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Mucosal immunity and host defense Chemosensory brush cells are found at nearly all mucosal sites, including the nose, trachea, intestine, gallbladder, urethra, and pancreas, where they are referred to as [...], Airway epithelial cells, once considered a simple barrier layer, are now recognized as providing an active site for antigen sensing and immune response initiation. Most mucosal sites contain chemosensory epithelial cells, rare and specialized cells gaining recognition for their unique functions in sensing and directing the immune response symphony. In this issue of the JCI, Hollenhorst, Nandigama, et al. demonstrated that tracheal chemosensory brush cells detected bitter-tasting substances, including quorum-sensing molecules (QSMs) generated by pathogenic Pseudomonas aeruginosa. The authors used various techniques, including genetic deletion of brush cells, genetic manipulation of brush cell signaling, deletion of sensory neurons, in vivo imaging, and infection models with P. aeruginosa, to show that QSMs increased vascular permeability and innate immune cell influx into the trachea. These findings link the recognition of bacterial QSMs to the innate immune response in the airways, with translational implications for airway inflammation and infectious pathology.

Introduction Constant exposure of the airways to inhaled particles and pathogens requires efficient early immune responses protecting against infections. These processes are largely mediated by the airway epithelium that is [...], Constant exposure of the airways to inhaled pathogens requires efficient early immune responses protecting against infections. How bacteria on the epithelial surface are detected and first-line protective mechanisms are initiated are not well understood. We have recently shown that tracheal brush cells (BCs) express functional taste receptors. Here we report that bitter taste signaling in murine BCs induces neurogenic inflammation. We demonstrate that BC signaling stimulates adjacent sensory nerve endings in the trachea to release the neuropeptides CGRP and substance P that mediate plasma extravasation, neutrophil recruitment, and diapedesis. Moreover, we show that bitter tasting quorum-sensing molecules from Pseudomonas aeruginosa activate tracheal BCs. BC signaling depends on the key taste transduction gene TrpmS, triggers secretion of immune mediators, among them the most abundant member of the complement system, and is needed to combat P. aeruginosa infections. Our data provide functional insight into firstline defense mechanisms against bacterial infections of the lung.

Author(s): Ruaidhrí Jackson [sup.1] , Lina Kroehling [sup.1] , Alexandra Khitun [sup.2] , Will Bailis [sup.1] , Abigail Jarret [sup.1] , Autumn G. York [sup.1] , Omair M. Khan [sup.1] [...], The annotation of the mammalian protein-coding genome is incomplete. Arbitrary size restriction of open reading frames (ORFs) and the absolute requirement for a methionine codon as the sole initiator of translation have constrained the identification of potentially important transcripts with non-canonical protein-coding potential.sup.1,2. Here, using unbiased transcriptomic approaches in macrophages that respond to bacterial infection, we show that ribosomes associate with a large number of RNAs that were previously annotated as 'non-protein coding'. Although the idea that such non-canonical ORFs can encode functional proteins is controversial.sup.3,4, we identify a range of short and non-ATG-initiated ORFs that can generate stable and spatially distinct proteins. Notably, we show that the translation of a new ORF 'hidden' within the long non-coding RNA Aw112010 is essential for the orchestration of mucosal immunity during both bacterial infection and colitis. This work expands our interpretation of the protein-coding genome and demonstrates that proteinaceous products generated from non-canonical ORFs are crucial for the immune response in vivo. We therefore propose that the misannotation of non-canonical ORF-containing genes as non-coding RNAs may obscure the essential role of a multitude of previously undiscovered protein-coding genes in immunity and disease.In mouse macrophages, a range of short and non-ATG-initiated open reading frames that can generate proteins are identified, one of which is shown to be essential for host immunity to enteric mucosal infection and inflammation.

Author(s): Sarah Kronheim [sup.1] , Martin Daniel-Ivad [sup.1] , Zhuang Duan [sup.1] , Sungwon Hwang [sup.1] , Andrew I. Wong [sup.1] , Ian Mantel [sup.1] , Justin R. Nodwell [sup.1] [...], The arms race between bacteria and the phages that infect them drives the continual evolution of diverse anti-phage defences. Previously described anti-phage systems have highly varied defence mechanisms.sup.1-11; however, all mechanisms rely on protein components to mediate defence. Here we report a chemical anti-phage defence system that is widespread in Streptomyces. We show that three naturally produced molecules that insert into DNA are able to block phage replication, whereas molecules that target DNA by other mechanisms do not. Because double-stranded DNA phages are the most numerous group in the biosphere and the production of secondary metabolites by bacteria is ubiquitous.sup.12, this mechanism of anti-phage defence probably has a major evolutionary role in shaping bacterial communities.Streptomyces secondary metabolites provide a chemical defence against phage predation.

L. monocytogenes is a facultative intracellular pathogen responsible for human listeriosis, a severe food-borne disease, and has emerged as a model for the study of host-pathogen interactions (4). This bacterium [...], During infection, pathogenic bacteria manipulate the host cell in various ways to allow their own replication, propagation and escape from host immune responses. Post-translational modifications are unique mechanisms that allow cells to rapidly, locally and specifically modify activity or interactions of key proteins. Some of these modifications, including phosphorylation and ubiquitylation (1,2), can be induced by pathogens. However, the effects of pathogenic bacteria on SUMOylation, an essential post-translational modification in eukaryotic cells (3), remain largely unknown. Here we show that infection with Listeria monocytogenes leads to a decrease in the levels of cellular SUMO-conjugated proteins. This event is triggered by the bacterial virulence factor listeriolysin O (LLO), which induces a proteasome-independent degradation of Ubc9, an essential enzyme of the SUMOylation machinery, and a proteasomedependent degradation of some SUMOylated proteins. The effect of LLO on Ubc9 is dependent on the pore-forming capacity of the toxin and is shared by other bacterial pore-forming toxins like perfringolysin O (PFO) and pneumolysin (PLY). Ubc9 degradation was also observed in vivo in infected mice. Furthermore, we show that SUMO overexpression impairs bacterial infection. Together, our results reveal that Listeria, and probably other pathogens, dampen the host response by decreasing the SUMOylation level of proteins critical for infection.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.anbehav.2009.10.012 Byline: Shelley A. Adamo, Amy Bartlett, Jeffrey Le, Nora Spencer, Kenneth Sullivan Abstract: Animals from across the animal kingdom decrease feeding during an infection. Superficially this response seems maladaptive because the decline in food intake occurs at the same time as immune activation increases energy expenditure. However, illness-induced anorexia could be beneficial by decreasing trade-offs between the immune system and digestion. For example, in insects (i.e. crickets) there is a trade-off between lipid transport and immune function. We predicted that increasing the need for lipid transport (e.g. when digesting a high fat meal) would reduce immune function. After consuming a high fat meal, crickets (Gryllus texensis) showed an increase in haemolymph lipid concentration. Crickets also showed a decrease in resistance to bacterial infection (Serratia marcescens). After an immune challenge, crickets not only ate less, they also preferred foods containing less fat. This occurred whether the target food was an ecologically valid food item (dead cricket), natural foods (e.g. lettuce and ground meat) or an artificial diet containing different amounts of lipid. Therefore, the change in feeding behaviour after an immune challenge is consistent with the need to reduce lipid transport in order to maximize immune function. Illness-induced anorexia may be one method by which animals can bias physiological pathways towards enhanced immune function. Some behaviours may be adaptive because they can bias the direction of physiological trade-offs. Author Affiliation: Department of Psychology and Neuroscience, Dalhousie University, Canada Article History: Received 31 July 2009; Revised 10 September 2009; Accepted 22 September 2009 Article Note: (miscellaneous) MS. number: A09-00511

The contribution of CCR6 and phagocyte recruitment to the initiation of T cell responses to a local pathogen is unclear. CD4 T cell activation to an injected soluble antigen occurred rapidly and was completely CCR6-independent. In marked contrast, the tempo of pathogen-specific CD4 T cell activation depended on whether the antigen was secreted or cell-associated. Furthermore, lymph node pathogen-specific CD4 T cell activation required CCR6 and cell migration from the site of infection. Surprisingly, adoptive transfer of wild-type blood phagocytes rescued bacteria-specific T cell activation in CCR6-deficient mice, even when these cells were unable to participate in direct antigen presentation. These data demonstrate that T cell responses to a local bacterial infection follow a distinct tempo, largely determined by bacterial protein secretion, and that CCR6-mediated blood phagocyte recruitment to the site of infection is a critical step in the initiation of pathogen-specific immune responses in skin draining lymph nodes. antigen presentation | bacteria

One of the first responses of plants to microbial attack is the production of extracellular superoxide surrounding infection sites. Here, we report that Magnaporthe grisea, the causal agent of rice blast disease, undergoes an oxidative burst of its own during plant infection, which is associated with its development of specialized infection structures called appressoria. Scavenging of these oxygen radicals significantly delayed the development of appressoria and altered their morphology. We targeted two superoxide-generating NADPH oxidase-encoding genes, Nox1 and Nox2, and demonstrated genetically, that each is independently required for pathogenicity of M. grisea. [DELTA]nox1 and Anox2 mutants are incapable of causing plant disease because of an inability to bring about appressorium-mediated cuticle penetration. The initiation of rice blast disease therefore requires production of superoxide by the invading pathogen. appressorium | pathogen | plant disease | superoxide | virulence

This study was aimed at testing the hypothesis that serotoninergic receptors in the locus coeruleus (LC) play a role in bacterial lipopolysaccharide-induced fever. To this end, 5-H[T.sub.1A] (WAY-100635; 3 µg/100 [...]

Previously, we observed an enhanced renal protein synthesis and increased de novo arginine production in the early response to endotoxemia in wild-type Swiss mice (Hallemeesch MM, Soeters PB, and Deutz NE. Am J Physiol Renal Physiol 282: F316-F323, 2002). To establish whether these changes are regulated by nitric oxide (NO) synthesized by NO synthase isoforms NOS2 and NOS3, we studied C57BL6/J wild-type (WT), NOS2-deficient ([NOS2.sup.-/-]), and NOS3-deficient ([NOS3.sup.-/-]) mice under baseline (unstimulated) and LPS-treated conditions. The metabolism of renal protein, amino acid, and arginine was studied at the whole body level and across the kidney by infusing the stable isotopes L-[phenyl-[sup.2][H.sub.5]]phenylalanine, L-[phenyl-[sup.2][H.sub.2]]tyrosine, L-guanidino-[[sup.15][N.sub.2]]arginine, and L-[ureido-[sup.13] C,[sup.2][H.sub.2]]citrulline. Renal blood flow was measured using radioactive PAH extraction. Under baseline conditions, renal blood flow was significantly reduced in [NOS2.sup.-/-] mice (0.29 [+ or -] 0.01 vs. 0.48 [+ or -] 0.07 ml x 10 g body [wt.sup.-1] x [min.sup.-l] in WT) (P < 0.05), and de novo arginine production was lower in [NOS2.sup.-/-] mice. After LPS challenge, renal protein turnover and arginine production increased in all three groups (P < 0.05), even though renal de novo arginine synthesis did not increase. The expected increase in renal citrulline production and disposal after LPS was not observed in [NOS2.sup.-/-] mice (P = 0.06). Collectively, these data show that NOS2 is constitutively expressed in the kidney and remarkably functional as it affects renal blood flow and de novo arginine production under baseline conditions and is important for the increase in renal citrulline turnover during endotoxemia. NOS3, in contrast, appears less important for renal metabolism. The increase in renal protein turnover during endotoxemia does not depend on NOS2 or NOS3 activity. kidney; nitric oxide; nitric oxide synthase; sepsis

Using a murine model of sepsis, we found that the balance of tissue pro- to anti-inflammatory cytokines directly correlated with severity of infection and mortality. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP). Liver tissue was analyzed for levels of IL-1 [beta], IL-1 receptor antagonist (IL-1ra), tumor necrosis factor (TNF)-[alpha], and soluble TNF receptor l by ELISA. Bacterial DNA was measured using quantitative real-time PCR. After CLP, early predominance of proinflammatory cytokines (6 h) transitioned to anti-inflammatory predominance at 24 h. The elevated anti-inflammatory cytokines were mirrored by increased tissue bacterial levels. The degree of anti-inflammatory response compared with proinflammatory response correlated with the bacterial concentration. To modulate the timing of the anti-inflammatory response, mice were treated with IL-1ra before CLP. This resulted in decreased proinflammatory cytokines, earlier bacterial load, and increased mortality. These studies show that the initial tissue proinflammatory response to sepsis is followed by an anti-inflammatory response. The anti-inflammatory phase is associated with increased bacterial load and mortality. These data suggest that it is the timing and magnitude of the anti-inflammatory response that predicts severity of infection in a routine model of sepsis. bacterial load; anti-inflammation

Ghrelin, a newly identified endogenous ligand for growth hormone secretagogue receptor 1a (GHSR-1a, i.e., ghrelin receptor), was recently demonstrated to be a potent vasoactive peptide. Although sepsis is characterized by an early, hyperdynamic phase, it remains unknown whether ghrelin or GHSR-1a plays a role in the cardiovascular response to sepsis. To determine this, polymicrobial sepsis was induced by cecal ligation and puncture in male adult rats. At 5 h (i.e., early sepsis) or 20 h (i.e., late sepsis) after cecal ligation and puncture, blood and tissue samples were collected. Ghrelin levels and ghrelin and GHSR-1a mRNA expression were assessed by RIA and RT-PCR, respectively. In addition, GHSR-1a protein levels in aorta, heart, and small intestine were determined by Western blotting. The vascular response to ghrelin was determined by using an isolated gut preparation. A primary rat aortic smooth muscle cell culture was used to determine the effects of LPS on GHSR-1a expression. The results indicate that although ghrelin levels decreased at early and late sepsis, its receptor was markedly elevated in early sepsis. Moreover, ghrelin-induced relaxation in resistance blood vessels of the isolated small intestine increased significantly during early sepsis but was not altered in late sepsis. Furthermore, GHSR-1a expression in smooth muscle cells was significantly increased at mRNA and protein levels with stimulation by LPS at 10 ng/ml. These results demonstrate that GHSR-1a expression is upregulated and vascular sensitivity to ghrelin stimulation is increased in the hyperdynamic phase of sepsis. vasoactive peptide; growth hormone secretagogue receptor; polymicrobial sepsis; aorta; gut

Sepsis frequently occurs after hemorrhage, trauma, burn, or abdominal surgery and is a leading cause of morbidity and mortality in severely ill patients. We performed experiments to delineate intestinal molecular and functional motility consequences of polymicrobial sepsis in the clinically relevant cecal ligation and puncture (CLP) sepsis model. CLP was performed on male Sprague-Dawley rats. Gastrointestinal transit, colonic in vivo pressure recordings, and in vitro muscle contractions were recorded. Histochemistry was performed for macrophages, monocytes, and neutrophils. Inflammatory gene expressions were quantified by real-time RT-PCR. CLP delayed gastrointestinal transit, decreased colonic pressures, and suppressed in vivo circular muscle contractility of the jejunum and colon over a 4-day period. A leukocytic infiltrate of monocytes and neutrophils developed over 24 h. Real-time RT-PCR demonstrated a significant temporal elevation in IL-6, IL-1[beta], monocyte chemoattractant protein-1, and inducible nitric oxide synthase, with higher expression levels of IL-6 and inducible nitric oxide synthase in colonic extracts compared with small intestine. Polymicrobial CLP sepsis induces a complex inflammatory response within the intestinal muscularis with the recruitment of leukocytes and elaboration of mediators that inhibit intestinal muscle function. Differences were elucidated between endotoxin and CLP models of sepsis, as well as a heterogeneous regional response of the gastrointestinal tract to CLP. Thus the intestine is not only a source of bacteremia but also an important target of bacterial products with major functional consequences to intestinal motility and the generation of cytokines, which participate in the development of multiple organ failure. inducible nitric oxide synthase; cecal ligation and puncture; lipopolysaccharide; proinflammatory cytokines

Although sepsis is the major cause of mortality and morbidity in the critically ill, precise mechanism(s) causing multiorgan dysfunction remain unclear. Findings of impaired oxygen utilization in septic patients and animals implicate nitric oxide-mediated inhibition of the mitochondrial respiratory chain. We recently reported a relationship between skeletal muscle mitochondrial dysfunction, clinical severity, and poor outcome in patients with septic shock. We thus developed a long-term, fluid-resuscitated, fecal peritonitis model utilizing male Wistar rats that closely replicates human physiological, biochemical, and histological findings with a 40% mortality. As with humans, the severity of organ dysfunction and eventual poor outcome were associated with nitric oxide overproduction and increasing mitochondrial dysfunction (complex I inhibition and ATP depletion). This was seen in both vital (liver) and nonvital (skeletal muscle) organs. Likewise, histological evidence of cell death was lacking, suggesting the possibility of an adaptive programmed shutdown of cellular function. This study thus supports the hypothesis that multiorgan dysfunction induced by severe sepsis has a bioenergetic etiology. Despite the well-recognized limitations of laboratory models, we found clear parallels between this long-term model and human disease characteristics that will facilitate future translational research. nitric oxide: adenosine 5'-triphosphate; respiratory chain; complex I; glutathione

Growth hormone (GH) stimulates insulin-like growth factor I (IGF-I) synthesis in both liver and muscle. During sepsis, proinflammatory cytokines inhibit GH action in liver, but it is unknown whether sepsis also produces GH resistance in muscle. Sepsis was induced by cecal ligation and puncture, and 18 h later the effect of GH on signal transducer and activator of transcription (STAT) phosphorylation and IGF-I mRNA content was assessed in rat gastrocnemius and liver. The relative abundance of phosphorylated (p)STAT5a, pSTAT5b, pSTAT3, and pSTAT1 was increased in liver from control rats after GH. Sepsis alone also increased hepatic pSTAT5a, pSTAT3, and pSTAT1. Sepsis dramatically impaired the ability of GH to stimulate the phosphorylation of STAT5a and -5b, as well as to increase IGF-I mRNA in liver. In muscle from control rats, GH increased pSTAT5a and -5b, whereas content of pSTAT3 and pSTAT1 was not affected. Sepsis increased basal content of pSTAT3 but not pSTAT5a, pSTAT5b, or pSTAT1 in muscle. The GH-induced increase of pSTAT5a and -5b in muscle from septic rats was not inhibited, suggesting that muscle was not GH resistant. In contrast to these changes in pSTAT5, the ability of GH to increase IGF-I mRNA was completely absent in muscle from septic rats. Because the suppressor of cytokine signaling (SOCS) proteins may function as negative regulators of GH signaling, we examined the content of these proteins. Sepsis produced small (30-50%), albeit statistically significant, increases in SOCS-1, -2, and -3 protein in muscle. In contrast to muscle, the SOCS proteins in the liver did not change under the various experimental conditions, suggesting that these proteins are not responsible for the impaired phosphorylation of STAT5 by GH. In conclusion, sepsis produces GH resistance in both muscle and liver, with the locus of this impairment in muscle differing from that in liver and being independent of a defect in STAT5 phosphorylation. growth hormone; signal transducer and activator of transcription; suppressors of cytokine signaling; insulin-like growth factor I; liver; endotoxin

ABBREVIATIONS. VLBW, very low birth weight; MPV, mean platelet volume; CONS, coagulase-negative Staphylococcus; NEC, necrotizing enterocolitis; ELA, endotoxin-like activity; CBC, complete blood count; PAF, platelet-activating factor; SD, standard deviation. Sepsis [...], Objective. Thrombocytopenia is commonly observed in very low birth weight (VLBW) neonates with sepsis. Specific platelet responses to different infectious agents have not been extensively characterized. The objectives of this study were to examine platelet counts and platelet indices in preterm neonates with culture-proven sepsis to determine if there are organism-specific platelet responses. Study Design. We analyzed a cohort of all VLBW neonates (birthweight Results. Sepsis was diagnosed in 154 (16%) of 943 patients in the study population. Of the sepsis episodes, 54% were associated with thrombocytopenia and 61% with an elevation in MPV. Infections were grouped by organism type: Gram-positive bacteria (117/154, 76%), Gram-negative bacteria (24/154, 16%), and fungi (13/154, 8%). When compared with patients with Gram-positive sepsis, those with Gram-negative or fungal sepsis had a significantly lower initial platelet count, a lower platelet nadir, a higher incidence of thrombocytopenia, and a greater duration of thrombocytopenia. The decrease in platelet count from baseline was also significantly less in the Gram-positive infections than in the fungal infections. Although there was an overall increase in MPV from baseline, there were no differences between groups. Conclusions. In our population of VLBW infants, sepsis is frequently associated with thrombocytopenia and an elevation in MPV. However, fungal and Gram-negative pathogens are associated with a lower platelet count and more prolonged thrombocytopenia compared with Gram-positive pathogens. We conclude that common pathogens causing sepsis have different effects on platelet kinetics. Pediatrics 2003;111:1411-1415; platelet, sepsis, very low birth weight infant, preterm infant, thrombocytopenia.

In sepsis, both necrotic and apoptotic cell death can occur. Apoptotic cells induce anergy that could impair the host response, whereas necrotic cells cause immune activation that might result in enhanced antimicrobial defenses. We determined whether adoptive transfer of apoptotic or necrotic cells impacted survival in a clinically relevant sepsis model. We also evaluated the effects of adoptive transfer of apoptotic or necrotic cells on the prototypical TH1 and TH2 cytokines IFN-[gamma] and IL-4, respectively. C57BL6/J mice had adoptive transfer of apoptotic (irradiated) or necrotic (freeze thaw) splenocytes. Controls received saline. Apoptotic cells greatly increased mortality, whereas necrotic splenocytes markedly improved survival, P [less than or equal to] 0.05. The contrasting effects that apoptotic or necrotic cells exerted on survival were mirrored by opposite effects on splenocyte IFN-[gamma] production with greatly decreased and increased production, respectively. Importantly, either administration of anti-IFN-[gamma], antibodies or use of IFN-[gamma] knockout mice prevented the survival benefit occurring with necrotic cells. This study demonstrates that the type of cell death impacts survival in a clinically relevant model and identifies a mechanism for the immune suppression that is a hallmark of sepsis. Necrotic cells (and likely apoptotic cells) exert their effects via modulation of IFN-[gamma].

Sepsis can predispose the lung to insults such as mechanical ventilation (MV). It was hypothesized that treating the lung with exogenous surfactant early in the development of sepsis will reduce the lung dysfunction associated with MV 18 h later. Mice underwent sham or cecal ligation and perforation (CLP) surgery. Immediately after surgery, mice were either untreated or given 100 mg/kg of bovine lipid extract surfactant intratracheally. Eighteen hours later, the lungs were removed and analyzed either immediately or following ventilation ex vivo for 2 h by an 'injurious' mode of ventilation (20 ml/kg, 0 cm positive end-expiratory pressure). In nonventilated lungs, exogenous surfactant had no impact on compliance or IL-6 concentrations in the lungs. In the ventilated groups, the administered surfactant had a significant protective effect on the lung dysfunction induced by MV, but only in the CLP lungs. We conclude that administration of exogenous surfactant at the time of a systemic insult can protect the lung from the damaging effects of MV 18 h later. acute respiratory distress syndrome; mechanical ventilation; sepsis

Acute respiratory distress syndrome (ARDS) is an acute lung injury of high mortality rate, and sepsis syndrome is one of the most frequent causes of ARDS. Metabolites of arachidonic acid, including thromboxanes and leukotrienes, are proinfiammatory mediators and potentially involved in the development of ARDS. A key enzyme for the production of these inflammatory mediators is cytosolic phospholipase [A.sub.2] (cPL[A.sub.2]). Recently, it has been reported that arachidonyl trifiuoromethyl ketone (ATK) is a potent inhibitor of cPL[A.sub.2]. In the present study, we hypothesized that pharmacological intervention of cPL[A.sub.2] could affect acute lung injury. To test this hypothesis, we examined the effects of ATK in a murine model of acute lung injury induced by septic syndrome. The treatment with ATK significantly attenuated lung injury, polymorphonuclear neutrophil sequestration, and deterioration of gas exchange caused by lipopolysaccharide and zymosan administration. The current observations suggest that pharmacological intervention of cPL[A.sub.2] could be a novel therapeutic approach to acute lung injury caused by sepsis syndrome. acute respiratory distress syndrome; lipopolysaccharide; sepsis; eicosanoid; leukotriene

The decrease in glomerular filtration rate (GFR) that is characteristic of sepsis has been shown to result from inhibition of glomerular endothelial nitric oxide synthase (eNOS) by nitric oxide (NO) generated from the inducible isoform of NOS (iNOS). Although L-arginine is the sole precursor for NO biosynthesis, its intracellular availability in glomeruli from septic animals has never been investigated. Arginine uptake was measured in freshly harvested glomeruli from the following experimental groups: 1) untreated rats; 2) rats pretreated with LPS (4 mg/kg body wt, 4 h before experiments); 3) rats treated with LPS as above with either L-[N.sup.6]-(1-iminoethyl)lysine hydrochloride (L-NIL), a selective iNOS antagonist, or 7-nitroindazole, a selective neuronal NOS antagonist; and 4) rats treated with L-NIL only. Both glomeular and mesangial arginine transport characteristics were found compatible with a [y.sup.+] system. Arginine uptake was augmented in glomeruli from LPS-treated rats. Treatment with L-NIL completely abolished this effect whereas L-NIL alone had no effect. Similar results were obtained when primary cultures of rat mesangial cells were preincubated with LPS (10 [micro]g/ml for 24 h) with or without L-NIL. Using RT-PCR, we found that in vivo administration of LPS resulted in a significant increase in glomerular cationic amino acid transporter-2 (CAT-2) mRNA expression whereas CAT-1 mRNA was undetected. Northern blotting further confirmed a significant increase in glomerular CAT-2 by LPS. In mesangial cells, the expression of both CAT-1 and CAT-2 mRNA was augmented after incubation with LPS. In conclusion, in vivo administration of LPS augments glomerular arginine transport through upregulation of steady-state CAT-2 mRNA while downregulating CAT-1 mRNA. These results may correspond to the changes in glomerular iNOS and eNOS activity in sepsis. cationic amino acid transporter-2; sepsis; nitric oxide; arginine transport

We assessed the hypothesis that the epinephrine surge present during sepsis accelerates aerobic glycolysis and lactate production by increasing activity of skeletal muscle [Na.sup.+]-[K.sup.+]-ATPase. Healthy volunteers received an intravenous bolus of endotoxin or placebo in a randomized order on two different days. Endotoxemia induced a response resembling sepsis. Endotoxemia increased plasma epinephrine to a maximum at t = 2 h of 0.7 [+ or -] 0.1 vs. 0.3 [+ or -] 0.1 nmol/l (P < 0.05, n = 6-7). Endotoxemia reduced plasma [K.sup.+] reaching a nadir at t = 5 h of 3.3 [+ or -] 0.1 vs. 3.8 [+ or -] 0.1 mmol/l (P < 0.01, n = 6-7), followed by an increase to placebo level at t = 7-8 h. During the declining plasma [K.sup.+], a relative accumulation of [K.sup.+] was seen reaching a maximum at t = 6 h of 8.7 [+ or -] 3.8 mmol/leg (P < 0.05). Plasma lactate increased to a maximum at t = 1 h of 2.5 [+ or -] 0.5 vs. 0.9 [+ or -] 0.1 mmol/l (P < 0.05, n = 8) in association with increased release of lactate from the legs. These changes were not associated with hypoperfusion or hypoxia. During the first 24 h after endotoxin infusion, renal [K.sup.+] excretion was 27 [+ or -] 7 mmol, i.e., 58% higher than after placebo. Combination of the well-known stimulatory effect of catecholamines on skeletal muscle [Na.sup.+]-[K.sup.+]-ATPase activity, with the present confirmation of an expected [Na.sup.+]-[K.sup.+]-ATPase-induced decline in plasma [K.sup.+], suggests that the increased lactate release was due to increased [Na.sup.+]-[K.sup.+]-ATPase activity, supporting our hypothesis. Thus increased lactate levels in acutely and severely ill patients should not be managed only from the point of view that it reflects hypoxia. [K.sup.+] homeostasis; sepsis; glycolysis

Many patients who experience surgical stress, including burn injury, become susceptible to severe sepsis and septic organ dysfunction, which remains the primary contributor to morbidity and mortality in burn patients. In the present study, we developed a murine model of burn-primed sublethal endotoxemia by producing a 15% BSA full-thickness burn on the dorsum of BALB/c mice under ether anesthesia and administering 10 mg/kg of LPS intravenously on day 11 to model endotoxemia. The prior burn injury in this model induced two-peaked production of cytokines, TNF-[alpha], and macrophage inflammatory protein-2 at 2 and 12 h after LPS administration, and it was associated with increased mortality. We also assessed the effect of pharmacological modulation with cytokine synthesis inhibitors prednisolone and JTE-607 and found that pretreatment with them attenuated later cytokine production and decreased mortality after LPS administration. We speculate that the prior burn injury primed the mice for the secondary insult via cytokine production. These results also suggested that an anticytokine strategy might serve as a novel prophylactic therapy for septic organ dysfunction in burn-primed patients. two-hit phenomenon; priming; systemic inflammatory response syndrome; chemokine; JTE-607