Involvement of platelet-activating factor (PAF) in endotoxin-induced intestinal motor disturbances in rats

Intestinal myoelectrical activity was investigated in conscious fasted rats chronically implanted with Nichrome electrodes in the duodeno-jejunum. Motility of the small intestine was characterized by the presence of migrating myoelectric complex (MMC) occurring regularly at 16.2 +/- 5.8 minute intervals. Intravenous administration of endotoxin (E. coli S.0111:B4) at a dose of 50 micrograms/kg increased the interval between MMC to 112.6 +/- 26.8 min, the duration of these effects being dose-related between 10 to 100 micrograms/kg. Such a typical myoelectrical alteration, corresponding to rapidly propagated groups of spike bursts, was mimicked by the IP administration of PAF at doses of 10 to 50 micrograms/kg. Previous administration of BN 52021, a specific PAF antagonist at a dose of 50 mg/kg abolished the motor alterations induced by IP injection of PAF (25 micrograms/kg) and significantly (p less than 0.01) reduced by 61.2% those induced by IV endotoxin (50 micrograms/kg). Indomethacin (10 mg/kg IP) as well as SC 19220 (5 mg/kg IV), a PGE2 antagonist, injected prior to endotoxin (50 micrograms/kg IV) or PAF (25 micrograms/kg IP) also reduced significantly (p less than 0.01) the duration of MMC inhibition. It is concluded that endogenous release of PAF is partly responsible for the intestinal motor alterations induced by endotoxin; these effects, strongly reduced after treatment with BN 52021, are also mediated through the release of prostaglandins.     

Calcium overload in endotoxemia

E.coli endotoxin stimulates endogenous lipolysis in the $\text{in vitro}$ perfused rat heart. Verapamil^® inhibits endotoxin- (as well as glucagon-) stimulated lipolysis. This suggests that the endotoxin used increases the availability of Ca⁺⁺ to the lipolytic system in the cardiocytes. This conclusion is supported by the observed stimulation of contractility of the heart, especially during perfusion at a low Ca⁺⁺ concentration.The endotoxin was found to inhibit ATP-dependent Ca⁺⁺ accumulation in sarcolemma vesicles prepared from rat heart. A direct Ca⁺⁺ ionophoric action of the endotoxin on these vesicles could be excluded.It is discussed that Ca⁺⁺ overload may not be confined to the cardiovascular system during endotoxemia.     

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.     

Hypoferremia in mice and its application to the bioassay of endotoxin

Baker, Phillip J. (University of Wisconsin, Madison), and J. B. Wilson. Hypoferremia in mice and its application to the bioassay of endotoxin. J. Bacteriol. 90:903-910. 1965.-The ability of endotoxin to induce hypoferremia in mice was used for the bioassay of endotoxin. A marked depression in the serum-iron levels of mice occurred 12 hr after the intraperitoneal injection of 0.01 to 100 mug of Escherichia coli endotoxin; similar results were obtained with 1.0 to 100 mug of Brucella abortus endotoxin. This biological response to endotoxin appeared to be specific, reproducible, and dose-dependent. As heat-killed cells of B. abortus and E. coli were also able to induce hypoferremia, this bioassay could be employed for the determination of the endotoxin content of killed-cell preparations. Treatment of endotoxin by acid hydrolysis, acetylation, or pyridine-formic acid greatly diminished the hypoferremic response as well as its lethality for mice. Pretreatment of mice with Thorotrast had little effect upon the ability of endotoxin to induce hypoferremia; however, a stimulation of the activity of the reticuloendothelial system (RES) by treatment of mice with triolein markedly reduced the ability of endotoxin to induce hypoferremia. The relationship between the hypoferremic response to endotoxin and alterations in the activity of the RES are discussed.     

Influence of intestinal myoelectrical activity on the growth of Escherichia coli

Intestinal bacteria, particularly those adhering to intestinal epithelial cells, are exposed to electric fields and currents generated by the muscular activity of the small intestine. This activity displays a regular pattern known as the myoelectrical migrating complex (MMC). In order to explore the possibility that these endogenous electric fields could affect bacterial growth, a digitised duodenal signal obtained via serosal electrodes from a healthy calf was recorded and then applied via platinum electrodes to Escherichia coli cultures. The culture tubes were placed within a Faraday shield, incubated at 37 degrees C with shaking, and stimulated by the electric current for 5 or 8 h. The growth of E. coli stimulated by the electric current was significantly altered compared to those of non-stimulated controls: after a period of intensive growth, inhibition of cell division was observed. This was not the case when the bacteria with lon mutation were used. Moreover, synchronic bacterial culture could not be achieved in the presence of the MMC-related electric field. These results suggest that the myoelectrical activity of the duodenum, through action on cell membrane, can affect cell division of intestinal bacteria.     

The effect of Escherichia coli endotoxin on luteal function in Holstein heifers

This study was undertaken to elucidate the possible role of endotcxin in mediating premature luteolysis in the well- documented phenomenon of short estrous cycles in postpartum dairy cows. Four groups of Holstein heifers (n = 4 to 6 each) received either intrauterine infusion of sterile culture medium (Group I); intrauterine infusion of Escherichia coli (E. coli ) endotoxin (5 mug/kg) in sterile culture medium (Group II); intrauterine administration of 10 ml of a 24-h culture of a strain of E. coli isolated from the uterus of a cow with metritis (approximately 10(9) colony forming units/ml; Group III); or intravenous administration of E. coli endotoxin (5 mug/kg; Group IV) on Day 7-9 of the estrous cycle. Blood samples were collected every 48 h during the pretreatment estrous cycle and up to the administration of the experimental treatment, thereafter 4-h samples were collected for 5 d. Sample collection was then performed every 48 h for the remainder of the treatment cycle and the post treatment cycle. Serum concentrations of progesterone and plasma concentrations of 15-keto-13, 14-dihydroprostaglandin F(2alpha) (PGFM) were determined by radionmmunoassay. Intrauterine infusion of endotoxin had no effect on the cycle length or on hormone concentrations, while infusion of viable E. coli organisms tended to shorten the estrous cycle. Intravenous administration of endotoxin produced a sharp increase in both progesterone and PGFM concentrations, followed by a transient decrease in progesterone concentrations. Cycle length remained unchanged. It was concluded that the intact endometrium prevents the uptake of endotoxin although pathogenic E. coli organisms may disrupt the endometrial integrity sufficiently to shorten the estrous cycle by premature luteolysis. It is postulated that intravenous administration of endotoxin influences luteal function by the activation of the arachidonic acid cascade, by a direct effect on the corpus luteum, or via other mediators.     

Hirulog effect in rat endotoxin shock

Hirulog^TM is a thrombin catalytic site inhibitor which exhibits specificity for the anionic binding exosite of alpha thrombin. Here, we have evaluated the effect of Hirulog^TM (1, 5 and 10 mg/kg, 30 min pretreatment) in a rat model of endotoxemia. Intravenous injection of lipopolysaccharide from E. coli (25 mg/kg; serotype 0127:B8) caused decreases in blood pressure which were significantly reduced (about 60%) in animals pretreated with Hirulog^TM. Rat survival to endotoxin was significantly increased in Hirulog^TM pretreated group (5 and 10 mg/kg) up to 24 hours. Hirulog^TMat the dose of 10 mg/kg inhibited both endotoxin-induced leukopenia at 30 and 60 minute points and thrombocytopenia at 30 minute point but not at 90 and 120 minute points. Fibrinogen levels were significantly reduced after 2 hours following endotoxin administration. Pretreatment with Hirulog^TM (5–10 mg/kg i.v.) 30 min prior to administration of endotoxin prevented changes in fibrinogen plasma levels. These results demonstrate that Hirulog^TM-induced inhibition of thrombin is effective in reducing toxic and lethal effects of endotoxin.     

Circulating cytokines and endotoxin are not necessary for the activation of the sickness or corticosterone response produced by peripheral E. coli challenge.

Peripheral administration of a variety of inflammatory stimuli, such as endotoxin or cytokines, induces an orchestrated set of brain-mediated events referred to as the sickness response. The mechanism for how immune products signal the brain is not clear, but accumulating evidence supports the existence of neural as well as blood-borne pathways. Although endotoxin or cytokine administration results in sickness responses, few data exist regarding the role of circulating endotoxin or cytokines in the induction of sickness during a real bacterial infection. Thus the present studies examined whether subcutaneously administered Escherichia coli can activate sickness responses and whether circulating endotoxin and/or proinflammatory cytokines are a prerequisite for these responses. Male Sprague-Dawley rats were injected subcutaneously with one of three doses (2.5 x 10(7), 2.5 x 10(8), 2.5 x 10(9) colony-forming units) of replicating E. coli, a ubiquitous bacterial strain, or vehicle. Core body temperature (Tc) and activity were measured for 3 days after the injection. A second set of groups of animals were killed 3, 6, 12, 18, 24, and 48 h after the injection, and blood samples and brains were collected. Injections dose dependently and consistently increased Tc and decreased activity, with increases in Tc beginning 4 h after the injection. In addition, E. coli significantly increased serum interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha and brain IL-1beta levels beginning at the 6-h time point. Corticosterone and endotoxin were first elevated in the circulation at 3 and 18 h after the injection, respectively. Because fever onset preceded brain cytokine induction, we also examined cytokine levels in the serum, brain, and inflammation site 2 and 4 h after injection. Cytokines were elevated at the inflammation site but were not detectable in the serum or brain at 2 and 4 h. We conclude that subcutaneous injection of replicating E. coli induces a consistent and naturalistic infection that includes features of the sickness response as well as increases in circulating, brain, and inflammation site tissue cytokines. In addition, injection of replicating E. coli produces a robust fever and corticosterone response at a time when there are no detectable increases in circulating cytokines or endotoxin. These results suggest that elevated levels of circulating cytokines and endotoxin are not necessary for the activation of the sickness or corticosterone response. Therefore, fever, activity reduction, and corticosterone elevation induced by E. coli infection may have been evoked by a neural, rather than a humoral, pathway from the periphery to the brain.     

Pregnancy interrupting effects of some bacterial toxins.

Embryotoxic properties of Shigella dysenteriae and Clostridium perfringens toxins, of E. coli endotoxin, V. cholerase and E. coli enterotoxins were compared in mice. E. coli endotoxin has embryotoxic effects at all stages of pregnancy. E. coli enterotoxin V. cholerae enterotoxin and Shigella dysenteriae toxin are most effective mainly at earlier stages of pregnancy. Clostridium perfringens toxin has no embryotoxic effect.     

Stress-induced ACTH release after removal of the hypothalamus in rats with atrophied neural lobe.

Twenty-four hours after isolation of the pituitary by surgical removal of the medial hypothalamus, i.e. in rats with pituitary island, E. coli endotoxin significantly increased the plasma corticosterone level. Atrophy of the neural lobe, due to pituitary stalk section performed one month prior to removal of the medial hypothalamus, did not prevent the increase of ACTH release by E. coli endotoxin. E. coli endotoxin-induced ACTH release in MBH-deprived animals does not appear to be a function of mechanisms operating only in the innervated lobe.     

Effect of the activation of macrophages on the course of regeneration of rat liver following partial hepatectomy

Stimulation of the Kupffer cells with E. coli endotoxin (the purified lipopolysaccharide) or with prodigiosan (a polysaccharide from Serratia marcescens) 24 h before partial hepatectomy (resection of 65-70% of the liver) stimulated and intensified the onset of liver regenerative activity (evaluated from changes in liver DNA synthesis, the H5 labelling index and the mitotic activity of the hepatocytes). Liver DNA synthesis increased together with the dose of endotoxin (i.v., from 25 to 1000 micrograms/kg body weight). If E. coli endotoxin was injected during or 3 h after partial hepatectomy, partial inhibition of liver DNA synthesis was observed. In mice stimulated with zymosan (a polysaccharide isolated from yeast), administered 5 days before performing partial hepatectomy, proliferation of the hepatocytes (evaluated from changes in the 3H labelling index and in the mitotic activity of the hepatocytes) was evaluated. The results confirm that proliferation is correlated to the state of reactivity of the Kupffer cells.     

Physical activity alters the brain Hsp72 and IL-1beta responses to peripheral E. coli challenge

Physically active rats have facilitated heat shock protein 72 (Hsp72) responses after stressor exposure in both brain and peripheral tissues compared with sedentary rats. This study verifies that physically active animals do not have elevated Hsp72 levels compared with sedentary animals in the hypothalamus, pituitary, or dorsal vagal complex. We then examined whether 1) physically active rats respond more efficiently than sedentary rats to a bacterial challenge; 2) peripheral immune challenge elicits brain induction of Hsp72; 3) this induction is facilitated by prior freewheel running; and 4) Hsp72 upregulation produced by peripheral immune challenge results in a commensurate decrease in the proinflammatory cytokine IL-1beta. Adult male Fischer 344 rats were housed with either a mobile or locked running wheel. Six weeks later, rats were injected intraperitoneally with saline or Escherichia coli and killed 30 min, 2.5 h, 6 h, and 24 h later. Serum endotoxin and IL-1beta, and peritoneal fluid endotoxin and E. coli colony-forming units (CFUs) were measured. Hsp72 and IL-1beta were measured in hypothalamus, pituitary, and dorsal vagal complex. The results were that physically active rats had a faster reduction in endotoxin and E. coli CFUs and lower levels of circulating endotoxin and cytokines compared with sedentary rats. E. coli challenge elicited significantly greater time-dependent increases of both Hsp72 and IL-1beta in hypothalamus, pituitary, and dorsal vagal complex of physically active animals but not sedentary animals. Contrary to our hypothesis, increases in Hsp72 were positively correlated with IL-1beta. This study extends our findings that physical activity facilitates stress-induced Hsp72 to include immunological stressors such as bacterial challenge and suggests that brain Hsp72 and IL-1beta responses to peripheral immune challenge may contribute to exercise-mediated resistance to long-term sickness.     

Edema of the paws of white mice--test for assessing the activity of E. coli enterotoxins]

The authors studied the paw edema test in mice for detection of the E. coli (strain P-99) enterotoxins activity. This test proved to be simple, sensitive and reproducible; it permitted to determine the activity of thermostable and thermolabile enterotoxins and endotoxin; the mentioned test was particularly useful in testing various preparations of enterotoxins obtained during their extraction and purification.     

Effect of exogenous interferon on the transplantation reactions of mice]

The authors studied the influence of the serum obtained at various periods after the administration of interferon inductors (New castle disease virus, amino ethylisothiouronium, E. coli endotoxin) on the rate of rejection of the skin or cell transplant of mice C3H and CBA, and also CC57Br. The allogenous skin transplant perished more rapidly; there was also an acceleration of elimination of allogenous lymphoid cells, suppression of colony formation by the cells of allogenous bone marrow in the spleen of the irradiated recipient in administration of the serum obtained at the period of maximal content of interferon induced by the Newcastle disease virus and by amino ethylisothiouronium. The cytotoxic activity of lymphocytes of mice CC57Br against the allogenous target cells rose in the presence of these sera. The serum containing interferon induced with E. coli endotoxin failed to influence the rate of the allotransplant rejection and did not increase the cytotoxic activity of lymphocytes.     

Lowered pulmonary arterial pressure prevents edema after endotoxin in sheep

Escherichia coli endotoxin causes increased capillary membrane permeability and increased pulmonary arterial pressure (PAP) in sheep. If the pulmonary hypertension extends to the level of the microvasculature, then the increased microvascular pressure may contribute to the pulmonary edema caused by endotoxin. We tested the hypothesis that reducing the pulmonary hypertension would reduce the amount of edema caused by endotoxin. Twelve sheep were chronically instrumented with catheters to measure PAP, left atrial pressure, and central venous pressure. The sheep were divided into two groups. One group (E) of six sheep received an intravenous infusion of 4 micrograms/kg of E. coli endotoxin. The second group (E + SNP) received the same dose of endotoxin as well as a continuous infusion of sodium nitroprusside (SNP) to reduce PAP. Three hours after the endotoxin infusions, the sheep were terminated and the extravascular fluid-to-blood-free dry weight ratios of the lungs were determined (EVF). The base-line PAP was 17.5 +/- 2.7 mmHg. A two-way analysis of variance demonstrated a significant difference (P less than 0.01) in PAP between the E and E + SNP groups. Although PAP in each group varied as a function of time, the difference between the two groups did not. The mean PAP for the E + SNP group (20.9 +/- 1.5 mmHg) was lower than the E group PAP of 27.3 +/- 2.1 mmHg after the endotoxin spike. Furthermore, the E + SNP group EVF (3.9 +/- 0.2) was significantly less than the EVF of the E group (4.7 +/- 0.5).(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of the Intestinal Flora on the Development of Immune Reactions in Infants

Breast-fed and artifically fed infants are in contact with the O antigen of Escherichia coli from the first days after birth. From the mother, the infant obtains antibodies against nonpathogenic E. coli strains in low titer, and the infant begins to form its own antibodies during the 2nd month of life. The transition is known to be continuous even though the transferred antibodies could not be differentiated from the infant's own antibodies. Contact with endotoxin caused sensitization which was detected by the skin test at about 2.5 months, and thereafter the skin test data correlated with the presence of serum antibodies against endotoxin. The newborn infant can be colonized with a different E. coli serotype; such an antigenic stimulus evokes the formation of antibodies sooner and at a significantly higher titer than (i) the level of maternal antibodies transferred or (ii) the infant's antibodies normally formed later on against other random E. coli serotypes.     

Relative potencies of four reference endotoxin standards as measured by the Limulus amoebocyte lysate and USP rabbit pyrogen tests.

Four commonly used reference endotoxin standards, Escherichia coli O113:H10:K0, E. coli O55:B5, Salmonella abortusequi, and Shigella dysenteriae were compared by the USP rabbit pyrogen and the Limulus amoebocyte lysate tests. By the rabbit pyrogen test, S. abortus equi was identified as the most potent endotoxin, followed closely by E. coli O113:H10:K0 and E. coli O55:B5.     

Comparison of several control standard endotoxins to the National Reference Standard Endotoxin--an HIMA collaborative study.

A collaborative study, initiated under the auspices of the Health Industry Manufacturers Association (HIMA), was designed to establish the relationship of Escherichia coli O55:B5 endotoxin (the control standard endotoxin of HIMA and the Food and Drug Administration's Office of Medical Devices) to the U.S. National Reference Standard Endotoxin and to two internationally used control standard endotoxins. By using two Limulus amoebocyte lysate test systems, it was established that the E. coli O55:B5 endotoxin lot originally used by HIMA and the Office of Medical Devices to establish Limulus amoebocyte lysate release test criteria for pyrogen testing of medical devices contains approximately 4.5 endotoxin units (EU) per ng. Thus, the 1.0-ng/kg endotoxin dose limit currently established for medical devices is approximately the same as the 5.0-EU/kg endotoxin limit (on an activity basis) established by several other Food and Drug Administration agencies for human and animal parenteral drugs and biological products.     

Lipid metabolism in endotoxic rats: decrease in hepatic triglyceride lipase activity

Studies were conducted to investigate the effect of E. coli endotoxin administration on hepatic triglyceride lipase (H-TGL) activity in rats, since H-TGL activity is known to behave differently from lipoprotein lipase (LPL) activity in various situations. Plasma triglyceride and free fatty acid concentrations were markedly elevated in animals after injection of endotoxin. Cholesterol and phospholipids were also increased significantly. Lipoprotein analysis by ultracentrifugation showed that the most pronounced increase of lipoproteins was in the VLDL and IDL fractions. Triglyceride lipase activities in post-heparin plasma were markedly decreased. A selective assay for H-TGL activity using a specific antibody revealed that this enzyme as well as LPL is significantly decreased (26% of control) in endotoxic animals. Thus, the increase of VLDL and IDL appears to result from the decrease of both of LPL and H-TGL.     

Comparison of several control standard endotoxins to the National Reference Standard Endotoxin--an HIMA collaborative study

A collaborative study, initiated under the auspices of the Health Industry Manufacturers Association (HIMA), was designed to establish the relationship of Escherichia coli O55:B5 endotoxin (the control standard endotoxin of HIMA and the Food and Drug Administration's Office of Medical Devices) to the U.S. National Reference Standard Endotoxin and to two internationally used control standard endotoxins. By using two Limulus amoebocyte lysate test systems, it was established that the E. coli O55:B5 endotoxin lot originally used by HIMA and the Office of Medical Devices to establish Limulus amoebocyte lysate release test criteria for pyrogen testing of medical devices contains approximately 4.5 endotoxin units (EU) per ng. Thus, the 1.0-ng/kg endotoxin dose limit currently established for medical devices is approximately the same as the 5.0-EU/kg endotoxin limit (on an activity basis) established by several other Food and Drug Administration agencies for human and animal parenteral drugs and biological products.