Immunization against intestinal bacterial endotoxin prevents alcoholic fatty liver in rats

Accumulating evidences indicate that an endotoxin originating from intestinal gram-negative bacteria may be involved in alcohol-induced liver injury including fatty liver. Therefore, whether immunization against intestinal bacterial endotoxin blocked fatty liver induced by chronic alcohol and diet including much-unsaturated fatty acid was investigated in rats. The titer of antibody against the endotoxin increased significantly after 13 weeks of continuous immunization. Daily alcohol treatment was initiated at 12 weeks and continued for 4 weeks. Plasma glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT) and triglyceride (TG) levels increased significantly in non-immunized rats receiving alcohol, but not in immunized rats. Continuous alcohol treatment gradually decreased the survival rate to 60% from 13 days after beginning administration in non-immunized, but not immunized, rats. A histochemical study revealed that continuous treatment with alcohol and unsaturated fatty acids caused fatty liver in non-immunized, but not immunized, rats. This study strongly supports the hypothesis that alcohol-induced fatty liver is due to a circulating endotoxin, and suggests that immunization for endotoxin prevent the alcoholic fatty liver.     

Growth and Development Symposium: Endotoxin, inflammation, and intestinal function in livestock

Endotoxin, also referred to as lipopolysaccharide (LPS), can stimulate localized or systemic inflammation via the activation of pattern recognition receptors. Additionally, endotoxin and inflammation can regulate intestinal epithelial function by altering integrity, nutrient transport, and utilization. The gastrointestinal tract is a large reservoir of both gram-positive and gram-negative bacteria, of which the gram-negative bacteria serve as a source of endotoxin. Luminal endotoxin can enter circulation via two routes: 1) nonspecific paracellular transport through epithelial cell tight junctions, and 2) transcellular transport through lipid raft membrane domains involving receptor-mediated endocytosis. Paracellular transport of endotoxin occurs through dissociation of tight junction protein complexes resulting in reduced intestinal barrier integrity, which can be a result of enteric disease, inflammation, or environmental and metabolic stress. Transcellular transport, via specialized membrane regions rich in glycolipids, sphingolipids, cholesterol, and saturated fatty acids, is a result of raft recruitment of endotoxin-related signaling proteins leading to endotoxin signaling and endocytosis. Both transport routes and sensitivity to endotoxin may be altered by diet and environmental and metabolic stresses. Intestinal-derived endotoxin and inflammation result in suppressed appetite, activation of the immune system, and partitioning of energy and nutrients away from growth toward supporting the immune system requirements. In livestock, this leads to the suppression of growth, particularly suppression of lean tissue accretion. In this paper, we summarize the evidence that intestinal transport of endotoxin and the subsequent inflammation leads to decrease in the production performance of agricultural animals and we present an overview of endotoxin detoxification mechanisms in livestock.     

Hypoperfusion of the small intestine during slow infusion of a low dosage of endotoxin in anesthetized horses

The effects of intravenous infusion of endotoxin for 30 minutes at a cumulative dosage of 0.03 micrograms/kg on average carotid arterial pressure, and on average arterial pressure, capillary pressure, venous pressure, total vascular resistance, precapillary resistance, postcapillary resistance, and capillary filtration coefficient in the jejunum were compared to the effects of intravenous infusion of 0.9% sodium chloride solution in 6 anesthetized horses. Endotoxin significantly reduced intestinal venous blood flow by inducing vasoconstriction. Increased vascular resistance resulted from increased precapillary resistance. The capillary filtration coefficient was unchanged by endotoxin. These results suggest that intestinal vasoconstriction occurs during the compensatory stages of endotoxemia.     

Escherichia coli associated endotoxemia in dogs with parvovirus infection

Escherichia coli bacteremia and endotoxemia were observed in 3 adult mongrel dogs which had been prediagnosed as canine parvoviral disease. The endotoxin level was 46.5 pg/ml in the plasma of clinical cases, while 2.3 pg/ml in healthy controls. The microflora of the feces was confused in the clinical cases. The percentage of E. coli was major in the feces. Serologically similar strains were isolated from the blood. These strains did not produce enterotoxins such as heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT). Histopathologically, the lesions in the small intestine consisted of epithelial degeneration and necrosis. Viral inclusion bodies were frequently observed in the epithelial cells. Disseminated intravascular coagulation was observed in various tissues including the liver and small intestinal submucosa. After experimental infection with CPV, all dogs showed various clinical signs. CPV was positive in the feces. Endotoxin level in the plasma gradually increased and high level continued for long period from 10 to 30 days. Mean maximum level of endotoxin in the experimental dogs was 73.6 pg/ml. These results indicate that intestinal flora plays a important role in the pathogenesis of CPV infection and that endotoxin is one of the factors which predispose to severe disease after the infection.     

Pathogenesis of experimental Leptospira interrogans serotype icterohaemorrhagiae infection in the guinea pigs: possible role of endotoxin of intestinal bacteria in the development of lesions.

Germ-free guinea pigs were infected with a virulent strain of Leptospira interrogans serotype icterohaemorrhagiae to evaluate the possible role of endotoxin of intestinal bacteria in the development of hemorrhages and hepatic and renal failure. Clinical manifestations, necropsy findings and histological lesions were similar to those seen in control animals. In the second part of the experiment, endotoxin was not detected by the Limulus test in the blood of ten guinea pigs during the five day course of leptospirosis.     

Equine endotoxemia: pathomorphological aspects of endotoxin-induced damage in equine mesenteric arteries

To evaluate the effects of endotoxin on the morphology of the equine mesenteric vasculature, each of two thoroughbred horses were given two intravenous injections (24 h apart) of a sublethal dose of endotoxin (10 microg/kg). Each injection produced results similar to those of clinical cases of equine colic with obstructive nature of the loop of bowel: diarrhoea within 2 h after administration, followed by cessation of both faecal excretion and sounds of intestinal peristalsis. The most prominent morphological change was the development of moniliform appearance of small mesenteric arteries, in which there were contracted and dilated segments of the small mesenteric arteries. This was accompanied by parietal hyalinization and intramural and extramural haemorrhage. These mesenteric vascular changes appear to reflect dynamic vasoconstriction in the living animal, resulting in reduction of mesenteric and intestinal blood flow and possibly inducing alterations of gastrointestinal function such as cessation of intestinal peristalsis.     

内毒素血症对大鼠肠黏膜杯状细胞的影响

本研究选用39只140~150g SPF级SD大鼠作为动物内毒素血症模型,运用组织学常规HE染色和PAS染色技术,采取定性、定位和定量相结合的方法,通过观察不同试验组大鼠肠黏膜杯状细胞(GC)的数量和分布的动态变化来探讨内毒素(ET)对肠道黏膜免疫系统的影响。结果表明,ET能够破坏肠道黏膜的正常结构,影响肠黏膜的正常功能,同时能够在不同程度上降低GC数量,从而破坏肠道黏膜免疫系统。     

Kinetics of endotoxin concentration and tumor necrosis factor-alpha,interleukin-1beta,and interleukin-6 activities in the systemic and portal circulation during small intestinal ischemia and reperfusion in dogs

OBJECTIVE: To determine whether small intestinal ischemia and reperfusion induces bacterial translocation and proinflammatory cytokine response in either the systemic or portal circulation in dogs. ANIMALS: 17 healthy adult Beagles. PROCEDURE: The superior mesenteric artery (SMA) was occluded for 0 (group-3 dogs), 30 (group-1 dogs), or 60 (group-2 dogs) minutes, followed by reperfusion for 180 minutes; serum lactate and endotoxin concentrations and tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 activities in the systemic and portal circulation and intramucosal pH were measured at various time points. RESULTS: In group-2 dogs, TNFalpha activity was found to be significantly increased in the portal circulation, peaking at 60 minutes of reperfusion; TNF-alpha activity, in the systemic circulation, gradually increased from 60 minutes of reperfusion to the end of the experiment; however, the increase was not significant. In group-1 and -2 dogs, IL-6 activities significantly and gradually increased in the systemic and portal circulation during the reperfusion phase, and the magnitude of these increases was dependent on the duration of the ischemic phase. There were no significant changes in IL-1beta activity or endotoxin concentration in any dog group. CONCLUSIONS AND CLINICAL RELEVANCE: Results of the our study indicate that intestinal ischemia and reperfusion leads to significant increases of the circulating TNF-alpha and IL-6 activities, depending on the duration of the ischemia phase, in the absence of detectable endotoxin in the circulation. This finding suggests that intestinal ischemia and reperfusion induces a systemic proinflammatory cytokine response in dogs.     

Effects of dietary butyrate supplementation on intestinal integrity of heat‐stressed cockerels

Butyrate modulates intestinal epithelial cell structure and function. Three hundred and sixty Lohmann LSL‐Classic layer cockerels were used to investigate the effect of butyrate on heat stress‐induced intestinal injury and intestinal integrity. The experiment was conducted from day 85 to 105 of age. The birds were divided randomly into three treatments: control, heat stress (HS), and heat stress provided with butyrate (HSB) at a level of 0.35 g/kg of diet. The control was reared at 21 ± 1 °C throughout the experiment. The HS and HSB treatments were exposed to a cyclic HS (35 ± 1 °C from 09:00 to 13:00 and 21 ± 1 °C from 13:00 to 09:00). Intestinal and mucosal weights, villus height, villus surface area (VSA), absorptive epithelial cell area and intestinal beneficial bacteria were lower in the HS treatment than in the other two treatments (p < 0.05). Heat‐stressed cockerels exhibited the highest (p < 0.05) villi injury scores and serum endotoxin levels compared with the other treatments. Dietary inclusion of butyrate increased (p < 0.05) intestinal and mucosal weights, villus height, VSA, absorptive epithelial cell area and intestinal beneficial bacteria counts and reduced (p < 0.05) HS‐induced injury in intestinal epithelia as well as intestinal permeability to endotoxin. In conclusion, dietary butyrate exerted protective effects against intestinal damage induced by HS and improved intestinal health and integrity.     

紫锥菊多糖对LPS损伤后IEC-6细胞分泌IL-1ɑmRNA的影响简

为了研究紫锥菊多糖对LPS损伤后IEC一6细胞分泌IL-1αmRNA的影响，以体外培养大鼠小肠上皮细胞为模型，进行细胞分组及收集，并给予LPS刺激造成内毒素损伤，根据GenBank中发布的IL-1α基因序列设计引物，以基因组R-NA为模板进行RT-PCR扩增，发现紫锥菊多糖对LPS刺激小肠上皮细胞产生的IL-1αmRNA的表达减少。     

谷氨酰胺的生物学功能及其在断奶仔猪中的应用

文中概述了谷氨酰胺的营养生理功能及对动物机体免疫功能的影响。谷胺酰胺对早期断奶仔猪胰腺及小肠胰蛋白酶活性,肠道结构和功能,血浆内毒素水平,饲喂生大豆后肠道通透性的影响及对早期断奶仔猪腹泻的影响,以及对骨骼肌中DNA、RNA浓度的影响。     

Binding of 125I-labeled endotoxin to bovine, canine, and equine platelets and endotoxin-induced agglutination of canine platelets

Endotoxin from Escherichia coli O127:B8, Salmonella abortus-equi and S minnesota induced clumping of some canine platelets (PLT) at a final endotoxin concentration of 1 microgram/ml. Endotoxin-induced clumping of canine PLT was independent of PLT energy-requiring processes, because clumping was observed with canine PLT incubated with 2-deoxy-D-glucose and antimycin A. The PLT responded to adenosine diphosphate before, but not after, incubation with the metabolic inhibitors. Endotoxin induced a slight and inconsistant clumping of bovine and equine PLT at high (mg/ml) endotoxin concentration. High-affinity binding sites could not be demonstrated on canine, bovine, and equine PLT, using 125I-labeled E coli O127:B8 endotoxin. Nonspecific binding was observed and appeared to be due primarily to an extraneous coat on the PLT surface that was removed by gel filtration. The endotoxin that was bound to PLT did not appear to modify PLT function. An attempt to identify plasma proteins that bound physiologically relevant amounts of endotoxin was not successful. The significance of the endotoxin-induced clumping or lack of it on the pathophysiology of endotoxemia is discussed.     

Hematological and Blood Biochemical Effects of Fasting and Subsequent Oral Administration of Endotoxin in Prepubertal Gilts

The main objective of the present study was to investigate the effects of short-term fasting in gilts on endocrinological and blood biochemical parameters and, further, the effects of subsequent oral endotoxin (ET) administration. Group 1 was fasted for 30 h and then received feed with ET added. Group 2 was fasted for 30 h but received standard feed at refeeding. In group 3, gilts were fed every 6 h for 30 h. The major effects of fasting were: gradually increased concentration of plasma prostaglandin F_2α metabolite, serum total bilirubin, serum free fatty acids, and decreased serum glucose. The values were normalized within 1–4 h of refeeding. Twelve hours after refeeding, the ET-refed gilts showed higher levels of serum total bile acids and polymorphonuclear leukocytes than those in group 2. It is possible that the observed changes during fasting reflect either an increased intestinal uptake of naturally present endotoxin or a reduced endotoxin detoxifying capacity of the liver. The increased bile acid concentration and polymorphonuclear leukocyte count following refeeding with ET-feed may indicate that orally administered ET is to some extent absorbed from the gut.     

Induction and characterization of endotoxin tolerance in equine peripheral blood mononuclear cells in vitro

Endotoxemia is responsible for severe illness in horses. Individuals can become clinically unresponsive to the endotoxin molecule after an initial exposure; a phenomenon referred to as 'endotoxin tolerance' (ET). ET has been observed clinically in horses in vivo; however, cytokine expression associated with ET has not been investigated. The purpose of this study was to develop and validate a method for inducing ET in equine peripheral blood mononuclear cells (PBMC) in vitro, and to describe selected cytokine responses which are associated with ET. ET was induced by culturing cells with three concentrations of endotoxin, and evaluated after a second dose of endotoxin given to challenge the cells. The relative mRNA expression of IL-10 and IL-12 was measured by use of quantitative PCR. ET was induced in all cells exposed to the 2-step endotoxin challenge. The relative mRNA expression of IL-10 in tolerized cells was not different from positive control cells. In contrast, the relative mRNA expression of IL-12 in tolerized cells was decreased by 15-fold after the second endotoxin challenge. This experiment demonstrated a reliable method for the ex vivo induction of ET in equine PBMC. A marked suppression of IL-12 production is associated with ET.     

Isolation of a protective, non-toxic capsular antigen from Pasteurella multocida, types B and E.

Separation of the capsular antigen and endotoxin from saline extracts of Pasteurella multocida type B was achieved by fractional precipitation from aqueous solution by addition of polar organic solvents. Biological tests for the presence of endotoxin showed that it was absent from capsular antigen preparations so obtained. Properties of the capsular antigen suggested that it was a high molecular weight acidic polysaccharide. The solvent fractionation method was found to be equally applicable to separation of capsular antigen and endotoxin of P multocida type E. The type B capsular antigen in the presence of aluminium hydroxide gel adjuvant, was poorly immunogenic in rabbits. In cattle, however, a dose-dependent serological response was obtained as demonstrated by the mouse passive protection test.     

Endotoxin activity in whole blood measured by neutrophil chemiluminescence is applicable to canine whole blood

The dog is widely used as a translational experimental model studying the host response and new treatments for human endotoxemia. The present study evaluated the applicability of a novel patient-near neutrophil chemiluminescence assay for the measurement of endotoxin activity in human blood when applied to canine blood samples. The assay was observed to be analytically sensitive and specific to endotoxin when tested in vitro, spiked with purified Escherichia coli lipopolysaccharide and live E. coli. The diagnostic sensitivity was sustained during Gram-positive contamination. Finally, it also demonstrated diagnostic potential when able to discriminate dogs with spontaneously occurring endotoxemia from both healthy dogs and diseased dogs without endotoxemia. The rapid patient-near assessment of endotoxin activity in canine blood should facilitate future studies on endotoxemia in both spontaneous disease and in experimental settings.     

Studies with Radioactive Endotoxin I. Clearance of 51Cr-Labelled Endotoxin from the Blood of Calves

The clearance of ⁵¹Cr-labelled Pseudomonas endotoxin from the blood was studied in calves in a nontolerant and in an endotoxin-tolerant state. Calves were rendered tolerant to the toxic effects of the endotoxin by four daily intravenous injections of endotoxin at the dose rate of 5 µg/kg body weight.

Clearance of a small amount of ⁵¹Cr-endotoxin from the blood of nontolerant calves was almost complete within three minutes of injection and was not significantly faster in tolerant calves. The lungs and liver were the major organs involved in clearance of endotoxin from the blood. The ⁵¹Cr label was slowly excreted by the kidneys. Neither platelets nor leukocytes were demonstrated to participate in endotoxin clearance in calves. ⁵¹CrCl₃ was injected into control calves. Relative to the distribution and loss of labelled endotoxin, the ⁵¹CrCl₃ was cleared slowly from the blood, was distributed uniformly throughout the body and was excreted rapidly.

     

Evaluation of in vitro properties of di-tri-octahedral smectite on clostridial toxins and growth

REASONS FOR PERFORMING STUDY: Clostridial colitis and endotoxaemia of intestinal origin are significant causes of morbidity and mortality in horses. Intestinal adsorbents are available for treatment of these conditions; however, little information exists supporting their use. OBJECTIVES: To evaluate the ability of di-tri-octahedral smectite to bind to Clostridium difficile toxins A and B, C. perfringens enterotoxin and endotoxin, inhibit clostridial growth and the actions of metronidazole in vitro. METHODS: Clostridium difficile toxins, C. perfringens enterotoxin and endotoxin were mixed with serial dilutions of di-tri-octahedral smectite, then tested for the presence of clostridial toxins or endotoxin using commercial tests. Serial dilutions of smectite were tested for the ability to inhibit growth of C. perfringens in culture broth, and to interfere with the effect of metronidazole on growth of C. perfringens in culture broth. RESULTS: Clostridium difficile toxins A and B, and C. perfringens enterotoxin were completely bound at dilutions of 1:2 to 1:16. Partial binding of C. difficile toxins occurred at dilutions up to 1:256 while partial binding of C. perfringens enterotoxin occurred up to a dilution of 1:128. Greater than 99% binding of endotoxin occurred with dilutions 1:2 to 1:32. No inhibition of growth of C. difficile or C. perfringens was present at any dilution, and there was no effect on the action of metronidazole. CONCLUSIONS: Di-tri-octahedral smectite possesses the ability to bind C. difficile toxins A and B, C. perfringens enterotoxin and endotoxin in vivo while having no effect on bacterial growth or the action of metronidazole. POTENTIAL RELEVANCE: In vivo studies are required to determine whether di-tri-octahedral smectite might be a useful adjunctive treatment of clostridial colitis and endotoxaemia in horses.     

Effects of flunixin meglumine in dogs following experimentally induced endotoxemia

Twelve dogs were randomly divided into three groups. Group 1 dogs were given Escherichia coli endotoxin and then treated with flunixin meglumine. Group 2 dogs were given endotoxin as group 1, but untreated. Group 3 dogs were given flunixin meglumine alone. The dogs were monitored clinically and urine and serum samples were collected at regular intervals for 72 hours. All surviving dogs were humanely killed after 72 hours and examined for gross and histologic lesions. Group 1 dogs all survived 72 hours, but showed prerenal azotemia, hepatocellular damage, hemorrhagic enteritis, and numerous gastric ulcerations. Three of the four dogs in group 2 died before 72 hours. Group 2 dogs showed many of the same chemical and hemodynamic changes as group 1. They had severe hemorrhage into the intestinal lumen; however, there were no gastric ulcerations. Group 3 dogs all survived and showed little physical or hematologic change. The study suggested the following: 1) flunixin meglumine was an effective drug in ameliorating the fatal effects of canine endotoxemia, 2) the effects of endotoxin in combination with flunixin meglumine, at 1.1 mg/kg body weight, caused gastric ulcerations, and 3) in normal dogs flunixin meglumine at 1.1 mg/kg body weight did not cause severe side effects or gross lesions.     

The pathogenesis of the post-weaning syndrome in weaned piglets: a review.

This review deals with the pathogenesis of the post-weaning syndrome. This syndrome includes post-weaning diarrhoea (PWD), oedema disease (OD) and endotoxin shock (ES). The role of different enteropathogenic Escherichia coli bacteria and some other predisposing factors relating to this post-weaning syndrome (PWS) are discussed. Based on intestinal pathophysiological mechanisms, some suggestions for the prevention of PWS and prospects for future research are given.