Development of Procathepsin L (pCTS-L)-Inhibiting Lanosterol-Carrying Liposome Nanoparticles to Treat Lethal Sepsis.

The pathogenesis of microbial infections and sepsis is partly attributable to dysregulated innate immune responses propagated by late-acting proinflammatory mediators such as procathepsin L (pCTS-L). It was previously not known whether any natural product could inhibit pCTS-L-mediated inflammation or could be strategically developed into a potential sepsis therapy. Here, we report that systemic screening of a NatProduct Collection of 800 natural products led to the identification of a lipophilic sterol, lanosterol (LAN), as a selective inhibitor of pCTS-L-induced production of cytokines [e.g., Tumor Necrosis Factor (TNF) and Interleukin-6 (IL-6)] and chemokines [e.g., Monocyte Chemoattractant Protein-1 (MCP-1) and Epithelial Neutrophil-Activating Peptide (ENA-78)] in innate immune cells. To improve its bioavailability, we generated LAN-carrying liposome nanoparticles and found that these LAN-containing liposomes (LAN-L) similarly inhibited pCTS-L-induced production of several chemokines [e.g., MCP-1, Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted (RANTES) and Macrophage Inflammatory Protein-2 (MIP-2)] in human blood mononuclear cells (PBMCs). In vivo, these LAN-carrying liposomes effectively rescued mice from lethal sepsis even when the first dose was given at 24 h post the onset of this disease. This protection was associated with a significant attenuation of sepsis-induced tissue injury and systemic accumulation of serval surrogate biomarkers [e.g., IL-6, Keratinocyte-derived Chemokine (KC), and Soluble Tumor Necrosis Factor Receptor I (sTNFRI)]. These findings support an exciting possibility to develop liposome nanoparticles carrying anti-inflammatory sterols as potential therapies for human sepsis and other inflammatory diseases.

Cold-inducible RNA-binding protein mediates neuroinflammation in cerebral ischemia.

BACKGROUND: Neuroinflammation is a key cascade after cerebral ischemia. Excessive production of proinflammatory mediators in ischemia exacerbates brain injury. Cold-inducible RNA-binding protein (CIRP) is a newly discovered proinflammatory mediator that can be released into the circulation during hemorrhage or septic shock. Here, we examine the involvement of CIRP in brain injury during ischemic stroke. METHODS: Stroke was induced by middle cerebral artery occlusion (MCAO). In vitro hypoxia was conducted in a hypoxia chamber containing 1% oxygen. CIRP and tumor necrosis factor-α (TNF-α) levels were assessed by RT-PCR and Western blot analysis. RESULTS: CIRP is elevated along with an upregulation of TNF-α expression in mouse brain after MCAO. In CIRP-deficient mice, the brain infarct volume, induction of TNF-α, and activation of microglia are markedly reduced after MCAO. Using microglial BV2 cells, we demonstrate that hypoxia induces the expression, translocation, and release of CIRP, which is associated with an increase of TNF-α levels. Addition of recombinant murine (rm) CIRP directly induces TNF-α release from BV2 cells and such induction is inhibited by neutralizing antisera to CIRP. Moreover, rmCIRP activates the NF-κB signaling pathway in BV2 cells. The conditioned medium from BV2 cells exposed to hypoxia triggers the apoptotic cascade by increasing caspase activity and decreasing Bcl-2 expression in neural SH-SY5Y cells, which is inhibited by antisera to CIRP. CONCLUSION: Extracellular CIRP is a detrimental factor in stimulating inflammation to cause neuronal damage in cerebral ischemia. GENERAL SIGNIFICANCE: Development of an anti-CIRP therapy may benefit patients with brain ischemia.

Elevated Circulating Procathepsin L as a Potential Biomarker of Inflamm-aging.

Inflamm-aging is a condition of low-grade and chronic systemic inflammation characterized by a systemic increase in multiple inflammatory biomarkers such as tumor necrosis factor (TNF), interleukin 6 (IL-6), C-reactive protein (CRP), and CXCL9 (MIG) in experimental and clinical settings. However, despite the recent identification of extracellular procathepsin L (pCTS-L) as a novel mediator of inflammatory diseases such as sepsis, its possible role in inflamm-aging was previously not investigated. In the present study, we compared blood levels of pCTS-L and other 62 cytokines and chemokines between young and aged Balb/C mice by Western blotting and Cytokine Antibody Arrays. In light of the surprising finding of a marked increase in blood pCTS-L levels in aged mice, we propose that blood pCTS-L levels may serve as another biomarker of inflamm-aging. Given the capacity of pCTS-L in inducing various cytokines (e.g., TNF and IL-6), it will be important to test the hypothetic role of pCTS-L in inflamm-aging under experimental and clinical conditions.

Therapeutic potential of procathepsin L-inhibiting and progesterone-entrapping dimethyl-ß-cyclodextrin nanoparticles in treating experimental sepsis.

The pathogenic mechanisms of bacterial infections and resultant sepsis are partly attributed to dysregulated inflammatory responses sustained by some late-acting mediators including the procathepsin-L (pCTS-L). It was entirely unknown whether any compounds of the U.S. Drug Collection could suppress pCTS-L-induced inflammation, and pharmacologically be exploited into possible therapies. Here, we demonstrated that a macrophage cell-based screening of a U.S. Drug Collection of 1360 compounds resulted in the identification of progesterone (PRO) as an inhibitor of pCTS-L-mediated production of several chemokines [e.g., Epithelial Neutrophil-Activating Peptide (ENA-78), Monocyte Chemoattractant Protein-1 (MCP-1) or MCP-3] and cytokines [e.g., Interleukin-10 (IL-10) or Tumor Necrosis Factor (TNF)] in primary human peripheral blood mononuclear cells (PBMCs). In vivo, these PRO-entrapping 2,6-dimethal-ß-cyclodextrin (DM-ß-CD) nanoparticles (containing 1.35 mg/kg PRO and 14.65 mg/kg DM-ß-CD) significantly increased animal survival in both male (from 30% to 70%, n = 20, P = 0.041) and female (from 50% to 80%, n = 30, P = 0.026) mice even when they were initially administered at 24 h post the onset of sepsis. This protective effect was associated with a reduction of sepsis-triggered accumulation of three surrogate biomarkers [e.g., Granulocyte Colony Stimulating Factor (G-CSF) by 40%; Macrophage Inflammatory Protein-2 (MIP-2) by 45%; and Soluble Tumor Necrosis Factor Receptor I (sTNFRI) by 80%]. Surface Plasmon Resonance (SPR) analysis revealed a strong interaction between PRO and pCTS-L (KD = 78.2 ± 33.7 nM), which was paralleled with a positive correlation between serum PRO concentration and serum pCTS-L level (ρ = 0.56, P = 0.0009) or disease severity (Sequential Organ Failure Assessment, SOFA; ρ = 0.64, P = 0.0001) score in septic patients. Our observations support a promising opportunity to explore DM-ß-CD nanoparticles entrapping lipophilic drugs as possible therapies for clinical sepsis.

A two-decade journey in identifying high mobility group box 1 (HMGB1) and procathepsin L (pCTS-L) as potential therapeutic targets for sepsis.

INTRODUCTION: Microbial infections and resultant sepsis are leading causes of death in hospitals, representing approximately 20% of total deaths worldwide. Despite the difficulties in translating experimental insights into effective therapies for often heterogenous patient populations, an improved understanding of the pathogenic mechanisms underlying experimental sepsis is still urgently needed. Sepsis is partly attributable to dysregulated innate immune responses manifested by hyperinflammation and immunosuppression at different stages of microbial infections. AREAS COVERED: Here we review our recent progress in searching for late-acting mediators of experimental sepsis and propose high mobility group box 1 (HMGB1) and procathepsin-L (pCTS-L) as potential therapeutic targets for improving outcomes of lethal sepsis and other infectious diseases. EXPERT OPINION: It will be important to evaluate the efficacy of HMGB1- or pCTS-L-targeting agents for the clinical management of human sepsis and other infectious diseases in future studies.

Identification of procathepsin L (pCTS-L)-neutralizing monoclonal antibodies to treat potentially lethal sepsis.

Antibody-based strategies have been attempted to antagonize early cytokines of sepsis, but not yet been tried to target inducible late-acting mediators. Here, we report that the expression and secretion of procathepsin-L (pCTS-L) was induced by serum amyloid A (SAA) in innate immune cells, contributing to its late and systemic accumulation in experimental and clinical sepsis. Recombinant pCTS-L induced interleukin-6 (IL-6), IL-8, GRO-α/KC, GRO-ß/MIP-2, and MCP-1 release in innate immune cells and moderately correlated with blood concentrations of these cytokines/chemokines in clinical sepsis. Mechanistically, pCTS-L interacted with Toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE) to induce cytokines/chemokines. Pharmacological suppression of pCTS-L with neutralizing polyclonal and monoclonal antibodies attenuated pCTS-L-mediated inflammation by impairing its interaction with TLR4 and RAGE receptors, and consequently rescued animals from lethal sepsis. Our findings have suggested a possibility of developing antibody strategies to prevent dysregulated immune responses mediated by late-acting cytokines.

Monoclonal antibodies capable of binding SARS-CoV-2 spike protein receptor-binding motif specifically prevent GM-CSF induction.

A severe acute respiratory syndrome (SARS)-like coronavirus 2 (SARS-CoV-2) has recently caused a pandemic COVID-19 disease that infected approximately 94 million and killed more than 2,000,000 people worldwide. Like the SARS-CoV, SARS-CoV-2 also employs a receptor-binding motif (RBM) of its envelope spike protein for binding the host angiotensin-converting enzyme 2 (ACE2) to gain viral entry. Currently, extensive efforts are being made to produce vaccines against a surface fragment of a SARS-CoV-2, such as the spike protein, in order to boost protective antibodies that can inhibit virus-ACE2 interaction to prevent viral entry. It was previously unknown how spike protein-targeting antibodies would affect innate inflammatory responses to SARS-CoV-2 infections. Here we generated a highly purified recombinant protein corresponding to the RBM of SARS-CoV-2, and used it to screen for cross-reactive monoclonal antibodies (mAbs). We found two RBM-binding mAbs that competitively inhibited its interaction with human ACE2, and specifically blocked the RBM-induced GM-CSF secretion in both human peripheral blood mononuclear cells and murine macrophage cultures. Our findings have suggested a possible strategy to prevent SARS-CoV-2-elicited "cytokine storm," and revealed a potentially anti-inflammatory and protective mechanism for SARS-CoV-2 spike-based vaccines.

Peripheral administration of human adrenomedullin and its binding protein attenuates stroke-induced apoptosis and brain injury in rats.

Stroke is a leading cause of death and the primary medical cause of acquired adult disability worldwide. The progressive brain injury after acute stroke is partly mediated by ischemia-elicited inflammatory responses. The vasoactive hormone adrenomedullin (AM), upregulated under various inflammatory conditions, counterbalances inflammatory responses. However, regulation of AM activity in ischemic stroke remains largely unknown. Recent studies have demonstrated the presence of a specific AM binding protein (that is, AMBP-1) in mammalian blood. AMBP-1 potentiates AM biological activities. Using a rat model of focal cerebral ischemia induced by permanent middle cerebral artery occlusion (MCAO), we found that plasma levels of AM increased significantly, whereas plasma levels of AMBP-1 decreased significantly after stroke. When given peripherally early after MCAO, exogenous human AM in combination with human AMBP-1 reduced brain infarct volume 24 and 72 h after MCAO, an effect not observed after the treatment by human AM or human AMBP-1 alone. Furthermore, treatment of human AM/AMBP-1 reduced neuron apoptosis and morphological damage, inhibited neutrophil infiltration in the brain and decreased serum levels of S100B and lactate. Thus, human AM/AMBP-1 has the ability to reduce stroke-induced brain injury in rats. AM/AMBP-1 can be developed as a novel therapeutic agent for patients with ischemic stroke.

Upregulation of Kupffer cell α2A-Adrenoceptors and downregulation of MKP-1 mediate hepatic injury in chronic alcohol exposure.

Alcohol-induced liver disease is associated with unacceptable morbidity and mortality. When activated, Kupffer cells (KCs), the resident macrophages in the liver, release proinflammatory cytokine TNF-α, a key mediator of hepatic damage. Although chronic alcohol causes increase in norepinephrine (NE) release leading to hepatic dysfunction, the mechanism of NE-induced hepatic injury in chronic alcohol exposure has not been elucidated. This study was conducted to determine whether chronic alcohol exposure increases NE and upregulates KC α(2A)-adrenoceptors (α(2A)-AR) to cause TNF-α release. We also examined the role of mitogen activated protein kinase (MAPK) phosphatase-1 (MKP-1) in this process. Male adult rats were fed the Lieber-DeCarli liquid diet containing alcohol as 36% of total calories. The animals were sacrificed after 6 weeks and blood and liver samples were harvested for further analysis. KCs from healthy male rats were cultured with alcohol for 7 days, and cells then harvested for RNA and protein analyses. Chronic alcohol exposure resulted in hepatic damage. Alcohol caused a 276% increase in circulating NE and 86% increase in TNF-α in the liver. There was a 75% and 62% decrease in MKP-1 mRNA and protein levels, respectively in the liver. In-vitro experiments revealed 121% and 98% increase in TNF-α and α(2A)-AR mRNA levels with alcohol exposure, respectively, and a 32% decrease in MKP-1 mRNA compared to controls. In summary, chronic alcohol exposure elevates NE and upregulates KC α(2A)-AR to release TNF-α. Alcohol induced downregulation of MKP-1 leads to further release of TNF-α and hepatic injury.

Endogenous Regulation and Pharmacological Modulation of Sepsis-Induced HMGB1 Release and Action: An Updated Review.

Sepsis remains a common cause of death in intensive care units, accounting for approximately 20% of total deaths worldwide. Its pathogenesis is partly attributable to dysregulated inflammatory responses to bacterial endotoxins (such as lipopolysaccharide, LPS), which stimulate innate immune cells to sequentially release early cytokines (such as tumor necrosis factor (TNF) and interferons (IFNs)) and late mediators (such as high-mobility group box 1, HMGB1). Despite difficulties in translating mechanistic insights into effective therapies, an improved understanding of the complex mechanisms underlying the pathogenesis of sepsis is still urgently needed. Here, we review recent progress in elucidating the intricate mechanisms underlying the regulation of HMGB1 release and action, and propose a few potential therapeutic candidates for future clinical investigations.

Human Dermcidin Protects Mice Against Hepatic Ischemia-Reperfusion-Induced Local and Remote Inflammatory Injury.

Background: Hepatic ischemia and reperfusion (I/R) injury is commonly associated with surgical liver resection or transplantation, and represents a major cause of liver damage and graft failure. Currently, there are no effective therapies to prevent hepatic I/R injury other than ischemic preconditioning and some preventative strategies. Previously, we have revealed the anti-inflammatory activity of a sweat gland-derived peptide, dermcidin (DCD), in macrophage/monocyte cultures. Here, we sought to explore its therapeutic potential and protective mechanisms in a murine model of hepatic I/R. Methods: Male C57BL/6 mice were subjected to hepatic ischemia by clamping the hepatic artery and portal vein for 60 min, which was then removed to initiate reperfusion. At the beginning of reperfusion, 0.2 ml saline control or solution of DCD (0.5 mg/kg BW) or DCD-C34S analog (0.25 or 0.5 mg/kg BW) containing a Cys (C)→Ser (S) substitution at residue 34 was injected via the internal jugular vein. For survival experiments, mice were subjected to additional resection to remove non-ischemic liver lobes, and animal survival was monitored for 10 days. For mechanistic studies, blood and tissue samples were collected at 24 h after the onset of reperfusion, and subjected to measurements of various markers of inflammation and tissue injury by real-time RT-PCR, immunoassays, and histological analysis. Results: Recombinant DCD or DCD-C34S analog conferred a significant protection against lethal hepatic I/R when given intravenously at the beginning of reperfusion. This protection was associated with a significant reduction in hepatic injury, neutrophilic CXC chemokine (Mip-2) expression, neutrophil infiltration, and associated inflammation. Furthermore, the administration of DCD also resulted in a significant attenuation of remote lung inflammatory injury. Mechanistically, DCD interacted with epidermal growth factor receptor (EGFR), a key regulator of liver inflammation, and significantly inhibited hepatic I/R-induced phosphorylation of EGFR as well as a downstream signaling molecule, protein kinase B (AKT). The suppression of EGFR expression by transducing Egfr-specific shRNA plasmid into macrophages abrogated the DCD-mediated inhibition of nitric oxide (NO) production induced by a damage-associated molecular pattern (DAMP), cold-inducible RNA-binding protein, CIRP. Conclusions: The present study suggests that human DCD and its analog may be developed as novel therapeutics to attenuate hepatic I/R-induced inflammatory injury possibly by impairing EGFR signaling.

Milk fat globule EGF factor 8 attenuates sepsis-induced apoptosis and organ injury in alcohol-intoxicated rats.

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.

Monoclonal Antibodies Capable of Binding SARS-CoV-2 Spike Protein Receptor Binding Motif Specifically Prevent GM-CSF Induction.

A severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) has recently caused a pandemic COVID-19 disease that infected more than 25.6 million and killed 852,000 people worldwide. Like the SARS-CoV, SARS-CoV-2 also employs a receptor-binding motif (RBM) of its envelope spike protein for binding the host angiotensin-converting enzyme 2 (ACE2) to gain viral entry. Currently, extensive efforts are being made to produce vaccines against a surface fragment of a SARS-CoV-2, such as the spike protein, in order to boost protective antibody responses. It was previously unknown how spike protein-targeting antibodies would affect innate inflammatory responses to SARS-CoV-2 infections. Here we generated a highly purified recombinant protein corresponding to the RBM of SARS-CoV-2, and used it to screen for cross-reactive monoclonal antibodies (mAbs). We found two RBM-binding mAbs that competitively inhibited its interaction with human ACE2, and specifically blocked the RBM-induced GM-CSF secretion in both human monocyte and murine macrophage cultures. Our findings have suggested a possible strategy to prevent SARS-CoV-2-elicited "cytokine storm", and provided a potentially useful criteria for future assessment of innate immune-modulating properties of various SARS-CoV-2 vaccines. ONE SENTENCE SUMMARY: RBM-binding Antibodies Inhibit GM-CSF Induction.

Identification of tetranectin-targeting monoclonal antibodies to treat potentially lethal sepsis.

For the clinical management of sepsis, antibody-based strategies have only been attempted to antagonize proinflammatory cytokines but not yet been tried to target harmless proteins that may interact with these pathogenic mediators. Here, we report an antibody strategy to intervene in the harmful interaction between tetranectin (TN) and a late-acting sepsis mediator, high-mobility group box 1 (HMGB1), in preclinical settings. We found that TN could bind HMGB1 to reciprocally enhance their endocytosis, thereby inducing macrophage pyroptosis and consequent release of lactate dehydrogenase and apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain. The genetic depletion of TN expression or supplementation of exogenous TN protein at subphysiological doses distinctly affected the outcomes of potentially lethal sepsis, revealing a previously underappreciated beneficial role of TN in sepsis. Furthermore, the administration of domain-specific polyclonal and monoclonal antibodies effectively inhibited TN/HMGB1 interaction and endocytosis and attenuated the sepsis-induced TN depletion and tissue injury, thereby rescuing animals from lethal sepsis. Our findings point to a possibility of developing antibody strategies to prevent harmful interactions between harmless proteins and pathogenic mediators of human diseases.

Human adrenomedullin and its binding protein attenuate organ injury and reduce mortality after hepatic ischemia-reperfusion.

OBJECTIVE: To determine whether administration of a vasoactive peptide, human adrenomedullin (AM), in combination with its binding protein (ie, AMBP-1), prevents or minimizes hepatic ischemia-reperfusion (I/R) injury. SUMMARY BACKGROUND DATA: Hepatic I/R injury results from tissue hypoxia and subsequent inflammatory responses. Even though numerous pharmacological modalities and substances have been studied to reduce I/R-induced mortality, none have been entirely successful. We have shown that administration of AM/AMBP-1 produces significant beneficial effects under various pathophysiological conditions. However, it remains unknown if human AM/AMBP-1 has any protective effects on hepatic I/R-induced tissue damage and mortality. METHODS: Seventy percent hepatic ischemia was induced in male adult rats by placing a microvascular clip across the hilum of the left and median lobes for 90 minutes. After removing the clip, human AM alone, human AMBP-1 alone, human AM in combination with human AMBP-1 or vehicle was administered intravenously over a period of 30 minutes. Blood and tissue samples were collected 4 hours after reperfusion for various measurements. In additional groups of animals, the nonischemic liver lobes were resected at the end of 90-minute ischemia. The animals were monitored for 7 days and survival was recorded. RESULTS: After hepatic I/R, plasma levels of AM were significantly increased, whereas AMBP-1 levels were markedly decreased. Likewise, gene expression of AM in the liver was increased significantly, whereas AMBP-1 expression was markedly decreased. Administration of AM in combination with AMBP-1 immediately after the onset of reperfusion down-regulated inflammatory cytokines, decreased hepatic neutrophil infiltration, inhibited liver cell apoptosis and necrosis, and reduced liver injury and mortality in a rat model of hepatic I/R. On the other hand, administration of human AM alone or human AMBP-1 alone after hepatic I/R failed to produce significant protection. CONCLUSIONS: Human AM/AMBP-1 may be a novel treatment to attenuate tissue injury after an episode of hepatic ischemia.

Orexigenic hormone ghrelin ameliorates gut barrier dysfunction in sepsis in rats.

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.

Association between fecal microbiota and generalized anxiety disorder: Severity and early treatment response.

BACKGROUND: Associations between abnormal gut microbiome compositions and anxiety-like behaviors are well established. However, it is unknown whether the gut microbiome composition is associated with the severity of generalized anxiety disorder (GAD) and relief from clinical symptoms in patients. METHODS: Stool samples from 36 patients with active GAD (A-GAD group) and 24 matched healthy control subjects (HC group) were analyzed by 16S rRNA gene sequencing. Anxiety was assessed with the Hamilton Anxiety Rating Scale and the Self-rating Anxiety Scale, and global assessments of functioning were performed at baseline and 1 month after drug treatment. RESULTS: Gut microbiome compositions were altered in A-GAD patients, with fewer operational taxonomic units and lower fecal bacterial α-diversity. Specifically, Firmicutes and Tenericutes abundances were lower in A-GAD patients, and several genera were differentially represented in the A-GAD and HC groups. The abundances of Eubacterium_coprostanoligenes_group, Ruminococcaceae_UCG-014, and Prevotella_9 correlated negatively with the anxiety severity and positively with anxiety reduction, whereas the abundances of Bacteroides and Escherichia-Shigella were positively associated with anxiety severity. Sex, smoking, and alcohol intake influenced the gut microbiome composition. LIMITATIONS: The sample sizes were small and the stool samples were collected only at baseline; therefore, a causal association between changes in intestinal flora and disease remission was not established. Moreover, the effects of different drugs on gut microbiome composition were not investigated. CONCLUSIONS: Altered gut microbiome composition may contribute to GAD pathogenesis and remission.

Enhanced Macrophage Pannexin 1 Expression and Hemichannel Activation Exacerbates Lethal Experimental Sepsis.

We have recently reported an important role of Connexin 43 (Cx43) hemichannels in the pathogenesis of lethal sepsis through facilitating ATP efflux to potentiate the double-stranded RNA-activated protein kinase R (PKR)-dependent macrophage activation. Here we further elucidated the possible role of Pannexin 1 (Panx1) hemichannel in lethal sepsis by assessing its expression along with the impact of a Panx1-specific mimetic inhibitory peptide, 10Panx, on macrophage hemichannel activity in vitro and animal sepsis lethality in vivo. Both crude bacterial lipopolysaccharide (LPS) and purified serum amyloid A (SAA) effectively induced the expression and extracellular release of Panx1 by macrophages or monocytes as judged by Western blotting and immunocytochemistry assays. In animal model of lethal sepsis, Panx1 expression levels were significantly elevated in the heart, but reduced in the kidney, lung, spleen, and blood. At relatively lower doses (10, 50, and 100 mg/kg), the Panx1 mimetic peptide, 10Panx, reproducibly exacerbated the sepsis-induced animal lethality, reducing survival rates from 60-70% to 0-10%. Consistently, 10Panx did not inhibit, but rather promoted, the LPS-induced elevation of Lucifer Yellow dye uptake, ATP release, and Nitric Oxide (NO) production. Collectively, these findings suggested that elevated macrophage Panx1 expression and hemichannel activation contribute to the pathogenesis of lethal sepsis.

Human vasoactive hormone adrenomedullin and its binding protein rescue experimental animals from shock.

We recently discovered that vascular responsiveness to adrenomedullin (AM), a vasoactive hormone, decreases after hemorrhage, which is markedly improved by the addition of its binding protein AMBP-1. One obstacle hampering the development of AM/AMBP-1 as resuscitation agents in trauma victims is the potential immunogenicity of rat proteins in humans. Although less potent than rat AM, human AM has been shown to increase organ perfusion in rats. We therefore hypothesized that administration of human AM/AMBP-1 improves organ function and survival after severe blood loss in rats. To test this, male Sprague-Dawley rats were bled to and maintained at an MAP of 40 mmHg for 90 min. They were then resuscitated with an equal volume of shed blood in the form of Ringer's lactate (i.e., low-volume resuscitation) over 60 min. At 15 min after the beginning of resuscitation, human AM/AMBP-1 (12/40 or 48/160 microg/kg BW) were administered intravenously over 45 min. Various pathophysiological parameters were measured 4h after resuscitation. In additional groups of animals, a 12-day survival study was conducted. Our result showed that tissue injury as evidenced by increased levels of transaminases, lactate, and creatinine, was present at 4h after hemorrhage and resuscitation. Moreover, pro-inflammatory cytokines TNF-alpha and IL-6 were also significantly elevated. Administration of AM/AMBP-1 markedly attenuated tissue injury, reduced cytokine levels, and improved the survival rate from 29% (vehicle) to 62% (low-dose) or 70% (high-dose). However, neither human AM alone nor human AMBP-1 alone prevented the significant increase in ALT, AST, lactate and creatinine at 4h after the completion of hemorrhage and resuscitation. Moreover, the half-life of human AM and human AMBP-1 in rats was 35.8 min and 1.68 h, respectively. Thus, administration of human AM/AMBP-1 may be a useful approach for attenuating organ injury, and reducing mortality after hemorrhagic shock.

Connexin 43 Hemichannel as a Novel Mediator of Sterile and Infectious Inflammatory Diseases.

Cytoplasmic membrane-bound connexin 43 (Cx43) proteins oligomerize into hexameric channels (hemichannels) that can sometimes dock with hemichannels on adjacent cells to form gap junctional (GJ) channels. However, the possible role of Cx43 hemichannels in sterile and infectious inflammatory diseases has not been adequately defined due to the lack of selective interventions. Here we report that a proinflammatory mediator, the serum amyloid A (SAA), resembled bacterial endotoxin by stimulating macrophages to up-regulate Cx43 expression and double-stranded RNA-activated protein kinase R (PKR) phosphorylation in a TLR4-dependent fashion. Two well-known Cx43 mimetic peptides, the GAP26 and TAT-GAP19, divergently affected macrophage hemichannel activities in vitro, and differentially altered the outcome of lethal sepsis in vivo. By screening a panel of Cx43 mimetic peptides, we discovered that one cysteine-containing peptide, P5 (ENVCYD), effectively attenuated hemichannel activities, and significantly suppressed endotoxin-induced release of ATP and HMGB1 in vitro. In vivo, the P5 peptide conferred a significant protection against hepatic ischemia/reperfusion injury and lethal microbial infection. Collectively, these findings have suggested a pathogenic role of Cx43 hemichannels in sterile injurious as well as infectious inflammatory diseases possibly through facilitating extracellular ATP efflux to trigger PKR phosphorylation/activation.