Oxidative stress adaptation improves postischemic ventricular recovery

Adaptation to various forms of stress has been found to be associated with increased cellular tolerance to myocardial ischemia. In this study, the effects of myocardial adaptation to oxidative stress was examined by injecting rats with endotoxin (0.5 mg/kg) and its non-toxic derivative, lipid A (0.5 mg/kg). Both compounds exerted oxidative stress within 1 h of treatment as evidenced by enhanced malonaldehyde formation. The oxidative stress disappeared steadily and progressively with time in concert with the appearance of the induction of glutathione and antioxidative enzymes that included superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase. After 24 h of endotoxin or lipid A treatment, the amount of oxidative stress and antioxidant enzyme levels were significantly lower and higher, respectively, compared to those at the baseline levels. Corroborating these results, both endotoxin and lipid A provided protection against myocardial ischemia and reperfusion injury as evidenced by significantly improved postischemic recovery of left ventricular functions. The data presented here demonstrates that a controlled amount of oxidative stress induces the expression of intracellular antioxidants that can result in enhanced myocardial tolerance to ischemia. This suggests that myocardial adaptation to oxidative stress may be a potential tool for reduction of ischemic/reperfusion injury.     

Heat shock proteins and heat adaptation of the whole organism

Moseley, Pope L. Heat shock proteins andheat adaptation of the whole organism. J. Appl.Physiol. 83(5): 1413-1417, 1997.Adaptation toheat may occur through acclimatization or thermotolerance; however, thelinkage of these phenomena is poorly understood. The importance of heatshock proteins (HSPs) in thermotolerance and differences in theiraccumulation in organisms adapted to the heat suggest a role for HSPsin acclimatization as well. The role of HSPs in heat adaptation of thewhole organism and the interrelationships among heat adaptation,endotoxin tolerance, and cytokine resistance through HSPs are reviewed.

     

Effects of lipid A content, hydrocortisone and polymyxin B on endotoxin-induced decreases in in vivo drug metabolism

Using hexobarbital sleeping and zoxazolamine paralysis time as indices of in vivo hepatic drug metabolism, the effects of endotoxin on drug action appear to be time- and dose-dependent and the lipid A moiety of endotoxin appears to be responsible for its inhibitory effects. These studies have also demonstrated that polymyxin B can ameliorate the adverse effects of endotoxin on drug metabolism. Since hydrocortisone protects mice from endotoxin lethality, but does not alter the prolongation of hexobarbital sleeping time caused by endotoxin, it is possible to separate the lethal effects of endotoxin from its effects on drug metabolism.     

Endotoxin extends survival of adult mice in hyperoxia

Research on endotoxin protection from oxygen toxicity is presently limited to the rat model since only rats have been protected by endotoxin. This study reports that endotoxin also extends survival of adult male mice in hyperoxia (greater than 99% oxygen at 1 ATA). Initially, 4-month-old male mice were treated with Boivin-extracted E. coli endotoxin and placed in hyperoxia. Zymosan-primed mice receiving 2 or 10 micrograms endotoxin, and unprimed mice receiving 10-40 micrograms endotoxin, showed moderate protection against hyperoxia; 11/15 Boivin-treated mice survived 120 hours exposure to hyperoxia with time-of-death in hyperoxia = 126.7 +/- 4.4 hours (mean +/- SEM, n = 15). This contrasts with untreated male mice; 0/4 survived 120 hours exposure to hyperoxia with mean survival = 103.5 +/- 3.5 hours. Mice receiving 20 or 60 micrograms Westphal-extracted endotoxin were not protected nor were older female mice receiving 20 micrograms Boivin-extracted endotoxin. This study suggests that age, sex, the extraction method used to obtain endotoxin, and possibly the time of year when endotoxin is administered, are important variables in allowing endotoxin to extend survival of mice in hyperoxia.     

The effect of hydrophobic interaction on endotoxin adsorption by polymeric affinity matrix

Endotoxin, a major pyrogen of concern to the biological industry, is a lipopolysaccharide containing a highly hydrophobic region, lipid A, in its structure. The effect of hydrophobic interaction on endotoxin adsorption from an aqueous solution was studied by covalently bonding aminoalkyl groups with varying hydrocarbon lengths to a cellulose and acrylic composite matrix. The amount of endotoxin adsorbed on the matrix increased with the increasing length of alkyl groups, demonstrating the contribution of hydrophobic interaction between endotoxin and the solid matrix. Both the hydrophobic and the charge interaction prove to be effective for endotoxin adsorption, and a synergistic effect from the dual chemical forces is achievable under specified conditions. The effect of solvent, pH and salts on endotoxin adsorption provides further evidence for the importance of hydrophobic force as a means of removing endotoxin from aqueous solutions.     

Endotoxin as modifier of particulate matter toxicity: a review of the literature

It is well known that particulate matter (PM) and endotoxin are able to trigger inflammatory responses in the lung. Most studies have focused on the components separately and on the identification of chemical components associated with PM. However, since biological components may represent around 20% of airborne PM, and endotoxin may reach concentrations as high as 30 EU/mg, recent studies have focused attention on the characterization of endotoxin present in PM and health effects. Most of the literature has suggested that endotoxin adsorbed in PM is able to elicit immunological responses associated with increase in pro-inflammatory cytokine expression. The aim of this paper is to provide an up to date review of the findings involving toxicity effects of endotoxin associated with PM.     

Inhibition of ascorbic acid uptake by endotoxin: evidence of mediation by serum factor(s)

The effect of bacterial endotoxin on the ascorbic acid uptake by 3T6 fibroblasts was studied. Endotoxin inhibited ascorbic acid uptake by fibroblasts in a dose dependent manner. The inhibition by endotoxin takes place only in the presence of unheated serum; decomplementing serum by heat inactivation at 56 degrees C for 30 minutes eliminates endotoxin's inhibitory effect on ascorbic acid uptake. The effect of endotoxin appears to be instantaneous since the inhibition seen in the cells without any preexposure was similar to the cells preexposed to endotoxin for up to 6 hours. Polymyxin B sulfate which is known to bind the lipid A portion of endotoxin did not reverse the inhibition of ascorbic acid uptake caused by endotoxin.     

Applicability of bacterial endotoxins test to various blood products by the use of endotoxin-specific lysates

Endotoxin contamination is a serious threat to the safety of parenteral drugs, and the rabbit pyrogen test has played a crucial role in controlling this contamination. Although the highly sensitive endotoxin test has replaced the pyrogen test for various pharmaceuticals, the pyrogen test is still implemented as the control test for most blood products in Japan. We examined the applicability of the endotoxin test to blood products for reliable detection and quantification of endotoxin. Nineteen types of blood products were tested for interfering factors based on spike/recovery of endotoxin by using 2 types of endotoxin-specific lysate reagents for photometric techniques. Interfering effects on the endotoxin test by the products could be eliminated by diluting from 1/2 to 1/16, with the exception of antithrombin III. However, conventional lysate reagents that also react with non-pyrogenic substances, such as (1–3)-β-d-glucan, produced results that were not relevant to endotoxin content or pyrogenicity. Our results showed that the endotoxin test would be applicable to most blood products if used with appropriate endotoxin-specific lysate reagents.     

High serum IL-6 level reflects susceptible status of the host to endotoxin and IL-1/tumor necrosis factor.

Patients with high level of serum endotoxin did not necessarily develop into lethal shock, whereas some patients died of septic shock even when their serum endotoxin levels were low. These results indicate that limiting factor which determines the host to be endotoxin shock principally depends on the host susceptibility to endotoxin instead of serum endotoxin level. To understand this susceptible status of the host to endotoxin, we used Propionibacterium acnes primed mouse endotoxin shock model. We found that P. acnes-primed mice responded to low dose of LPS by enhanced production of IL-1 and TNF. And such mice were highly susceptible to the lethal shock inducing effect of IL-1 and/or TNF, which also induced high level of serum IL-6 in these mice. Therefore, measurement of serum IL-6 level provides us with the information of the preceding exposure of the host to either LPS or IL-1 and/or TNF and the highly susceptible status of the host to these stimuli. Based on these results obtained from animal model, we investigated the relationship between serum IL-6 levels and serum endotoxin levels in the patients with malignant hematologic disorders. We found that these patients fell into two groups; an endotoxin susceptible group, equivalent to P. acnes-primed mice, showing high level of serum IL-6 with low level of serum endotoxin, and a nonendotoxin susceptible group, equivalent to P. acnes-nonprimed mice, showing low or undetectable level of serum IL-6 with high level of serum endotoxin. We propose that the measurement of serum IL-6 level in the patients positive for endotoxin is a useful tool in evaluating diagnosis and prognosis of endotoxin shock.     

The Gram-negative bacterium Chlamydia trachomatis L2 stimulates tumor necrosis factor secretion by innate immune cells independently of its endotoxin

The endotoxin of Chlamydia trachomatis L(2), the causative agent of lymphogranuloma venerum, has been described as an endotoxin with an atypical structure and weak stimulatory activity. It is, however, unclear whether chlamydial endotoxin plays a role in the stimulation of innate immune cells upon contact with the whole microorganism C. trachomatis L(2). We show here that chlamydial endotoxin and, as expected, Escherichia coli O55:B5 endotoxin depend on Toll-like receptor 4 without depending on Toll-like receptor 2 to stimulate bone marrow-derived dendritic cells to secrete tumor necrosis factor (TNF). In contrast, the whole microorganism C. trachomatis L(2) induces TNF secretion by innate immune cells independently of Toll-like receptor 4, while stimulation by E. coli O55:B5 depends on Toll-like receptor 4. Furthermore, although TNF secretion of the macrophage cell line RAW264.7 with chlamydial or E. coli O55:B5 endotoxin as well as with the bacterium E. coli O55:B5 is inhibited by the endotoxin-neutralizing compound polymyxin B, C. trachomatis L(2)-induced secretion of TNF cannot be reduced. In accordance with the literature, the potential of chlamydial endotoxin is more than 100-fold weaker than E. coli O55:B5 endotoxin on all cell types tested. We conclude that chlamydial endotoxin is unlikely to be involved in C. trachomatis L(2)-induced release of TNF by innate immune cells.     

Endotoxin removal using a synthetic adsorbent of crystalline calcium silicate hydrate

A synthetic adsorbent of crystalline calcium silicate hydrate, the product LRA by Advanced Minerals Corp., has been studied for endotoxin removal from aqueous solutions. This adsorbent removes endotoxin effectively, and the removal is greatly enhanced by the presence of an electrolyte such as NaCl, Tris-HCl, or Na2HPO4. It has an endotoxin removal capacity as high as 6 million endotoxin units (EU) per gram. Its endotoxin removal kinetics is fast, and for instance, over 99.9% endotoxin in a 5000 EU/mL solution was removed by mixing for 2 min at an adsorbent usage of 10 g/L. Using the chromatographic column method to treat a 5000 EU/mL solution, an endotoxin log-reduction factor of 6.2 was achieved with a single pass. This adsorbent also demonstrated significantly better performance when compared to many commonly used endotoxin removal agents, such as ActiClean Etox Endotoxin Removal Resin, Affi-Prep Polymyxin Support, Detroxi-Gel Endotoxin Removing Gel, Q Sepharose Fast Flow Media, and Sigma Endotoxin Removal Solution. Furthermore, it demonstrated a high selective removal of endotoxin from a solution of lambda DNA. This adsorbent provides opportunities for developing disposable, scaleable, and cost-effective methods for endotoxin reduction in many biotechnological and pharmaceutical processes.     

Level of bacterial endotoxins in Haemophilus influenzae type b vaccines conjugated with toxoids (td, Di)

Quantitative evaluation of bacterial endotoxin was performed in the following vaccines: Act-Hib, Hiberix, Hib-Titer. The aim of this study was to assess the accuracy and precision of chromogenic LAL test with S-2423 substrate for this particular biopreparations and after that to determine the amounts of endotoxin as a factor of vaccine safety. Because of the lack of information concerning the presence of endotoxin in Act-Hib vaccine, we also tried to establish the limits for the presence of endotoxin in this type of vaccine. The estimated level of endotoxin was as follows: 110 EU/ml in Act-Hib, 1.64 EU/ml in Hiberix and 2.4 EU/ml in Hib-Titer. The results of this study showed that the amounts of endotoxin was dependent on the molecular size of polysaccharide PRP and on the presence of protein component. The limit of endotoxin presence in Act-Hib vaccine recommended by us is max. 150 EU/ml.     

Distribution of Chromate-labeled Brucella abortus Endoxin in Tolerant Mice

Endotoxin tolerance as manifested by a lesser degree of hypoferremia was demonstrated in mice when both pretreatment (10 mug per injection) and challenge (100 mug) does of Brucella abortus endotoxin were administered intraperitoneally. Qualitative and quantitative studies on the distribution of chromate-labeled endotoxin in normal mice revealed that the endotoxin localized predominately in the liver and hypoferremia could be related to a high uptake of endotoxin by this organ. In tolerant mice, the labeled endotoxin was found mainly in the mesenteric lymph nodes (MLN) with smaller quantities in the blood, spleen, and liver. Experiments with splenectomized mice provided supporting evidence that the liver was the target organ of the hypoferremic response to endotoxin. High localization of endotoxin in the MLN with lower quantities in the blood, livers, and spleens of tolerant mice indicated that tolerance may be the result of a blockage by the MLN, preventing the endotoxin from reaching the liver. This inference was supported by the finding that hypoferremic tolerance did not occur when the hypoferremia-provoking dosage of endotoxin (100 mug) was given intravenously to mice pretreated intraperitoneally. There was less hypoferremia in normal mice injected with a mixture of antiserum and 100 mug of endotoxin than in mice given the same dosage of endotoxin in saline. Distribution studies on endotoxin treated with specific antiserum revealed that the endotoxin localized principally in the MLN, thus preventing most of the endotoxin from reaching the liver, the target organ of the hypoferremic response.     

Effect of bacterial endotoxin on the respiratory function of liver mitochondria

It is shown that LD50 of bacterial endotoxin exerts an uncoupling effect on the mouse liver mitochondria. The effect of endotoxin is observed 3 h after its administration. Direct addition of endotoxin to isolated mitochondria has induced a rotenone-like effect and an uncoupling action when using succinate as a substrate of oxidation.     

Effects of ONO-6240, a platelet-activating factor antagonist, on endotoxin shock in unanesthetized sheep

To determine the role of platelet-activating factor (PAF) in endotoxin shock, we studied the effects of ONO-6240, a PAF antagonist, on endotoxin shock in unanesthetized sheep. Changes in hemodynamics, lung lymph balance, leukocyte and platelet counts, and arterial blood gas tensions were measured in four groups; endotoxin alone; endotoxin plus ONO-6240; ONO-6240 alone; vehicle control. Pretreatment with ONO-6240 in sheep given endotoxin significantly prevented the decreases in systemic arterial pressure, left atrial pressure and cardiac output observed in sheep given endotoxin alone. A partial effect on diminishing the magnitude of peripheral leukopenia was also noted. However, pretreatment with ONO-6240 had little effect on pulmonary hypertension and lung lymph balance. We conclude that endotoxin causes two different effects: vascular collapse and direct lung injury; and that PAF is involved only in the circulatory manifestations.     

亲和介质及溶液条件对蛋白质溶液中内毒素去除的影响

生物制品中内毒素的去除是一项十分重要的工作。为了更好地去除各种生物制品中的内毒素,采用合成的多粘菌素B琼脂糖亲和介质,通过静态吸附的方法去除蛋白质溶液中的内毒素。重点考察了介质的间臂长度、配基密度以及各种溶液条件(pH值、盐种类和浓度、蛋白质种类和浓度、内毒素浓度、添加剂等)对内毒素去除率及蛋白质回收率的影响。分别采用动态浊度法和Lowry法检测内毒素含量和蛋白质浓度。结果表明该介质具有载量高、去除速度快、去除率高、可重复使用的特点。此外,配基密度、pH值、盐浓度和蛋白质特性(等电点和疏水性)对内毒素去除效果均有重要影响。在优化的条件下,血红蛋白、人血清白蛋白和溶菌酶的回收率分别达到87.2%、73.4%和97.3%,相应的内毒素去除率分别达到99.8%、97.9%和99.7%。阐明了各种因素对内毒素去除率和蛋白质回收率的影响规律,为生物制品中内毒素的高效去除提供了参考。     

Effect of endotoxin on protein degradation and lipid peroxidation of erythrocytes.

Sepsis has often been associated with infection due to endotoxin (LPS) produced from gram-negative bacteria. Microcirculatory failure is one of the ultimate causes of septic shock. We studied the effect of endotoxin on the protein breakdown and lipid peroxidation of erythrocyte. In vivo (20 ug LPS/100 g) studies in rats showed increased tyrosine production from erythrocyte, as an index of protein degradation in erythrocyte. In vitro studies using 25 microg to 250 microg LPS per ml also showed similar type of increased effect of endotoxin in protein degradation. Washed erythrocyte devoid of plasma and leucocytes did not show any increased effect after endotoxin treatment. Lipid peroxidation was also increased after endotoxin treatment. However, protein degradation was more prominent than lipid peroxidation. We concluded therefore that the protein degradation and lipid peroxidation of erythrocytes caused by endotoxin are probably related to the production of septic shock.     

Reexamination of the effect of endotoxin on cell proliferation and transfection efficiency

Plasmid DNA purified from bacterial cells can be contaminated with endotoxin to different extents, depending on the purification method. Earlier reports indicate that endotoxin can decrease transfection efficiency in many eukaryotic cell lines; however, the amount of endotoxin required for inhibition is unclear. We determined endotoxin effects in several cell lines and observed that endotoxin levels greater than or equal to 10,000 endotoxin units (EU) were needed to significantly affect cell proliferation and viability; levels greater than 2000 EU/mu g DNA were required to significantly inhibit transfection for all but one (Huh-7) of the cell lines tested. These endotoxin levels are significantly higher than endotoxin contamination in plasmid DNA purified by anion exchange, CsCl2 gradient and endotoxin-free purification technology, but not as high as a crude alkaline lysis preparatory method. Plasmid DNA prepared using anion exchange technology was comparable to endotoxin-free technology in terms of transfection efficiency. Even Huh-7 cells, which are markedly more sensitive to endotoxins, have comparable transfection efficiencies using plasmid DNA purified by either of these two methods. We conclude that for those cell lines commonly used for transfection studies, endotoxin-free, quality DNA is not necessary because significantly higher levels of bacterial endotoxins are required to inhibit either cell proliferation or transfection.     

Potentiation of lethal endotoxin shock by streptococcal pyrogenic exotoxin in rabbits: Possible relevance of hyperreactivity of macrophages to endotoxin

Abstract Streptococcal pyrogenic exotoxin (SPE) potentiates lethal shock induced by endotoxin. We have previously reported that macrophages derived from SPE-treated rabbits showed hyperreactivity to endotoxin, and that the effect of SPE on macrophages was mediated by a lymphokine(s). Here we show that culture supernatants of SPE-stimulated lymphocytes, when administered into rabbits three hours before or together with endotoxin, potentiate a variety of endotoxin-induced pathophysiological changes and even lethal shock. These results suggest that SPE-induced lymphokine(s) mediates the potentiating effect of SPE on the lethal endotoxin shock through enhancing endotoxin reactivity of macrophages which play the central role in mediating endotoxin toxicity.     

Mitogenic effect of endotoxin on lung and tolerance of rats to hyperoxia

Treatment of rats with endotoxin, as late as 24 h after beginning exposure to greater than 95 O2 at 1 atm, increases survival at 72 h from 20-30% to greater than 95% (J. Clin. Invest. 65: 1104, 1980), whereas treatment with corticosteroids reduces survival (Toxicol. Appl. Pharmacol. 47: 367, 1979). Since endotoxin is mitogenic to some cells and glucocorticosteroids decrease DNA synthesis by lung cells, we asked 1) is endotoxin mitogenic to the lung, and, if so, 2) is the mitogenic effect required for endotoxin to produce tolerance to hyperoxia? We found endotoxin administered in vivo does have a mitogenic effect on the lung as indicated by an increased rate of DNA synthesis by lung slices; dexamethasone blocked this effect. However, although dexamethasone given alone markedly diminished survival in hyperoxia, dexamethasone did not impair the protection conferred to rats by endotoxin against the edemogenicity and lethality of hyperoxia. Furthermore, dexamethasone did not diminish the rise of antioxidant enzyme activity in the lungs of endotoxin-treated O2-exposed rats. We conclude endotoxin can produce tolerance to hyperoxia even when its mitogenic action on the lung is substantially diminished.