Micrograms Of Endotoxin Research Articles

Leukocyte and cytokine accumulation in the ovine teat and udder during endotoxin-induced inflammation.

The accumulation of leukocytes, ovine serum albumin and the cytokines interleukin-1 beta(IL-1 beta), tumour necrosis factor-alpha(TNF-alpha), interleukin-8 (IL-8), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma(IFN-gamma) was studied during endotoxin-induced inflammation in lactating and dry ovine udders, and in the teat cisterns of dry ewes after surgical closure of the passage between the teat and udder cisterns. Samples were taken before infusion and hourly up to 10 h after infusion of 0.1, 1 or 10 micrograms of endotoxin, or infusion of pyrogen-free saline (PFS) as a control. Rectal temperatures were measured. A significant dose- and time-dependent accumulation of leukocytes, mainly neutrophils, was observed in the lactating udders and in the teat cisterns. In the dry udders, the leukocyte accumulation was significant for time but not for dose. Peak numbers of cells were reached at 3-4 h in the dry udders and in the teat cisterns, but not until 10 h after infusion in the lactating udders. The changes in the ovine serum albumin concentrations mostly paralleled changes in leukocyte numbers. A role was indicated for TNF-alpha, IL-8 and GM-CSF, but not for IL-1 beta and IFN-gamma, during endotoxin-induced inflammation in the ovine udder. Release of TNF-alpha, IL-8 and GM-CSF was most prominent in lactating udders, peaking at 2 or 3 h after infusion, but was also detected in dry udders and teat cisterns. Detectable levels of IL-1 beta and IFN-gamma were occasionally found in all three groups.

Partially-hydrophobized polymer particles derived from N,N-dimethylaminopropylacrylamide for endotoxin removal from acidic protein solution.

Novel copolymeric adsorbents for the selective removal of endotoxin from an acidic protein solution were prepared. The adsorbents comprise spherical copolymers derived from N,N-dimethylaminopropylacrylamide (DMAPAA) and divinylbenzene (DVB). When the molar ratio of DMAPAA to DVB was 80/20 (amino-group content: 5.1 meq/g) and the pore size (molecular mass exclusion of polysaccharide, Mlim) was 4000 to 10000, DMAPAA/DVB showed high endotoxin-adsorbing activity at pH 5.0 to 9.0 and ionic strengths of mu = 0.05 to 0.4. The capacity of the adsorbent (Mlim: 4000) was 390 micrograms of endotoxin (lipopolysaccharide purified from E. coli O111:B4) per ml of the adsorbent using the batchwise method. The apparent dissociation constant between endotoxin and the adsorbent was 2.2 x 10(-12) M. On the other hand, the adsorption of bovine serum albumin, an acidic protein, by the adsorbent increased with an increase in Mlim from 4000 to 10000, but decreased with an increase in ionic strength (mu) from 0.05 to 0.2. As a result, DMAPAA/DVB (80/20) (Mlim: 4000) selectively removed endotoxin from various acidic protein solutions at pH 7.0 and mu = 0.05. The residual concentration of endotoxin in the protein solution always decreased to a concentration lower than 0.1 ng/ml, and recovery of the protein was more than 97%.

Endotoxin-mediated dendritic cell release from the intestine. Characterization of released dendritic cells and TNF dependence.

Dendritic cells (DC) acquire Ag in peripheral tissues and transport it to lymph nodes where they efficiently activate resting T cells. We have shown that i.v. endotoxin causes increased release of intestinal DC into lymph. In this paper we further characterize the release of DC and the properties of the released cells. A total of 50 micrograms of endotoxin injected i.v. causes an increase in DC output within 6 h that peaks between 12 and 24 h, with a maximum output of 8 to 15 times normal. At the same time lymphocyte output is markedly decreased. The increased output of DC is followed by a decrease to subnormal levels. The stimulated release of DC is almost totally blocked by a monoclonal anti-TNF-alpha Ab. A second injection of TNF-alpha does not result in further DC release. DC are not released from lymph nodes into efferent lymph by endotoxin. DC collected from lymph after endotoxin treatment show increased expression of the p55 IL-2 receptor and the OX48 Ag but otherwise resemble normal lymph DC. In functional assays they show no significant differences from normal in their ability to stimulate a MLR or to present Ags to sensitized T cells. Immunocytochemistry with the use of MRC OX62 suggests that the DC are released into lymph from the lamina propria of the small intestine. The stimulated release of DC mediated by TNF-alpha may be important in regulating Ag presentation in lymph nodes draining inflammatory sites.

Prothrombotic effects of endothelin-2 (ET-2).

In this report we demonstrate that ET-2, in addition to its powerful vasoconstrictor properties and its role in hypertension, is capable of creating a hypercoagulable (prothrombotic) state. This study was facilitated by the utilization of a sensitive modified recalcification time (MRT) test that can measure coagulation at all points in the coagulation spectrum. The MRT was determined on aliquots (1 ml) of citrated blood which were added to saline (MRTS), to 10 micrograms of endotoxin (MRTE), and to 10 pg of ET-2 (MRT ET-2). The mean values of MRTS, MRTE, and MRT ET-2 were 4.3 +/- 0.8, 3.7 +/- 1.0, and 3.9 +/- 0.8 min, respectively. There was a statistically significant difference between MRTE vs. MRTS and MRT ET-2 vs. MRTS using the two-tailed t-test (p < 0.02 and p < 0.005, respectively).

Endotoxin-induced vasodilatation in anaesthetized rat skin involves nitric oxide and prostaglandin synthesis.

1. The effect of intradermally injected endotoxin on skin blood flow was investigated in anaesthetized male Wistar rats in vivo. 2. Local skin blood flow changes were measured hourly for 6 h in the shaved dorsal skin with a laser-Doppler flow probe and compared to changes in control sites which had been injected with 100 microliters of phosphate-buffered saline. By 3 h, skin blood flow increased above basal by 129 +/- 27% and 186 +/- 29% with 1 and 10 micrograms of endotoxin respectively. Blood flow remained significantly elevated at 6 h, the corresponding figures being 129 +/- 24% and 154 +/- 31% (P less than 0.05, n = 6 rats, mean +/- s.e.mean). 3. In further experiments, the response to 3 micrograms of endotoxin was measured at 4 h and treatment with a cyclo-oxygenase inhibitor, nitric oxide synthase inhibitors or a topical steroid all significantly inhibited this response (P less than 0.05 in each case, n = 6 rats in each group with duplicate sites in each animal). 4. Indomethacin 3 x 10(-9) mol per site injected 3.5 h after injection of endotoxin suppressed the mean 4 h response to endotoxin by 78%; NG-nitro-L-arginine methyl ester (L-NAME) 10(-7) mol per site suppressed the response by 95%; NG-monomethyl-L-arginine (L-NMMA) 10(-7) mol per site suppressed the response by 50%; whereas the D-isomer of NG-monomethyl-arginine 10(-7) mol per site had no significant effect.5. Topical application of the corticosteroid, betamethasone 17-valerate (1% solution) 18 h before injection of endotoxin inhibited the mean 4 h response to endotoxin by 66% and the 6 h response by 48%.6. In the same model, the vasodilator response to arachidonic acid was inhibited by both indomethacin and nitric oxide synthase inhibitors (P<0.05 in each case).7. These data suggest that the microcirculatory vasodilator response to endotoxin and arachidonic acid injected locally involves both nitric oxide synthase and cyclo-oxygenase in this in vivo model.

Toxic shock syndrome toxin 1 enhances synthesis of endotoxin-induced tumor necrosis factor in mice

Toxic shock syndrome toxin 1 (TSST-1) was tested for its ability to enhance the production of endotoxin-induced tumor necrosis factor (TNF) in C3H/HeN mice. The TNF level in serum was quantified by a sandwich enzyme-linked immunosorbent assay (ELISA). It was found that when mice were injected with 20 micrograms of TSST-1 12 h before exposure to 1 micrograms of endotoxin, the serum endotoxin-induced TNF was 20 times as high as that found in mice exposed to endotoxin alone. Although 20 micrograms of TSST-1 did induce a maximum level of near 1 ng of TNF per ml of serum 1.5 h after exposure, the TNF concentration was greatly diminished after 5 to 6 h and was no longer detectable after 12 h. Pretreatment of mice with 20 micrograms of TSST-1 or 1 micrograms of endotoxin did not influence TNF induction by TSST-1 12 h later. Also, pretreatment of mice with 1 micrograms of endotoxin did not enhance TNF induction by endotoxin 12 h later. Enhancement was achieved only when mice were exposed to TSST-1 more than 4 h and less than 24 h before injection of endotoxin. Despite the relatively high serum TNF levels (30 to 50 ng/ml), no mortality was observed in the mice treated with both TSST and endotoxin.

Sympathico-adrenal effects of endotoxaemia in cattle.

Intradermal injection of 46 micrograms E coli endotoxin had no effect on the plasma cortisol and noradrenaline concentrations of four dairy cows. Mean values were similar to normal values reported in the literature. Intravenous injection of 75 micrograms of endotoxin on the following day caused a massive increase in plasma cortisol concentrations which lasted for seven hours. Plasma noradrenaline concentrations increased rapidly after the intravenous administration of endotoxin and remained high for at least one hour. A possible relationship between endotoxaemia and the pathogenesis of acute laminitis is discussed.

Effect of bacterial endotoxin on lipoxygenase and cyclo-oxygenase metabolites by rat neutrophils and correlation with cellular functional parameters

The effect of Salmonella enteritidis endotoxin on in vitro rat neutrophil cyclo-oxygenase and lipoxygenase metabolism, phagocytic activity, superoxide (O2-) generation, and microbicidal activity was investigated. Incubation of polymorphonuclear leukocytes (PMN) with 5, 25, and 50 micrograms of endotoxin significantly enhanced synthesis of immunoreactive (i) leukotriene (LT)C4/D4 and thromboxane (Tx)B2 (P less than 0.001) as compared to control cells. Endotoxin 5 micrograms/ml produced optimal stimulation of the arachidonic acid metabolites. Calcium ionophore, A23187, significantly enhanced iLTC4/D4 and iTxB2 synthesis more than that elicited with endotoxin. Although phagocytic function was not significantly altered by endotoxin, intracellular killing of C. albicans demonstrated enhanced microbicidal activity at 5 micrograms/ml of endotoxin. Superoxide generation was significantly enhanced in neutrophils stimulated with phorbol myristate acetate (PMA). Endotoxin (5 micrograms/ml) further potentiated superoxide generation by these cells when stimulated by PMA. These findings demonstrate that endotoxin directly enhances neutrophil iLTC4/D4 and iTxB2 synthesis. The enhanced arachidonic acid metabolism elicited by endotoxin in these cells parallels increased microbicidal activity and superoxide generation.

Physiology of the potentiation of lethal endotoxin shock by streptococcal pyrogenic exotoxin in rabbits.

Streptococcal pyrogenic exotoxin (SPE) dramatically potentiates the lethal shock induced by gram-negative bacterial endotoxin. To provide further understanding of the mechanism underlying the potentiating effect, the physiological basis for the toxic synergism of the two toxins was investigated. Pretreatment of rabbits with an intravenous (i.v.) dose (10 micrograms/kg of body weight) of SPE greatly enhanced the endotoxin lethality and reduced the 50% lethal dose to less than 5 micrograms of endotoxin per kg. The SPE pretreatment dose caused severe pathophysiological changes in combination with a small i.v. dose of endotoxin (1 microgram/kg). These changes included transient hyperglycemia followed by profound hypoglycemia, elevation of the blood lipoperoxide level, and an acute increase in plasma beta-glucuronidase activity. These changes were comparable with those in animals given a large i.v. dose of endotoxin (100 micrograms/kg) alone. An injection of SPE alone did not alter any of the parameters described above. These results suggest that SPE renders rabbits more sensitive to extensive pathophysiologic effects of endotoxin, and the potentiating effect on endotoxin lethality may thus involve a general potentiation of physiologic failures. The SPE pretreatment depressed the vascular clearance of a large dose of endotoxin (100 micrograms/kg) but failed to affect that of a small dose of endotoxin (1 microgram/kg). The data suggest that the potentiating effect is not readily explained solely on the basis of the decreased clearance of endotoxin.

Protection from endotoxemia: a rat model of plasmapheresis and specific adsorption with polymyxin B.

Polymyxin B (PB), a cyclic polypeptide antibiotic, has potent antiendotoxin properties but is associated with significant toxicity when given parenterally. As an alternative, PB was immobilized on a solid phase (Sepharose 4B; Pharmacia, Uppsala, Sweden), and a system of plasmapheresis was developed in the conscious rat, with specific on-line plasma adsorption of endotoxin by a PB-Sepharose column. PB-Sepharose columns removed 94% of a challenge dose of 5 micrograms of endotoxin in vitro. Rats were pretreated with lead acetate so that they were sensitized to endotoxin and then given 10 micrograms of endotoxin/kg intraarterially. After 15 min plasmapheresis was begun and continued for 90 min. Animals whose plasma was perfused over PB-Sepharose were protected from endotoxin-induced leukopenia (P less than .01), thrombocytopenia (P less than .001), and death (four of four survivors compared with none of four controls). Thus plasmapheresis with on-line removal of endotoxin is a safe and highly effective means of protecting animals from the effects of endotoxemia.

Clearance of endotoxin from blood of rabbits injected with staphylococcal toxic shock syndrome toxin-1.

This study was undertaken to examine some of the properties of staphylococcal toxic shock syndrome toxin 1 (TSST-1) with regard to the clearance of endotoxin from blood. The concentration of endotoxin in blood was measured by using a chromogenic limulus test and modified perchloric acid method. When TSST-1, which produces fever in rabbits, was injected (100 ng/ml or 100 micrograms/ml per kg) intravenously (i.v.) into the animals, no measurable level of endotoxin was detected in the blood. In control animals, which were given 5 micrograms of endotoxin per ml per kg i.v., endotoxin could be detected in the blood at rapidly declining levels. These results suggested that TSST-1 might not lead bacterial endotoxin from other body sites into the blood. When the animals were given TSST-1 (1 to 100 ng/ml per kg) i.v. and then endotoxin (5 micrograms/ml per kg) i.v. 4 h later, endotoxin was detected in the blood at a high level, depending on the dose of TSST-1 injected. These results showed that TSST-1 inhibited the clearance of endotoxin in the blood; this clearance is thought to be mainly done by the reticuloendothelial system. In the animals given TSST-1 (100 ng/ml per kg) and endotoxin (5 micrograms/ml per kg) simultaneously, the endotoxin level in the blood was found to be higher than that in control animals given endotoxin only but lower than that in the animals given TSST-1 and then endotoxin at the same doses.

Endotoxin contamination of enzyme conjugates used in enzyme-linked immunosorbent assays.

The specificity of the enzyme-linked immunosorbent assay(s) is thought to depend on the specificity of the antibody used in the assay system. Therefore, the association of broadly reactive antigens like endotoxin with enzyme conjugates or other enzyme-linked immunosorbent assay reagents has the potential of altering the specificity of reactions in the enzyme-linked immunosorbent assay. Using the Limulus amoebocyte lysate assay, we demonstrated that commercially prepared conjugates of goat anti-human immunoglobulin G peroxidase, goat anti-rabbit immunoglobulin G alkaline phosphatase, rabbit anti-human immunoglobulin G, and other enzyme conjugates contained endotoxin. Furthermore, the staphylococcal protein A, horseradish peroxidase, and bovine alkaline phosphatase used to prepare enzyme conjugates also contained endotoxin. Commercially obtained bovine alkaline phosphatase contained as much as 1.0 microgram of endotoxin per ml of enzyme solution. Both commercially prepared enzyme conjugates and those prepared by us contained endotoxin as determined by their absorption to immobilized monoclonal antibody to lipid A or to immobilized Limulus amoebocyte lysate. The results of this study further suggest that the endotoxin was associated with the enzyme component of the conjugate. In a competitive inhibition enzyme immunoassay, 10 micrograms of lipid A per ml inhibited binding of the enzyme conjugate to adsorbed Limulus amoebocyte lysate, thereby confirming that endotoxin mediated the binding of the conjugate in that system. The potential significance of endotoxin bound to enzyme conjugates may be far reaching because of the ubiquity of endotoxin in conjugates and the prevalence of antibodies to endotoxin in mammalian serum.

Absence of endotoxin fever but not prostaglandin E2 fever in the Brattleboro rat.

Changes in colonic temperature following intracerebroventricular injection (icv) of bacterial endotoxin or prostaglandin E2 (PGE2) were measured in Long-Evans (LE) and Brattleboro (DI) rats. Indwelling cannulas were implanted into the brains of rats for subsequent microinjection into a lateral cerebral ventricle. Microinjection of 1 microgram of bacterial endotoxin into a lateral cerebral ventricle produced a fever in the LE rat but not in the DI rat. Daily injections of 1 microgram of endotoxin icv in the DI rat did not result in a fever. Intraperitoneal injections of 50 micrograms of bacterial endotoxin resulted in a fever in the LE rat, but the DI rat showed no such response. Both groups of animals did produce a fever in response to icv administration of 200 ng of PGE2. The lack of arginine vasopressin in the DI rat may be related to the animal's failure to show a febrile response to endotoxin.

Leucocytic changes in cows given intravenous injections of Escherichia coli endotoxin

Single intravenous injections of Escherichia coli endotoxin into normal cows produced dose dependent leucocytic and febrile responses. Injections of 5 to 20 microgram endotoxin induced a neutrophilia between 4 and 8 h, whereas 50 to 500 microgram produced a severe neutropenia for 4 to 6 h followed by a gradual increase in neutrophils along with slight to moderate left shift. Higher doses caused progressively severe lymphopenia and greater increase in fever than the lower doses. It was concluded that blood leucocytic changes during the early part of endotoxin-induced mastitis are partly related to systemic absorption of endotoxin, and that the size of the bone marrow neutrophil reserve in the cow could be estimated by injecting 5 to 10 microgram of endotoxin intravenously.

Unilateral Shwartzman reaction: Cortical necrosis in one kidney following in vivo perfusion with endotoxin

Unilateral renal cortical necrosis was selectively induced by in situ perfusion of the rabbit kidney with a perfusate containing 50 microgram of endotoxin followed by the i.v. administration of 250 microgram of endotoxin 24 hr later. The results strongly support the idea that the initial event in the genesis of renal cortical necrosis during the Shwartzman reaction is a specific local effect of endotoxin on the vascular endothelium.