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An important mechanism by which IFN-[gamma] primes macrophages for enhanced innate immune responses is abrogation of feedback inhibitory pathways. Accordingly, IFN-[gamma] abrogates endotoxin tolerance, a major negative feedback loop that silences expression of inflammatory cytokine genes in macrophages previously exposed to endotoxin/Toll-like receptor (TLR) ligands. Mechanisms by which IFN-[gamma] inhibits endotoxin tolerance have not been elucidated. Here, we show that pretreatment with IFN-[gamma] prevented tolerization of primary human monocytes and restored TLR4-mediated induction of various proinflammatory cytokines, including IL-6 and TNF[alpha]. Surprisingly, IFN-[gamma] did not alter proximal TLR4 signaling defects in tolerized monocytes. Instead, IFN-[gamma] blocked tolerance-associated down-regulation of IL6 and TNF transcription, RNA polymerase II recruitment, and NF-[kappa]B and CCAAT/enhancer-binding protein [beta] transcription factor binding to the IL6 and TNF promoters in tolerized monocytes. The mechanism by which IFN-[gamma] restored IL6 expression was by facilitating TLR4-induced recruitment of chromatin remodeling machinery to the IL6 promoter and promoting IL6 locus accessibility in tolerized monocytes. Our results suggest that IFN-[gamma] overcomes endotoxin tolerance by facilitating TLR-induced chromatin remodeling to allow expression of proinflammatory genes. These results identify a mechanism by which IFN-[gamma] promotes activation of macrophages and highlight the importance of chromatin remodeling and transcriptional control in the regulation of inflammatory cytokine production in tolerant and activated macrophages. inflammation | innate immunity | macrophage doi/ 10.1073/pnas.1007816107

Mares and geldings in good body condition selected for hyperleptinemia vs. normal leptin concentrations were studied to determine whether the hyperleptinemic condition affected various characteristics of the hematologic and hormonal systems after a challenge with lipopolysaccharide endotoxin. Four mares and 4 geldings that were determined to be hyperleptinemic (mean plasma leptin concentrations of 10.0 to 15.5 ng/mL) and 4 mares and 4 geldings with mean plasma leptin concentrations between 2.4 and 5.5 ng/ mL were administered Escherichia coli O55:B5 endotoxin (35 ng/kg of BW in 500 mL of saline over a 30-min infusion), or saline only, in pairs in a single-switchback design, with horses and treatments randomly assigned for the first infusion. Physiological variables and blood components were monitored for 24 h after the onset of infusions. Treatments were switched and the second infusions were administered 8 d later. Relative to vehicle infusion, endotoxin infusion increased (P < 0.01) the rectal temperature, heart rate, respiration rate, plasma total protein concentration, and blood packed cell volume; there was an interaction of leptin status, endotoxin treatment, and time for heart rate (P = 0.039), respiration rate (P = 0.018), and plasma total protein concentration (P = 0.054). Blood concentrations of leukocytes, lymphocytes, and neutrophils all decreased (P < 0.001) after endotoxin infusion; there was an interaction (P = 0.0057) between sex and leptin status for blood platelet concentration. Plasma leptin concentrations increased (P = 0.013) after endotoxin infusion in both hyperleptinemic horses and those with reduced leptin concentrations. There were interactions (P < 0.037) of sex with endotoxin treatment and time for plasma concentrations of cortisol and prolactin, whereas plasma GH concentrations were affected (increased; P < 0.001) only by time after infusion. Given that the effects of hyperleptinemia were generally minor, it was concluded that the hyperleptinemic condition, and its associated type-2 diabetic symptoms, has a minimal impact on the components of the hematologic and hormonal systems studied. Key words: endotoxin, gelding, hematologic, hyperleptinemia, mare doi:10.2527/jas.2009-2337

Listeriolysin O (LLO) is a pore-forming toxin that mediates phagosomal escape and cell-to-cell spread of the intracellular pathogen Listeria monocytogenes. In order to identify factors that control the production, activity, or secretion of this essential virulence factor, we constructed a Himar1 mariner transposon delivery system and screened 50,000 mutants for a hypohemolytic phenotype on blood agar plates. Approximately 200 hypohemolytic mutants were identified, and the 51 most prominent mutants were screened ex vivo for intracellular growth defects. Eight mutants with a phenotype were identified, and they contained insertions in the following genes: lmo0964 (similar to yjbH), lmo1268 (clpX), lmol401 (similar to ymdB), 1mo1575 (similar to ytqI), lmo1695 (mprF), lmo1821 (similar to prpC), lmo2219 (prsA2), and lmo2460 (similar to cggR). Some of these genes are involved in previously unexplored areas of research with L. monocytogenes: the genes yjbH and clpX regulate the disulfide stress response in Bacillus subtilis, and the prpC phosphatase has been implicated in virulence in other gram-positive pathogens. Here we demonstrate that prsA2, an extracytoplasmic peptidyl-prolyl cis/trans isomerase, is critical for virulence and contributes to the folding of LLO and to the activity of another virulence factor, the broad-range phospholipase C (PC-PLC). Furthermore, although it has been shown that prsA2 expression is linked to PrfA, the master virulence transcription factor in L. monocytogenes pathogenesis, we demonstrate that prsA2 is not directly controlled by PrfA. Finally, we show that PrsA2 is involved in flagellum-based motility, indicating that this factor likely serves a broad physiological role.

In sepsis, systemic inflammatory response syndrome (SIRS), and multiorgan dysfunction syndrome (MODS), a severe prognostically relevant cardiac autonomic dysfunction exists, as manifested by a strong attenuation of sympathetically and vagally mediated heart rate variability (HRV). The mechanisms underlying this attenuation are not limited to the nervous system. They also include alterations of the cardiac pacemaker cells on a cellular level. As shown in human atria] cardiomyocytes, endotoxin interacts with cardiac hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels, which mediate the pacemaker current [I.sub.f] and play an important role in transmitting sympathetic and vagal signals on heart rate and HRV. Moreover, endotoxin sensitizes cardiac HCN channels to sympathetic signals. These findings identify endotoxin as a pertinent modulator of the autonomic nervous regulation of heart function. In MODS, the vagal pathway of the autonomic nervous system is particularly compromised, leading to an attenuation of the cholinergic antiinflammatory reflex. An amelioration of the blunted vagal activity appears to be a promising novel therapeutic target to achieve a suppression of the inflammatory state and thereby an improvement of prognosis in MODS patients. Preliminary data revealed therapeutic benefits (increased survival rates and improvements of the depressed vagal activity) of the administration of statins, β-blockers, and angiotensin-converting enzyme inhibitors in patients with MODS. Key words: Autonomic dysfunction, heart rate variability (HRV), endotoxin, HCN channel, cardiac pacemaker current If, sepsis, systemic inflammatory response syndrome (SIRS), multiorgan dysfunction syndrome (MODS), statins, P-blockers, angiotensin-converting enzyme inhibitors (ACEI), multiorgan failure (MOF). Resume : La sepsie, le syndrome de reponse inflammatoire systemique (SRIS) et le syndrome de defaillance multiviscerale (SDMV) sont associes a un grave dysfonctionnement d'interet diagnostique de la fonction autonome cardiaque, qui est illustre par une forte attenuation de la variabilite de la frequence cardiaque (VFC), vehiculee par voie sympathique et vagale. Les mecanismes a l'origine de cette attenuation ne se limitent pas au systeme nerveux. Ils incluent aussi des modifications des cellules regulatrices du rythme cardiaque au niveau cellulaire. Comme demontre dans les cardiomyocytes auriculaires humains, l'endotoxine interagit avec les canaux (HCN) actives par l'hyperpolarisation, modules par les nucleotides cycliques et non selectifs aux cations, qui medient le courant de pacemaker If et jouent un role important dans la transmission des signaux sympathiques et vagaux sur la frequence cardiaque et sa variabilite. De plus, l'endotoxine sensibilise les canaux HCN aux signaux sympathiques. Ces resultats identifient l'endotoxine comme un modulateur pertinent de la regulation nerveuse autonome de la fonction cardiaque. Dans le SDMV, la voie vagale du systeme nerveux autonome en particulier est affaiblie, entrainant une attenuation du reflexe cholinergique anti-inflammatoire. La stimulation de l'activite vagale semble etre une cible therapeutique prometteuse afin d'obtenir la suppression de l'inflammation et, par consequent, un meilleur pronostic chez les patients SDMV. Les premiers resultats preliminaires revelent des benefices therapeutiques (augmentation des taux de survie et stimulation de l'activite vagale deprimee) de l'administration de statines, de beta-bloquants et d'inhibiteurs de l'enzyme de conversion de l'angiotensine chez les patients souffrant du SDMV. Mots-cles : dysfonctionnement autonome, variabilite de la frequence cardiaque (VFC), endotoxine, canal HCN, courant pacemaker If, sepsie, syndrome de reponse inflammatoire systemique (SRIS), syndrome de defaillance multiviscerale (SDMV), statines, beta-bloquants, inhibiteurs de l'enzyme de conversion de l'angiotensine (IECA), defaillance polyviscerale (DFV). [Traduit par la Redaction], Sepsis, SIRS, and MODS--major problems in intensive care medicine Sepsis, noninfectious systemic inflammatory response syndrome (SIRS), and multiorgan dysfunction syndrome (MODS) (Fig. 1) pose tremendous problems in our health care [...]

This study was performed to investigate the glomerular permeability alterations responsible for the microalbuminuria occurring in endotoxemia and during anaphylactic shock. In anesthetized Wistar rats, the left ureter was catheterized for urine collection while, simultaneously, blood access was achieved. Endotoxemia was induced by lipopolysaccharide (LPS) from Escherichia coli, and glomerular permeability was assessed at 60 and 90 (n = 7) and 120 (n = 7) min. Anaphylaxis was induced by a bolus dose of Dextran-70, and glomerular permeability assessed at 5 min (n = 8) and 40 min (n = 9). Sham animals were followed for either 5 or 120 min. The glomerular sieving coefficients ([theta]) to fluorescein isothiocyanate-Ficoll (70/400) were determined from plasma and urine samples and assessed using size-exclusion chromatography (HPLC). After start of the LPS infusion (2 h), but not at 60 or 90 min, [theta] for [Ficoll.sub.70A] had increased markedly [from 2.91 x [10.sup.-5] [+ or -] 6.33 x [10.sup.-6] to 7.78 x [10.sup.-5] [+ or -] 6.21 x [10.sup.-6] (P < 0.001)]. In anaphylaxis, there was a large increase in [theta] for Ficolls >60 [Angstrom] in molecular radius already at 5 min, but the glomerular permeability was completely restored at 40 min. In conclusion, there was a transient, immediate increment of glomerular permeability in dextran-induced anaphylaxis, which was completely reversible within 40 min. By contrast, endotoxemia caused an increase in glomerular permeability that was manifest first after 2 h. In both cases, [theta] to large Ficoll molecules were markedly increased, reflecting an increase in the number of large pores in the glomerular filter. endotoxin; systemic inflammatory response syndrome

Oxytocin is a hormone and neurotransmitter found to have anti-inflammatory functions in rodents. Here we used experimental bacterial endotoxinemia to examine the role of exogenous oxytocin administration on innate immune responses in humans. Ten healthy men received, in a randomized, placebo-controlled, crossover design, placebo, oxytocin, LPS, and LPS + oxytocin. Oxytocin treatment resulted in a transient or prolonged reduction of endotoxin-induced increases in plasma ACTH, cortisol, procalcitonin, TNF-[alpha], IL-1 receptor antagonist, IL-4, IL-6, macrophage inflammatory protein-1[alpha], macrophage inflammatory protein-l[beta], monocyte chemoattractant protein-1 (MCP-1), interferon-inducible protein 10, and VEGF. In vitro, oxytocin had no impact on LPS effects in releasing TNF-[alpha], IL-6, and MCP-1 in monocytes and peripheral blood mononuclear cells from healthy human donors. In summary, oxytocin decreases the neuroendocrine and cytokine activation caused by bacterial endotoxin in men, possibly due to the pharmacological modulation of the cholinergic anti-inflammatory pathway. Oxytocin might be a candidate for the therapy of inflammatory diseases and conditions associated with high cytokine and VEGF levels. neuroendocrinology; hypothalamic-pituitary-adrenal; cytokines

This study was designed to test the hypothesis that the transient receptor potential vanilloid type 1 (TRPV1) channel, expressed primarily in sensory nerves, and substance P (SP), released by sensory nerves, play a protective role against lipopolysaccharide (LPS)-induced hypotension. LPS (10 mg/kg iv) elicited tachycardia and hypotension in anesthetized male Wistar rats, which peaked at 10 min and gradually recovered 1 h after the injection. Blockade of TRPV1 with its selective antagonist capsazepine (CAPZ, 3 mg/kg iv) impaired recovery given that the fall in mean arterial pressure (MAP) was greater 1 h after CAPZ plus LPS injections compared with LPS injection alone (45 [+ or -] 5 vs. 25 [+ or -] 4 mmHg, P < 0.05). Blockade of the neurokinin 1 (NK1) receptor with its selective antagonists RP-67580 (5 mg/kg iv) or L-733,060 (4 mg/kg iv) prevented recovery, considering that falls in MAP were not different 1 h after injections of NKI antagonists plus LPS from their peak decreases (66 [+ or -] 9 vs. 74 [+ or -] 5 mmHg or 60 [+ or -] 7 vs. 69 [+ or -] 3 mmHg, respectively, P > 0.05). LPS increased plasma SP, norepinephrine (NE), and epinephrine (Epi) levels compared with vehicles, and the increases in plasma SP, NE, and Epi were significantly inhibited by CAPZ or RP-67580. The survival rate at 24 or 48 h after LPS injection (20 mg/kg ip) was lower in conscious rats pre-treated with CAPZ or RP-67580 compared with rats treated with LPS alone (P < 0.05). Thus our results show that the TRPV1, possibly via triggering release of SP which activates the NK1 and stimulates the sympathetic axis, plays a protective role against endotoxin-induced hypotension and mortality, suggesting that TRPV1 receptors are essential in protecting vital organ perfusion and survival during the endotoxic condition. endotoxin; transient receptor potential vanilloid type 1 channel; substance

Gentamicin is a mainstay in treating gram-negative sepsis. However, it also may potentiate endotoxin (LPS)-driven plasma TNF-[alpha] increases. Because gentamicin accumulates in renal tubules, this study addressed whether gentamicin directly alters LPS-driven tubular cell TNF-[alpha] production. HK-2 proximal tubular cells were incubated for 18 h with gentamicin (10-2,000 [micro]g/ml). Subsequent LPS-mediated TNF-[alpha] increases (at 3 or 24 h; protein/mRNA) were determined. Gentamicin effects on overall protein synthesis ([[sup.35]S]methionine incorporation), monocyte chemoattractant protein-1 (MCP-1) levels, and LPS-stimulated TNF-[alpha] generation by isolated mouse proximal tubules also were assessed. Finally, because gentamicin undergoes partial biliary excretion, its potential influence on gut TNF-[alpha]/MCP-1 mRNAs was probed. Gentamicin caused striking, dose-dependent inhibition of LPS-driven TNF-[alpha] production (up to 80% in HK-2 cells/isolated tubules). Surprisingly, this occurred despite increased TNF-[alpha] mRNA accumulation. Comparable changes in MCP-I were observed. These changes were observed at clinically relevant gentamicin concentrations and despite essentially normal overall protein synthetic rates. Streptomycin also suppressed LPS-driven TNF-[alpha] increases, suggesting an aminoglycoside drug class effect. Gentamicin doubled basal TNF-[alpha] mRNA in cecum and in small intestine after LPS. Gentamicin can suppress LPS-driven TNF-[alpha] production in proximal tubule cells, likely by inhibiting its translation. Overall preservation of protein synthesis and comparable MCP-1 suppression suggest a semiselective blockade within the LPS inflammatory mediator cascade. These results, coupled with increases in gut TNF-[alpha]/ MCP-1 mRNAs, imply that gentamicin may exert protean, countervailing actions on systemic cytokine/chemokine production during gram-negative sepsis. HK-2 cells; aminoglycosides; acute renal failure; tumor necrosis factor-[alpha]

The pathogenesis of acute renal failure (ARF) occurring during the course of sepsis is incompletely understood. Intercellular adhesion molecule-1 (ICAM-1) is a key cell adhesion molecule upregulated by LPS, which binds to the integrins CD11a/CD18 and CD11b/CD18 present on the surface of leukocytes. We hypothesized that ICAM-1 facilitates renal injury in LPS-induced ARF. To test this, three groups of mice (n = 8 per group) were injected intraperitoneally with 6 mg/kg LPS: 1) normal C57BL/6 mice, 2) mice with a targeted deficiency of ICAM-1 ([ICAM-1.sup.-/-]), and 3) mice expressing very low levels of CD18 (CD18-def). [ICAM-1.sup.-/-] mice were significantly resistant to LPS-mediated ARF, as opposed to CD18-def mice, which developed severe ARF, as did wild-type controls (48 h blood urea nitrogen 143 [+ or -] 31.5, 70.8 [+ or -] 24.4, and 185 [+ or -] 16.6 mg/dl in wild-type, [ICAM-1.sup.-/-], and CD18-def mice, respectively, P < 0.05). At death, [ICAM-1.sup.-/-] mice had significantly less renal neutrophil infiltration than the other two groups, as well as less histological tubular injury. Depletion of neutrophils with mAb Gr-1 led to a profound exaggeration of tumor necrosis factor (TNF) release and high mortality, but neutrophil-depleted mice receiving 10-fold less LPS were protected against ARF despite TNF release similar to what is normally associated with LPS-induced ARF. LPS caused a significant increase in renal expression of chemokines; however, this increase was significantly exaggerated in CD18-def mice, which may account for their lack of protection. In conclusion, these data show that ICAM-1 plays a key role in LPS-induced ARF. sepsis; neutrophils; vascular cell adhesion molecule-1; chemokines

The objective of this study was to determine whether Toll-like receptor 4 (TLR4) has a role in alcohol-mediated acetaminophen (APAP) hepatotoxicity. TLR4 is involved in the inflammatory response to endotoxin. Others have found that ethanol-mediated liver disease is decreased in C3H/HeJ mice, which have a mutated TLR4 resulting in a decreased response to endotoxin compared with endotoxin-responsive mice. In the present study, short-term (1 wk) pretreatment with ethanol plus isopentanol, the predominant alcohols in alcoholic beverages, caused no histologically observed liver damage in either C3H/HeJ mice or endotoxin-responsive C3H/HeN mice, despite an increase in nitrotyrosine levels in the livers of C3H/HeN mice. In C3H/HeN mice pretreated with the alcohols, subsequent exposure to APAP caused a transient decrease in liver nitrotyrosine formation, possibly due to competitive interaction of peroxynitrite with APAP producing 3-nitroacetaminophen. Treatment with APAP alone resulted in steatosis in addition to congestion and necrosis in both C3H/HeN and C3H/HeJ mice, but the effects were more severe in endotoxin-responsive C3H/HeN mice. In alcohol-pretreated endotoxin-responsive C3H/HeN mice, subsequent exposure to APAP resulted in further increases in liver damage, including severe steatosis, associated with elevated plasma levels of TNF-[alpha]. In contrast, alcohol pretreatment of C3H/HeJ mice caused little to no increase in APAP hepatotoxicity and no increase in plasma TNF-[alpha]. Portal blood endotoxin levels were very low and were not detectably elevated by any of the treatments. In conclusion, this study implicates a role of TLR4 in APAP-mediated hepatotoxicity. endotoxin; nitrotyrosine

Byline: Vladimir B. Serikov (1), T. V. Glazanova (2), E. H. Jerome (3), N. W. Fleming (4), Haruki Higashimori (5), N. C. Sr. Staub (6) Keywords: endotoxin; sepsis; shock; Tyloxapol; receptor blockade Abstract: We have previously demonstrated that the detergent Tyloxapol is effective in preventing reactions to endotoxin. We studied the effects of Tyloxapol on the morbidity and mortality from endotoxemia in rabbits and on the mortality in rats with sepsis. The effects of Tyloxapol on endotoxin binding and macrophage activation were studied in the macrophage cell line RAW264.7 and CHO cells expressing CD14. Isolated human leukocytes were used to study the effects of Tyloxapol on immune reactions, leukocyte motility, and phagocytosis. Intravenous Tyloxapol (200 mg/kg), given prior to or at the time of endotoxin infusion protected rabbits from developing shock. In rats with peritoneal sepsis, a lipid-rich diet and Tyloxapol given at the time of induction of peritonitis protected them from septic death. In vitro, Tyloxapol blocked the binding of endotoxin to murine macrophages and CHO cells expressing CD14, activation of macrophages, and also some antigen--antibody immune reactions (mediated by CD2, CD4, CD22, HLA-DR). Tyloxapol may prevent the reaction to endotoxin by desensitizing endotoxin-recognizing receptors. Author Affiliation: (1) Children's Hospital Oakland Research Institute, California (2) Department of Immunology, St. Petersburg Institute of Blood, St. Petersburg, Russia (3) Cardiovascular Research Institute, UCSF, San Francisco, California (4) Department of Anesthesiology, UC Davis, School of Medicine, Davis, California (5) Department of Human Physiology, UC Davis School of Medicine, Davis, California (6) Cardiovascular Research Institute, UCSF, San Francisco, California Article History: Registration Date: 20/10/2004

Byline: M. H. Iversen (1), R. G. Hahn (1) Keywords: Key words Endotoxin; Leucopenia; Pharmacokinetics; Retinyl; Retinol; Sepsis Abstract: Background: Vitamin A reduces the pathophysiological effects of endotoxin in animals, but the mechanism and the lowest effective dose are not clear.APMethods: An intravenous bolus of endotoxin 20 ug * kg.sup.--1 was given to 30 rabbits. In 10 of them, 1000 IE * kg.sup.--1 retinyl palmitate was injected intravenously 1 h before the endotoxin and in another 10 rabbits 1 h after the endotoxin. A one-compartment open model was fitted to the time-concentration profile of endotoxin in plasma.APResults: The half-life of endotoxin was half as long when vitamin A was given for prophylaxis (median 35 min) and for treatment (33 min) than in the controls (67 min p < 0.004). The plasma concentrations of immunoglobulin G and M endotoxin-core antibodies, the leucocyte count and the acid-base balance did not differ between the groups during the experiment, but the pyrogenic reaction was more pronounced in the controls.APConclusion: A fairly low dose of vitamin A reduced the half-life of endotoxin. Author Affiliation: (1) Department of Anaesthesia, Stockholm Soder Hospital, S-11 883 Stockholm, Sweden e-mail: Robert.Hahn@anest.sos.sll.se Tel. + 46(8)6 16 22 26 Fax + 46(8)6 16 22 34, SE Article note: Received: 16 October 1998 Final revision received: 25 May 1999 Accepted: 25 June 1999

Byline: Le Feng Wang (1), Katsuyuki Tomita (1), Takao Sasaki (1) Abstract: Tumor necrosis factor alpha (TNF-[alpha]) responses of alveolar macrophages (AMs) and peritoneal macrophages (PMs) were studied in rats after intravenous injection of lipopolysaccharide (LPS). High levels of plasma TNF-[alpha], increased pulmonary myeloperoxidase activity, and leukopenia occurred within 2 h after LPS injection. Alveolar spaces exhibited a strict compartment property, as manifested by only slightly increased LPS and TNF-[alpha] levels in alveolar lavage fluid and an unchanged capacity of AMs to produce TNF-[alpha]. By contrast, the peritoneal cavity had greatly increased local LPS and TNF-[alpha] levels and a diminished PMs TNF-[alpha] response to LPS. The amount of LPS in the alveolar spaces was less than 0.2% of the level in peritoneal fluid. These results indicate that activation of resident macrophages is dependent on the amounts of local LPS and, in addition, suggest that resident AMs neither participate in the plasma TNF-[alpha] response nor contribute to neutrophil sequestration in the lung during the early stages of endotoxemia. Author Affiliation: (1) Third Department of Internal Medicine, Faculty of Medicine, Tottori University, 36--1, Nishi-machi, Yonago-shi, 683--8504, Japan Article History: Registration Date: 03/10/2004