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PubMed2001

Role of the hepatic function in the development of the pyrogenic tolerance to muramyl dipeptide.

We have demonstrated that the hepatic function may have an important role in the development of tolerance to the pyrogenic effect induced by endotoxin. To further investigate if the role of the hepatic function in the development of tolerance also extends to that induced by other pyrogenic stimuli, we investigated the effect of galactosamine, a specific inhibitor of the hepatic protein synthesis, on the development of tolerance to the pyrogenic effect induced by muramyl dipeptide (MDP) in rats. Pyrogenic tolerance was observed after the second intravenous or intraperitoneal injection of MDP (500 microgram/kg), 24 h after the first injection, similar to what was observed with endotoxin. Pyrogenic tolerance was abolished when galactosamine (300 mg/kg ip) was injected simultaneously with MDP (500 microgram/kg iv) on the first day. When uridine (600 mg/kg ip) was administered simultaneously with galactosamine (300 mg/kg ip) and the first injection of MDP (500 microgram/kg ip), pyrogenic tolerance was again observed after the second injection of the peptidoglycan. In conclusion, the hepatic function may not be important only for the development of tolerance to endotoxin, but also to a totally different pyrogenic stimulus such as MDP.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1985

Comparison of febrile responsiveness of rats and rabbits to endogenous pyrogen.

The fever responses of rats and rabbits were compared in detail using a single common source of semipurified endogenous pyrogen prepared from human monocytes. The characteristics and dynamics of the fever-response curves for each species were examined and their dose-response curves were determined and compared. The fevers displayed by rats were qualitatively similar to those of rabbits, but, typically, they developed and terminated more rapidly than those of rabbits. Rabbits were much more sensitive to the endogenous pyrogen than rats. The threshold dose of pyrogen required to elicit a fever was 5 times lower in the rabbit, and the slope of the rabbit's dose-response curve was 1.5 times steeper than that of the rat. The maximum fevers attainable in rabbits were approximately twice those attainable in rats. It was also shown that the more rapid febrile responses of the rat were not due to the 10-fold smaller mass of the rat; instead, we proposed that this difference was more likely due to a closer diffusional proximity of the pyrogen receptor sites to the circulation in rats. The lower sensitivity of the rat to endogenous pyrogen was attributed to a relative insensitivity of the pyrogen receptor sites in rats in the translation of the endogenous pyrogen stimulus into fever.

Animals↗

PubMed1977

Factors affecting pyrogen testing in rabbits.

A response of rabbits to toxins causing fever and originating from microorganisms may be related to different factors. In this respect the race and the age of the animals were examined as variables. Three rabbit strains of local origin were used: New Zealand White, White of Dendermonde and Holland race. As pyrogen preparations sterile surface water and preparations of E. coli strains were used. A bacterial count, as for ordinary drinking water examination, was carried out in order to evaluate the presence of bacteria to pyrogenicity. An attempt was made to remove the pyrogens by absorption on charcoal and by filtration through asbestos filters. A decrease of pyrogenicity was obtained but the fever toxins did not completely disappear with the simple procedure. The stability of the toxins found in surface waters was examined over a short range of preservation. Only a slow decrease in pyrogenic activity was seen. Pyrogens of E. coli were prepared in vitro, but used unpurified. A slight difference in the strains was observed, but all E. coli's were pyrogenic.

Age Factors↗

PubMed1988

Characteristics and applications of adsorbents for pyrogen removal.

Characteristics and applications of immobilized histidine and immobilized histamine for pyrogen removal were investigated. Immobilized histidine showed a high affinity for pyrogen at low ionic strength and over a wide pH range. The adsorption capacity was 0.53 mg of lipopolysaccharide per milliliter of the adsorbent. The apparent dissociation constant was 1.57 X 10(-9) M. The adsorption of pyrogen to immobilized histidine decreased with increasing ionic strength, but pyrogen could be adsorbed even at ionic strengths of gamma/2 = 0.05-0.1, at which other substances were little adsorbed; that is, specific adsorption of pyrogen was observed. The adsorption of pyrogen could be increased at ionic strengths of gamma/2 = 0.05-0.1 by using a lower flow rate or a longer column length. Immobilized histidine and immobilized histamine could be used for the removal of natural pyrogens contaminating various useful low-molecular-weight compounds as well as high-molecular-weight compounds such as proteins.

Adsorption↗

PubMed1974

Pyrogen release in vitro by lymphoid tissues from patients with Hodgkin's disease.

The mechanism of fever in patients with Hodgkin's disease was investigated by examining endogenous pyrogen production by blood, spleen, and lymph node cells incubated in vitro. Blood leucocytes from febrile or afebrile patients with Hodgkin's disease did not produce pyrogen spontaneously. Spleen cells, however, frequently released pyrogen during initial incubations, unlike spleen cells from patients with non-malignant diseases. Pyrogen production occurred from spleens without observed pathologic infiltrates of Hodgkin's disease. Lymph nodes involved with Hodgkin's disease produced pyrogen more frequently than did nodes involved with other diseases. Pyrogen production by tissue cells was prolonged, required protein synthesis, and in some cases was due to mononuclear cells; it did not correlate with fever in the patient. These studies demonstrate spontaneous production of endogenous pyrogen in vitro by lymphoid tissue cells from patients with Hodgkin's disease.

Animals↗

PubMedDate not supplied

Destruction of typical endotoxins by dry heat as determined using LAL assay and pyrogen assay.

The kinetics of destruction by dry heat of two typical endotoxins, Escherichia coli 055:B5 (E. coli endotoxin) and Salmonella abortus equi (S. abortus equi endotoxin), were determined using the Limulus Amebocyte Lysate (LAL) and pyrogen assays. The efficiency of recovery of these endotoxins from carriers using a pyrogen assay was also determined simultaneously. In the LAL assay 0.1-10,000 EU was used and 10-1000 EU in the pyrogen assay. Recoveries of E. coli endotoxin and S. abortus equi endotoxin were, respectively, 49.7-92.0% and 27.0-70.1% by the LAL assay, and 31.1% and 60.6% by the pyrogen assay. Fourier transformation infrared (FT-IR) spectra demonstrated the presence of chemical structural differences between the two endotoxins. By dry heat (200 or 250 degrees C), there were no significant differences in the destruction kinetics between the two endotoxins; either endotoxin can therefore be adapted for use in the endotoxin challenge test. Destruction in the pyrogen assay was significantly quicker than that predicted by the LAL assay for each of the two endotoxins. In this endotoxin destruction system, 3 log cycle reduction (the United State Pharmacopeia (USP) recommendation for the depyrogenation process) could not be obtained by challenge with 10,000 EU of endotoxin under the depyrogenation conditions of 200 degrees C for 60 min (a set of conditions described in the European Pharmacopoeia (EP)), though little pyrogenicity remained. On the other hand, at 250 degrees C for 30 min (a set of conditions described in the EP, USP and Pharmacopoeia of Japan (JP)),a 3 log cycle reduction was achieved without any pyrogenicity remaining.

Drug Contamination↗

PubMed1982

Studies on the active site of human leukocytic pyrogen.

Leukocytic pyrogen, a polypeptide produced by phagocytic mononuclear cells, is thought to be the endogenous mediator of fever. In addition to its effects on thermoregulation, leukocytic pyrogen has been shown to induce synthesis of acute-phase proteins, increase lymphocyte blastogenesis to mitogens, and cause release of neutrophil-specific granule contents. Despite its important role in biologic responses, little is known concerning the structure-function relationship of the molecule. In the present studies several protein-modifying conditions were used in order to examine specific amino acid participation at the active site. Because the state of purity of leukocytic pyrogen may be critical during certain reaction conditions, highly purified preparations were used. Experiments suggest that the active site requires the gamma-carboxyl group of glutamic acid and that blocking arginine reduces both the pyrogenic and neutrophil releasing properties of the molecule. Other studies demonstrate that the pyrogenicity of human leukocytic pyrogen is not due to serine esterase or carboxypeptidase B activity and that the 15,000-dalton molecule may be a glycoprotein. These experiments provide further evidence that the lymphocyte-activating and neutrophil-granule-releasing properties of human leukocytic pyrogen require the same active site which produces fever.

Arginine↗

PubMed2001

Pyrogen retention by highly permeable synthetic membranes during in vitro dialysis.

Pyrogen permeability of the new highly permeable synthetic membrane polyethersulfone (DIAPES) was compared to polysulfone in vitro dialysis experiments with heparinized human donor blood in the blood compartment. After sterile dialysis for 5 min, dialysate was contaminated with a culture filtrate of Pseudomonas aeruginosa using high and moderate challenge doses (Limulus assay reactivity 20,000 EU/ml and 50 EU/ml, respectively). Whole blood samples were separated from the blood compartment during the sterile (5 min) and contaminated (60 min) phases of dialysis and incubated for 6 h at 37 degrees C. Blood samples were lysed, and interleukin-1beta and tumor necrosis factor alpha were measured by specific ELISAs. Moderate dialysate contamination (50 EU/ml) did not induce detectable cytokine production in whole blood. High challenge dose (20,000 EU/ml) induced whole blood interleukin-1beta and tumor necrosis factor alpha production in the blood compartment, which was higher with DIAPES than with polysulfone after 30 min. After 60 min, membrane-dependent differences were no longer detectable. Pyrogen concentrations in the dialysate decreased with time indicating adsorption of cytokine-inducing substances to the dialyzer membrane. Pyrogens adsorbed to dialyzer membranes were resuspended during recirculation of sterile phosphate-buffered saline in the dialysate compartment and retained cytokine-inducing activity as seen from whole blood incubation experiments. DIAPES and polysulfone adsorbed pyrogens in the presence of whole blood. Pyrogen adsorption to the membrane polymer and/or to the protein coat on the membrane prevented the passage of pyrogens in the presence of moderately contaminated dialysate. High grade dialysate contamination caused breakthrough of pyrogens into the blood with DIAPES and polysulfone. In order to reduce the risk of a dialysis-dependent inflammatory response, dialysate of high bacteriological quality (ultrapure dialysate) should be mandatory.

Adsorption↗

PubMed1982

Chemical characterization of a rabbit leukocytic pyrogen.

Crude pyrogen was obtained by stimulating 1.4 x 10(12) peritoneal-exudate leukocytes from rabbits in vitro with Escherichia coli endotoxin. A pyrogen molecule was isolated by ammonium-sulfate precipitation, polyacrylamide gel electrophoresis at pH 5.5 and 8.9, and gel filtration on Bio-Gel P-100. These procedures yielded a pyrogenic fraction which was electrophoretically and immunologically homogeneous. The pyrogenicity was 50 ng/kg of rabbit. This leukocyte pyrogen consisted of a protein with a molecular weight of approximately 14 000. Amino-acid analysis showed high amounts of glutamic acid and glycine; methionine and tryptophan were present in small quantities. Arginine was found to be the N-terminal amino acid. The molecule contained about 0.8% neutral sugar and 0.7% lipids. Its approximate isoelectric point in citrate/phosphate buffer was 4.2. The activity was lost by lyophilization and storage at - 18 degrees C for six months. The data presented here are the first detailed characterization of a rabbit leukocyte pyrogen. The properties in common to leukocytic pyrogen and interleukin 1 are discussed.

Amino Acids↗

PubMed1984

Involvement of central action of lipopolysaccharide in pyrogen fever.

To elucidate the mechanisms of fever response by bacterial pyrogen (lipopolysaccharide, LPS), we investigated both pyrogenicity and Limulus amoebocyte lysate (LAL) gelation activity of cerebrospinal fluid (CSF) withdrawn from febrile rabbits induced by i.v. injection of LPS. One ml of CSF was withdrawn from donor animals 2 hr after i.v. injection of E. coli LPS at graded doses of O, 1, 10 and 50 micrograms/kg, and its pyrogenicity was checked by administration into cisterna magna of recipient animals. Pyrogenicity was revealed only in the CSF withdrawn from the group injected with 50 micrograms/kg of LPS. Differences of both protein content and concentrations of ions (Na, K and Ca) were not obtained among the CSF withdrawn from control and LPS-injected groups. All of the above CSF had no LAL gelation activity, but the activity could be detected in the CSF withdrawn from animals injected with a higher dose of LPS (500 micrograms/kg, i.v.). LAL gelation activity of LPS dissolved in normal CSF was 2 orders less potent in comparison with that of LPS dissolved in saline, which suggested the presence of inhibitor(s) in CSF for LAL assay. Pyrogenicity was not revealed in the CSF withdrawn from hyperthermic rabbits induced by administration of reserpine after pretreatment with a monoamine oxidase inhibitor. These findings suggest that the central action of LPS is involved in pyrogen fever and that monoamines are not closely related to pyrogen fever.

Animals↗

PubMed1998

[Development of a pyrogen test based on rabbit blood]

The testing of drugs for pyrogen contamination e.g. the exclusion of fever inducing impurities, was standardised in the Forties in the form of animal pyrogen assay. We recently proposed an alternative method using human whole blood from healthy donors. The substances to be tested are incubated in the presence of blood and the generation of endogenous pyrogens (Interleukin 1, IL-1) are measured reflecting the primary fever reaction in man. With this method it is possible to detect the reaction of the relevant species. However, pronounced differences were reported for the potency of several bacterial pyrogens in different species (e.g. up to factor 10.000 for an individual endotoxin in different mammals). As a part of the validation of the new method discrepancies between the whole blood system and the rabbit test might occur. In these cases it is not possible to distinguish between an in-vitro-artefact and species differences. Therefore, the goal of this project is the development of an in-vitro-test based on rabbit blood. For this purpose rabbit IL-1 was expressed. At present chickens and mice are immunized and possibly crossreacting anti-human-IL-1-antibodies are tested. In this way, an ELISA for the rabbit cytokin will be established. Using a temporarily commercially available rabbit TNF-ELISA, the feasibility of the test-system could already be shown: Pyrogens induced the generation of TNF in rabbit blood. In the human model, the endpoints IL-1 and TNF were very similar. The sensitivity of human blood and rabbit blood appeared to be very similar. However, individual pyrogens (only different endotoxins were tested up to now) showed considerable deviations. However, these results require the verification in the final test. The rabbit test in preparation will be implemented immediately into the ongoing evaluations by the Paul-Ehrlich-Institute (BMBF) and at the University of Heidelberg (European Pharmakopoea). Furthermore, such a test also represents an interesting alternative in the veterinarian medical field using the correct target species for pyrogenicity testing. This project war supported by the Foundation Research 3R, CH-Muensingen.

Journal Article↗

PubMed1988

Decrease in pyrogenicity of muramyl dipeptide after coupling with luteinizing hormone-releasing hormone.

Muramyl dipeptide (MDP) and its adjuvant active derivative lysine-MDP (Lys-MDP) have been demonstrated to be pyrogenic and to induce endogenous pyrogen (EP) production in vivo and in vitro. It has recently been shown that immunologic castration can be achieved in mice by immunization with luteinizing hormone-releasing hormone (LHRH) directly conjugated by carbodiimide to Lys-MDP, termed LHRH-Lys-MDP (cdi), or with a linear monomeric MDP-linked molecule obtained by total synthesis, termed LHRH-Lys-MDP (s). These preparations were tested in the rabbit for their capacity to induce fever and were found to be devoid of pyrogenicity at dosage levels of Lys-MDP that induced fever. This decrease of pyrogenicity of Lys-MDP after coupling to LHRH seems to be related to the structure of the conjugate because the derivative LHRH-LysNH2-MDP exhibited the same pyrogenic activity as the free glycopeptide. Surprisingly, nonpyrogenic LHRH-Lys-MDP induced production of EP and interleukin-1 (IL-1) in vitro and increased in vivo modifications of metal levels attributed to the action of IL-1. Moreover, LHRH-Lys-MDP reduced the pyrogenic effect of an exogenous dose of EP.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1995

Pyrogens specifically disrupt the acquisition of a task involving cognitive processing in the rat.

In addition to changes in body temperature and other metabolic and physiological responses corresponding to immune activation, pyrogens can induce profound behavioral changes referred to collectively as sickness behavior. One feature of sickness behavior, sometimes reported in clinical settings, but rarely exposed to experimental analysis, is depressed cognitive functioning. The present series of five experiments sought to demonstrate the existence of specific cognitive deficits in rats, independently of any confounding performance effects of pyrogen injections. The behavioral task used, called autoshaping, consisted of presenting hungry Wistar rats with a stimulus (introduction of a retractable lever) that predicted food delivery. Control rats quickly learned to press the lever, although this response does not influence the probability of food delivery. When pyrogens (250 micrograms/kg lipopolysaccharide, 4 micrograms/rat interleukin-1 beta, or 300 mg/rat yeast) were injected to rats during acquisition of this task, they severely disrupted acquisition while the pyrogen was active. The same treatments were, however, without effect on performance when injected later, when performance had stabilized. It is argued that these results demonstrate specific, performance-independent effects of pyrogens on the cognitive processes needed for the acquisition of this task. The results are discussed in terms of the relationship between these effects and the cytokines induced in the brain by pyrogens, and in terms of the exact nature of the cognitive process likely to be affected.

Animals↗

PubMed1982

Production of lymphocyte activating factor in the absence of endogenous pyrogen by rabbit or human leukocytes stimulated by a muramyl dipeptide derivative.

N-acetylmuramyl-L-alanyl-D-isoglutamine or MDP is a synthetic immunoadjuvant capable of inducing a febrile response in the rabbit and of eliciting production of endogenous pyrogen (EP) in vivo and in vitro. It was also shown to stimulate in vitro lymphocyte activating factor (LAF) release. After conjugation to a synthetic carrier, the pyrogenic effect of MDP is strongly enhanced. Recently, adjuvant active derivatives of MDP devoid of pyrogenicity such as MDP(Gln)-OnBu (AcMur-L-Ala-D-Gln-alpha-nbutyl ester) have become available. In the present study, their stimulating properties on rabbit peritoneal exudate cells or human peripheral blood mononuclear cells were simultaneously evaluated by testing lymphocyte activating property and pyrogenic effect of the culture supernatant. In both types of cell cultures, whereas MDP elicited dual effects, the nonpyrogenic analog MDP(Gln)-OnBu produced LAF without detectable pyrogenicity even when a very sensitive assay (intracerebroventricular administration) was used. In contrast, conjugated MDP was capable of inducing the production of endogenous pyrogen in rabbit cell cultures with no or little LAF release. These results argue against the possibility that a single factor is responsible for both LAF and EP activities.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1997

Human monocytoid cell lines as indicators of endotoxin: comparison with rabbit pyrogen and Limulus amoebocyte lysate assay.

The aim of this study was to develop an in vitro test system for pyrogenic substances. Three clones derived from human monocytoid cell lines, which were selected by their high sensitivity to lipopolysaccharide (LPS), were assessed for tumor necrosis factor (TNF) production. Their response to pyrogen-containing samples was compared with that in a Limulus amoebocyte lysate assay and the rabbit pyrogen test. We show here that the induction of TNF in these clones is a valid in vitro alternative to determine endotoxin in commercial preparations requiring pyrogenicity testing. Cell clones derived from Mono Mac 6 (MM6 2H8 and MM6 4B5) responded to sub-ng/ml concentrations of complete rough-strain and smooth-strain LPS, to ng/ml concentrations of diphosphoryl-lipid A, and to microgram/ml concentrations of monophosphoryl-lipid A and to detoxified LPS. Cells reacted to > or = 1 microgram/ml lipoteichoic acid by TNF production, and were relatively insensitive to toxic shock syndrome toxin-1 (TSST-1) and to muramyl dipeptide adjuvant peptide. The reaction pattern of a clone derived from THP-1 (THP-1 1G3) was in general, similar to that of the MM6 clones, except that THP-1 1G3 failed to react to diphosphoryl-lipid A. When tested on commercial samples destined for parenteral use, there was a close correlation between a sensitive Limulus amoebocyte lysate (LAL) test and the cell culture test on the one hand, and between the pyrogen test and the cell culture test on the other hand. The data suggest that this cell-based test is able to recognize pyrogens derived from gram-negative organisms in test samples with appropriate sensitivity and specificity. This test appears to be able to eliminate some of the false-positive data obtained in the LAL test.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1955

Studies on the pathogenesis of fever. I. The presence of transferable pyrogen in the blood stream following the injection of typhoid vaccine.

The rate of clearance of intravenously injected typhoid vaccine was studied in unsensitized, sensitized, and pyrogen-tolerant rabbits by means of a passive transfer technique. The blood of unsensitized rabbits which had not been previously exposed to bacterial pyrogen remained pyrogenic for normal recipients throughout a period of 2 hours following the injection. In contrast, rabbits sensitized by having received either one or two injections of the vaccine at least 3 weeks prior to the experiment cleared their blood of the test vaccine within 30 minutes despite the fact that they exhibit the same febrile response as unsensitized rabbits. After 1 hour, however, a transferable pyrogenic substance was again demonstrable in the sera of this group. Reasons are discussed for believing that this newly appearing substance may be of endogenous origin and may be the factor which directly affects the thermoregulatory centers of the brain. Rabbits which are made tolerant by repeated daily injections of vaccine have a characteristically depressed febrile response. Not only were the blood streams of such animals cleared of the injected vaccine within less than 5 minutes, but samples of their sera obtained 1 and 2 hours after the injection also failed to contain demonstrable quantities of the secondary pyrogen observed in sensitized animals. The latter observation is in keeping with the suggestion that the secondary pyrogen may play a critical role in the production of fever.

Animals↗

PubMed1967

Release of an endogenous pyrogen in vitro from rabbit mononuclear cells.

The capacity of rabbit mononuclear cells to release an endogenous pyrogen (EP) in vitro has been studied. After incubation with tuberculin, preparations of predominantly monocytic cells, derived from the respiratory passages of the lungs of rabbits sensitized with BCG, were activated to release EP. Pyrogen production occurred more slowly with lung monocytes than with blood leukocytes of similarly sensitized rabbits and 9 to 10 hr incubation in a fully supportive medium was required to produce clear-cut results. As previously reported with blood leukocytes, mononuclear cells from the lungs of normal animals were also activated by tuberculin but to a lesser degree than were those from specifically sensitized rabbits. Under a variety of conditions, mononuclear cells from either spleen or lymph nodes of the same sensitized rabbits failed to release detectable amounts of pyrogen when incubated with tuberculin in vitro but were activated in a majority of instances when phagocytosis of heat-killed staphylococci was used as the stimulus. Release of pyrogen from lung monocytes appears to be an active process that is both temperature-dependent and requires protein synthesis. Neither serum antibody nor complement appears to play a role in this process. Evidence is presented that the granulocyte is the main source of pyrogen evolved by blood leukocytes incubated in vitro with OT or heat-killed staphylococci, whereas the lung macrophage and/or monocyte is responsible for most of the pyrogen released from the lung cell preparations. From these studies, it is concluded that mononuclear cells can be activated in vitro by several microbial stimuli and must be considered an additional cellular source of EP. The clinical implications of these findings for the pathogenesis of fever in granulomatous diseases where the monocyte is the predominant cell are discussed.

Animals↗

PubMed1969

Species specificity of leukocytic pyrogens.

Polymorphonuclear neutrophilic leukocytes of the dog, cat, and goat release leukocytic pyrogen under the same conditions as the heterophile polymorphonuclear leukocytes of the rabbit. The characteristics of the febrile response to an intravenous injection of homologous leukocytic pyrogen in all four species are very similar: a brisk monophasic fever reaching a peak between 30 and 50 min with smooth defervescence to the baseline by 3 hr. Shivering, which is not obvious in the rabbit, is noted in the dog, cat, and goat during the first 30 min. Quantitative differences in response reveal the cat to be the most sensitive of of these species to homologous leukocytic pyrogen, followed by the rabbit, dog, and goat. The response to heterologous pyrogen is in most cases markedly diminished compared to that after equal doses of homologous protein, suggesting the operation of species specificity, although canine and feline pyrogen behaved very similarly in all tests. Species specificity of leukocytic pyrogen is probably related to amino acid substitutions in different species of a common mammalian protein effector molecule.

Animals↗