Nitric oxide and reactive nitrogen intermediates during lethal and nonlethal strains of murine malaria.

The virulence of Plasmodia depends partly on the strain of parasite and partly on the host. In this study, Plasmodium berghei N/13/1A/4/203 caused the death of mice, whereas Plasmodium chabaudi chabaudi AS was not lethal. Current opinion is that nitric oxide (NO) and other reactive nitrogen intermediates (RNI) are produced in several host organs during malaria to resist infection or produce tissue damage. NO and RNI production in blood or plasma, brain, liver and spleen in MF1 mice was investigated during P. berghei and P. c. chabaudi infection, in order to help determine whether changes in NO production are beneficial or detrimental to the host in vivo. NO production was measured both directly and indirectly as nitrites and nitrates, to represent RNI. No changes in blood NO were detected in P. berghei infected mice, but increases were observed in brain, liver and spleen. In P. c. chabaudi infected mice, rises in NO concentration were observed in blood and spleen, whereas a decline in liver NO was seen, but there were no changes in brain. Liver contained the highest concentration of RNI, but increasing concentrations were seen in both plasma and spleen in both P. berghei and P. c. chabaudi infected mice. These results show that NO and RNI production alters during murine malaria. The changes depend upon the tissue, the day of infection, the degree of parasitaemia, the strain of Plasmodia and the method of measuring NO biosynthesis. Lethal P. berghei induced NO production in the mid and late stages of infection in mice when parasitaemia was high, whereas in nonlethal P. c. chabaudi infection, NO production was increased in the early and late stages when parasitaemia was low. These data are consistent with a role for NO in the protection of the MF1 mouse against Plasmodia. Failure to clear the parasite is associated with evidence of increased NO production in brain and liver, which may contribute to the pathology of malaria, but this hypothesis requires confirmation from other experimental approaches.

Immunomodulatory effects of pharmacological elevation of cyclic AMP in T lymphocytes proceed via a protein kinase A independent mechanism.

The role of the cAMP pathway as an immunomodulatory system has been an area of intensive research. Pharmacological elevation of the cAMP pathway inhibits T lymphocyte proliferation and production of Th1-type cytokines. The effects of cAMP are thought to be mediated via activation of the intracellular receptor, protein kinase A (PKA). We investigated the inhibitory effects of cAMP elevation on human lymphocyte proliferation and function by utilising a range of selective inhibitors of PKA. Elevation of cAMP activity by dbcAMP, Sp-cAMPS and forskolin induced significant decreases of Con A stimulated PBMC proliferation. Co-incubation with the selective PKA inhibitors HA1004, KT5720 and Rp-cAMPS showed these antiproliferative effects to persist, despite measurable PKA activity being inhibited to that of untreated cells or less. IL-2 production was also inhibited by dbcAMP in the presence of HA1004 and Rp-cAMPS. It has been demonstrated that the inhibitory effects of pharmacological elevations in cAMP on human T cell proliferation and IL-2 production do not require PKA activity. These observations indicate that control of lymphocyte proliferation and functional status by cAMP proceeds through PKA-independent events. Identification of the underlying mechanisms behind these effects would increase our understanding of the cAMP cascade and may provide a potentially novel target for immunomodulation.

Inhibitors of glutathione reductase as potential antimalarial drugs. Kinetic cooperativity and effect of dimethyl sulphoxide on inhibition kinetics.

We have developed inhibitors of glutathione reductase that improve on the inhibition of literature lead compounds by up to three orders of magnitude. Thus, analogues of Safranine O and menadione were found to be strong, reversible inhibitors of yeast glutathione reductase. Safranine O exhibited partial, uncompetitive inhibition with Ki and alpha values of 0.5 mM and 0.15, respectively. Thionine O was a partial (hyperbolic) uncompetitive inhibitor with Ki and alpha values of 0.4 microM and 0.15, respectively. LY83583 and 2-anilino-1,4-naphthoquinone also showed (hyperbolic) partial, uncompetitive inhibition with micromolar Ki values. For Nile Blue A a model for two-site binding with (parabolic) uncompetitive inhibition fitted the data with a Ki value of 11 microM and a kinetic cooperativity between the sites of 0.12, increased to 0.46 by preincubation of the enzyme and Nile Blue A in the presence of glutathione disulphide. Analysis of the effects of preincubation on the kinetics and cooperativity indicated the possibility of a slow conformational change in the homodimeric enzyme, the first such indication of kinetic cooperativity in the native enzyme to our knowledge. Further evidence of conformational changes for this enzyme came from studies of the effects of dimethyl sulphoxide which indicated that this co-solvent, which at low concentrations has no apparent effect on initial velocities under normal assay conditions, induced a slow conformational change in the enzyme. Thionine O, Nile Blue A and LY83583 were redox-cycling substrates producing superoxide ion, detectable by means of cytochrome c reduction, but leading to no loss of glutathione reductase activity, under aerobic or anaerobic conditions. The water-soluble Safranine analogues Methylene Blue, Methylene Green, Nile Blue A and Thionine O (5 mg/kg i.p. x 5) were effective antimalarial agents in vivo against P. berghei, but their effect was small and a higher dose (50 mg/kg i.p. x 1) was toxic in mice. Comparison was made with human glutathione reductase and its literature-reported interactions with several tricyclic inhibitors as studied by X-ray diffraction. It is possible that the conformational changes detected in the present study from alterations in detailed kinetic inhibition mechanisms may shed light on information transfer through the glutathione reductase molecule from the dimer interface ligand pocket to the active-site.

Novel aryl-bis-quinolines with antimalarial activity in-vivo.

Three rationally designed isomeric aryl-bridged bis-quinolines, N1,Nx-bis(7-chloroquinolin-4-yl)phenylene-1,x-diamines, where x=2, 3 or 4, i.e. o-, m- and p-substituted analogues respectively, were synthesized and evaluated against Plasmodium berghei in-vivo. The compound with x=2 had an ID50 of 30 mg kg(-1), whereas the p-substituted analogue (x=4) was not statistically schizonticidal at either of the two dose levels tested in olive oil-dimethylsulphoxide (5 and 25 mg kg(-1), ID50=60 mg kg(-1) approx.). When the delivery vehicle was changed to saline-DMSO, antimalarial potency increased for the p-substituted compound (ID50 17 mg kg(-1)). In contrast, the m-substituted analogue had marked antimalarial activity (ID50 1.2 mg kg(-1)), which compares favourably with that of chloroquine diphosphate (ID50 = 4.3 mg kg(-1)). The data presented show that the aminomethylene side chain in amodiaquine can be successfully replaced by a 7-halo-4-aminoquinoline, establishing that carbon bridges containing less than four contiguous carbon atoms can be present within highly active aryl-substituted 4-aminoquinoline antimalarials. These results confirm that the presence of an OH group in the aryl bridge is not necessary for antimalarial activity and substantiate the view that, despite the appearance of resistant strains, new and existing aminoquinolines still have an important role in treating malaria.

Rational drug design approach for overcoming drug resistance: application to pyrimethamine resistance in malaria.

Pyrimethamine acts by selectively inhibiting malarial dihydrofolate reductase-thymidylate synthase (DHFR-TS). Resistance in the most important human parasite, Plasmodium falciparum, initially results from an S108N mutation in the DHFR domain, with additional mutation (most commonly C59R or N51I or both) imparting much greater resistance. From a homology model of the 3-D structure of DHFR-TS, rational drug design techniques have been used to design and subsequently synthesize inhibitors able to overcome malarial pyrimethamine resistance. Compared to pyrimethamine (Ki 1.5 nM) with purified recombinant DHFR fromP. falciparum, the Ki value of the m-methoxy analogue of pyrimethamine was 1.07 nM, but against the DHFR bearing the double mutation (C59R + S108N), the Ki values for pyrimethamine and the m-methoxy analogue were 71.7 and 14.0 nM, respectively. The m-chloro analogue of pyrimethamine was a stronger inhibitor of both wild-type DHFR (with Ki 0.30 nM) and the doubly mutant (C59R +S108N) purified enzyme (with Ki 2.40 nM). Growth of parasite cultures of P. falciparum in vitro was also strongly inhibited by these compounds with 50% inhibition of growth occurring at 3.7 microM for the m-methoxy and 0.6 microM for the m-chloro compounds with the K1 parasite line bearing the double mutation (S108N + C59R), compared to 10.2 microM for pyrimethamine. These inhibitors were also found in preliminary studies to retain antimalarial activity in vivo in P. berghei-infected mice.

Nitroglycerine reduces neutrophil activation and acute damage in latissimus dorsi muscle grafts.

BACKGROUND: Damage to the latissimus dorsi muscle (LDM) may jeopardize a successful outcome to dynamic cardiomyoplasty. We and others have demonstrated muscle damage in LDM in various species including humans. Ischemia is now recognized to be an important contributory factor. We postulated that glyceryl trinitrate, a nitric oxide donor, might protect against ischemic endothelial dysfunction and so reduce resultant muscle damage. METHODS: In 20 adult rats the left LDM was mobilized on its thoracodorsal neurovascular pedicle and maintained as an orthotopic graft. Half of the animals received glycerol trinitrate intraoperatively and postoperatively for 24 hours. The other half served as untreated controls. Each group was further subdivided into two groups (n = 5 in each): animals in which the LDM was excised after 4 hours for myeloperoxidase studies, and animals in which the LDM was excised at 24 hours for analysis of muscle damage by histology and enzyme macrohistochemistry. Blood samples were taken at 24 hours for assay of plasma nitrite and nitrate as nitric oxide metabolites. RESULTS: Glycerol trinitrate-treated animals had higher plasma nitric oxide metabolite levels after 24 hours (after nitrate reductase treatment, total nitrite, 78.3+/-11.8 nmol/mL, mean +/- SEM) than controls (42.1+/-3.7 nmol/ mL, p = 0.008). The proportion of viable LDM in glycerol trinitrate-treated animals was greater than in untreated animals, mainly in the middle and distal regions of the graft (middle region, 96.3%+/-0.5% versus 75.7%+/-4.1%, p<0.001; distal region, 94.4%+/-0.8% versus 40.9%+/-3.1%, p<0.001). Macrohistochemical findings correlated well with the histologic findings. Myeloperoxidase activity (U/g) was markedly lower in glycerol trinitrate-treated LDMs, mainly in the distal part of the graft (glycerol trinitrate versus control, 20.5+/-2.1 versus 40.9+/-3.1 U/g, p<0.001). CONCLUSIONS: Glycerol trinitrate significantly reduced acute damage to the distal two-thirds of the mobilized LDM, possibly by modifying leukocyte activation and endothelial dysfunction associated with ischemic injury.

Febrile response to tissue inflammation involves both peripheral and brain IL-1 and TNF-alpha in the rat.

We investigated the role and interaction between tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, and IL-6 in the development of fever and their involvement in brain and systemic pathways in response to localized tissue inflammation caused by injection of turpentine (TPS) in the rat. Intramuscular injection of 10 microl TPS caused significant increases in body temperature, of up to 2 degrees C, compared with saline-treated animals. Fevers were maximal 7-8 h after injection and were preceded by significant increases in plasma bioactive IL-6. No changes in circulating bioactive IL-1 or TNF-alpha were detected. Systemic injection of IL-1 receptor antagonist (IL-1ra, 2 mg/kg i.p.) or anti-TNF-alpha antiserum (0.4 ml i.v.) almost completely abolished the febrile responses to TPS over 8 h and markedly inhibited the rise in plasma IL-6 bioactivity measured 6 h after TPS. To test the involvement of brain cytokines, anti-TNF-alpha antiserum and IL-1ra were injected intracerebroventricularly. Injections of anti-TNF-alpha antiserum (3 microl/rat i.c.v.) or IL-1ra (400 microg/kg i.c.v.) significantly (P < 0.01 and P < 0.05, respectively) inhibited fever induced by TPS. These data suggest that both localized peripheral and brain IL-1 and TNF-alpha are involved directly in the pyrogenic response to inflammation. The results indicate that, in the periphery, IL-1 and TNF-alpha cause increased production of IL-6, the most likely candidate as a circulating endogenous pyrogen.

An exploration of the structure-activity relationships of 4-aminoquinolines: novel antimalarials with activity in-vivo.

The structure-activity relationships of bisquinolines, a potentially important group of novel antimalarial drugs, were studied. The high-temperature (180-250 degrees C) synthesis of 4-aminoquinolines, including bisquinolines, by nucleophilic displacement was both fast and efficient Several bisquinolines including (+/-)-trans-N1,N2-bis(7-trifluoroquinolin-4-yl)cyclohexane-1, 2-diamine and 1R,2R-(-)-, 1S,2S-(+)-, (+/-)-trans- and cis-N1, N2-bis(7-chloroquinolin-4-yl)cyclohexane-1,2-diamine exhibited potent activity against Plasmodium berghei in mice; (+/-)-trans-N1,N2-bis(7-chloroquinolin-4-yl)cyclohexane-1, 2-diamine was orally active. Our results indicate that these compounds conform to a putative receptor for quinoline antimalarials. In addition, a 7-haloquinoline linked by a heterocyclic bridge, at the 4-position, to another heterocycle (such as an acridine at the 9-position) maximally occupies the active site of our postulated target.

Interleukin-1 receptor antagonist inhibits endotoxin fever and systemic interleukin-6 induction in the rat.

Although a number of studies indicate that the pyrogenic activity of lipopolysaccharide (LPS) and/or interleukin (IL)-1 is mediated via induction of IL-6, this has been questioned by recent evidence demonstrating a dissociation between fever and circulating IL-6. The present study reexamines this relationship by use of human recombinant interleukin-1 receptor antagonist (IL-1ra). Injection of LPS (100 micrograms/kg ip) into rats induced fever (2.0 degrees C) that was significantly inhibited (P < 0.05) when IL-1ra (16 mg/kg ip) was given 1 and 2 h after LPS. The rise in plasma IL-6 preceded the febrile response by 1-1.5 h and, although the concentrations of bioactive IL-6 in plasma and cerebrospinal fluid (CSF) were not reduced at 4 h, at 2 h plasma and CSF IL-6 bioactivity was inhibited by 80 and 70%, respectively, after a single injection of IL-1ra (16 mg/kg ip). Intracerebroventricular injection of IL-1ra (200 micrograms/rat) inhibited LPS fever but did not affect the plasma IL-6 bioactivity measured 2 or 4 h after intraperitoneal LPS. These data show that peripheral IL-1 plays a part in the induction of both fever and the rise in plasma IL-6 that precedes it, and that IL-1 within the brain is also important in the induction of fever by LPS.

Adrenocorticotropic hormone as a potential enhancer of T-lymphocyte function in the rat mixed lymphocyte reaction.

The effects of adrenocorticotropic hormone (ACTH) (10(-6) to 1 U/ml) on T-lymphocyte proliferation and function were studied in the rat mixed lymphocyte reaction (MLR). ACTH produced a modest increase in lymphocyte proliferation on day 3 in lymph node (LN) cells and on day 5 in spleen cells. In addition, LN MLR cells, activated in the presence of ACTH, showed a higher proliferative response to restimulation on day 5 and on day 11 of the primary culture. Cytotoxic activity and the number of IL-2R- cells were increased in ACTH-treated LN MLR cultures in experiments where control MLR levels were low. ACTH also overcame the generation of low levels of suppressor activity in spleen MLR cells. These findings indicate that ACTH could play a role in increasing the priming of T-lymphocytes and enhancing, in particular, suboptimal primary responses.

Blood-brain barrier damage in experimental African trypanosomiasis.

African sleeping sickness is characterized by progressive central nervous system (CNS) involvement, leading to the so-called secondary or late stage in which there are widespread inflammatory changes with lymphoplasmocytic infiltration. A study was made of blood-brain barrier (BBB) integrity in the late stages of a rodent model by assessing the uptake of the fluorescent fluid-phase marker sulphorhodamine B into the brain tissue. Brain oedema was estimated from brain weight, density and electrolyte concentrations. Trypanosome distribution was studied by light and electron microscopy. At 35 days post-infection (p.i.) fluorescent dye penetration occurred in several brain regions, including thalamus and hypothalamus. At 40 days p.i., BBB damage was extensive, with dye penetration throughout both the grey and the white matter of the cortex. Infected rats had significantly higher brain water content than uninfected controls and altered sodium and potassium concentrations characteristic of vasogenic oedema. The morphological studies showed early accumulation of parasites within, and associated damage to the choroid plexus, and, in the late stages, the presence of small numbers of trypansomes scattered in the nerve tissue of the brain and spinal cord, similar to previous descriptions. The findings show that chronic trypanosomiasis in the rat model is accompanied by BBB damage and vasogenic oedema.

Cyclic AMP inhibits macrophage suppressor function and enhances lymphocyte proliferation.

The effects of increasing the level of cyclic AMP (cAMP) activity on lymphocyte proliferation in the rat mixed lymphocyte reaction (MLR) were investigated. Dibutyryl cAMP (dbcAMP) and prostaglandin E2 (PGE2) produced a dose-dependent reduction in proliferation in the lymph node (LN) MLR, but produced a substantial increase in proliferation in the spleen MLR at the lower concentrations used (10(-5)-10(-4) M dbcAMP; 10(-7)-10(-6) M PGE2). Enhancement of proliferation was dependent on the presence of macrophages and was probably due to inhibition of macrophage activation. This was based on the following findings: (1) spleen MLR proliferation was lower than that in the LN MLR; (2) depletion of spleen macrophages increased proliferation in the spleen MLR and addition of these macrophages to the LN MLR reduced proliferation; (3) macrophage depletion from the spleen MLR abolished the proliferation-enhancing effect of dbcAMP. In conclusion, cAMP enhances lymphocyte proliferation in this system, apparently as a consequence of suppressing the inhibitory influence of macrophages.

Importance of brain IL-1 type II receptors in fever and thermogenesis in the rat.

Interleukin-1 (IL-1) acts centrally to induce fever and thermogenesis in rodents. The central actions of IL-1 alpha and IL-1 beta apparently involve different mechanisms, and the effects of IL-1 beta are not consistent with interaction with a type I (IL-1RI) 80-kDa receptor. In the present study the involvement of the type II IL-1 receptor (IL-1RII) was tested in the rat by examining the effects of central injection of a monoclonal antibody (ALVA-42), which blocks the IL-1RII. Pretreatment of rats with ALVA-42 (6 micrograms icv) inhibited the thermogenic and pyrogenic responses to intracerebroventricular injection of 5 ng (but not 50 ng) of IL-1 beta in conscious rats but did not significantly modify responses to IL-1 alpha. ALVA-42 also failed to modify the responses to peripherally administered IL-1 beta (1 microgram) but significantly attenuated the pyrogenic and thermogenic responses to peripheral (125 micrograms) or central (1 microgram) injection of endotoxin. These data indicate that IL-1RII mediates the central effects of a low dose of IL-1 beta, but not IL-1 alpha, on fever and thermogenesis in the rat. They also imply that responses to endotoxin are due, at least in part, to the activation of IL-1RII by IL-1 beta released within the brain and that effects of peripherally injected IL-1 beta involve different mechanisms, probably associated with IL-1RI.

Chronic effects of interleukin-1 beta on fever, oxygen consumption and food intake in the rat.

Chronic subcutaneous infusion (from osmotic minipumps) of IL-1 beta (1 microgram/d) in male rats over seven days caused transient (1-3 d) increases in body temperature and reductions in body weight gain and food intake. By day 3, when colonic temperature was similar for vehicle and IL-1 infused groups, the acute responses (increases in temperature and VO2) to a maximal dose (1 microgram, sc) of IL-1 beta was almost identical in all animals. In a separate study intraperitoneal infusion of the same dose of IL-1 beta (1 microgram/d) increased the duration of changes in body temperature, weight and food intake, compared to subcutaneous infusion. In further groups of rats, pyrogenic responses to daily injections of IL-1 beta (1 microgram ip) were sustained for the entire 7 d period, but this treatment did not affect body weight. These data demonstrate that tolerance to infusion of IL-1 is not accompanied by reduced maximal responses to acute administration of IL-1, and indicate that more sustained effects of IL-1 are achieved by intraperitoneal rather than subcutaneous infusions, or by repetitive daily injections of the cytokine. These observations indicate that low levels of IL-1 release, maintained over periods of several days could be responsible for changes in body temperature and energy balance during chronic infections or inflammation.

Adrenalectomy reverses the impaired pyrogenic responses to interleukin-beta in obese Zucker rats.

Interleukin-1 is an important endogenous pyrogen which stimulates thermogenesis in normal animals by a central action which is dependent on release of corticotrophin releasing factor (CRF). Central injection of murine recombinant interleukin-1 beta (IL-1 beta, 5 ng) in conscious lean (+/?) Zucker rats produced significant increases in resting oxygen consumption (VO2, 26 per cent), colonic temperature (1.3 degrees C) and thermogenic activity (mitochondrial GDP binding) of brown adipose tissue (BAT, 24 per cent). In contrast, genetically obese (fa/fa) Zucker rats showed nonsignificant changes in VO2 (4 per cent), temperature (0.5 degrees C) and BAT activity (0 per cent). Bilateral surgical adrenalectomy (ADX) dramatically enhanced the effects of IL-1 beta on VO2 (45 per cent) body temperature (1.8 degrees C) and BAT activity (44 per cent) in obese mutants, but only slightly increased responses in lean rats. These data suggest that impaired responses to IL-1 beta in obese mutants may be due to inhibitory actions of glucocorticoids on either prostaglandin synthesis or CRF release within the hypothalamus.

Effects of malaria on O2 consumption and brown adipose tissue activity in mice.

Increased energy expenditure often occurs during illness or after injection of endotoxin and can contribute to the generation of fever. In laboratory rats and mice the thermogenic response has been attributed to the sympathetic activation of brown adipose tissue (BAT), although mice often fail to show pyrexia. In this study the effects of malaria on O2 consumption and BAT were studied in mice inoculated with Plasmodium berghei. Parasitemia was maximal (greater than 50% of erythrocytes showing positive Leishman staining) 72 h after inoculation. Up to this time body weight and food intake were similar to values for control mice, although colonic temperatures were slightly depressed in infected mice. Thereafter, infected mice showed marked hypophagia, loss of body weight, and severe hypothermia; colonic temperature was less than 31 degrees C at 96 h when the experiment was terminated. Resting O2 consumption (VO2) measured at 24 degrees C was slightly elevated in infected mice 12 h after inoculation and reached a peak value (31% above controls) at 48 h. VO2 returned to the same value as controls at 96 h. In vitro thermogenic activity of BAT (assessed from binding of guanosine diphosphate to isolated mitochondria) was not significantly altered in infected mice. These data demonstrate a marked thermogenic response to malarial infection, but this is not accompanied by fever in mice and is dissociated from stimulation of BAT activity.

Ergometrine and 5-hydroxytryptamine binding sites in rat brain and myometrium.

Functional studies suggest that ergometrine is a partial agonist involving 5-hydroxytryptamine (5-HT) receptors in rat uterus. Ergometrine displaced [3H]5-HT from specific binding sites in rat brain, but did not displace [3H]5-HT at functionally important concentrations in rat myometrium. These binding studies indicate that the agonist and antagonist actions of ergometrine in rat uterus arise from its initial interaction with binding sites other than those for 5-HT.

Central activation of thermogenesis and fever by interleukin-1 beta and interleukin-1 alpha involves different mechanisms.

Interleukin-1 exists in two forms (alpha and beta) which are assumed to act on the same receptor. Both forms of the molecule stimulated fever and thermogenesis in the rat when injected into the brain, but interleukin-1 beta was more effective, and combined injection of alpha and beta elicited additive responses. The actions of interleukin-1 beta were inhibited by pretreatment of the animals with either a receptor antagonist or monoclonal antibody to corticotrophin releasing factor. The effects of interleukin-1 alpha were unaltered by these treatments. The results indicate that brain corticotrophin releasing factor mediates thermogenesis and fever induced by interleukin-1 beta but not by interleukin-1 alpha.

Impaired effects of interleukin-1 beta on fever and thermogenesis in genetically obese rats.

Interleukin-1 beta (IL-1 beta) is an endogenous peptide which induced fever (1.8 degrees C rise in colonic temperature) when injected interacerebroventricularly (i.c.v., 80ng human recombinant IL-1 beta) into conscious lean rats. IL-1 beta also stimulated resting oxygen consumption (VO2) by 38 percent, in vitro thermogenic activity (mitochondrial GDP binding) of brown adipose tissue (BAT) by almost two-fold, and blood flow to brown fat (assessed from the distribution of radiolabelled microspheres) by nine-fold in lean animals. Genetically obese Zucker rats showed only small increases in temperature (0.5 degrees C), VO2 (15 percent) and blood flow to BAT (less than two-fold), and no change in GDP binding, but exhibited normal thermogenic responses to i.c.v. injection of corticotrophin releasing factor (GRF). The results indicate that the obese Zucker is insensitive to the central effects of interleukin; this may explain the reduced febrile responses to endotoxin which have previously been reported.