes of parenteral medicinal products. Many of these products did not have a specified endotoxin limit concentration that was established as the maximum valid dilution to comply with the test. The results of the in vitro tests for pyrogens were compared with the results from the Limulus amoebocyte lysate (LAL) and rabbit pyrogen tests. The Second International Standard for endotoxin was used to calibrate all of the assays and the International Standard for IL-6 was used to calibrate the IL-6 ELISA which provided the readout for the in vitro tests for pyrogens. Preparatory tests were conducted to ensure that the "criteria for validity and precision of the standard curve" were satisfied and that the drugs being tested did not interfere in the tests. The PBMNC/IL-6 test had a detection limit of 0.06 EU/ml and spike recoveries were 62-165%. The whole blood/IL-6 test also had a detection limit of 0.06 EU/ml and spike recoveries were 58-132%. The application to the detection of non-endotoxin pyrogens needs to be evaluated in more detail, but the two in vitro tests for pyrogens showed good agreement overall, both with each other and with the LAL test and the rabbit pyrogen test for the detection of endotoxins.

At first, we investigated whether both beta-endorphin release level in the hypothalamus and body temperature can be altered after intracerebroventricular (i.c.v.) injection of either lipopolysaccharide (LPS), interleukin-1beta (IL-1beta), or prostaglandin E(2) (PGE(2)) in rats. It was found that in the rat, i.c.v. administration of either LPS (0.5 microg in 10 microl), IL-1beta (10 ng in 10 microl), or PGE(2) (200 ng in 10 microl), in addition to producing fever, upregulated the immunoreactivity of beta-endorphin in the preoptic anterior hypothalamus of rat brain. Secondarily, we assessed whether the fever induced by either LPS, IL-1beta, or PGE(2) can be altered by pretreatment with buprenorphine (an opioid receptor antagonist). The results revealed that i.c.v. administration of buprenorphine (1 - 10 microg in 10 microl) alone had an insignificant effect on the body temperature. However, the fever induced by i.c.v. injection of either LPS, IL-1beta, or PGE(2) was significantly attenuated by pretreatment with i.c.v. injection of buprenorphine 1 h before the pyrogen injection in rats. The results suggest that pyrogens enhance beta-endorphin release in the hypothalamus and trigger fever which can be attenuated by buprenorphine, an opioid receptor antagonist.

The objective of this study was to explore whether and how ovarian hormones interact with the febrile response to pyrogens. Estrogen and progesterone treatment of ovariectomized rats was associated with a reduction in lipopolysaccharide (LPS)-induced fever, compared with ovariectomized controls. LPS-fever reduction was accompanied by reduced levels of the inducible cyclooxygenase-2 (COX-2) protein expression in the hypothalamus as well as reduced plasma levels of IL-1beta. The amount of LPS-induced IL-6 in the plasma was not affected by ovarian hormone replacement. In contrast, hypothalamic COX-2 expression in response to intraperitoneal injection of IL-1beta was potentiated by the ovarian hormone replacement. IL-1beta induced a moderate increase in plasma levels of IL-6 that was suppressed by ovarian hormone replacement. These data suggest that ovarian hormone replacement attenuated the proinflammatory response to LPS by suppressing the LPS-induced IL-1beta production and COX-2 expression in the hypothalamus. The markedly different action of ovarian hormones on IL-1beta and LPS effects suggests that this sex hormone modulation of the immune response is a function of the nature of infection and provides further evidence that LPS actions are different from those of IL-1beta.

The European Pharmacopeia (EP) and the United States Pharmacopeia (USP) pyrogen test for parenterals involve the measurement of the temperature rise in rabbits following the intravenous injection of the test solution. Between the two methods, the conditions for the result "pyrogen free" or "pyrogen", as well as the number of rabbits tested, differ substantially. Therefore, both methods were compared with respect to the differences in temperature rise interpretations with special attention to the selectivity of the tests. To exclusively study the temperature rise interpretations, the temperature measurements were simulated with models. With an increasing mean temperature rise, an intermediate range exists where the probability of "pyrogen free" and of "pyrogen" cannot be ignored. A single pyrogen test still gives an unambiguous result, but in this range, the probability is rather high that additional tests will not reproduce the same result. This range is approximately 0.3 degrees C wide for the USP test and 0.2 degrees C wide for the EP test. Simulating the temperature rise with a normally distributed model, the intermediate range is narrower for the interpretation according to the EP than the USP. For a USP test, the probability of the test result "pyrogen free" decreases as lower mean temperature rises. However, the two methods are similar in the high probability range for the presence of pyrogens. At the edge of the intermediate range, a slight mean temperature rise of 0.05 degrees C significantly changes the probability of a pyrogen result. Within a rather wide mean temperature rise range (approximately 0.15 degrees C wide), a product will reliably fulfil the EP requirements but not the USP requirements. These main statements are rather robust and do not depend significantly on the model used. Different temperature rise distributions only result in negligible variations in the interpretation of the tests. Information more detailed than mean temperature rise and standard deviation of the temperature rises is not absolutely necessary. For product quality, it is much more useful to review and interpret all individual temperature rises measured than to interpret the results of the pyrogen test only [corrected].

A rapid, 'one-plate' monocyte-activation test is described for detecting endotoxin and non-endotoxin pyrogens in parenteral medicinal products. The one-plate test offers useful gains over conventional 'two-plate' (cell culture plate+ELISA plate) tests in terms of its limit of detection, robustness, speed and cost. The 'one-plate' test is likely to be applicable to a wide range of products because it allows less time for product interference in the test. The 'one-plate' test utilises pyrogen-free anti-cytokine (interleukin (IL)-6 or tumour necrosis factor alpha (TNFalpha)) antibodies (Ab), coated and stabilised onto (pyrogen-free) 96-well plates. Monocytes/monocytic cells, endotoxin (lipopolysaccharides, LPS) standard or sample and (pyrogen-free) second (labelled) Ab are cultured together (usually for 2-4 h) on the Ab-coated plate and then the plate is washed and the ELISA completed. There is no transfer from one plate to another and no (further) incubations of (released) cytokine with, first, coating Ab and, then, developing Ab since these steps have already taken place during the initial cell culture. The rapid, 'one-plate' test is readily automated. The preferred readout is IL-6, which gives a limit of detection of 0.015 endotoxin units (EU)/ml with peripheral blood mononuclear cell (PBMNC), 0.03 EU/ml with diluted whole blood and 0.05 EU/ml with a monocytic cell line (MONO MAC 6).

A comparison between humans and rabbits was performed based on stimulation of whole blood with well-known pyrogens from Gram-negative and Gram-positive bacteria, such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA), respectively. The reactivity was measured as release of IL-1 beta and IL-8 by ELISA. The reactivity of the two species towards LPS was found to be similar, whereas their reactivity towards LTA differed considerably. Differences between the levels of IL-1 beta and IL-8 release were observed in both species. This finding suggests that the In vitro Pyrogen Test (IPT) which uses human blood to detect contaminations, e.g. of injectable drugs, might predict the human reaction to the contamination better than the "gold standard" rabbit pyrogen test.

The Limulus amoebocyte lysate (LAL) test has replaced about 80% of the use of the rabbit pyrogen test. Ideally, human-based in vitro tests are needed, to replace the remaining use of the rabbit test and the use of the LAL test. The progress of an EU-funded project is described, in which a number of in vitro tests, based on the human fever reaction are passing through a prevalidation study on the way to evaluation in a formal validation study.

It has been proposed that prostaglandin (PG)E(2) production via a process catalyzed by the inducible isoform of cyclooxygenase (COX)-2 and activation of specific PGE(2) receptor subtypes within the preoptic/anterior hypothalamus (AH/POA) is the last step and unique pathway in the induction of a fever. However, many data support the existence of a PG-independent pathway. That is, other more rapid mechanisms, which involve the constitutive COX-1 isozyme, may be more critical for a PG-dependent fever. Thus, we examined the role of both COX isoforms in the AH/POA in fevers induced by macrophage inflammatory protein (MIP)-1beta, a PG-independent pyrogen, and RANTES (regulated on activation, normal T-cells expressed and secreted), a PG-dependent pyrogen. In freely moving rats, two independent polyclonal antibodies were used which neutralize COX-1 and COX-2. The microinjection of either MIP-1beta or RANTES into the pyrogen-sensitive region of the AH/POA induced an intense fever of rapid onset. Peripheral pretreatment with an antipyretic dose of dexamethasone which prevents COX-2 expression, or the microinjections into the AH/POA of either anti-COX-1 or anti-COX-2, blocked the febrile response induced by RANTES but not that induced by MIP-1beta. These results provide strong evidence for the existence of rapid mechanisms in the AH/POA which involve both COX isozymes during the fever induced by RANTES, and further support the existence of an alternative PG-independent pathway in the febrile response., (Copyright 2002 Elsevier Science Inc.)