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.

Automated low flow pump system for the treatment of refractory ascites: a single-center experience.

INTRODUCTION: Ascites is a common complication of liver cirrhosis and represents the main cause of hospitalization among patients with cirrhosis. First-line therapy for those patients is the use of diuretics and dietary sodium restriction. However, 10 % of patients per year become therapy refractory to diuretic treatment with the need of repeated high-volume paracentesis or transjugular intrahepatic portosystemic shunt (TIPS). For these patients, an automated pump system (Alfapump/Sequana Medical) was developed. Here, we describe our single-center experience of ten consecutively implanted pump systems. PATIENTS AND METHODS: Between 08/13 and 11/14, ten Alfapump systems were implanted in patients with refractory ascites all suffering from liver cirrhosis. Those patients were treated as a bridge to transplant (4/10) or as an end-stage therapy (6/10). Median follow-up was 165 days (23-379 days). RESULTS: Postimplant, the need of paracentesis could be markedly reduced to a mean of 0.45 (0-4/month) per month. In eight patients, paracentesis was not needed after implantation of the pump system. The median daily output volume was 1000 ml/day (450-2000 ml/day). Prerenal insufficiency was a recurrent complication in the postoperative period. DISCUSSION: The Alfapump system is a useful system in the treatment of patients suffering from therapy refractory ascites. However, due to the high level of comorbidities, careful patient selection and postoperative monitoring are required.

Acute postinfectious glomerulonephritis associated with Campylobacter jejuni enteritis - a case report and review of the literature on C. jejuni's potential to trigger immunologically mediated renal disease.

Kidney disease is a rare complication of Campylobacter jejuni (C. jejuni) enteritis. We here present the case of an 18-year-old male patient with crampy abdominal pain, vomiting, diarrhea, and fever. Three weeks later urinalysis revealed mild proteinuria and hematuria and a marked raise in serum creatinine was observed. Renal biopsy demonstrated acute endocapillary glomerulonephritis with mesangial IgM (immunoglobuline M) deposits. Extensive workup revealed no signs of skin or joint disease, thus excluding Henoch-Schönlein purpura. Due to persistent abdominal discomfort further gastro-enterological tests were performed and eventually Campylobacter jejuni was isolated from the patient's feces. In the absence of other precipitating factors for renal diseases we presumed an association between the bacterial infection and this postinfectious glomerulonephritis. Over a time period of 6 months the patient's kidney function normalized completely. However, long-term prognosis remains unclear. In addition to the case report, we conducted a review of the literature with results underlining Campylobacter jejuni's potential to trigger various types of immune mediated kidney diseases.

A novel technique for selective NF-kappaB inhibition in Kupffer cells: contrary effects in fulminant hepatitis and ischaemia-reperfusion.

BACKGROUND AND AIMS: The transcription factor nuclear factor kappa B (NF-kappaB) has risen as a promising target for anti-inflammatory therapeutics. In the liver, however, NF-kappaB inhibition mediates both damaging and protective effects. The outcome is deemed to depend on the liver cell type addressed. Recent gene knock-out studies focused on the role of NF-kappaB in hepatocytes, whereas the role of NF-kappaB in Kupffer cells has not yet been investigated in vivo. Here we present a novel approach, which may be suitable for clinical application, to selectively target NF-kappaB in Kupffer cells and analyse the effects in experimental models of liver injury. METHODS: NF-kappaB inhibiting decoy oligodeoxynucleotides were loaded upon gelatin nanoparticles (D-NPs) and their in vivo distribution was determined by confocal microscopy. Liver damage, NF-kappaB activity, cytokine levels and apoptotic protein expression were evaluated after lipopolysaccharide (LPS), d-galactosamine (GalN)/LPS, or concanavalin A (ConA) challenge and partial warm ischaemia and subsequent reperfusion, respectively. RESULTS: D-NPs were selectively taken up by Kupffer cells and inhibited NF-kappaB activation. Inhibition of NF-kappaB in Kupffer cells improved survival and reduced liver injury after GalN/LPS as well as after ConA challenge. While anti-apoptotic protein expression in liver tissue was not reduced, pro-apoptotic players such as cJun N-terminal kinase (JNK) were inhibited. In contrast, selective inhibition of NF-kappaB augmented reperfusion injury. CONCLUSIONS: NF-kappaB inhibiting decoy oligodeoxynucleotide-loaded gelatin nanoparticles is a novel tool to selectively inhibit NF-kappaB activation in Kupffer cells in vivo. Thus, liver injury can be reduced in experimental fulminant hepatitis, but increased at ischaemia-reperfusion.

JNK inhibition sensitises hepatocellular carcinoma cells but not normal hepatocytes to the TNF-related apoptosis-inducing ligand.

BACKGROUND: cJun terminal kinase (JNK) is constitutively activated in most hepatocellular carcinomas (HCCs), yet its exact role in carcinogenesis remains controversial. While tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known as a major mediator of acquired immune tumour surveillance, and is currently being tested in clinical trials as a novel cancer therapy, the resistance of many tumours to TRAIL and concerns about its toxicity in vivo represent obstacles to its clinical application. In this study we investigated whether JNK activity in HCC could contribute to the resistance to apoptosis in these tumours. METHODS: The effect of JNK/Jun inhibition on receptor-mediated apoptosis was analysed by pharmacological inhibition or RNA interference in cancer cells and non-tumour cells isolated from human liver or transgenic mice lacking a phosphorylation site for Jun. RESULTS: JNK inhibition caused cell cycle arrest, enhanced caspase recruitment, and greatly sensitised HCC cells but not normal hepatocytes to TRAIL. TRAIL-induced activation of JNK could be effectively interrupted by administration of the JNK inhibitor SP600125. CONCLUSIONS: Expression and TRAIL-dependent feedback activation of JNK likely represent a mechanism by which cancer cells escape TRAIL-mediated tumour surveillance. JNK inhibition might represent a novel strategy for specifically sensitising HCC cells to TRAIL thus opening promising therapeutic perspectives for safe and effective use of TRAIL in cancer treatment.

[64-year old patient with tachycardia and signs of heart failure]. / 64-jähriger Patient mit Herzrasen und Zeichen einer Herzinsuffizienz.

We report on a 64-year old patient with known Morbus Osler and high cardiac output failure due to distinct arterio-venous malformations of the liver. Since the patient suffered from severe right heart insufficiency despite optimized medical therapy, we decided to conduct an interventional occlusion of the hepatic shunts in three single sessions. The transient elevation of transaminases was reversible. After interventional therapy cardiac output decreased from 20 l/min to 15 l/min (25%) leading to a reduction of diuretic dosage and a sustained stabilization of the clinical condition.

Immunoreactive human chorionic gonadotropin and its free beta subunit in serum and ascites of patients with malignant tumors.

Human chorionic gonadotropin (hCG) is a clinically relevant marker of trophoblastic and nontrophoblastic malignancies. In the present studies, in addition to determining serum hCG, we investigated the presence and properties of hCG immunoreactivity in ascites of patients with nontrophoblastic malignant tumors and, for comparison, in ascites caused by cirrhotic liver disease. Total hCG immunoreactivity [hCG (+hCG-beta)] was found to be elevated above the reference value (greater than 5 IU/liter) in the serum of 2 of 20 patients with cirrhosis and 11 of 20 patients with malignant tumors. For comparison, in ascites, hCG (+hCG-beta) concentrations were frequently higher than in the corresponding serum samples and exceeded 10 IU/liter in 0 of 20 cirrhotic samples and in 16 of 20 malignant samples. In order to elucidate the nature of the hCG immunoreactive material, all samples were then assessed by immunoradiometric assays specific for the intact hCG molecule (holo-hCG) and the free hCG-beta subunit, respectively. In the holo-hCG assay, elevated values were detected in 0 of 20 (0 of 20) cirrhotic ascites (serum) samples and 0 of 20 (1 of 20) malignant ascites (serum) samples. In the free hCG-beta assay, on the other hand, no positive results were obtained in the ascites or serum of 20 patients with liver cirrhosis; however, 8 of 20 serum samples and 16 of 20 ascites samples derived from tumor patients were positive. In accord with the immunological data, gel chromatographical studies of malignant ascites revealed the abundance of free hCG-beta subunit rather than that of holo-hCG. In contrast to malignancy-related ascites, in ascites of patients receiving hCG injections for treatment of infertility, holo-hCG was more abundant than free hCG-beta immunoreactivity. Incubation experiments of purified holo-hCG in ascites for 24 h at -20, 20, or 37 degrees C showed no substantial dissociation of the hCG molecule and release of free hCG-beta immunoreactivity, thus arguing against production of free hCG-beta by degradation of holo-hCG and in favor of its tumor-related secretion. In conclusion, hCG-beta immunoreactivity is frequently elevated in malignancy-related ascites and appears to be related to the presence of free beta subunit of hCG rather than that of the intact hCG molecule. Interestingly, hCG-beta determination in ascites proved to be clearly superior to serum measurement in discriminating between tumor and cirrhosis.(ABSTRACT TRUNCATED AT 400 WORDS)

The role of atrial natriuretic peptide (ANP) in chronic liver disease.

The role of atrial natriuretic peptide (ANP) and potential defects of ANP in liver disease are reviewed. Patients with cirrhosis of the liver show no decrease of ANP plasma concentrations nor changes in the pattern of ANP immunoreactivity nor changes of splanchnic ANP clearance. The renal effects of exogenously administered as well as endogenously released ANP are blunted in cirrhosis, in particular in patients with ascites. This seems due to increased activity of sodium-retaining hormonal systems and changes of the renal ANP receptor status. Pharmacological inhibition of ANP-degradation or clearance may yield therapeutic potential.

Tumour size is an important predictor for the outcome after liver transplantation for hepatocellular carcinoma.

AIMS: Recently, there is a tendency to expand tumour sizes qualifying for OLT. The present study re-evaluates tumour size and histopathological features as selection criteria for OLT. METHODS: Retrospective analysis of 93 adult HCC patients underwent OLT between June 1985 and December 2003. Median follow-up was 28 months (1-222 months). The Milan criteria were routinely applied since 1994. RESULTS: Five year survival rate of HCC patients was significantly lower than in patients transplanted for benign diseases, 41 and 71%, respectively (p<0.0001). Multivariate analysis revealed that the presence of vascular invasion represents the most significant predictor (p<0.001) affecting the survival rate. Survival was also significantly impaired when the tumour size was >5 cm (p<0.05), whereas the number of nodules had no significant effect on survival. Consequently, the survival rate for HCC fulfilling the Milan criteria histologically improved to 70% since 1994. CONCLUSION: Tumour size has been shown to be the most important pre-operatively detectable predictor for patient survival after OLT.

Isolation, identification, and continuous culture of coronary endothelial cells from guinea pig hearts.

Viable and homogeneous endothelial cells were obtained from isolated guinea pig hearts by application of a special perfusion technique of the coronary system with an isotonic collagenase-trypsin solution and subsequent purification of the dissociated cells by Percoll density gradient centrifugation. The coronary endothelial cells were grown in tissue culture for periods up to 7 months. Serial passage proved to be possible. During logarithmic growth, generation time was found to be 18 h; it could be reduced to 16 h by addition of thrombin to the culture medium. Light, phase contrast and scanning electron microscopy as well as autoradiography revealed that cultured coronary endothelial cells grew as strict monolayers of closely apposed, polygonal large cells. By scanning electron microscopy, it could be demonstrated that the morphology of the cultured cells changes characteristically during attachment of the cells to their substratum. The changes observed were very similar to those of proliferating endothelial cells of isolated coronary vessels kept in organ culture. According to transmission electron microscopy studies, cultured coronary endothelial cells proved to contain only an extremely small number of Weibel-Palade bodies. Nucleoside phosphorylase (EC 2.4.2.5.) and 5'-nucleotidase (EC 3.1.3.5.) were identified in freshly isolated as well as in cultured endothelial cells. Their specific and total activities proved to be much higher than in myocardial tissue, thus indicating a prominent role of nucleotide metabolism in the coronary endothelium.

Detection of C-type natriuretic peptide (CNP) transcript in the rat heart and immune organs.

Previous studies suggested the expression of mRNA, coding for CNP, exclusively in the central nervous system. In the present study, using the polymerase chain reaction (PCR) technique instead of the less sensitive Northern blot hybridization, CNP-specific sequences have also been detected in rat atria and ventricles of the heart as well as in organs of the immune system (thymus, spleen and lymph nodes). Parallel PCR-assays documented ANP-mRNA in these tissues. To verify specificity of the PCR-products, Southern blots have been hybridized with a third internal oligonucleotide and amplification products have been sequenced. The relative level of CNP-mRNA in these tissues was estimated to be in the range of 1-9% of total brain CNP transcripts. The results suggest that the peptide may have a peripheral as well as a central site of action. In light of its pronounced effect on cell proliferation, particular interest should focus on a possible role of CNP in the immune system.

Transcription of brain natriuretic peptide and atrial natriuretic peptide genes in human tissues.

We have compared the expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) genes in various human tissues using a quantitative polymerase chain reaction technique. Tissues of three human subjects, obtained at autopsy, were analyzed. BNP transcripts could be detected in the central nervous system, lung, thyroid, adrenal, kidney, spleen, small intestine, ovary, uterus, and striated muscle. ANP transcripts could also be demonstrated in various human extracardiac tissues including several endocrine organs. In all peripheral tissues, the level of both natriuretic peptide transcripts was approximately 1-2 orders of magnitude lower than in cardiac ventricular tissues. This distribution is in marked contrast to the much lower level of ANP and BNP transcripts present in extracardiac rat tissues (generally less than 1/1000 of ventricles). These data suggest differential expression of the two natriuretic peptide genes in cardiac and extracardiac tissues in man. Furthermore, the presence of local synthesis of ANP and BNP in various peripheral organs suggests paracrine and/or autocrine function of these natriuretic peptides.

Microcirculatory failure after rat liver transplantation is related to Kupffer cell-derived oxidant stress but not involved in early graft dysfunction.

BACKGROUND: Microcirculatory failure, activation of Kupffer cells (KC), and the formation of reactive oxygen species (ROS) are considered pivotal mechanisms of reperfusion injury after orthotopic liver transplantation. However, the sequence of these events and their impact on early graft function remain controversial. We therefore investigated whether KC induce microcirculatory disturbances through ROS release and whether microcirculatory failure contributes to early graft function after liver transplantation. METHODS: Donor livers of Lewis rats were pretreated either with saline or with gadolinium chloride (GdCl3), an inhibitor of KC function (n=8 each). Syngeneic OLT was performed after 24 hr of hypothermic preservation in University of Wisconsin solution. RESULTS: Intravital microscopy revealed significantly higher sinusoidal perfusion rates in GdCl3-treated allografts (92+/-1.1% vs. 75.7+/-0.8%; P<0.001) compared with untreated controls; permanent leukocyte sticking in sinusoids (23.5+/-2.1 vs. 62.6+/-3.3 cells/lobule, P<0.001) and in postsinusoidal venules (153.1+/-10.4 vs. 446.6+/-46.4 cells/mm(2), P<0.001) were markedly attenuated in GdCl3-treated allografts. Improvement of microcirculatory parameters in GdCl3-treated livers was correlated with a significant reduction of plasma glutathione disulfide formation by KC-derived ROS (0.96+/-0.1 microM vs. 1.79+/-0.5 microM; P<0.01). Despite these beneficial effects, GdCl3-pretreatment failed to improve postischemic alanine aminotransferase release and bile flow. CONCLUSIONS: Microcirculatory failure after liver transplantation is related to KC-derived oxidant stress but not involved in early graft dysfunction.

The actions of human atrial natriuretic factor on hepatic arterial and portal vascular beds of the anaesthetized dog.

1. The vascular actions of atrial natriuretic factor (ANF) have been assessed with other vasoactive agents on the hepatic arterial and portal vascular beds of the anaesthetized dog. 2. Intra-arterial bolus injections of ANF (0.1-50 nmol) caused graded increases in hepatic arterial blood flow representing a vasodilatation of relatively short duration. Vasoconstriction was never observed. 3. The maximum increase in hepatic arterial blood was the same for ANF and isoprenaline (Iso) i.e. approximately 60-70% increase over control flow. 4. On a molar basis, ANF was less potent than Iso although over the higher dose range (10(-9)-10(-7) mol) its vasodilator activity exceeded that of the endogenous vasodilator adrenaline. 5. Intraportal bolus injections (1.0-50 nmol) of ANF did not alter portal inflow resistance since no changes in portal inflow pressure occurred when the portal circuit was perfused at constant inflow volume. 6. This differential action of ANF on the hepatic arterial and portal vascular beds may provide a change in total liver blood flow in favour of the arterial component. 7. ANF, by altering hepatic haemodynamics to favour formation of trans-sinusoidal fluid exchange, may provide a temporary expansion of the extravascular fluid reservoir to buffer any increased venous pressure. However, chronically elevated plasma levels of ANF would encourage the formation of ascitic fluid.