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The examination of 52 patients with different forms of intestinal dysbacteriosis revealed a considerable decrease in the indices of immunity to endotoxin of gram-negative bacteria. In particular, a decrease in the content of antibodies to chemotype Re glycolipid, as well as in the content of polymorphonuclear leukocytes binding antibodies in the blood stream of the examined patients and leukocytes capable of binding endotoxin in endotoxin-treated blood smears, was registered, which was indicative of the loss of endotoxin binding reserves by leukocytes.     

Antiendotoxin therapies for septic shock

Gram-negative shock is thought to result primarily from the effects of endotoxin, a component of the bacterial outer membrane. Accordingly, therapies aimed at inhibiting, neutralizing, or clearing endotoxin have been extensively explored. Despite over 30 years of research, no antiendotoxin approach to the treatment of human septic shock is of proven benefit. In recent randomized clinical trials of monoclonal antibodies against endotoxin, therapeutic efficacy was not convincingly demonstrated. This result, however, does not eliminate the possibility that other antiendotoxin therapies may be effective. The antibodies used in these clinical trials do not appear to neutralize endotoxin in vitro and are not reproducibly protective in animal models of sepsis. Newer agents with well-defined mechanisms of antiendotoxin activity may help clarify the role of endotoxin in septic shock and prove useful therapy for some patients.     

Prevalence of antibodies to lipid A in Danish cattle

A cross-sectional study was performed on the occurrence of IgG antibodies to lipid A of the Gram-negative bacterial lipopolysaccharide (LPS, endotoxin) on serum of 2272 cattle distributed on 19 Danish dairy herds. The relationship between the concentration of antibodies to lipid A (ALI) and age, herd, pregnancy rate and occurrence of mastitis, bovine virus diarrhoea (BVD), reproductive and digestive disorders, diarrhoea, pneumonia, foot disorders, various infections and traumatic udder lesions was investigated. ALI generally was low in calves and increased during their first 1.5 years of life to a steady state, which could be altered by the occurrence of disease. There were significant differences in the mean ALI among the herds (P < 0.001). High ALI was associated with a low herd pregnancy rate, to preceding occurrence of mastitis (P < 0.048), BVD (P < 0.01), reproduction diseases (P < 0.041) and digestion disorders (P < 0.064) in animals older than 2 years. The calf mortality rate was not associated to ALI and there was no correlation between the ALI in calves and their dams. The occurrence of high ALI levels on a herd basis may be an indication of increased challenge or enhanced immunological defense to Gram-negative bacteria or endotoxin.     

Interactions between metallothionein inducers in rat liver and primary cultures of rat hepatocytes

The interaction of Zn, stress and endotoxin on liver metallothionein (MT) regulation has been studied in the rat. Zn, stress and endotoxin increased liver MT levels significantly, by 12-, 5- and 8-fold, respectively. The previous administration of Zn to stress or endotoxin treatments increased MT levels by 35- and 42-fold, respectively, indicating a synergistic effect in both cases. In contrast, when liver MT was preinduced by stress, MT levels were further increased by endotoxin only in an additive manner. In another experiment where liver MT induction by stress was studied in control rats and in rats with preinduced MT by Zn, endotoxin or stress, it was found that Zn pretreated animals had higher MT-I mRNA levels than endotoxin- or stress-pretreated ones. No synergisms between dexamethasone, Zn, TNF and IFN were observed in primary culture of hepatocytes. These results suggest that the observed synergisms between Zn and other MT inducers in vivo in the liver is a consequence of increased Zn levels in the body and mobilization capacity, with concomitant MT synthesis.     

Tobacco use and other risk behaviors among adolescents in an STD clinic

OBJECTIVE: To test efficacy of murine monoclonal, rabbit polyclonal recombinant equine or human tumor necrosis factor-alpha (rETNF or rHTNF, respectively) antibodies to inhibit native equine tumor necrosis factor (TNF) activity. ANIMALS: 8 and 18 healthy adult horses for parts 1 and 2 of the study, respectively. PROCEDURES: In part 1, supernates from endotoxin-activated peritoneal macrophages were incubated with various dilutions of each rETNF antibody and subsequently tested for TNF activity. Serum was also obtained from a horse 1 hour after infusion with 20 ng of endotoxin/kg of body weight and was incubated with various dilutions of rabbit polyclonal rHTNF antibody. In part 2, 20 ng of endotoxin/kg was infused in horses during a 30-minute period. Fifteen minutes after the endotoxin infusion was initiated, 1 of 3 preparations was infused: 0.1 mg of rabbit polyclonal (rHTNF antibody/kg, 0.1 mg of human IgG/kg, or 500 ml of 5% dextrose. Clinical and hematologic data were collected for 24 hours. RESULTS: Compared with the monoclonal antibody, the rabbit polyclonal rETNF antibody was more effective in inhibiting TNF activity. The 50% effective doses of the murine monoclonal rETNF, rabbit polyclonal rETNF, and rabbit rHTNF antibodies were 1.8, 0.8, and 0.6 micrograms of antibody/ml, respectively. In part 2, endotoxin infusion resulted in significant alternations in all variables; however, differences among treatment groups were not significant. CONCLUSIONS AND CLINICAL RELEVANCE: Although murine monoclonal and rabbit polyclonal rETNF or rHTNF antibodies are capable of inhibiting native equine TNF activity in vitro, when given after initiation of endotoxemia, administration of 0.1 mg of rabbit polyclonal rHTNF/kg does not alter the response to infusion of endotoxin.     

Inhibitory effect on LPS-induced tumor necrosis factor in calves treated with chlorpromazine or pentoxifylline

The inhibitory effect of chlorpromazine (CPZ), pentoxifylline (PTX) and dexamethasone (DEX) was investigated in a model of endotoxin shock in Holstein calves following an intravenous administration of Esherichia coli endotoxin (LPS). Initial correlations with its effects on the levels of tumor necrosis factor (TNF), a pivotal mediator of endotoxin shock, and clinical signs were obtained. The pretreatment of CPZ or DEX significantly decreased the serum levels of TNF, and reduced endotoxic shock. But the pretreatment of PTX hardly reduced the increase of serum TNF levels and endotoxin shock. The levels of serum endotoxin were not significantly different a minute of postinjection of LPS in calves. The results of this study indicate that pretreatment of CPZ or DEX inhibit various biological effects on endotoxin in calves.     

Sensitive quantitation of endotoxin by enzyme-linked immunosorbent assay with monoclonal antibody against Limulus peptide C

Limulus peptide C, a 28-amino-acid fragment of coagulogen formed by the reaction of endotoxin with Limulus amebocyte lysate, was synthesized, and a monoclonal antibody against it was raised. A new microassay for endotoxin was developed, using this antibody in an enzyme-linked immunosorbent assay for generated peptide C-like immunoreactivity. A linear relationship between absorbance and endotoxin concentration was obtained. Control standard endotoxin in water could be detected to a level of 0.001 endotoxin unit per ml. The endotoxin levels in plasma samples from normal humans, rabbit, mice, and guinea pigs were generally found to be below the detection limit of 0.01 endotoxin unit per ml of plasma. The color and turbidity of specimens did not interfere with the assay. The consumption of Limulus amebocyte lysate in the assay was less than 5% of that in the gel-clot and chromogenic assays. With raw lysate, which was much more stable in solution than chloroform-treated lysate, the assay was still highly sensitive to endotoxin but was totally unresponsive to natural glucans. The monoclonal antibody cross-reacted with peptide C-like immunoreactivity generated in Tachypleus amebocyte lysate, which gave equal sensitivity in the endotoxin assay.     

Plasma endotoxin and cytokine concentrations in patients with hemorrhagic shock

OBJECTIVES: The roles of cytokines and endotoxin in hemorrhagic shock, particularly the translocation of endotoxin and bacteria during hemorrhagic shock, were investigated. DESIGN: Prospective study. SETTING: Critical care and emergency center of a university hospital. PATIENTS: Twenty-nine patients with hemorrhagic shock and 20 healthy controls. INTERVENTIONS: Serial blood samples were collected from both study and control patients. Standard resuscitation techniques were used. MEASUREMENTS AND MAIN RESULTS: Plasma levels of endotoxin and various cytokines were determined repeatedly during hemorrhagic shock. Endotoxin was measured using an endotoxin-specific assay in addition to a new perchloric acid method for pretreatment of plasma. Cytokines were measured by commercial enzyme-linked immunosorbent assays. Plasma endotoxin concentrations remained within the normal range for 7 days after admission. Although levels of tumor necrosis factor-alpha and several interleukins increased slightly in some patients, these cytokines did not reach the levels seen in septic shock. CONCLUSIONS: Translocation of bacteria or endotoxin from the gastrointestinal tract into the bloodstream has been noted in animal experiments; however, translocation was not detected in our patients with hemorrhagic shock.     

Ciliogenesis in the mucous cells of the quail oviduct. II. Hormonal control

Endotoxin was infused into normal rabbits and C6 deficient rabbits prepared with cortisone for the generalized Shwartzman reaction. Endotoxin produced profound granulocytopenia and moderate thrombocytopenia in both normal and C6 deficient rabbits. In normal rabbits endotoxin consistently produced extensive intravascular clotting. In C6 deficient animals endotoxin resulted in intravascular clotting of variable extent. In one group of eight C6 deficient rabbits mean fibrinogen levels fell 0.67 g per 1 over 6 hrs after endotoxin and four of eight animals developed a generalized Shwartzman reaction. In a second group of seven C6 deficient rabbits mean fibrinogen level fell only 0.17 g per 1 over 6 hrs and one animal developed a generalized Shwartzman reaction. Values for mean fibrinogen consumption, calculated from plasma fibrinogen levels and rate of disappearance of 25I-fibrinogen, were as follows: normal animals infused with saline, 10 mg per kg; C6 deficient animals infused with endotoxin, 58 mg per kg. Fibrinogen consumption after endotoxin was found to be related to granulocyte levels prior to endotoxin, which determined the number of granulocytes disappearing from the blood after endotoxin. The data indicate that C6 deficiency in the rabbit does not prevent intravascular clotting and the generalized Shwartzman reaction.     

Initial transient phase of salivary secretion by the submandibular glands of rats under anoxic conditions

In 148 patients after major surgical procedures urinary endotoxin levels were determined and compared with bacteriological results. The study was designed as a screening study. Urine samples were collected once by suprapubic or transurethral catheters. In a first series of 49 patients urine bacteriology was positive (mainly, Gram-negative rods were found) in 3 cases. However, endotoxin determination was positive in these 3 patients and in a further 10 patients receiving antibiotic therapy for other reasons. Therefore, the following 99 patients were studied also by urinalysis by reagent strips for leukocytes and nitrite. In the second series, 12 urine cultures positive for bacteria were observed. Eleven samples were also endotoxin positive. Five more patients were endotoxin positive and had pathological but unspecific reagent strip results. These patients were treated with antibiotics for other reasons. Patients with candida found in the urine culture (n = 5) were endotoxin negative. Thus, endotoxin determination in urine obtained by suprapubic or transurethral catheters proved to be a very sensitive method for diagnosis of bacterial contamination, even during antibiotic treatment.     

Gut is not a source of cytokines in a porcine model of endotoxicosis

BACKGROUND: We sought to determine whether ischemic gut is a source of endotoxin, tumor necrosis factor (TNF), and interleukin-6 (IL-6) in a porcine model of endotoxicosis. METHODS: Under general anesthesia pigs underwent neck dissection and laparotomy for placement of catheters in the carotid artery and portal vein and application of an ultrasonic flow probe around the portal vein. Endotoxin, TNF, and IL-6 levels were measured from the carotid artery and the portal vein during a 4 hour period in animals given endotoxin (50 mg/kg; n = 6) and in animals in the control group (n = 6). Gut fluxes of the substances of interest were calculated as the product of concentration and portal venous flow. A tonometer placed in the terminal ileum was used to monitor mucosal pH. RESULTS: Small bowel mucosal pH was significantly depressed in endotoxemic animals (6.8 +/- 0.1) when compared with baseline (7.1 +/- 0.1; p < 0.05) and control levels. In the control group portal venous levels of endotoxin, TNF, and IL-6 did not change significantly from baseline levels (1.5 +/- 0.4 endotoxin units (EU)/ml, 24 +/- 3 pU/ml, and 1.3 +/- 0.4 nU/ml, respectively). In the endotoxemic animals portal venous endotoxin and TNF levels peaked immediately after the endotoxin infusion (2186 +/- 437 EU/ml, and 293 +/- 125 pU/ml, respectively), and portal venous IL-6 levels peaked at 180 minutes (168 +/- 21 nU/ml). At no time did endotoxin, TNF, or IL-6 levels differ between arterial and portal venous blood, and at no time did efflux from the gut significantly exceed gut influx in either the control or endotoxemic animals. CONCLUSIONS: Ischemic gut as indicated by decreased mucosal pH is not associated with gut release of endotoxin, IL-6, or TNF in this porcine model of endotoxicosis.     

Ursodeoxycholic acid modifies gut-derived endotoxemia in neonatal rats

We developed a model for the translocation of intraluminal endotoxin in the neonatal animal and used it to examine the capacity of a nonhepatotoxic bile acid, ursodeoxycholic acid (UDCA), to modify endotoxin translocation and cytokine response. Three-d-old Sprague-Dawley rats were randomized to receive enterally either no drug, lipopolysaccharide (LPS, 1 mg/animal), or UDCA (400 micrograms/animal) alone, or UDCA followed by LPS 1 h later. One h after LPS administration, the rats were killed and plasma endotoxin and tumor necrosis factor (TNF) were measured. Control animals had low circulating endotoxin (21.2 +/- 7.6 endotoxin units) and TNF (0.06 +/- 0.02 ng/mL). Enteral administration of LPS 1 h before the rats were killed resulted in significant elevation of endotoxin (249.5 +/- 71.3, p = 0.008) and TNF (3.6 +/- 1.3, p = 0.019). UDCA alone did not alter endotoxin levels (8.7 +/- 2.1). UDCA 1 h before LPS prevented the rise in endotoxin (38.9 +/- 11.2 endotoxin units) and TNF (0.2 +/- 0.05) significantly. Chenodeoxycholic acid was studied in a similar group of experiments and prevented neither the translocation of LPS nor the development of increased TNF levels in animals receiving LPS. In conclusion, LPS can cross the intestinal barrier in the normal neonatal rat. UDCA, administered before LPS, can decrease the translocation of LPS and prevent the cytokine response as measured by TNF levels. We speculate that UDCA, administered prophylactically, might reduce morbidity in clinical conditions leading to gut-derived endotoxemia.     

Blunted cortisol response after administration of corticotropin releasing hormone in endotoxemic dogs

To evaluate the effects of a standard inflammatory challenge on the dynamics of the hypothalamic-pituitary-adrenal (HPA) axis, we studied the effects of low-dose endotoxin (1.0 microgram/kg) on plasma adrenocorticotropic hormone (ACTH) and cortisol concentrations in a saline-controlled study in five awake dogs. Four hours after endotoxin or saline challenge human corticotrophin-releasing hormone (hCRH; 1.0 microgram/kg) was administered. Plasma ACTH and cortisol levels increased considerably in response to endotoxin, from 13 +/- 1 ng/l to 360 +/- 85 ng/l (p < 0.01) and from 60 +/- 20 nmol/l to 710 +/- 80 nmol/l (p < 0.01). Despite a considerable difference in ACTH and cortisol levels prior to CRH administration between both studies (p < 0.01), the absolute increase in ACTH levels induced by hCRH was not different (231 +/ 43 ng/l vs 238 +/- 45 ng/l, control vs endotoxin). Plasma cortisol levels increased significantly in the control study (from 40 +/- 10 nmol/l to 330 +/- 40 nmol/l, p < 0.01), whereas they did not change in the endotoxin study after hCRH administration (from 710 +/- 80 nmol/l to 730 +/- 70 nmol/l, ns). We conclude that the HPA-axis reacts initially to endotoxin in such a way that cortisol, but not ACTH, secretion is maximized. Therefore, a blunted cortisol response to CRH testing is part of the initial response to infection.     

Effects of removing circulatory tumor necrosis factor by immunoadsorption on experimental endotoxin shock animals

OBJECTIVE: To evaluate the effects of removing circulatory tumor necrosis factor (TNF) by immunoadsorption on endotoxin shock animals. METHODS: Sixty New Zealand white rabbits were injected intravenously with lethal dose of endotoxin (10 Billion cfu/kg E. Coli endotoxin) and randomly divided into 3 groups: perfusion group, hemoperfusion started at 1 hour after injecting endotoxin through immunoadsorbent columns against TNF; pseudoperfusion group, hemoperfusion through blank columns; and control group, injected with endotoxin only. The arterial pressure, microcirculation of the mesentery, plasma levels of TNF, IL-1, IL-6, IL-8, nitrite, endothelin-1 (ET-1), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine were measured and analyzed and finally the survival rate was observed. RESULTS: Plasma levels of TNF were sharply reduced after immunoadsorption. Moreover, release of IL-1, IL-6, IL-8, NO and ET-1 were also attenuated. Hemodynamic abnormalities could be improved and survival rate ameliorated significantly. CONCLUSION: Specific immunoadsorption of circulating TNF might be a new and effective therapy for endotoxin shock.     

Obstructive jaundice in rats results in exaggerated hepatic production of tumor necrosis factor-alpha and systemic and tissue tumor necrosis factor-alpha levels after endotoxin

BACKGROUND: Obstructive jaundice (OJ) predisposes patients to postoperative sepsis. We determined whether OJ led to an increased endotoxin stimulated tumor necrosis factor-alpha (TNF-alpha) production by macrophage-rich organs and whether a lack of intraluminal gut bile contributed to this increased sensitivity. METHODS: Rats underwent laparotomy and common bile duct ligation and division (CBDL) or sham operation after they were given low-dose endotoxin or saline solution (NS). TNF-alpha levels in plasma, perfusate from the isolated perfused rat liver, and tissue from lung, spleen, and liver were measured 90 minutes later. An additional group underwent creation of a choledochal-vesical fistula and endotoxin administration. RESULTS: The plasma TNF-alpha, liver perfusate TNF-alpha, and the tissue TNF-alpha levels in liver, lung, and spleen were significantly elevated in the CBDL + endotoxin (CBDL + ETX) group compared with the SHAM + ETX and CBDL + NS groups (p < 0.05). The choledochal-vesical fistula group after endotoxin had plasma TNF-alpha levels only 27% that of the CBDL + ETX group (p < 0.05). CONCLUSIONS: OJ sensitizes macrophage-rich organs to produce larger amounts of TNF-alpha in response to endotoxin. This sensitization is not solely due to decreased intraluminal gut bile.     

A spontaneous induction of fetal membrane prostaglandin production precedes clinical labour

Fetal membranes from term human pregnancies produce prostaglandins, and may respond to bacterial endotoxin or interleukin-1 beta (IL-1 beta) with increased prostaglandin E2 (PGE2) production. The effects of endotoxin persisted for up to 24 h, whereas those of IL-1 beta were maximal 4-8 h after addition. The maximum levels of PGE2 (200-350 pg/ml) were similar in all experiments, and were independent of the stimulus used. Not all tissues responded to these stimuli; those which did not had basal levels of PGE2 production of 200-350 pg/ml, which was not further increased by endotoxin or IL-1 beta. The basal production from these tissues was therefore similar to the maximal production from those tissues which responded to endotoxin or IL-1 beta. The high basal production of PGE2 was attributed to prior in vivo activation of the membranes such that PGE2 synthesis could not be further stimulated in vitro. Overnight pretreatment with aspirin decreased basal PGE2 production from these activated membranes to < 100 pg/ml/4 h during subsequent culture in aspirin-free medium. Both endotoxin and IL-1 beta increased PGE2 production from the activated aspirin-pretreated membranes during this culture time, but this was transient as after 12 h of culture basal PGE2 production rose to over 200 pg/ml despite aspirin pretreatment.     

The polymerase chain reaction in pathology

Membrane-fixed CD14 acts as a receptor for the protein-bound endotoxin (LPS) complex and mediates the cellular effects of endotoxin. Soluble CD14 (sCD14) is suggested to neutralize circulating LPS, i.e., acting as an endotoxin antagonist. The aim of this study was to elucidate the release of both sCD14 and endotoxin in traumatized patients, starting from the earliest phase after trauma. A total of 15 patients (O ISS = 19, 9-75) suffering major trauma were enrolled in this prospective study. Blood samples were collected as early as immediately at the site of accident, on hospital admission, and thereafter hourly, then daily. For patients (O ISS = 47) died within 24 h because of their severe injuries. Immediately after the accident as well as during the first 2 h after hospital admission, the mean sCD14 levels of surviving patients did not differ from those of healthy volunteers (n = 53). Thereafter, however, sCD14 increased continuously in the trauma group. The concentrations remained elevated throughout the entire observation period. There was, however, no relation between the sCD14 release and the pattern or the severity of injury. In contrast, endotoxin levels revealed a pattern-specific release. The highest plasma concentrations of LPS were observed in patients suffering from (additional) thoracic injury. On the basis of these results we conclude that the release of sCD14 after trauma does not reflect a strict principle such as action/reaction caused by the appearance of endotoxin immediately after the injury. Soluble CD14 is more likely release by an endotoxin-independent mechanism.     

Concerted regulation of low density lipoprotein receptor gene expression by follicle-stimulating hormone and insulin-like growth factor I in porcine granulosa cells: promoter activation, messenger ribonucleic acid stability, and sterol feedback

The consequences of glucocorticoid receptor (GR) dysfunction for neuroimmunoendocrine responses to an inflammatory challenge were studied in transgenic mice expressing antisense RNA directed against the GR [GR-impaired (GR-i) mice]. Mice were implanted intraperitoneally with a biotelemetry transmitter to monitor body temperature and locomotion. GR-i mice showed decreased locomotion and body temperature during the dark phase of the diurnal cycle. Intraperitoneal administration of saline caused a rapid increase in body temperature in control mice, which was terminated within 90 min. In GR-i mice, however, body temperature remained elevated for about 6 h. Intraperitoneal injection of endotoxin (10 micrograms/mouse) produced a biphasic fever in control mice. However, in endotoxin-injected GR-i mice, body temperature was not significantly different from their saline-injected controls during the first 6 h. Body temperature then increased and remained elevated during the night period. Both strains showed hypolocomotion after endotoxin. In a second experiment, mice were injected intraperitoneally with saline or endotoxin and killed after 1, 3, 6 or 24 h. In GR-i mice, endotoxin caused an augmented rise in plasma ACTH, but not in corticosterone levels. The endotoxin-induced increase in serum levels of interleukin-1 beta and interleukin-6 was not different between the strains. However, whereas in control mice tumour necrosis factor-alpha levels were below detection at the time points studied, substantial levels of this cytokine were found in the serum of GR-i mice 1 h after endotoxin administration. It may be concluded that life-long impairment of GR evolves in aberrant physiological and humoral responses to an acute inflammatory challenge. These findings expand our understanding about the neuroendocrine and physiological disturbances associated with stress-related disorders.     

Epidural injection of ketamine for perineal analgesia in the horse

Work-related respiratory symptoms, acute lung function changes and personal endotoxin exposure were studied in 61 workers from a potato processing plant. According to their job title mean endotoxin exposure level, workers were divided into low (AM = 21 EU/m3) and high (AM = 56 EU/m3) exposure categories. Shortness of breath and chest tightness during work were reported by 18% and 16% of the workers, respectively, mainly in the low endotoxin exposure category. A total of 148 across-shift lung function changes were measured during three consecutive afternoon shifts. The mean FEV1 and MMEF showed a decrease over the work shift, being largest on the first working day after a 3-day absence from work. Workers exposed to high endotoxin levels showed a larger across-shift decrease in lung function than workers exposed to low endotoxin exposures, the effect being most pronounced on the first day after a 3-day absence from work. At the start of the second work shift, FVC, FEV1 and MMEF were lower than at the start of the first work shift. This difference was larger for high exposed workers. High exposed workers with work-related respiratory symptoms showed an 8-10% across-shift change in FVC, FEV1 and MMEF We conclude that significant across-shift decreases in lung function of potato processing workers is related to endotoxin exposure levels above 53 EU/m3 over 8 hr.     

Clinical experience in detecting endotoxemia with the limulus test in typhoid fever and other Salmonella infections

Semiquantitative estimates of circulating endotoxin were performed by the limulus test in patients suffering from typhoid fever and other salmonelloses. The test was positive in a large number of cases. However, no clearcut correlation was found between existence of endotoxemia, as such, and pyrexia. A correlation with recent bacteremia was found for highest levels of endotoxin activity. In minor salmonelloses a striking prevalence of positive cases was observed in the age group under one year. These findings were discussed in relation to the diagnostic and pathogenetic facets of the problem.