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Purpose Low-grade endotoxin (lipopolysaccharide; LPS) exposure may contribute to the development of exaggerated acute postoperative pain. In the present study, we investigated the possible impact of intraoperative administration of dexmedetomidine (DEX) on LPS-induced postoperative hyperalgesia in a rat incisional pain model. Methods The surgical and sham-surgical animals were randomly divided into saline-treated control, 5.0 mg/kg LPS-treated, 10 [micro]g/kg DEX-treated, and 5.0 mg/kg LPS + 10 [micro]g/kg DEX-treated groups. In the surgical animals, a 1-cm-long plantar incision was made through the skin and fascia under isoflurane anesthesia. The sham-surgical rats were only anesthetized. All treatments were administered by a single intraperitoneal (i.p.) injection 60 min before surgery. Acute postoperative pain was assessed using the Rat Grimace Scale (RGS) one day before surgery (baseline) and at 2 h post incision. In another experiment, the involvement of the [alpha].sub.2-adrenergic receptor was tested using atipamezole, an [alpha].sub.2-adrenergic receptor antagonist. Results In the sham-surgical animals, the RGS did not increase at 2 h after sham surgery compared with the corresponding baseline values in all groups. In the surgical rats, however, the postoperative RGS value of the LPS group was significantly higher than the control group, indicating LPS-induced postoperative hyperalgesia. Administration of intraoperative DEX could prevent the development of such LPS-induced exacerbated post-incisional pain. In addition, the preventive effects of intraoperative DEX were inhibited by pretreatment with atipamezole. Conclusion Our findings indicate that intraoperative DEX treatment can prevent LPS-induced exacerbated post-incisional pain via the [alpha].sub.2-adrenergic receptor signaling pathway., Author(s): Daiki Yamanaka [sup.1] , Takashi Kawano [sup.1] , Atsushi Nishigaki [sup.1] , Bun Aoyama [sup.1] , Hiroki Tateiwa [sup.1] , Marie Shigematsu-Locatelli [sup.1] , Fabricio M. Locatelli [sup.1] , [...]

Disease tolerance is the ability of the host to reduce the effect of infection on host fitness. Analysis of disease tolerance pathways could provide new approaches for treating infections and other inflammatory diseases. Typically, an initial exposure to bacterial lipopolysaccharide (LPS) induces a state of refractoriness to further LPS challenge (endotoxin tolerance). We found that a first exposure of mice to LPS activated the ligand-operated transcription factor aryl hydrocarbon receptor (AhR) and the hepatic enzyme tryptophan 2,3-dioxygenase, which provided an activating ligand to the former, to downregulate early inflammatory gene expression. However, on LPS rechallenge, AhR engaged in long-term regulation of systemic inflammation only in the presence of indoleamine 2,3-dioxygenase 1 (IDO1). AhR-complex-associated Src kinase activity promoted IDO1 phosphorylation and signalling ability. The resulting endotoxin-tolerant state was found to protect mice against immunopathology in Gram-negative and Gram-positive infections, pointing to a role for AhR in contributing to host fitness., Although lipopolysaccharide (LPS)-induced proinflammatory molecules are indispensable for counteracting the growth and dissemination of Gram-negative bacteria, overproduction can lead to sepsis syndrome, also known as endotoxin shock. However, a prior [...]

Plasma levels of asymmetric dimethylarginine (ADMA) are known to be elevated under pathological conditions, but reports on intracellular ADMA levels are scarce. In this study, we investigated whether lipopolysaccharide (LPS)- induced endotoxemia alters the intra-and extra-cellular partition of L-arginine and ADMA. The effect of [H.sub.2]S pretreatmentwas also researched. Wistar rats were given sodium hydrogen sulfide (NaHS, 1 mg x [(kg body mass).sup.-1]) one hour before the LPS injections (20 mg x [kg.sup.-1]). Six hours after the LPS treatment, the animals were sacrificed. Myeloperoxidase (MPO) and dimethylarginine dimethylaminohydrolase (DDAH) activities and levels of hypoxia- inducible factor (HIF)-1α were measured in the liver. ADMA and arginine levels were determined using HPLC. LPS injection caused liver injury, as evidenced by the activities of alanine transaminase, aspartate transaminase, and arginase. LPS increased L-arginine content and decreased DDAH activity in the rat liver. MPO activity and HIF-1α levels indicated inflammation and hypoxia. Despite the accumulation of ADMA in the plasma, the level remained unchanged in the liver. NaHS pretreatment restored both the DDAH activity and intracellular L-arginine levels. It is concluded that increased [H.sub.2]S generation has a potency to restore hepatic L-arginine levels and ADMA handling in endotoxemia. Extra- and intra-cellular partitions of ADMA seem to depend on transport proteins as well as the DDAH activity. Key words: hydrogen sulfide, lipopolysaccharide, arginine, asymmetric dimethylarginine, hypoxia-inducible factor (HIF)-1α. On sait que les niveaux de la dimethylarginine asymetrique (ADMA) plasmatiques sont eleves dans differentes conditions pathologiques, mais les donnees portant sur les niveaux intracellulaires d'ADMA sont rares. Dans cette etude, nous avons examine si l'endotoxemie induite par le LPS modifie le partage intracellulaire et extracellulaire de L-arginine et d'ADMA. Les effets d'un pretraitement au [H.sub.2]S ont aussi ete etudies. Des rats Wistar ont recu du sulfure d'hydrogene de sodium (NaHS, 1 mg x [(kg de masse corporelle).sup.-1]) une heure avant les injections de lipopolysaccharide (LPS; 20 mg x [kg.sup.-1]). Six heures apres le traitement au LPS, les animaux ont ete sacrifies. L'activite de la myeloperoxydase (MPO) et de la dimethylarginine dimethylaminohydrolase (DDAH) ainsi que les niveaux du facteur HIF-1α (hypoxia-inducible factor alpha) ont ete mesures dans le foie. Les niveaux d'ADMA et d'arginine ont ete determines par HPLC. L'injection de LPS provoquait un dommage au foie, mis en evidence par l'activite de l'alanine transaminase, de l'aspartate transaminase et d'arginase. Elle accroissait le contenu en L-arginine et diminuait l'activite de la DDAH dans le foie. L'activite de la MPO et les niveaux de HIF-1a indiquaient la presence d'inflammation et d'hypoxie. Malgre l'accumulation d'ADMA dans le plasma, son niveau hepatique demeurait inchange. Un pretraitement au NaHS retablissait tant l'activite de la DDAH que les niveaux intracellulaires de L-arginine. En conclusion, la generation accrue de [H.sub.2]S a le potentiel de retablir les niveaux de L-arginine et la prise en charge d'ADMA lors de l'endotoxemie. Le partage extracellulaire et intracellulaire d'ADMA semble dependre de proteines de transport de meme que de l'activite de la DDAH. [Traduit par la Redaction] Mots-cles: sulfure d'hydrogene, lipopolysaccharide, arginine, asymetrique, dimethylarginine, facteur HIF-1a., Introduction Hydrogen sulfide ([H.sub.2]S), a colorless gas with a characteristic rotten-egg odor, has emerged as an important biologically active compound owing to its physiological role in health and disease. In [...]

During sepsis, activation of phagocytes leads to the overproduction of proinflammatory cytokines, causing systemic inflammation. Despite substantial information regarding the underlying molecular mechanisms that lead to sepsis, several elements in the pathway remain to be elucidated. We found that the enzyme sphingosine kinase 1 (SphK1) is up-regulated in stimulated human phagocytes and in peritoneal phagocytes of patients with severe sepsis. Blockade of SphK1 inhibited phagocyte production of endotoxin-induced proinflammatory cytokines. We observed protection against sepsis in mice treated with a specific SphK1 inhibitor that was enhanced by treatment with a broad-spectrum antibiotic. These results demonstrated a critical role for SphK1 in endotoxin signaling and sepsis-induced inflammatory responses and suggest that inhibition of SphK1 is a potential therapy for septic shock. 10.1126/science.1188635

The impact of endotoxemia on cerebral endothelium and cerebral blood flow (CBF) regulation was studied in conscious newborn lambs. Bacterial endotoxin [LPS, 2 [micro]g/kg iv] was infused on 3 consecutive days. Cerebrovascular function was assessed by monitoring CBF and cerebral vascular resistance (CVR) over 12 h each day and by the endothelium-dependent vasodilator bradykinin (BK) (n = 10). Inflammatory responses were assessed by plasma tumor necrosis factor-[alpha] (TNF-[alpha], n = 5). Acutely, LPS disrupted the cerebral circulation within l h, with peak cerebral vasoconstriction at 3 h (CBF -28 and CVR +118%, P < 0.05) followed by recovery to baseline by 12 h. TNF-[alpha] and body temperature peaked ~1 h post-LPS. BK-induced vasodilatation (CVR -20%, P < 0.05) declined with each LPS infusion, was abolished after 3 days, and remained absent for at least the subsequent 5 days. Histological evidence of brain injury was found in four of five LPS-treated newborns. We conclude that endotoxin impairs cerebral perfusion in newborn lambs via two mechanisms: 1) acute vasoconstriction (over several hours); and 2) persistent endothelial dysfunction (over several days). Endotoxin-induced circulatory impairments may place the newborn brain at prolonged risk of CBF dysregulation and injury as a legacy of endotoxin exposure. cerebral blood flow; cerebral endothelial function doi:10.1152/ajpregu.00398.2009

IL-10 produced by dendritic cells (DC) can limit or terminate ongoing inflammatory responses by inhibiting the proinflammatory cytokine production. Currently, the molecular mechanism by which IL-10 suppresses cytokine production is still ill-defined. In this study, we showed that IL-10 produced by DC dampens myeloid differentiation factor (MyD)88-dependent, but not MyD88-independent signaling. At the molecular level, IL-10 induces ubiquitination and subsequent protein degradation of MyD88-dependent signaling molecules, including IL-1 receptor-associated kinase 4 and TNF-receptor associated factor 6. Protein degradation by IL-10 was associated with decreased phosphorylation of p38, JNK, and IKK. All of these events were prevented by either blocking IL-10 receptor signaling or inhibiting proteasome degradation. IL-10 induced LPS hyporesponsiveness using the same mechanisms, i.e., ubiquitination and protein degradation. Thus, a previously undescribed regulatory mechanism by which IL-10-mediated protein degradation contributes to the inhibition of inflammatory cytokine production and endotoxin tolerance in DC. cytokine | ubicluitination | endotoxin tolerance doi/10.1073/pnas.0905815106

Heart rate variability (HRV) falls in humans with sepsis, but the mechanism is not well understood. We utilized a mouse model of endotoxemia to test the hypothesis that cytokines play a role in abnormal HRV during sepsis. Adult male C57BL/6 mice underwent surgical implantation of probes to continuously monitor electrocardiogram and temperature or blood pressure via radiotelemetry. Administration of high-dose LPS (Escherichia coli LPS, 10 mg/kg, n = 10) caused a biphasic response characterized by an early decrease in temperature and heart rate at 1 h in some mice, followed by a prolonged period of depressed HRV in all mice. Further studies showed that LPS doses as low as 0.01 mg/kg evoked a significant decrease in HRV. With high-dose LPS, the initial drops in temperature and HR were temporally correlated with peak expression of TNF[alpha] 1 h post-LPS, whereas maximal depression in HRV coincided with peak levels of multiple other cytokines 3-9 h post-LPS. Neither hypotension nor hypothermia explained the HRV response. Pretreatment with dexamethasone prior to LPS significantly blunted expression of 7 of the 10 cytokines studied and shortened the duration of depressed HRV by about half. Interestingly, dexamethasone treatment alone caused a dramatic increase in both low- and highfrequency HRV. Administration of recombinant TNF[alpha] caused a biphasic response in HR and HRV similar to that caused by LPS. Understanding the role of cytokines in abnormal HRV during sepsis could lead to improved strategies for detecting life-threatening nosocomial infections in intensive care unit patients. sepsis; lipopolysaccharide; dexamethasone; tumor necrosis factor alpha doi: 10.1152/ajpregu.00132.2009

OBJECTIVE: Exposure to endotoxin, a component of gram-negative bacteria! cell walls, is widespread in many industrial settings and in the ambient environment. Heavy-exposure environments include livestock farms, cotton textile facilities, [...]

Lipopolysaccharide (LPS) is an important factor in sepsis. LPS given by intraperitoneal injection induces intestinal hyperpermeability and bacterial translocation in animals and stimulates hepatic Kupffer cells to release TNF-[alpha] into the bile. This study aims to test the hypothesis that in response to LPS stimulation, hepatic Kupffer cells and extrahepatic macrophages release a large amount of the inflammatory cytokine high-mobility group box 1 (HMGB1) into the bile and that bile containing HMGB 1 contributes to gut barrier dysfunction in experimental endotoxemia. To test this, rat common bile ducts were catheterized and bile flow rate was monitored before and during the LPS administration. Eight hours after LPS challenge, anti-HMGB1 neutralizing antibody or nonimmune (sham) IgG was injected into the duodenal lumen of endotoxemic rats; normal mice were also gavaged with normal or endotoxemic rat bile (bile collected from LPS-treated rats). We found that after LPS challenge, the bile flow rate in rats was significantly decreased at the 4- to 12-h time points, TNF-[alpha] concentration in the bile was markedly elevated at the 3- to 4-h time points, and bile HMGB1 levels were significantly increased at the 8- to 12-h time points. Duodenal injection with anti-HMGB1 antibody reversed LPS-induced gut barrier dysfunction in rats. In addition, feeding endotoxemic rat bile to normal mice significantly increased both mucosal permeability and bacterial translocation. The increase in permeability and bacterial translocation was reversible following removal of HMGB1 from the endotoxemic rat bile. These findings document that bile HMGB1 mediates gut barrier dysfunction in experimental endotoxemia. endotoxemia; gut barrier function

Pathogenic bacteria have evolved numerous mechanisms to evade the human immune system and have developed widespread resistance to traditional antibiotics. We studied the human pathogen Neisseria meningitidis and present evidence of novel mechanisms of resistance to the human antimicrobial peptide LL-37. We found that bacteria attached to host epithelial cells are resistant to 10 [micro]M LL-37 whereas bacteria in solution or attached to plastic are killed, indicating that the cell microenvironment protects bacteria. The bacterial endotoxin lipooligosaccharide and the polysaccharide capsule contribute to LL-37 resistance, probably by preventing LL-37 from reaching the bacterial membrane, as more LL-37 reaches the bacterial membrane on both lipooligosaccharide-deficient and capsule-deficient mutants whereas both mutants are also more susceptible to LL-37 killing than the wild-type strain. N. meningitidis bacteria respond to sublethal doses of LL-37 and upregulate two of their capsule genes, siaC and siaD, which further results in upregulation of capsule biosynthesis.

Maternal bacterial infections adversely affect lung development by crossing the placental barrier and infecting the developing fetus. The underlying mechanism negatively affecting pulmonary development remains unknown. Herein, we investigated whether a systemic maternal infection affects postnatal inflammation and alveolar development. Pregnant rats were injected with 2.5 mg/kg LPS on day 20 and 21 (term = 22 days). Postnatal (PN0-21) mRNA and protein expression of cytokines (IL-1[beta], IL-6, IL-10, CXCL1/2, TNF[alpha]) and genes implicated in alveologenesis [tropoelastin, lysyl oxidase (LOX), lysyl oxidase-like (LOXL)1, tenascin-C (TNC), fibulin 5, vascular endothelial growth factor (VEGF-A), VEGF receptor (VEGFR)2, VEGFR1, platelet-derived growth factor (PDGF)A, PDGFB, and PDGFR[alpha]] were quantified by real-time PCR and beadlyte technology. Lung transcript and protein levels of IL-1[beta], IL-6, and CXCL1/2 were significantly greater in LPS-exposed pups than those of control pups at PN0, 2, 6, 10, and 14. Bronchoalveolar lavage fluid (BALF) of LPS-exposed animals contained significantly more macrophages at PN2 and 14 than BALF of control pups. Morphometric analysis revealed that LPS-exposed animals had fewer and larger alveoli, fewer secondary septa, and decreased peripheral vessel density when compared with control pups. This morphological delay in alveolar development disappeared after PN14. Tropoelastin, LOXL1, VEGF, VEGFR2, and PDGFR[alpha] mRNA expression of LPS-exposed animals was significantly greater than those of control animals in PN2-14 lungs. TNC, LOX, fibulin 5, VEGFR1, PDGFA, and PDGFB expression was not affected by maternal LPS exposure. Together, the data demonstrate that maternal exposure to endotoxin results in a prolonged pulmonary inflammation postnatally, altered gene expression of molecules implicated in alveologenesis, and delayed morphological maturation of the lung. antenatal endotoxin; pulmonary inflammation

BACKGROUND: The relationship of domestic endotoxin exposure to allergy and asthma has been widely investigated. However, few studies have evaluated predictors of household endotoxin, and none have done so for [...]

Byline: Stephanie Wolff (1), Sabine Klatt (1), Jens C. Wolff (2), Jochen Wilhelm (3), Ludger Fink (3), Manfred Kaps (1), Bernhard Rosengarten (1) Keywords: Sepsis; Delirium; Encephalopathy; Hippocampus; Chemokines; Microarrays Abstract: Purpose We studied gene expression differences in brain homogenate, hippocampus, somatosensory cortex and cerebellum of rats suffering from sepsis-associated delirium and analyzed the effects of norepinephrine and 1,400 W (specific inhibitor of the inducible nitric-oxide synthase). Methods We applied microarray screenings to rat brain homogenate 1, 3 and 4.5 h after lipopolysaccharide (LPS, 5 mg/kg) or 0.9% NaCl treatment. Therapy groups were analyzed after 4.5 h. Validations and compartment specific investigations were carried out by real-time PCR. Results Most striking gene expression differences were seen 4.5 h after LPS administration, especially within the hippocampus (chemokines and endothelial cell-specific molecule 1). Norepinephrine resulted in a discrete chemokine up-regulation, while 1,400 W had hardly any effect. Conclusion Strongest gene regulations were found within the hippocampus. Norepinephrine showed a tendency of having a proinflammatory influence, while 1,400 W had no clear-cut effect onto the gene expression level. Author Affiliation: (1) Department of Neurology, Justus-Liebig-University Giessen, Am Steg 14, 35392, Giessen, Germany (2) Max-Planck-Institute for Heart and Lung Research, 61231, Bad Nauheim, Germany (3) Department of Pathology, Justus-Liebig-University Giessen, 35392, Giessen, Germany Article History: Registration Date: 05/01/2009 Received Date: 06/06/2008 Accepted Date: 10/11/2008 Online Date: 21/01/2009 Article note: This article is discussed in the editorial available at: doi: 10.1007/s00134-009-1395-6.

Endotoxins are amphipathic lipopolysaccharides (LPSs), major constituents of the outer membrane of gram-negative bacteria. They consist of a lipid region, covalently linked to a core oligosaccharide, to which may be linked a repetitive glycosidic chain carrying antigenic determinants. Most of the biological activities of endotoxins have been associated with the lipid moiety of the molecule: unique to gram-negative bacteria, LPS is a ligand of the mammalian TLR4-MD2-CD14 pathogen recognition receptor complex. Lipid A preparations are often heterogeneous with respect to both the numbers and the lengths of fatty acids and the natures of substituents on the phosphate groups when present. The variants can significantly affect host immune responses. Nine species in the Bordetella genus have been described, and the fine LPS structures of seven of them have been published. In this report, lipids A from Bordetella pertussis Tohama I and B. bronchiseptica strain 4650 were further characterized and revealed to have a glucosamine substituting both lipid A phosphate groups of the diglucosamine backbone. These substitutions have not been previously described for bordetellae. Moreover, a B. pertussis transposon mutation that maps within a gene encoding a Bordetella ArnT (formerly PmrK) glycosyl transferase ortholog does not carry this substitution, thus providing a genetic basis for the modification. Reverse transcriptase PCR of this locus showed that it is Bvg regulated, suggesting that the ability of Bordetella to modify lipid A via this glucosamine modification is a potential virulence trait.

Endothelial hyperpermeability leading to vascular leak is an important consequence of sepsis and sepsis-induced lung injury. We previously reported that heat shock protein (hsp) 90 inhibitor pretreatment improved pulmonary barrier dysfunction in a murine model of sepsis-induced lung injury. We now examine the effects of hsp90 inhibitors on LPS-mediated endothelial hyperpermeability, as reflected in changes in transendothelial electrical resistance (TER) of bovine pulmonary arterial endothelial cells (BPAEC). Vehicle-pretreated cells exposed to endotoxin exhibited a concentration-dependent decrease in TER, activation of [pp60.sup.src], phosphorylation of the focal adhesion protein paxillin, and reduced expression of the adherens junction proteins, vascular endothelial (VE)-cadherin and [beta]-catenin. Pretreatment with the hsp90 inhibitor, radicicol, prevented the decrease in TER, maintained VEcadherin and [beta]-catenin expression, and inhibited activation of [pp60.sup.Src] and phosphorylation of paxillin. Similarly, when BPAEC hyperpermeability was induced by endotoxin-activated neutrophils, pretreatment of neutrophils and/or endothelial cells with radicicol protected against the activated neutrophil-induced decrease in TER. Increased paxillin phosphorylation and decreased expression of [beta]-catenin and VE-cadherin were also observed in mouse lungs 12 h after intraperitoneal endotoxin and attenuated in mice pretreated with radicicol. These results suggest that hsp90 plays an important role in sepsisassociated endothelial barrier dysfunction. endothelial permeability; radicicol; endotoxin; acute respiratory distress syndrome

BACKGROUND: Damp building--related respiratory illnesses are an important public health issue. OBJECTIVE: We compared three respiratory case groups defined by questionnaire responses [200 respiratory cases, 123 of the respiratory cases [...]

Endotoxin [or lipopolysaccharide (LPS)] increases levels of superoxide in blood vessels and impairs vasomotor function. Angiotensin II plays an important role in the generation of superoxide in several disease states, including hypertension and heart failure. The goal of this study was to determine whether the activation of the renin-angiotensin system contributes to oxidative stress and endothelial dysfunction after endotoxin. We examined the effects of enalapril (an angiotensin-converting enzyme inhibitor) or L-158809 (an angiotensin receptor blocker) on increases of superoxide and vasomotor dysfunction in mice treated with LPS. C57BL/6 mice were treated with either enalapril (60 mg x [kg.sup.-1] x [day.sup.-1]) or L-158809 (30 mg x [kg.sup.-1] x [day.sup.-1]) for 4 days. After the third day, LPS (10-20 mg/kg) or vehicle was injected intraperitoneally, and one day later, vasomotor function of the aorta was examined in vitro. After precontraction with [PGF.sub.2[alpha]], the maximal responses to sodium nitroprusside were similar in the aorta from normal and LPS-treated mice. In contrast, the relaxation to acetylcholine was impaired after LPS (54 [+ or -] 5% at [10.sup.-5], mean [+ or -] SE) compared with vessels treated with vehicle (88 [+ or -] 1%; P < 0.05). Enalapril improved (P < 0.05) relaxation in response to acetylcholine to 81 [+ or -] 6% after LPS. L-158809 also improved relaxation in response to acetylcholine to 77 [+ or -] 4% after LPS. Superoxide (measured with lucigenin and hydroethidine) was increased (P < 0.05) in aorta after LPS, and levels were reduced (P < 0.05) following enalapril and L-158809. Thus, after LPS, enalapril and L-158809 reduce superoxide levels and improve relaxation to acetylcholine in the aorta. The findings suggest that activation of the renin-angiotensin system contributes importantly to oxidative stress and endothelial dysfunction after endotoxin. endotoxin; enalapril; L-158809; endothelial function

To identify new components that regulate the inflammatory cascade during sepsis, we characterized the functions of myeloid-related protein-8 (Mrp8, S100A8) and myeloid-related protein-14 (Mrp14, S100A9), two abundant cytoplasmic proteins of phagocytes. We now demonstrate that mice lacking Mrp8-Mrp14 complexes are protected from endotoxin-induced lethal shock and Escherichia coli-induced abdominal sepsis. Both proteins are released during activation of phagocytes, and Mrp8-Mrp14 complexes amplify the endotoxin-triggered inflammatory responses of phagocytes. Mrp8 is the active component that induces intracellular translocation of myeloid differentiation primary response protein 88 and activation of interleukin-1 receptor-associated kinase-1 and nuclear factor-[kappa]B, resulting in elevated expression of tumor necrosis factor-[alpha] (TNF-[alpha]). Using phagocytes expressing a nonfunctional Toll-like receptor 4 (TLR4), HEK293 cells transfected with TLR4, CD14 and MD2, and by surface plasmon resonance studies in vitro, we demonstrate that Mrp8 specifically interacts with the TLR4-MD2 complex, thus representing an endogenous ligand of TLR4. Therefore Mrp8-Mrp14 complexes are new inflammatory components that amplify phagocyte activation during sepsis upstream of TNF[alpha]-dependent effects., Author(s): Thomas Vogl [1]; Klaus Tenbrock [2, 3]; Stephan Ludwig [4]; Nadja Leukert [1]; Christina Ehrhardt [4]; Marieke A D van Zoelen [5]; Wolfgang Nacken [1]; Dirk Foell [2, 3]; [...]

A major toxicity of the cancer chemotherapeutic agent cisplatin is acute renal failure. Sepsis is a common cause of acute renal failure in humans and patients who receive cisplatin are at increased risk for sepsis. Accordingly, this study examined the interactions between cisplatin and endotoxin in vivo with respect to renal function and cytokine production. Mice were treated with either a single dose of cisplatin or two doses of LPS administered 24 h apart, or both agents in combination. Administration of 10 mg/kg cisplatin had no effect on blood urea nitrogen or creatinine levels throughout the course of the study. LPS resulted in a modest rise in blood urea nitrogen at 24 and 48 h, which returned to normal by 72 h. In contrast, mice treated with both cisplatin and LPS developed severe renal failure and an increase in mortality. Urine, but not serum, TNF-[alpha] levels showed a synergistic increase by cisplatin and LPS. Urinary IL-6, MCP-1, KC, and GM-CSF also showed a synergistic increase with cisplatin+LPS treatment. The renal dysfunction induced by cisplatin+LPS was completely dependent on TLR4 signaling and partially dependent on TNF-[alpha] production. Increased cytokine production was associated with a moderate increase in infiltrating leukocytes which was not different between cisplatin+LPS and LPS alone. These results indicate that cisplatin and LPS act synergistically to produce nephrotoxicity which may involve proinflammatory cytokine production. tumor necrosis factor-[alpha]; Toll-like receptor 4; sepsis; acute renal failure; cytokines doi:10.1152/ajprenal.00158.2007

Gram-negative sepsis with release of endotoxin is a frequent cause of cachexia that develops partly because of resistance to growth hormone (GH) with reduced insulin-like growth factor-I (IGF-I) expression. We set out to more fully characterize the mechanisms for the resistance and to determine whether in addition to a defect in the janus kinase 2 (JAK2)-signal transducer and activator of transcription (STAT) 5b pathway, required for GH-induced IGF-I expression, there might also be a more distal defect. Conscious rats were given endotoxin and studied 4 h later. In liver of these animals, GH-induced JAK2 and STAT5 phosphorylation was impaired and appeared to be caused, at least in part, by a marked increase in hepatic tumor necrosis factor-[alpha] and interleukin-6 mRNA expression accompanied by elevated levels of inhibitors of GH signaling, namely cytokine-inducible suppressors of cytokine signaling-1 and -3 and cytokine-inducible SH2 protein (CIS). Nuclear phosphorylated STAT5b levels were significantly depressed to 61% of the control values and represent a potential cause of the reduced GH-induced IGF-I expression. In addition, binding of phosphorylated STAT5b to DNA was reduced to an even greater extent and averaged 17% of the normal control value. This provides a further explanation for the impaired IGF-I gene transcription. Interestingly, when endotoxin-treated rats were treated with GH, there was a marked increase in proinflammatory cytokine gene expression in the liver. If such a response were to occur in humans, this might provide a partial explanation for the adverse effect of GH treatment reported in critically ill patients. lipopolysaccharide; insulin-like growth factor I; inflammation; cytokines doi: 10.1152/ajpendo.00581.2006

Over the last three decades, experiments in several mammalian species have shown that the febrile response to bacterial endotoxin is attenuated late in pregnancy. More recent evidence has established that the expression of nitric oxide synthase (NOS) enzymes is increased in the brain late in pregnancy. The current study investigated the possible role of brain nitric oxide in mediating the phenomenon of fever suppression. Core body temperature ([T.sub.b]) of near-term pregnant rats (day 19 and 20) was measured following inhibition of brain NOS and intraperitoneal injection of LPS (50 [micro]g/kg); they were compared with both day 15 pregnant and virgin animals. Intracerebroventricular injection with an inhibitor of NOS, [N.sup.G]-monomethyl-L-arginine citrate (L-NMMA; 280 [micro]g), in near-term pregnant rats restored the febrile response to LPS. As expected, near-term dams that received intracerebroventricular vehicle + IP LPS did not increase [T.sub.b], in contrast to the 1.0 [+ or -] 0.2[degrees]C rise in [T.sub.b] in dams treated with ICV L-NMMA + IP LPS (P < 0.01). In virgin females and day 15 pregnant controls receiving this treatment, the increases in [T.sub.b] were 1.5 [+ or -] 0.3[degrees]C and 1.6 [+ or -] 0.4[degrees]C, respectively. Thus, blockade of brain NOS restored the febrile response to LPS in near-term dams; at 5 h postinjection, [T.sub.b] was 60-70% of that observed in virgins and day 15 pregnant animals. Intracerebroventricular L-NMMA alone did not induce a significant change in [T.sub.b] in any group. These results suggest that the mechanism underlying the suppression of the febrile response in near-term pregnancy is mediated by nitric oxide signaling in the brain. nitric oxide synthase; [N.sup.G]-monomethyl-L-arginine citrate; lipopolysaccharide; core body temperature; thermoregulation doi: 10.1152/ajpregu.00032.2007

We investigated whether LPS-induced hypothermia develops in a serotype-specific manner in biotelemetered conscious rats. Two different Escherichia coli serotypes of LPSs were injected at a dose of 250 [micro]g/kg ip. E. coli O55:B5 LPS elicited an initial hypothermia and subsequent fever, but E. coli O111:B4 LPS caused more potent monophasic hypothermia. Serum tumor necrosis factor (TNF)-[alpha] levels were dramatically elevated at the initial phase of the hypothermia induced by both LPSs. This elevation tended to subside at the nadir of E. coli O55:B5 LPS-induced response but progressively increased at the nadir of E. coli O111:B4 LPS hypothermia. Serum IL-10 levels were moderately elevated at the initial phase of the hypothermia and persisted at the same level at the nadir of each LPS-induced response. No change was observed at the serum IL-18 levels. A selective cyclooxygenase (COX)-1 enzyme inhibitor, valeryl salicylate (20 mg/kg sc), abolished the hypothermia without any effect on the elevated cytokine levels. Another COX-1-selective inhibitor, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC-560; 1 mg/kg sc) inhibited hypothermic responses as well. Meanwhile, cytokine levels were also reduced by SC-560 treatment. These findings suggest that LPS-induced hypothermia may have serotype-specific characteristics in rats. E. coli O111:B4 LPS has more potent hypothermic activity than E. coli O55:B5 LPS; that may presumably be related to its higher or sustained capability to release antipyretic cytokines, such as TNF-et. COX-1 enzyme may be involved in the generation of the hypothermia, regardless of the type of LPS administered. acute-phase response; fever; endotoxin; antipyretic cytokines; cyclooxygenase-1 selective inhibitors doi:10.1152/ajpregu.00786.2006

Occupational hygiene was investigated in two Finnish combined-drum-and-tunnel composting plants, Plant A (composting sewage sludge) and Plant B (composting source-separated biowaste), in 1998-2000. The concentrations of viable mesophilic and thermophilic microbes (fungi, bacteria, and actinomycetes), the total number of microbes (viable + dead), endotoxin concentrations, and noise level were determined for each plant. In addition, dust concentrations were investigated in Plant B. In Plant A, working areas were aired before the measurements were taken. Differences in microbe concentrations between the plants were statistically significant. There were more problems with microbes in Plant B, where the working areas were not aired. Also, endotoxins were a problem in Plant B; the threshold value of 200 endotoxin units per cubic meter was exceeded in several measurements., Introduction Partly voluntarily and partly by order of the waste act, Finland is moving toward source separation of kitchen biowastes. At present, about 70 percent of Finns are sorting their [...]

Soluble guanylyl cyclase (sGC) is a cGMP-generating enzyme implicated in the control of smooth muscle tone that also regulates platelet aggregation. Moreover, sGC activation has been shown to reduce leukocyte adherence to the endothelium. Herein, we investigated the expression of sGC in a murine model of LPS-induced lung injury and evaluated the effects of sGC inhibition in the context of acute lung injury (ALI). Lung tissue sGC [alpha] and [beta] subunit protein levels were determined by Western blot and immunohistochemistry, and steady-state mRNA levels for the [beta] subunit were assessed by real-time PCR. LPS inhalation resulted in a decrease in [beta] mRNA levels, as well as a reduction in both sGC subunit protein levels. Decreased [alpha]1 and [beta]1 expression was observed in bronchial smooth muscle and epithelial cells. TNF-[alpha] was required for the LPS-triggered reduction in sGC protein levels, as no change in [alpha]l and [beta]1 levels was observed in TNF-[alpha] knockout mice. To determine the effects of sGC blockade in LPS-induced lung injury, mice were exposed to 1H-[1,2,4]oxodiazolo [4,3-a]quinoxalin-1-one (ODQ) prior to the LPS challenge. Such pretreatment led to a further increase in total cell number (mainly due to an increase in neutrophils) and protein concentration in the bronchoalveoalar lavage fluid; the effects of ODQ were reversed by a cell-permeable cGMP analog. We conclude that sGC expression is reduced in LPS-induced lung injury, while inhibition of the enzyme with ODQ worsens lung inflammation, suggesting that sGC exerts a protective role in ALI. endotoxin; guanosine 3',5'-cyclic monophosphate; acute lung injury

Byline: Durgul Ozdemir (1), Nazan Uysal (2), Kazim Tugyan (3), Sevil Gonenc (2), Osman Acikgoz (2), Ilkay Aksu (2), Hasan Ozkan (1) Keywords: Endotoxemia; Infant rats; Lipid peroxidation; Antioxidant enzymes; Melatonin; Intestinal apoptosis Abstract: Objective To evaluate the effect of melatonin on the intestinal apoptosis along with oxidative damage in endotoxemic infant rats. Design and setting Prospective animal study in a university-based experimental research laboratory. Subjects and interventions Wistar albino 7-day-old rat pups (na-=a-21). The animals were randomized into three experimental groups: (1) controls (2) endotoxemia (3) endotoxemia treated with melatonin (10a-mg/kg). Endotoxemia was induced in rats by intraperitoneal injection of lipopolysaccharide (Escherichia coli serotype 0111:B4 3a-mg/kg). Measurements and results Four hours after LPS injection, the antioxidant enzyme activities, including superoxide dismutase (SOD), glutathione peroxidase (GPx), and thiobarbituric acid reactive substance (TBARS) levels as an indicator of lipid peroxidation, were determined. Intestinal apoptosis was assessed by hematoxylin-eosin staining and terminal deoxynucleotide transferase-mediated fluorescein-dUTP nick end labeling. The administration of melatonin into endotoxemic rats prevented the increase in the TBARS levels, and increased the activities of antioxidant enzymes and attenuated apoptotic cell death in both intestinal epithelium and lamina propria. Conclusions Melatonin diminished the intestinal oxidative stress and apoptotic damage induced by endotoxemia in infant rats. Author Affiliation: (1) Department of Pediatrics, School of Medicine, Dokuz Eylul University, Inciralti, 35340, Izmir, Turkey (2) Department of Physiology, School of Medicine, Dokuz Eylul University, Inciralti, 35340, Izmir, Turkey (3) Department of Histology and Embryology, School of Medicine, Dokuz Eylul University, Inciralti, 35340, Izmir, Turkey Article History: Registration Date: 21/11/2006 Received Date: 20/01/2006 Accepted Date: 20/11/2006 Online Date: 22/12/2006

A small percentage of pathologically obese subjects with fatty livers develop histological signs of necroinflammation and fibrosis, suggesting a variety of cofactors in the pathogenesis of obesity-related liver diseases including nonalcoholic steatohepatitis. Since several observations have linked bacterial endotoxins to liver damage, the aim of this study was to determine the effect of obesity on intestinal mucosal integrity and portal blood endotoxemia in two strains of obese mice: leptin-deficient (ob/ob) and hyperleptinemic (db/db) mice. Murine intestinal mucosal barrier function was assessed using a Ussing chamber, whereas ileum tight junction proteins were analyzed by immunocytochemistry and Western blot analysis. Circulating proinflammatory cytokines and portal blood endotoxin levels were measured by ELISA and the limulus test, respectively. The inflammatory and fibrogenic phenotype of murine hepatic stellate cells (HSCs) was determined by ELISA and quantitative RT-PCR. Ob/ob and db/db mice showed lower intestinal resistance, profoundly modified distribution of occludin and zonula occludens-1 in the intestinal mucosa, and higher circulating levels of inflammatory cytokines and portal endotoxemia compared with lean control mice. Moreover, HSCs isolated from ob/ob and db/db mice showed higher membrane CD14 mRNA levels and more pronounced lipopolysaccharide-induced proinflammatory and fibrogenic responses than HSCs from lean animals. In conclusion, genetically obese mice display enhanced intestinal permeability leading to increased portal endotoxemia that makes HSCs more sensitive to bacterial endotoxins. We suggest that in metabolic syndrome, patients may likewise have a greater intestinal mucosa permeability and increased lipopolysaccharide levels in portal blood that can contribute to the liver inflammatory damage. Toll-like receptors; endotoxins; fibrosis; tight junction proteins; hepatic stellate cells

A study identified, cloned and sequenced a 2.2-kb fragment containing a replicon from pBtoxis, the large plasmid that encodes the insecticidal endotoxins of Bacillus thuringiensis subsp. israelensis. This fragment consists of cis elements, including iterons, found in replication origins of other large plasmids and suggests that pBtoxis replicates by a type A theta mechanism.

Acute renal failure (ARF) in septic patients drastically increases the mortality to 50-80%. Sepsis induces several proinflammatory cytokines including tumor necrosis factor-[alpha] (TNF-[alpha]), a major pathogenetic factor in septic ARF. Pentoxifylline has several functions including downregulation of TNF-[alpha] and endothelia-dependent vascular relaxation. We hypothesized that pentoxifylline may afford renal protection during endotoxemia either by downregulating TNF-[alpha] and/or by improving endothelial function. In wild-type mice, pentoxifylline protected against the fall in glomerular filtration rate (GFR; 105.2 [+ or -] 6.6 vs. 50.2 [+ or -] 6.6 [micro]l/min, P < 0.01) at 16 h of LPS administration (2.5 mg/kg ip). This renal protective effect of pentoxifylline was associated with an inhibition of the rise in serum TNF-[alpha] (1.00 [+ or -] 0.55 vs. 7.02 [+ or -] 2.40 pg/ml, P < 0.05) and serum IL-l[beta] (31.3 [+ or -] 3.6 vs. 53.3 [+ or -] 5.9 pg/ml, P < 0.01) induced by LPS. Pentoxifylline also reversed the LPS-related increase in renal iNOS and ICAM-1 and rise in serum nitric oxide (NO). Enhanced red blood cell deformability by pentoxifylline may have increased shear rate and upregulated eNOS. Studies were therefore performed in eNOS knockout mice. The renal protection against endotoxemia with pentoxifylline was again observed as assessed by GFR (119.8 [+ or -] 18.0 vs. 44.5 [+ or -] 16.2 [micro]l/min, P < 0.05) and renal blood flow (0.86 [+ or -] 0.08 vs. 0.59 [+ or -] 0.05 ml/min, P < 0.05). Renal vascular resistance significantly decreased with the pentoxifylline (91.0 [+ or -] 5.8 vs. 178.0 [+ or -] 7.6 mmHg x [ml.sup.-1] x [min.sup.-1], P < 0.01). Thus pentoxifylline, an FDA-approved drug, protects against endotoxemia-related ARF and involves a decrease in serum TNF-[alpha], IL-1[beta], and NO as well as a decrease in renal iNOS and ICAM-1. sepsis; tumor necrosis factor

Acute renal failure (ARF) markedly sensitizes mice to endotoxin (LPS), as evidenced by exaggerated renal cytokine/chemokine production. This study sought to further characterize this state by testing the following: 1) does anti-inflammatory heme oxygenase-1 (HO-1) upregulation in selected ARF models prevent this response? 2) Is the ARF hyperresponsive state specifically triggered by LPS? 3) Does excess iNOS activity/protein nitrosylation participate in this phenomenon? and 4) are upregulated Toll receptors involved? Mice with either 1) rhabdomyolysis-induced ARF (massive HO-1 overexpression), 2) cisplatin nephrotoxicity, 3) or HO-1 inhibition (Sn protoporphyrin) were challenged with either LPS (a TLR4 ligand), lipoteichoic acid (LTA; a TLR2 ligand), or vehicle. Two hours later, renal and plasma TNF-[alpha]/mRNA, MCP-1/ mRNA, renal nitrotyrosine/iNOS mRNA, and plasma cytokines were assessed. Renal TLR4 was gauged by mRNA and Western blot analysis. Both ARF models markedly hyperresponded to both LPS and LTA, culminating in exaggerated TNF-[alpha], MCP-1, and iNOS/ nitrotryosine increments. This was despite the fact that HO-1 exerted anti-inflammatory effects. TLR4 levels were either normal (cisplatin), or markedly depressed (~50%; rhabdomyolysis) in the ARF kidneys, despite the LPS hyperresponsive state. 1) The ARF kidney can hyperrespond to chemically dissimilar Toll ligands; 2) HO-1 does not prevent this response; 3) excess NO/protein nitrosylation can result; and 4) this hyperresponsiveness can be expressed with either normal or reduced renal TLR4 expression. This suggests that diverse signaling pathways may be involved. endotoxin; lipoteichoic acid; iNOS; tumor necrosis factor-[alpha]; monocyte chemoattractant protein-1 doi:10.1152/ajprenal.00072.2006

Neutrophil extravasation from sinusoids is a critical step for acute inflammatory tissue injury. However, the role of sinusoidal endothelial cells (SECs) in this process remains unclear. Matrix metalloproteinases (MMPs) have been shown to involve gap formation in SECs in several liver diseases. Therefore, the present study examined SEC modifications elicited by galactosamine (Ga1)/endotoxin (ET). Treatment of male C3Heb/FeJ mice with Gal/ET or Gal/TNF caused the formation of numerous gaps in SECs at 4 h when no neutrophil extravasation occurred. Six hours after Gal/ET or Gal/TNF treatment, blood elements started to penetrate to the extrasinusoidal space through large gaps. Treatment with ET alone caused sinusoidal neutrophil accumulation but no gap formation, neutrophil extravasation, or hemorrhage. Gal/ET treatment increased hepatic MMP-2 and MMP-9 mRNA expression (6.7- and 11-fold, respectively). Pretreatment with 2-[(4-biphenylsultbnyl) amino]-3-phenyl-propionic acid, an MMP-2/MMP-9 inhibitor (5 mg/kg), minimized gap formation after Gal/ET and Gal/TNF treatment. The MMP inhibitor reduced injury only in the Gal/ET model mainly due to reduced TNF formation. The MMP inhibitor attenuated sinusoidal neutrophil accumulation at 6 h but failed to attenuate Gal/TNF-induced liver injury at 7 h due to excessive apoptosis. These results suggest that Gal/ET or Gal/TNF activates MMPs, which are responsible for SEC gap formation. Although the initial appearance of gap formation is independent of neutrophils, the gaps allow initial contact of neutrophils with damaged hepatocytes. In addition, MMP activation promotes neutrophil accumulation in sinusoids. neutrophil; transmigration; matrix metalloproteinase; apoptosis; tumor necrosis factor doi:10.1152/ajpgi.00312.2005

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

The purpose of the present study was to determine whether catabolic stimuli that induce muscle atrophy alter the muscle mRNA abundance of insulin-like growth factor binding protein (IGFBP)-4 and -5, and if so determine the physiological mechanism for such a change. Catabolic insults produced by endotoxin (LPS) and sepsis decreased IGFBP-5 mRNA time- and dose-dependently in gastrocnemius muscle. This reduction did not result from muscle disuse because hindlimb immobilization increased IGFBP-5. Continuous infusion of a nonlethal dose of tumor necrosis factor-[alpha] (TNF-[alpha]) decreased IGFBP-5 mRNA 70%, whereas pretreatment of septic rats with a neutralizing TNF binding protein completely prevented the reduction in muscle IGFBP-5. The addition of LPS or TNF-[alpha] to cultured [C.sub.2][C.sub.12] myoblasts also decreased IGFBP-5 expression. Although exogenously administered growth hormone (GH) increased IGFBP-5 mRNA 2-fold in muscle from control rats, muscle from septic animals was GH resistant and no such elevation was detected. In contrast, exogenous administration of IGF-I as part of a binary complex composed of IGF-I/IGFBP-3 produced comparable increases in IGFBP-5 mRNA in both control and septic muscle. Concomitant determinations of IGF-I mRNA content revealed a positive linear relationship between IGF-I and IGFBP-5 mRNA in the same muscle in response to LPS, sepsis, TNF-[alpha], and GH treatment. Although dexamethasone decreased muscle IGFBP-5, pretreatment of rats with the glucocorticoid receptor antagonist RU486 did not prevent the sepsis-induced decrease in IGFBP-5 mRNA. In contrast, muscle IGFBP-4 mRNA abundance was not significantly altered by LPS, sepsis, or hindlimb immobilization. In summary, these data demonstrate that various inflammatory insults decrease muscle IGFBP-5 mRNA, without altering IGFBP-4, by a TNF-dependent glucocorticoid-independent mechanism. Finally, IGF-I appears to be a dominant positive regulator of IGFBP-5 gene expression in muscle under both normal and catabolic conditions. endotoxin; insulin-like growth factor binding protein-3/insulin-like growth factor-I binary complex; glucocorticoids; rats

Respiratory epithelial cells play a crucial role in the inflammatory response in endotoxin-induced lung injury, an experimental model for acute lung injury. To determine the role of epithelial cells in the upper respiratory compartment in the inflammatory response to endotoxin, we exposed tracheobronchial epithelial cells (TBEC) to lipopolysaccharide (LPS). Expression of inflammatory mediators was analyzed, and the biological implications were assessed using chemotaxis and adherence assays. Epithelial cell necrosis and apoptosis were determined to identify LPS-induced cell damage. Treatment of TBEC with LPS induced enhanced protein expression of cytokines and chemokines (increases of 235-654%, P < 0.05), with increased chemotactic activity regarding neutrophil recruitment. Expression of the intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-I (VCAM-I) was enhanced by 52-101% (P < 0.0001). This upregulation led to increased adhesion of neutrophils, with >95% adherence to TBEC after LPS stimulation, which could be blocked by either ICAM-1 (69%) or VCAM-1 antibodies (55%) (P < 0.05). Enhanced neutrophil-induced necrosis of TBEC was observed when TBEC were exposed to LPS. Reduced neutrophil adherence by ICAM-1 or VCAM-1 antibodies resulted in significantly lower TBEC death (52 and 34%, respectively, P < 0.05). Therefore, tight adherence of neutrophils to TBEC appears to promote epithelial cell killing. In addition to indirect effector cell-induced TBEC death, direct LPS-induced cell damage was seen with increased apoptosis rate in LPS-stimulated TBEC (36% increase of caspase-3, P < 0.01). These data provide evidence that LPS induces TBEC killing in a necrosis- and apoptosis-dependent manner. lipopolysaccharide; inflammatory mediators; leukocytes

Given the high morbidity and mortality rates associated with pulmonary inflammation in sepsis, there is a pressing need for new therapeutic modalities to prevent acute respiratory distress. The enzyme heine oxygenase-1 (HO-1) provides potent cytoprotection against lung injury; however, the mechanism by which it does so is unclear. HO-1 catabolizes heme into biliverdin (BV), which is rapidly converted to bilirubin by BV reductase. We tested the hypothesis that BV administration could substitute for the effects observed with HO-1. Using the well-described rat model of LPS-induced shock, we demonstrate that exposure to BV imparts a potent defense against lethal endotoxemia systemically, as well as in the lungs, and effectively abrogates the inflammatory response. BV administration before a lethal dose of LPS leads to a significant improvement in long-term survival: 87% vs. 20% in sham-treated controls. BV treatment suppressed LPS-induced increases in lung permeability and lung alveolitis and significantly reduced serum levels of the LPS-induced proinflammatory cytokine IL-6. Moreover, bilirubin administered just alter LPS also abrogated lung inflammation. BV treatment also augmented expression of the anti-inflammatory cytokine IL-10. Similar effects on production were observed with BV treatment in vitro in mouse lung endothelial cells and RAW 264.7 macrophages treated with LPS. In conclusion, these data demonstrate that BV can modulate the inflammatory response and suppress pathophysiological changes in the lung and may therefore have therapeutic application in inflammatory disease states of the lung. biliverdin reductase; oxidative stress; inflammation; heme oxygenase-1

Clinical and laboratory data indicate that the liver plays an important role in the incidence, pathogenesis, and outcome of acute lung injury/acute respiratory distress syndrome. To distinguish direct effects of endotoxin on the lungs from liver-dependent effects during the early phase of the response to endotoxemia, we used an in situ perfused piglet preparation in which only the ventilated lung or both the lung and liver could be included in a blood perfused circuit. We monitored pulmonary vascular resistance, oxygenation, neutrophil count, lung edema as reflected by wet-dry weights of lung tissue, perfusate concentrations of TNF-[alpha], IL-6, and 8-isoprostane (a marker of oxidative stress), and activation of the transcription factor (NF-[kappa]B) in lung tissue before and for 2 h after endotoxin. When only the lung was perfused, endotoxin caused pulmonary hypertension and neutropenia; but oxygenation was maintained; TNF-[alpha], IL-6, and 8-isoprostane levels were minimally elevated; and there was no lung edema. When both the liver and lung were perfused, endotoxin caused marked hypoxemia, large increases in perfusate TNF-[alpha], IL-6, and 8-isoprostane concentrations, and severe lung edema. NF-[kappa]B activation in the lung was greatest when the liver was in the perfusion circuit. We conclude that the direct effects of endotoxemia on the lungs include vasoconstriction and leukocyte sequestration, but not lung injury. Intense activation of the inflammatory response and oxidative injury that results in pulmonary edema and hypoxemia (acute lung injury) requires interaction of the lungs with the liver. liver-lung interaction; oxidative stress; acute respiratory distress syndrome

Gastrointestinal injury usually starts in the superficial mucosa. We investigated whether leukocyte-endothelial interactions were greater in the gastrointestinal mucosa than the submucosa and muscularis in control tissue and after upregulation of adhesion molecules with endotoxin and after chemical insult with nonsteroidal anti-inflammatory drugs. Inactin-anesthetized rats were given either endotoxin, flurbiprofen, or nitric oxide (NO)-flurbiprofen, after which ICAM-1 and P-selectin expression was measured with the dual-label antibody technique. Leukocyte-endothelial interactions in the different gastric layers were assessed after endotoxin using intravital microscopy. Endotoxin caused a two- to threefold increase in ICAM-1 expression in the stomach and duodenum. There was, however, a gradient in expression across the gut wall with the level of expression in the superficial mucosa (per g) being only 10-25% of that in the deeper layers in both control and endotoxin-treated animals. Constituitive expression of P-selectin in control animals was barely detectable. Endotoxin caused a modest increase in mucosal P-selectin but a very significant increase in the deeper layers. Flurbiprofen caused a slight upregulation of ICAM-1 in the gastric mucosa and duodenum, whereas NO-flurbiprofen had no affect on expression. Intravital microscopy revealed no adhesion and virtually no leukocyte rolling in the vessels of the gastric mucosa despite endotoxin treatment. There was, however, some adhesion and significant leukocyte rolling in the submucosa and muscularis. Thus the superficial gastric and duodenal mucosal microcirculations have a much lower density of ICAM-1 and P-selectin and less leukocyte-endothelial interactions than occurs in the deeper layers of the gut wall even during stimulated upregulation with endotoxin. intracellular adhesion molecule 1; P-selectin; endotoxin; flurbiprofen; intravital microscopy

Heat shock modulates cellular proinflammatory responses, and we have been interested in elucidating the mechanisms that govern this modulation. The dual specific phosphatase, MAP kinase phosphatase-1 (MKP-1), is an important modulator of cellular inflammatory responses, and we recently reported that heat shock increases expression of MKP-1. Herein we sought to elucidate the mechanisms by which heat shock modulates MKP-1 gene expression. Subjecting RAW264.7 macrophages to heat shock increased MKP-1 gene expression in a time-dependent manner. Transfection with a wild-type routine MKP-1 promoter luciferase reporter plasmid demonstrated that heat shock activates the MKP-1 promoter. When the reporter plasmid was transfected into heat shock factor-1 (HSF-1)-null fibroblasts, the MKP-1 promoter was activated in response to heat shock in a manner similar to that of wild-type fibroblasts with intact HSF-1. Site-directed mutagenesis of two potential heat shock elements in the MKP-1 promoter demonstrated that both sites are required for basal promoter activity. mRNA stability assays demonstrated that heat shock increased MKP-1 mRNA stability compared with cells maintained at 37[degrees]C. Inhibition of p38 MAP kinase activity inhibited heat shock-mediated expression of MKP-1. These data demonstrate that heat shock regulates MKP-1 gene expression at both the transcriptional and posttranscriptional levels. Transcriptional mechanisms are HSF-1 independent but are dependent on putative heat shock elements in the MKP-1 promoter. Posttranscriptional mechanisms involve increased stability of MKP-1 mRNA that is partially dependent on p38 MAP kinase activity. These data demonstrate another potential mechanism by which heat shock can modulate inflammation-related signal transduction. endotoxin; phosphatase; inflammation; heat shock factor; p38

The specific systems and mechanisms affected by sleep deprivation that may perpetuate disease processes in humans still are speculative. In laboratory rats, prolonged sleep deprivation induces a state marked by abnormal control over indigenous bacteria that results in transient infections of internal tissues and eventual lethal septicemia. The present studies investigated changes in blood, serum, and bone marrow parameters that may provide diagnostic clues to immunopathology. Prolonged sleep deprivation was produced in rats by the disk-over-water method, a well-established and selective means that does not interfere with normal waking behaviors. Measurements included bone and blood differential white blood cell counts, multiple serum cytokines and chemokines, several major Ig classes and subclasses, and serum endotoxin concentrations. The results indicated mild, regenerative neutrophilia in sleep-deprived rats, initially accompanied by immature neutrophils and later by monocytosis. The corresponding serum cytokine profile revealed an evolving proinflammatory state, particularly by high incidence of interleukin-1[beta], implicating mononuclear phagocytes and resident tissue cells as main intermediary sources. In addition, multiple serum Ig classes were increased by sleep deprivation without experimental administration of an exogenous antigen. Despite this immune activation, there was failure to eradicate invading bacteria and toxins, suggesting competing anti-inflammatory processes or interference with immune effector functions during sleep deprivation. Nearly all of the immune-related events that emerged as responses to sleep deprivation have been implicated as etiological or provocative factors in other disease processes and may provide means by which sleep deprivation as a risk factor in disease may become understood. interleukin-1[beta]; inflammation; endotoxin

Sublethal exposure to Escherichia coli endotoxin [lipopolysaccharide (LPS)] attenuates the lethal effects of subsequent insults associated with oxidative stress, such as higher LPS dose, septic peritonitis, and ischemia. Because administration of the antioxidant ascorbate protects against these same insults and injection of dehydroascorbic acid (DHAA) protects against ischemia, the hypothesis that sublethal LPS increases endogenous ascorbate concentration and recycling (i.e., synthesis from DHAA) was tested. Injection of LPS [5 x [10.sup.6] endotoxin units/kg body weight, i.p.] in mice caused a temporary inhibition of food intake, which was significant by 20 h and recovered within 3 d. LPS increased ascorbate concentration in adrenal gland, heart, kidney, and liver. LPS had similar effects in wild-type and Sic23a2+/- mice despite the latter's deficiency in the ascorbate transporter SVCT2. In liver, the ascorbate response to LPS was not accompanied by change in glutathione concentration. LPS decreased gulonolactone oxidase activity, which is rate-limiting for de novo synthesis of ascorbate from glucose, but increased the rate of DHAA reduction to ascorbate. In conclusion, sublethal endotoxin increases ascorbate recycling in liver and ascorbate concentration in liver, adrenal gland, heart, and kidney. The enhanced rate of ascorbate production from DHAA may protect these organs against the reactive oxygen species produced by subsequent, potentially lethal challenges. KEY WORDS: * ascorbate * dehydroascorbic acid * lipopolysaccharide * gulonolactone oxidase * transport

Studies are conducted to demonstrate a tight interaction between plasma gelsolin, a highly conserved human protein, and lipopolysaccharide (LPS) that affects both gelsolin's actin binding function and some aspects of the effects of LPS on cells. The results show a strong effect of LPS on plasma gelsolin function and suggest that some effects of endotoxin in vivo may be mediated or inhibited by plasma gelsolin.