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PubMed1977

Significance of endotoxaemia in experimental "galactosamine-hepatitis" in the rat.

The course of galactosamine hepatitis induced by 1.0 g/kg i.p. injected galactosamine (Ga1N) was investigated a sequential study in normal rats, in colectomized rats, and in rats being endotoxin resistent against both exogenous and endogenous endotoxin. Clinical symptoms of Ga1N-hepatitis such as pyrogen reaction, disseminated intravascular coagulation, arterial hypotension, and hypoglycaemia correlated significantly with the development of endotoxaemia, which was detected by means of the limulus gelation test (L.G.T.) Ga1N refractoriness was found after colectomy, a situation, in which gram negative bacterias and their endotoxins were eliminated. Ga1N refractoriness was also observed in case of endotoxin resistence. It is concluded that endotoxins contribute significantly to the pathogenesis of "Ga1N-hepatitis" and its clinical symptoms.

Animals↗
PubMed1996

Intestinal permeability, gastric intramucosal pH, and systemic endotoxemia in patients undergoing cardiopulmonary bypass.

OBJECTIVE: To examine the relationship between gastric intramucosal pH, intestinal permeability, endotoxemia, and oxygen delivery in patients undergoing cardiopulmonary bypass (CPB). DESIGN: Prospective, observational study. SETTING: Tertiary care center. PATIENTS: Fifty patients undergoing elective cardiac surgery and 10 patients awaiting elective cardiac surgery. INTERVENTIONS: Patients received chromium 51-labeled ethylenediaminetetraacetic acid (51Cr-EDTA) as a marker of intestinal permeability; insertion of a nasogastric tonometer to measure intramucosal pH (pHi); insertion of a pulmonary artery catheter to measure systemic oxygen delivery and consumption variables; arterial blood sampling for plasma endotoxin by the Limulus amebocyte lysate assay; and blood and urine sampling for measurement of 51Cr-EDTA. MAIN OUTCOME MEASURES: Systemic oxygen delivery, duration of gastric mucosal acidosis, absorption of 51Cr-EDTA, appearance of systemic endotoxemia, renal dysfunction, and duration of hospital stay. RESULTS: Median (range) 24-hour urinary recovery of 51Cr-EDTA in patients was 10.6% (2.1% to 40.2%) while that in controls was 1.2% (0.7% to 2.0%, P<.001). Intestinal permeability increased during CPB. The median (range) for the lowest pHi after bypass was 6.98 (6.74 to 7.17). The pHi did not decline until CPB was discontinued and the heart took over the load of the circulation. Endotoxin was detectable (>0.2 endotoxin unit per milliliter) in the plasma of 21 patients (42%) during the study, most of whom were endotoxemic by the end of CPB. There was no evident relationship between the degree of gut permeability, endotoxemia, gut ischemia, or systemic oxygen dynamics. CONCLUSIONS: Cardiopulmonary bypass is associated with increases in gut permeability, which precede gut mucosal ischemia. In cardiac surgical patients, a low pHi is not necessarily indicative of an adverse clinical outcome. Endotoxemia as measured by the Limulus amebocyte lysate assay is common. The increased intestinal absorption of 51Cr-EDTA and gastric mucosal acidosis occur as independent phenomena and are not related in severity or time of onset.

Acidosis↗
PubMed1996

Stimulation of cytokine production via a special standardized mistletoe preparation in an in vitro human skin bioassay.

The mistletoe preparation Lektinol is standardized with respect to bioactive mistletoe lectin, the active component of mistletoe. This standardized mistletoe preparation and its active components (mistletoe lectins) were compared in the skin2 bioassay in vitro for their capacity to stimulate interleukin 1 alpha and interleukin 6 release from skin analogue tissue, composed of human cells in their naturally secreted matrix. The standardized mistletoe preparation, its basic ingredient, aqueous mistletoe extract, and pure mistletoe lectins all stimulated IL-1 alpha and IL-6 release from skin2 tissues during 24 h incubation. The amounts of cytokines released from various skin2 tissue lots by mistletoe lectin I (ML I) (0.75-8.0 ng/ml) and by the standardized mistletoe preparation remained relatively constant across a series of different batches. Concentration-response curves to the standardized mistletoe preparation and ML I were similar for IL-1 alpha and IL-6 release. The importance of the concentration of mistletoe lectins for the cytokine-releasing action of the standardized mistletoe preparation was confirmed using a neutralizing anti-mistletoe lectin antiserum. CONCLUSIONS. Using the skin2 method it was shown that reproducible stimulation of cytokine release by a standardized mistletoe preparation from batch to batch is one of the notable features of its pharmaceutical quality. This standardized mistletoe preparation therefore represents a preparation with constant immunobiological effects. Mistletoe lectins of the standardized mistletoe preparation are the active substances in the skin2 bioassay. The skin2 method is a reliable quantitative bioassay for determination of immunopharmacological effects.

Biological Assay↗
PubMedDate not supplied

Effect of steam-heat treatment with/without divalent cations on the inactivation of lipopolysaccharides from several bacterial species.

The inactivation of endotoxin from six species of smooth gram-negative bacteria (S-form) by steam-heat treatment was investigated using the Limulus amebocyte lysate (LAL) assay. Biphasic decreases of endotoxins from four species of bacteria were observed upon steam-heat treatment of 1 microgram/ml endotoxin solution at 121 degrees C in a steam sterilizer. A lag time, however, was observed in the inactivation profiles of V. cholerae and P. aeruginosa. Distinct differences in heat resistance were observed among the bacterial species. The decrease rate was found to be concentration-dependent, and endotoxins at low concentrations (less than 10 ng/ml) were inactivated by the treatment to below the detection limit of the LAL assay. The time-course of the decrease of endotoxin from rough strains (R-form) resembled that of the respective S-form. The inactivation of R-form, especially Rc mutant, endotoxin was markedly affected by divalent cations such as Mg2+ and Ca2+, which appear to promote reaggregation of the endotoxin.

Calcium Chloride↗
PubMed1996

Plasma concentrations of endotoxin and antiendotoxin antibodies in patients with multiple injuries: a prospective clinical study.

OBJECTIVE: To investigate the time course of endotoxaemia and its effects on the synthesis of antiendotoxin antibodies in patients with multiple injuries. DESIGN: Prospective clinical study. SETTING: University hospital, Germany. PATIENTS: 40 Patients with multiple injuries and 20 healthy volunteers who served as controls. INTERVENTIONS: Blood samples were collected up to 10 days after injury and the concentrations of endotoxin, antiendotoxin antibodies to four endotoxins, and for anti-alpha-haemolysin of Staphylococcus aureus were measured. The kinetics of endotoxaemia and antiendotoxin antibodies were investigated. RESULTS: Endotoxaemia peaked 0-3 hours after injury at 0.425 EU/ml and decreased thereafter to 0.04 EU/ml within five days. Total concentrations of IgM, IgA, and IgG increased continuously after day 3 (p < 0.05), but the specific IgM response to all endotoxins was only temporary and the relative content of specific antibodies to all endotoxins peaked at day 3 (p < 0.05). Antiendotoxin antibodies of IgM class cross-reacted among themselves. There was no general increase in specific antiendotoxin antibodies of IgA and IgG class. The relative content of specific antibodies to alpha-haemolysin of S aureus of all classes of immunoglobulins (IgM, IgA, IgG) remained on the same level from day 0-10. CONCLUSION: Multiple injuries are associated with early and temporary endotoxaemia which causes specific increases in antiendotoxin antibodies of the IgM-class. IgM antibodies to endotoxins cross-react among themselves.

Adolescent↗
PubMed1996

Stimulation of neutropoiesis by a special standardized mistletoe preparation after cyclophosphamide chemotherapy in mice.

Lektinol, is a special mistletoe preparation (SMP) with immunostimulatory activity, standardized with respect to bioactive mistletoe lectin, one of the most active components of mistletoe. In the present study, stimulation of leukopoiesis by SMP was investigated in mice after induction of myelosuppression by the cytostatic agent cyclophosphamide (CP). Under the experimental conditions described, CP induced severe leukopenia followed by a recovery phase where leukopoiesis was distinctly enhanced in mice treated additionally with SMP at daily intravenous doses in a range of 30 to 5000 microliters/kg body weight. This phenomenon was mainly due to a stimulation of granulopoiesis resulting in a stronger increase of metamyelocyte and mature neutrophil counts in the peripheral blood.

Animals↗
PubMed1997

Uromodulin and Tamm-Horsfall protein induce human monocytes to secrete TNF and express tissue factor.

Effects of uromodulin (URO) and Tamm-Horsfall protein (THP), the most abundant proteins in the urine of pregnant and normal women, respectively, on the induction of TNF-alpha secretion and tissue factor (TF) expression of human monocytes were studied. THP, URO, and its fragments stimulated human mononuclear cells to proliferate and secrete TNF-alpha. The release of URO and THP-induced TNF-alpha in monocytes was dependent upon protein tyrosine kinase activation that results in tyrosine phosphorylation. URO and THP also induced TF expression of human monocytes and monocytic cell line U937 in a dose-dependent manner. TF expression was transient, reached its peak at 6 h and declined toward basal levels by 24 h. Reverse transcriptase-PCR and dot-blot analysis confirmed the induction of TF mRNA synthesis. URO and THP-induced TF expression were inhibited by actinomycin D and pentoxifylline further supporting the requirement of de novo TF mRNA synthesis. The possibility of LPS contamination of URO and THP was excluded because: 1) URO and THP-induced TF expression were inhibited by specific Ab; 2) URO was less capable of inducing TF in HUVEC as compared with LPS; 3) polymyxin B blocked the induction of Limulus clotting by LPS but not by URO and THP; 4) both LPS-sensitive (C3H/HeN) and -resistant (C3H/HeJ) mice produced little or no TNF-alpha after URO challenge. Therefore, our findings suggest that URO and THP play a significant role in the innate immunity of the urinary system and that the immunostimulatory activity of URO is potentially useful for immunotherapy.

Animals↗
PubMed1997

The lipid A moiety of Porphyromonas gingivalis lipopolysaccharide specifically mediates the activation of C3H/HeJ mice.

The lipid A preparation isolated from Porphyromonas gingivalis was found to induce splenocyte mitogenicity and TNF-alpha release from peritoneal macrophages in LPS-unresponsive C3H/HeJ mice to the same extent as in LPS-responsive mice. In order to clarify whether the activation of C3H/HeJ mice was specifically caused by the lipid A and not by contaminating protein, two strategies were employed. The lipid A fraction from P. gingivalis was subjected to either hydrochloric acid or alkaline treatment to eliminate either glycosylated phosphate or O-acylated fatty acids from the lipid A structure, and the biologic activities of the derivatives were compared in both LPS-responsive and unresponsive C3H/HeJ mice. De-1-O-phosphorylated P. gingivalis lipid A showed partial loss, and de-O-acylated lipid A complete loss of splenocyte mitogenic and TNF-alpha-inductive activities from peritoneal macrophages in both LPS-responsive and unresponsive mice. The relative activities of the intact and treated lipid A compounds in splenocyte mitogenicity and TNF-alpha-inductive activity in macrophages were similar to the relative activities of these preparations in Limulus gelation activities. The LPS-specific antagonist, succinylated lipid A precursor, inhibited P. gingivalis lipid A-mediated splenocyte mitogenicity and TNF-alpha induction in macrophages in a similar manner in LPS-responsive and unresponsive mice. These results strongly suggest that the activation of LPS-unresponsive C3H/HeJ mice by P. gingivalis lipid A was specifically mediated by the lipid A portion and not by contaminating protein. The characteristic action of P. gingivalis lipid A on LPS-unresponsive C3H/HeJ mice was thought to reflect the unique chemical properties of this compound.

Amino Acids↗
PubMed1979

Endotoxin determination in viscous opaque solutions of iron dextran by Limulus amebocyte lysate.

LAL would not form a clot when mixed with a viscous, opaque parenteral preparation of iron dextran spiked with endotoxin. However, recoverable precipitate could be obtained by diluting the LAL iron dextran mixture with PBS and centrifuging. Although the pellet so formed was red colored the protein present could be quantitated by dissolving it in a Coomassie Blue stain solution. The very rapid change in color from reddish black to deep blue was measured quantitatively in a spectrophotometer and was sigmoidally related to the amount of endotoxin used to spike the iron dextran. This method is suggested to be generally useful to measure quantitatively endotoxin concentrations too low to form a clot with LAL but high enough to precipitate recoverable protein from LAL.

Animals↗
PubMedDate not supplied

Enhancing effect of nonionic surfactant on the inactivation of lipopolysaccharide by steam-heat treatment II.

n-alkylpolyoxyethylene surfactants (CnH2n+1O(CH2CH2O)mH; CnEm) showed a strong enhancing effect on the inactivation of lipopolysaccharide (LPS) by heat treatment over a wide range of temperatures. The effect of CnE8 (n = 10-16) was observed above the critical micelle concentration (CMC) and above the cloud point, and was influenced by the length of the alkyl chains. The efficacy of the surfactants was in the order C10E8 < C12E8, C16E8 < C14E8. However, the hydrophilic moiety seemed to have no influence. An 80-95% solution of n-butanol showed a similar effect, indicating that LPS was more effectively inactivated in the oily phase of the surfactants than in water. The effect of surfactant on the hydrodynamic diameter of LPS was the same before and after steam-heat treatment for 20 minutes at 121 degrees C. Each surfactant disaggregated LPS without alteration of the activity of LPS before the heat treatment. We consider that the surfactants interact with LPS in the region of lipid A in a manner that favors loss of the activities of LPS during heating.

Chemical Phenomena↗
PubMedDate not supplied

[Endotoxic contamination of biological products (ribosomal vaccines, viral vaccines and interferon)].

The A.A. have examined by the Limulus assay the possible endotoxin contamination in some biological products (ribosomal vaccines, viral vaccines, interferon). While the preparations of influenza vaccines and a partially purified fraction of ribosomal vaccine from Pseudomonas aeruginosa exhibit a gelation of lysate with high levels of endotoxin, rubeola vaccines, interferon and a purified fraction of ribosomal vaccine, presented a negligible amount of endotoxin. The results are discussed with the aim to examine the possible role of contaminating endotoxin in the mediation of some adverse effects and of the unsuspected extrinsic adjuvant activities developed in clinical and experimental use of these preparations.

Bacterial Vaccines↗