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We develop a universal method for spray-deposition of polymeric semiconductor blends, based on blends of polyfluorenes (F8TBT), polythiophenes (P3HT) and fullerenes (PCBM), as suitable for large areas. A multi-faceted characterisation approach, studying photoluminescence quenching, together with atomic force and optical microscopy, illustrates favourable results in terms of layer thickness, uniformity, and mesoscale morphology. With key engineering tolerances in mind, thermal (melt) and solvent-vapour annealing are investigated as post-processing methods, for improving the planarity of craterform layers and blend photophysical characteristics. [ABSTRACT FROM AUTHOR]

• A system model for liver function is used to study the effect of fructose on NAFLD. • Model supports clinical evidence that high fructose causes hepatic lipid deposition. • Synergistic effect of three interventions has been predicted to be the strongest. • Systems models in combination with in vivo experiments help explore an organ system. Metabolic disturbances to the liver system can induce lipid deposition and subsequently cause non-alcoholic fatty liver disease (NAFLD). Increasing consumption of fructose has been proposed as a crucial risk component in the development of NAFLD. Three potential therapeutic targets in the network were explored using a composite model of liver function. Introducing a fructose enriched diet under insulin resistance conditions was simulated to evaluate the effectiveness of the model in novel therapy design. In vitro experiments were conducted on rat liver samples to assess the robustness of the model predictions. Synergistic application of all three interventional points in silico has been predicted as the most effective treatment to reduce lipid production under both moderate and severe insulin resistance conditions. This study demonstrates how we can use system models together with in vivo experiments to explore the behaviour of the liver system in response to fructose variation and use it to help identify possible drug targets. [ABSTRACT FROM AUTHOR]

Background & Aims: Increased intrahepatic resistance in cirrhosis is associated with reduced endothelial NO synthase (eNOS) activity and exacerbated by superimposed inflammation. NOSTRIN induces intracellular translocation of eNOS and reduces NO generation. Our aims were to quantify and compare hepatic expression of eNOS, NOSTRIN, NOSIP, and caveolin-1 in alcoholic cirrhosis with or without superimposed alcoholic hepatitis and in normal livers. Methods: Biopsy specimens from 20 decompensated alcoholic cirrhotic patients with portal hypertension (10 with alcoholic hepatitis) and 6 normal livers were analyzed: real-time polymerase chain reaction for quantification of messenger RNA; Western blotting; and enzyme assays of eNOS in normal and diseased liver were performed. Localization and interaction of eNOS and NOSTRIN in liver was assessed by immunohistochemistry and co-immunoprecipitation. Results: eNOS mRNA was significantly increased and eNOS activity decreased in alcoholic hepatitis patients, despite no differences in eNOS protein expression among the patients. Patients with alcoholic hepatitis had significantly higher hepatic levels of NOSTRIN and caveolin-1 mRNA compared with cirrhosis alone or normal biopsy specimens. A NOSTRIN splice variant, not present in normal tissue, was detected on mRNA and protein levels in all alcoholic patients. Coimmunoprecipitation demonstrated association among NOSTRIN, eNOS, and caveolin-1. Conclusions: An increase in mRNA and protein of NOSTRIN and its shortened variant in alcoholic hepatitis may partly account for the paradox of increased mRNA levels and normal protein expression but decreased enzymatic activity of eNOS in diseased liver. Such intracellular regulators of NO production may be important in the development of increased intrahepatic resistance in alcoholic hepatitis patients. [Copyright &y& Elsevier]

Abstract: Electron paramagnetic resonance (EPR) spectra of complex biological systems contain information about the paramagnetic centres present. Retrieving such information is important since paramagnetic species are common intermediates of all redox reactions in both normal and abnormal metabolism. However, it is often difficult to determine the nature and content of all paramagnetic species present because the EPR signals from individual centres overlap. Here, we apply our deconvolution method based on spectra subtraction with variable coefficient to quantify individual paramagnetic components of human muscle biopsies taken from critically ill patients with severe sepsis. We use low temperature EPR spectroscopy to identify and quantify nine different paramagnetic species in the tissue. These include the majority of the mitochondrial iron–sulfur centres and the first in vivo report of a mitochondrial radical assigned to a spin-coupled pair of semiquinones (SQ·–SQ·). We have previously demonstrated in these same muscle biopsies that biochemical assays of mitochondrial dysfunction correlate with clinical outcomes (D. Brealey, M. Brand, I. Hargreaves, S. Heales, J. Land, R. Smolenski, N.A. Davies, C.E. Cooper, M. Singer, Association between mitochondrial dysfunction and severity and outcome of septic shock. Lancet 360 (2002) 219–223.). Analysis of the paramagnetic centres in the muscle confirms and extends these findings: the (SQ·–SQ·) radical species negatively correlates with the illness severity of the patient (APACHE II score) and a decreased concentration of mitochondrial Complex I iron–sulfur redox centres is linked to mortality. [Copyright &y& Elsevier]

Abstract: We compared nitric oxide production and nitrosyl hemoglobin steady state concentrations during the early phases of endotoxemic and hemorrhagic shock of equivalent severity. Sprague–Dawley rats were randomly assigned to (1) sham-operated control, (2) hemorrhage, and (3) intravenous endotoxin. Electron paramagnetic resonance spectroscopy was used to measure NO in the vasculature (binding to hemoglobin) and in the liver (binding to cytochrome P450). Despite similar changes in cardiorespiratory variables and identical microvascular pO2, nitrosyl hemoglobin concentrations were significantly higher in endotoxemic rats than in rats in hemorrhagic shock, suggesting increased rates of NO production. A substantial venous minus arterial concentration gradient was observed for nitrosyl hemoglobin. This increased in line with the plasma total nitrite + nitrate concentration. Nitrosyl hemoglobin formation is likely to occur predominantly in the venous pool, suggesting that removal of NO from hemoglobin in the presence of oxygen may be faster than previously thought. In the liver, an increase in intracellular heme–NO complexes was detected in endotoxemic rats compared with rats in hemorrhagic shock; this was associated with increased reduction of the mitochondrial respiratory chain and is suggestive of NO inhibition of mitochondrial respiration. [Copyright &y& Elsevier]

Mitochondrial cytochrome oxidase is competitively and reversibly inhibited by inhibitors that bind to ferrous heme, such as carbon monoxide and nitric oxide. In the case of nitric oxide, nanomolar levels inhibit cytochrome oxidase by competing with oxygen at the enzyme''s heme–copper active site. This raises the Km for cellular respiration into the physiological range. This effect is readily reversible and may be a physiological control mechanism. Here we show that a number of in vitro and in vivo conditions result in an irreversible increase in the oxygen Km. These include: treatment of the purified enzyme with peroxynitrite or high (μM) levels of nitric oxide; treatment of the endothelial-derived cell line, b.End5, with NO; activation of astrocytes by cytokines; reperfusion injury in the gerbil brain. Studies of cell respiration that fail to vary the oxygen concentration systematically are therefore likely to significantly underestimate the degree of irreversible damage to cytochrome oxidase. [Copyright &y& Elsevier]

Hepatic encephalopathy (HE) incorporates a spectrum of neuropsychiatric abnormalities seen in patients with liver dysfunction with a potential for full reversibility. Distinct syndromes are identified in acute liver failure and cirrhosis. Rapid deterioration in consciousness level and increased intracranial pressure that may result in brain herniation and death are a feature of acute liver failure whereas manifestations of HE in cirrhosis include psychomotor dysfunction, impaired memory, increased reaction time, sensory abnormalities, poor concentration and in severe forms, coma. For over a 100 years ammonia has been considered central to its pathogenesis. In the brain, the astrocyte is the main site for ammonia detoxification, during the conversion of glutamate to glutamine. An increased ammonia level raises the amount of glutamine within astrocytes, causing an osmotic imbalance resulting in cell swelling and ultimately brain oedema. The present review focuses upon the molecular mechanisms involved in the pathogenesis of HE. Therapy of HE is directed primarily at reducing ammonia generation and increasing its detoxification. [Copyright &y& Elsevier]

People living with dementia commonly experience anxiety, which is often challenging to manage. We investigated the effectiveness of treatments for the management of anxiety in this population. We conducted a systematic review and meta-analysis of randomised controlled trials, and searched EMBASE, CINAHL, MEDLINE and PsycInfo. We estimated standardised mean differences at follow-up between treatments relative to control groups and pooled these across studies using random-effects models where feasible. Thirty-one studies were identified. Meta-analysis demonstrated non-pharmacological interventions were effective in reducing anxiety in people living with dementia, compared to care as usual or active controls. Specifically, music therapy (SMD–1.92(CI:−2.58,−1.25)), muscular approaches (SMD-0.65(CI:−1.02,−0.28)) and stimulating cognitive and physical activities (SMD–0.31(CI:−0.53,−0.09)). Pharmacological interventions with evidence of potential effectiveness included Ginkgo biloba, probiotics, olanzapine, loxapine and citalopram compared to placebo, olanzapine compared to bromazepam and buspirone and risperidone compared to haloperidol. Meta-analyses were not performed for pharmacological interventions due to studies' heterogeneity. This has practice implications when promoting the use of more non-pharmacological interventions to help reduce anxiety among people living with dementia. • Non-pharmacological interventions were effective in reducing anxiety. • Including music therapy, muscular and stimulating cognitive and physical activities. • Cognitive approaches and sensory stimulation were not effective. • Some pharmacological interventions demonstrated potential effectiveness. [ABSTRACT FROM AUTHOR]

Background & Aims Liver failure is characterized by endothelial dysfunction, which results in hemodynamic disturbances leading to renal failure. Albumin infusion improves hemodynamics and prevents renal dysfunction in advance liver failure. These effects are only partly explained by the oncotic properties of albumin. This study was designed to test the hypothesis that albumin exerts its beneficial effects by stabilising endothelial function. Methods In vivo : systemic hemodynamics, renal function, markers of endothelial dysfunction (ADMA) and inflammation were studied in analbuminaemic and Sprague–Dawley rats, 6-weeks after sham/bile duct ligation surgery. In vitro : human umbilical vein endothelial cells were stimulated with LPS with or without albumin. We studied protein expression and gene expression of adhesion molecules, intracellular reactive oxygen species, and cell stress markers. Results Compared to controls, analbuminaemic rats had significantly greater hemodynamic deterioration after bile duct ligation, resulting in worse renal function and shorter survival. This was associated with significantly greater plasma renin activity, worse endothelial function, and disturbed inflammatory response. In vitro studies showed that albumin was actively taken up by endothelial cells. Incubation of albumin pre-treated endothelial cells with LPS was associated with significantly less activation compared with untreated cells, decreased intracellular reactive oxygen species, and markers of cell stress. Conclusions These results show, for the first time, that absence of albumin is characterised by worse systemic hemodynamics, renal function and higher mortality in a rodent model of chronic liver failure and illustrates the important non-oncotic properties of albumin in protecting against endothelial dysfunction. [ABSTRACT FROM AUTHOR]

Background & Aims Portal hypertension is characterized by reduced hepatic eNOS activity. Asymmetric-dimethylarginine (ADMA), an eNOS inhibitor, is elevated in cirrhosis and correlates with the severity of portal hypertension. Dimethylarginine dimethylaminohydrolase-1 (DDAH-1) is the key enzyme metabolizing hepatic ADMA. This study characterized DDAH-1 in cirrhosis, and explored hepatic DDAH-1 reconstitution through farnesoid X receptor (FXR) agonism and DDAH-1 gene therapy. Methods DDAH-1 immunohistochemistry was conducted on human cirrhosis and healthy liver tissue. Subsequently, sham-operated or bile-duct-ligated (BDL) cirrhosis rats were treated with the FXR agonist obeticholic acid (OA, 5 mg/kg) or vehicle for 5 days. Further, animals underwent hydrodynamic injection with DDAH-1-expressing plasmid or saline control, which resulted in the following groups: sham + saline, BDL + saline, BDL + DDAH-1-plasmid. Portal pressure (PP) measurements were performed. Plasma ALT was measured by COBAS INTEGRA, DDAH-1 expression by qPCR and Western blot, eNOS activity by radiometric assay. Results Immunohistochemistry and Western-blotting confirmed hepatic DDAH-1 was restricted to hepatocytes, and expression decreased significantly in cirrhosis. In BDL rats, reduced DDAH-1 expression was associated with elevated hepatic ADMA, reduced eNOS activity and high PP. OA treatment significantly increased DDAH-1 expression, reduced hepatic tissue ADMA, and increased liver NO generation. PP was significantly reduced in BDL + OA vs. BDL + vehicle (8 ± 1 vs. 13.5 ± 0.6 mmHg; p <0.01) with no change in the mean arterial pressure (MAP). Similarly, DDAH-1 hydrodynamic injection significantly increased hepatic DDAH-1 gene and protein expression, and significantly reduced PP in BDL + DDAH-1 vs. BDL + saline ( p <0.01). Conclusions This study demonstrates DDAH-1 is a specific molecular target for portal pressure reduction, through actions on ADMA-mediated regulation of eNOS activity. Our data support translational studies, targeting DDAH-1 in cirrhosis and portal hypertension. [ABSTRACT FROM AUTHOR]

Background & Aims: In cirrhosis, superimposed inflammation often culminates in acute-on-chronic liver failure (ACLF) but the mechanism underlying this increased sensitivity is not clear. Cx43 is a ubiquitous gap junction protein that allows transmission of signals between cells at a much higher rate than the constitutively expressed gap junctions. The aims of the study were to test the hypothesis that inflammation drives the increased expression of hepatic Cx43 and to determine its role by Cx43 inhibition. Methods: Four weeks after bile-duct ligation (BDL) or sham operation, rats were treated with an anti-TNF antibody, or saline; with or without LPS (1mg/kg); given 3h prior to termination. Biochemistry and cytokines were measured in the plasma and hepatic protein expression (NFkB, TNFα, iNOS, 4HNE, Cx26, 32, and 43) and confocal microscopy (Cx26, 32, and 43) were performed. The effect of a Cx43-specific inhibitory peptide was studied in a mouse BDL model. Results: BDL animals administered LPS developed typical features of ACLF but animals administered infliximab were relatively protected. Cx26/32 expression was significantly decreased in BDL animals while Cx43 was significantly increased and increased further following LPS. Infliximab treatment prevented this increase. However, inhibiting Cx43 in BDL mice produced detrimental effects with markedly greater hepatocellular necrosis. Conclusions: The results of this study show for the first time an increased expression of hepatic Cx43 in cirrhosis and ACLF, which was related to the severity of inflammation. This increased Cx43 expression is likely to be an adaptive protective response of the liver to allow better cell-to-cell communication. [ABSTRACT FROM AUTHOR]

Background & Aims: Superimposed infection and/or inflammation precipitate renal failure in cirrhosis. This study aimed at testing the hypothesis that increased gut bacterial translocation in cirrhosis primes the kidney to the effect of superimposed inflammation by upregulating expression of Toll-like receptor 4 (TLR4), NFκB, and cytokines. A well-characterized bile-duct ligated (BDL) model of cirrhosis, which develops renal failure following superimposed inflammatory insult with lipopolysaccharide (LPS), was used and selective gut decontamination was performed using norfloxacin. Methods: Sprague–Dawley rats were studied: Sham, Sham+LPS; BDL, BDL+LPS; an additional BDL and BDL+LPS groups were selectively decontaminated with norfloxacin. Plasma biochemistry, plasma renin activity (PRA) and cytokines and, protein expression of TLR4, NFκB, and cytokines were measured in the kidney homogenate. The kidneys were stained for TLR4, TLR2, and caspase-3. Endotoxemia was measured using neutrophil burst and Limulus amoebocyte lysate (LAL) assays. Results: The groups treated with norfloxacin showed significant attenuation of the increase in plasma creatinine, plasma and renal TNF-α and renal tubular injury on histology. The increased renal protein expression of TLR4, NFκB, and caspase-3 in the untreated animals was significantly attenuated in the norfloxacin treated animals. PRA was reduced in the treated animals and severity of endotoxemia was also reduced. Conclusions: The results show for the first time that kidneys in cirrhosis show an increased expression of TLR4, NFκB, and the pro-inflammatory cytokine TNF-α, which makes them susceptible to a further inflammatory insult. This increased susceptibility to LPS can be prevented with selective decontamination, providing novel insights into the pathophysiology of renal failure in cirrhosis. [ABSTRACT FROM AUTHOR]

Background & Aims: Deranged neutrophil function in alcoholic hepatitis has been shown to be transmissible to normal neutrophils by patient plasma. The aims of this study were (i) to evaluate whether patients with stable cirrhosis have a similar transmissible neutrophil defect and (ii) to explore the possible mechanisms. Methods: Plasma samples from 108 stable cirrhotic patients (Child A or B: 58; Child C: 50) and matched controls were incubated with normal neutrophils. Neutrophil resting respiratory burst, phagocytosis, and toll-like receptors 2, 4, and 9 expressions as well as plasma endotoxin, bacterial DNA, and cytokines were measured. In a separate study, eight patients and five controls were studied using a novel ‘skin-window’ technique to evaluate neutrophil function in an area of induced sterile inflammation. Results: Patient plasma induced neutrophil phagocytic dysfunction was greater in patients with more severe disease and was associated with increased expression of toll-like receptors 2 and 4. An increased resting respiratory burst was observed in a subset of patients, showing higher levels of inflammatory cytokines and more pronounced phagocytic impairment. No correlation was found with endotoxemia or bacterial DNA. In patients with compensated cirrhosis and apparently normal neutrophil function, the ‘skin-window’ study disclosed a severe phagocytic defect at the site of inflammation. Significantly higher levels of neutrophil elastase and IL-8 were found in the blister fluid. Conclusions: Stable cirrhosis is characterized by neutrophil phagocytic dysfunction which may be subtle and only revealed in inflamed peripheral tissues where excessive inflammatory mediators continue to be released. [ABSTRACT FROM AUTHOR]

Background & Aims: Liver failure is associated with progressive cytotoxic brain oedema (astrocyte swelling), which underlies hepatic encephalopathy (HE). Ammonia and superimposed inflammation are key synergistic factors in HE, but the mechanism(s) involved remain unknown. We aimed to determine whether aquaporin-4 (AQP4), an astrocyte endfeet bi-directional water channel, is associated with the brain oedema of HE. Method: Rats (n =60) received sham-operation (sham), 5days hyperammonaemia-inducing diet (HD), galactosamine (GALN) induced acute liver failure (ALF), 4weeks bile duct-ligation (BDL) induced cirrhosis, or caecal ligation and puncture (CLP), a 24h model of bacterial peritonitis. Rats from every group (except CLP) were randomised to receive intraperitoneal injections of lipopolysaccharide (LPS; 1mg/kg) or saline, prior to termination 3h later. Brain water, AQP4 protein expression (western blot) and AQP4 localisation by immunogold electron microscopy were investigated. Results: Significant hyperammonaemia was observed in saline-injected BDL (p <0.05), GALN (p <0.01), and HD (p <0.01), compared to sham rats. LPS injection did not affect arterial ammonia or plasma biochemistry in any of the treatment groups. Increased brain water was observed in saline-injected GALN (p <0.05), HD (p <0.01), and CLP (p <0.001) compared to sham rats. Brain water was numerically increased in BDL rats, but this failed to reach significance (p =0.09). LPS treatment further increased oedema significantly in all treatment groups (p <0.05, respectively). AQP4 expression was significantly increased in saline-injected BDL (p <0.05), but not other treatment groups, compared to sham rats. Membrane polarisation was maintained in BDL rats. Conclusion: The results suggest that AQP4 is not directly associated with the development of brain oedema in liver failure, hyperammonaemia, or sepsis. In cirrhosis, there is increased AQP4 protein expression, but membrane polarisation, is maintained, possibly in a compensatory attempt to limit severe brain oedema. [Copyright &y& Elsevier]

Background/Aim: Endotoxaemia contributes to neutrophil dysfunction, infection risk and mortality in patients with alcoholic cirrhosis. As probiotics may decrease Gram-negative gut organisms, we hypothesised that probiotic treatment would restore neutrophil function. Methods: In an open-label study, patients with alcoholic cirrhosis (n =12) received Lactobacillus casei Shirota (6.5×109) 3 times daily for 4 weeks. Data were compared to healthy controls (n =13) and cirrhotic patients (n =8) who did not receive probiotics. Neutrophil oxidative burst, phagocytosis, toll-like-receptor (TLR) expression, plasma cytokines and ex vivo endotoxin-stimulated cytokine production were measured. Results: Baseline neutrophil phagocytic capacity in patients was significantly lower compared to healthy controls (73% versus 98%, p <0.05), but normalised at the end of the study (n =10, 100%, p <0.05). No improvement was seen in disease controls. Soluble TNF-receptor (sTNFR)-1 and-2 and interleukin (IL)10 were significantly elevated in patients’ plasma but did not change during the study. Ex vivo endotoxin-stimulated levels of sTNFR1, sTNFR2 and IL10 were significantly lower at the end of the study (p <0.05). TLR2, 4 and 9 were overexpressed in patients. TLR4 expression normalised by the end of the study. Conclusions: Our data provide a proof-of-concept that probiotics restore neutrophil phagocytic capacity in cirrhosis, possibly by changing IL10 secretion and TLR4 expression, warranting larger randomised controlled and mechanistic studies. [Copyright &y& Elsevier]

Unlike other Toll-like receptors (TLRs), the role of toll like receptor 2 (TLR-2) in the pathogenesis of chronic liver disease and hepatocellular carcinoma (HCC) is not well studied. We, therefore, set out to investigate the expression of TLR-2 in different chronic liver disease states along with other markers of cell death, cellular proliferation and tissue vascularisation Immunohistochemistry was performed on liver tissue microarrays comprising hepatitis, cirrhosis and HCC patient samples using antibodies against TLR-2, Ki-67, Caspase-3 and VEGF. This was done in order to characterise receptor expression and translocation, apoptosis, cell proliferation and vascularisation. Cytoplasmic TLR-2 expression was found to be weak in 5/8 normal liver cases, 10/19 hepatitis cases and 8/21 cirrhosis patients. Moderate to strong TLR-2 expression was observed in some cases of hepatitis and cirrhosis. Both, nuclear and cytoplasmic TLR-2 expression was present in HCC with weak intensity in 11/41 cases, and moderate to strong staining in 19/41 cases. Eleven HCC cases were TLR-2 negative. Surprisingly, both cytoplasmic and nuclear TLR-2 expression in HCC were found to significantly correlate with proliferative index (r = 0.24 and 0.37), Caspase-3 expression (r = 0.27 and 0.38) and vascularisation (r = 0.56 and 0.23). Further, nuclear TLR-2 localisation was predominant in HCC, whereas cytoplasmic expression was more prevalent in hepatitis and cirrhosis. Functionally, treatment of HUH7 HCC cells with a TLR-2 agonist induced the expression of cellular proliferation and vascularisation markers CD34 and VEGF. Our results demonstrate a positive correlation between the expression of TLR-2 and other markers of proliferation and vascularisation in HCC which suggests a possible role for TLR-2 in HCC pathogenesis [ABSTRACT FROM AUTHOR]

A correction to the article "Hepatic Dimethylarginine-Dimethylaminohydrolase1 is Reduced in Cirrhosis and is a Target for Therapy in Portal Hypertension" published in a previous issue of the journal is presented.