Micro-RNA profiling in human serum reveals compartment-specific roles of miR-571 and miR-652 in liver cirrhosis

Background and Aims

Micro-RNAs (miRNAs) have recently emerged as crucial modulators of molecular processes involved in chronic liver diseases. The few miRNAs with previously proposed roles in liver cirrhosis were identified in screening approaches on liver parenchyma, mostly in rodent models. Therefore, in the present study we performed a systematic screening approach in order to identify miRNAs with altered levels in the serum of patients with chronic liver disease and liver cirrhosis.

Methods

We performed a systematic, array-based miRNA expression analysis on serum samples from patients with liver cirrhosis. In functional experiments we evaluated the relationship between alterations of miRNA serum levels and their role in distinct cellular compartments involved in hepatic cirrhosis.

Results

The array analysis and the subsequent confirmation by qPCR in a larger patient cohort identified significant alterations in serum levels of miR-513-3p, miR-571 and miR-652, three previously uncharacterized miRNAs, in patients with alcoholic or hepatitis C induced liver cirrhosis. Of these, miR-571 serum levels closely correlated with disease stages, thus revealing potential as a novel biomarker for hepatic cirrhosis. Further analysis revealed that up-regulation of miR-571 in serum reflected a concordant regulation in cirrhotic liver tissue. In isolated primary human liver cells, miR-571 was up-regulated in human hepatocytes and hepatic stellate cells in response to the pro-fibrogenic cytokine TGF-β. In contrast, alterations in serum levels of miR-652 were stage-independent, reflecting a concordant down-regulation of this miRNA in circulating monocytes of patients with liver cirrhosis, which was inducible by proinflammatory stimuli like bacterial lipopolysaccharide.

Conclusion

Alterations of miR571 and miR-652 serum levels in patients with chronic liver disease reflect their putative roles in the mediation of fibrogenic and inflammatory processes in distinct cellular compartments involved in the pathogenesis of liver cirrhosis.     

Increased diacylglycerols characterize hepatic lipid changes in progression of human nonalcoholic fatty liver disease; comparison to a murine model

Background and Aims

The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and progression to cirrhosis. While differences in liver lipids between disease states have been reported, precise composition of phospholipids and diacylglycerols (DAG) at a lipid species level has not been previously described. The goal of this study was to characterize changes in lipid species through progression of human NAFLD using advanced lipidomic technology and compare this with a murine model of early and advanced NAFLD.

Methods

Utilizing mass spectrometry lipidomics, over 250 phospholipid and diacylglycerol species (DAGs) were identified in normal and diseased human and murine liver extracts.

Results

Significant differences between phospholipid composition of normal and diseased livers were demonstrated, notably among DAG species, consistent with previous reports that DAG transferases are involved in the progression of NAFLD and liver fibrosis. In addition, a novel phospholipid species (ether linked phosphatidylinositol) was identified in human cirrhotic liver extracts.

Conclusions

Using parallel lipidomics analysis of murine and human liver tissues it was determined that mice maintained on a high-fat diet provide a reproducible model of NAFLD in regards to specificity of lipid species in the liver. These studies demonstrated that novel lipid species may serve as markers of advanced liver disease and importantly, marked increases in DAG species are a hallmark of NAFLD. Elevated DAGs may contribute to altered triglyceride, phosphatidylcholine (PC), and phosphatidylethanolamine (PE) levels characteristic of the disease and specific DAG species might be important lipid signaling molecules in the progression of NAFLD.     

Serum Autotaxin Is a Parameter for the Severity of Liver Cirrhosis and Overall Survival in Patients with Liver Cirrhosis – A Prospective Cohort Study

Background

Autotaxin (ATX) and its product lysophosphatidic acid (LPA) are considered to be involved in the development of liver fibrosis and elevated levels of serum ATX have been found in patients with hepatitis C virus associated liver fibrosis. However, the clinical role of systemic ATX in the stages of liver cirrhosis was unknown. Here we investigated the relation of ATX serum levels and severity of cirrhosis as well as prognosis of cirrhotic patients.

Methods

Patients with liver cirrhosis were prospectively enrolled and followed until death, liver transplantation or last contact. Blood samples drawn at the day of inclusion in the study were assessed for ATX content by an enzyme-linked immunosorbent assay. ATX levels were correlated with the stage as well as complications of cirrhosis. The prognostic value of ATX was investigated by uni- and multivariate Cox regression analyses. LPA concentration was determined by liquid chromatography-tandem mass spectrometry.

Results

270 patients were enrolled. Subjects with liver cirrhosis showed elevated serum levels of ATX as compared to healthy subjects (0.814±0.42 mg/l vs. 0.258±0.40 mg/l, P<0.001). Serum ATX levels correlated with the Child-Pugh stage and the MELD (model of end stage liver disease) score and LPA levels (r = 0.493, P = 0.027). Patients with hepatic encephalopathy (P = 0.006), esophageal varices (P = 0.002) and portal hypertensive gastropathy (P = 0.008) had higher ATX levels than patients without these complications. Low ATX levels were a parameter independently associated with longer overall survival (hazard ratio 0.575, 95% confidence interval 0.365–0.905, P = 0.017).

Conclusion

Serum ATX is an indicator for the severity of liver disease and the prognosis of cirrhotic patients.     

Correlation of MicroRNA-16, MicroRNA-21 and MicroRNA-101 Expression with Cyclooxygenase-2 Expression and Angiogenic Factors in Cirrhotic and Noncirrhotic Human Hepatocellular Carcinoma

Background

Hepatocellular carcinoma (HCC) is a classical example of inflammation-linked cancer and is characterized by hypervascularity suggesting rich angiogenesis. Cycloxygenase-2 (COX-2) is a potent mediator of inflammation and is considered to upregulate angiogenesis. The aims of the study are (1) to analyze expression of Cox-2 mRNA, Cox-2 protein, miR-16, miR-21 and miR-101 in HCC and adjacent liver parenchyma in cirrhotic and noncirrhotic liver, (2) to investigate the relation between COX-2 expression, miR-21 expression and angiogenic factors in these tissues and (3) to investigate the association between miR-16 and miR-101 and COX-2 expression.

Methods

Tissue samples of HCC and adjacent liver parenchyma of 21 noncirrhotic livers and 20 cirrhotic livers were analyzed for COX-2 expression at the mRNA level (qRT-PCR) and at the protein level by Western blot and immunohistochemistry. Gene expression of VEGFA, VEGFR1, VEGFR2, Ang-1, Ang-2 and Tie-2 were correlated with COX-2 levels. miR-16, miR-21 and miR-101 gene expression levels were quantified in HCC tumor tissue.

Results

COX-2 mRNA and protein levels were lower in HCC as compared to adjacent liver parenchyma both in cirrhotic and noncirrhotic liver. COX-2 protein localized mainly in vascular and sinusoidal endothelial cells and in Kupffer cells. At the mRNA level but not at the protein level, COX-2 correlated with mRNA levels of angiogenic factors VEGFR1, Ang-1, and Tie2. miR-21 expression was higher in cirrhotic tissues versus noncirrhotic tissues. MiR-101 expression was lower in cirrhotic versus noncirrhotic adjacent liver parenchyma. None of the miRNAs correlelated with COX-2 expression. miR-21 correlated negatively with Tie-2 receptor in adjacent liver parenchyma.

Conclusions

In human HCC, COX-2 mRNA but not COX-2 protein levels are associated with expression levels of angiogenic factors. MiR-21 levels are not associated with angiogenic molecules. MiR-16 and miR-101 levels do not correlate with COX-2 mRNA and protein levels.     

Functional gene arrays-based analysis of fecal microbiomes in patients with liver cirrhosis

Background

Human gut microbiota plays an important role in the pathogenesis of cirrhosis complications. Although the phylogenetic diversity of intestinal microbiota in patients with liver cirrhosis has been examined in several studies, little is known about their functional composition and structure.

Results

To characterize the functional gene diversity of the gut microbiome in cirrhotic patients, we recruited a total of 42 individuals, 12 alcoholic cirrhosis patients, 18 hepatitis B virus (HBV)-related cirrhosis patients, and 12 normal controls. We determined the functional structure of these samples using a specific functional gene array, which is a combination of GeoChip for monitoring biogeochemical processes and HuMiChip specifically designed for analyzing human microbiomes. Our experimental data showed that the microbial community functional composition and structure were dramatically distinctive in the alcoholic cirrhosis. Various microbial functional genes involved in organic remediation, stress response, antibiotic resistance, metal resistance, and virulence were highly enriched in the alcoholic cirrhosis group compared to the control group and HBV-related cirrhosis group. Cirrhosis may have distinct influences on metabolic potential of fecal microbial communities. The abundance of functional genes relevant to nutrient metabolism, including amino acid metabolism, lipid metabolism, nucleotide metabolism, and isoprenoid biosynthesis, were significantly decreased in both alcoholic cirrhosis group and HBV-related cirrhosis group. Significant correlations were observed between functional gene abundances and Child-Pugh scores, such as those encoding aspartate-ammonia ligase, transaldolase, adenylosuccinate synthetase and IMP dehydrogenase.

Conclusions

Functional gene array was utilized to study the gut microbiome in alcoholic and HBV-related cirrhosis patients and controls in this study. Our array data indicated that the functional composition of fecal microbiomes was heavily influenced by cirrhosis, especially by alcoholic cirrhosis. This study provides new insights into the functional potentials and activity of gut microbiota in cirrhotic patients with different etiologies.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-753) contains supplementary material, which is available to authorized users.     

Pathological Changes in Pulmonary Circulation in Carbon Tetrachloride (ccl4)-Induced Cirrhotic Mice

Rationale

Lack of an experimental model of portopulmonary hypertension (POPH) has been a major obstacle in understanding of pathophysiological mechanisms underlying the disease.

Objective

We investigated the effects of CCl₄-mediated cirrhosis on the pulmonary vasculature, as an initial step towards an improved understanding of POPH.

Methods And Results

Male C57BL/6 mice received intraperitoneal injection of either sterile olive oil or CCl₄ 3 times/week for 12 weeks. Cirrhosis and portal hypertension were confirmed by evidence of bridging fibrosis and nodule formation in CCl₄-treated liver determined by trichrome/picrosirius red staining and an increase in spleen weight/body weight ratio, respectively. Staining for the oxidative stress marker, 4-hydroxynonenal (4-HNE), was strong in the liver but was absent in the lung, suggesting that CCl₄ did not directly induce oxidative injury in the lung. Pulmonary acceleration time (PAT) and the ratio of PAT/pulmonary ejection time (PET) measured by echocardiography were significantly decreased in cirrhotic mice. Increase in right ventricle (RV) weight/body weight as well as in the weight ratio of RV/(left ventricle + septum) further demonstrated the presence of pathological changes in the pulmonary circulation in these mice. Histological examination revealed that lungs of cirrhotic mice have excessive accumulation of perivascular collagen and thickening of the media of the pulmonary artery.

Conclusion

Collectively, our data demonstrate that chronic CCl₄ treatment induces pathological changes in pulmonary circulation in cirrhotic mice. We propose that this murine cirrhotic model provides an exceptional tool for future studies of the molecular mechanisms mediating pulmonary vascular diseases associated with cirrhosis and for evaluation of novel therapeutic interventions.     

NT Pro BNP Plasma Level and Atrial Volume Are Linked to the Severity of Liver Cirrhosis

Background and Aims

Plasma levels of NT-pro-BNP, a natriuretic peptide precursor, are raised in the presence of fluid retention of cardiac origin and can be used as markers of cardiac dysfunction. Recent studies showed high levels of NT pro BNP in patients with cirrhosis. We assessed NT pro-BNP and other parameters of cardiac dysfunction in patients with cirrhosis, with or without ascites, in order to determine whether the behaviour of NT pro BNP is linked to the stage of liver disease or to secondary cardiac dysfunction.

Methods

Fifty eight consecutive hospitalized patients mostly with viral or NAFLD-related cirrhosis were studied. All underwent abdominal ultrasound and upper GI endoscopy. Cardiac morpho-functional changes were evaluated by echocardiography and NT-pro-BNP plasma levels determined upon admission. Twenty-eight hypertensive patients, without evidence of liver disease served as controls.

Results

Fifty eight cirrhotic patients (72% men) with a median age of 62 years (11% with mild arterial hypertension and 31% with type 2 diabetes) had a normal renal function (mean creatinine 0.9 mg/dl, range 0.7–1.06). As compared to controls, cirrhotic patients had higher NT pro-BNP plasma levels (365.2±365.2 vs 70.8±70.6 pg/ml; p<0.001). Left atrial volume (LAV) (61.8±26.3 vs 43.5±14.1 ml; p = 0.001), and left ventricular ejection fraction (62.7±6.9 vs. 65.5±4%,; p = 0.05) were also altered in cirrhotic patients that in controls. Patients with F2-F3 oesophageal varices as compared to F0/F1, showed higher e'' velocity (0.91±0.23 vs 0.66±0.19 m/s, p<0.001), and accordingly a higher E/A ratio (1.21±0.46 vs 0.89±0.33 m/s., p = 0.006).

Conclusion

NT-pro-BNP plasma levels are increased proportionally to the stage of chronic liver disease. Advanced cirrhosis and high NT-pro-BNP levels are significantly associated to increased LAV and to signs of cardiac diastolic dysfunction. NT pro-BNP levels could hence be an useful prognostic indicators of early decompensation of cirrhosis.     

Altered Levels of Acetylcholinesterase in Alzheimer Plasma

Background

Many studies have been conducted in an extensive effort to identify alterations in blood cholinesterase levels as a consequence of disease, including the analysis of acetylcholinesterase (AChE) in plasma. Conventional assays using selective cholinesterase inhibitors have not been particularly successful as excess amounts of butyrylcholinesterase (BuChE) pose a major problem.

Principal Findings

Here we have estimated the levels of AChE activity in human plasma by first immunoprecipitating BuChE and measuring AChE activity in the immunodepleted plasma. Human plasma AChE activity levels were ∼20 nmol/min/mL, about 160 times lower than BuChE. The majority of AChE species are the light G₁+G₂ forms and not G₄ tetramers. The levels and pattern of the molecular forms are similar to that observed in individuals with silent BuChE. We have also compared plasma AChE with the enzyme pattern obtained from human liver, red blood cells, cerebrospinal fluid (CSF) and brain, by sedimentation analysis, Western blotting and lectin-binding analysis. Finally, a selective increase of AChE activity was detected in plasma from Alzheimer''s disease (AD) patients compared to age and gender-matched controls. This increase correlates with an increase in the G₁+G₂ forms, the subset of AChE species which are increased in Alzheimer''s brain. Western blot analysis demonstrated that a 78 kDa immunoreactive AChE protein band was also increased in Alzheimer''s plasma, attributed in part to AChE-T subunits common in brain and CSF.

Conclusion

Plasma AChE might have potential as an indicator of disease progress and prognosis in AD and warrants further investigation.     

The Role of miRNA-34a as a Prognostic Biomarker for Cirrhotic Patients with Portal Hypertension Receiving TIPS

Background

Circulating miRNA-34a is increased in blood of patients with different liver diseases when compared to healthy controls. However, the origin of miRNA-34a and its possible relationship with hemodynamics and outcome in cirrhotic patients with portal hypertension is unknown. We analyzed the levels of miRNA-34a in cirrhotic patients with severe portal hypertension.

Methods

We included 60 cirrhotic patients receiving TIPS for prevention of rebleeding and/or therapy-refractory ascites. miRNA-34a levels were measured using qPCR and normalized by SV-40 in the portal and hepatic venous blood of these patients taken at TIPS procedure. Hemodynamic and clinical parameters were assessed before TIPS and during follow-up.

Results

Levels of miRNA-34a were higher in the hepatic vein than in the portal vein. Circulating miRNA-34a in the hepatic vein correlated with ALT, CHE and sodium excretion after TIPS. miRNA-34a showed no correlation with portal pressure, but its levels in the portal vein correlated inversely with the congestion index. Interestingly, the levels of miRNA-34a in the portal and hepatic vein showed inverse correlation with arterial pressure. Furthermore, levels of miRNA-34a in the hepatic vein had a predictive value for survival, but MELD, creatinine at short-time follow-up 14 days after TIPS-insertion and portal pressure after TIPS performed better.

Conclusion

This study demonstrates for the first time, that miRNA-34a may originate to a large extent from the liver. Even though higher levels of miRNA-34a are possibly associated with better survival at long-term follow-up in cirrhotic patients with severe portal hypertension receiving TIPS, classical prognostic parameters predict the survival better.     

Altered Functionality of Anti-Bacterial Antibodies in Patients with Chronic Hepatitis C Virus Infection

Background

Using comparative glycoproteomics, we have previously identified a glycoprotein that is altered in both amount and glycosylation as a function of liver cirrhosis. The altered glycoprotein is an agalactosylated (G0) immunoglobulin G molecule (IgG) that recognizes the heterophilic alpha-gal epitope. Since the alpha gal epitope is found on gut enterobacteria, it has been hypothesized that anti-gal antibodies are generated as a result of increased bacterial exposure in patients with liver disease.

Methods

The N-linked glycosylation of anti-gal IgG molecules from patients with fibrosis and cirrhosis was determined and the effector function of anti-bacterial antibodies from over 100 patients examined. In addition, markers of microbial exposure were determined.

Results

Surprisingly, the subset of agalactosylated anti-gal antibodies described here, was impaired in their ability to mediate complement mediated lysis and inhibited the complement-mediated destruction of common gut bacteria. In an analysis of serum from more than 100 patients with liver disease, we have shown that those with increased levels of this modified anti-gal antibody had increased levels of markers of bacterial exposure.

Conclusions

Anti-gal antibodies in patients with liver cirrhosis were reduced in their ability to mediate complement mediated lysis of target cells. As bacterial infection is a major complication in patients with cirrhosis and bacterial products such as LPS are thought to play a major role in the development and progression of liver fibrosis, this finding has many clinical implications in the etiology, prognosis and treatment of liver disease.     

Cirrhosis,Liver Transplantation and HIV Infection Are Risk Factors Associated with Hepatitis E Virus Infection

Background

Acute and chronic hepatitis E have been associated with high mortality and development of cirrhosis, particularly in solid-organ recipients and patients infected by human immunodeficiency virus. However, data regarding the epidemiology of hepatitis E in special populations is still limited.

Aims

Investigate seroprevalence and possible factors associated with HEV infection in a large cohort of immunosuppressed patients.

Methods

Cross-sectional study testing IgG anti-HEV in serum samples from 1373 consecutive individuals: 332 liver-transplant, 296 kidney-transplant, 6 dual organ recipients, 301 non-transplanted patients with chronic liver disease, 238 HIV-infected patients and 200 healthy controls.

Results

IgG anti-HEV was detected in 3.5% controls, 3.7% kidney recipients, 7.4% liver transplant without cirrhosis and 32.1% patients who developed post-transplant cirrhosis (p<0.01). In patients with chronic liver disease, IgG anti-HEV was also statistically higher in those with liver cirrhosis (2% vs 17.5%, p<0.01). HIV-infected patients showed an IgG anti-HEV rate of 9.2%, higher than those patients without HIV infection (p<0.03). Multivariate analysis showed that the factors independently associated with anti-HEV detection were liver cirrhosis, liver transplantation and HIV infection (OR: 7.6, 3.1 and 2.4). HCV infection was a protective factor for HEV infection (OR: 0.4).

Conclusions

HEV seroprevalence was high in liver transplant recipients, particularly those with liver cirrhosis. The difference in anti-HEV prevalence between Liver and Kidney transplanted cases suggests an association with advanced liver disease. Further research is needed to ascertain whether cirrhosis is a predisposing factor for HEV infection or whether HEV infection may play a role in the pathogeneses of cirrhosis.     

Decreased Serum Hepcidin Concentration Correlates with Brain Iron Deposition in Patients with HBV-Related Cirrhosis

Purpose

Excessive brain iron accumulation contributes to cognitive impairments in hepatitis B virus (HBV)-related cirrhotic patients. The underlying mechanism remains unclear. Hepcidin, a liver-produced, 25-aminoacid peptide, is the major regulator of systemic iron metabolism. Abnormal hepcidin level is a key factor in some body iron accumulation or deficiency disorders, especially in those associated with liver diseases. Our study was aimed to explore the relationship between brain iron content in patients with HBV-related cirrhosis and serum hepcidin level.

Methods

Seventy HBV-related cirrhotic patients and forty age- sex-matched healthy controls were enrolled. Brain iron content was quantified by susceptibility weighted phase imaging technique. Serum hepcidin as well as serum iron, serum transferrin, ferritin, soluble transferrin receptor, total iron binding capacity, and transferrin saturation were tested in thirty cirrhotic patients and nineteen healthy controls. Pearson correlation analysis was performed to investigate correlation between brain iron concentrations and serum hepcidin, or other iron parameters.

Results

Cirrhotic patients had increased brain iron accumulation compared to controls in the left red nuclear, the bilateral substantia nigra, the bilateral thalamus, the right caudate, and the right putamen. Cirrhotic patients had significantly decreased serum hepcidin concentration, as well as lower serum transferring level, lower total iron binding capacity and higher transferrin saturation, compared to controls. Serum hepcidin level negatively correlated with the iron content in the right caudate, while serum ferritin level positively correlated with the iron content in the bilateral putamen in cirrhotic patients.

Conclusions

Decreased serum hepcidin level correlated with excessive iron accumulation in the basal ganglia in HBV-related cirrhotic patients. Our results indicated that systemic iron overload underlined regional brain iron repletion. Serum hepcidin may be a clinical biomarker for brain iron deposition in cirrhotic patients, which may have therapeutic potential.     

The Roles of Albumin Levels in Head and Neck Cancer Patients with Liver Cirrhosis Undergoing Tumor Ablation and Microsurgical Free Tissue Transfer

Objective

To evaluate the changes of serum albumin levels during the peri-operative period, and correlate these changes to surgical outcomes, postoperative morbidity and mortality in head and neck cancer patients with cirrhosis.

Methods

57 patients with liver cirrhosis out of 3,022 patients who underwent immediate free flap reconstruction after surgical ablation of head and neck cancer performed over a 9-year period were included in the study. Two sets of groups were arranged based on the preoperative albumin (>3.5 g/dL vs. ≤ 3.5 g/dL) and POD1 albumin (>2.7 g/dL vs. ≤ 2.7 g/dL) levels and were compared with respect to patient-related variables, surgical outcomes, medical and surgical complications, and mortalities.

Results

All patients had significant decreases in albumin levels postoperatively. Hypoalbuminemia, both preoperative and postoperative, was associated with the model for end-stage liver disease (MELD) score, the amount of blood loss, the duration of ICU stay and hospital stay, and postoperative medical and surgical complications. In particular, preoperative hypoalbuminemia (serum albumin ≤ 3.5 g/dL) was associated strongly with medical complications and mortality, while postoperative hypoalbuminemia (serum albumin ≤ 2.7 g/dL) with surgical complications.

Conclusion

Our study demonstrated the prognostic values of albumin levels in head and neck cancer patient with liver cirrhosis. The perioperative albumin levels can be utilized for risk stratification to potentially improve surgical and postoperative management of these challenging patients.     

Reversal of Succinylcholine Induced Apnea with an Organophosphate Scavenging Recombinant Butyrylcholinesterase

Background

Concerns about the safety of paralytics such as succinylcholine to facilitate endotracheal intubation limit their use in prehospital and emergency department settings. The ability to rapidly reverse paralysis and restore respiratory drive would increase the safety margin of an agent, thus permitting the pursuit of alternative intubation strategies. In particular, patients who carry genetic or acquired deficiency of butyrylcholinesterase, the serum enzyme responsible for succinylcholine hydrolysis, are susceptible to succinylcholine-induced apnea, which manifests as paralysis, lasting hours beyond the normally brief half-life of succinylcholine. We hypothesized that intravenous administration of plant-derived recombinant BChE, which also prevents mortality in nerve agent poisoning, would rapidly reverse the effects of succinylcholine.

Methods

Recombinant butyrylcholinesterase was produced in transgenic plants and purified. Further analysis involved murine and guinea pig models of succinylcholine toxicity. Animals were treated with lethal and sublethal doses of succinylcholine followed by administration of butyrylcholinesterase or vehicle. In both animal models vital signs and overall survival at specified intervals post succinylcholine administration were assessed.

Results

Purified plant-derived recombinant human butyrylcholinesterase can hydrolyze succinylcholine in vitro. Challenge of mice with an LD₁₀₀ of succinylcholine followed by BChE administration resulted in complete prevention of respiratory inhibition and concomitant mortality. Furthermore, experiments in symptomatic guinea pigs demonstrated extremely rapid succinylcholine detoxification with complete amelioration of symptoms and no apparent complications.

Conclusions

Recombinant plant-derived butyrylcholinesterase was capable of counteracting and reversing apnea in two complementary models of lethal succinylcholine toxicity, completely preventing mortality. This study of a protein antidote validates the feasibility of protection and treatment of overdose from succinylcholine as well as other biologically active butyrylcholinesterase substrates.     

IgM-linked SerpinB3 and SerpinB4 in sera of patients with chronic liver disease

Background

Epidemiological studies indicate that a growing number of cirrhotic patients will develop hepatocellular carcinoma (HCC) in the next decade. Recent findings have demonstrated that Squamous cell carcinoma antigen 1 (SCCA1) and 2 (SCCA2) isoforms, now classified as serpinB3 and serpinB4, are over-expressed in HCC, but not in normal liver. As reported, high levels of circulating SCCA-IgM immunocomplexes in patients with cirrhosis are significantly associated with HCC development.

Aim

To ascertain whether IgM-linked SCCA isoforms circulate in patients with chronic liver disease, compared to total SCCA-IgM levels.

Methodology and Findings

79 patients with chronic liver disease were studied, including 17 patients with chronic hepatitis, 36 patients with cirrhosis and 26 with HCC. 28 blood donors were used as control. Monoclonal antibodies against serpinB3 and serpinB4 were used as catcher antibodies to set up specific ELISA assays, while total SCCA-IgM immunocomplexes were detected by commercially available ELISA assay. Overall, the results revealed a better diagnostic sensitivity of total SCCA-IgM assay, compared to both serpinB3 and serpinB4 IgM-linked assays. SerpinB4-IgM median values obtained with SCC103 antibody were moderately higher in patients with cirrhosis than in those with HCC, median values: 0.168 (IQR 0.140–0.427) vs. 0.140 (IQR 0.140–0.278), (p = 0.177). A trend toward decreasing serpinB4-IgM/serpinB3-IgM median ratio was observed in patients with advanced liver disease, being 1.08 in patients with HCC, 1.10 in patients with cirrhosis and 1.40 in patients with chronic hepatitis (p = 0.079).

Conclusions

IgM-linked SCCA isoforms in serum of patients with chronic liver diseases were quantified for the first time. Although the number of patients was limited, this preliminary study reveals that the relative balance of the two serpin isoforms is altered in HCC and it is characterized by a lower serpinB4-IgM/serpinB3-IgM ratio, determined by lower serpinB4 levels.     

Novel Bioimaging Techniques of Metals by Laser Ablation Inductively Coupled Plasma Mass Spectrometry for Diagnosis Of Fibrotic and Cirrhotic Liver Disorders

Background and Aims

Hereditary disorders associated with metal overload or unwanted toxic accumulation of heavy metals can lead to morbidity and mortality. Patients with hereditary hemochromatosis or Wilson disease for example may develop severe hepatic pathology including fibrosis, cirrhosis or hepatocellular carcinoma. While relevant disease genes are identified and genetic testing is applicable, liver biopsy in combination with metal detecting techniques such as energy-dispersive X-ray spectroscopy (EDX) is still applied for accurate diagnosis of metals. Vice versa, several metals are needed in trace amounts for carrying out vital functions and their deficiency due to rapid growth, pregnancy, excessive blood loss, and insufficient nutritional or digestive uptake results in organic and systemic shortcomings. Established in situ techniques, such as EDX-ray spectroscopy, are not sensitive enough to analyze trace metal distribution and the quantification of metal images is difficult.

Methods

In this study, we developed a quantitative biometal imaging technique of human liver tissue by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) in order to compare the distribution of selected metals in cryo-sections of healthy and fibrotic/cirrhotic livers.

Results

Most of the metals are homogeneous distributed within the normal tissue, while they are redirected within fibrotic livers resulting in significant metal deposits. Moreover, total iron and copper concentrations in diseased liver were found about 3-5 times higher than in normal liver samples.

Conclusions

Biometal imaging via LA-ICP-MS is a sensitive innovative diagnostic tool that will impact clinical practice in identification and evaluation of hepatic metal disorders and to detect subtle metal variations during ongoing hepatic fibrogenesis.     

Spironolactone lowers portal hypertension by inhibiting liver fibrosis, ROCK-2 activity and activating NO/PKG pathway in the bile-duct-ligated rat

Objective

Aldosterone, one of the main peptides in renin angiotensin aldosterone system (RAAS), has been suggested to mediate liver fibrosis and portal hypertension. Spironolactone, an aldosterone antagonist, has beneficial effect on hyperdynamic circulation in clinical practice. However, the mechanisms remain unclear. The present study aimed to investigate the role of spionolactone on liver cirrhosis and portal hypertension.

Methods

Liver cirrhosis was induced by bile duct ligation (BDL). Spironolactone was administered orally (20 mg/kg/d) after bile duct ligation was performed. Liver fibrosis was assessed by histology, Masson''s trichrome staining, and the measurement of hydroxyproline and type I collagen content. The activation of HSC was determined by analysis of alpha smooth muscle actin (α-SMA) expression. Protein expressions and protein phosphorylation were determined by immunohistochemical staining and Western blot analysis, Messenger RNA levels by quantitative real time polymerase chain reaction (Q-PCR). Portal pressure and intrahepatic resistance were examined in vivo.

Results

Treatment with spironolactone significantly lowered portal pressure. This was associated with attenuation of liver fibrosis, intrahepatic resistance and inhibition of HSC activation. In BDL rat liver, spironolactone suppressed up-regulation of proinflammatory cytokines (TNFα and IL-6). Additionally, spironolactone significantly decreased ROCK-2 activity without affecting expression of RhoA and Ras. Moreover, spironolactone markedly increased the levels of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS and the activity of NO effector- protein kinase G (PKG) in the liver.

Conclusion

Spironolactone lowers portal hypertension by improvement of liver fibrosis and inhibition of intrahepatic vasoconstriction via down-regulating ROCK-2 activity and activating NO/PKG pathway. Thus, early spironolactone therapy might be the optional therapy in cirrhosis and portal hypertension.     

Non-Invasive Evaluation of Cystic Fibrosis Related Liver Disease in Adults with ARFI, Transient Elastography and Different Fibrosis Scores

Background

Cystic fibrosis-related liver disease (CFLD) is present in up to 30% of cystic fibrosis patients and can result in progressive liver failure. Diagnosis of CFLD is challenging. Non-invasive methods for staging of liver fibrosis display an interesting diagnostic approach for CFLD detection.

Aim

We evaluated transient elastography (TE), acoustic radiation force impulse imaging (ARFI), and fibrosis indices for CFLD detection.

Methods

TE and ARFI were performed in 55 adult CF patients. In addition, AST/Platelets-Ratio-Index (APRI), and Forns'' score were calculated. Healthy probands and patients with alcoholic liver cirrhosis served as controls.

Results

Fourteen CF patients met CFLD criteria, six had liver cirrhosis. Elastography acquisition was successful in >89% of cases. Non-cirrhotic CFLD individuals showed elastography values similar to CF patients without liver involvement. Cases with liver cirrhosis differed significantly from other CFLD patients (ARFI: 1.49 vs. 1.13 m/s; p = 0.031; TE: 7.95 vs. 4.16 kPa; p = 0.020) and had significantly lower results than individuals with alcoholic liver cirrhosis (ARFI: 1.49 vs. 2.99 m/s; p = 0.002). APRI showed the best diagnostic performance for CFLD detection (AUROC 0.815; sensitivity 85.7%, specificity 70.7%).

Conclusions

ARFI, TE, and laboratory based fibrosis indices correlate with each other and reliably detect CFLD related liver cirrhosis in adult CF patients. CF specific cut-off values for cirrhosis in adults are lower than in alcoholic cirrhosis.     

Circulating microRNAs as a Fingerprint for Liver Cirrhosis

Background

Sensitive and specific detection of liver cirrhosis is an urgent need for optimal individualized management of disease activity. Substantial studies have identified circulation miRNAs as biomarkers for diverse diseases including chronic liver diseases. In this study, we investigated the plasma miRNA signature to serve as a potential diagnostic biomarker for silent liver cirrhosis.

Methods

A genome-wide miRNA microarray was first performed in 80 plasma specimens. Six candidate miRNAs were selected and then trained in CHB-related cirrhosis and controls by qPCR. A classifier, miR-106b and miR-181b, was validated finally in two independent cohorts including CHB-related silent cirrhosis and controls, as well as non−CHB-related cirrhosis and controls as validation sets, respectively.

Results

A profile of 2 miRNAs (miR-106b and miR-181b) was identified as liver cirrhosis biomarkers irrespective of etiology. The classifier constructed by the two miRNAs provided a high diagnostic accuracy for cirrhosis (AUC = 0.882 for CHB-related cirrhosis in the training set, 0.774 for CHB-related silent cirrhosis in one validation set, and 0.915 for non−CHB-related cirrhosis in another validation set).

Conclusion

Our study demonstrated that the combined detection of miR-106b and miR-181b has a considerable clinical value to diagnose patients with liver cirrhosis, especially those at early stage.