A moderate dose of cycloheximide does not prevent the febrile response to endotoxin but interfere with induction of pyrogenic tolerance in rabbit.

The purpose of the present study was to examine the effect of cycloheximide (Cx)--inhibitor of protein synthesis, on the development of pyrogenic tolerance to LPS. It has been observed that Cx at a dose of 1 mg/kg given intravenously 1 h prior to LPS did not prevent fever response, however it modified the induction of pyrogenic tolerance. It was manifested in existence of the second phase of fever after the following administrations of LPS into rabbits pretreated with Cx. In control group of rabbits the induction of pyrogenic tolerance was accompanied with decaying of the second peak of fever visible as early as the second dose of LPS.     

The pathophysiological effect of scarlet fever toxin

The intravenous injection of scarlet fever toxin leads to acute changes in the white blood picture. The changes are correlated to the dose; a small dose causes lymphopenia and granulocytosis, while higher doses are followed by initial granulocytopenia. Tolerant rabbits do not have granulocytopenia, but lymphopenia persists. Tolerance to the leucopenic effect of the toxin probably develops somewhat sooner and lasts longer than tolerance to its pyrogenic effect. The blood picture of a cortisone-treated rabbit after the intravenous injection of scarlet fever toxin resembles the blood picture of a tolerant rabbit. The significance and associations of these observations are briefly discussed.     

Studies on pyrogenic tolerance to bacterial lipopolysaccharide in rabbits.

The development of pyrogenic tolerance was studied in rabbits treated with varying doses of E. coli lipopolysaccharide (LPS). The following results were obtained. 1) Development of pyrogenic tolerance seemed to proceed in two steps: that is, the first in which tolerance appeared rapidly and the second in which tolerance proceeded more gradually or steadily in response to repeated injections of a constant dose. 2) Tolerance induced by the latter method was not absolute; the rabbits were still sensitive to increased doses of LPS. 3) Rabbits immunized with E. coli vaccine lost their pyrogenic sensitivity of parent LPS to some extent. 4) Following intracisternal injection of LPS into tolerant rabbit, pyrogenic response was not decreased but rather enhanced in comparison with control. 5) The contents of nor-epinephrine and serotonin in the brain did not differ between normal and tolerant rabbits. 6) The mechanisms of endotoxin tolerance remain to be further studied.     

Detoxified bacterial endotoxins. I. Preparation and biological properties of an acetylated crude endotoxin from Salmonella typhimurium

Martin, William J. (University of Utah, Salt Lake City), and Stanley Marcus. Detoxified bacterial endotoxins. I. Preparation and biological properties of an acetylated crude endotoxin from Salmonella typhimurium. J. Bacteriol. 91:1453-1459. 1966.-Acetylation of a crude endotoxin prepared by the Roschka-Edwards (RE) procedure from a strain of Salmonella typhimurium yields a product with reduced pyrogenicity in rabbits as well as one which is nontoxic in mice. A reduction in pyrogenicity of approximately 100 times was noted with this acetylated crude endotoxin when compared with the parent RE preparation. A comparison was made of immunogenicity with mice of Boivin, RE, and acetylated Roschka-Edwards (Acet-RE) preparations with a heat-killed, phenol-preserved (HP) vaccine prepared from the same strain of S. typhimurium. Less than pyrogenic doses of all vaccines were not protective. The least pyrogenic preparation (Acet-RE) was immunogenically effective in about five times the minimal pyrogenic dose. The data suggest that the Acet-RE preparation should be considered further in the search for enteric fever vaccines with lowered potential for undesirable systemic responses.     

Neuropeptide alpha-MSH antagonizes IL-6- and TNF-induced fever.

The pro-opiomelanocortin-derived peptide melanocyte stimulating hormone (alpha-MSH) antagonizes the fever induced by several stimuli including endotoxin, endogenous pyrogen, and certain cytokines. To determine if alpha-MSH can antagonize the pyrogenic action of recombinant IL-6 and TNF directly within the central nervous system, the cytokines were injected with and without alpha-MSH (200 ng) into a lateral cerebral ventricle of rabbits and rectal temperature was monitored continuously. Central administration of both cytokines caused fever. However, when alpha-MSH was injected after cytokine administration, the fevers were markedly reduced. The results are consistent with previous observations on the antipyretic effect of alpha-MSH and they show that the peptide can act within the brain to antagonize pyrogenic actions of specific cytokines believed to be important in CNS mediation of fever.     

Sensitivity of hypothalamic sites to salicylate and prostaglandin.

This study was designed to determine the dose of salicylate necessary to produce substantial antipyresis, and to determine the relationship between the response to salicylate and prostaglandin infused into the same region of the preoptic area of the rabbit. The effect of preoptic infusions of three doses of sodium salicylate, or a control solution, on the fever produced by an intravenous injection of endogenous pyrogen was measured. The pyrogenic response to prostaglandin E1 injected into the same preoptic sites in the same rabbits was also monitored. The results showed that the 50 microgram/microL per hour dose of salicylate did not produce significant antipyresis but that the 100 and 200 microgram/microL per hour doses did. The results also showed a significant correlation between the magnitude of fever produced by prostaglandin E1 and the magnitude of antipyresis produced by sodium salicylate at a particular site. Those sites at which infusion of salicylate produced the most effective antipyresis were also the ones at which prostaglandin E1 produced the largest fevers.     

Role of cAMP in the mechanism of the fever reaction

It has been shown that accumulation of endogenous cAMP caused by theophylline increases the body sensitivity to the minimum doses of leukocytic and bacterial pyrogens and to prostaglandin E1. The increasing effect of theophylline was abolished by administering the higher doses of the pyrogenic agents. Repeated daily administration of bacterial pyrogen in conjunction with theophylline does not induce the development of tolerance. The possible reasons for enhanced fever in response to the minimum doses of the pyrogenic agents during accumulation of endogenous cAMP are discussed.     

Staphylococcal enterotoxin C1-induced pyrogenic cytokine production in human peripheral blood mononuclear cells is mediated by NADPH oxidase and nuclear factor-kappa B

The staphylococcal enterotoxins produced by Staphylococcus aureus are associated with pyrogenic response in humans and primates. This study investigates the role of NADPH oxidase and nuclear factor-kappa B (NF-kappaB) on enterotoxin staphylococcal enterotoxin C1 (SEC1)-induced pyrogenic cytokine production in human peripheral blood mononuclear cells (PBMC). The results indicate that the febrile response to the supernatant fluids of SEC1-stimulated PBMC in rabbits was in parallel with the levels of interleukin-1beta and interleukin-6 in the supernatants. The release of interleukin-1beta and interleukin-6, nuclear translocation of NF-kappaB and its DNA binding activity in the SEC1-stimulated PBMC were time-dependent and were completely eliminated by pyrrolidine dithiocarbamate or SN-50 (NF-kappaB inhibitors). The release of reactive oxygen species in the supernatants and translocation of the NADPH oxidase p47(phox) subunit to the plasma membrane of SEC1-stimulated PBMC were time-dependent. Administration of apocynin (NADPH oxidase inhibitor) attenuated the febrile response to the supernatants in rabbits and decreased the translocation of NADPH oxidase p47(phox) subunit and NF-kappaB activity in the SEC1-stimulated PBMC, and suppressed reactive oxygen species and pyrogenic cytokine production in the supernatants. Taken together, SEC1 may act through an NADPH oxidase mechanism to release reactive oxygen species, which activate NF-kappaB in PBMC to stimulate the synthesis of pyrogenic cytokines that trigger a fever response in rabbits.     

Candida albicans and Saccharomyces cerevisiae cell wall mannans produce fever in rats: role of nitric oxide and cytokines

Mannan components of C. albicans (5 mg/kg, i.p.) and S. cerevisiae (2.5 mg/kg, i.p.) cell walls produced pyrogenic responses which were completely inhibited by indomethacin (5 mg/kg, s.c.) pretreatment in rats. A non-selective NOS inhibitor, L-NAME (10 mg/kg, s.c.), also inhibited the pyrogenic effectiveness of C. albicans mannan, whereas it was ineffective on the fever induced by S. cerevisiae mannan. A selective elevation in the serum TNF-alpha levels was observed at the initial phase of the fever due to S. cerevisiae mannan, whereas there was no significant change on the serum levels of TNF-alpha, IL-1beta and IFN-gamma during the latent period or at the initial phase of the fever induced by C. albicans mannan. Injections of N-linked and/or O-linked oligomannosides of the either mannan did not cause any significant change in the body temperature and serum cytokine levels. These data suggest that the mannan components of C. albicans and S. cerevisiae cell walls produce a prostaglandin-dependent fever in rats. The initial signal for fever seems to be different for each mannan. Data also indicate that integrity of the mannans is necessary for the pyrogenic response.     

Biological activity of synthetic subunits of streptococcus peptidoglycan. I. Pyrogenic and thrombocytolytic activity.

The ability of some synthetically prepared analogues of Streptococcus peptidoglycan subunits (dipeptide, tetrapeptide, glycodipeptide and glycotetrapeptide) to cause fever in rabbits and lysis of rabbit blood platelets was studied. While di- and tetrapeptides did not exhibit these activities, glycodipeptides and glycotetrapeptides displayed pyrogenic and thrombocytolytic activities comparable with those of natural peptidoglycans.     

Fever produced by intrahypothalamic injection of interleukin-1 and interleukin-6

Pure human interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6), both of natural origin, were found to cause fever in rabbits when injected into the PO/AH region of the brain. The threshold dose required for this effect was between 0.4 and 4 U, equivalent to 0.04 to 0.4 ng for IL-1 beta, and around 50 U, equivalent to 0.05 ng for IL-6. From this it was estimated that this area of the brain responds to a local concentration of approximately 1 ng/ml of these cytokines, a level which can easily be reached after intravenous administration of threshold pyrogenic doses of either cytokine. The observation supports the view that fever induced by systemic endogenous production of IL-1 and IL-6 is due to a direct effect on the thermoregulatory center and may not require production of mediators, such as prostaglandins, at sites distant from the center.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

The neutral fraction (neutral CPS-K) of Klebsiella pneumoniae capsular polysaccharide (CPS-K) from type 1, Kasuya strain, has already been reported as the active substance responsible for the strong adjuvant effect of CPS-K. The present results demonstrate that neutral CPS-K exhibits further common biological activities with lipopolysaccharide (LPS) isolated from Salmonella enteritidis. The intensity of the lethality in mice of neutral CPS-K by the intraperitoneal route is very similar to that of LPS. Its lethality for mice by the intravenous (i.v.) route is significantly stronger than that of LPS, because the degree of increase in the sensitivity to their lethality by i.v. challenge is smaller for LPS than for neutral CPS-K. The intensity of the pyrogenicity of neutral CPS-K in rabbits is approximately one-tenth of that of LPS as judged by the minimal pyrogenic doses and fever indices. The skin-preparatory potency of neutral CPS-K for the dermal Shwartzman phenomenon in rabbits is also approximately one-tenth of that of LPS compared on the basis of the minimal skin-preparatory doses. When injected i.v., neutral CPS-K exhibits a provocative effect on hemorrhagic reactions in skin sites prepared with neutral CPS-K or LPS.     

Pyrogenic Responses to Staphylococcal Enterotoxins A and B in Cats

Pyrogenic responses, ranging up to 4.8 F, were induced in cats by oral administration of highly purified staphylococcal enterotoxin B in doses from 10 to 100 mug/kg. Fever was a more sensitive indicator of intoxication than was emesis. Highly purified preparations of enterotoxin A, whether administered intravenously (0.01 to 1.0 mug/kg), orally (10 to 25 mug/kg), or into the cerebral ventricles (0.005 to 0.020 mug in 0.20 ml), were also pyrogenic in cats. Tolerance to the pyrogenic activity was produced by repeated intravenous injection of a given dose of enterotoxin A but not by repeated intracerebroventricular injection. Enterotoxin A was more potent than enterotoxin B after intravenous injection in causing both fever and emesis. Cross-tolerance could not be demonstrated between enterotoxin A and enterotoxin B or Salmonella typhosa endotoxin. This lack of cross-tolerance plus the inability of large oral doses (100 to 4,700 mug/kg) of endotoxin to cause fever or emesis indicate that the reported responses were attributable to the specific toxins administered and not to contamination by other pyrogens.     

Muramyl dipeptide does not induce slow-wave sleep or fever in rats

The synthetic muramyl dipeptide, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), is reported to increase slow-wave sleep and body temperature in cats, rabbits, and squirrel monkeys. The present study examined the ability of MDP to induce sleep and fever in rats. MDP was administered IP at 50, 250 and 500 micrograms/kg. Sleep and body temperature were monitored for 12 hr. MDP failed to affect the duration of wakefulness, S1, S2, or total (S1 + S2) slow-wave sleep. There was also no change in the latency to the first episode of S2 sleep. In contrast, rapid-eye-movement (REM) sleep was significantly suppressed for the first 6 hr after 250 and 500 microgram/kg doses of MDP. There was, however, a rebound increase in REM sleep after the initial period of suppression which resulted in no overall change in the amount of REM sleep. Body temperature was unaffected by MDP. Thus, we conclude that MDP has neither sleep-promoting nor pyrogenic actions in the rat when administered systemically at doses reported to be effective in several other species.     

Biological activity of synthetic subunits of Streptococcus peptidoglycan. II. Relation of peptidoglycan subunits and analogues to fever effect and induction of tolerance

A series of synthetic subunits and analogues of streptococcal peptidoglycan was prepared and used in fever and tolerance experiments on rabbits. The lengthening of the chain of the peptide moiety of peptidoglycan did not result in pyrogenic activity, except for hexapeptide. Attachment of the muramyl residue rendered the peptides pyrogenic. The activity of such materials varied in degree and was rather in an indirect relation to peptide chain length. A change in the configuration of C4-OH or C3-OR in the muramyl residue resulted in a profound decrease in pyrogenicity. No inhibitory effect of N-acetylmuramyl-D-alanyl-D-isoglutamine on muramyldipeptide (MDP) pyrogenicity could be demonstrated. Repeated administration of MDP resulted in the induction of tolerance to the pyrogenicity of this substance in rabbits. These animals were not tolerant to the pyrogenicity of peptidoglycan. Nontolerance was also observed in reciprocal experiments with these materials as well as in trials with hexapeptide and peptidoglycan given in either order. The data are consistent with the assumption that peptidoglycan contains more than one biologically active subunit. There is a structure-to-function relationship. The knowledge of the biological effects of the synthetic analogues is essential for the prospect of their use under model or human conditions.     

Effect of interleukin-18 on mouse core body temperature

We have studied, using a telemetry system, the pyrogenic properties of recombinant murine interleukin-18 (rmIL-18) injected into the peritoneum of C57BL/6 mice. The effect of IL-18 was compared with the febrile response induced by human IL-1beta, lipopolysaccharide (LPS), and recombinant murine interferon-gamma (rmIFN-gamma). Both IL-1beta and LPS induced a febrile response within the first hour after the intraperitoneal injection, whereas rmIL-18 (10-200 microg/kg) and rmIFN-gamma (10-150 microg/kg) did not cause significant changes in the core body temperature of mice. Surprisingly, increasing doses of IL-18, injected intraperitoneally 30 min before IL-1beta, significantly reduced the IL-1beta-induced fever response. In contrast, the same pretreatment with IL-18 did not modify the febrile response induced by LPS. IFN-gamma does not seem to play a role in the IL-18-mediated attenuation of IL-1beta-induced fever. In fact, there was no elevation of IFN-gamma in the serum of mice treated with IL-18, and a pretreatment with IFN-gamma did not modify the fever response induced by IL-1beta. We conclude that IL-18 is not pyrogenic when injected intraperitoneally in C57BL/6 mice. Furthermore, a pretreatment with IL-18, 30 min before IL-1beta, attenuates the febrile response induced by IL-1beta.     

Experimental protective action of kanamycin, ampicillin and their combination with methyluracil and pyrogenal]

Efficacy of kanamycin, ampicillin and their combinations with methyluracyl and pyrogenal in experimental Coli infections was studied. The antibiotics were administered an hour after the infection. Methyluracyl and pyrogenal were used according to 2 schemes. Scheme No. I: the drug is used daily for 7 days in increasing doses, the last dose is administered 24 hours before the infection. Scheme No. 2: the drug is used once at the moment of the infection. The methyluracyl doses were: 0.5, 1.0, 2.5 mg and 5 mg and 5 mg per a mouse during the following 4 days. The pyrrogenal doses were: 5, 10, 15, 25, 30 and 35 minimum pyrogenic doses. 5 mg of methyluracyl and 35 minimum pyrogenic doses of pyrogenal were used according to scheme No. 2. The most pronounced increase in the efficacy of kanamycin, ampicillin and their combination was observed in the animals treated simultaneously with methyluracyl and pyrogenal according to scheme No. 1. The efficacy of kanamycin and ampicillin increased 3 and 2.68 times respectively. ED50 of kanamycin and ampicillin used in combination in the animals treated with methyluracyl and pyrogenal was lowered 4 and 2.9 times respectively as compared to that in the animal groups treated only with the antibiotic combination and 21 and 15.2 times respectively when the antibiotics were used alone. Sanation of the animal organs was also rather successful. A single administration of methyluracyl and pyrogenal simultaneously with the infection (scheme No. 2) had a lower effect on the efficacy.     

Amphotericin B Formulated in Liposomes and Lipid Based Systems: A Review

Abstract

The therapeutic index (T.I.) of amphotericin B (amB) is significantly increased when the drug is formulated in a variety of liposome and lipidic systems. This increase is due to a dramatic decrease in toxicity with maintenance of antifungal activity. Three formulations are currently being commercialized: ABLC?, AmBisome and Amphocil?. ABLC? is a ribbon-type lipid complex with a diameter in the 2-5 micron range consisting of dimyristoylphosphatidylcholine, dimyristoylphosphatidylglycerol and amB (7/3/10 mole ratio). AmBisome? is a liposome with a particle diameter less than 100 nm and composed of hydrogenated soy phosphatidylcholine, cholesterol, distearoylphosphatidylglycerol and amB (2/1/0.8/0.4 mole ratio). Amphocil? is a lipidic particle with a diameter of 115 nm and consists of cholesterylsulfate and amB (1/1 mole ratio). In spite of the differences, each formulation reduces the transfer of amB into a sensitive site of toxicity while affecting transfer of the drug into the fungal target to a lesser extent. Two principal mechanisms account for this: vehicle composition decreases the transfer rate of amB into cholesterol containing mammalian membranes (sites of toxicity) more than into ergosterol containing fungal membranes (sites of efficacy). Vehicle diameter influences the pharmacokinetics and disposition of the drug into the reticuloendothelial system. Immediate toxicity is reduced because the drug is rapidly eliminated from the central compartment and directed into macrophages. Subsequent release from macrophages at sites of infection can improve die efficacy. The chapters in this volume review the current understanding of the mechanism of amB and cover in detail die biophysical, pharmaceutical and therapeutic aspects of amphotericin B lipid formulations.     

Age-related differences in thermoregulatory responses to endotoxin in rabbits.

E. coli endotoxin evokes fever in rabbits immediately after birth. In 0--3 day-old rabbits the fever is monophasic and brown fat thermogenesis is mainly responsible for the reaction. In 6--10 day-old animals the fever is usually biphasic and increased heat conservation also contributes to the response. An inverse relationship exists between the endotoxin dose and the latent period before the onset of fever, while the height of the fever is independent of the endotoxin dose. The response is similar as that of adult rabbits, except that after all endotoxin doses the latent period is longer and the magnitude of the response slightly smaller in the newborn.     

The production of migration inhibitory factor (MIF) in rabbit lymphocytes by other than immunological mechanism

The ability of certain substances to activate lymphocytes in terms of the production of biologically active substances was studied. These substances were tested by following their migration inhibitory activity. The capacity of concanavalin A and commercial preparations of phytohaemagglutinin (PHA-M and PHA-P) to induce formation of MIF was confirmed. In addition, similar activities were found even in antirabbit antilymphocyte serum (ALS) and erythrogenic toxin (ET). The production of MIF was usually found in material obtained from rabbits treated with complete Freund adjuvant (CFA). On the other hand, material obtained from non-treated rabbits, was found to be inactive with the exception of thymus from young rabbits. The results support the significance of an increasing pharmacological potency of cells in the course of sensitization with CFA. The evidence concerning the release of MIF by ET was extended by similar finding in rabbits made tolerant to the pyrogenic activity of this particular toxin. Lymphocytes obtained from rabbits that were unable to respond to ET by fever, could not liberate MIF (at the same time) upon incubation with ET, even though these lymphocytes produced MIF normally after incubation with PPD tuberculin or concanavalin A. The importance of these results is discussed in terms of the immunologically nonspecific activation of lymphocytes that can mimic immunologically specific events.