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Biliary tract infection (BTI)-derived sepsis remains a serious problem with significant morbidity and mortality in the modern era of critical care management. Current animal models of BTI have relied mostly on injecting purified bacteria or their toxins into the biliary tract. These models do not fully reflect pathophysiology or disease processes of clinical cholangitis or cholecystitis. In the current study, we developed a novel model of BTI by performing cholecystocolonic anastomosis (CCA) in rabbits and characterized pathophysiologic changes in this model. This model is intended to mimic the clinical process of cholecystocolonic fistula with reflux cholangitis, a severe form of BTI. Adult male rabbits were subjected to BTI-derived sepsis through an anastomosis of the gall bladder to the colon (i.e., CCA). The animals were monitored for 7 days to record survival. In additional groups of animals, various bacterial, hemodynamic, histological and biochemical parameters were measured at 12, 24, 48 and 72 h after CCA. The anastomosis between the gallbladder and the colon required about 5-8 min to finish. The median survival time for rabbits after CCA was 96 h. The positive rates of bacterial culture at 72 h after CCA were 83.3% and 100% in the blood and liver, respectively. The most common microorganism was Escherichia coli followed by Enterococcus. Plasma Tumor Necrosis Factor-α (TNF-α), Lnterleukin-10 (IL-10), Lnterleukin-6 (IL-6), and High-mobility group box 1 protein (HMGB-1) levels were greatly elevated after CCA. The cardiac index and heart rate increased slightly at 12 h after CCA and then continued to decrease. Systemic hypotension developed 48 h after CCA. Histological studies showed reflux cholangitis with acute lung and kidney injury. Cholecystocolonic anastomosis produces polymicrobial sepsis in rabbits, which mimics many aspects of human BTI-derived sepsis. It is reproducible and easy to perform and may serve as an excellent model for future sepsis research.

A systemic inflammatory response is observed in patients undergoing hemorrhagic shock and sepsis. Here we report increased levels of cold-inducible RNA-binding protein (CIRP) in the blood of individuals admitted to the surgical intensive care unit with hemorrhagic shock. In animal models of hemorrhage and sepsis, CIRP is upregulated in the heart and liver and released into the circulation. In macrophages under hypoxic stress, CIRP translocates from the nucleus to the cytosol and is released. Recombinant CIRP stimulates the release of tumor necrosis factor-α (TNF-α) and HMGB1 from macrophages and induces inflammatory responses and causes tissue injury when injected in vivo. Hemorrhage-induced TNF-α and HMGB1 release and lethality were reduced in CIRP-deficient mice. Blockade of CIRP using antisera to CIRP attenuated inflammatory cytokine release and mortality after hemorrhage and sepsis. The activity of extracellular CIRP is mediated through the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex. Surface plasmon resonance analysis indicated that CIRP binds to the TLR4-MD2 complex, as well as to TLR4 and MD2 individually. In particular, human CIRP amino acid residues 106-125 bind to MD2 with high affinity. Thus, CIRP is a damage-associated molecular pattern molecule that promotes inflammatory responses in shock and sepsis.

Background: Cardiovascular dysfunction, characterized by reduced cardiac contractility and depressed endothelium-dependent vascular relaxation, is common in severe sepsis. Although it is known that ghrelin produces beneficial effects following various adverse circulatory conditions, it remains unknown whether ghrelin increases cardiac contractility and improves vascular responsiveness to vasoactive agents in severe sepsis., Methods: Male adult rats were subjected to sepsis by cecal ligation and puncture (CLP). At 5 h after CLP, a bolus intravenous injection of 2 nmol ghrelin was followed by a continuous infusion of 12 nmol ghrelin via a primed mini-pump over 15 h. At 20 h after CLP (i.e., severe sepsis), the maximal rates of ventricular pressure increase (+dP/dt(max)) and decrease (-dP/dt(max)) were determined in vivo. In additional groups of animals, the thoracic aortae were isolated at 20 h after CLP. The aortae were cut into rings, and placed in organ chambers. Norepinephrine (NE) was used to induce vascular contraction. Dose responses for an endothelium-dependent vasodilator, acetylcholine (ACh), and an endothelium-independent vasodilator, nitroglycerine (NTG) were carried out., Results: +dP/dt(max) and -dP/dt(max) decreased significantly at 20 h after CLP. Treatment with ghrelin significantly increased +dP/dt(max) and -dP/dt(max) by 36% (P < 0.05) and 35% (P < 0.05), respectively. Moreover, NE-induced vascular contraction and endothelium-dependent (ACh-induced) vascular relaxation decreased significantly at 20 h after CLP. Administration of ghrelin, however, increased NE-induced vascular contraction and ACh-induced vascular relaxation. In contrast, no significant reduction in NTG-induced vascular relaxation was seen in rats with severe sepsis irrespective of ghrelin treatment., Conclusions: Ghrelin may be further developed as a useful agent for maintaining cardiovascular stability in severe sepsis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Purpose: Animal milk fat globule-EGF factor 8 (MFG-E8) has been shown to be beneficial in attenuating the inflammatory response in sepsis. In this study, we examined the effect of recombinant human MFG-E8 (rhMFG-E8) in an animal model of sepsis in an effort to develop it as a potential therapy against sepsis in humans., Methods: Rats were subjected to sepsis by cecal ligation and puncture (CLP), and at 5 h post-CLP, they were given different doses of rhMFG-E8 (20, 40, 80, 160 μg/kg BW) in normal saline. At 20 h post-CLP, samples were collected for further analysis. A 10-day survival study was also performed., Results: At 20 h after CLP, organ injury indicators, serum IL-6 and TNF-α, and plasma HMGB-1 levels were significantly increased as compared to sham-operated animals. Treatment with 20 μg/kg rhMFG-E8 significantly reduced these levels. With higher doses, further reductions in AST and ALT (59-62%), creatinine (65-68%), and lactate (46-57%), and serum IL-6 and TNF-α were obtained. The 160 μg/kg dose produced the greatest reduction in serum TNF-α. With treatment with 20 μg/kg rhMFG-E8, HMGB-1 levels decreased by 80%, returning back to sham values. In a 10-day survival study, vehicle-treated animals produced a 36% survival rate, while rhMFG-E8 significantly improved the survival rate to 68-72%. Treatment with increasing doses of rhMFG-E8 significantly reduced the number of apoptotic cells detected and markedly attenuated the tissue damages observed in the lungs., Conclusions: These data suggest that recombinant human MFG-E8 is beneficial in ameliorating sepsis in an animal model of sepsis.

Sepsis is a devastating and complex syndrome and continues to be a major cause of morbidity and mortality among critically ill patients at the surgical intensive care unit setting in the United States. The occurrence of sepsis and septic shock has increased significantly over the past two decades. Despite of highly dedicated basic research and numerous clinical trials, remarkable progress has not been made in the development of novel and effective therapeutics. The sepsis-induced physiologic derangements are due largely to the host responses to the invading microorganism in contrast to the direct effects of the microorganism itself. Sepsis, the systemic inflammatory response to infection, is marked by dysregulated production of pro-inflammatory cytokines. Although pro-inflammatory cytokine production is normally indispensable to protect against pathogens and promote tissue repair, the dysregulated and prolonged production of these cytokines can trigger a systemic inflammatory cascade mediated by chemokines, vasoactive amines, the complement and coagulation system, and reactive oxygen species (ROS), amongst others. These mediators collectively lead to multiple organ failure, and ultimately to death. In this regard, the role of inflammation in the pathophysiology of sepsis, although still incompletely understood, is clearly critical. Recent findings resulting from vigorous investigations have contributed to delineate various novel directions of sepsis therapeutics. Among these, this review article is focused on new promising mechanisms and concepts that could have a key role in anti-inflammatory strategies against sepsis, including 1) "inflammasome": a multiprotein complex that activates caspase-1; 2) "the cholinergic anti-inflammatory pathway": the efferent arm of the vagus nerve-mediated, brain-to-immune reflex; 3) "stem cells": unspecialized and undifferentiated precursor cells with the capacity for self-renewal and potential to change into cells of multiple lineages; 4) "milk fat globule-EGF factor VIII (MFG-E8)": a bridging molecule between apoptotic cells and phagocytes, which promotes phagocytosis of apoptotic cells.

Apoptosis plays an important role in the patho-biology of sepsis. The opsonizing protein milk fat globule-EGF factor VIII (MFG-E8) is involved in apoptotic cell clearance. Our previous studies have shown that administration of rat MFG-E8-containing exosomes or recombinant murine MFG-E8 (rmMFG-E8) is protective in a rat model of sepsis induced by cecal ligation of puncture (CLP). However, one obstacle hampering the development of MFG-E8 as a therapeutic agent for septic patients is the potential immunogenicity of animal proteins in humans. The purpose of this study, therefore, was to express recombinant human MFG-E8 (rhMFG-E8) and characterize its biological activity. Using an E. coli system, we successfully expressed and purified the mature molecule of human MFG-E8 (Leu24-Cys387). The purity of rhMFG-E8 was over 99% and it was immunoreactive for specific anti-human MFG-E8 antibodies. Amino acid sequence analysis by LC-MS/MS identified the purified protein as human MFG-E8. Using primary rat peritoneal macrophages, we showed that rhMFG-E8 markedly increased peritoneal macrophage phagocytosis of apoptotic thymocytes, which was as effective as commercial rmMFG-E8. To determine the biological activity of rhMFG-E8 in vivo, male adult rats were subjected to sepsis by CLP. rhMFG-E8 or rmMFG-E8 were administered intravenously at the time of CLP. Our results demonstrated that both rhMFG-E8 and rmMFG-E8 reduced thymocyte apoptosis and plasma levels of lactate and IL-6 at 20 h after CLP, and improved the 10-day survival rate. Thus, we have successfully expressed and purified biologically active rhMFG-E8. Our newly-expressed rhMFG-E8 is highly effective in the rat model of sepsis.

Sepsis and ischemia-reperfusion (I/R) injury are among the leading causes of death in critically ill patients at the surgical intensive care unit setting. Both conditions are marked by the excessive inflammatory response which leads to a lethal disease complex such as acute lung injury, systemic inflammatory response syndrome and multiple organ dysfunction syndrome. Despite the advances in the understanding of the pathophysiology of those conditions, very little progress has been made toward therapeutic interventions. One of the key aspects of these conditions is the accumulation of apoptotic cells that have the potential to release toxic and proinflammatory contents due to secondary necrosis without appropriate clearance by phagocytes. Along with the prevention of apoptosis, that is reported to be beneficial in sepsis and I/R injury, thwarting the development of secondary necrosis through the active removal of apoptotic cells via phagocytosis may offer a novel therapy. Milk fat globule-EGF factor VIII (MFG-E8), which is mainly produced by macrophages and dendritic cells, is an opsonin for apoptotic cells and acts as a bridging protein between apoptotic cells and phagocytes. Recently, we have shown that MFG-E8 expression is decreased in experimental sepsis and I/R injury models. Exogenous administration of MFG-E8 attenuated the inflammatory response as well as tissue injury and mortality through the promotion of phagocytosis of apoptotic cells. In this review, we describe novel information available about the involvement of MFG-E8 in the pathophysiology of sepsis and I/R injury, and the therapeutic potential of exogenous MFG-E8 treatment for those conditions.

Background: Despite advances in our understanding of excessive alcohol-intake-related tissue injury and modernization of the management of septic patients, high morbidity and mortality caused by infectious diseases in alcohol abusers remain a prominent challenge. Our previous studies have shown that milk fat globule epidermal growth factor-factor VIII (MFG-E8), a protein required to opsonize apoptotic cells for phagocytosis, is protective in inflammation. However, it remains unknown whether MFG-E8 ameliorates sepsis-induced apoptosis and organ injury in alcohol-intoxicated rats. The purpose of this study was to determine whether recombinant murine MFG-E8 (rmMFG-E8) attenuates organ injury after acute alcohol exposure and subsequent sepsis., Methods: Acute alcohol intoxication was induced in male adult rats by a bolus injection of intravenous alcohol at 1.75 g/kg BW, followed by an intravenous infusion of 300 mg/kg BW/h of alcohol for 10 hours. Sepsis was induced at the end of 10-hour alcohol infusion by cecal ligation and puncture (CLP). rmMFG-E8 or vehicle (normal saline) was administered intravenously 3 times (i.e., at the beginning of alcohol injection, the beginning of CLP, and 10 hours post-CLP) at a dose of 20 microg/kg BW each. Blood and tissue samples were collected 20 hours after CLP in alcoholic animals for various measurements., Results: Acute alcohol exposure per se did not affect the production of MFG-E8; however, it primed the animal and enhanced sepsis-induced MFG-E8 downregulation in the spleen. Administration of rmMFG-E8 reduces alcohol/sepsis-induced apoptosis in the spleen, lungs, and liver. In addition, administration of rmMFG-E8 after alcohol exposure and subsequent sepsis decreases circulating levels of TNF-alpha and interleukin-6 and attenuates organ injury., Conclusions: rmMFG-E8 attenuates sepsis-induced apoptosis and organ injury in alcohol-intoxicated rats.

Sepsis is a serious complication for patients with obstructive jaundice. Although administration of adrenomedullin (AM) in combination with its binding protein (AMBP-1) is protective after injury, it remains unknown whether AM/AMBP-1 ameliorates sepsis-induced organ injury and mortality in the setting of biliary obstruction. The aim of this study is, therefore, to test the efficacy of human AM/AMBP-1 in a rat model of obstructive jaundice and polymicrobial sepsis. To study this, obstructive jaundice was induced in male adult rats (275-325g) by common bile duct ligation (BDL). One week after BDL, the rats were subjected to sepsis by cecal ligation and puncture (CLP). Plasma levels of AM and AMBP-1 were measured at 20h after CLP. In additional groups of BDL+CLP rats, human AM/AMBP-1 (24/80microg/kg body weight (BW)) or vehicle (i.e., human albumin) was administered intravenously at 5h after CLP. Blood and tissue samples were collected at 20h after CLP for various measurements. To determine the long-term effect of human AM/AMBP-1 after BDL+CLP, the gangrenous cecum was removed at 20h after CLP and 7-day survival was recorded. Our results showed that plasma levels of AM were significantly increased while AMBP-1 levels were markedly decreased after BDL+CLP (n=8, P<0.05). Administration of human AM/AMBP-1 attenuated tissue injury and inflammatory responses after BDL+CLP. Moreover, human AM/AMBP-1 significantly increased the survival rate from 21% (n=14) to 53% (n=15). Thus, human AM/AMBP-1 ameliorates sepsis-induced organ injury and mortality in jaundiced rats. Human AM/AMBP-1 can be further developed as a novel treatment for sepsis in jaundiced patients., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Sepsis is a systemic inflammatory response syndrome (SIRS) when an infection is the etiology of SIRS. Our previous studies have indicated that the release of the sympathetic neurotransmitter, norepinephrine (NE), from the gut is increased in sepsis, and that NE potentiates endotoxin-induced tumor necrosis factor (TNF)-alpha upregulation via the A subtype of alpha(2)-adrenoceptors (i.e., alpha(2A)-AR) expressed on the surface of Kupffer cells. A specific antagonist for alpha(2A)-AR, 2-[(4,5-dihydro-1H-imidazol-2-yl) methyl]-2,3-dihydro-1-methyl-1H-isoindole maleate (BRL-44408 maleate), reduces TNF-alpha secretion in cultured Kupffer cells. We, therefore, hypothesize that administration of BRL-44408 maleate inhibits inflammatory responses and reduces organ injury in sepsis. To study this, sepsis was induced in male rats by cecal ligation and puncture (CLP). At 5 h after CLP, BRL-44408 maleate (0.3125, 0.625, 1.25, 2.5, or 5.0 mg/kg BW) or vehicle (1-ml normal saline) were administered intravenously over a period of 30 min. Blood and intestinal samples were collected at 20 h after CLP. Serum levels of TNF-alpha, interleukin (IL)-6, IL-10, keratinocyte-derived chemokine (KC), macrophage inflammatory protein-2 (MIP-2), liver enzymes (i.e., aspartate aminotransferase (AST) and alanine aminotransferase (ALT)), and lactate were measured. The intestinal levels of TNF-alpha, IL-6, and myeloperoxidase (MPO) activities were also analyzed. In additional groups of animals, the necrotic cecum was excised at 20 h post-CLP, and the 10-day survival was recorded. Our results showed that serum levels of proinflammatory cytokines (TNF-alpha and IL-6), anti-inflammatory cytokine (IL-10), chemokines (KC, MIP-2), liver enzymes (AST and ALT), lactate, and intestinal levels of TNF-alpha, IL-6, and MPO were significantly elevated at 20 h after CLP. Administration of BRL-44408 maleate significantly reduced serum levels of proinflammatory cytokines, chemokines, liver enzymes, and lactate, and dramatically decreased TNF-alpha, IL-6, and MPO levels in the gut. However, it has no statistical effects on the elevated serum levels of IL-10. Moreover, BRL-44408 maleate at the doses of 2.5 or 5.0 mg/kg BW significantly increased the survival rate after CLP and cecal excision. In conclusion, modulation of the sympathetic nervous system by blocking alpha(2A)-AR appears to be a novel treatment for inflammatory conditions such as sepsis.

Hepatocellular dysfunction occurs early in sepsis and this appears to be caused by Kupffer cell-derived TNF-alpha production from the liver as a result of the increased release of the sympathetic neurotransmitter, norepinephrine, from the gut. Ghrelin, a novel stomach-derived peptide, is down-regulated in sepsis and administration of ghrelin into rodents decrease pro-inflammatory cytokines, attenuates hepatic and other organ injuries and improves survival. Ghrelin's beneficial effect in sepsis is mediated by the inhibition of the sympathetic nervous system (SNS), as evidenced by the reduced gut-derived norepineprine (NE) release in sepsis after ghrelin treatment. Recent data suggest that MKP-1, the MAPK phosphatase-1, is involved in the innate immune responses. To determine that the beneficial effect of ghrelin in sepsis is mediated by MKP-1, rats were subjected to sepsis by cecal ligation and puncture (CLP) alone, or treated with ghrelin, beginning at 5-h post-CLP and liver tissues were harvested and examined for MKP-1 mRNA and protein expression. CLP alone produced a significant decrease in MKP-1 gene expression in liver tissues at 20 h after CLP (P<0.05). MKP-1 mRNA was decreased by 30-40% at 2 and 5 h after CLP, but not statistically significant. MKP-1 protein expression was significantly decreased as early as 2 h after CLP and remained low at 5-20 h after CLP. While septic rats treated with vehicle produced significant decreases from sham rats, ghrelin treatment improved both mRNA and protein from vehicle group (0.58+/-0.069 vs. 0.91+/-0.16, P<0.05; 0.14+/-0.027 vs. 0.22+/-0.017, P=0.013), respectively. Since ghrelin's inhibitory effect is mediated by the SNS, we hypothesized that NE treatment in Kupffer cells may downregulate MKP-1. Kupffer cells were treated with NE and examined for MKP-1. Treatment with NE for 60 min showed an average of 46.9% decrease in MKP-1 mRNA expression compared to untreated cells (P<0.001). Likewise, NE treatment in RAW264.7 cells produced significantly lower MKP-1 mRNA than that of control cells. To further confirm the effect of NE on MKP-1, normal rats were infused with NE for 2 h through the portal vein and MKP-1 mRNA from the liver was examined. Infusion with NE produced a significant 73.7% decrease in MKP-1 mRNA. Therefore, ghrelin's inhibitory effect on gut-derived NE release in sepsis leading to the downregulation of pro-inflammatory cytokines is mediated by MKP-1.

In the terrorist radiation exposure scenario, radiation victims are likely to suffer from additional injuries such as sepsis. Our previous studies have shown that ghrelin is protective in sepsis. However, it remains unknown whether ghrelin ameliorates sepsis-induced organ injury and mortality after radiation exposure. The purpose of this study is to determine whether human ghrelin attenuates organ injury and improves survival in a rat model of radiation combined injury (RCI) and, if so, the potential mechanism responsible for the benefit. To study this, adult male rats were exposed to 5-Gy whole body irradiation followed by cecal ligation and puncture (CLP, a model of sepsis) 48 h thereafter. Human ghrelin (30 nmol/rat) or vehicle (saline) was infused intravenously via an osmotic minipump immediately after radiation exposure. Blood and tissue samples were collected at 20 h after RCI (68 h after irradiation or 20 h after CLP) for various measurements. To determine the longterm effect of human ghrelin after RCI, the gangrenous cecum was removed at 5 h after CLP and 10-d survival was recorded. In addition, vagotomy or sham vagotomy was performed in sham and RCI animals immediately prior to ghrelin administration, and various measurements were performed at 20 h after RCI. Our results showed that serum levels of ghrelin and its gene expression in the stomach were decreased markedly at 20 h after RCI. Administration of human ghrelin attenuated tissue injury markedly, reduced proinflammatory cytokine levels, decreased tissue myeloperoxidase activity, and improved survival after RCI. Furthermore, elevated plasma levels of norepinephrine (NE) after RCI were reduced significantly by ghrelin. However, vagotomy prevented ghrelin's beneficial effects after RCI. In conclusion, human ghrelin is beneficial in a rat model of RCI. The protective effect of human ghrelin appears to be attributed to re-balancing the dysregulated sympathetic/parasympathetic nervous systems.

Sepsis, a highly lethal systemic inflammatory syndrome, is associated with increases of proinflammatory cytokines (e.g., TNF-alpha, HMGB1) and the accumulation of apoptotic cells that have the potential to be detrimental. Depending on the timing and tissue, prevention of apoptosis in sepsis is beneficial; however, thwarting the development of secondary necrosis through the active removal of apoptotic cells by phagocytosis may offer a novel anti-sepsis therapy. Immature dendritic cells (IDCs) release exosomes that contain milk fat globule EGF factor VIII (MFGE8), a protein required to opsonize apoptotic cells for phagocytosis. In an experimental sepsis model using cecal ligation and puncture, we found that MFGE8 levels decreased in the spleen and blood, which was associated with impaired apoptotic cell clearance. Administration of IDC-derived exosomes promoted phagocytosis of apoptotic cells and significantly reduced mortality. Treatment with recombinant MFGE8 was equally protective, whereas MFGE8-deficient mice suffered from increased mortality. IDC exosomes also attenuated the release of proinflammatory cytokines in septic rats. Liberation of HMGB1, a nuclear protein that contributes to inflammation upon release from unengulfed apoptotic cells, was prevented by MFGE8-mediated phagocytosis in vitro. We conclude that IDC-derived exosomes attenuate the acute systemic inflammatory response in sepsis by enhancing apoptotic cell clearance via MFGE8.

Objectives: We have recently shown that ghrelin, a novel orexigenic hormone, is reduced in sepsis. Ghrelin treatment, mediated through ghrelin receptors in the brain, attenuates sepsis-induced inflammation and mortality. Gut barrier dysfunction is common in sepsis. High-mobility group B1 (HMGB1) increases gut permeability both in vitro and in vivo. However, it remains unknown whether ghrelin has any effects on HMGB1 and gut barrier function in sepsis. We hypothesized that ghrelin decreases HMGB1 release and attenuates sepsis-induced gut barrier dysfunction through central ghrelin receptors., Design: Prospective, controlled, and randomized animal study., Setting: A research institute laboratory., Subjects: Male adult Sprague-Dawley rats (275-325 g)., Interventions: Cecal ligation and puncture (CLP) followed by injection/infusion of ghrelin., Measurements and Main Results: Five hours after CLP, a bolus intravenous injection of 2 nmol of ghrelin was followed by a continuous infusion of 12 nmol of ghrelin via an osmotic mini-pump for 15 hrs. Twenty hours after CLP, brain ghrelin levels, serum HMGB1 levels, ileal mucosal permeability to fluorescein isothiocyanate dextran, bacterial counts in the mesenteric lymph nodes complex, and gut water content were determined. In additional groups of animals, bilateral trunk vagotomy was performed at 5 hrs after CLP before ghrelin injection. Furthermore, to confirm the role of central ghrelin receptors in ghrelin's effect, ghrelin (1 nmol) was administered through intracerebroventricular injection at 5 hrs after CLP. Our results showed that brain levels of ghrelin decreased by 34% at 20 hrs after CLP. Intravenous administration of ghrelin completely restored brain levels of ghrelin, significantly reduced the elevated HMGB1 levels, and attenuated gut barrier dysfunction. Vagotomy eliminated ghrelin's inhibition of HMGB1 and attenuation of gut barrier dysfunction. Intracerebroventricular injection of ghrelin decreased serum HMGB1 levels and ameliorated gut barrier dysfunction., Conclusions: Ghrelin reduces serum HMGB1 levels and ameliorates gut barrier dysfunction in sepsis by vagus nerve activation via central ghrelin receptors. Ghrelin can be further developed as a novel agent to protect gut barrier function in sepsis.

We recently demonstrated that early administration of rat adrenomedullin (AM), a vasoactive peptide, in combination with its binding protein (human AMBP-1) produces various beneficial effects in sepsis. Human AM is a 52-amino acid peptide, but rat AM differs from human AM, having only 50 amino acid residues, with two amino acid deletions and six substitutions. It remains unknown whether a combination of human AM and human AMBP-1 (AM/AMBP-1) is also beneficial in sepsis and, if so, whether human AM/AMBP-1 reverses established sepsis in rats. To test the effects of human AM/AMBP-1, we induced sepsis in male adult rats by cecal ligation and puncture (CLP). At 10 h after CLP (i.e., severe sepsis), human AM (12-48 microg/kg body weight) was administered in combination with human AMBP-1 (40-160 microg/kg body weight). Vehicle-treated animals received a nonspecific human plasma protein (albumin). Blood and intestinal samples were collected at 20 h for various measurements. In additional groups of septic animals, the gangrenous cecum was surgically excised at 20 h after CLP. The 10-day survival was recorded. Our results showed that tissue injury, as evidenced by increased levels of transaminases and lactate, was present at 20 h after CLP. Proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6 were significantly elevated. Gut barrier dysfunction, manifested by increased mucosal permeability to hydrophilic macromolecules and increased bacterial translocation to mesenteric lymph nodes, also occurred at 20 h after CLP. Administration of human AM/AMBP-1 in established sepsis markedly attenuated tissue injury, reduced proinflammatory cytokine levels, ameliorated intestinal-barrier dysfunction, and improved the survival rate from 47% to 67%-80%. Thus, human AM/AMBP-1 can be further developed as a safe and effective therapy for patients with established sepsis.

Background: Norepinephrine (NE) modulates the responsiveness of macrophages to proinflammatory stimuli through the activation of adrenergic receptors (ARs). Being part of the stress response, early increases of NE in sepsis sustain adverse systemic inflammatory responses. The intestine is an important source of NE release in the early stage of cecal ligation and puncture (CLP)-induced sepsis in rats, which then stimulates TNF-alpha production in Kupffer cells (KCs) through the activation of the alpha(2)-AR. It is important to know which of the three alpha(2)-AR subtypes (i.e., alpha(2A), alpha(2B) or alpha(2C)) is responsible for the upregulation of TNF-alpha production. The aim of this study was to determine the contribution of alpha(2A)-AR in this process., Methodology/principal Findings: Adult male rats underwent CLP and KCs were isolated 2 h later. Gene expression of alpha(2A)-AR was determined. In additional experiments, cultured KCs were incubated with NE with or without BRL-44408 maleate, a specific alpha(2A)-AR antagonist, and intraportal infusion of NE for 2 h with or without BRL-44408 maleate was carried out in normal animals. Finally, the impact of alpha(2A)-AR activation by NE was investigated under inflammatory conditions (i.e., endotoxemia and CLP). Gene expression of the alpha(2A)-AR subtype was significantly upregulated after CLP. NE increased the release of TNF-alpha in cultured KCs, which was specifically inhibited by the alpha(2A)-AR antagonist BRL-44408. Equally, intraportal NE infusion increased TNF-alpha gene expression in KCs and plasma TNF-alpha which was also abrogated by co-administration of BRL-44408. NE also potentiated LPS-induced TNF-alpha release via the alpha(2A)-AR in vitro and in vivo. This potentiation of TNF-alpha release by NE was mediated through the alpha(2A)-AR coupled Galphai protein and the activation of the p38 MAP kinase. Treatment of septic animals with BRL-44408 suppressed TNF-alpha, prevented multiple organ injury and significantly improved survival from 45% to 75%., Conclusions/significance: Our novel finding is that hyperresponsiveness to alpha(2)-AR stimulation observed in sepsis is primarily due to an increase in alpha(2A)-AR expression in KCs. This appears to be in part responsible for the increased proinflammatory response and ensuing organ injury in sepsis. These findings provide important feasibility information for further developing the alpha(2A)-AR antagonist as a new therapy for sepsis.

Introduction: Protein disulfide isomerase (PDI) is an important factor for the protein modification step in the post-translational event. PDI plays an essential role in cell survival under various stress conditions. It has been reported that PDI can serve as a negative regulator of nuclear factor-kappa-B (NF-kappaB) and that it can inhibit lipopolysaccharide (LPS)-induced proinflammatory cytokine production in macrophages. Thus, PDI may be an intracellular anti-inflammatory molecule. Although we have previously shown that Kupffer cell-derived proinflammatory cytokines cause liver injury in sepsis, the effect of sepsis on PDI expression as well as the effect of PDI inhibition on cytokine production have not been investigated. We therefore hypothesized that sepsis downregulates PDI expression and that the inhibition of PDI promotes proinflammatory cytokine production., Method: Adult male rats were subjected to sepsis by cecal ligation and puncture (CLP) or endotoxemia (continuous infusion of 1 microg/kg body weight LPS by an osmotic pump) for 20 hours. Hepatic tissues were collected and PDI gene expression was determined. In additional experiments, cells from a macrophage-like cell line, RAW 264.7, were treated with 100 ng/mL LPS for 4 hours and protein expressions were measured. RAW 264.7 cells were also treated with bacitracin, a specific PDI inhibitor, for 24 hours, and tumor necrosis factor-alpha (TNF-alpha) gene and protein expression as well as its release in the cell supernatant were determined. To further confirm the beneficial effect of PDI in sepsis, RAW 264.7 cells were transfected with PDI short interfering RNA (siRNA) and PDI gene expression and TNF-alpha release were measured by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively., Results: PDI gene expression was significantly decreased by 28% and 69% at 20 hours after CLP or LPS infusion, respectively. LPS also decreased PDI protein expression by 33% in RAW 264.7 cells. Incubation of RAW 264.7 cells with bacitracin significantly increased TNF-alpha gene expression and TNF-alpha release as well as its cellular levels in a dose-dependent manner. Transfection of RAW 264.7 cells with PDI siRNA produced an average 36.8% inhibition of the PDI gene expression. This downregulation was correlated with a 3.19-fold increase in TNF-alpha release into the cell supernatant., Conclusion: Taken together, these results suggest that downregulation of PDI by sepsis significantly increases proinflammatory cytokine production. Thus, prevention of PDI downregulation in sepsis may be a novel approach to attenuate hyperinflammation and to reduce tissue injury under such conditions.

The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is anti-inflammatory in a cell-based system and in animal models of endotoxemia. We have shown that PPAR-gamma gene expression is downregulated in macrophages after lipopolysaccharide (LPS) stimulation. However, it remains unknown whether hepatic PPAR-gamma is altered in sepsis and, if so, whether LPS directly downregulates PPAR-gamma. To study this, rats were subjected to sepsis by cecal ligation and puncture (CLP). Hepatic tissues were harvested at 5, 10, and 20 h after CLP. PPAR-gamma gene expression and protein levels were determined by RT-PCR and Western blot analysis, respectively. The results showed that PPAR-gamma gene expression decreased at 10 and 20 h and that its proteins levels were reduced at 20 h after CLP. PPAR-gamma levels were also decreased in animals that were administered LPS. To determine the direct effects of LPS on PPAR-gamma downregulation, LPS binding agent polymyxin B (PMB) was administered intramuscularly after CLP. The administration of PMB significantly reduced plasma levels of endotoxin, but it did not prevent the downregulation of PPAR-gamma expression. We found that circulating levels of TNF-alpha still remained significantly elevated in PMB-treated septic animals. We, therefore, hypothesize that the decrease of PPAR-gamma expression is TNF-alpha dependent. To investigate this, Kupffer cells (KCs) were isolated from normal rats and stimulated with LPS or TNF-alpha. TNF-alpha significantly attenuated PPAR-gamma gene expression in KCs. Although LPS decreased PPAR-gamma in KCs, the downregulatory effect of LPS was blocked by the addition of TNF-alpha-neutralizing antibodies. Furthermore, the administration of TNF-alpha-neutralizing antibodies to animals before the onset of sepsis prevented the downregulation of PPAR-gamma in sepsis. We, therefore, conclude that LPS downregulates PPAR-gamma expression during sepsis via an increase in TNF-alpha release.

Our previous studies have shown that norepinephrine (NE) upregulates proinflammatory cytokines by activating alpha(2)-adrenoceptor. Therefore, modulation of the sympathetic nervous system represents a novel treatment for sepsis. We have also shown that a novel stomach-derived peptide, ghrelin, is downregulated in sepsis and that its intravenous administration decreases proinflammatory cytokines and mitigates organ injury. However, it remains unknown whether ghrelin inhibits sympathetic activity through central ghrelin receptors [i.e., growth hormone secretagogue receptor 1a (GHSR-la)] in sepsis. To study this, sepsis was induced in male rats by cecal ligation and puncture (CLP). Ghrelin was administered through intravenous or intracerebroventricular injection 30 min before CLP. Our results showed that intravenous administration of ghrelin significantly reduced the elevated NE and TNF-alpha levels at 2 h after CLP. NE administration partially blocked the inhibitory effect of ghrelin on TNF-alpha in sepsis. GHSR-la inhibition by the administration of a GHSR-la antagonist, [d-Arg(1),d-Phe(5), d-Trp(7,9),Leu(11)]substance P, significantly increased both NE and TNF-alpha levels even in normal animals. Markedly elevated circulating levels of NE 2 h after CLP were also significantly decreased by intracerebroventricular administration of ghrelin. Ghrelin's inhibitory effect on NE release was completely blocked by intracerebroventricular injection of the GHSR-1a antagonist or a neuropeptide Y (NPY)/Y(1) receptor antagonist. However, ghrelin's downregulatory effect on TNF-alpha release was only partially diminished by these agents. Thus ghrelin has sympathoinhibitory properties that are mediated by central ghrelin receptors involving a NPY/Y1 receptor-dependent pathway. Ghrelin's inhibitory effect on TNF-alpha production in sepsis is partially because of its modulation of the overstimulated sympathetic nerve activation.

Rationale: Our study has shown that plasma levels of ghrelin, a stomach-derived peptide, are significantly reduced in sepsis, and that ghrelin administration improves organ blood flow via a nuclear factor (NF)-kappaB-dependent pathway. However, it remains unknown whether ghrelin has any protective effects on severe sepsis-induced acute lung injury (ALI) and, if so, whether inhibition of NF-kappaB plays any role in it., Objectives: To test the hypothesis that ghrelin reduces severe sepsis-induced ALI and mortality through inhibition of NF-kappaB., Methods: Sepsis was induced in rats by cecal ligation and puncture (CLP). Five hours after CLP, a bolus intravenous injection of 2 nmol of ghrelin was followed by continuous infusion of 12 nmol of ghrelin via a minipump for 15 hours. Samples were harvested 20 hours post-CLP (i.e., severe sepsis). Pulmonary levels of ghrelin and proinflammatory cytokines were measured by ELISA. NF-kappaB p65 and IkappaBalpha expression and NF-kappaB activity were measured by Western blot analysis and ELISA, respectively. Pulmonary blood flow was measured with radioactive microspheres. In additional animals, the necrotic cecum was excised 20 hours post-CLP and 10-day survival was recorded., Measurements and Main Results: Pulmonary levels of ghrelin decreased significantly 20 hours post-CLP. Ghrelin administration restored pulmonary levels of ghrelin, reduced lung injury, increased pulmonary blood flow, down-regulated proinflammatory cytokines, inhibited NF-kappaB activation, and improved survival in sepsis. Administration of a specific ghrelin receptor antagonist worsened the survival rate after CLP and cecal excision., Conclusions: Ghrelin can be developed as a novel treatment for severe sepsis-induced ALI. The protective effect of ghrelin is mediated through inhibition of NF-kappaB.

Objective: To test the hypothesis that administration of ghrelin attenuates inflammatory responses in sepsis through vagal nerve stimulation., Summary Background Data: Ghrelin has been demonstrated to possess multiple functions, including stimulation of the vagus nerve. Our recent study has shown that plasma levels of ghrelin were significantly reduced in sepsis; and ghrelin administration improved organ perfusion and function. However, it remained unknown whether ghrelin also decreases proinflammatory cytokines in sepsis and, if so, whether the down-regulatory effect of ghrelin is mediated by activation of the vagus nerve., Methods: Male rats were subjected to sepsis by cecal ligation and puncture (CLP). At 5 hours after CLP, a bolus intravenous injection of 2 nmol ghrelin was followed by a continuous infusion of 12 nmol ghrelin via a primed 200-microL Alzet mini-pump for 15 hours. At 20 hours after CLP, plasma and peritoneal fluid levels of TNF-alpha and IL-6 were determined. The direct effect of ghrelin on cytokine production was studied using cultured normal rat Kupffer cells or peritoneal macrophages stimulated by lipopolysaccharide (LPS). In additional animals, vagotomy or sham vagotomy was performed in sham and septic animals immediately prior to ghrelin administration and cytokine levels were then measured., Results: Ghrelin significantly reduced TNF-alpha and IL-6 levels in sepsis. In contrast, ghrelin did not inhibit TNF-alpha and IL-6 release from LPS-stimulated Kupffer cells or peritoneal macrophages. However, vagotomy, but not sham vagotomy, prevented ghrelin's down-regulatory effect on TNF-alpha and IL-6 production., Conclusions: Ghrelin down-regulates proinflammatory cytokines in sepsis through activation of the vagus nerve. Pharmacologic stimulation of the vagus nerve may offer a novel approach of anti-sepsis therapy.

Background: The "double-hit" model of hemorrhage and sepsis mimics the critically ill patient admitted to the surgical intensive care unit. Although the protein expression of a cytochrome (CYP) P-450 isoform CYP1A2 is reduced in the late stage of sepsis, the effect of hemorrhage on CYP isoforms and the anti-inflammatory nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has not been investigated. We hypothesized that hemorrhage down-regulates CYP isoforms and PPAR-gamma in the liver, which plays an important role in producing tissue injury and proinflammatory responses after the subsequent sepsis (i.e., double-hit)., Materials and Methods: Male Sprague Dawley rats were divided into four groups. Animals in the double-hit group underwent hemorrhage (40 +/- 2 mmHg for 90 min) followed by fluid resuscitation. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) 20 h after hemorrhage, and the animals were sacrificed 4 h after CLP. Rats in the hemorrhage-alone group were sacrificed 20 h after the insult. Rats in the CLP-alone group were sacrificed 4 h after the onset of sepsis. Animals in the sham-operated group underwent neither hemorrhage nor CLP. The gene expression of P-450 isoforms (i.e., CYP1A2 and 2C11) and PPAR-gamma in the liver was determined using RT-PCR. Serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate, and proinflammatory cytokines (i.e., IL-6, TNF-alpha) were also assessed., Results: In the hemorrhage-alone group, hepatic mRNA expression of CYP1A2, CYP2C11, and PPAR-gamma was significantly down-regulated 20 h after the initial stress compared with sham-operated rats. Double-hit did not appear to further decrease CYP and PPAR-gamma gene expression. In contrast, serum levels of AST, ALT, lactate, IL-6, and TNF-alpha did not change significantly in either hemorrhaged or septic animals. Those organ injury indicators and cytokines, however, were significantly elevated after the double-hit of hemorrhage and sepsis., Conclusions: Hepatic CYP1A2, CYP2C11, and PPAR-gamma were down-regulated after the initial stress (hemorrhage). These down-regulated CYPs and PPAR-gamma seem to work as important factors contributing to the progression of organ injury and proinflammatory responses after the second stress (CLP).

Objective: Although phytochemical curcumin has been shown to possess anti-inflammatory properties, it remains unknown whether this agent has any beneficial effects in sepsis. The purpose of this study was to demonstrate whether curcumin protects septic animals and, if so, whether activation of peroxisome proliferator-activated receptor (PPAR)-gamma, an anti-inflammatory nuclear receptor, plays any role., Design: Prospective, controlled, and randomized animal study., Setting: A research institute laboratory., Subjects: Male Sprague-Dawley rats., Interventions: A bolus injection of 0.2 micromol of curcumin was given intravenously to male adult rats, followed by continuous infusion of curcumin (0.24 micromol/day) for 3 days via a primed 2-mL mini-pump. The rats were then subjected to sepsis by cecal ligation and puncture (CLP)., Measurements and Main Results: Serum levels of liver enzymes (alanine aminotransferase and aspartate aminotransferase), lactate, albumin, and tumor necrosis factor (TNF)-alpha were measured at 20 hrs after CLP (i.e., late stage of sepsis). In addition, a 10-day survival curve was conducted following CLP and cecal excision with or without curcumin treatment. Furthermore, macrophages cell line RAW 264.7 cells were treated with curcumin followed by stimulation with endotoxin. TNF-alpha and PPAR-gamma expression were then measured. The results indicate that intravenous administration of curcumin before the onset of sepsis attenuated tissue injury, reduced mortality, and decreased the expression of TNF-alpha in septic animals. Similar results were also found when curcumin was administered after the onset of sepsis. Moreover, the down-regulated PPAR-gamma in the liver at 20 hrs after CLP was significantly improved by curcumin treatment. Concurrent administration of curcumin and GW9662, a specific PPAR-gamma antagonist, completely abolished the beneficial effects of curcumin under such conditions. In cultured RAW 264.7 cells, curcumin inhibited endotoxin-induced increases in TNF-alpha expression and markedly up-regulated PPAR-gamma expression without affecting cell viability. Curcumin also prevented morphologic alterations in macrophages induced by endotoxin., Conclusions: The protective effect of curcumin makes it or its analogues strong candidates as a novel therapy for sepsis. The beneficial effect of curcumin appears to be mediated by up-regulation of nuclear receptor PPAR-gamma.

In sepsis, several cell types (e.g., lymphocytes) undergo apoptosis and have the potential to harm the host if not cleared by professional phagocytes. Apoptotic cells display "eat me" signals such as phosphatidylserine that can be readily recognized by phagocytes. For full engulfment of these cells, binding to integrin alpha(v)beta(3), mediated by the bridging protein, milk fat globule epidermal growth factor-factor VIII (MFG-E8), is necessary. We hypothesized that, in sepsis, phagocytosis of apoptotic cells is impaired due to decreased MFG-E8 expression and that adoptive transfer of exosomes containing MFG-E8 is beneficial. Sepsis was induced in rats by cecal ligation and puncture (CLP) and MFG-E8 expression assessed by Western blot 20 h later. Dendritic cells were generated from bone marrow cells, and secreted exosomes were collected and injected into CLP animals. Plasma cytokines (enzyme-linked immunosorbent assay) and thymocyte apoptosis (TC-Ao, annexin V) were assessed. The ability of peritoneal macrophages from septic animals to engulf apoptotic cells was determined in an ex vivo phagocytosis assay. A 10-day survival study was conducted. Cecal ligation and puncture reduced MFG-E8 protein levels in the spleen and liver by 48% and 70%, respectively, and increased TC-Ao by 1.6-fold. Injection of MFG-E8-containing exosomes, however, led to a 33% reduced detection of TC-Ao, without directly inhibiting apoptosis. In fact, peritoneal macrophages from exosome-treated rats displayed a 2.8-fold increased ability to phagocytose apoptotic thymocytes. Inhibition of MFG-E8 before injection of exosomes completely abrogated the enhanced phagocytosis. Treatment with bone marrow dendritic cell-derived exosomes also reduced plasma tumor necrosis factor alpha and interleukin (IL)-6 levels and improved survival from 44% to 81%. We conclude that, by providing the indispensable factor MFG-E8 for complete engulfment of apoptotic cells, these exosomes lead to an attenuation of the systemic inflammatory response and overall beneficial effect in sepsis.

We have previously shown that administration of adrenomedullin (AM) and AM binding protein-1 (AMBP-1) in combination maintains cardiovascular stability and reduces mortality in a rat model of sepsis. However, it is unknown whether AMBP-1 is reduced under the septic condition and, if so, whether lipopolysaccharide (LPS) plays a role in down-regulating AMBP-1. To determine this, male adult Sprague-Dawley rats were subjected to either polymicrobial sepsis by cecal ligation and puncture (CLP), or endotoxemia by intraperitoneal injection of LPS (15 mg/kg body weight). In an additional group of animals, LPS neutralizing agent polymyxin B (PMB) was given intramuscularly at 0.5 h before and 9 h after CLP. At 20 h after CLP (i.e. the late stage of sepsis) or endotoxemia, hepatic tissue and blood samples were collected. Hepatic AMBP-1 gene expression along with hepatic and plasma AMBP-1 protein levels were measured by RT-PCR and Western blot analysis, respectively. Our results showed that hepatic AMBP-1 gene expression decreased by 65%, hepatic AMBP-1 protein levels decreased by 72%, and plasma levels of AMBP-1 decreased by 59% at 20 h after CLP. Similar results were also seen in the animals receiving LPS injection. Administration of PMB, however, prevented the downregulation of AMBP-1 expression at 20 h after CLP. Thus, AMBP-1 is downregulated in the late phase of sepsis, and LPS plays a critical role in the reduction of AMBP-1.

Objectives: Severe sepsis is associated with increased total peripheral resistance (TPR) and decreased organ blood flow, in which endothelin-1 (ET-1) plays an important role. Plasma levels of ghrelin, a newly-identified endogenous ligand for growth hormone secretagogue receptor and a potent vasodilatory peptide, are significantly reduced in sepsis. Ghrelin downregulation heralds the hypodynamic response in severe sepsis. Therefore, we hypothesized that the administration of exogenous ghrelin improves organ blood flow by downregulation of ET-1 under such conditions., Methods: Male adult Sprague-Dawley rats were subjected to sepsis by cecal ligation and puncture (CLP). At 5 h post-CLP, a bolus intravenous injection of 2 nmol ghrelin was followed by a continuous infusion of 12 nmol ghrelin via a primed mini-pump over 15 h. At 20 h post-CLP (i.e., severe sepsis), cardiac output (CO), stroke volume (SV), TPR, and organ blood flow were measured using (141)Ce-microspheres. Plasma ET-1 levels and preproET-1 gene expression in the liver, small intestine, and kidneys were measured by ELISA and RT-PCR, respectively. The direct effect of ghrelin on ET-1 production was studied using cultured human umbilical vein endothelial cells (HUVECs) treated with tumor necrosis factor-alpha (TNF-alpha)., Results: Ghrelin administration reduced TPR, increased CO, SV, and organ blood flow, downregulated preproET-1 gene expression, and decreased plasma levels of ET-1 in sepsis. Ghrelin inhibited TNF-alpha-induced ET-1 release from HUVECs in a dose-dependent manner. Moreover, ghrelin inhibited TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) in HUVECs., Conclusions: The improvement of tissue perfusion by ghrelin in severe sepsis appears to be mediated by downregulation of ET-1 involving a NF-kappaB-dependent pathway.

In the history of medicine, the interaction between mind and body has been repeatedly proposed. However, the influence of the nervous system on the immune regulation has, until now, drawn little attention. In this regard, the adrenergic system has been explored, and mainly catecholamine-mediated anti-inflammatory effects have been described. These inhibitory effects of epinephrine and norepinephrine were found to be mediated by beta2-adrenoceptors expressed on mononuclear cells. Recently, the role of the parasympathetic nervous system in the local anti-inflammatory reflex has been investigated. Stimulation of the vagus nerve decreases the pro-inflammatory response of macrophages via alpha7-cholinergic receptors. Thus, both the sympathetic and parasympathetic nervous systems are thought to work hand in hand in their anti-inflammatory responses. Here we discuss the deteriorating effects of the release of norepinephrine in sepsis. We have discovered that organ dysfunction in severe sepsis is mediated at least in part by an increase in pro-inflammatory cytokine release from Kupffer cells, which is caused by a priming via gut-derived norepinephrine. The sympathetic nervous system and gut-derived norepinephrine mediate the pro-inflammatory effects by activating alpha2A-adrenoceptor on Kupffer cells. In this review, we will focus on the differential function of the noradrenergic system on local and systemic inflammatory responses and the possibilities of the modulation of sympathetic outflow by centrally active inhibitors such as the novel peptide ghrelin or NMDA-receptor blockers. Furthermore, we will introduce the new concept of "sympathetic excitotoxicity in sepsis" characterized by the neurogenic priming of the systemic pro-inflammatory response.

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.

Objective: We have recently shown that administration of human inter-alpha inhibitor proteins (IalphaIp) very early after the onset of sepsis maintains cardiovascular stability and reduced mortality. However, it remains unknown whether injection of IalphaIp at later time points of sepsis has any beneficial effects. We therefore hypothesized that IalphaIp and its active component bikunin are reduced in sepsis and that the delayed administration of IalphaIp also improves survival rate., Design: : Prospective, controlled, and randomized animal study., Setting: A research institute laboratory., Subjects: : Male adult Sprague-Dawley rats., Interventions: Rats were subjected either to polymicrobial sepsis by cecal ligation and puncture (CLP) or to sham operation followed by the administration of normal saline solution (i.e., fluid resuscitation)., Measurements and Main Results: : Bikunin gene expression in the liver was measured by reverse transcription polymerase chain reaction. Plasma concentrations of IalphaIp were determined by Western blot at 5 and 20 hrs after CLP. IalphaIp clearance was assessed by injecting radioactive IalphaIp at 12 hrs post-CLP, and the half-life was determined. In addition, IalphaIp (30 mg/kg of body weight) or vehicle was administered at 1, 5, or 10 hrs (single treatment) or at both 10 and 20 hrs (double treatment) post-CLP. The necrotic cecum was excised at 20 hrs post-CLP, and 10-day survival was recorded. The results indicate that bikunin gene expression decreased significantly at 20 hrs post-CLP. Moreover, IalphaIp concentrations decreased significantly at 5 and 20 hrs post-CLP, and its half-life increased from 5.6 +/- 0.3 hrs to 11.8 +/- 2.7 hrs (p <.05), suggesting down-regulation of IalphaIp in sepsis despite the decreased clearance. Administration of IalphaIp at 1 hr post-CLP improved the survival rate from 50% to 92% (p <.05), whereas there was no significant improvement when IalphaIp was administrated at 5 or 10 hrs post-CLP. However, double injection of IalphaIp at 10 and 20 hrs post-CLP (i.e., severe sepsis) increased the survival rate from 44% to 81% (p <.05)., Conclusion: Since delayed but repeated administration of human IalphaIp improves survival after CLP, this compound appears to be a useful agent for the treatment of severe sepsis.

Objective: Sepsis is characterized by an early, hyperdynamic phase and a late, hypodynamic phase. Although studies have shown that cytochrome P450 (CYP) plays an important role in the regulation of vascular reactivity, alterations of vascular CYP isoforms in sepsis remain unknown. Since CYP2C11 and CYP2J4 convert arachidonic acid to vasodilative epoxyeicosatrienoic acids, and CYP4A3 metabolizes arachidonic acid to both epoxyeicosatrienoic acids and vasoconstrictive 19,20-hydroxyeicosatetraenoic acid, the aim of this study was to examine the expression of these isoforms in sepsis and their association with hemodynamic changes., Design: Prospective, controlled, and randomized animal study., Setting: An institute research laboratory., Subjects: Male adult Sprague-Dawley rats were subjected either to polymicrobial sepsis by cecal ligation and puncture or to sham operation followed by the administration of normal saline solution (i.e., fluid resuscitation)., Interventions: At 5 hrs (early sepsis) or 20 hrs (late sepsis) after cecal ligation and puncture, blood vessel-rich tissues (i.e., lungs) were harvested. The expression of CYP isoforms at both messenger RNA and protein levels was determined by reverse transcription polymerase chain reaction and Western blot analysis (CYP2C11), respectively. Hemodynamic variables were measured by radioactive microspheres., Main Results: The results indicate that the gene expression of CYP2C11 and CYP2J4 was significantly down-regulated at 20 hrs after cecal ligation and puncture, whereas the expression of CYP4A3 was markedly up-regulated at 5 hrs. The protein concentrations of CYP2C11 also decreased significantly at 20 hrs after cecal ligation and puncture. Although total peripheral resistance markedly increased, mean arterial pressure did not change significantly at 20 hrs after the onset of sepsis. In contrast, cardiac output and pulmonary perfusion markedly decreased in late sepsis., Conclusions: Since the up-regulated CYP4A3 is associated with the early, hyperdynamic phase of sepsis and the down-regulated CYP2C11 and CYP2J4 are associated with the late, hypodynamic phase, vascular CYP isoforms that metabolize arachidonic acid may be involved in regulating the cardiovascular response during the progression of sepsis.

The cardiovascular response to sepsis is characterized by an early, hyperdynamic phase followed by a late, hypodynamic phase. Ghrelin, a newly-identified endogenous ligand for growth hormone secretagogue receptor (i.e., ghrelin receptor), was recently demonstrated to be a potent vasoactive peptide in addition to its effects on growth hormone release and energy homeostasis. We have shown that ghrelin (via its receptor) may play an important role in regulating cardiovascular responses in the progression of polymicrobial sepsis. However, it remains unknown whether the clearance of this peptide is altered in sepsis. To determine this, male adult rats were injected with 125I-ghrelin through the jugular vein at 5 or 20 h after cecal ligation and puncture (CLP, i.e., sepsis model) or sham operation. The blood sample was collected every 2 min for 30 min for determining half-life (t1/2). Tissue samples (i.e., kidneys, liver, brain, heart, lungs, spleen, stomach, small intestine, large intestine, skin and muscle) were then harvested. The radioactivities of samples were counted. The results indicate that 125I-ghrelin's t1/2 and its distribution were not significantly altered in early sepsis (5 h after CLP). However, the t1/2 increased significantly in late sepsis (20 h after CLP). Tissue distribution of 125I-ghrelin was far greater in the kidneys than in any other tissues tested in both sham and septic animals. Moreover, the kidneys and liver had significantly less radioactive uptake at 20 h after CLP, but the radioactivity in blood was much higher at the same time point. There were no significant changes in 125I-ghrelin distribution in other organs at the late stage of sepsis. These results indicate that the kidneys are the primary site of ghrelin clearance, which is significantly diminished in late sepsis. In addition, the liver also plays a role in the clearance of ghrelin, which was also reduced in late sepsis. The decreased clearance of ghrelin by the kidneys and liver may be due to renal and hepatic dysfunctions under such conditions.

Objective: To study the gene expression of adhesion molecules in pulmonary and hepatic microvascular endothelial cells during sepsis in mice., Methods: Male mice were subjected to cecal ligation and puncture (CLP) and microvascular endothelial cells in pulmonary and hepatic tissues were harvested at 3 hours (early sepsis) and 12 hours (late sepsis) after CLP, respectively. Gene expression of the adhesion molecules was assessed by reverse transcription polymerase chain reaction (RT-PCR). Simultaneously, the alterations of myeloperoxidase (MPO) activity in pulmonary and hepatic tissues were also examined., Results: E-selectin mRNA levels markedly increased at 3 hours after CLP in both pulmonary and hepatic microvascular endothelial cells, then they returned to the normal level at 12 hours after CLP. Increases in intercellular adhesion molecule-1 (ICAM-1) mRNA levels were found at 3 hours after CLP in both pulmonary and hepatic microvascular endothelial cells, and these levels became higher at 12 hours after CLP. Adhesion molecule-1 (VCAM-1) mRNA expression of vascular cells also increased significantly at 3 hours and 12 hours after CLP in both pulmonary and hepatic microvascular endothelial cells. The level of VCAM-1 mRNA in hepatic microvascular endothelial cells was higher at 3 hours than that at 12 hours after CLP, while the level of VCAM-1 mRNA in pulmonary microvascular endothelial cells was higher at 12 hours than that at 3 hours after CLP. The MPO activity in pulmonary and hepatic tissues increased at 3 hours after CLP, compared with that of the sham group. They both declined significantly at 12 hours after CLP, but they were still higher than that of the sham group., Conclusions: The up-regulation of the gene expression of adhesion molecules in pulmonary and hepatic microvascular endothelial cells is an important step for the migration and accumulation of leukocytes at the site of inflammation, which plays a critical role in organ damage during sepsis. And the contribution of the heterogeneity of endothelial cells in organs' vulnerability during sepsis is worth a further investigation.

Background: Sepsis remains a major health issue without an effective therapy. Ferroptosis, an iron‐dependent programmed cell death, has been proposed to be related to the pathogenesis of sepsis. Irisin, a myokine released during exercise, improves mitochondrial function under various conditions. Ferroptosis is closely related to mitochondrial function. However, the role of irisin in sepsis‐induced ferroptosis and mitochondrial dysfunction in the liver remained unknown. Thus, we hypothesize that irisin treatment suppresses ferroptosis and improves mitochondrial function in sepsis. Methods: To study this, we first explored the role of serum irisin levels in patients with sepsis, and then determined the effect of irisin administration on ferroptosis and mitochondrial function in the liver of septic mice. Results: Serum irisin levels were decreased and negatively correlated with the APACHE II scores in patients with sepsis. In mice subjected to cecal ligation and puncture (CLP), exogenous irisin administration suppressed ferroptosis, inhibited inflammatory response, decreased reactive oxygen species (ROS) production, restored abnormal mitochondrial morphology, and increased mtDNA copy number and adenosine triphosphate (ATP) content. The effect of irisin on ferroptosis was confirmed in LPS‐treated hepatocytes and CLP‐induced septic mice. Inhibition of glutathione peroxidase 4 (GPX4), a central regulator of ferroptosis, reduced irisin's protective effects in LPS‐treated hepatocytes and CLP‐induced septic mice, while blocking the irisin receptor with RGD peptide or Echistain decreased irisin‐induced GPX4 expression. Conclusions: Serum irisin levels are decreased and negatively correlated with disease severity in patients with sepsis, and irisin treatment suppresses ferroptosis and restores mitochondrial function in experimental sepsis. Irisin may offer therapeutic potential in the management of sepsis. [ABSTRACT FROM AUTHOR]

The threat of nuclear terrorism has led to growing worldwide concern about exposure to radiation. Acute radiation syndrome, or radiation sickness, develops after whole-body or a partial-body irradiation with a high dose of radiation. In the terrorist radiation exposure scenario, however, radiation victims likely suffer from additional injuries such as trauma, burns, wounds or sepsis. Thus, high-dose radiation injuries and appropriate therapeutic interventions must be studied. Despite advances in our understanding of the pathophysiology of radiation injury, very little information is available on the therapeutic approaches to radiation combined injury. In this review, we describe briefly the pathological consequences of ionizing radiation and provide an overview of the animal models of radiation combined injury. We highlight the combined radiation and sepsis model we recently established and suggest the use of ghrelin, a novel gastrointestinal hormone, as a potential therapy for radiation combined injury. [ABSTRACT FROM AUTHOR]

Abstract: Polymicrobial sepsis is characterized by an early, hyperdynamic phase followed by a late hypodynamic phase. Adrenomedullin (AM), a vasodilatory peptide, inhibits this transition from the early phase to the late phase. Adrenomedullin binding protein-1 (AMBP-1) enhances AM-mediated activities. The decrease of AMBP-1 levels in late sepsis reduces the vascular response to AM and produces the hypodynamic phase. Studies have indicated that the administration of LPS downregulates AMBP-1 production in the liver. Since hepatocytes are the primary source of AMBP-1 biosynthesis in the liver, we employed a co-culture strategy using hepatocyte and Kupffer cells to determine whether LPS directly or by increasing pro-inflammatory cytokines from Kupffer cells downregulates AMBP-1 production. Hepatocytes and Kupffer cells isolated from rats were co-cultured and treated with LPS for 24 h. LPS significantly attenuated AMBP-1 protein expression in a dose-dependent manner. Since AMBP-1 is basically a secretory protein, cell supernatants from co-culture cells treated with LPS were examined for AMBP-1 protein levels. LPS treatment caused a dose related decrease in AMBP-1 protein secretion. Similarly, LPS treatment produced a significant decrease in AMBP-1 protein expression in hepatocytes and Kupffer cells cultured using transwell inserts. LPS had no direct effect on AMBP-1 levels in cultured hepatocytes or Kupffer cells alone. To confirm that the observed effects in co-culture were due to the cytokines released from Kupffer cells, hepatocytes were treated with IL-1β or TNF-α for 24 h and AMBP-1 expression was examined. The results indicated that both cytokines significantly inhibited AMBP-1 protein levels. Thus, pro-inflammatory cytokines released from Kupffer cells are responsible for downregulation of AMBP-1. [Copyright &y& Elsevier]

Background: Acute renal failure secondary to ischemia and reperfusion (I/R) injury poses a significant burden on both surgeons and patients. It carries a high morbidity and mortality rate and no specific treatment currently exists. Major causes of renal I/R injury include trauma, sepsis, hypoperfusion, and various surgical procedures. We have demonstrated that adrenomedullin (AM), a novel vasoactive peptide, combined with AM binding protein-1 (AMBP-1), which augments the activity of AM, is beneficial in various disease conditions. However, it remains unknown whether human AM/AMBP-1 provides any beneficial effects in renal I/R injury. The objective of our study therefore was to determine whether administration of human AM/AMBP-1 can prevent and/or minimize damage in a rat model of renal I/R injury. Methods: Male adult rats were subjected to renal I/R injury by bilateral renal pedicle clamping with microvascular clips for 60 min followed by reperfusion. Human AM (12 μg/kg BW) and human AMBP-1 (40 μg/kg BW) or vehicle (52 μg/kg BW human albumin) were given intravenously over 30 min immediately following the clip removal (i.e., reperfusion). Rats were allowed to recover for 24 h post-treatment, and blood and renal tissue samples were collected. Plasma levels of AM were measured using a radioimmunoassay specific for rat AM. Plasma AMBP-1 was measured by Western analysis. Renal water content and serum levels of systemic markers of tissue injury were measured. Serum and renal TNF-α levels were also assessed. Results: At 24 h after renal I/R injury, plasma levels of AM were significantly increased while plasma AMBP-1 was markedly decreased. Renal water content and systemic markers of tissue injury (e.g., creatinine, BUN, AST, and ALT) were significantly increased following renal I/R injury. Serum and renal TNF-α levels were also increased post injury. Administration of human AM/AMBP-1 decreased renal water content, and plasma levels of creatinine, BUN, AST, and ALT. Serum and renal TNF-α levels were also significantly decreased after AM/AMBP-1 treatment. Conclusion: Treatment with human AM/AMBP-1 in renal I/R injury significantly attenuated organ injury and the inflammatory response. Thus, human AM combined with human AMBP-1 may be developed as a novel treatment for patients with acute renal I/R injury. [Copyright &y& Elsevier]