Pyrogen retention: Comparison of the novel medium cut-off (MCO) membrane with other dialyser membranes.

Haemodialysis effectively removes small solutes and smaller-sized middle molecules from the blood; however, the clearance of larger middle molecules, which have been associated with negative effects, is poor. The novel medium cut-off (MCO) dialysis membrane has larger pore sizes and a more open structure than other high-flux membranes, providing improved removal of larger middle molecules while retaining albumin. However, larger pore sizes may potentially increase permeability to pyrogens, including endotoxins and other bacterial contaminants, that could be present in the dialysis fluid. In this study, we tested the capacity of low-flux, high-flux, MCO and high cut-off dialyser membranes with different pore sizes to prevent pyrogens crossing from dialysate to the blood side in a closed-loop test system, differentiating among lipopolysaccharides, peptidoglycans and bacterial DNA using a toll-like receptor assay. Even though the bacterial contamination levels in our test system exceeded the acceptable pyrogen dose for standard dialysis fluid, levels of lipopolysaccharides, peptidoglycans and bacterial DNA in the blood-side samples were too low to identify potential differences in pyrogen permeability among the membranes. Our results suggest that MCO membranes are suitable for haemodialysis using ISO standard dialysis fluid quality, and retain endotoxins at a similar level as other membranes.

Pyrogens, a polypeptide produces fever by metabolic changes in hypothalamus: Mechanisms and detections.

Fever is one of the cardinal symptoms of onset of an infection or inflammation and is the common clinical indicator for medical consultation in mammalian host worldwide. Simply, fever manifested with elevation of body temperature from normal physiological range represents adaptive response of immune system on challenge with an infectious and non-infectious circumstance. Fever usually initiated in the periphery as a result of interaction of immune cells with exogenous or endogenous pyrogens. Peripheral pyrogenic signals gain access to the central nervous system via humoral and neural route. Humoral pathway was initiated with production of pyrogenic cytokines and prostaglandins from immune cells of blood as well as liver, transmitted directly to pre-optic area of hypothalamus through the circumventricular organ of brain. On the other hand an alternative pathway was initiated by the same cytokines indirectly via stimulating the vagal sensory neurons result in pyrogenic fever; so-called neuronal pathway. If the magnitude of pyrogens associated fever is very high, it will lead to severe illness ranging from septic shock to death. So it is necessary to evaluate the presence of pyrogens in implants, medical devices, drugs and biological materials to ensure safety in biomedical applications and therapeutics. Classification, route of administration, mechanism of action and detection of pyrogens and associated products are the major subject of this review.

Cardiogenic Causes of Fever.

BACKGROUND: Persistent fever of unknown cause is only rarely of cardiac origin, but heart disease must be considered in the differential diagnosis. Aside from endocarditis, pericarditis and various other conditions may be responsible. METHODS: This review is based on pertinent articles retrieved by a selective search in PubMed and Google Scholar employing the term "fever" in combination with "myocardial infarction," "pericarditis," "endocarditis," and "postcardiac injury," with additional consideration of current cardiological guidelines. RESULTS: Endocarditis is associated with fever in 90% of cases, but 25-50% of patients also develop high body temperatures after acute myocardial infarction. In pericarditis, a temperature above 38°C indicates a poorer prognosis; if accompanied by other warning signs, it is an indication for hospitalization and pericardiocentesis. Fever can arise after cardiac surgical procedures as a manifestation of post - cardiotomy syndrome, a special type of perimyocarditis. There may be a latency period of up to 3 months. CONCLUSION: Fever can have both infectious and non-infectious cardiac causes. Its interpretation depends on the clinical context. The evidence base for treatment is sparse, and controlled trials are needed.

Surface contamination of dental implants assessed by gene expression analysis in a whole-blood in vitro assay: a preliminary study.

AIM: We aimed at evaluating pyrogen contamination of dental implants made of titanium and zirconia by using gene expression analysis in a whole-blood in vitro assay. MATERIAL AND METHODS: Titanium and zirconia implants (five each) were incubated in human whole blood. Samples were assayed for gene expression levels of toll-like receptor 4 (TLR4), TLR9, interleukin (IL)-1ß, nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-kB), tumour necrosis factor (TNF)-α, and Fas-associated protein with death domain (FADD) as indicators of surface contamination resulting in lipopolysaccharides (LPS)-stimulated TLR- or TNF-mediated immune responses. Gene expression was assayed using real-time quantitative polymerase chain reaction (RT-qPCR). Non-stimulated blood from the same donor served as a negative control, and blood stimulated with LPS served as a positive control. After dry-heat treatment with dry heat, all implants were re-analysed as described above. RESULTS: Both implant systems contained surface contaminants evoking a pro-inflammatory response similar to that induced by LPS. After dry-heat treatment, gene expression was significantly decreased to levels similar to those of negative control samples. CONCLUSIONS: The results demonstrated LPS-like surface-bound contaminants in both tested implant systems. Depyrogenation with dry heat seems to be an effective means of reducing such contamination in dental implants.

Virulence-suppressing effects of linezolid on methicillin-resistant Staphylococcus aureus: possible contribution to early defervescence.

In the present study, immunomodulatory effects of linezolid (LZD) on methicillin-resistance Staphylococcus aureus (MRSA) infections were evaluated. We have retrospectively reviewed treatment effects of LZD on 52 patients with severe MRSA infections. Sixty-four percent of the febrile patients demonstrated significant defervescence within 3 days, despite the presence of positive culture results. We speculated that this finding might be due to early anti-inflammatory effects of LZD, and to investigate this further we initiated in vivo experiments using mice MRSA pneumonia models. Mice were treated with either LZD or vancomycin (VCM) immediately after intranasal administration of MRSA. Bacterial numbers and levels of inflammatory cytokines in the lungs were determined. Although the bacterial burden in the lungs was not apparently different between the two groups, LZD but not VCM treatment significantly reduced induction of inflammatory cytokines in the lungs (P < 0.05). To evaluate whether this anti-inflammatory response was due to suppression of virulence factor expression, filter-sterilized supernatants of MRSA incubated in broth overnight with sub-MICs of LZD were subcutaneously administered to mice. To clarify whether LZD possesses direct host-modulating activity, cytokine responses to the supernatants were examined in mice pretreated with LZD. Interestingly, MRSA solutions prepared in the presence of sub-MICs of LZD revealed significant suppression of interleukin 6 (IL-6) in a dose-dependent manner (P < 0.05), but pretreatment of mice with LZD revealed no changes in cytokines. These findings suggest that sub-MICs of LZD might suppress virulence factors of MRSA, which may be associated with a reduction in endogenous pyrogens. These data may explain at least in part early defervescence observed in LZD-treated individuals.

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 characterization of the zinc site coordinating histidine residues of staphylococcal enterotoxin C2.

The bacterial toxin staphylococcal enterotoxin C2 (SEC2) can cause staphylococcal toxic shock syndrome and food poisoning. Although the previously determined crystal structure of SEC2 revealed that some histidine residues (His47, His118 and His122) contribute to the binding of zinc ions, little is known about their biological roles in SEC2. This prompted us to investigate the role of the zinc site coordinating histidine residues in the biological activities of SEC2. The mutants with substitutions at positions 118 and 122 all retained T-cell stimulatory activity, whereas the histidine mutants at position 47 were defective in the ability to stimulate T-cell proliferation. Further toxicity assays in vivo indicated that mutants SEC2-H118A and SEC2-H122A were defective in emetic and febrile activities. However, mutant SEC2-H47A could cause significant emetic and febrile responses in comparison with the other two histidine mutants. These findings suggested that the zinc-coordinating histidine residues play significant roles in superantigen and toxic activities of SEC2 and further implied that superantigen and febrile activities could be separable in staphylococcal enterotoxins. The results also show that it should be possible to design new SEC2 immunotherapeutic agents that have superantigen activity and low toxicity.

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

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

Isolation, purification and antitumor activity of lipopolysaccharide from cow placenta.

Lipopolysaccharide from cow placenta (LPS-CP-2) has been isolated and purified by hot phenol-water extraction, enzyme hydrolysis, chloroform-petroleum ether method, ion-exchange and gel-filtration chromatography. Also, LPS-PS-2 was evaluated for antitumor activity against Ehrlich ascites carcinoma (EAC)-bearing Swiss albino mice. LPS-PS-2 caused significant (P<0.05) decrease in tumor volume, and viable cell count; and it prolonged the life span of EAC-tumor-bearing mice. Hematological profile indicates that LPS-CP-2 possessed protective action on the haemopoietic system. Further, administration of LPS-CP-2 reduced the tumor volume of both DLA and EAC cell lines in a dose-dependent way. The LPS-PS was found to be devoid of pyrogenic response in the rabbits. These results indicate that LPS-PS exhibited significant antitumor activity without pyrogenic response, suggesting its potential as antitumor agent.

Endogenous opioids: role in prostaglandin-dependent and -independent fever.

This study evaluated the participation of mu-opioid-receptor activation in body temperature (T(b)) during normal and febrile conditions (including activation of heat conservation mechanisms) and in different pathways of LPS-induced fever. The intracerebroventricular treatment of male Wistar rats with the selective opioid mu-receptor-antagonist cyclic d-Phe-Cys-Try-d-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP; 0.1-1.0 microg) reduced fever induced by LPS (5.0 microg/kg) but did not change T(b) at ambient temperatures of either 20 degrees C or 28 degrees C. The subcutaneous, intracerebroventricular, and intrahypothalamic injection of morphine (1.0-10.0 mg/kg, 3.0-30.0 microg, and 1-100 ng, respectively) produced a dose-dependent increase in T(b). Intracerebroventricular morphine also produced a peripheral vasoconstriction. Both effects were abolished by CTAP. CTAP (1.0 microg icv) reduced the fever induced by intracerebroventricular administration of TNF-alpha (250 ng), IL-6 (300 ng), CRF (2.5 microg), endothelin-1 (1.0 pmol), and macrophage inflammatory protein (500 pg) and the first phase of the fever induced by PGF(2alpha) (500.0 ng) but not the fever induced by IL-1beta (3.12 ng) or PGE(2) (125.0 ng) or the second phase of the fever induced by PGF(2alpha). Morphine-induced fever was not modified by the cyclooxygenase (COX) inhibitor indomethacin (2.0 mg/kg). In addition, morphine injection did not induce the expression of COX-2 in the hypothalamus, and CTAP did not modify PGE(2) levels in cerebrospinal fluid or COX-2 expression in the hypothalamus after LPS injection. In conclusion, our results suggest that LPS and endogenous pyrogens (except IL-1beta and prostaglandins) recruit the opioid system to cause a mu-receptor-mediated fever.

Endogenous antipyretics.

The febrile increase of body temperature is regarded as a component of the complex host response to infection or inflammation that accompanies the activation of the immune system. Late phases of fever appear mediated by pro-inflammatory cytokines called endogenous pyrogens. The rise of body temperature is beneficial because it accelerates several components of the activated immune system. To prevent an excessive and dangerous rise of body temperature the febrile response is controlled, limited in strength and duration, and sometimes even prevented by the actions of endogenous antipyretic substances liberated systemically or within the brain during fever. In most cases the antipyretic effects are achieved by an inhibitory influence on the formation or action of endogenous pyrogens, or by effects on neuronal thermoregulatory circuits that are activated during fever. Endogenous antipyretic substances include steroid hormones, neuropeptides, cytokines and other molecules. It is the purpose of this review to consider the current state in the research on endogenous antipyretic systems.

Changes in quinolinic acid production and its related enzymes following D-galactosamine and lipopolysaccharide-induced hepatic injury.

Increases in quinolinic acid (QUIN), a neurotoxic L-tryptophan metabolite, have been observed in human serum and cerebrospinal fluid and in animal models of severe hepatic injury. The aim of this study was to evaluate the changes in QUIN accumulation and its related enzymes after acute hepatic injury induced by D-galactosamine and endotoxin. Gerbils were given an intraperitoneal injection of pyrogen-free saline alone as control, lipopolysaccharide (LPS) alone (150 ng/kg), D-galactosamine alone (500 mg/kg) or a combination of D-galactosamine with LPS. Concentrations of QUIN, its related metabolites, and related enzyme activities were determined. D-Galactosamine treatment significantly decreased activities of hepatic aminocarboxymuconate-semialdehyde decarboxylase (ACMSDase) resulting in increased QUIN concentrations in serum and tissues. The magnitude of QUIN responses was markedly increased by endotoxin due to the increased availability of L-kynurenine, a rate-limiting substrate for QUIN synthesis. Further, infiltration of monocytes/macrophages, which is a possible major source of QUIN production in the liver, was shown by immunohistochemistry after hepatic injury induced by D-galactosamine and endotoxin. Increased serum QUIN concentrations are probably due to the increased substrate availability and the decreased activity of aminocarboxymuconate-semialdehyde decarboxylase in the liver, accompanying the increased monocyte/macrophage infiltration into the liver after hepatic injury.

Possible relationship between streptococcal pyrogenic exotoxin A and Kawasaki syndrome in patients older than six months of age.

BACKGROUND: We previously investigated antibody titers against four kinds of superantigens [streptococcal pyrogenic exotoxin A (SPEA), streptococcal pyrogenic exotoxin C, toxic shock syndrome toxin-1 and staphylococcal enterotoxin B] in patients with Kawasaki syndrome (KS) younger than 6 months of age and reported a relationship between toxic shock syndrome toxin-1 and KS patients. In this study we have investigated antibody titers in KS patients older than 6 months of age. METHODS: Serum of 81 patients with KS older than 6 months of age, before intravenous gamma-globulin therapy, and 88 normal age-matched children were used in this study. The IgG antibody titers against four kinds of superantigens were measured with an enzyme-linked immunosorbent assay. RESULTS: The KS patients showed significantly elevated mean SPEA titer (P = 0.006) and significantly higher incidence of high SPEA (P = 0.0024) compared with the controls. The SPEA titer in KS patients showed a significant positive correlation with the number of days from onset of illness (P = 0.0002). CONCLUSIONS: The elevated antibody titer against superantigens of KS patients older than 6 months of age was different from that of KS patients younger than 6 months of age. Our results suggest that KS patients' exposure to SPEA occurred a few weeks before the onset of KS. SPEA may be one of the possible etiologic agents of KS among patients older than 6 months of age in Kagoshima, Japan.

Tyrosine kinase is involved in hemin-induced pyresis.

To investigate the mechanisms involved in hemin-induced febrile response, the rectal temperature of rats were measured after intracerebroventricular (i.c.v.) injections of hemin, with or without antagonists. Hemin (10 microg) elicited a significant febrile response, which lasted from 30 min, to more than 6 h, after its administration, but this was not the case with biliverdin (i.c.v.) and bilirubin (i.c.v.). The hemin-induced febrile response was significantly inhibited by pretreatment with an inhibitor of tyrosine kinase (genistein), but not by pretreatment with an inhibitor of protein kinase C (chelerythrine) and a scavenger of iron (deferoxamine). These results suggest that tyrosine kinase is involved in the hemin-induced febrile response.

Endotoxins and medical devices: the significance of dead bacteria.

There has been some recent speculation that endotoxins, the toxic molecules that are present in the cell wall of certain bacteria, could play a role in the development of tissue reactions to medical devices. This article discusses the basis for this speculation.

Characterization of two novel pyrogenic toxin superantigens made by an acute rheumatic fever clone of Streptococcus pyogenes associated with multiple disease outbreaks.

The pathogenesis of acute rheumatic fever (ARF) is poorly understood. We identified two contiguous bacteriophage genes, designated speL and speM, encoding novel inferred superantigens in the genome sequence of an ARF strain of serotype M18 group A streptococcus (GAS). speL and speM were located at the same genomic site in 33 serotype M18 isolates, and no nucleotide sequence diversity was observed in the 33 strains analyzed. Furthermore, the genes were absent in 13 non-M18 strains tested. These data indicate a recent acquisition event by a distinct clone of serotype M18 GAS. speL and speM were transcribed in vitro and upregulated in the exponential phase of growth. Purified SpeL and SpeM were pyrogenic and mitogenic for rabbit splenocytes and human peripheral blood mononuclear cells in picogram amounts. SpeL preferentially expanded human T cells expressing T-cell receptors Vbeta1, Vbeta5.1, and Vbeta23, and SpeM had specificity for Vbeta1 and Vbeta23 subsets, indicating that both proteins had superantigen activity. SpeL was lethal in two animal models of streptococcal toxic shock, and SpeM was lethal in one model. Serologic studies indicated that ARF patients were exposed to serotype M18 GAS, SpeL, and SpeM. The data demonstrate that SpeL and SpeM are pyrogenic toxin superantigens and suggest that they may participate in the host-pathogen interactions in some ARF patients.

Differential sensitivities of pyrogenic chemokine fevers to cyclooxygenase isozymes antibodies.

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.

Association of pyrogenic exotoxin genes with pharyngitis and rheumatic fever/rheumatic heart disease among Indian isolates of Streptococcus pyogenes.

AIM: To monitor the presence of various pyrogenic exotoxin genes in strains of Streptococcus pyogenes isolated in India. METHODS & RESULTS: Isolates recovered from pharyngitis (52) and rheumatic fever (RF)/ rheumatic heart disease (RHD) (8) patients were analysed for the presence of toxin genes, speA, speB and speF, by PCR. The specificity of the products was confirmed by restriction enzyme digestion and Southern hybridization. Among the 60 isolates studied, the incidence of speA, speB and speF were 5(8.3%), 56(93.3%) and 53(88.3%), respectively. The expression of these genes was established in representative isolates by RT-PCR. CONCLUSIONS: Comparative analysis of frequency of the speA, speB and speF genes, among pharyngitis and RF/RHD associated isolates, showed higher incidence in RF/RHD (25%, 100%,100%) as compared to pharyngitis patients (5.8%, 92.3%, 86.5%), respectively. SIGNIFICANCE OF STUDY: The presence of the speA gene, which is usually associated with scarlet fever or toxic shock-like syndrome, within few Indian isolates may be indicative of new virulent strains circulating within the Indian community. High distribution of toxin genes among RF/RHD compared to pharyngitis isolates indicate their possible role in increased virulence.

Evidence of recurrent atypical meningioma with rhabdoid transformation and expression of pyrogenic cytokines in a child presenting with a marked acute-phase response: case report and review of the literature.

Children presenting with acute systemic illnesses that lack specific clinical or serological defining features may be diagnosed as having a chronic infection, an atypical systemic vasculitis or a connective tissue disease, but often turn out to have occult neoplasias. Cytokines have been implicated in causing many of the systemic effects in such cases. In this study, we describe the case of a 9-year-old boy presenting at an interval of 18 months with a marked acute-phase response due to a recurrent atypical meningioma with rhabdoid transformation of the tentorium cerebelli. Resection of the recurrent tumor was curative. We evaluated in detail the local and systemic production of cytokines released by the primary and the recurrent tumor. Blood and CSF samples were taken pre-, intra-, and postoperatively, and the production of IL-6, IL-1beta, and TNF-alpha was measured by enzyme-linked immunosorbent assays (ELISA). The level of IL-6 in CSF was about 150-fold increased before tumor resection, normalizing postoperatively. On the contrary, the levels of IL-1beta and TNF-alpha in CSF and of IL-6, IL-1beta, and TNF-alpha in serum were pre-, intra-, and postoperatively within normal limits. Cytokine production was also evaluated immunohistochemically, and confirmed strong IL-6 and TNF-alpha expression in the primary and the recurrent tumor, while expression of IL-1beta was lacking. The scattered MHC class II- and leukocyte common antigen (LCA)-expressing inflammatory cells, which were infiltrating exclusively the tumoral stroma, had no detectable cytokine immunoreactivity. We conclude that chronic IL-6 and TNF-alpha production by the tumor cells in this patient was responsible for the severe systemic illness with which he presented.

Cyclooxygenase-2 mediates the febrile response of mice to interleukin-1beta.

Various lines of evidence have implicated cyclooxygenase (COX)-2 as a modulator of the fever induced by the exogenous pyrogen lipopolysaccharide (LPS). Thus, treatment with specific inhibitors of COX-2 suppresses the febrile response without affecting basal body (core) temperature (T(c)). Furthermore, COX-2 gene-ablated mice are unable to develop a febrile response to intraperitoneal (i.p.) LPS, whereas their COX-1-deficient counterparts produce fevers not different from their wild-type (WT) controls. To extend the apparently critical role of COX-2 for LPS-induced fevers to fevers produced by endogenous pyrogens, we studied the thermal responses of COX-1- and COX-2 congenitally deficient mice to i.p. and intracerebroventricular (i.c.v.) injections of recombinant murine (rm) interleukin (IL)-1beta. We also assessed the effects of one selective COX-1 inhibitor, SC-560, and two selective COX-2 inhibitors, nimesulide (NIM) and dimethylfuranone (DFU), on the febrile responses of WT and COX-1(-/-) mice to LPS and rmIL-1beta, i.p. Finally, we verified the integrity of the animals' responses to PGE2, i.c.v. I.p. and i.c.v. rmIL-1beta induced similar fevers in WT and COX-1 knockout mice, but provoked no rise in the T(c)s of COX-2 null mutants. The fever produced in WT mice by i.p. LPS was not affected by SC-560, but it was attenuated and abolished by NIM and DFU, respectively, while that caused by i.p. rmIL-1beta was converted into a T(c) fall by DFU. There were no differences in the responses to i.c.v. PGE2 among the WT and COX knockout mice. These results, therefore, further support the notion that the production of PGE2 in response to pyrogens is critically dependent on COX-2 expression.