Fouzia Sadiq

Amino acids and insulin act additively to regulate components of the ubiquitin-proteasome pathway in C2C12 myotubes

BackgroundThe ubiquitin-proteasome system is the predominant pathway for myofibrillar proteolysis but a previous study in C2C12 myotubes only observed alterations in lysosome-dependent proteolysis in response to complete starvation of amino acids or leucine from the media. Here, we determined the interaction between insulin and amino acids in the regulation of myotube proteolysisResultsIncubation of C2C12 myotubes with 0.2 × physiological amino acids concentration (0.2 × PC AA), relative to 1.0 × PC AA, significantly increased total proteolysis and the expression of 14-kDa E2 ubiquitin conjugating enzyme (p < 0.05). The proteasome inhibitor MG132 blocked the rise in proteolysis observed in the 0.2 × PC AA media. Addition of insulin to the medium inhibited proteolysis at both 0.2 and 1.0× PC AA and the expression of 14-kDa E2 proteins and C2 sub unit of 20 S proteasome (p < 0.05). Incubation of myotubes with increasing concentrations of leucine in the 0.2 × PC AA media inhibited proteolysis but only in the presence of insulin. Incubation of rapamycin (inhibitor of mTOR) inhibited amino acid or insulin-dependent p70 S6 kinase phosphorylation, blocked (P < 0.05) the inhibitory effects of 1.0 × PC AA on protein degradation, but did not alter the inhibitory effects of insulin or leucineConclusionIn a C2C12 myotube model of myofibrillar protein turnover, amino acid limitation increases proteolysis in a ubiquitin-proteasome-dependent manner. Increasing amino acids or leucine alone, act additively with insulin to down regulate proteolysis and expression of components of ubiquitin-proteasome pathway. The effects of amino acids on proteolysis but not insulin and leucine, are blocked by inhibition of the mTOR signalling pathway.

Hirmi Valley liver disease: a disease associated with exposure to pyrrolizidine alkaloids and DDT.

BACKGROUND & AIMS Hirmi Valley liver disease was first reported in 2001 in Tigray, Ethiopia. 591 cases, including 228 deaths, were reported up to December 2009. The pyrrolizidine alkaloid acetyllycopsamine was detected in stored grain and residents reported adding the pesticide DDT (dichlorodiphenyldichloroethylene) directly to their food stores. We aimed to characterise the clinical features of the disease, and explore the role of these chemicals in its aetiology. METHODS 32 cases were examined and full clinical histories taken. Nine cases underwent liver biopsy in hospitals. Serum and urine samples were collected from cases and controls. Urine was analysed for acetyllycopsamine by UPLC-MS. Total DDT in serum was measured by ELISA. Hepatotoxicity of DDT and acetyllycopsamine alone or in combination was explored in C57BL/6J mice. RESULTS Clinical presentation included epigastric pain, abdominal swelling, bloody diarrhoea, hepatomegaly, splenomegaly, and ascites. Histology revealed acute injury characterised by centrilobular necrosis or chronic injury with bile ductular reaction, cytomegaly and fibrosis but no hepatic vein occlusion. Acetyllycopsamine was detected in urine samples taken in the affected area with significantly greater concentrations in 45 cases than in 43 controls (p=0.02). High levels of DDT (>125 ppb) were detected in 78% of serum samples. In mice, DDT (3 × 75 mg/kg) significantly increased the hepatotoxicity (plasma ALT, p=0.0065) of acetyllycopsamine (750 mg/kg), and in combination induced liver pathology similar to Hirmi Valley liver disease including centrilobular necrosis and cytomegaly. CONCLUSIONS This novel form of disease appears to be caused by co-exposure to acetyllycopsamine and DDT.

Defective monocyte oxidative burst predicts infection in alcoholic hepatitis and is associated with reduced expression of NADPH oxidase

Objective In order to explain the increased susceptibility to serious infection in alcoholic hepatitis, we evaluated monocyte phagocytosis, aberrations of associated signalling pathways and their reversibility, and whether phagocytic defects could predict subsequent infection. Design Monocytes were identified from blood samples of 42 patients with severe alcoholic hepatitis using monoclonal antibody to CD14. Phagocytosis and monocyte oxidative burst (MOB) were measured ex vivo using flow cytometry, luminometry and bacterial killing assays. Defects were related to the subsequent development of infection. Intracellular signalling pathways were investigated using western blotting and PCR. Interferon-γ (IFN-γ) was evaluated for its therapeutic potential in reversing phagocytic defects. Paired longitudinal samples were used to evaluate the effect of in vivo prednisolone therapy. Results MOB, production of superoxide and bacterial killing in response to Escherichia coli were markedly impaired in patients with alcoholic hepatitis. Pretreatment MOB predicted development of infection within two weeks with sensitivity and specificity that were superior to available clinical markers. Accordingly, defective MOB was associated with death at 28 and 90 days. Expression of the gp91phox subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was reduced in patients with alcoholic hepatitis demonstrating defective MOB. Monocytes were refractory to IFN-γ stimulation and showed high levels of a negative regulator of cytokine signalling, suppressor of cytokine signalling-1. MOB was unaffected by 7 days in vivo prednisolone therapy. Conclusions Monocyte oxidative burst and bacterial killing is impaired in alcoholic hepatitis while bacterial uptake by phagocytosis is preserved. Defective MOB is associated with reduced expression of NADPH oxidase in these patients and predicts the development of infection and death.

Effect of prolonged intravenous glucose and essential amino acid infusion on nitrogen balance, muscle protein degradation and ubiquitin-conjugating enzyme gene expression in calves

BackgroundIntravenous infusions of glucose and amino acids increase both nitrogen balance and muscle accretion. We hypothesised that co-infusion of glucose (to stimulate insulin) and essential amino acids (EAA) would act additively to improve nitrogen balance by decreasing muscle protein degradation in association with alterations in muscle expression of components of the ubiquitin-proteasome proteolytic pathway.MethodsWe examined the effect of a 5 day intravenous infusions of saline, glucose, EAA and glucose + EAA, on urinary nitrogen excretion and muscle protein degradation. We carried out the study in 6 restrained calves since ruminants offer the advantage that muscle protein degradation can be assessed by excretion of 3 methyl-histidine and multiple muscle biopsies can be taken from the same animal. On the final day of infusion blood samples were taken for hormone and metabolite measurement and muscle biopsies for expression of ubiquitin, the 14-kDa E2 ubiquitin conjugating enzyme, and proteasome sub-units C2 and C8.ResultsOn day 5 of glucose infusion, plasma glucose, insulin and IGF-1 concentrations were increased while urea nitrogen excretion and myofibrillar protein degradation was decreased. Co-infusion of glucose + EAA prevented the loss of urinary nitrogen observed with EAA infusions alone and enhanced the increase in plasma IGF-1 concentration but there was no synergistic effect of glucose + EAA on the decrease in myofibrillar protein degradation. Muscle mRNA expression of the ubiquitin conjugating enzyme, 14-kDa E2 and proteasome sub-unit C2 were significantly decreased, after glucose but not amino acid infusions, and there was no further response to the combined infusions of glucose + EAA.ConclusionProlonged glucose infusion decreases myofibrillar protein degradation, prevents the excretion of infused EAA, and acts additively with EAA to increase plasma IGF-1 and improve net nitrogen balance. There was no evidence of synergistic effects between glucose + EAA infusion on muscle protein degradation or expression of components of the ubiquitin-proteasome proteolytic pathway.

Hepatitis B testing and treatment in HIV patients in The Gambia—Compliance with international guidelines and clinical outcomes

Background Compliance with WHO guidelines on HBV screening and treatment in HIV-coinfected patients is often challenging in resource limited countries and has been poorly assessed in sub-Saharan Africa. Methods Between 2015 and 2016, we assessed physician’s compliance with WHO guidelines on HIV-HBV coinfection in the largest HIV clinic in The Gambia, and the hepatic outcomes in HIV-HBV coinfected patients as compared to randomly selected HIV-monoinfected controls. Results 870 HIV-infected patients regularly seen in this clinic agreed to participate in our study. Only 187 (21.5%, 95% CI 18.8–24.3) had previously been screened for HBsAg, 23 (12.3%, 95% CI 8.0–17.9) were positive of whom none had liver assessment and only 6 (26.1%) had received Tenofovir. Our HBV testing intervention was accepted by all participants and found 94/870 (10.8%, 95% CI 8.8–13.1) positive, 78 of whom underwent full liver assessment along with 40 HBsAg-negative controls. At the time of liver assessment, 61/78 (78.2%) HIV-HBV coinfected patients received ART with 7 (11.5%) on Tenofovir and 54 (88.5%) on Lamivudine alone. HIV-HBV coinfected patients had higher APRI score compared to controls (0.58 vs 0.42, p = 0.002). HBV DNA was detectable in 52/53 (98.1%) coinfected patients with 14/53 (26.4%) having HBV DNA >20,000 IU/L. 10/12 (83.3%) had at least one detectable 3TC-associated HBV resistance, which tended to be associated with increase in liver fibrosis after adjusting for age and sex (p = 0.05). Conclusions Compliance with HBV testing and treatment guidelines is poor in this Gambian HIV programme putting coinfected patients at risk of liver complications. However, the excellent uptake of HBV screening and linkage to care in our study suggests feasible improvements.

OC-021 Monocyte oxidative burst defect is associated with susceptibility to infection in severe alcoholic hepatitis

Introduction Infection is a common cause of mortality in severe alcoholic hepatitis (SAH). Monocytes are innate immune cells that play a key role in fighting infection. We sought to characterise monocyte phenotype and function in SAH and relate defects to the development of infection. Method We analysed pre-treatment blood samples from 73 patients admitted to hospital suffering from SAH (DF >32), 34 healthy controls (HC) and 17 abstinent compensated cirrhotic patients (CLD). Flow cytometry was used to measure monocyte phenotype, cytokine production, phagocytosis and oxidative burst ex vivo . Production of superoxide (O2 -) was measured by luminometry and bacterial killing was assessed by counting viable E. coli colonies growing on agar plates after incubation with monocytes. Additionally, we evaluated the impact of in vivosteroid therapy on monocyte function via the Steroids or Pentoxyfilline for Alcoholic Hepatitis (STOPAH) clinical trial. Results The proportion of CD14+ +CD16+ + monocytes was increased in SAH and CLD vs HC (P < 0.003). These cells expressed elevated levels of activation marker HLA-DR (P < 0.01) and chemokine receptor CCR-5 (.01). In addition, they produced greater TNF-α in response to lipopolysaccharide vs CD14+ +CD16- cells (.03). Conversely, patrolling CD14+CD16+ + monocytes were reduced in SAH vs CLD and HC (.01). Phagocytosis was preserved in SAH for all subsets but monocyte oxidative burst (mOB) (P < 0.001), O2 -production (.02) and bacterial killing (.01) were markedly impaired vs CLD and HC. Importantly, pre-treatment mOB defect predicted the development of infection within the subsequent two weeks of sampling with area under receiver-operator curve of 0.86 (P < 0.02) (Figure 1). Accordingly, defective mOB was associated with death at 28 and 90 days (one-tailed .04 for both). Ex vivomOB was unchanged in patients who had received 7 days steroid therapy.Abstract OC-021 Figure 1 Conclusion Defective mOB is prevalent in patients with SAH and is strongly associated with the development of infection within the subsequent two weeks. In addition, CD14+ +CD16+ +monocytes are expanded in SAH and bear features suggestive of an inflammatory role in disease pathogenesis. Further work should seek the molecular mechanisms of these defects and components that might be amenable to therapeutic intervention. Disclosure of interest None Declared.

P104 Coagulation proteins in liver fibrosis: a role for tissue factor and fibrin/fibrinogen

Introduction Recent evidence suggests a role for the coagulation cascade in promoting liver fibrosis, but with the exception of thrombin the expression and role of individual coagulation proteins in the pathogenesis of liver fibrosis is poorly understood. Furthering our understanding of the role of specific coagulation proteins is essential when considering viable targets for anti-fibrotic therapies. Aim To quantify and qualify the expression of tissue factor (TF) and fibrin/fibrinogen in both murine liver fibrosis and human hepatitis C (HCV) related liver fibrosis. Method C57BL/6J mice (n=7), aged 8 weeks old, were treated with carbon tetrachloride by intraperitoneal injection for a period of 4 weeks to induce liver fibrosis. Animals were then culled and livers extracted and fixed in formalin. Mice injected with normal saline acted as normal controls. For human tissue, archived liver biopsy specimens (n=11) performed for the clinical staging of chronic HCV infection were used. An indirect immunohistochemical detection technique was employed with digital image analysis to qualify and semi-quantify expression of TF and fibrin/fibrinogen in tissue sections. Results In murine liver tissue, TF and fibrin/fibrinogen were expressed in hepatic sinusoids, peri-fibrotic areas and fibrotic septa. Digital image analysis demonstrated significant upregulation of TF (p=0.002) and fibrin/fibrinogen (p=0.009) in fibrotic vs normal control liver tissue. In HCV human liver tissue, TF and fibrin/fibrinogen were expressed in hepatic sinusoids and fibrotic areas. Digital image analysis demonstrated a significant correlation between TF expression and both fibrosis grade (r=0.71; p=0.015) and inflammatory score (r=0.79; p=0.004). Fibrin/fibrinogen expression was significantly correlated with inflammatory score (r=0.82; p=0.007), with a borderline correlation with grade of fibrosis (r=0.66; p=0.056). A significant correlation between TF and fibrin/fibrinogen expression was demonstrated (r=0.82; p=0.024). Conclusion The hepatic expression of TF and fibrin/fibrinogen is upregulated with fibrosis and inflammation. These findings suggest that activation of the coagulation cascade occurs in and may contribute to the generation of hepatic fibrosis. The therapeutic potential of targeted inhibition of specific coagulation proteins need to be evaluated in fibrotic liver disease.

PTH-096 Monocyte oxidative burst defect in alcoholic hepatitis is resistant to interferon gamma

Introduction An important cause of mortality in patients with severe alcoholic hepatitis (SAH) is infection. We and others have found that the oxidative burst response of phagocytic cells to E. coliis impaired in SAH which may contribute to susceptibility to infection. This is analogous to Chronic Granulomatous Disease, an inherited condition caused by a mutation in the enzyme NADPH oxidase which leads to predisposition to recurrent life-threatening infections. Treatment with interferon gamma (IFN-γ) reduces the frequency of severe infections in these patients. We sought to investigate the molecular mechanism underlying the monocyte oxidative burst (mOB) defect in SAH and determine whether IFN-γ can restore this defect in vitro . Method Peripheral blood mononuclear cells (PBMCs) were isolated from 6 patients with SAH and 4 healthy controls (HCs). Monocytes were isolated from PBMCs using magnetic MACS beads and negative selection. Monocytes were incubated with 50 ng/ml IFN-γ supplemented by 10% healthy serum for 24 h and the mOB to E. coliwas quantified by oxidation of fluorogenic rhodamine and flow cytometry as previously described. Western blotting was used to quantify protein using monoclonal antibodies to components of the IFN-γ signalling pathway (pSTAT-1 and negative regulator SOCS-1) and glucose-6-phosphate dehydrogenase (G6PD, which generates NADPH substrate required for NAPDH oxidase function), in the monocytes of HCs and AH patients. G6PD enzyme activity was measured using a colorimetric kit. Quantitative PCR was used to determine expression of STAT-1 in monocytes stimulated for 24 h in 50 ng/ml IFN-γ. Results Surprisingly, SAH mOB defect was resistant to 24 h’ stimulation by 50 ng/mlIFN-γ. The level of unstimulated phosphorylated STAT-1 protein, a transcription factor activated by IFN-γ signalling was significantly reduced in monocytes of SAH patients compared to healthy controls (pSTAT-1: housekeeping gene 0.90 HC vs 0.14 SAH, p < 0.05). q-PCR confirmed STAT-1 expression following stimulation with IFN-γ was significantly reduced in SAH compared to HCs (ΔCT 0.97 HC vs SAH 0.54, p < 0.05). The level of SOCS-1 protein was significantly increased in monocytes of SAH patients compared to HCs (SOCS-1: housekeeping gene 0.98 HC vs 2.24 AH, p < 0.05). There was no difference in G6PD enzyme activity or protein expression between groups. Conclusion Our study suggests that elevated levels of the inhibitory factor SOCS-1 may reduce phosphorylation and activation of STAT-1. Reduced pSTAT-1 may explain the resistance to IFN-γ observed and hence the burst defect in SAH. This work provides a novel insight into the impaired mOB in SAH and furthers our knowledge of the predisposition to infection observed in this patient group. Disclosure of interest None Declared.

390 TARGETED INHIBITION OF TISSUE FACTOR AND THROMBIN ON CD31 EXPRESSING CELLS SUPPRESSES HEPATIC FIBROSIS IN CCL4 TREATED MICE

Introduction Recent evidence suggests a role for the coagulation cascade in promoting liver fibrosis. However, the cellular basis for this relationship is unclear. In order to explore this relationship we employed two unique transgenic mice strains expressing membrane–tethered tissue factor pathway inhibitor (TFPI) or hirudin (anti-thrombin) fusion proteins driven by a CD31 promoter. These strains allow for the selective inhibition of tissue factor (TF) or thrombin on endothelial cells, monocytes and platelets expressing CD31. Aims To evaluate the impact of the targeted inhibition of tissue factor and thrombin on effector cells of liver fibrosis in murine hepatic fibrosis induced by CCl 4 . Methods Liver fibrosis was induced in CD31-TFPI, CD31-Hirudin and wild type control mice with 4 weeks carbon tetrachloride (CCl 4 ) administered by intraperitoneal injection. Animals were culled and livers extracted for histological and biochemical analysis. Fibrosis was scored using a four point semi-quantitative system and quantified by digital image analysis to determine percentage area of fibrosis. Immunohistochemistry to determine α-SMA expression, a marker of hepatic stellate cell activation was performed and the mean number of activated stellate cells was quantified per high power field. Results The percentage area of fibrosis was significantly less in CD31-TFPI (1.89%±0.26, p=0.001) and CD31-Hirudin mice (1.04%±0.16, p=0.00003) in comparison to control mice (3.66%±0.39). Semiquantitative fibrosis scoring showed a significant difference between the CD31-TFPI (median 2/4) and CD31-Hirudin (median 3/4) mice in comparison with control mice (median 3/4, p=0.009) but the difference between the two transgenic strains was not significant. Both transgenic strains demonstrated a significantly reduced mean number of α-SMA stellate cells per high power field in comparison to control mice (CD31-TFPI vs control, 7.4 vs 29.4, p=0.0002; CD31-hirudin vs control, 5.25 vs 29.4, p=0.0002). Conclusion CD31 targeted inhibition of TF and thrombin significantly reduces liver fibrosis and stellate cell activation in a murine CCl 4 model. The data supports the use of this novel murine model as a tool for investigating the cellular biology of the role of coagulation in liver fibrogenesis. It will also provide information for developing new treatments of human liver fibrosis. Competing interests None declared.

P56 Factor Xa inhibition suppresses thioacetamide induced liver fibrosis

Introduction In addition to its role in activating fibrinogen, thrombin mediates cellular activation of macrophages, platelets and hepatic stellate cells via the protease-activated receptor, PAR-1. Thrombin antagonists demonstrate anti-fibrotic properties. Factor Xa (FXa), a protease which is activated earlier in the coagulation cascade, promotes connective tissue growth factor, and activates fibroblasts via PAR receptors. Direct FXa inhibition has recently been shown to significantly reduce lung fibrosis, a paradigm for hepatic fibrosis, in a bleomycin mouse model. Specific inhibition of FXa may offer additional efficacy as an anti-fibrotic in models of chronic liver injury. Aim To evaluate the impact of FXa inhibition and thrombin antagonism on hepatic fibrosis using a thioacetamide (TAA) mouse model. Method 45 C57BL/6J mice were administered TAA (300 mg/l) via drinking water for a period of 8 weeks to induce liver fibrosis. A subset of these animals were given Rivaroxaban, a direct FXa inhibitor (n=15), or Dagibatran, a direct thrombin antagonist (n=15). Both drugs were administered daily by oral gavage at doses to achieve prolongation of the prothrombin time. The remaining animals (n=15) received no anticoagulation, and acted as the control group. At 8 weeks livers were extracted and liver sections stained with picorsirus red and visually scored for fibrosis using an adapted Ishak Modified Histology Activity Index by a blinded histopathologist. Digital image analysis was performed to calculate the mean percentage area of fibrosis per section. Results In control mice the mean fibrosis score was 4.08 and the mean percentage area of fibrosis was 3.76%. In comparison mice treated with FXa inhibition had a mean fibrosis score of 2.46 (p=0.008) and mean percentage area of fibrosis of 2.02% (p=0.012). In contrast mice treated with thrombin inhibition had a mean fibrosis score of 3.25 (p=0.68 vs controls), and mean percentage area of fibrosis of 3.70% (p=0.68 vs controls). Factor Xa inhibition was significantly more effective than thrombin in reducing percentage area of fibrosis (p=0.031). Conclusion FXa inhibition significantly decreased the rate of hepatic fibrosis in a TAA model of liver fibrosis. It is likely that direct thrombin inhibition is less effective than FXa inhibition because thrombin inhibitors fail to block PAR-mediated stellate cell activation by FXa. FXa inhibition is a potential novel anti-fibrotic approach and warrants further investigation in human studies.