Complete Genome Sequence of Stenotrophomonas maltophilia Siphophage Salva.

Stenotrophomonas maltophilia is a Gram-negative pathogen causing severe and often refractory illnesses such as pneumonia and bacteremia. We present the genome of phage Salva, a novel S. maltophilia phage that is not closely related to any phages currently deposited in GenBank. The genome is 60,789 bp, containing 102 putative protein-coding genes.

Inhibition of thrombospondin-1 reduces glutathione activity and worsens acute liver injury during acetaminophen hepatotoxicity in mice.

Acetaminophen (N-Acetyl-p-Aminophenol or APAP)-induced hepatotoxicity is the most common cause of acute liver failure in the United States and Western Europe. Previous studies have shown that TGFß1 is elevated during APAP-induced hepatotoxicity and promotes liver injury by reducing liver regeneration while inducing hepatocyte senescence. At this time, little is known about the role of proteins that activate latent TGFß1 and their effects during APAP-induced hepatotoxicity. Thrombospondin-1 (TSP1) is a homotrimeric protein that can not only activate latent TGFß1 but can also interact with other proteins including Nrf2 to induce antioxidant signaling. The aim of the current study was to assess the role of thrombospondin-1 (TSP1) in both TGFß1 activation and its contribution to APAP-induced liver injury. C57Bl/6 mice or TSP1 null mice (TSP1-/-) were administered 300 mg/kg or 600 mg/kg of APAP. TGFß1 signaling, TSP1 expression, measures of hepatic injury, Nrf2 expression, measures of oxidative/nitrosative stress and GSH metabolism were assessed. The expression of TGFß1, TSP1 and phosphorylation of SMAD proteins increased in APAP-treated mice compared to controls. TSP1-/- mice had reduced TGFß1 expression and phosphorylation of SMAD proteins but increased liver injury. Hepatocyte cell death was increased in TSP1-/- mice and this was associated with decreased Nrf2 activity, decreased GSH levels and increased oxidative stress in comparison to wild-type C57Bl/6 mice. Together, these data demonstrate that elimination of TSP1 protein in APAP-treated mice reduces TGFß1 signaling but leads to increased liver injury by reducing Nrf2 expression and GSH activity, ultimately resulting in increased cell death.

Thrombospondin-1 Exacerbates Acute Liver Failure and Hepatic Encephalopathy Pathology in Mice by Activating Transforming Growth Factor ß1.

Severe hepatic insults can lead to acute liver failure and hepatic encephalopathy (HE). Transforming growth factor ß1 (TGFß1) has been shown to contribute to HE during acute liver failure; however, TGFß1 must be activated to bind its receptor and generate downstream effects. One protein that can activate TGFß1 is thrombospondin-1 (TSP-1). Therefore, the aim of this study was to assess TSP-1 during acute liver failure and HE pathogenesis. C57Bl/6 or TSP-1 knockout (TSP-1-/-) mice were injected with azoxymethane (AOM) to induce acute liver failure and HE. Liver damage, neurologic decline, and molecular analyses of TSP-1 and TGFß1 signaling were performed. AOM-treated mice had increased TSP-1 and TGFß1 mRNA and protein expression in the liver. TSP-1-/- mice administered AOM had reduced liver injury as assessed by histology and serum transaminase levels compared with C57Bl/6 AOM-treated mice. TSP-1-/- mice treated with AOM had reduced TGFß1 signaling that was associated with less hepatic cell death as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and cleaved caspase 3 expression. TSP-1-/- AOM-treated mice had a reduced rate of neurologic decline, less cerebral edema, and a decrease in microglia activation in comparison with C57Bl/6 mice treated with AOM. Taken together, TSP-1 is an activator of TGFß1 signaling during AOM-induced acute liver failure and contributes to both liver pathology and HE progression.

The TGFß1 Receptor Antagonist GW788388 Reduces JNK Activation and Protects Against Acetaminophen Hepatotoxicity in Mice.

Acute liver failure is a serious consequence of acetaminophen (APAP)-induced hepatotoxic liver injury with high rates of morbidity and mortality. Transforming growth factor beta 1 (TGFß1) is elevated during liver injury and influences hepatocyte senescence during APAP-induced hepatotoxicity. This study investigated TGFß1 signaling in the context of inflammation, necrotic cell death, and oxidative stress during APAP-induced liver injury. Male C57Bl/6 mice were injected with 600 mg/kg APAP to generate liver injury in the presence or absence of the TGFß receptor 1 inhibitor, GW788388, 1 h prior to APAP administration. Acetaminophen-induced liver injury was characterized using histological and biochemical measures. Transforming growth factor beta 1 expression and signal transduction were assessed using immunohistochemistry, Western blotting and ELISA assays. Hepatic necrosis, liver injury, cell proliferation, hepatic inflammation, and oxidative stress were assessed in all mice. Acetaminophen administration significantly induced necrosis and elevated serum transaminases compared with control mice. Transforming growth factor beta 1 staining was observed in and around areas of necrosis with phosphorylation of SMAD3 observed in hepatocytes neighboring necrotic areas in APAP-treated mice. Pretreatment with GW788388 prior to APAP administration in mice reduced hepatocyte cell death and stimulated regeneration. Phosphorylation of SMAD3 was reduced in APAP mice pretreated with GW788388 and this correlated with reduced hepatic cytokine production and oxidative stress. These results support that TGFß1 signaling plays a significant role in APAP-induced liver injury by influencing necrotic cell death, inflammation, oxidative stress, and hepatocyte regeneration. In conclusion, targeting TGFß1 or downstream signaling may be a possible therapeutic target for the management of APAP-induced liver injury.

Elevated circulating TGFß1 during acute liver failure activates TGFßR2 on cortical neurons and exacerbates neuroinflammation and hepatic encephalopathy in mice.

BACKGROUND: Acute liver failure resulting from drug-induced liver injury can lead to the development of neurological complications called hepatic encephalopathy (HE). Hepatic transforming growth factor beta 1 (TGFß1) is upregulated due to liver failure in mice and inhibiting circulating TGFß reduced HE progression. However, the specific contributions of TGFß1 on brain cell populations and neuroinflammation during HE are not known. Therefore, the aim of this study was to characterize hepatic and brain TGFß1 signaling during acute liver failure and its contribution to HE progression using a combination of pharmacological and genetic approaches. METHODS: C57Bl/6 or neuron-specific transforming growth factor beta receptor 2 (TGFßR2) null mice (TGFßR2ΔNeu) were treated with azoxymethane (AOM) to induce acute liver failure and HE. The activity of circulating TGFß1 was inhibited in C57Bl/6 mice via injection of a neutralizing antibody against TGFß1 (anti-TGFß1) prior to AOM injection. In all mouse treatment groups, liver damage, neuroinflammation, and neurological deficits were assessed. Inflammatory signaling between neurons and microglia were investigated in in vitro studies through the use of pharmacological inhibitors of TGFß1 signaling in HT-22 and EOC-20 cells. RESULTS: TGFß1 was expressed and upregulated in the liver following AOM injection. Pharmacological inhibition of TGFß1 after AOM injection attenuated neurological decline, microglia activation, and neuroinflammation with no significant changes in liver damage. TGFßR2ΔNeu mice administered AOM showed no effect on liver pathology but significantly reduced neurological decline compared to control mice. Microglia activation and neuroinflammation were attenuated in mice with pharmacological inhibition of TGFß1 or in TGFßR2ΔNeu mice. TGFß1 increased chemokine ligand 2 (CCL2) and decreased C-X3-C motif ligand 1 (CX3CL1) expression in HT-22 cells and reduced interleukin-1 beta (IL-1ß) expression, tumor necrosis factor alpha (TNFα) expression, and phagocytosis activity in EOC-20 cells. CONCLUSION: Increased circulating TGFß1 following acute liver failure results in activation of neuronal TGFßR2 signaling, driving neuroinflammation and neurological decline during AOM-induced HE.

Bile Acid-Mediated Sphingosine-1-Phosphate Receptor 2 Signaling Promotes Neuroinflammation during Hepatic Encephalopathy in Mice.

Hepatic encephalopathy (HE) is a neuropsychiatric complication that occurs due to deteriorating hepatic function and this syndrome influences patient quality of life, clinical management strategies and survival. During acute liver failure, circulating bile acids increase due to a disruption of the enterohepatic circulation. We previously identified that bile acid-mediated signaling occurs in the brain during HE and contributes to cognitive impairment. However, the influences of bile acids and their downstream signaling pathways on HE-induced neuroinflammation have not been assessed. Conjugated bile acids, such as taurocholic acid (TCA), can activate sphingosine-1-phosphate receptor 2 (S1PR2), which has been shown to promote immune cell infiltration and inflammation in other models. The current study aimed to assess the role of bile-acid mediated S1PR2 signaling in neuroinflammation and disease progression during azoxymethane (AOM)-induced HE in mice. Our findings demonstrate a temporal increase of bile acids in the cortex during AOM-induced HE and identified that cortical bile acids were elevated as an early event in this model. In order to classify the specific bile acids that were elevated during HE, a metabolic screen was performed and this assay identified that TCA was increased in the serum and cortex during AOM-induced HE. To reduce bile acid concentrations in the brain, mice were fed a diet supplemented with cholestyramine, which alleviated neuroinflammation by reducing proinflammatory cytokine expression in the cortex compared to the control diet-fed AOM-treated mice. S1PR2 was expressed primarily in neurons and TCA treatment increased chemokine ligand 2 mRNA expression in these cells. The infusion of JTE-013, a S1PR2 antagonist, into the lateral ventricle prior to AOM injection protected against neurological decline and reduced neuroinflammation compared to DMSO-infused AOM-treated mice. Together, this identifies that reducing bile acid levels or S1PR2 signaling are potential therapeutic strategies for the management of HE.

Melatonin inhibits hypothalamic gonadotropin-releasing hormone release and reduces biliary hyperplasia and fibrosis in cholestatic rats.

Melatonin is a hormone produced by the pineal gland with increased circulating levels shown to inhibit biliary hyperplasia and fibrosis during cholestatic liver injury. Melatonin also has the capability to suppress the release of hypothalamic gonadotropin-releasing hormone (GnRH), a hormone that promotes cholangiocyte proliferation when serum levels are elevated. However, the interplay and contribution of neural melatonin and GnRH to cholangiocyte proliferation and fibrosis in bile duct-ligated (BDL) rats have not been investigated. To test this, cranial levels of melatonin were increased by implanting osmotic minipumps that performed an intracerebroventricular (ICV) infusion of melatonin or saline for 7 days starting at the time of BDL. Hypothalamic GnRH mRNA and cholangiocyte secretion of GnRH and melatonin were assessed. Cholangiocyte proliferation and fibrosis were measured. Primary human hepatic stellate cells (HSCs) were treated with cholangiocyte supernatants, GnRH, or the GnRH receptor antagonist cetrorelix acetate, and cell proliferation and fibrosis gene expression were assessed. Melatonin infusion reduced hypothalamic GnRH mRNA expression and led to decreased GnRH and increased melatonin secretion from cholangiocytes. Infusion of melatonin was found to reduce hepatic injury, cholangiocyte proliferation, and fibrosis during BDL-induced liver injury. HSCs supplemented with BDL cholangiocyte supernatant had increased proliferation, and this increase was reversed when HSCs were supplemented with supernatants from melatonin-infused rats. GnRH stimulated fibrosis gene expression in HSCs, and this was reversed by cetrorelix acetate cotreatment. Increasing bioavailability of melatonin in the brain may improve outcomes during cholestatic liver disease.NEW & NOTEWORTHY We have previously demonstrated that GnRH is expressed in cholangiocytes and promotes their proliferation during cholestasis. In addition, dark therapy, which increases melatonin, reduced cholangiocyte proliferation and fibrosis during cholestasis. This study expands these findings by investigating neural GnRH regulation by melatonin during BDL-induced cholestasis by infusing melatonin into the brain. Melatonin infusion reduced cholangiocyte proliferation and fibrosis, and these effects are due to GNRH receptor 1-dependent paracrine signaling between cholangiocytes and hepatic stellate cells.

Retrograde facilitation under midazolam: the role of general and specific interference.

In a double-blind, placebo-controlled experiment that used midazolam, a benzodiazepine that creates temporary amnesia, we compared acquisition and retention of paired associates of different types. Some word pairs were studied before the injection of saline or midazolam, and two lists of word pairs were studied after the injection. Critical comparisons involved retention of pairs that were practiced on all three lists, pairs studied on only one list, and pairs that involved recombining cue and response terms from one list to the next, as a function of drug condition. Previous research with benzodiazepines had found retrograde facilitation for material acquired prior to injection, compared with the control condition. One explanation for this facilitation is that the anterograde amnesia produced by the benzodiazepine frees up the hippocampus to better consolidate previously learned material (Wixted, 2004, 2005). We accounted for a rich data set using a simple computational model that incorporated interference effects (cue overload) at retrieval for both general (experimental context) interference and specific (stimulus term) interference without the need to postulate a role for consolidation. The computational model as an Excel spreadsheet may be downloaded from www.psychonomic.org/archive.