Low-dose Low-dose Lipopolysaccharide Research Articles

Mild portal endotoxaemia induces subacute hepatic inflammation and pancreatic β‐cell dysfunction in rats

Portal endotoxaemia has been speculated to be crucially involved in the pathogenesis of chronic hepatic inflammation, which is highly associated with the development of type 2 diabetes mellitus. This study tests whether portal endotoxaemia is a pathogenic link between chronic subacute hepatic inflammation and pancreatic beta-cell dysfunction. Rats were randomly assigned into two groups: rats with intraportal saline or low-dose lipopolysaccharide (LPS) infusion for 4 weeks. Pathological changes in the liver were evaluated via histological and biochemical examination. Pancreatic insulin secretion was evaluated by in vivo hyperglycaemic clamp study. White blood cell count was significantly increased after intraportal LPS infusion for 4 weeks. Plasma amylase and chemoluminescence counts indicating superoxide levels were significantly increased after LPS treatments for 2 and 4 weeks. Intraportal low-dose LPS infusion significantly increased tumour necrosis factor-alpha and interleukin-6 contents in liver and pancreas. Circulating C-reactive protein, thiobarbituric acid reactive substances (TBARS) and endotoxin levels were not different among groups. The first- and second-phase insulin secretions in hyperglycaemic clamp were significantly decreased in LPS-treated rats. The histopathological scores, de novo production of reactive oxygen substrate and TBARS contents in the liver and pancreas were significantly increased in LPS-infused rats. Leucocyte infiltration was clearly visible in pancreatic islets of LPS-treated rats. The present study demonstrated that mild portal endotoxaemia caused subacute hepatic inflammation and impaired pancreatic insulin secretion, implicating that portal endotoxaemia is a potential risk factor to link chronic subacute hepatic inflammation and pancreatic beta-cell dysfunction.

COADMINISTRATION OF LOW-DOSE LIPOPOLYSACCHARIDE AND SOLUBLE TISSUE FACTOR INDUCES THROMBOSIS AFTER RADIOSURGERY IN AN ANIMAL ARTERIOVENOUS MALFORMATION MODEL

Radiosurgery for arteriovenous malformations is limited to small lesions and may take 3 years to produce total occlusion. It has recently been shown that coadministration of low-dose lipopolysaccharide (LPS) and soluble tissue factor (sTF) selectively induces thrombosis in murine tumor models, attributable perhaps to the prothrombotic phenotype of tumor vasculature. Radiosurgery may induce changes in endothelial prothrombotic molecules similar to those found in tumors. This study aimed to determine if a similar strategy could be used to stimulate thrombus formation in an animal arteriovenous malformation model. Seventeen rats underwent creation of a carotid-to-jugular anastomosis. Animals were intravenously injected with sTF, low-dose LPS, a combination of both, or placebo 24 hours after stereotactic irradiation of the anastomosis. Control animals received both agents after sham irradiation. Coadministration of sTF and LPS led to the formation of thrombi in up to 69% of small vessels and 39% of medium-sized vessels within the target region. The irradiated vasculature demonstrated intermediate rates of thrombosis after treatment with either sTF or LPS alone as did vessels within the fistula in the control group. Logistic regression analysis demonstrated significant associations between development of thrombi and treatment with radiation, sTF, or LPS (P < 0.005). There was no evidence of systemic thrombus formation or toxicity in any group. Treatment with sTF and LPS selectively induces thrombosis of irradiated vessels in a rat model of arteriovenous malformation. Stimulation of thrombosis may improve the efficacy of radiosurgery, increasing the treatable lesion size and reducing latency.

Effects of low-dose lipopolysaccharide (LPS) pretreatment on LPS-induced intra-uterine fetal death and preterm labor

Lipopolysaccharide (LPS) has been associated with adverse developmental outcome, including embryonic resorption, intra-uterine fetal death (IUFD), intra-uterine growth retardation (IUGR) and preterm delivery in rodents. The purpose of the present study was to investigate whether administration of a low-dose LPS to the pregnant mice induce a reduced sensitivity to subsequent high-dose LPS-induced IUFD and preterm labor. We found that LPS-induced IUFD was obviously attenuated when the pregnant mice were pretreated with low-dose LPS (10 μg/kg, i.p.) 24 h before high-dose LPS (120 μg/kg, i.p.). Consistent with its protective effect, when administered 24 h before high-dose LPS, low-dose LPS pretreatment obviously inhibited the releases of tumor necrosis factor alpha (TNF-α) in maternal serum and amniotic fluid and attenuated LPS-induced placental lipid peroxidation and GSH depletion. However, when administered 4 h before high-dose LPS, low-dose LPS pretreatment did not induced a reduced sensitivity to subsequent high-dose LPS-induced release of TNF-α in maternal serum and amniotic fluid. Actually, low-dose LPS pretreatment 4 h before high-dose LPS worsened LPS-induced oxidative stress in mouse placenta and increased nitric oxide production in maternal serum and amniotic fluid. Correspondingly, low-dose LPS pretreatment 4 h before high-dose LPS aggravated LPS-induced IUFD. Taken together, these results indicate that whether a low-dose LPS exposure during pregnancy produce LPS hyporesponsiveness depends on the interval between the two doses of LPS. When administered 24 h before high-dose LPS, a low-dose LPS pretreatment induces a reduced sensitivity to subsequent high-dose LPS-induced IUFD, TNF-α production and oxidative stress.

Interleukin 10 Regulates Inflammatory Cytokine Synthesis to Protect Against Lipopolysaccharide-Induced Abortion and Fetal Growth Restriction in Mice1

Interleukin 10 (IL10) is a potent immune-regulating cytokine and inhibitor of inflammatory cytokine synthesis. To evaluate the anti-inflammatory role of IL10 in pregnancy, the response of genetically IL10-deficient mice to low-dose lipopolysaccharide (LPS)-induced abortion was examined. When IL10-null mutant C57Bl/6 (Il10(-/-)) and control (Il10(+/+)) mice were administered low-dose LPS on Day 9.5 of gestation, IL10 deficiency predisposed to fetal loss accompanied by growth restriction in remaining viable fetuses, with an approximately 10-fold reduction in the threshold dose for 100% abortion. After LPS administration, inflammatory cytokines tumor necrosis factor-alpha (TNFA) and IL6 were markedly increased in serum, uterine, and conceptus tissues in Il10(-/-) mice compared with Il10(+/+) mice, with elevated local synthesis of Tnfa and Il6 mRNAs in the gestational tissues. IL1A and IL12p40 were similarly elevated in serum and gestational tissues, whereas interferon gamma (IFNG) and soluble TNFRII content were unchanged in the absence of IL10. Recombinant IL10 rescued the increased susceptibility to LPS-induced fetal loss in Il10(-/-) mice but did not improve outcomes in Il10(+/+) mice. IL10 genotype also influenced the responsiveness of mice to a TNFA antagonist, etanercept. Fetal loss in Il10(-/-) mice was partly alleviated by moderate or high doses of etanercept, whereas Il10(+/+) mice were refractory to high-dose etanercept, consistent with attenuation by IL10 status of TNFA bioavailability after etanercept treatment. These data show that IL10 modulates resistance to inflammatory stimuli by downregulating expression of proinflammatory cytokines TNFA, IL6, IL1A, and IL12, acting to protect against inflammation-induced pathology in the implantation site.

Neutrophil Elastase Inhibitor, Sivelestat, Attenuates Acute Lung Injury After Cardiopulmonary Bypass in the Rabbit Endotoxemia Model

Neutrophil elastase probably contributes to the development of acute lung injury after cardiopulmonary bypass (CPB) in patients with infection or shock. We evaluated whether pretreatment with sivelestat sodium hydrate, a neutrophil elastase inhibitor (EI), can prevent acute lung injury caused by CPB. Rabbits were assigned four groups: CPB for 60 minutes, control CPB group; low-dose lipopolysaccharide (LPS) administration without CPB, LPS group; CPB after lipopolysaccharide administration, LPS+CPB group; or preparation with continuous infusion of sivelestat and CPBs after lipopolysaccharide administration, EI group. Blood samples to determine blood gas concentration, plasma elastase activity, and plasma interleukin-8 levels were obtained. Histopathologic examinations of the lung were performed. The decreased arterial oxygen pressure at the end of CPB was observed in the LPS+CPB group only, but was suppressed in the EI group (p < 0.01). Elastase activity was markedly elevated at 120 minutes after CPB, and interleukin-8 levels were markedly elevated at 180 minutes in the LPS+CPB group but were much lower (p < 0.05) in the EI group. Histopathology demonstrated accumulation of polymorphonuclear neutrophils in bronchoalveolar areas in the LPS+CPB group (p < 0.01). Pulmonary myeloperoxidase activity was significantly lower in the LPS+CPB group than in the other groups (p < 0.01). These changes were minimal in the EI group. The combination of low dose LPS+60 minutes of CPB, but neither intervention alone, produced evidence of acute lung injury in a rabbit model. This did not occur when the animals were pretreated with sivelestat.

Pro-inflammatory cytokine and acute phase protein responses to low-dose lipopolysaccharide (LPS) challenge in pigs

AbstractThe objective of this study was to establish the pro-inflammatory cytokine and acute phase protein responses to low-dose lipopolysaccharide (LPS) challenge in pigs and to determine whether these immune parameters could also be measured in saliva. Possible gender differences in the acute phase reaction were also assessed. At 6 weeks of age, 24 male and 24 female pigs were injected intraperitoneally with a single dose of 0 or 5 μg/kg live weight (LW) of LPS fromEscherichia coli(treatment). Matched saliva and blood samples were taken at 0, 2, 4, 8, 12 or 24 h after treatment administration. Samples were analysed for concentrations of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β), the acute phase proteins C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp), and cortisol. Low-dose LPS administration increased plasma levels of TNF-α (P&lt;0·001), CRP (P&lt;0·05) and SAA (P&lt;0·05) but did not affect plasma concentrations of IL-1β or Hp (P&gt;0·1). Treatment by time interactions showed that plasma levels of TNF-α and CRP in LPS-treated pigs peaked at 2 h (P&lt;0·001) and 12 h (P&lt;0·01), respectively. Low-dose LPS injection tended to increase plasma concentrations of cortisol (P=0·056) and the response to LPS differed between genders (P&lt;0·05), with females showing higher cortisol responsiveness to the challenge (P&lt;0·01). Males showed higher levels of both cytokines regardless of the treatment (P&lt;0·05), probably due to the inhibition of cytokine synthesis by cortisol. Concentrations of both pro-inflammatory cytokines were consistently detectable in saliva and were present in higher concentrations than in plasma (P&lt;0·001). Hence, plasma TNF-α, CRP and SAA are useful indicators of sub-acute inflammation/infection in pigs as simulated by a low-dose LPS challenge and gender differences exist in the pro-inflammatory cytokine response after a low dose of LPS.

Effects of surgical castration on the behavioural and physiological responses of weaned pigs

Surgical castration is an acute stressor that can affect the behaviour, endocrine and immune responses of pigs [1,2]. Therefore, it may impair the health and welfare of these animals. This could be of particular concern at weaning, which constitutes a severe nutritional, physical and psychological stressor often associated with increased disease susceptibility [3]. Thus, an experiment was carried out to evaluate the effect of surgical castration on postweaning behaviour and on the behavioural, endocrine and immune responses elicited by a low-dose lipopolysaccharide (LPS) challenge after weaning. At 5 days-ofage, 64 male piglets were randomly assigned to undergo surgical castration or were left untreated. Pigs were weaned at 28 days-of-age. Behaviour post-weaning and mixing was assessed during a 1-h period, during which agonistic interactions were recorded. One day post-weaning, pigs were injected with a single dose of 0 or 5 μg/kg of body weight (BW) of LPS from Escherichia coli. Sickness behaviour was studied by scan sampling every 5 minutes for 45 minutes at 0, 1, 2, 3, 4, 6 and 8 h after the challenge was initiated. Blood samples were taken at 0, 2, 12 and 24 h after injection and were analysed for plasma concentrations of tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1β), C-reactive protein (CRP), serum amyloid A (SAA) and cortisol. Saliva samples were also taken when pigs were 3, 4, 5, 6 and 8 weeks-of-age for subsequent determination of salivary testosterone concentrations. Results showed that entire males were more aggressive than castrated pigs immediately after weaning (P < 0.05). Administration of LPS provoked behaviours characteristic of sickness including a reduction in general activity, as well as decreased eating and exploratory behaviours (P < 0.05). Significant treatment by challenge interactions showed that castration reduced the occurrence of sickness behaviours induced by LPS, such as depressed general activity (P < 0.01), anorexia (P < 0.01) and reduced exploratory behaviours (P < 0.05). LPS administration increased TNF-α levels (P < 0.05), with peak concentrations 2 h after injection (P < 0.01). CRP levels of LPStreated pigs were higher that saline-treated animals at 12 h (P < 0.05). LPS administration tended to increase plasma SAA levels (P < 0.1), but did not increase cortisol levels. Nonetheless, castration did not affect the response of pro-inflammatory cytokines, acute phase proteins and cortisol to the challenge. Concentrations of testosterone in saliva were higher in entire males (P < 0.001). Furthermore, salivary testosterone levels in entire males varied with age and were markedly elevated at 4 weeks-of-age (P < 0.001), which coincided with weaning. These results show that surgical castration reduces aggressiveness at weaning. It is likely that peak levels of testosterone are in part responsible for the higher frequency of agonistic encounters among entire male pigs [4]. In addition, castration affects the coping mechanisms of weaned pigs against a low-dose LPS challenge. It is also possible that entire males may be more efficient in overcoming the bacterial endotoxin challenge, taking into consideration the beneficial effects of sickness behaviour in the recovery from infection [5]. In conclusion, surgical castration reduces aggressiveness at weaning and also impairs the behavioural, but not the endocrine and immune responses elicited by low-dose LPS challenge in pigs. from Prevention of Boar Taint in Pig Production: The 19th Symposium of the Nordic Committee for Veterinary Scientific Cooperation Gardermoen, Norway. 21–22 November 2005

Multiple bioimaging modalities in evaluation of an experimental osteonecrosis induced by a combination of lipopolysaccharide and methylprednisolone

Aims: The present study employed both static and dynamic imaging modalities to study both intra- and extravascular events attributing to steroid-associated osteonecrosis (ON) using an experimental protocol with a single low-dose lipopolysaccharide (LPS) injection and subsequently three injections of high-dose methylprednisolone (MPS). Methods: Fourteen 28-week-old male New Zealand white rabbits received one intravenous injection of LPS (10 μg/kg). 24 h later, three injections of 20 mg/kg of MPS were given intramuscularly at a time interval of 24 h. Additional 6 rabbits were used as controls. Dynamic MRI was performed on bilateral femora for local intraosseous perfusion before and after LPS injection. Blood samples were collected for hematological examinations before and after LPS injection. Bilateral femora were dissected and decalcified for microCT-based microangiography. ON lesion, intravascular thrombus and extravascular marrow fat cell size were examined histopathologically. Results: Intravascular thrombus was observed in all ON rabbits. Extravascular marrow fat cell size was significantly increased in ON rabbits than that of the controls ( P < 0.05). Compared to baseline, a significant decrease in ratio of tissue-type plasminogen activator/plasminogen activator inhibitor 1, activated partial thromboplatin time and a significant increase in ratio of low-density lipoprotein/high-density lipoprotein were only found in ON rabbits ( P < 0.05). Dynamic MRI showed a significant decrease in the perfusion index ‘maximum enhancement’ in the ON rabbits ( P < 0.05), and microCT-based microangiography showed blocked stem vessels in ON samples. Overall, 93% of the rabbits (13/14) developed ON, and no rabbits died throughout the experiment period. Conclusion: Both intra- and extravascular events were found attributing to the steroid-associated ON based on our experimental protocol with a single low-dose LPS injection and subsequent three injections of high-dose MPS. Both high ON incidence and no mortality in rabbits treated with this inductive protocol suggested its effectiveness for future studies on evaluation of therapeutic efficacy of interventions developed for prevention of steroid-associated ON.

Effects of Hyperglycemia, Hyperinsulinemia, and Hyperosmolarity on Neutrophil Apoptosis

Hyperglycemia is an independent risk factor for increased mortality of critically ill surgical patients, but despite the recognized clinical benefits of early insulin treatment, there is a lack of understanding of the cellular and molecular mechanisms behind this phenomenon. We hypothesized that polymorphonuclear neutrophils, the first line of the innate immune defense system, suffer from altered apoptotic turnover when exposed to hyperglycemic conditions, ultimately decreasing the number of viable cells active at a site of infection. Venous blood samples were drawn from 10 volunteers and incubated for 0.5 or 24 h in a 1:10 dilution with RPMI 1640 medium at various glucose and insulin concentrations. Mannitol was used to control for increased osmolarity. In addition, all samples were incubated either with low-dose lipopolysaccharide (LPS) (1 ng/mL) or without LPS. Neutrophils were extracted using Ficoll-Hypaque density centrifugation and stained with annexin V and propidium iodide. Fluorescence was detected by flow cytometry and analyzed using CellQuest software. The mean percentage of apoptotic neutrophils after 24 h of incubation at physiologic glucose concentrations (100 mg/dL) was 42.2 +/- 4.1%; exposure to low-dose LPS decreased this number to 18.4 +/- 2.5% (p < 0.01). Neither the exposure to low (10 mg/dL) nor increasingly high (200 or 400 mg/dL) glucose concentrations altered these percentages significantly. Exposing whole blood to increasing osmolarity (addition of 5.5 mM and 16.5 mM mannitol to simulate 200 and 400 mg/dL glucose) led to a mean absolute reduction of the percentage of apoptotic neutrophils to 34.6 +/- 3.6% (+5.5 mOsm; p < 0.05) and 32.3 +/- 4.5% (16.5 mOsm; p < 0.01), respectively. The ability of neutrophils to enter their apoptotic program in cultured whole blood withstands short-term changes in glucose and insulin concentrations. Neither hyperglycemia nor hypoglycemia led to a significant alteration of the apoptotic turnover of these cells, suggesting that the increased rate of infectious complications in short-term hyperglycemic critically ill patients may not be traced to increased apoptosis of neutrophils. However, isolated hyperosmolarity reduces neutrophil apoptosis, an observation that may warrant future investigation.

Protective effect of pretreatment with low-dose lipopolysaccharide on D-galactosamine-induced acute liver failure.

The effect of low-dose lipopolysaccharide (LPS) induced nitric oxide (NO) on liver damage and survival in rats with acute liver failure caused by a lethal dose of D-galactosamine (D-gal) was studied. Ninety percent of control animals died within 4 days after D-gal injection, but pretreatment with low-dose LPS significantly decreased mortality to 5%. There was marked elevation in serum aspartate aminotransferase and alanine aminotransferase levels 24 h after D-gal injection. These aminotransferases were significantly improved in low-dose LPS pretreated rats 24 h after the administration of D-gal. NG-Nitro-L-arginine-methyl ester, but not NG-nitro-D-arginine-methyl ester, reversed this cytoprotection. Pretreatment with low-dose LPS prevents experimental liver failure induced by D-gal through activation of endogenous NO synthesis.

Acute endotoxin tolerance downregulates superoxide anion release by the perfused liver and isolated hepatic nonparenchymal cells

This work is based on the hypothesis that low-dose lipopolysaccharide (LPS) suppresses the stimulatory and priming effects of a subsequent high-dose endotoxin on the formation of toxic oxygen-derived radicals by the perfused liver and isolated hepatic nonparenchymal cells. Such effects may in turn contribute to hyposensitivity to the lethal effect of large doses of endotoxin. Male Sprague-Dawley rats received a nonlethal ("low-dose") intravenous injection of Escherichia coli LPS (0.5 mg/kg body weight) 12 to 120 hours before they were challenged by a "large dose" of endotoxin (10 mg/kg). Three hours after LPS challenge, the livers were perfused, and superoxide release was determined. Nonparenchymal cells were also isolated for the determination of superoxide anion formation in vitro. There was a low rate (0.14 +/- 0.1 nmol/min/g liver weight) of superoxide generated by the perfused livers from rats that received the low-dose LPS 1 to 5 days previously. Control livers generated less than 0.08 nmol superoxide. A high rate (1.3 +/- 0.1 nmol/min/g) of superoxide release was measured in the perfused liver 4 hours after treatment of previously untreated control rats with large-dose LPS. This was attenuated to 0.7 +/- 0.04 nmol/min/g by an injection of low-dose LPS before challenge. This attenuation was time dependent; it failed to manifest at 12, 24, or 120 hours after low-dose LPS. Isolated endothelial cells, Kupffer cells, and sequestered hepatic neutrophils from rats given a high-dose LPS also generated significant amounts of superoxide both in the presence or absence of agonists, i.e., phorbol myristate acetate or opsonized zymosan.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute endotoxin tolerance downregulates superoxide anion release by the perfused liver and isolated hepatic nonparenchymal cells

This work is based on the hypothesis that low-dose lipopolysaccharide (LPS) suppresses the stimulatory and priming effects of a subsequent high-dose endotoxin on the formation of toxic oxygen-derived radicals by the perfused liver and isolated hepatic nonparenchymal cells. Such effects may in turn contribute to hyposensitivity to the lethal effect of large doses of endotoxin. Male Sprague-Dawley rats received a nonlethal (“low-dose”) intravenous injection of Escherichia coli LPS (0.5 mg/kg body weight) 12 to 120 hours before they were challenged by a “large dose” of endotoxin (10 mg/kg). Three hours after LPS challenge, the livers were perfused, and superoxide release was determined. Nonparenchymal cells were also isolated for the determination of superoxide anion formation in vitro. There was a low rate (0.14 ± 0.1 nmol/min/g liver weight) of superoxide generated by the perfused livers from rats that received the low-dose LPS 1 to 5 days previously. Control livers generated less than 0.08 nmol superoxide. A high rate (1.3 ± 0.1 nmol/min/g) of superoxide release was measured in the perfused liver 4 hours after treatment of previously untreated control rats with large-dose LPS. This was attenuated to 0.7 ± 0.04 nmol/min/g by an injection of low-dose LPS before challenge. This attenuation was time dependent; it failed to manifest at 12, 24, or 120 hours after low-dose LPS. Isolated endothelial cells, Kupffer cells, and sequestered hepatic neutrophils from rats given a high-dose LPS also generated significant amounts of superoxide both in the presence or absence of agonists, i.e., phorbol myristate acetate or opsonized zymosan. Pretreatment of rats with the low dose of LPS 48 hours before the large dose attenuated the spontaneous and primed release of superoxide by isolated nonparenchymal cells. Serum transaminase activities were also reduced in these LPS-tolerant rats. The suppressed oxygen-derived radical formation by the liver and nonparenchymal cells during acute LPS tolerance was associated with down-regulation of tumor necrosis factor (TNF) release in vivo. TNF has been shown to stimulate and prime neutrophils and Kupffer cells for increased respiratory burst. These results suggest that suppression of free radical formation may contribute to the amelioration of hepatic injury during acute endotoxin tolerance.