Dose Of Endotoxin Research Articles

Abstract 353: Chronic Polarization of Low-grade Inflammatory Monocytes During the Pathogenesis of Atherosclerosis

Background: Chronic inflammation mediated by low-grade inflammatory monocytes may serve as a key culprit for atherosclerosis. However, the cellular and molecular mechanisms responsible for the low-grade inflammatory polarization of monocytes are not well understood. We hypothesize that the selective clearance of homeostatic molecular switches may pre-dispose innate monocytes for the establishment of non-resolving low-grade inflammation. Methods and Results: By comparing high-fat-diet (HFD) fed ApoE deficient mice chronically challenged with either PBS or a subclinical dose endotoxin, we observed that subclinical endotoxin potently induced the establishment of low-grade inflammation, as manifested in elevated levels of systemic inflammatory mediators, accumulation of low-grade inflammatory circulating monocytes and neutrophils, as well as lipids. Immunohistochemical staining of liver and aorta tissues revealed significantly elevated steatosis and atherosclerosis in ApoE deficient mice chronically challenged with subclinical dose of endotoxin. At the mechanistic level, the polarization of low-grade inflammatory monocytes were due to the down-regulation and removal of key homeostatic molecules such as IRAK-M and Tollip. ApoE and IRAK-M double deficient mice had enhanced inflammatory polarization of innate monocytes, and developed severe atherosclerosis. Conclusions: Our data suggest that the clearance of homeostatic suppressors such as IRAK-M and Tollip may cause the memory establishment of low-grade inflammatory monocytes that are conducive for the chronic pathogenesis of atherosclerosis. Key words: Low-grade inflammation, monocyte polarization, innate memory, atherosclerosis

LPS alters pattern of sickness behavior but does not affect glutathione level in aged male rats.

Behavioral symptoms of sickness, such as fever and motor activity are a coordinated set of changes that develop during infection. The aim of study was to compare the sickness behaviour (SB) in healthy old and young rats treated with pyrogenic dose of endotoxin and to check their glutathione level. Before experimentation male Wistar rats were selected according to standard body mass, motor activity, and white blood cells count. Intraperitoneal injection of lipopolysaccharide (LPS) from E. coli was used to provoke SB. The level of liver glutathione, interleukin (IL) -6, deep body temperature (Tb) and motor activity were measured. Glutathione level in old and young rats did not differ significantly. In both young and old rats LPS administration provoked fever (the mean value of Tb was 38.06 ± 0.01 °C in old rats, and 38.19 ± 0.06 °C in young rats). LPS injection affected night-time activity in both groups (12 h averages were 1.56 ± 0.40 counts in old LPS-treated rats vs 2.74 ± 0.53 counts in not-treated old rats and 3.44 ± 0.60 counts for young LPS-treated vs 4.28 ± 0.57 counts for young not-treated rats). The injection of LPS provoked an elevation of plasma IL-6 concentration (from values below the lowest detectable standard in not-treated groups of animals to 6322.82 ± 537.00 pg/mL in old LPS-treated rats and 7415.62 ± 451.88 pg/mL in young LPS-treated rats). Based on these data, we conclude that good health of aged rats prevents decrease in the glutathione level. Old rats are still able to develop SB in response to pyrogenic dose of LPS, although its components have changed pattern compared to young animals.

IMPACT OF OBESITY ON ENDOTOXIN-INDUCED DISSEMINATED INTRAVASCULAR COAGULATION

An early activation of coagulation and fibrinolysis occurs during sepsis, leading to the syndrome of disseminated intravascular coagulation (DIC). Obesity has been demonstrated to be a hypercoagulable and hypofibrinolytic state, but its impact on DIC has never been studied. In this study, we aimed to determine if obesity impairs DIC in an acute endotoxic shock model using minipigs. This was a prospective, comparative, and experimental ancillary study approved by the Animal Ethics Committee. Pigs were chosen as a clinically relevant species, resembling humans in coagulation reactions. Four groups of five "Yucatan" minipigs were studied: lean and obese control groups, a lean lipopolysaccharide (LPS) group receiving Escherichia coli endotoxin (LPS), and an obese LPS group receiving the same endotoxin dose. We measured standard coagulation parameters (prothrombin time [PT], platelet count, and fibrinogen levels), thrombin-antithrombin complexes, tissue-type plasminogen activator, and plasminogen activator inhibitor-1. All measurements were performed at baseline and 30, 60, 90, 150, and 300 min. Results were given as median with interquartile ranges. At baseline, platelet count (477 [428 - 532] G/L vs. 381 [307 - 442] G/L; P = 0.005) and fibrinogen levels (4.6 [3.8 - 5.2] g/L vs. 2 [1.8 - 2.9] g/L; P < 0.001) were significantly higher, whereas PT (80% [76% - 92%] vs. 96% [89% - 100%]; P = 0.01) was significantly lower in obese pigs compared with lean pigs. In the LPS groups, administration of endotoxin resulted in a typical hypokinetic shock with DIC. The decrease in coagulation parameters (PT, platelet count, and fibrinogen levels) and the increase in thrombin-antithrombin complexes (581 [382 - 1,057] μg/mL vs. 247 [125 - 369] μg/mL at 150 min; P = 0.03) were significantly more important in the obese LPS group compared with those in the lean LPS group. Concerning the fibrinolytic reaction, we found a slightly more elevated increase of plasminogen activator inhibitor-1 in the obese LPS group at 300 min (481 [365 - 617] ng/mL vs. 355 [209 - 660] ng/mL; P = 0.66). In our model of endotoxic shock, obese pigs developed a more severe DIC with a more severe procoagulant response.

Novel approach to simultaneously monitor local and systemic in vivo effects of varying doses of intradermally injected LPS (VET1P.1123)

Abstract We recently established the dermis (pulp) of growing feathers (GF) as a minimally invasive test-site to monitor in vivo tissue effects of test substances. The objective of this study was to use the GF test-system to simultaneously monitor local and systemic effects of varying doses of endotoxin (LPS) injected into GF on leukocyte profiles and activities. Growing feathers of 16 chickens were injected with either 10 µL of endotoxin-free PBS or 10, 100, or 1000 ng of LPS per feather (18 GF/chicken). Blood and three GF were collected at each time point: 0 (before injection), 2, 4, 8, and 24 h post-injection. The concentration of leukocytes in the blood and proportions of leukocytes within the feather pulp were determined for each time point. Both LPS 1000 and 100 exhibited similar patterns of leukocyte recruitment in the blood and GF. Specifically, GF injection of LPS resulted in an initial drop (2 h) in blood leukocytes followed by increasing levels at 4 and 8 h and returning to near control levels by 24 h. The drop in blood leukocytes at 2 h was not observed with the LPS 10 treatment. At the site of injection (GF), increased levels of heterophils were already observed at 2 h for LPS 10 and 100, whereas this increase was delayed for LPS 1000. At 8 h post LPS injection, leukocyte levels were greatly elevated for all LPS treatments. Overall, based on leukocyte recruitment patterns in blood and tissue, the lowest dose of LPS may serve as a model for the local inflammatory response.

Application of a discovery to targeted LC-MS proteomics approach to identify deregulated proteins associated with idiosyncratic liver toxicity in a rat model of LPS/diclofenac co-administration.

Increasing experimental and clinical evidence suggest a contribution of non-drug related risk factors (e.g., underlying disease, bacterial/viral infection) to idiosyncratic drug reactions (IDR). Our previous work showed that co-treatment with bacterial endotoxin (LPS) and therapeutic doses of diclofenac (Dcl), an analgesic associated with drug idiosyncrasy in patients, induced severe hepatotoxicity in rats. Here, we used an integrated discovery to targeted LC-MS proteomics approach to identify mechanistically relevant liver and plasma proteins modulated by LPS/Dcl treatment, potentially applicable as early markers for IDRs. Based on pre-screening results and their role in liver toxicity, 47 liver and 15 plasma proteins were selected for targeted LC-MS analysis. LPS alone significantly changed the levels of 19 and 3 of these proteins, respectively. T-kininogen-1, previously suggested as a marker of drug-induced liver injury, was markedly elevated in plasma after repeated Dcl treatment in the absence of hepatotoxicity, possibly indicating clinically silent stress. Dcl both alone and in combination with LPS, caused up-regulation of the ATP synthase subunits (ATP5J, ATPA, and ATPB), suggesting that Dcl may sensitize cells against additional stress factors, such as LPS through generation of mitochondrial stress. Additionally, depletion of plasma fibrinogen was observed in the co-treatment group, consistent with an increased hepatic fibrin deposition and suspected contribution of the hemostatic system to IDRs. In contrast, several proteins previously suggested as liver biomarkers, such as clusterin, did not correlate with liver injury in this model. Taken together, these analyses revealed proteomic changes in a rat model of LPS/Dcl co-administration that could offer mechanistic insight and may serve as biomarkers or safety alert for a drug's potential to cause IDRs.

Physiologic variability at the verge of systemic inflammation: multiscale entropy of heart rate variability is affected by very low doses of endotoxin.

Human injury or infection induces systemic inflammation with characteristic neuroendocrine responses. Fluctuations in autonomic function during inflammation are reflected by beat-to-beat variation in heart rate, termed heart rate variability (HRV). In the present study, we determine threshold doses of endotoxin needed to induce observable changes in markers of systemic inflammation, investigate whether metrics of HRV exhibit a differing threshold dose from other inflammatory markers, and investigate the size of data sets required for meaningful use of multiscale entropy (MSE) analysis of HRV. Healthy human volunteers (n = 25) were randomized to receive placebo (normal saline) or endotoxin/lipopolysaccharide (LPS): 0.1, 0.25, 0.5, 1.0, or 2.0 ng/kg administered intravenously. Vital signs were recorded every 30 min for 6 h and then at 9, 12, and 24 h after LPS. Blood samples were drawn at specific time points for cytokine measurements. Heart rate variability analysis was performed using electrocardiogram epochs of 5 min. Multiscale entropy for HRV was calculated for all dose groups to scale factor 40. The lowest significant threshold dose was noted in core temperature at 0.25 ng/kg. Endogenous tumor necrosis factor α and interleukin 6 were significantly responsive at the next dosage level (0.5 ng/kg) along with elevations in circulating leukocytes and heart rate. Responses were exaggerated at higher doses (1 and 2 ng/kg). Time domain and frequency domain HRV metrics similarly suggested a threshold dose, differing from placebo at 1.0 and 2.0 ng/kg, below which no clear pattern in response was evident. By applying repeated-measures analysis of variance across scale factors, a significant decrease in MSE was seen at 1.0 and 2.0 ng/kg by 2 h after exposure to LPS. Although not statistically significant below 1.0 ng/kg, MSE unexpectedly decreased across all groups in an orderly dose-response pattern not seen in the other outcomes. By using repeated-measures analysis of variance across scale factors, MSE can detect autonomic change after LPS challenge in a group of 25 subjects using electrocardiogram epochs of only 5 min and entropy analysis to scale factor of only 40, potentially facilitating MSE's wider use as a research tool or bedside monitor. Traditional markers of inflammation generally exhibit threshold dose behavior. In contrast, MSE's apparent continuous dose-response pattern, although not statistically verifiable in this study, suggests a potential subclinical harbinger of infectious or other insult. The possible derangement of autonomic complexity prior to or independent of the cytokine surge cannot be ruled out. Future investigation should focus on confirmation of overt inflammation following observed decreases in MSE in a clinical setting.

Cerebral metabolism during experimental endotoxin shock and after preconditioning with monophosphoryl lipid A

PurposePreconditioning with low doses of monophosphoryl lipid A (MPL) has been shown to induce endotoxin tolerance and to reduce the metabolic and hemodynamic consequences of endotoxin shock. However, no data are available about the effects of endotoxin preconditioning on cerebral metabolism during endotoxemia. The study was designed to determine the effects of endotoxin preconditioning with MPL on cerebral metabolism via microdialysis compared to muscle tissue metabolism during experimental endotoxemia. MethodsIn a controlled animal study, continuous endotoxin infusion (1μg/kg b.w. per h) was administrated to 7 female mixed-breed pigs after pretreatment with MPL in incremental doses of endotoxin during days 5–2 before the experiments. In the control group, 7 animals received a saline pretreatment. In addition to hemodynamic monitoring and blood gas analyses, interstitial lactate, pyruvate, glucose and glycerol concentrations in muscle and cerebral tissue were measured using in vivo microdialysis. ResultsThere were no significant differences between the two groups with respect to hemodynamic parameters, while mixed venous oxygen saturation (SvO2), arterial blood pH and mean pulmonary arterial pressure (MPAP) were significantly higher in the preconditioned group. Cerebral perfusion pressure (CPP) and brain tissue oxygen pressure (ptiO2) values stayed stable throughout the experiment with no inter-group differences. While interstitial concentrations of lactate and glycerol as well as the lactate/pyruvate (LP) and the lactate/glucose (LG) ratio in muscle tissues were significantly increased in control animals compared to those who had been pretreated with MPL; the results of cerebral microdialysis showed no significant changes in interstitial lactate or glycerol levels in both groups. However, the lactate/glucose (LG) ratio in the control group showed a significantly higher increase than in the preconditioned group. ConclusionsPreconditioning with low doses of MPL ameliorates the negative metabolic effects of endotoxin shock in muscle tissue. With regard to cerebral metabolism, the present study suggests that MLP preconditioning provides moderate advantages, at least in an experimental model of endotoxin shock.

Modeling immunity and inflammation in stroke: can mice be trusted?

Sixty-five million years have passed since mouse and man shared a common ancestor. The principles of evolution, as well as the scientific literature, suggest that there are many similarities between both mammal species but also significant differences. Humans are definitely no 75 kg mice,1 but is it true that “the mouse model has been totally misleading for at least three major killers—sepsis, burns, and trauma,” as the New York Times concluded after the publication of a study by Seok et al?2 By looking at transcriptional responses of blood cells, this study found “that, although acute inflammatory stresses from different etiologies result in highly similar genomic responses in humans, the responses in corresponding mouse models correlate poorly with the human conditions and also, one another.” The results of the Seok et al2 study are by no means surprising or new,3 but they nevertheless carry an important message for experimental stroke research. The authors used male mice of the C57 BL/6J strain, which is exquisitely resistant to sepsis: more than one million-fold doses of endotoxin are required to cause shock …

Activation of central muscarinic receptor type 1 prevents development of endotoxin tolerance in rat liver

Endotoxin tolerance is a mechanism in which cells receiving low doses of endotoxin, enter a transient phase with less inflammatory response to the next endotoxin challenges. Central nervous system is known to modulate systemic inflammation through activation of the cholinergic system; however, the role of central anti-inflammatory pathway in pathophysiology of hepatic endotoxin tolerance is unknown. Our study was designed to assess the effect central muscarinic type 1 receptor (M1) activation on development of endotoxin tolerance in rat liver. Endotoxin tolerance was induced by daily intraperitoneal injection of endotoxin (1mg/kg) for 5 days. Animals were randomly divided into two groups which received intracerebroventricular injection of either MCNA-343 (an M1 agonist, 5ng/kg) or saline 1h after intraperitoneal injection of saline or endotoxin. The responsiveness to endotoxin was assessed by measuring hepatic MCP-1 (monocyte chemotactic protein-1), iNOS (inducible nitric oxide synthase) and TNF-α (tumor necrosis-α) mRNA expression 3h after intraperitoneal administration of endotoxin using quantitative RT-PCR. A significant reduction in hepatic expression of MCP-1, iNOS and TNF-α was observed in rats with 5 days endotoxin challenge in comparison with rats given a single dose of endotoxin. There was no significant difference in hepatic expression of MCP-1, iNOS or TNF-α between acute and chronic LPS-treated groups in rats given MCNA-343. Central MCNA-343 stimulation could prevent the induction of hepatic endotoxin tolerance in animals receiving repeated doses of endotoxin. This indicates that M1 cholinergic receptor activation in the central nervous system can modulate endotoxin tolerance in rat liver.

Impact of Endotoxin Challenge in Obese Pigs

Studies exploring the influence of obesity on septic shock remain limited and controversial. Pigs were chosen as a clinically relevant species, resembling to humans in various functions. We hypothesize obesity may impair porcine acute endotoxic shock. Four groups of five "Yucatan" minipigs were studied: lean and obese control groups, lean lipopolysaccharide (LPS) group receiving Escherichia coli endotoxin (LPS) and obese LPS group receiving the same endotoxin dose. We measured hemodynamic and oxygenation parameters, skin microvascular blood flow at rest and during reactive hyperemia, von Willebrand factor, tumor necrosis factor α, and interleukin 6. All measurements were performed at baseline and at 30, 60, 90, 150, and 300 min. Results were given as median with 25th to 75th interquartile range. Control groups remained stable during the study period. In LPS groups, administration of endotoxin resulted in a typical hypokinetic shock. In obese LPS group at 300 min, we observed a significant impairment of cardiac index (1.2 [1.06-1.45] vs. 1.7 [1.57-1.97] L/min per m, P = 0.008) compared with the lean LPS group; moreover, pulmonary hypertension (mean arterial pressure: 42 [39-47] vs. 32 [28-34] mmHg, P = 0.008), hypoxemia (partial pressure of oxygen: 216 [178-262] vs. 325 [285-414] mmHg, P = 0.02), and lactate levels (5.8 [4.2-6.8] vs. 3.9 [2.2-5.5] mmol/L, P = 0.04) were significantly higher compared with the lean LPS group. Throughout the study, rest flow and peak flow during reactive hyperemia were more decreased in the obese LPS group. Compared with the lean LPS group, tumor necrosis factor α levels at 60 min (269 [178-428] vs. 126 [105-166] ng/mL, P = 0.03) and interleukin 6 levels at 300 min (101 [61-142] vs. 52 [36-64] ng/mL, P = 0.03) were significantly higher in the obese LPS group. In our model of endotoxic shock, obese pigs developed a more severe hemodynamic failure with pronounced microcirculatory dysfunction and proinflammatory response.

Molecular and Cellular Mechanisms Responsible for Cellular Stress and Low-grade Inflammation Induced by a Super-low Dose of Endotoxin

Super-low-dose endotoxemia in experimental animals and humans is linked to low-grade chronic inflammatory diseases. However, the underlying molecular and cellular mechanisms are not well understood. In this study, we examined the effects of a super-low dose of LPS on low-grade inflammation in macrophages as well as underlying mechanisms. We observed that a super-low dose of LPS induces mitochondrial fission and cell necroptosis in primary murine macrophages, dependent upon interleukin 1 receptor-associated kinase (IRAK-1). Mechanistically, our study reveals that a super-low dose of LPS causes protein ubiquitination and degradation of mitofusin 1 (Mfn1), a molecule required for maintaining proper mitochondrial fusion. A super-low dose of LPS also leads to dephosphorylation and activation of Drp1, a molecule responsible for mitochondrial fission and cell necroptosis. Furthermore, we demonstrated that a super-low dose of LPS activates receptor interacting protein 3 kinase (RIP3), a key molecule critical for the assembly of the necrosome complex, the initiation of Drp1 dephosphorylation, and necroptosis. The effects of a super-low dose of LPS are abolished in macrophages harvested from IRAK-1-deficient mice. Taken together, our study identified a novel molecular pathway that leads to cellular stress and necroptosis in macrophages challenged with a super-low dose of endotoxin. This may reconcile low-grade inflammation often associated with low-grade endotoxemia.

Cellular Metabolic Regulators

The Toll-like receptor 4 (TLR4) ligand endotoxin triggers robust systemic inflammatory responses in humans at doses equal to or greater than 1 ng/kg. In this study, we tested the hypothesis that evidence of TLR4-induced responses would be detectable in leukocytes challenged with endotoxin doses that are below the threshold needed to trigger a characteristic systemic inflammatory phenotype in humans. Subjects were challenged with endotoxin at 1, 0.5, or 0.1 ng/kg (n = 5 per dose). Systemic responses were monitored for 24 hours. Blood samples, collected at designated intervals, were used to determine plasma cytokines levels, total and differential leukocyte counts, expression of leukocyte cell surface receptors, and changes in the leukocyte transcriptome. Western blotting was used to determine changes in leukocyte protein expression. We found that in vivo endotoxin at doses below 1.0 ng/kg triggers weak and variable responses in humans. In marked contrast, we show that endotoxin at a concentration as low as 0.1 ng/kg triggers a transient decline in cellular ATP levels in leukocytes. This is associated with the appearance of a unique protein expression signature in leukocytes. The protein expression signature includes 3 prominent features: (i) AMP-activated protein kinase subunit α (AMPKα) degradation, (ii) increased hypoxia inducible factor-1 (HIF-1) α expression, and (iii) autophagy, collectively indicative of a regulated metabolic response. An indistinguishable response phenotype was observed in human leukocytes treated with endotoxin in vitro. These data demonstrate for the first time in humans that a TLR4 ligand concentration that is below the threshold needed to trigger clinically evident systemic inflammatory manifestations initiates a transient decline in ATP levels, AMPKα degradation, HIF-1α expression, and autophagy in leukocytes. This establishes that low-grade TLR4 activation exerts control over leukocyte metabolism in the absence of systemic inflammatory indicators.

Moving Forward in Sepsis Research

To the Editor: We thank Dr. Perlman and colleagues for their timely and thoughtful perspective on the Proceedings of the National Academy of Sciences of the United States of America study describing large discrepancies between murine and human leukocyte transcriptional profiles obtained in critical illness settings (1, 2). The gulf between human and mouse in sepsis research was vividly illustrated by the recent negative phase 3 trial of Toll-like receptor 4 (TLR4) blockade (3). Yet, the discovery of TLR4 as the body’s sensor for gram-negative endotoxin is rightly considered foundational in immunology and was driven in large part by murine studies recognized by the 2011 Nobel Prize in Physiology or Medicine (4). How should the scientific community resolve this paradox? The cross-species comparison of expression arrays by Seok and colleagues has injected new, important data into this ongoing conversation (2). Proving the absence of an association between mouse and human is difficult, as highlighted by the methodological issues raised in Perlman and colleagues’ editorial (1). Given the profound implications of Seok and colleagues’ article, clinicians and researchers interested in critical illness would have benefited from an even more intense effort by the authors to compare “apples to apples.” We propose that two additional experiments would have strengthened their conclusions, particularly regarding the dissimilarity in leukocyte responses to endotoxin. First, Seok and colleagues showed that the mouse transcriptional response to endotoxin was markedly attenuated immediately downstream of TLR4 (2). Because the murine version of TLR4 is considered the true ortholog of the human gene, this surprising result made us wonder whether an adequate exposure to endotoxin was achieved or whether the transcriptional signature of high-TLR4-expressing cells in mice (e.g., monocytes) was swamped out by lower-expressing cells (e.g., lymphocytes). Had Seok and colleagues been able to adjust endotoxin dose or duration to achieve comparable TLR4 signaling responses in their own readout, the subsequent negative findings would have been greatly bolstered. Second, Seok and colleagues could have compared transcriptional responses to endotoxin of homologous cell types between species—for example, monocytes isolated from each species stimulated with the same endotoxin serotype analyzed at comparable doses and durations of exposure. Had the ex vivo profiles been unexpectedly similar, readers could then have interpreted the in vivo dissimilarity more deeply, for example, by raising the possibility that mouse and human responses to endotoxin do not diverge at the initial molecular events, but rather, in their subsequent effects on the intercellular network found within the body. Overall, we commend Seok and colleagues on their nuanced approach to the mouse-as-sepsis-model question. We believe that ample evidence exists to support use of the mouse as a discovery tool, but that future mechanistic studies in sepsis should emphasize early validation in the human setting (5). Rather than delaying proof-of-concept human studies, basic and clinical researchers in this field should collaborate early and extensively to ensure that the most “translatable” science is moving forward.

Inflammatory response and oxidative stress in developing rat brain and its consequences on motor behavior following maternal administration of LPS and perinatal anoxia

Cerebral palsy (CP) is a disorder of locomotion, posture and movement that can be caused by prenatal, perinatal or postnatal insults during brain development. An increased incidence of CP has been correlated to perinatal asphyxia and maternal infections during gestation. The effects of maternal exposure to low doses of bacterial endotoxin (lipopolysaccharide, LPS) associated or not with perinatal anoxia (PA) in oxidative and inflammatory parameters were examined in cerebral cortices of newborns pups. Concentrations of TNF-α, IL-1, IL-4, SOD, CAT and DCF were measured by the ELISA method. Other newborn rats were assessed for neonatal developmental milestones from day 1 to 21. Motor behavior was also tested at P29 using open-field and Rotarod. PA alone only increased IL-1 expression in cerebral cortex with no changes in oxidative measures. PA also induced a slight impact on development and motor performance. LPS alone was not able to delay motor development but resulted in changes in motor activity and coordination with increased levels of IL-1 and TNF-α expression associated with a high production of free radicals and elevated SOD activity. When LPS and PA were combined, changes on inflammatory and oxidative stress parameters were greater. In addition, greater motor development and coordination impairments were observed. Prenatal exposure of pups to LPS appeared to sensitize the developing brain to effects of a subsequent anoxia insult resulting in an increased expression of pro-inflammatory cytokines and increased free radical levels in the cerebral cortex. These outcomes suggest that oxidative and inflammatory parameters in the cerebral cortex are implicated in motor deficits following maternal infection and perinatal anoxia by acting in a synergistic manner during a critical period of development of the nervous system.

High‐fat meal induced postprandial inflammation

Raised levels of circulating inflammatory markers are associated with coronary artery disease, obesity and type II diabetes. It has been proposed that the ingestion of high-fat meals may serve as a stimulus to raise systemic inflammatory tone, although interventional studies have yielded conflicting results. We here review 57 studies of high-fat meal induced acute postprandial inflammation to identify the most frequently reported markers of postprandial inflammation and to compare these results with the highly consistent low-grade endotoxaemia model in man. Most plasma borne markers of inflammation, such as cytokines and soluble adhesion molecules, were not consistently raised after a high-fat meal. However, pro-inflammatory leukocyte surface markers, mRNA and proteins were elevated in almost all studies in which they were measured. These markers followed kinetics similar to those observed following intravenous injection of low doses of endotoxin in man, were positively associated with likelihood of contamination of test meals with pro-inflammatory bacterial molecules and were reduced in several studies examining parallel meals supplemented with foodstuffs containing anti-inflammatory phytochemicals. Future studies of postprandial inflammation may yield more consistent evidence by focusing on leukocyte, rather than plasma-borne, markers of inflammation and by considering the test meal content of pro- and anti-inflammatory dietary constituents.

Effects of oral clenbuterol on the clinical and inflammatory response to endotoxaemia in the horse

Pro-inflammatory cytokines, such as IL-1β and TNFα, play a major role in activating leukocytes and endothelial cells during the systemic inflammatory response to endotoxin in the horse. β2 agonist drugs, such as clenbuterol, inhibit leukocyte activation. This study aimed to determine the effects of oral clenbuterol on clinical and leukocyte responses, including production of TNFα, in an in vivo endotoxin challenge model. In a randomised crossover design, horses received either clenbuterol or a placebo product prior to the administration of low dose endotoxin (30ng/kg over 30min). Clinical signs were measured and leukocyte counts and serial blood samples were obtained over 6h. Pre-treatment with oral clenbuterol (0.8μg/kg) significantly reduced (P=0.046) the peak rectal temperature and the peak plasma TNFα concentration (P=0.026) following endotoxin challenge. These data suggest that oral clenbuterol at the therapeutic dose has anti-inflammatory effects in horses challenged with a low dose of endotoxin.

Neutrophil recruitment in response to intradermal endotoxin challenge in man

BackgroundNeutrophil recruitment is a key component of the innate immune response, but it is difficult to study in vivo in man, where multi-time-point samples from disease or challenge models are hard to obtain. In-vitro data from our group suggest that neutrophilic inflammation in response to endotoxin will be initiated by a cytokine cascade in which interleukin (IL) 1 has an early and important role. To study this in human beings, we have established a model of intradermal challenge. MethodsEndotoxin and control solution (0·9% NaCl) were injected intradermally in the volar surface of the forearm of healthy volunteers. Serum was taken for blood count and C-reactive protein (CRP) measurements at 0, 1, 6, and 24 h. Temperature, blood pressure, and pulse were monitored. Sites were sampled by 4 mm punch biopsies at varying time points and after varying doses. Biopsy samples were analysed by immunohistochemistry and qPCR. FindingsIntradermal erythema and mild oedema were seen over a current dose range of 0·01–15 ng endotoxin per site. Total dose per individual was 1 to 45 ng. No volunteer had significant systemic reactions. After 5 h, a peripheral blood neutrophilia was seen in some individuals. Erythema area increased up to 6 h, and there was a trend to larger area with larger dose. Above 1 ng endotoxin per site, neutrophilic infiltration of the skin was visible microscopically, but this was much greater at doses of 5 ng or greater. A significant difference was seen between neutrophil elastase stained biopsy area at 2 h and 6 h post administration of endotoxin for the higher doses. There were no changes in serum cytokine levels post injection. By contrast, qPCR revealed a rapid local generation of IL-8 mRNA, and more sustained expression of IL1α/β. InterpretationIntradermal administration of endotoxin within the limits described seems to be safe. It is an effective method of inducing significant local neutrophilic inflammation at an accessible site. Neutrophil infiltration of tissues starts before 2 h and continues beyond 6 h in response to a single dose of endotoxin. Neutrophilia (without CRP rise or symptoms), appears to be associated with a higher total endotoxin dose. IL-8 production at the inflamed site peaks early, whereas IL1α/β mRNA levels remain high at 6 h. FundingNational Institute for Health Research.

Pathophysiology of endotoxin tolerance: mechanisms and clinical consequences

Endotoxin tolerance was first described in a study that exposed animals to a sublethal dose of bacterial endotoxin. The animals subsequently survived a lethal injection of endotoxin. This refractory state is associated with the innate immune system and, in particular, with monocytes and macrophages, which act as the main participants. Several mechanisms are involved in the control of endotoxin tolerance; however, a full understanding of this phenomenon remains elusive. A number of recent reports indicate that clinical examples of endotoxin tolerance include not only sepsis but also diseases such as cystic fibrosis and acute coronary syndrome. In these pathologies, the risk of new infections correlates with a refractory state. This review integrates the molecular basis and clinical implications of endotoxin tolerance in various pathologies.

Interleukin-8 induces an elevation in the endotoxin activity assay (EAA) level: does the EAA truly measure the endotoxin level?

The endotoxin activity assay (EAA) is a FDA-approved blood endotoxin assay that is reported as a useful tool for the diagnosis of gram-negative bacterial infection. However, discrepancies between the results of the EAA and those of the limulus amebocyte lysate (LAL) assay have been reported. Thus, we verified these methods. Blood was incubated with anti-endotoxin antibody, the resultant polymorphonuclear activation to produce oxidants was measured and the EAA level calculated. As a reference endotoxin assay, we used an endotoxin-specific LAL assay. Significant increases in plasma LAL assay levels were observed only in patients with sepsis caused by gram-negative bacterial infections, whereas higher EAA levels were observed in almost all the sepsis cases and the SIRS cases, especially those with acute pancreatitis. Graded amounts of LPS (1-10,000pg/ml) were spiked into normal blood to obtain dose-response curves: a good dose-response curve, from 1 to 1,000pg/ml, was obtained for the LAL assay. A good dose-response curve was barely obtained for the EAA; the lowest detection limit seemed to be 1,000pg/ml. Addition of methylprednisolone decreased the EAA levels. Interleukin-8 (IL-8) induced elevation in EAA levels when IL-8 was added to volunteers' blood samples. Overall, the EAA kit could not measure clinically relevant doses of endotoxin. Because IL-8 induced an increase in EAA level, it is suggested that the EAA level reflects the primed state of polymorphonuclear leukocytes.

Global sensitivity analysis of endotoxin-induced acute inflammatory responses predicts the multimodal dependence of global tissue damage both on host IL-6 responses and endotoxin dose

22 Tissue-specific patterns of caspase-1 and cytokines in excisional wounds are altered by shock in rat skin and muscle Ravi Starzl , Dolores Wolfram , Ruben Zamora , Bahiyyah Jefferson , Derek Barclay , Chien Ho , Gerald Brandacher , Stefan Schneeberger , W.P. Andrew Lee , Jaime Carbonell , Yoram Vodovotz c,d Language Technologies Institute, Carnegie Mellon University, Pittsburgh, PA, USA Department of Plastic and Reconstructive Surgery, Innsbruck Medical University, Innsbruck, Tyrol, Austria Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA Center for Inflammation and Regenerative Modeling, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA Objectives: Skin and muscle wounds often lead to significant inflammation in the affected tissue. The primary mechanism by which inflammation is initiated, sustained, and terminated is cytokine-mediated immune signaling, but this can be altered by cardiogenic shock. The complexity and context sensitivity of immune signaling in general stymied a clear understanding of these signaling dynamics. We hypothesized that advanced numerical and biological function analysis methods would help elucidate the inflammatory response to skin and muscle wounds in rats, both with and without concomitant shock. Methods: We studied 2 experimental groups: wound only (“wound group”) and wound with cardiogenic shock (“shock group”). In the “wound group,” 4 Lewis rats were anesthetized and an excision biopsy was taken from the lateral aspect of the thigh on one of the hind limbs in each of the rats. In the “shock group,” 4 Lewis rats were sacrificed, and excision biopsy (wound) was carried out 15 to 30 seconds after cessation of heartbeat. Skin and muscle tissue was then separated and assayed for total protein content by BCA assay, and the inflammation biomarkers interferon-γ, interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-18, monocyte chemotactic protein (MCP-1), growth-related oncogene/KC, tumor necrosis factor α, and granulocyte-macrophage colony-stimulating factor were assayed by Luminex (Luminex Corp, Austin, TX). Caspase-1, which drives activation of the NLRP-3 inflammasome, was assessed by Western blot assay. Statistical and computational analyses of cytokine network profiles (1-way balanced analysis of variance, unpaired 1-tailed heteroscedastic t test, principal components analysis, and confirmatory factor analysis) were performed in Matlab (The MathWorks, Inc, Natick, MA). Results: Granulocyte-macrophage colony-stimulating factor was elevated in the wound group (P b .05), whereas IL-4, IL-12p70, interferon-γ, and IL-18 were elevated in the shock group (P b .05). Interleukin-1α and IL-18 were more elevated in skin vs muscle (P b .05), which was suggestive of inflammasome activation in the skin. MCP-1, IL-1β, IL-2, IL-6, IL-10, IL-4, IL-12p70, and tumor necrosis factor α (P b .05) were also differentially higher in skin vs muscle. Immunoblotting revealed caspase-1 activation in skin but not muscle. Notably, IL-1α and IL-18, along with caspase-1, were greatly elevated in the skin after cardiogenic shock (P b .05). Principal components analysis suggested that more than 95% of observed variance could be explained by 2 principal components in the skin and muscle and suggested distinct groups of inflammatory mediators induced in the “wound group” vs the “shock group”. Interleukin-18 and IL-1α were primary contributors to the first principal component, whereas IL-6, MCP-1, growth-related oncogene/KC, and IL-1β were primary contributors to the second principal component. Principal components analysis results were reinforced by confirmatory factor analysis. Conclusions: Caspase-1 and the NLRP-3 inflammasome appear to be key factors in determining the type and severity of the inflammatory response to excisional wounding, especially in the presence of shock. Activated caspase-1 is associated with defined, compartmentalized patterns of cytokine production that may be discerned via data-driven modeling. http://dx.doi.org/10.1016/j.jcrc.2012.10.038