Course of fever response to repeated administration of sublethal doses of lipopolysaccharides, polyinosinic:polycytidylic acid and muramyl dipeptide to rabbits

The purpose of the present study was to examine the development of tolerance to three structurally dissimilar pyrogens, i.e., lipopolysaccharide (LPS), muramyl dipeptide (MDP) and polyinosinic:polycytidylic acid (poly I:C) in rabbits. The possibility of pyrogenic cross-tolerance among these agents has also been studied. It was observed that repeated injection of sublethal doses of LPS and MDP was connected with the changing of biphasic fever to monophasic. The consequence of this was a drop in the fever index. In contrast to LPS and MDP, the repeated administration of poly I:C did not result in such changes. Successive injections of this pyrogen always evoked biphasic fever. We also demonstrated that pyrogenic cross-tolerance between LPS and MDP did not occur. The cross-tolerance between LPS and MDP did not occur. The cross-tolerance among pyrogens was possible if they originated from the same class, for example endotoxin from Salmonella abortus eq. and endotoxin from Escherichia coli.     

Lack of pyrogenic tolerance transmission between brain and periphery in the rabbit

Summary Successive injections of lipopolysaccharide (LPS) either intravenously (i.v.) or intracerebroventricularly (i.c.v.) induced pyrogenic tolerance to LPS in rabbits. Tolerance was shown by a decrease of the magnitude of the fever response to repeated doses of LPS, irrespective of the route of pyrogen administration. A significantly greater and more dramatic decrease of the fever index, however, was observed in rabbits made tolerant to pyrogen given i.v. than when the pyrogen was given i.c.v. Transmission of the pyrogenic toleraance between brain and peripheral tissues, however, has not been ascertained.     

Lack of pyrogenic tolerance transmission between brain and periphery in the rabbit

Successive injections of lipopolysaccharide (LPS) either intravenously (i.v.) or intracerebroventricularly (i.c.v.) induced pyrogenic tolerance to LPS in rabbits. Tolerance was shown by a decrease of the magnitude of the fever response to repeated doses of LPS, irrespective of the route of pyrogen administration. A significantly greater and more dramatic decrease of the fever index, however, was observed in rabbits made tolerant to pyrogen given i.v. than when the pyrogen was given i.c.v. Transmission of the pyrogenic tolerance between brain and peripheral tissues, however, has not been ascertained.     

The effect of endotoxin on membrane fatty acid composition in BCG-sensitized mice

Summary The effects of endotoxin on mouse liver phospholipid fatty acid composition have been investigated. Administration of endotoxin fromSalmonella abortus equi led to a decrease in the polyunsaturated fatty acid content of livers from mice sensitized with Bacille Calmette Guérin (GCG). The content of arachidonic acid fell significantly in both the phosphatidylcholine and phosphatidylinositol fractions whereas in the phosphatidylethanolamine fraction the linoleic acid content was significantly reduced. The polyunsaturated fatty acids were replaced by increased amounts of oleic acid and palmitic acid, leading to a reduction in the polyunsaturated to saturated fatty acid ratio.     

Endogenous pyrogen formation by human blood monocytes stimulated by polyriboinosinic acid:Polyribocytidylic acid

The pyrogenic response to supernatants from human blood monocytes stimulated with polyriboinosinic acid:polyribocytidylic acid (poly I:C) was characteristic of a response to endogenous pyrogen in that it was brief and monophasic, and was destroyed by heating the supernatants at 70°C for 30 min. Pyrogen production was unimpaired when the incubations were carried out in the presence of cycloheximide (50 g/ml; an inhibitor of protein synthesis) or indomethacin (50 g/ml; an inhibitor of prostaglandin synthesis). Also, neither interferon, interleukins, tumor necrosis factor nor prostaglandin E₂ were detectable in the supernatants from the poly I:C-stimulated human monocytes.     

Type III (Arthus) allergic reactions and endotoxin toxicity in guinea-pigs

Summary Lipopolysaccharide (endotoxin) fromE. coli cells produced lethal effects in guinea-pigs. Endotoxin caused no visible dermal change in normal animals, but produced skin reactions characterized by specific Arthus-type (Type III immune hypersensitivity) vascular inflammation in immunized animals. It is concluded that Arthus allergic reactions were evoked by endotoxin, however, endotoxin lethal toxicity appears independent of this process.Acknowledgment. Supported by a grant from the National Health and Medical Research Council of Australia.     

Absence of endotoxin-fever but not hyperthermia in Brattleboro rats

I.c.v. administration of bacterial endotoxin produced a fever in the Long-Evans rat but not in the Brattleboro rat. Similar administration of arachidonic acid, prostaglandin E2, prostacyclin, dibutyryl cAMP, norepinephrine, morphine and beta-endorphin caused hyperthermia in both Long-Evans and Brattleboro rats. Variable doses of exogenous arginine vasopressin (AVP) when centrally administered with endotoxin caused fever in the Brattleboro rat. It is suggested that AVP may play an important role in the production and release of endogenous pyrogen.     

Radioprotective and haemopoietic effects of some lipopolysaccharides from Rhodospirillaceae species in mice

Summary Lipopolysaccharides (LPS) from variousRhodopseudomonas andRhodospirillum species were tested for their radioprotective efficiency against X-irradiation and for their influence on the growth of spleen colony forming units (CFU-s) in mice. The LPS fromRhodopseudomonas gelatinosa Dr₂ gave a high survival rate. It also favoured CFU-s formation and erythroid differentiation.     

Absence of endotoxin-fever but not hyperthermia in Brattleboro rats

Summary I. c. v. administration of bacterial endotoxin produced a fever in the Long-Evans rat but not in the Brattleboro rat. Similar administration of arachidonic acid, prostaglandin E₂, prostacyclin, dibutyryl cAMP, norepinephrine, morphine and -endorphin caused hyperthermia in both Long-Evans and Brattleboro rats. Variable doses of exogenous arginine vasopressin (AVP) when centrally administered with endotoxin caused fever in the Brattleboro rat. It is suggested that AVP may play an important role in the production and release of endogenous pyrogen.     

Depletion of hypothalamic norepinephrine reduces the fever induced by polyriboinosinic acid: polyribocytidylic acid (Poly I:Poly C) in rats

Administration of either Poly I:Poly C (0.05-0.50 micrograms) or norepinephrine (2-8 micrograms) into the anterior hypothalamic area produced a dose-related fever in rats. The fever induced by Poly I:Poly C was attenuated after selective depletion of norepinephrine in the hypothalamus. However, selective depletion of hypothalamic norepinephrine did not affect the fever induced by intrahypothalamic norepinephrine. The data indicate that Poly I:Poly C may act to induce fever through the endogenous release of norepinephrine from the rat's hypothalamus.     

Depletion of hypothalamic norepinephrine reduces the fever induced by polyriboinosinic acid: polyribocytidylic acid (Poly I:Poly C) in rats

Summary Administration of either Poly I:Poly C (0.05–0.50 g) or norepinephrine (2–8 g) into the anterior hypothalamic area produced a dose-related fever in rats. The fever induced by Poly I:Poly C was attenuated after selective depletion of norepinephrine in the hypothalamus. However, selective depletion of hypothalamic norepinephrine did not affect the fever induced by intrahypothalamic norepinephrine. The data indicate that Poly I:Poly C may act to induce fever through the endogenous release of norepinephrine from the rat's hypothalamus.This work was supported by grants from the National Science Council (Taipei, Republic of China).     

Colony stimulating activity in acute and chronic endotoxinemia in man

Summary Blood granulocyte-macrophage colony stimulating activity (GM CSF) was measured in 6 normal individuals challenged with low-dose endotoxin and in 63 unselected patients with nonhaematological disorders. 5/63 patients were febrile and 5 other patients showed detectable endotoxin levels, as measured by the Limulus assay. CSA levels showed a rapid increase in normal individuals following endotoxin administration, but were in the normal range in patients with chronic endotoxinemia or in those with febrile disorders. Thus, unlike acute endotoxinemia, chronic endotoxinemia is not associated with elevated activity that promotes growth of myeloid commited stem cells. In addition, fever per se did not coincide with elevated blood CSA levels.     

Colony stimulating activity in acute and chronic endotoxinemia in man.

Blood granulocyte-macrophage colony stimulating activity (GM CSF) was measured in 6 normal individuals challenged with low-dose endotoxin and in 63 unselected patients with nonhaematological disorders. 5/63 patients were febrile and 5 other patients whoed detectable endotoxin levels, as measured by the Limulus assay. CSA levels showed a rapid increase in normal individuals following endotoxin administration, but were in the normal range in patients with chronic endotoxinemia or in those with febrile disorders. Thus, unlike acute endotoxinemia, chronic endotoxinemia is not associated with elevated activity that promotes growth of myeloid commited stem cells. In addition, fever per se did not coincide with elevated blood CSA levels.     

Lack of tolerance development to tumor necrosis factor α inside the central nervous system

Tumor necrosis factor (TNF) was repeatedly microinfused into the lateral ventricle of guinea pig brains at a dose of 200 ng, 4 times within 150 min, at intervals of 3 days. In comparison to guinea pigs infused with solvent according to the same time schedule, the animals responded to TNF with pronounced fevers. The quantity of the fever response was the same after each of the 4 microinfusions of TNF. Three days after the last infusion of TNF or solvent all animals received an intramuscular injection of bacterial lipopolysaccharide (LPS). The fever in response to LPS was the same in both groups. Thus, the reported development of tolerance to repeated systemic administration of TNF^1–3 does not develop inside the blood-brain barrier. Also, the febrile response to LPS is not influenced by repeated central pre-treatment with TNF, whereas repeated peripheral treatment does have an effect.     

Resistance of in vivo-selected spontaneously transformed cells and Rous sarcoma virus-transformed cells to macrophage-mediated cytotoxicity

The cytotoxic activity (CTA) of activated peritoneal macrophages (MP) on variant lines of Syrian hamster embryo (HE) cells of differing malignant characteristics was studied. The target cells were a line of low-malignant cells resulting from spontaneous transformation of HE cells in vitro (STHE strain), and malignant variants selected from them in vivo (STHE-LM-4, STHE-LM-8, and STHE-75/18 strains). In addition, we used cells of the HET-SR-1 strain; these are HE cells transformed in vitro by a tumorigenic Rous sarcoma virus (Schmidt-Ruppin strain, RSV-SR), or the TU-SR strain induced by RSV-SR in vivo. Thioglycollate-elicited peritoneal MP from Syrian hamsters were activated in vitro with bacterial levan, LPS or MDP and used as effector cells. MP-mediated cytolysis was determined by means of a 42-h radioactivity release assay with³H-thymidine-labeled target cells. We found that only the parental STHE cells were susceptible towards fully-activated MP-mediated CTA. All three of the in vivo-selected malignant variants of the STHE cell sublines, as well as the tumorigenic RSV-SR transformants, were resistant to cytolysis by activated MP. Non-activated thioglycollate-elicited MP did not lyse any of the tumor cells studied.     

Development of DNA probes for cytotoxin and enterotoxin genes in enteric bacteria

Summary DNA probes to identify the genes encoding toxins in enteric bacteria have been developed. Use of these probes reduces the number of animals required for toxicity testing, as suspect bacteria can be directly tested for the presence of toxin. We have augmented the gene probes available by developing probes against theEscherichia coli enterotoxin LTII and shiga toxin fromShigella dysenteriae 1.The LTII gene fromE. coli 357900 was identified and characterised and a suitable internal probe was obtained. The LTII gene was found not to be common among enterobacteriae from various geographical locations. Isolates predominately of animal origin from Nigeria and Thailand hybridized with the probe.The shiga toxin gene was isolated fromS. dysenteriae 1 by a combination of in vivo and in vitro methods. An internal probe was identified and used against different serogroups ofShigella andE. coli isolated. The probe was found to hybridize withS. dysenteriae 1 isolates and also someS. flexneri andS. sonnei strains. Representatives were tested for toxin production and found to produce toxin at low levels.     

Resistance of in vivo-selected spontaneously transformed cells and Rous sarcoma virus-transformed cells to macrophage-mediated cytotoxicity.

The cytotoxic activity (CTA) of activated peritoneal macrophages (MP) on variant lines of Syrian hamster embryo (HE) cells of differing malignant characteristics was studied. The target cells were a line of low-malignant cells resulting from spontaneous transformation of HE cells in vitro (STHE strain), and malignant variants selected from them in vivo (STHE-LM-4, STHE-LM-8, and STHE-75/18 strains). In addition, we used cells of the HET-SR-1 strain; these are HE cells transformed in vitro by a tumorigenic Rous sarcoma virus (Schmidt-Ruppin strain, RSV-SR), or the TU-SR strain induced by RSV-SR in vivo. Thioglycollate-elicited peritoneal MP from Syrian hamsters were activated in vitro with bacterial levan, LPS or MDP and used as effector cells. MP-mediated cytolysis was determined by means of a 42-h radioactivity release assay with 3H-thymidine-labeled target cells. We found that only the parental STHE cells were susceptible towards fully-activated MP-mediated CTA. All three of the in vivo-selected malignant variants of the STHE cell sublines, as well as the tumorigenic RSV-SR transformants, were resistant to cytolysis by activated MP. Non-activated thioglycollate-elicited MP did not lyse any of the tumor cells studied.     

The implantation of sepharose beads in mouse livers as an aid in the study of hepatic schistosomal fibrosis

Summary An experimental model of schistosomal portal fibrosis is described. Sepharose beads the size of schistosome eggs, loaded or not with soluble egg antigen (SEA) fromSchistosoma mansoni, are injected into the coecal vein of C3H/Sn mice and become embolized in the liver. Only SEA-coated beads evoke a granulomatous reaction; this is enhanced by simultaneous priming of the mice with spleen cells fromSchistosoma mansoni-infected syngeneic animals. The fibrosis, which ensues around the beads, is stable and is much more evident after priming. Preliminary collagen tissue immunotyping reveals the presence of collagen deposits of types I and III collagen. Type IV collagen remains unchanged in the portal tracts. The model appears to be well suited for studies of the pathogenesis of portal fibrosis.This work was supported by a contract from INSERM (Action Spéciale No 3).     

Some comparative studies of two alkaline phosphatases fromAspergillus niger

Summary Alkaline phosphatases (ALP I and ALP II), isolated and purified fromAspergillus niger exhibited broad specificity towards a wide variety of substrates, and consistently ALP II was more active than ALP I. It is possible that the difference in levels of activity of the 2 enzymes may be of physiological importance in the mycelia ofA. niger.     

Antibody to tumor necrosis factor (TNF) reduces endotoxin fever

Antibody to tumor necrosis factor (TNF), injected intravenously, reduced endotoxin fever in the rabbit. The fever-reducing effect was apparent in the latter half of the febrile response.