Post-Transcriptional Regulation of Hepatic DDAH1 with TNF Blockade Leads to Improved eNOS Function and Reduced Portal Pressure In Cirrhotic Rats.

Portal hypertension (PH) is a major cause of morbidity and mortality in chronic liver disease. Infection and inflammation play a role in potentiating PH and pro-inflammatory cytokines, including TNF, are associated with severity of PH. In this study, cirrhotic bile duct ligated (BDL) rats with PH were treated with Infliximab (IFX, a monoclonal antibody against TNF) and its impact on modulation of vascular tone was assessed. BDL rats had increased TNF and NFkB compared to sham operated rats, and their reduction by IFX was associated with a reduction in portal pressure. IFX treatment also reduced hepatic oxidative stress, and biochemical markers of hepatic inflammation and injury. IFX treatment was associated with an improvement in eNOS activity and increased L-arginine/ADMA ratio and DDAH1 expression. In vitro analysis of HepG2 hepatocytes showed that DDAH1 protein expression is reduced by oxidative stress, and this is in part mediated by post-transcriptional regulation by the 3'UTR. This study supports a role for the DDAH1/ADMA axis on the effect of inflammation and oxidative stress in PH and provides insight for new therapies.

Neutrophil gelatinase-associated lipocalin is a biomarker of acute-on-chronic liver failure and prognosis in cirrhosis.

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is a syndrome that occurs in cirrhosis characterized by organ failure(s) and high mortality rate. There are no biomarkers of ACLF. The LCN2 gene and its product, neutrophil gelatinase-associated lipocalin (NGAL), are upregulated in experimental models of liver injury and cultured hepatocytes as a result of injury by toxins or proinflammatory cytokines, particularly Interleukin-6. The aim of this study was to investigate whether NGAL could be a biomarker of ACLF and whether LCN2 gene may be upregulated in the liver in ACLF. METHODS: We analyzed urine and plasma NGAL levels in 716 patients hospitalized for complications of cirrhosis, 148 with ACLF. LCN2 expression was assessed in liver biopsies from 29 additional patients with decompensated cirrhosis with and without ACLF. RESULTS: Urine NGAL was markedly increased in ACLF vs. no ACLF patients (108(35-400) vs. 29(12-73)µg/g creatinine; p<0.001) and was an independent predictive factor of ACLF; the independent association persisted after adjustment for kidney function or exclusion of variables present in ACLF definition. Urine NGAL was also an independent predictive factor of 28day transplant-free mortality together with MELD score and leukocyte count (AUROC 0.88(0.83-0.92)). Urine NGAL improved significantly the accuracy of MELD in predicting prognosis. The LCN2 gene was markedly upregulated in the liver of patients with ACLF. Gene expression correlated directly with serum bilirubin and INR (r=0.79; p<0.001 and r=0.67; p<0.001), MELD (r=0.68; p<0.001) and Interleukin-6 (r=0.65; p<0.001). CONCLUSIONS: NGAL is a biomarker of ACLF and prognosis and correlates with liver failure and systemic inflammation. There is remarkable overexpression of LCN2 gene in the liver in ACLF syndrome. LAY SUMMARY: Urine NGAL is a biomarker of acute-on-chronic liver failure (ACLF). NGAL is a protein that may be expressed in several tissues in response to injury. The protein is filtered by the kidneys due to its small size and can be measured in the urine. Ariza, Graupera and colleagues found in a series of 716 patients with cirrhosis that urine NGAL was markedly increased in patients with ACLF and correlated with prognosis. Moreover, gene coding NGAL was markedly overexpressed in the liver tissue in ACLF.

Role of Toll-like receptors 2, 4, and 9 in mediating neutrophil dysfunction in alcoholic hepatitis.

Neutrophil dysfunction in alcoholic hepatitis is associated with endotoxemia and an increased incidence of infection, but the mechanism is unclear. We aimed to investigate the role of Toll-like-receptors (TLR)2, 4, and 9 in mediating neutrophil dysfunction in alcoholic hepatitis. Neutrophils from healthy volunteers were incubated with alcoholic hepatitis patients' plasma (n = 12) with and without TLR2, 4, or 9 antagonists and with and without human albumin. TLR2, 4, and 9 expression, neutrophil oxidative burst, phagocytosis, and CXCR1+2 expression were measured by FACS analysis. Patients' plasma increased oxidative burst, decreased CXCR1+2 expression, and decreased phagocytosis of normal neutrophils in association with increased expression of TLR2, 4, and 9 and depletion of ATP. Inhibition of TLR2, 4, and 9 prevented the increase in oxidative burst and the decrease in CXCR1 and CXCR2 expression but did not prevent phagocytic dysfunction. Incubation with albumin completely prevented the patient plasma induced neutrophil dysfunction. Increased expression of TLR2, 4, and 9 is associated with neutrophil dysfunction, endotoxemia, and energy depletion. TLR2, 4, and 9 inhibition does not improve phagocytosis, indicating that TLR overexpression may be the result and not the cause of neutrophil activation. Albumin, an endotoxin scavenger, prevents the deleterious effect of patients' plasma on neutrophil phagocytosis, resting burst, and TLR expression.

Role of ammonia and inflammation in minimal hepatic encephalopathy.

BACKGROUND: Minimal hepatic encephalopathy (MHE) is common in cirrhosis but its pathophysiologic basis remains undefined. We evaluated whether the presence of MHE was associated with severity of liver disease, ammonia levels or the presence of inflammation and assessed factors determining neuropsychological deterioration accompanying induction of hyperammonemia. METHODS: Eighty four cirrhotics were studied. A neuropsychological test battery was performed and blood taken for ammonia, WCC, CRP, nitrate/nitrite, IL-6 and amino acids, before and after, induction of hyperammonemia by administration of a solution mimicking the amino acid composition of haemoglobin (60) or placebo (24). RESULTS: The presence and severity of MHE were independent of severity of liver disease and ammonia concentration but markers of inflammation were significantly higher in those with MHE compared with those without. Induction of hyperammonemia produced deterioration in one or more neuropsychological tests by > or =1 SD in 73.3%. This was independent of the magnitude of change in plasma ammonia and severity of liver disease but was significantly greater in those with more marked inflammation. CONCLUSION: Our data show that inflammation is an important determinant of the presence and severity of MHE. The change in neuropsychological function following induced hyperammonemia is greater in those with more severe inflammation.

Remote ischaemic preconditioning of the hind limb reduces experimental liver warm ischaemia-reperfusion injury.

BACKGROUND: Direct ischaemic preconditioning of the liver reduces ischaemia-reperfusion injury (IRI). Remote ischaemic preconditioning (RIPC) of a limb has been shown to reduce IRI to the heart. This study determined the effect of brief remote ischaemia to the limb in reducing early liver warm IRI. METHODS: Twenty-eight male rabbits were allocated to four groups: sham operated, RIPC alone, IRI alone, and RIPC plus IRI. RIPC was induced in the leg with a tourniquet, before liver IRI, by three alternate cycles of 10 min ischaemia followed by 10 min reperfusion. Liver IRI was produced by total inflow occlusion for 25 min. Markers of liver injury and systemic and hepatic haemodynamics were measured for 2 h after reperfusion. RESULTS: At 2 h, IRI alone was associated with increased serum levels of aminotransferases, and reduced mean arterial blood pressure, hepatic blood flow and peripheral oxygen saturation. There was significant improvement in these variables in animals that had RIPC before liver IRI, and hepatic venous nitrate/nitrite levels were also significantly higher. CONCLUSION: In this experimental model RIPC appeared to reduce liver IRI.

Tumour necrosis factor alpha is an important mediator of portal and systemic haemodynamic derangements in alcoholic hepatitis.

BACKGROUND: The role of proinflammatory cytokines in the pathogenesis of portal hypertension is unclear. AIMS AND METHODS: This study tests the hypothesis that tumour necrosis factor alpha (TNF-alpha) is an important mediator of the circulatory disturbances in alcoholic hepatitis (AH) and evaluates the acute and short term effect of a single infusion of the monoclonal chimeric anti-TNF-alpha antibody (Infliximab) on portal and systemic haemodynamics in 10 patients with severe biopsy proven AH. Cardiovascular haemodynamics, hepatic venous pressure gradient (HVPG), and hepatic and renal blood flow were measured before, 24 hours after Infliximab, and prior to hospital discharge. RESULTS: Serum bilirubin (p<0.05), C reactive protein (p<0.001), and white cell count (p<0.01) were reduced significantly, as were plasma levels of interleukin (IL)-6 and IL-8 after treatment. Of the 10 patients, nine were alive at 28 days. Mean HVPG decreased significantly at 24 hours (23.4 (2.8) to 14.3 (1.9) mm Hg; p<0.001) with a sustained reduction prior to discharge (12.8 (1.9) mm Hg; p<0.001). Mean arterial pressure and systemic vascular resistance increased significantly (p<0.001and p<0.01, respectively), mirrored by a reduction in cardiac index (5.9 (0.5) to 4.7 (0.5) l/min/m(2); p<0.05) prior to discharge. Hepatic and renal blood flow also increased significantly (506.2 (42.9) to 646.3 (49.2) ml/min (p=0.001) and 424.3 (65.12) to 506.3 (85.7) ml/min (p=0.001), respectively) prior to discharge. CONCLUSION: The results of this study illustrate that anti-TNF-alpha treatment in AH patients produces a highly significant, early, and sustained reduction in HVPG, possibly through a combination of a reduction in cardiac output and intrahepatic resistance. In addition, there was a reduction in hepatic inflammation and improved organ blood flow, suggesting an important role for TNF-alpha in mediating the circulatory disturbances in AH.

Combined hepatic artery and segmental portal vein occlusion in antiphospholipid syndrome.

Antiphospholipid syndrome can have various clinical presentations, two of the most common being arterial and venous thrombosis. It is, however, unusual for them to occur in combination. We report here a case of combined hepatic artery and segmental portal venous occlusion in a 32-year-old patient who was shown to have a lupus anticoagulant. There have been no previous reports of thrombosis occurring simultaneously in the coeliac axis and the portal vein. Computerised tomography, Doppler ultrasound scanning and selective visceral angiography were used to demonstrate the anatomical lesions. The patient was treated medically with unfractionated heparin leading to a favourable clinical outcome. The diagnosis and management of this case is discussed with reference to the current literature on visceral thrombosis and antiphospholipid antibody syndrome.

Indocyanine green clearance reflects reperfusion injury following liver transplantation and is an early predictor of graft function.

BACKGROUND/AIMS: Primary graft dysfunction is difficult to predict. We have previously shown that indocyanine green clearance measured at 24 h following orthotopic liver transplantation predicts graft survival and outcome. We prospectively evaluated the use of indocyanine green clearance (with a cut-off value of 200 ml/min) as a marker of graft function following orthotopic liver transplantation and investigated its relationship with the markers of reperfusion injury during orthotopic liver transplantation. METHODS: In all patients indocyanine green clearance was measured at 24 h. Repeated blood samples were taken before, during the anhepatic and reperfusion phase and up to 12 h following orthotopic liver transplantation to measure the levels of neutrophil elastase and reactive oxygen intermediates. All patients studied had normal hepatic arterial pulse on Doppler-ultrasound post orthotopic liver transplantation. RESULTS: All patients with indocyanine green clearance >200 ml/min recovered following orthotopic liver transplantation and remained well up to 3 months of follow up. Four patients had an indocyanine green clearance <200 ml/min; three were re-transplanted for graft failure within 3 days of the transplant, while one survived after prolonged intensive support and hospitalization. Indocyanine green clearance significantly correlated with reactive oxygen intermediates production and neutrophil elastase during orthotopic liver transplantation (r=-0.61, p<0.002 and r=-0.66, p<0.0009, respectively). Indocyanine green clearance was also significantly correlated with alanine aminotransferase and prothrombin time at 24 h post-transplantation (r=-0.35, p<0.02 and r=-0.4, p<0.0077, respectively). CONCLUSION: Indocyanine green reflects the degree of reperfusion injury and is a good early marker of primary graft function. Indocyanine green clearance over 200 ml/min is associated with favorable outcome.

Prediction of cirrhosis in patients with chronic hepatitis C infection by artificial neural network analysis of virus and clinical factors.

The diagnosis of cirrhosis in patients with hepatitis C virus (HCV) infection is currently made using a liver biopsy. In this study we have trained and validated artificial neural networks (ANN) with routine clinical host and viral parameters to predict the presence or absence of cirrhosis in patients with chronic HCV infection and assessed and interpreted the role of the different inputs on the ANN classification. Fifteen routine clinical and virological factors were collated from 112 patients who were HCV RNA positive by reverse transcriptase-polymerase chain reaction (RT-PCR). Standard and Ward-type feed-forward fully-connected ANN analyses were carried out both by training the networks with data from 82 patients and subsequently testing with data from 30 patients plus performing leave-one-out tests for the whole patient data set. The ANN results were also compared with those from multiple logistic regression. The performance of both ANN methods was superior compared with the logistic regression. The best performance was obtained with the Ward-type ANNs resulting in a sensitivity of 92% and a specificity of 98.9% together with a predictive value of a positive test of 95% and a predictive value of a negative test of 97% in the leave-one-out test. Hence, further validation of the ANN analysis is likely to provide a non-invasive test for diagnosing cirrhosis in HCV-infected patients.

Transjugular intrahepatic portosystemic stent-shunt (TIPSS) insertion as a prelude to orthotopic liver transplantation in patients with severe portal hypertension.

OBJECTIVE: To investigate the impact of preoperative transjugular intrahepatic portosystemic stent-shunt (TIPSS) on patients undergoing liver transplantation. DESIGN: A retrospective non-randomized comparative clinical study. SETTING: Tertiary referral institution. PATIENTS, PARTICIPANTS: Twenty-four patients with liver cirrhosis, portal hypertension and gastro-oesophageal varices who underwent liver transplantation. INTERVENTIONS: TIPSS insertion had been performed preoperatively in 12 patients. MAIN OUTCOME MEASURES: Operative dissection times and blood transfusion requirements during liver transplantation. Postoperative complication rate. Cumulative patient and graft survival. RESULTS: There were no significant differences in outcome measures between patients with and without previous TIPSS insertion with respect to recipient hepatectomy times (mean 192 min (126-280) versus 196 min (145-254)), total operating time (mean 484 min (330-690) versus 486 min (370-580)), intraoperative blood transfusion (mean 11 units (2-29) versus 12 units (2-30)), intraoperative fresh frozen plasma transfusion (mean 9 units (1-16) versus 11 (2-23) units), patient survival (83% versus 92% cumulative 1-year survival), graft survival (80% versus 83% cumulative 1-year survival), or postoperative complication rates. CONCLUSION: TIPSS insertion is feasible and relatively safe as a 'bridge to transplantation' in patients who have had a variceal haemorrhage. There is little evidence that preoperative TIPSS insertion directly affects the performance of liver transplantation as TIPSS neither, hinders nor facilitates surgery or post operative survival. Although it is important that the potential hazards of TIPSS extension into the inferior vena cava or superior mesenteric vein be recognized, liver transplant surgeons need not be unduly concerned about the overall impact of TIPSS as it becomes more universally available in the management of variceal haemorrhage.

Transjugular intrahepatic portosystemic stent-shunt (TIPSS) occlusion and the role of biliary venous fistulae.

BACKGROUND/AIM: The aim of this study was to assess the pathology and pathogenic mechanisms involved in the occlusion of transjugular intrahepatic portosystemic stent-shunts. METHODS: Thirty-four patients with transjugular intrahepatic portosystemic stent-shunt who had at least two portographic assessments of shunt function were the subjects of this study. The contents of any shunt demonstrating > 70% stenosis were biopsied before balloon dilatation. Further assessment was made of 10 livers obtained at either post mortem (8) or at liver transplantation (2). Cholangiography was performed in these explanted livers, which were then perfused and fixed with formaldehyde. The shunts were dissected out, sectioned, opened and the contents and the surrounding liver examined macroscopically, histopathologically and immunohistochemically. RESULTS: Fourteen patients with TIPSS developed shunt stenosis. In eight patients the stenosis was greater than 70% and significant re-stenosis occurred in all at repeat portography. Three of these patients who were managed by insertion of new shunts showed no further shunt-related problems, whereas the five who were treated solely by dilatation developed further re-stenosis. Organising thrombus was found in all eight patients and bile was incorporated in the thrombus in four. Biliary epithelium was found in two. Four of the ten explanted livers showed evidence of shunt stenosis, of which three were severe and one was mild (< 70%). The occluding material in patients with severe stenosis was composed of organising thrombus containing bile and a granulomatous inflammatory response. This was associated with a transected bile duct, and the degree of stenosis was related closely to the size of the bile duct transected. The shunts free of bile showed no stenosis. CONCLUSIONS: The results of this study suggest that transection of a major bile duct and bile leak play an important role in the stenosis and occlusion of the intraparenchymal portion of transjugular intrahepatic portosystemic stent-shunt. This has important implications for patient management and stent design.

A comparative study of emergency transjugular intrahepatic portosystemic stent-shunt and esophageal transection in the management of uncontrolled variceal hemorrhage.

OBJECTIVES: Transjugular intrahepatic portosystemic stent-shunts (TIPSS) have been shown to reduce portal hypertension consistently and have recently been reported to arrest active variceal hemorrhage. This retrospective and nonrandomized study compares the results of TIPSS with esophageal transection (ET) and devascularization in patients with uncontrolled variceal hemorrhage admitted to a single center with an interest in variceal bleeding. PATIENTS AND METHODS: Two hundred and sixty cirrhotic patients have been referred with variceal bleeding over the past 7 yr. In 41 patients (15.8%), hemorrhage was uncontrolled despite two treatments with sclerotherapy. Thirty-eight patients were eligible for analysis. Nineteen were considered for ET and 19 for TIPSS. Patients in the two groups were well matched for age, sex, etiology of liver disease, and its severity and complications. They have been followed for 13 patient years (TIPSS-7, longest 20 months; ET-6, longest 23 months). Data for survival and rebleeding were analyzed by the Kaplan-Meier method on an intention-to-treat basis. RESULTS: Seven of the 19 were considered unfit for surgery, and 12 underwent esophageal transection and devascularization. TIPSS was undertaken successfully in 17 patients, the Palmaz stent being used in 4 and the Wallstent in 13. Successful TIPSS reduced the mean portal pressure gradient from 22.2 (SE 1.2) to 9.7 (SE 0.7) mm Hg (p < 0.001). Mortality within 30 days of the initial bleed was 42% in the TIPSS group compared with 79% in the ET group (p < 0.05). Rebleeding occurred in 15.6% patients with TIPSS, compared with 26.2% in the ET group. Encephalopathy in the two groups of patients was not significantly different (TIPSS 25% and ET 22%). TIPSS was followed by active infection in 20% compared with 36% after ET. CONCLUSIONS: This study shows that the overall mortality in this group of patients is high whatever the type of treatment used. TIPSS can be performed successfully on these patients who are often not suitable for surgery. Mortality rates were significantly lower in patients treated by TIPSS. Compared with ET, TIPSS should be regarded as the preferred mode of treatment for uncontrolled variceal hemorrhage in patients with cirrhosis.

Transjugular intrahepatic portasystemic stent-shunt in the treatment of variceal haemorrhage.

The transjugular intrahepatic portasystemic stent-shunt (TIPSS) is a side-to-side portocaval shunt, performed by interventional radiological methods, linking the hepatic and portal veins through the liver parenchyma with an expandable metal stent. The technique can be performed successfully in over 90 per cent of patients. The procedure-related mortality rate is about 1 per cent, mainly from intraperitoneal bleeding. The main indications for TIPSS insertion are control of acute variceal bleeding in patients with cirrhosis that is refractory to sclerotherapy and recurrent variceal haemorrhage despite sclerotherapy or band ligation. TIPSS insertion is followed by variceal rebleeding in about 10-20 per cent of cases, encephalopathy in 10-20 per cent, transient deterioration of liver function in 25-35 per cent and subsequent shunt dysfunction over a 6-12-month period in 15-60 per cent. The final place of TIPSS insertion in the management of portal hypertension is being evaluated in controlled studies, but its use in the treatment of uncontrolled variceal haemorrhage seems assured.

Laparoscopy and histology in the diagnosis of chronic liver disease.

We compared laparoscopy with histology in identifying fatty change, fibrosis, the degree of inflammatory activity, cirrhosis and the cause of liver disease. Laparoscopic liver biopsy was performed in 145 consecutive patients. The laparoscopist and the histologist were provided with similar clinical and biochemical information. Both scored the appearances on respective examinations for the degree of fatty change, fibrosis and activity, presence or absence of cirrhosis; and provided a provisional diagnosis. The final diagnosis was determined by clinicopathological conference and clinical follow-up. Laparoscopy was successfully performed in 142 patients (97.9%). Compared with histology, the sensitivity and specificity of laparoscopy for identifying fatty change were 96.4% and 100%, 100% and 95% for fibrosis and 94% and 95% for inflammatory activity, respectively. For cirrhosis, laparoscopy was 100% sensitive and 97.1% specific. Histology missed 10 cases of cirrhosis (6.1%). Histology did, however, provide additional information in 9 patients (6.3%) which contributed to the final diagnosis. Overall, histology is required in addition to laparoscopy in cases where the aetiology is unclear. The sensitivity and specificity in identifying fatty change, fibrosis, activity and cirrhosis are similar for laparoscopy and histology. The combination of information gained on laparoscopy with histology provides the diagnosis in most patients. Laparoscopy may replace the need for liver biopsy in patients in whom the aetiological diagnosis is not in question and the biopsy is being performed to stage the disease. We used it as an integral part of the work-up of a patient with liver disease.

Laparoscopy in the diagnosis of chronic liver disease.

Although laparoscopy has been in use for a substantial length of time, it remains a much underused tool in the diagnosis of chronic liver disease. The purpose of this review is to assess the present practice of laparoscopy in order to define its place as a diagnostic tool for the hepatologist.

Transjugular intrahepatic portosystemic stent-shunt (TIPSS): long-term follow-up.

We assessed the long-term efficacy of transjugular intrahepatic portasystemic stent-shunt (TIPSS) in 64 patients. Insertion was successful in 56 patients (87.5%). The reasons for its use were: variceal bleeding (49); ascites (6); portal hypertensive gastropathy (6); hypersplenism (2); and embolization of a spontaneous shunt (1). Fourteen patients were Childs A, 20 Childs B and 28 Childs C cirrhotics. Two patients were non-cirrhotic; one with amyloidosis and one with non-cirrhotic portal fibrosis. Patients were followed clinically and radiologically (Doppler ultrasonography and routine portography at 6 months). During 33 patient-years of follow-up, 22 died, 12 during index admission (two were procedure-related) and nine were transplanted. Twenty-five patients are alive, with a mean survival of 7.1 (SD 7) months. Variceal rebleeding occurred in 10 patients (22.7%), one of whom died, and was always associated with shunt insufficiency (shunt thrombosis 2, hepatic vein stenosis (HVS) 1, intimal hyperplasia (IH) 4, dislocated stent 1, inadequate stent 2). Clinical encephalopathy was induced in seven patients (17.1%) following TIPSS. All responded to medical therapy, but two required reduction in shunt size. Ascites improved after TIPSS in 36 patients (87.8%), but reaccumulated in seven (17.5%), associated with shunt dysfunction in five (SBP 2, IH 3, HVS 2). Fatal sepsis occurred in two patients, and 14 other episodes of infection required antibiotics. TIPSS is a useful treatment for variceal bleeding, resistant ascites and portal hypertensive gastropathy. Shunt dysfunction and sepsis occur frequently, and regular surveillance is necessary.

Multiple pancreatic abscesses due to Candida albicans following ERCP.

A report is presented of a patient who developed multiple abscesses of the pancreas due to Candida albicans following an Endoscopic retrograde chole-pancreatography (ERCP) for acute pancreatitis. He was not immunocompromised, debilitated and had not had recent surgery. There was complete radiological and clinical resolution of the abscess on prolonged treatment with amphotericin alone. Only a few cases of candidal abscess of the pancreas have been reported, none of them having occurred after an ERCP.