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Effect of prostaglandin, pyrogen and noradrenaline, injected into the hypothalamus, on thermoregulation in newborn lambs.

Thirteen newborn lambs were implanted stereotaxically with an array of 4 guide tubes positioned bilaterally so that their tips lay above various hypothalamic loci. At postnatal ages 50-70 h, rectal temperature was monitored, and bilateral 1 microliter injections were made into the hypothalamus. Prostaglandin E1 (0.2 microgram) and prostaglandin E2 (0.2-2.0 microgram) did not cause fever on any occasion though injections were made into 44 sites throughout the hypothalamus. Nevertheless, lambs that did not develop fever following central injection were often, if sensitized by bacterial pyrogen given earlier able to develop fever after intravenous injection of bacterial pyrogen. Infusion of a bacterial pyrogen (S. abortus equi, 0.2 microgram) into these same sites caused fever only when injections were made into the anterior hypothalamic/preoptic area (AH/POA). Eight lambs were placed in a 10 degrees C environment and noradrenaline was injected. Rectal temperature fell by 0.5-1.4 degrees C after 7 bilateral injections into the AH/POA, but 9 injections into other hypothalamic areas caused little temperature change. When 6 lambs were placed at a temperature of 30 degrees C and noradrenaline was injected, no consistent temperature changes were observed after microinjection into the AH/POA or other parts of the hypothalamus.

Animals

Reduced hypothalamic thermosensitivity following intraventricular injection of pyrogen in conscious rabbits.

Hypothalamic temperature thresholds for vasodilatory and respiratory reactions were determined before and after i.c.v. injection of pyrogen in rabbits. During the rising phase of fever the increases in the hypothalamic thresholds for vasodilatory and respiratory reactions differed from those found in the pre-pyrogen preoptic anterior hypothalamic area (POAH) heating by 2.1 +/- 0.2 degrees C and 1.89 +/- 0.31 degrees C, respectively. During the plateau phase of fever the threshold for vasodilatory reaction was further increased (by 0.7 +/- 0.23 degrees C), whereas that for panting remained at the same level. It is concluded that pyrogen exerts a depressive action on POAH thermosensitivity.

Animals

Electrical stimulation of the paraventricular nucleus attenuates pyrogen fever in the rabbit.

Arginine vasopressin (AVP) perfused within the ventral septal area (VSA) suppresses fever normally evoked by pyrogenic substances, including Salmonella abortus equi (SAE). Neurons containing AVP and located within the paraventricular nucleus (PVN) or the nearby bed nucleus of the stria terminalis (BnST) are believed to have projections to this septal region. A series of experiments was undertaken to determine whether electrical stimulation of these areas, which might be expected to cause the release of AVP within the VSA, would affect similarly the pathogenesis of fever. A stainless steel cannula was implanted surgically in each of 22 male New Zealand White rabbits and a monopolar electrode was lowered through this guide cannula to the PVN or BnST areas. Electrical stimulation (20 Hz, 10 s on, 10 s off, 2.6-3.2 V) was initiated 30 min prior to and was continued until 90 min after the intravenous (i.v.) administration of 0.1-1.0 micrograms of SAE (1.0 ml carrier vehicle). While afebrile body temperature remained unchanged, electrical stimulation of sites located in the rostral extension of the PVN effectively attenuated the pyrogen-induced fever. Stimulation of sites outside these areas did not affect either the absolute magnitude or the duration of the fever. Although the reduction in fever was most pronounced during the period of electrical stimulation, in some cases the fever remained suppressed beyond the application of the current. These experiments provide the first evidence that electrical stimulation of paraventricular areas with AVP-containing cell bodies is effective in suppressing a fever evoked by systemic administration of a pyrogen. Although untested, it is possible that a stimulus-induced release of AVP within the VSA is responsible for the attenuation of the fever.

Animals

The detection of endotoxin by in vitro production of endogenous pyrogen: comparison with limulus amebocyte lysate gelation.

The sensitivities of leukocyte endogenous pyrogen (EP) production and limulus amebocyte lysate (LAL) gelation to endotoxin from E. coli (minimum i.v. pyrogenic dose 4 ng/kg in rabbits) were determined. Concentrations of 0.5-1.0 ng/ml could be detected by LAL. The minimum endotoxin concentrations which generated detectable EP from 2 X 10(6) monocytes was 10-fold lower (0.05-0.1 ng/ml). At an endotoxin concentration of 0.4 ng/ml the minimum number of monocytes required for detectable EP production was 5 X 10(5). It is concluded that the LAL gelation test cannot safely be used to exclude significant endotoxin contamination in a cellular system where EP production is being measured. The same conclusion applies even more forcibly to the in vitro production of lymphocyte activating factor (LAF, interleukin-1), since it appears that LAF and EP are identical and sub-pyrogenic amounts of EP are easily detectable in the LAF assay.

Animals

Ibuprofen inhibits pyrogen-dependent expression of VCAM-1 and ICAM-1 on human endothelial cells.

Leukocyte adhesion and transmigration through the endothelial cell (EC) layer plays a crucial role in inflammation. IL-1 alpha and TNF alpha increase EC-adhesiveness for leukocytes by stimulating surface expression of ICAM-1 (intercellular adhesion molecule 1, CD54), VCAM-1 (vascular cell adhesion molecule 1, CD106) and E-selectin (CD62E). In this study, the effects of ibuprofen on IL-1 alpha and TNF alpha-induced expression of ICAM-1, VCAM-1 and E-selectin on cultured human umbilical vein EC (HUVEC) were analyzed. Exposure to IL-1 alpha or TNF alpha resulted in an increased expression of VCAM-1, ICAM-1, and E-selectin. Ibuprofen was identified as a potent inhibitor of IL-1 alpha and TNF alpha-induced surface expression of VCAM-1 and a less potent inhibitor of pyrogen-induced expression of ICAM-1, whereas no effect on E-selectin was found. The effects of ibuprofen on VCAM-1 expression were dose-dependent (IC50 [IL-1 alpha]: 0.5 mM; IC50 [TNF alpha]: 0.5 mM) and time-dependent with maximum responses observed after 18 h. Moreover, ibuprofen abrogated pyrogen-dependent adhesion of leukocytes to HUVEC. Ibuprofen also inhibited VCAM-1 mRNA expression in pyrogen activated EC. VCAM-1-downregulation on EC by ibuprofen may contribute to the anti-inflammatory actions of the drug.

Base Sequence

Synthesis and pyrogenic effect of 3 alpha, 7 alpha-dihydroxy-5 beta-androstan-17-one and 3 alpha-hydroxy-5 beta-androstane-7, 17-dione.

The first chemical synthesis of 3 alpha, 7 alpha-dihydroxy-5 beta-androstan-17-one and 3 alpha-hydroxy-5 beta-androstane-7, 17-dione is reported. In this method, the 17 beta-side chain of commercial chenodesoxycholic acid was degraded in 6 steps after selective protection of the hydroxyl groups: 3 alpha-OH by a tert-butyldimethylsilyl group and 7 alpha-OH by an acetoxy group. The capacity of 3 alpha, 7 alpha-dihydroxy-5 beta-androstan-17-one and 3 alpha-hydroxy-5 beta-androstane-7, 17-dione to release a pyrogen by human leukocytes was investigated by two independent methods: supernatants from leukocytes incubated with a steroid are injected to rabbits whose fever is measured, or tested by the Limulus Test (a pyrogen detection technique). The 7-keto substituted etiocholanolone still possessed pyrogenic activity, while the 7 alpha-hydroxyl substituted one did not.

Animals

A novel process for preparing an acellular pertussis vaccine composed of non-pyrogenic toxoids of pertussis toxin and filamentous hemagglutinin.

A novel process for preparing non-pyrogenic toxoids of pertussis toxin (PT) and filamentous hemagglutinin (FHA) is described. The process consists of chromatographies on perlite then on hydroxylapatite. Purification yields for PT and FHA are 62 and 68%, respectively. The purification process takes advantage of the novel use of perlite (a filter aid) for the simultaneous purification of PT and FHA. The hydroxylapatite, in addition to removing the remaining contaminants, also concentrates the antigens. The resulting PT and FHA are approximately 95% pure, and are non-pyrogenic as judged by the rabbit pyrogen test. The purification process is simple, inexpensive, and does not use blood components or toxic substances. The mild conditions in which the PT and FHA are purified ensure the recovery of native protein. The purified PT and FHA are detoxified in the presence of glycerol using glutaraldehyde and formaldehyde, respectively, to produce antigenic components of an acellular pertussis vaccine. The final PT and FHA toxoids are immunogenic in guinea-pigs and have been shown to be protective in the mouse intracerebral challenge test.

Bordetella pertussis

Activation of acute-phase responses by intrapreoptic injections of endogenous pyrogen in guinea pigs.

The acute-phase reaction (APR) is the concatenation of events that develops in response to infectious or other acute inflammatory stimuli. It includes fever and changes in plasma trace metal and glycoprotein levels. Endogenous pyrogen (EP) is believed to be the mediator of the APR. It acts within the preoptic-anterior hypothalamus (PO) to initiate fever; prostaglandins E (PGE) may modulate this action. To determine whether the nonfebrile responses to EP also are mediated by the PO and through PGE, guinea pigs were injected bilaterally intra-PO (iPO) with homologous EP (1 microliter) or PGE2 (0.1 microgram), and their colonic temperatures (Tco) and plasma iron (Fe), zinc (Zn), copper (Cu), and N-acetylneuraminic acid (NANA) levels were measured. For comparison, EP (2 ml) also was injected intraperitoneally (IP). Heat-denatured EP (delta EP) or pyrogen-free saline (PFS) was the corresponding control. Fevers were induced by IP EP (1.0 +/- 0.1 degrees C [mean +/- SD]), iPO EP (1.1 +/- 0.2 degrees C), and iPO PGE2 (1.4 +/- 0.2 degrees C); neither delta EP nor PFS was pyrogenic. Plasma Fe and Zn levels were decreased significantly after IP EP, but unchanged after iPO EP and PGE2. Plasma Cu and NANA levels were elevated significantly following both IP and iPO EP, but not after iPO PGE2. delta EP or PFS did not cause any changes, by either route. It appears, therefore, that EP-induced fever and rises in plasma Cu and NANA are mediated by the PO, while the decreases of plasma Fe and Zn are direct, peripheral effects. On the other hand, PGE2 appears to be involved only in the central febrile response. Indeed, guinea pigs, pretreated with indomethacin (5 mg/kg, IP), and injected iPO with EP or IP with S. enteritidis endotoxin (2 micrograms/kg), did not develop fever, but exhibited the rise in plasma Cu and NANA.

Animals

Differential production of endogenous pyrogen by human peripheral blood leucocytes following interaction with H3N2 or H1N1 influenza viruses of differing virulence.

Fever and other constitutional effects of influenza (headache, myalgia, listlessness, nausea, shivering, anorexia and depression) result from liberation of endogenous pyrogen (EP) from phagocytes. These effects are milder for recent H1N1 influenza virus isolates than for H3N2 strains. Interaction with human peripheral blood leucocytes in vitro showed that H1N1 strains, A/USSR/90/77 and A/Fiji/15899/83, elicited significantly less EP (as assessed by the rabbit pyrogen assay) than two virulent clones, 7a and 64c, of the A/Puerto Rico/8/34-A/England/939/69 (H3N2) reassortant virus system. Similar observations were made with UV-inactivated A/Fiji/15899/69 and clone 64c. These results are in accord with the differential severity of fever produced by these strains in ferrets when intranasally infected or intracardially inoculated with live and inactivated viruses. They show that influenza virus strains differ in capacity to induce EP from phagocytes. Furthermore, the observations with inactivated virus show that certain virion components are pyrogenic and differ in quantity or nature between strains. These results are important in relation to the differential severity of influenza epidemics and the reactogenicity of vaccine strains.

Animals

[Limulus test using a chromogenic method: application to the control of pyrogens in blood derivatives].

We report here the application of the LAL Test to a chromogenic substrate to detect endotoxins in Human Blood Products. In order to reduce the cost, we used a microplate procedure with the Multiskan Reader. Quantitative results in the range of 0,01 to 0,1 ng/ml allowed for a good correlation with the Rabbit Pyrogen test. For 20% albumins and 4% albumins, the mean endotoxins levels of non pyrogenic lots were 0,38 +/- 0,18 and 0,09 +/- 0,03 ng/ml. All the lots which passed the Rabbit Pyrogen test had endotoxins levels lower than 1 ng/ml and 0,2 ng/ml, respectively. We can use this test for other plasma derivatives; Gammaglobulines and PPSB are easily tested. Dried Concentrated Antihemophilic Factor and Dried plasma contain citrate which inhibits the reaction. Dilution and Addition of Calcium Chloride overcome this inhibition. For dried plasma, we should destroy plasma inhibitors by heating at 75 degrees C. This sensitive and reproductible in vitro assay improves the control of pyrogenecity in Human Blood Products.

Blood Transfusion

Measurement of hypozincaemia in mice: a sensitive test for detection of pyrogens.

1. The effect of bacterial pyrogens on plasma zinc concentration in mice was studied as a method of bioassay for these substances. 2. A dose-related depression of plasma zinc concentrations was observed 4 h after intravenous injection of doses of 0.05-500 ng of purified endotoxins of Salmonella abortus equi, Shigella dysenteriae and Escherichia coli. Responses were dose-dependent and reproducible for each endotoxin in five strains of mice. 3. Tolerance to endotoxin could be induced in mice by injection of doses of 500 ng but was not seen with doses of less than 5 ng, even after repeated treatment. 4. Dinitrophenol, a metabolic inhibitor, and amphetamine, a metabolic enhancer, did not affect plasma zinc levels, indicating that changes in basal metabolic rate do not affect the outcome of this assay. The effects of pyrogenic materials other than endotoxin on plasma zinc levels were also tested and the results suggest that measurement of hypozincaemia in mice provides the basis for a simple, practical and inexpensive test for endotoxins and other pyrogens.

Amphetamine

Studies on the pathogenesis of fever. XIV. Further observations on the chemistry of leukocytic pyrogen.

Leukocytic pyrogen previously reported to contain an essential protein moiety, appears to be a lipid-protein complex having a molecular weight in the range of 10,000 to 20,000. Evidence that it contains essential lipid includes its inactivation by Cu(++), its lability in alkaline solutions (pH 8.5 and above), and its loss of pyrogenicity when extracted with acid-isooctane. Its solubility in 66% methanol, and the enhancing action of ethanol in freeing it from sonicated cells, suggest the presence of exposed lipid groups at its surface. Once the complex is separated from other proteins, its biological activity is readily destroyed. Although the lipid component is presumed to contain unesterified fatty acid(s), its precise composition is unknown. The finding of lipid in the active complex is in keeping with the hypothesis that the pyrogen is derived from leukocytic membranes.

Fever

Studies on the pathogenesis of fever. XV. The production of endogenous pyrogen by peritoneal macrophages.

Macrophages from oil-induced peritoneal exudates in rabbits produce endogenous pyrogen when first activated by incubation in 4 hr exudate fluid and then stimulated by incubation in potassium-free isotonic sodium chloride solution. The failure of earlier investigators to obtain pyrogen from macrophages is explained, and the relevance of macrophage pyrogen to fevers of agranulocytosis and other diseases, in which mononuclear rather than granulocytic exudates predominate, is discussed.

Animals

Studies on the pathogenesis of fever. 18. Activation of leukocytes for pyrogen production.

Blood leukocytes, in contrast to exudate leukocytes, release little or no pyrogen when incubated in 0.15 M NaCl unless previously activated by exposure to endotoxin or to a protein activator that is present in acute exudate fluid. The activation process, which also occurs during phagocytosis, involves the synthesis of cellular protein, presumably related to the pyrogen molecule. Evidence is presented that generation of pyrogen in sterile inflammatory lesions depends on both the activator and the anaerobic conditions in the exudate fluid.

Animals

Studies on the pathogenesis of fever. XX. Suppression and regeneration of pyrogen-producing capacity of exudate granulocytes.

Suppression of the pyrogen-producing capacity of exudate granulocytes results from incubation of the cells in plasma, serum, or Ringer's solution. When transferred in this state and incubated in isotonic NaCl, the cells release much less pyrogen than untreated exudate cells. The suppressive effect is reversible and appears to involve the cellular uptake of calcium ions. In contrast, regeneration of pyrogen-producing capacity in depleted exudate cells occurs only when the cells are incubated in serum. The process resembles activation and requires the cellular synthesis of protein.

Blood

Group B streptococcal toxic shock-like syndrome: report of a case and purification of an associated pyrogenic toxin.

A patient with streptococcal toxic shock-like syndrome (TSLS) caused by Streptococcus agalactiae (group B Streptococcus) is described. The patient had all of the defining criteria for toxic shock syndrome (TSS), including fever, hypotension, erythematous rash, desquamation, and involvement of multiple organs. Neither Staphylococcus aureus nor group A streptococci were recovered, but vaginal and urine cultures yielded group B streptococci. The group B streptococcal isolate gave negative results in antibody tests for the production of TSS toxin 1, staphylococcal enterotoxins, and streptococcal scarlet fever toxins. However, the strain produced a novel pyrogenic toxin. This toxin, purified from culture fluids by ethanol precipitation, isoelectric focusing, and reverse-phase high-pressure liquid chromatography, had a molecular weight of 12,000 and an isoelectric point of approximately 7.0. The purified toxin was pyrogenic in rabbits, enhanced the susceptibility of the animals to lethal endotoxin shock, and caused the proliferation of rabbit splenocytes; these properties define pyrogenic toxins. When given to three rabbits via a subcutaneous miniosmotic pump, the toxin caused TSS-like symptoms ending in death. Three additional group B streptococcal strains from patients with TSLS were tested and were found to produce a toxin with similar properties.

Adult

Effects of homologous O-antibody on host responses to lipopolysaccharide from Yersinia enterocolitica: neutralization of its pyrogenicity.

The pyrogenic activity of lipopolysaccharide (LPS) obtained from Yersinia enterocolitica was neutralized by the homologous O-antiserum at the optimal antigen/antibody ratio. The level of neutralization in either antigen excess or extreme antibody excess was significantly lower than that at the optimal ratio. These facts suggested that the structure of the so-called "lattice" of LPS/antibody complex might influence the neutralization of pyrogenic activity. Moreover, when LPS was gradually added to the antiserum to reach the optimal antigen/antibody ratio, the pyrogenic activity of LPS was only slightly neutralized by the antibody. The similar event to Danysz phenomenon that has been known in the diphtheria toxin-antitoxin system was also observed in the LPS-anti-LPS system.

Animals