A novel alternative for pyrogen detection based on a transgenic cell line.

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.

The Application of HL60-IL-6 Assay for in vitro Pyrogen Detection of CAR-T Cells Products.

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.

Effect of Homeopathic Medicines and a Nosode on Larvae of Cochliomyia hominivorax (Diptera: Calliphoridae).

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.

Application of a TLR overexpression cell model in pyrogen detection.

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.

The Place of Large Pore Membranes in the Treatment Portfolio of Patients on Hemodialysis.

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.

[Toxicity studies in vivo and in vitro for sheep skin acellular dermal matrix].

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.

Applicability of the Monocyte Activation Test (MAT) for hyperimmune sera in the routine of the quality control laboratory: Comparison with the Rabbit Pyrogen Test (RPT).

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.

Soluble ß-(1,3)-glucans enhance LPS-induced response in the monocyte activation test, but inhibit LPS-mediated febrile response in rabbits: Implications for pyrogenicity tests.

In the present study, we aimed to determine the influence of ß-(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 ß-(1,3)-d-glucans elicited the production of pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α, also known as endogenous pyrogens, but not enough to classify them as pyrogenic according to MAT. The same ß-(1,3)-d-glucans samples were non-pyrogenic by RPT. However, ß-(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, ß-(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 ß-(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.

The human whole blood pyrogen test - lessons learned in twenty years.

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.

Cryopreservation of human monocytes for pharmacopeial monocyte activation test.

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.

Normal temperature variation in New Zealand white rabbits during restraint for preliminary pyrogen test.

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.

Thermal preferences of wintering snails Planorbarius corneus (L.) exposed to lipopolysaccharide and zymosan.

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.

Detection of interleukin -1ß from isolated human lymphocyte in response to lipopolysaccharide and lipoteichoic acid.

AIM: To detect the interleukin -1ß 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 1ß (IL-1ß) release was measured by Sandwich ELISA method. RESULTS: The results indicated that the release of IL-1ß 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-1ß 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-1ß release. CONCLUSION: The isolated lymphocyte system can be used as an alternative to the in vivo rabbit pyrogen assay.

Oxidative stress and pyrogenic fever pathogenesis.

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.

Dissociation between learning and memory impairment and other sickness behaviours during simulated Mycoplasma infection in rats.

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)-1ß 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-1ß, 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.

The antipyretic effect of dipyrone is unrelated to inhibition of PGE(2) synthesis in the hypothalamus.

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.

Ccl22/MDC, is a prostaglandin dependent pyrogen, acting in the anterior hypothalamus to induce hyperthermia via activation of brown adipose tissue.

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.

[Biological activity of native and modified lipopolysaccharides Rahnella aquatilis].

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.

Neurons and glial cells of the rat organum vasculosum laminae terminalis directly respond to lipopolysaccharide and pyrogenic cytokines.

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-1ß (IL-1ß), 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-1ß 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-1ß. 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.

A new cell-based innate immune receptor assay for the examination of receptor activity, ligand specificity, signalling pathways and the detection of pyrogens.

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.